药学专业英语文章及翻译
Expression of livin in gastric cancer and induction of apoptosis in SGC-7901 cells by shRNA-mediated silencing of livin gene
Background-Because of increased resistance to apoptosis in tumor cells, inhibition of specific antiapoptotic factors may provide a rational approach for the development of novel therapeutic strategies.Livin, a novel inhibitor of apoptosis protein family, has been found to be expressed in various malignancies and is suggested to have poorly prognostic significance. However, no data are available concerning the significance of livin in gastric cancer. In this study, we detected the expression of livin in human gastric carcinoma and investigated the apoptotic susceptibility of SGC-7901 cell by shRNAmediated silencing of the livin gene.
Methods-The mRNA and protein expression of livin were analyzed by RT-PCR and western blot assay.The relationship between livin expression and clinical pathologic parameters was investigated. The small interfering RNA eukaryotic expression vector specific to livin was constructed by gene recombination, and the nucleic acid was sequenced. Then it was transfected into SGC-7901 cells by Lipofectamin 2000. RT-PCR and Western blot assay were used to validate gene-silencing efficiency of livin in SGC-7901 cells. Stable clones were obtained by G418 screening. The cell apoptosis was assessed by flow cytometry (FCM). Cell growth state and 50 % inhibition concentration (IC50) of 5-FU and cisplatin was determined by MTT method.
Results-The expression of livin mRNA and protein were detected in 19 of 40 gastric carcinoma cases (47.5%) and SGC-7901 cells. No expression of livin was detected in tumor adjacent tissues and benign gastric lesion. The positive correlation was found between livin expression and poor differentiation of tumors as well as lymph node metastases (P < 0.05). Four small interfering RNA eukaryotic expression vector specific to livin were constructed by gene recombination. And one of them can efficiently decrease the expression of livin, the inhibition of the gene was not less than 70% (P < 0.01). The recombinated plasmids were extracted and transfected gastric cancer cells. The stable clones were obtained by G418 screening, and were amplified and cultured. When livin gene was silenced, the reproductive activity of the gastric cancer cells was significantly lower than the control groups(P < 0.05). The study also showed that IC50 of 5-Fu and cisplatin on gastric cancer cells treated by shRNA was
decreased and the cells were more susceptible to proapoptotic stimuli (5-Fu and cisplatin) (P < 0.01).
Conclusions-C Livin is overexpressed in gastric carcinoma with a relationship to tumor differentiation and lymph node metastases, which is suggested to be one of the molecular prognostic factors for some cases of gastric cancer. ShRNA can inhibit livin expression in SGC-7901 cells and induce cell apoptosis.Livin may serve as a new target for apoptosis-inducing therapy of gastric cancer.
1. Introduction
Gastric cancer is one of the most common malignancies in the world. Most patients with this disease are diagnosed in advanced stages, and lose the chance of surgical eradication. Despite much progress in chemotherapy, the overall survival of the patients with gastric cancer in advanced stage is still poor. Resistance of cancer cells to chemoagents may contribute to failure of the treatment. Among the reasons of drug resistance, inhibited process of cell apoptosis may play an important role.
Cancer cells are often characterized by increased resistance to apoptosis [1], which mediates their increased resistance to various stimuli of cell apoptosis, such as DNA damage, hypoxia, nutrient-deprivation [2,3]. Moreover, apoptosis resistance is considered to be a major cause of therapeutic failure for tumors in clinical practice, since many chemo- and/or radiotherapeutic agents function through the induction of apoptotic tumor death [4].
Inhibitor of apoptosis protein (IAPs) is a novel family of intracellular proteins which suppress apoptosis induced by a variety of stimuli [5,6], including viral infection, chemotherapeutic drugs, staurosporin, growth factor withdrawal, and by components of the tumor necrosis factor-a (TNF-a)/Fas apoptotic signaling pathways [7¨C9]. The IAPs consists of a group of structurally related proteins with antiapoptotic properties [10], and may play a substantial role in preventing tumor cell from apoptosis, and has become the focus of research in recent years. A novel member of this family is ML-IAP/livin/KIAP/BIRC7 (in the following termed livin) which has two isoforms, livin a and livin b [11¨C14]. It has been shown that over-expression of the livin can block apoptosis induced by a variety of proapoptotic stimuli [12]. Interestingly, livin gene has been found to be restrictively expressed in tumor cells, but not, or to lesser amounts in most normal adult tissues [11¨C15], and may contribute to tumorigenesis by allowing malignant cell to avoid apoptotic cell death. So inhibition of livin expression may represent an interesting therapeutic strategy.
In the present study, we investigated the expression of livin in gastric cancinomas and their adjacent tissues. The relationship between livin expression and clinical pathologic parameters was analyzed. Furthermore, we explored the feasibility of shRNA in inhibiting livin gene expression and the apoptotic susceptibility of gastric cancer cell by shRNA-mediated silencing of the livin gene.
2. Patients and methods
2.1. Patients and tumor samples
Forty samples of gastric carcinoma and 13 samples of paracancerous tissues were collected from the patients who received gastrectomy (age of patients ranging from 29-77 years). Thirteen samples of benign gastric lesion (chronic superficial gastritis) were gained from the patients undergoing gastric endoscopic examination (age of patients ranging from 33-77 years). These samples were collected from patients admitted to the First Affiliated Hospital of Nanjing Medical University. The patients with gastric cancer were diagnosed as being in stage I to IV based on TNM classification (UICC, 2002). Tumor specimens were immediately frozen in liquid nitrogen after surgery and stored at -80℃until use. Informed consent was obtained from all patients.
2.2. RT-PCR procedure
Total RNA (2 mg) extracted from frozen tissues was reverse transcribed in a final volume of 25 ml with 100 pmol of oligo(dT)15 and 200U M-MLV reverse transcriptase (promega, USA), according to the manufacturer’s guidelines.
Aliquots corresponding to 2.5 ml cDNA were then amplified in PCR buffer containing 25pmol/ml each primer and 1 U Taq polymerase in a final volume of 50 ml. Each amplification was performed for 35 cycles, one cycle profile consisted of denaturationat 94 8C for 30 s, annealing at 59 8C (livin and b-actin) for 30 s and extension at 72 8C for 30 s. A sample without RNA was included in each RT¨CPCR as a negative control.
Sequences of livin and β-actin primers used are as follows:livina/b up stream,50-TCCACAGTGTGCAGGAGACT-30;livina/βdownstream,50-ACGGCACA AAGACGATGGAC-30;b-actinupstream,50-AGCGCAAGTACTCCGTGTG-30;β-acti n downstream, 50-AAGCAATGCTATCACCTCCC-30.The size of the amplified products were312/258 bp for livina/b and 501 bp for b-actin respectively.
2.3. Western Blot Analysis
Tissues were homogenized with lysis buffer [50 mM Tris-HCl(pH 7.5), 250 mM
NaCl, 0.1% NP40, and 5 mM EGTA containing 50 mM sodium fluoride, 60 mM b-glycerol-phosphate, 0.5 mM sodium vanadate, 0.1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml leupeptin. The total protein concentration was determined using Coomassie Brilliant Blue. Protein samples were electrophoresed in a 10% denaturing SDS gel and transferred to PVDF membrane (Roche, USA). The membranes were incubated with specific primary antibodies, reacted with a peroxidase-conjugated secondary antibody (Cell signaling technology,USA), and finally visualized by enhanced chemiluminescence (Cell signaling technology, USA). Monoclonal antibodies recognizing livin (1:250) and actin (1:400) were purchased from Alexesis Inc. (USA) and Santa Cruz Biotechnology (USA).
2.4. Cell lines and cell culture
We selected a human gastric adenocarcinoma cell lines for thisstudy. SGC-7901 (Shanghai Institute of Cell Research, Shanghai,China) is an adherent, moderately differentiated, human gastric adenocarcinoma cell line. The cell lines are gastric cancer epithelial cells and grow as adherent cells in RPMI 1640 (Hyclone Inc, USA)containing 10% FCS (Life Technologies, Inc.), 100 units/ml penicillin,and 100 mg/ml streptomycin (BioWhittaker). SGC-7901 cells were maintainedat37 8Cina humidified incubatorwithanatmosphere of 5% CO2. Cisplatin and 5-fluorouracil (Qilu pharmaceutical factory,China) were solublized in DMSO and stored at 4 8C.
2.5. ShRNA synthesis and construction of PGPU/GFP/Neo/livin plasmids
ShRNA sequences of livin were designed by software of siRNA Sequence-Selector and synthesized (Shanghai Biotech, Ltd.Corp., China). The sequences as following (Table 1)then were inserted into BbsI and BamH sites of the pGPU/GFP/Neo(Shanghai GenePharma Co. Ltd China) to generate pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control plasmids,respectively.
2.6. Establishment of SGC-7901 stable transfectants expressing pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control
For transfection experiments, SGC-7901 cells were plated into 6-well plates (3¡Á105 cells/well), 96-well plates (1×104cells/well) and 12-well plates (1.5×105 cells/well) for 24 h before transfection
The cells were transfected with 4 mg/well of empty pGPU/GFP/Neo/vector, pGPU/GFP/Neo/livin or pGPU/GFP/Neo/Control plasmid using Li-pofectAMINE 2000 (Life Technologies, Inc., Grand Island,NY) according to the manufacturer¡¯s instructions. Forty-eight hours after transfection, the cells were passaged at 1:15 (v/v)
and cultured in mediumsupplemented with Geneticin (G418) at 1000 g/ml for 4 weeks. Stably transfected clones were picked and maintained in medium containing 400 g/ml G418 for additional studies.
2.7. Assay of anchorage-dependent cell growth
Parent cells and cells stably expressing empty pGPU/GFP/Neo vector, pGPU/GFP/Neo/livin or pGPU/GFP/Neo/Control were seeded into 6-well plates. Cells from triplicate wells were collected every other day. Cell numbers were determined using a Coulter counter (Coulter Electronics, Miami, FL). The number of cells per well is reported as the average SD at the indicated number of days after plating.
2.8. MTT assay
Cytotoxicity was measured by MTT assay. Cells growing exponentially were plated onto 96-well plates at a density of 10000 cells/well for 24 h. The cells were then treated with different concentrations of drugs for 48 h. One hundred microliters of MTT stock solution (1 mg/ml) were added to each well, and the cells were further incubated at 37℃for 4 h. The supernatant was replaced with isopropyl alcohol to dissolve formazan production. The absorbance at wavelength 595 nm was measured with a micro-ELISA reader (ClinBio-128, SLT, Austria). The ratio of the absorbance of treated cells relative to that of the control cells was calculated and expressed as a percentage of cell death.
2.9. Flow cytometry
Cells were collected and fixed with ice-cold 70% ethanol in PBS and stored at -4℃until use. After resuspension, cells were incubated with 100 ml of RNase I (1 mg/ml) and 100 ml of PI (400 mg/ml) at 37℃ and analyzed by flow cytometry (BD, USA).
2.10. Statistical analysis
Data were expressed as the means of at least three different experiments SD. The results were analyzed by Student’s t-test, and P < 0.05 was considered statistically significant.
3. Results
3.1. Expression of livin in gastric carcinomas
In the present study, for the first time, we evaluated by RT¨CPCR and westen blot the presence of livin expression in 40 gastric cancinomas, 13 para-cancerous tissues and 13 benign lesions of gastric mucosa. In para-cancerous tissues and benign lesions of gastric mucosa, no detectable levels of either mRNA isoforms were revealed, while
among tumor tissues, 19/40(47.5%) showed mRNA and protein expression of livina and livinb (Figs. 1 and 2). Livin expression correlated with some of the known prognostic variables, such as histologic grade and lymph node metastasis, but not with age, sexuality, stage and tumor infiltration extent (Table 2).
3.2. Characterization of stable transfectants expressing pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control
We established SGC-7901 stable transfectants with either pGPU/GFP/Neo/livin, pGPU/GFP/Neo/Control plasmid, or empty pGPU/GFP/Neo/vector (Fig. 3). Some clones from each transfection were selected and analyzed by RT-PCR and Western blot to determine the livin mRNA and protein expression, and others were selected for expansion and additional studies. As shown in (Figs. 4 and 5), the level of livin mRNA and protein in SGC-7901 pGPU/GFP/Neo/livin2 transfectants was reduced by more than 90%. The suppression of livin expression was not observed in pGPU/GFP/Neo/livin1 transfectants and negative control. So SGC-7901 pGPU/GFP/Neo/livin2 transfectants was chosen for subsequent experiment.
3.3. Inhibition of cell growth in stable transfectants
The growth rate of SGC-7901 pGPU/GFP/Neo/livin2 transfectants was significantly inhibited. As shown in Fig. 6, SGC-7901 pGPU/GFP/Neo/livin2 transfectant cells number had significant decreases at 72 h and 96 h after plating (P < 0.01) compared with negative control and parent cells.
3.4. Stable transfectants were more susceptible to proapoptotic stimuli
We treated SGC-7901 pGPU/GFP/Neo/livin2 transfectants and negative control cells with cytotoxic drugs (5-fluorouracil and cisplatin). MTT assay showed that SGC-7901 pGPU/GFP/Neo/livin2 transfectants were more sensitive to cisplatin and 5-fluorouracil than negative control and parent cells (Figs. 7A, 6B). The number of apoptotic cells induced by cisplatin and 5-fluorouracil increased to about 2.5¨C3-fold in pGPU/GFP/Neo/livin2 transfectants compared with their control cells (P < 0.001; Fig. 7C). Furthermore, stable transfectants underwent spontaneous apoptosis more readily without proapoptotic stimuli than the control cells (P < 0.05; Fig. 7C).
4. Discussion
In this study, we show that livin, a new member of the IAP family, was found to be not expressed in any of the NOT cancerous gastric tissues, and expressed only in a proportion of gastric cancer patients (47.5%), and also show that suppressing livin expression or function causes spontaneous apoptosis and inhibition of SGC- 7901 cells
growth and make cells more susceptible to proapoptotic stimuli. It was thought that livin has two isoforms, a and b. Although both isoforms are involved in blocking apoptosis induced by TNF-a and anti-CD95 in vitro, they show some different antiapoptotic properties. livin b seems to be more effective than livin a in blocking apoptosis induced by DNA damaging agents[13].
Some study on tissue distribution of livin has recently shown that elevated levels of both livin isoforms a and b have been detected in heart, placenta, lung, spleen and ovary, while livin balone has been detected specifically in fetal tissues and dult kidney and livin a alone has been detected in brain, skeletal muscle and peripheral blood lymphocytes [11-14]. Furthermore, while livin expression was detected in a variety of cancerous cell lines and some tumor tissues [14-18] and anti-livin antibody was recognized in sera of gastric cancer and lung cancer patients [19,20], no data were available concerning the expression of livin isoforms in gastric tumor tissues. Our study for the first time demonstrates that livin isoforms a and b were almost both expressed in a proportion of gastric cancer tissues (47.5%) and livin expression correlate with some of the known prognostic variables, such as grade and lymphonode metastasis. Data from the literature have demonstrated that both livin isoforms are involved in blocking apoptosis and may give cells with livin overexpression a strong resistance to chemotherapy-induced apoptosis. Gastric cancer in general is highly resistant to chemoradiotherapy and moderately resistant to apoptosis [21]. These result suggested that overexpression of livin may effect the responsibility of chemotherapy on some gastric cancer patients and prognosis of patients.
The specific interference with factors contributing to the apoptosis resistance of tumor cells may provide a novel basis for the development of rational intervention strategies in cancer therapy [22,23]. Since the expression of livin could contribute to the apoptosis-resistant phenotype of cancer cells and its specific expression in tumors could make livin an interesting therapeutic target for tumor-specific intervention strategies, we chose the livin gene as a molecular target. The shRNA technology representiong an extremely powerful tool to inhibit endogenous gene expression [24,25] be made to inhibit livin gene and attempt to correct the apoptosis deficiency of gastric tumor cells. The efficacy of shRNAs to silence expression of a tageted gene is different, relation with the half-life and abundance of the gene product as well as with accessibility of target mRNA [24-27]. In this study, we observed that si-livin1 was regularly more strongly silence the livin gene than si-livin2. Our study results also
shown that silencing livin gene expression may strongly increase apoptotic response of SGC-7901 cells in the presence or absence of several proapoptotic agents and inhibit the cells growth, which indicate that the interference with livin leads to a sensitization to proapoptotic stimuli. The similar result on hela cell was reported by Crnkovic-Mertens [18].
In summary, our results showed that inhibition of livin expression and function resulted in spontaneous apoptosis and inhibitor cell growth enhanced sensitivity to cytotoxic drugs in vitro. Because of the preferential expression of livin in gastric cancer but not in normal tissues, these data suggest that targeting the livin pathway alone or with cytotoxic drugs may be useful in the treatment of gastric cancer. Despite their therapeutic potential, major technical hurdles still have to be overcome, in order to apply shRNAs as drugs. Under therapeutic aspects, will have to meet the general challenges of gene therapy approaches, such as efficient delivery into the target cells or the circumvention of immune responses. Notably, recent in vivo studies showed that shRNAs could be directly applied to organs of postnatal mice by highpressure injection into the tail vein, leading to the specific inhibition of target genes [28-30]. These data show that a direct application of active shRNAs via the bloodstream is principally feasible.
英文翻译
学生姓名徐蔓玲指导教师王宝庆专业制药工程学院药学院
2011年5 月25 日
介导的shRNA能抑制肺癌细胞中livin沉默基因的表达从而促进SGC-7901细胞凋亡
背景—由于肿瘤细胞抑制凋亡增殖,特定凋亡的抑制因素会对于发展新的治疗策略提供一个合理途径。Livin是一种凋亡抑制蛋白家族成员，在多种恶性肿瘤的表达中具有意义。但是, 在有关胃癌方面没有可利用的数据。在本研究中,我们发现livin基因在人类胃癌中的表达并调查了介导的shRNA能抑制肺癌细胞中livin沉默基因的表达，从而促进SGC-7901细胞凋亡。
方法—mRNA及蛋白质livin基因的表达用逆转录聚合酶链反应技术及西方吸干化验进行了分析。小干扰RNA真核表达载体具体到livin基因采用基因重组、测序核酸。然后用Lipofectamin2000转染进入SGC-7901细胞。逆转录聚合酶链反应技术和西方吸干化验用来验证的livin基因在SGC-7901细胞中使沉默基因生效。所得到的稳定的复制品用G418来筛选。细胞凋亡用应用流式细胞仪(FCM)来评估。细胞生长状态和5-FU的50%抑制浓度(IC50)和顺铂都由MTT比色法来决定。
结果—livin mRNA和蛋白质的表达检测40例中有19例(47.5%)有胃癌和SGC-7901细胞。没有livin基因表达的是在肿瘤邻近组织和良性胃溃疡病灶。相关发现在livin基因的表达和肿瘤的微小分化和淋巴结转移一样(P < 0.05)。4个小干扰RNA真核表达矢量具体到基因重组的livin基因建立。其中之一,能有效地减少livin基因的表达,抑制基因不少于70%(P < 0.01)。重组的质粒被提取和转染到胃癌细胞。G418筛选所得到的稳定的复制品被放大讲究。当livin基因沉默,胃癌细胞的生殖活动明显低于对照组(P < 0.05)。研究还表明,IC50上的5-Fu和顺铂在胃癌细胞的治疗上是通过shRNA减少以及刺激这些细胞(5-Fu proapoptotic和顺铂)(P < 0.01)。
结论—livin基因在胃癌中的过分表达与肿瘤分化与淋巴结转移建立联系,建议了治疗胃癌病例分子预后因素之一。ShRNA可以抑制在SGC-7901细胞中的livin基因表达,诱导细胞凋亡。Livin可以作为治疗胃癌凋亡的新目标。
1．介绍
胃癌是世界上最常见的恶性肿瘤之一。大多数患者被诊断为这个疾病的阶段,在最佳时间的机会错失了手术治愈。尽管有很大改善,但处于晚期胃癌的化疗患者的总体存活率仍然很低。癌症细胞化疗耐抗性可能导致手术失败。在耐药的原因中,抑制细胞凋亡的过程会起重要作用。癌细胞常有抗凋亡增长的特征[1],介导其增加的阻力不同来刺激的细胞凋亡,如DNA损伤、缺氧、营养损失[2、3]。此外, 在临床实践中细胞凋亡抵抗被认为是肿瘤手术失败的主要原因,因此许多化疗药物和/
或放射线疗法都是通过诱导凋亡肿瘤死亡实现的[4]。
酶抑制剂(IAPs),是一种新型的凋亡蛋白抑制基因家族[5,6],包括病毒感染,化疗药物, ,生长因子和肿瘤坏死因子-α(TNF-α凋亡信号通路)/ Fas信号通路[7 - 9]。IAPs是由一组有凋亡特性的结构相关的蛋白质构成[10]，在预防肿瘤细胞凋亡方面可能扮演一个重要的角色,并已成为近年来研究的热点。这个家庭的新成员是ML-IAP/livin/KIAP/BIRC7 (以下称为livin)有两个种类、livinα和livinβ[11-14]。
有证据表明, livin的过分表达能阻止由多种刺激诱导的细胞凋亡[12]。有趣的是, livin基因被发现在肿瘤细胞中限制性表达,但是不存在或是很少数量存在于正常成人体组织中[11-15],并且通过允许恶性细胞,以避免凋亡细胞死亡的方式导致肿瘤形成,。所以抑制livin基因表达可能会呈现出一个有趣的治疗策略。
在目前的研究中,我们调查了livin的表达在胃cancinomas及其邻近组织。livin 的表达和临床病理参数之间的关系进行了分析。此外,我们探索了在抑制livin基因表达的shRNA可行性和胃癌易感性的凋亡细胞由shRNA介导的livin沉默基因。
2患者和方法
2.1。患者和肿瘤样本
胃癌中四十个病例及接受胃切除手术的患者收集到的胃癌组织中13个病例(患者年龄从29~77岁)。其中良性胃溃疡的13个病例(慢性浅表胃炎)患者在接受了胃内视镜检查(患者年龄从33~77岁)。这些病例均来自南京医科大学第一附属医院。胃癌患者被诊断为TNM级的1~4阶段(UICC ，2002)。手术之后肿瘤标本就立即被冻结在液态氮中,储存在-80℃直到使用为止。这是在所有的病人知情同意的情况下获得的。
2.2逆转录聚合酶链反应技术程序
总共RNA(2毫克)提取冷冻组织反转录进行，最后的体积2微升是用100 pmol of oligo(dT)15和200U M-MLV与逆转录酶(promega、美国) ,根据制造商的说明。
Aliquots对应的250微升cDNA被放大在PCR缓冲容器中，在最终的50微升中含25pmol / ml处理剂和1U聚合酶。每一个放大了35周期,一个周期的变性曲线在30 s内到达94℃。热处理在30s内59℃（livin and b-actin）扩大到30s内72℃。没有RNA的病例作为阴性对照物包含在RT–PCR中。
一系列常用的的livin和β-actin处理剂如下：livinα / βupstream，5＇-TCCACAGTGTGCAGGAGACT-3＇;livinα/ βdownstream;5＇-TCCACAGTGTGCAGGAGACT-3＇;β-actin upstream,5＇-ACGGCACAAAGACGATGGAC-3＇β-actin downstream,5＇-AGCGCAAGTACTCCGTGTG-3＇。产品的尺寸分别为livinα/β是312/258 bp ，
β-actin 是501bp。
2.3 西方吸干技术分析
病变同质性与冲力缓冲50mM Tris-HCl (pH 7.5), 250 mM NaCl,0.1% NP40和5mM EGTA包含50mM氟化钠，60mM β-丙三醇-磷酸盐,0.5mM钒酸钠,0.1mM苯甲基磺醯化氟10μg/ml亮抑蛋白酶肽。用考马斯亮蓝微盘比色法测定蛋白质含量。蛋白质样品电泳10% 变性SDS凝胶并转移到PVDF膜(Roche、美国)。
膜是培养特定的主要的抗体,与过氧化物酶继发性抗体反应(细胞信号技术、美国),最后通过增强化学荧光达到可视化(细胞信号技术、美国)。Alexesis(美国) 和散塔克鲁兹生物技术(美国)购买的单克隆抗体livin (1:250) and actin (1:400)
24细胞系和细胞培养
我们选了一个人类胃腺癌细胞系在这一研究中。SGC-7901(上海细胞研究所,中国上海,)是一种附着中度分化胃腺的人类细胞株。线是胃癌细胞上皮细胞,并成长为附着细胞RPMI 1640 (Hyclone Inc, USA)含10%FCS (Life Technologies, Inc.),每毫升100个单位的青霉素和毫升100微克的链霉素(BioWhittaker)。
在含有5%CO2空气的条件下的37℃湿润培养器中保存SGC-7901细胞。溶解顺铂和氟尿嘧啶(齐鲁制药厂、中国)在DMSO并且4℃储存。
2.5。ShRNA的合成和PGPU / GFP /Neo/livin质粒的制造
通过siRNASequence-Selector软件设计并合成了Livin的ShRNA序列(上海生物技术有限责任公司。集团公司、中国)。序列如下(表1),然后被插入pGPU/GFP/Neo(上海GenePharma股份有限公司。中国) BbsI and BamH地址产生pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control质子。
2.6SGC-7901的建立稳定表达pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control转
染实验,SGC-7901细胞被镀成6孔板(3×105孔密度), , 96孔板(1 ×104孔密度)和12孔板(1.5 ×105孔密度)转染之前培养24小时。
按照制造商的说明这些细胞被调控子用Li-pofectAMINE 2000转染4毫克/孔空的pGPU/GFP/Neo/vector，pGPU/GFP/Neo/livin或pGPU/GFP/Neo/Control 质粒(生命技术公司、大岛屿,NY)。转染48小时后,这些细胞被转移在1:15 (v/v)并用Geneticin (G418) 1000克/毫升培养4周。稳定转染的克隆体取出并保存在媒介容器400 g/ml G418用作另外的研究。
2．7依赖贴壁细胞细胞生长的测定
亲本细胞和细胞稳定表达pGPU/GFP/Neo/vector, pGPU/GFP/Neo/livin or pGPU/GFP/Neo/Control被种到6孔盘子中。每隔一天收集三孔的细胞。使用计数器确定细胞数目(Coulter Electronics, Miami, FL).。种植一些天数后用平均SD记录每孔细胞的数量。
2.8MTT测定
通过MTT对细胞毒性进行了测量。呈几何数增长的细胞被镀在密度为10000细胞/孔的96孔板上作用24小时。接下来这些细胞被以不同浓度的药物治疗48小时。每孔加入100微升MTT溶液(1毫克/毫升),并且这些细胞放在37℃下培养四小时。上层清液用异丙醇代替溶解有色甲品。用micro-ELISA测量到吸光率的波长为595nm(ClinBio-128 SLT,,奥地利)。治疗细胞的吸光率相当于计算控制细胞的吸光率并用细胞死亡百分率显示出来。
2.9流式细胞术
细胞被收集并加入冰冷的70%乙醇于PBS缓冲液中储存在-4摄℃暖直到使用。悬浮后后，100 ml核糖核酸酶I (1 mg/ml) and 100 ml 的聚酰亚胺(400 μg/ml) 37℃下培养细胞并用流式细胞术(BD,美国)进行分析。
2.10统计分析
数据的展现要用至少三个不同实验±SD的方法。实验结果用学生的t检验来分析和当P < 0.05时被认为是具有统计学显著性。
3结果
3.1。livin在胃肠癌中的表达
在目前的研究中,我们第一次验证了逆转录聚合酶链反应技术和西方吸干技术的存在在40胃癌中,13 癌组织和13良性病变胃粘膜损伤。在癌组织和良性病变胃粘膜损伤中, 每个mRNA亚型不可见水平被发现后,在肿瘤组织中,19/40(47.5%)显示出mRNA及livinaα和livinβ蛋白质表达(Figs. 1 and 2). 。livin 表达与预后变量相关,如组织学恶性度和淋巴结转移,但包括年龄、性别、阶段和肿瘤细胞浸润程度(表2)。
3.2。稳定转染表达pGPU/GFP/Neo/livin与pGPU/GFP/Neo/Control的特征
我们建立了SGC-7901 与任一pGPU/GFP/Neo/livin稳定转染，pGPU/GFP/Neo /Control 质粒，或空pGPU/GFP/Neo/vector (图3)。用西方吸干技术和逆转录聚合酶链反应技术选择每个转染的克隆并分析决定livin mRNA,和蛋白质表达。
其他的都被选择作为扩展和另外的研究。(如图4及5)显示，livin mRNA和蛋白质的水平在SGC-7901pGPU/GFP/Neo/livin2中转染降低了90%以上。Livin表达抑制在pGPU/GFP/Neo/livin1转染和消极控制中没有被发现。所以SGC-7901
pGPU/GFP/Neo/livin2被用来做后续实验。
3.3稳定转染中抑制细胞生长
SGC-7901的增长率pGPU / GFP /Neo/ livin2均有显著的抑制转染。如图显示，SGC-7901 Pgpu/GFP/尼欧/ livin2 / GFP细胞数目有显著下降转染在72小时到96小时后镀(P < 0.01),而阴性对照和家长的细胞。
3.4。稳定的转染容易受细胞凋亡因子刺激
我们认为SGC-7901 pGPU/GFP/Neo/livin2 转染和阴性对照细胞同细胞毒顺铂的增长速率明显抑制，图表6中显示SGC-7901 pGPU/GFP/Neo/livin2转染细胞数量培养后72小时和96小时与消极控制和亲本细胞相比明显降低阴性对照细胞和细胞毒药物(5 -氟尿嘧啶和顺铂)。pGPU MTT测定表明,SGC-7901 /Neo/ livin2 / GFP更敏感转染顺铂和氟尿嘧啶比消极的控制和亲本细胞(Figs. 7A, 6B)。。由顺铂和5氟尿嘧啶诱导凋亡细胞数增加至约2.5 - 3倍的pGPU/GFP/Neo/livin2转相比，其控制细胞（P<0.001;图7C条。）。此外，经历了稳定转染无自发性凋亡更容易比对照细胞（P<0.05。图7C条）凋亡刺激。
讨论
在本研究中,我们表明,推荐的新成员:一个新的IAP家族成员，被认为是不符合非癌胃组织的表达，只有在胃癌患者（47.5％）的比例计算，也表明，抑制Livin 的表达或功能的原因自发性细胞凋亡和对SGC - 7901细胞生长的抑制，使细胞更容易凋亡刺激。据认为，活着有两个亚型，A和B虽然这两个亚型在阻止肿瘤坏死因子诱导细胞凋亡参与- A和抗CD95的在体外，他们表现出一些不同的抗凋亡的特性。活着b似乎是在阻断DNA损伤剂诱导细胞凋亡[13]超过活着有效。
一些组织中Livin分布研究表明，最近都活着升高亚型A和B已发现在心脏，胎盘，肺，脾，卵巢，而活着balone特别是在已检测到胎儿组织和dult肾脏和Livin一单是在脑，骨骼肌和外周血淋巴细胞的检测[11-14]。此外，虽然Livin的表达是在一个癌细胞的细胞株和肿瘤组织中的一些品种检测[14-18]和反活着抗体在胃癌和肺癌患者血清识别[19,20]，没有数据有关亚型中Livin的表达在胃癌肿瘤组织。
我们的第一次研究表明，活着亚型A和B几乎都在胃癌组织（47.5％）和Livin 表达与一些已知的预后因素，如分级，淋巴结转移，相关的比例计算。从文献资料表明，这两个活着亚型参与了阻止细胞凋亡，并可能给活着的过度表达与细胞的强烈抵抗化疗诱导细胞凋亡。胃癌一般具有高度抗癌症放化疗和中抗凋亡[21]。这些结果表明，Livin的高表达可能对某些癌症患者和胃癌患者预后化疗的责任。
与促进肿瘤细胞的凋亡抵抗可能提供一个合理干预策略在癌症治疗的基础上发展新的特定因素干扰[22,23]。由于Livin的表达可能有助于肿瘤细胞和肿瘤的特异性表达及其在细胞凋亡的抗性表型可以让活着的一个有趣的肿瘤治疗靶点的具体干预措施的战略，我们选择了作为一个分子靶点的Livin基因。的shRNA技术representiong一个极其有力的工具，抑制内源性基因表达[24,25]作出抑制Livin 基因，并试图纠正胃癌细胞凋亡的不足。作者：沉默的shRNA功效的tageted基因的表达是不同的，与半的生活和丰富的基因产物与靶mRNA作为[24-27]，以及
无障碍的关系。
在这项研究中，我们观察到硅livin1是经常更强烈的沉默比硅livin2 Livin基因。我们的研究结果还表明，沉默Livin基因的表达可能存在强烈的增加或几个凋亡的代理人在场下的SGC - 7901细胞凋亡反应，抑制细胞的生长，这表明，与美好生活的干扰导致了对凋亡刺激的敏感性。对HeLa细胞类似的结果报告了Crnkovic -梅坦斯[18]。
总之，我们的结果表明，Livin的表达和功能抑制自发性细胞凋亡和抑制细胞生长的体外敏感性增强化疗药物的结果。由于在胃癌中的表达，但活着的优惠在正常组织中，这些数据表明，针对活着途径单独或与细胞毒性药物可能在胃癌的治疗作用。尽管他们的治疗潜力，主要技术障碍仍有待克服，才能申请成为毒品的shRNA。
在治疗方面，将不得不满足基因治疗的办法，如高效输送到目标细胞的免疫反应或规避，一般的挑战。值得注意的是，在最近的研究表明，体内的shRNA 可以直接应用到出生后小鼠脏器highpressure尾静脉注射，导致靶基因特异性抑制[28-30]。这些数据表明，一个活跃的shRNA通过血液的直接应用是主要可行的。
制药工程专业英语课文翻译
Unit 1 Production of Drugs About 5000 antibiotics have already been isolated from microorganisms，but of these only somewhat fewer than 100 are in therapeutic use. It must be remembered，however，that many derivatives have been modified by partial synthesis for therapeutic use;some 50，000 agents have been semisynthetically obtained from户lactams alone in the last decade. Fermentations are carried out in stainless steel fermentors with volumes up to 400 m3. To avoid contamination of the microorganisms with phages etc. the whole process has to be performed under sterile conditions. Since the more important fermentations occur exclusively under aerobic conditions a good supply of oxygen or air(sterile)is needed. Carbon dioxide sources include carbohydrates，e. g. molasses，saccharides，and glucose. Additionally the microorganisms must be supplied in the growth medium with nitrogen-containing compounds such as ammonium sulfate，ammonia，or urea，as well as with inorganic phosphates. Furthermore，constant optimal pH and temperature are required. In the case of penicillin G，the fermentation is finished after 200 hours，and the cell mass is separated by filtration. The desired active agents are isolated from the filtrate by absorption or extraction processes. The cell mass，if not the desired product，can be further used as an animal feedstuff owing to its high protein content. 关于5000抗生素已经分离出的微生物，但其中只有不到100有些治疗使用。必须记住，但是，许多衍生工具已被用于治疗使用部分合成修改;约50,000剂已被semisynthetically取得户内酰胺在过去十年孤独。发酵都是在不锈钢发酵罐出来的量高达400立方米。为了避免与噬菌体等微生物污染的全过程都必须在无菌条件下进行。由于更重要的发酵只发生在有氧条件下的氧气或空气好电源（无菌）是必要的。二氧化碳的来源包括碳水化合物，大肠杆菌克糖蜜，糖和葡萄糖。另外必须提供的微生物在与含氮如硫酸铵，氨水或尿素化合物生长介质，以及与无机磷酸盐。此外，不断最适pH和温度是必需的。在青霉素G的情况下，发酵完成200小时后，细胞的质量是由过滤分离。所需的活性剂是隔离的滤液吸收或提取工艺。大规模的细胞，如果不理想的产品，可进一步用作动物，由于其蛋白质含量高的饲料。 By modern recombinant techniques microorganisms have been obtained which also allow production of peptides which were not encoded in the original genes. Modified E. coli bacteria make it thus possible to produce A- and B- chains of human insulin or proinsulin analogs. The disulfide bridges are formed selectively after isolation，and the final purification is effected by chromatographic procedures. In this way human insulin is obtained totally independently from any pancreatic material taken from animals. Other important peptides，hormones，and enzymes，such as human growth hormone (HGH)，neuroactive peptides，somatostatin，interferons，tissue plasminogen activator (TPA)，lymphokines，calcium regulators like calmodulin，protein vaccines，as well as monoclonal antibodies used as diagnostics，are synthesized in this way. 利用现代微生物重组技术已获得这也让其中不是在原来的基因编码多肽的生产。改性大肠杆菌从而使可能产生A型和B -人胰岛素或胰岛素原类似物链。二硫键形成的选择性分离后，最终由色谱净化工序的影响。通过这种方式获得的人类胰岛素完全独立采取任何从动物胰腺材料。 其他重要肽，激素和酶，如人类生长激素（hGH），神经活性肽，生长抑素，干扰素，组织型纤溶酶原激活物（tPA），淋巴因子，如钙调节钙调蛋白，蛋白疫苗，以及作为诊断用单克隆抗体是合成了这种方式。 The enzymes or enzymatic systems which are present in a single microorganism can be used for directed stereospecific and regiospecific chemical reactions. This principle is especially useful in steroid chemistry. Here we may refer only to the microbiological 11-a- hydro xylation of progesterone to 11-a-hydroxyprogesterone，a key product used in the synthesis of cortisone. Isolated enzymes are important today not only because of the technical importance of the enzymatic saccharification of starch，and the isomerization of glucose to fructose，They are also significant in the countless test procedures used in diagnosing illness，and in enzymatic analysis which is used in the monitoring of therapy. A number of enzymes are themselves used as active ingredients. Thus preparations containing proteases (e. g. chymotrypsin，pepsin，and trypsin)，amylases and lipases，mostly in combination with synthetic antacids，promote digestion. Streptokinase and urokinase are important in thrombolytics，and asparaginase is used as a cytostatic agent in the treatment of leukemia. 这些酶或微生物在一个单一的酶系统，目前可用于立体定向和regiospecific化学反应。这个原则是有用的，尤其是在化学类固醇。在这里，我们只能引用的微生物十一水电黄体酮xylation至11人羟，一个关键的产品在可的松合成。隔离酶是重要的，不仅因为淀粉的酶法糖化技术重要性的今天，和葡萄糖异构果糖，他们也都在无数次试验在诊断疾病所用的程序显着，在酶的分析，在使用监测治疗。 数量的酶本身作为活性成分。因此，含有蛋白酶制剂（如糜蛋白酶，胃蛋白酶和胰蛋白酶），淀粉酶和脂肪酶的合成主要是在与抗酸药相结合，促进消化。链激酶和尿激酶溶栓是重要的，是天冬酰胺酶在治疗白血病细胞生长剂。 Finally mention must be made of the important use of enzymes as `biocatalysts’in chemical reactions where their
药学英语课后翻译
Unit 1 1. A full appreciation of the physiology of a living organism must be based on a sound knowledge of its anatomy. Anatomy does not merely study the separation of parts, but the accurate description of the morphologies and functions of different organs. 对生物生理学的全面了解必须基于解剖学的系统知识;解剖学不仅仅是研究 人体各部分的分离；还要准确的描述各个器官的形态和生理功能; 2. Our daily food intake must match requirements and any excess must be excreted for balance to be maintained. 我们每天摄入的事物必须满足需要；任何多余的东西必须排出体外才能维持 平衡; 3. The process of stabilization of the internal environment is called homeostasis and is essential if the cells of the body are to function normally. 内环境稳定的过程称之为体内平衡；体内平衡也是机体的细胞正常发挥作用所必 不可少的; 4. Human cells have the ability to break down large molecules to smaller ones to liberate sufficient energy for their activities. 人类细胞有将大分子分解成小分子的能力；从而为自身活动释放足够的能量; 5. As long as normal conditions are maintained in this internal environment, the cells of the body continue to live and function properly. 只要这种内环境正常的条件得以维持；机体的细胞就能继续生存并发挥正常 功能; Unit 2 1. Biochemistry asks how the thousands of different biomolecules interact with each other to confer the remarkable properties of living organisms. 生物化学探寻的是数千种不同的生物分子如何相互作用；以赋予生物体具备显着的特性; 2. Enzymes are catalysts that accelerate the rates of biological reactions. Each enzyme is very specific in its function and acts only in a particular metabolic reaction. 酶是能加速生物学反应速率的催化剂;每一种酶都有专一的功能并且仅在特定代谢反应中发挥作用; 3. One of the most fruitful approaches to understand biological phenomena has been to purify an individual chemical component, such as a protein, from a living organism and to characterize its chemical structure or catalytic activity. 用以了解生物学现象的最有效的方法之一是从生物体中纯化出单一化学成分；例如蛋白质；并对其化学结构或催化活性进行表征; 4. The chemical principles that govern the properties of biological molecules include the covalent bonding of carbon with itself and with other elements and the functional groups that appear in common biological molecules, etc. 决定生物分子特性的化学原理包括碳与自身或其他元素的共价结合和一般生物分子中出现的功能基团等; 5. The basic unit of DNA is a linear polymer of four different monomeric subunits,
(完整版)药学英语第五版原文翻译
Introduction to Physiology Introduction Physiology is the study of the functions of living matter. It is concerned with how an organism performs its varied activities: how it feeds, how it moves, how it adapts to changing circumstances, how it spawns new generations. The subject is vast and embraces the whole of life. The success of physiology in explaining how organisms perform their daily tasks is based on the notion that they are intricate and exquisite machines whose operation is governed by the laws of physics and chemistry. Although some processes are similar across the whole spectrum of biology —the replication of the genetic code for or example specific to particular groups of organisms. For this reason it is necessary to divide the subject into various parts such as bacterial physiology, plant physiology, and animal physiology. To study how an animal works it is first necessary to know how it is built. A full appreciation of the physiology of an organism must therefore be based on a sound knowledge of its anatomy. Experiments can then be carried out to establish how particular parts perform their functions. Although there have been many important physiological investigations on human volunteers, the need for precise control over the experimental conditions has meant that much of our present physiological knowledge has been derived from studies on other animals such as frogs, rabbits, cats, and dogs. When it is clear that a specific physiological process has a common basis in a wide variety of animal species, it is reasonable to assume that the same principles will apply to humans. The knowledge gained from this approach has given us a great insight into human physiology and endowed us with a solid foundation for the effective treatment of many diseases. The building blocks of the body are the cells, which are grouped together to form tissues. The principal types of tissue are epithelial, connective, nervous, and muscular, each with its own characteristics. Many connective tissues have relatively few cells but have an extensive extracellular matrix. In contrast, smooth muscle consists of densely packed layers of muscle cells linked together via specific cell junctions. Organs such as the brain, the heart, the lungs, the intestines, and the liver are formed by the aggregation of different kinds of tissues. The organs are themselves parts of distinct physiological systems. The heart and blood vessels form the cardiovascular system; the lungs, trachea, and bronchi together with the chest wall and diaphragm form the respiratory system; the skeleton and skeletal muscles form the musculoskeletal system; the brain, spinal cord, autonomic nerves and ganglia, and peripheral somatic nerves form the nervous system, and so on. Cells differ widely in form and function but they all have certain common characteristics. Firstly, they are bounded by a limiting membrane, the plasma membrane. Secondly, they have the ability to break down large molecules to smaller ones to liberate energy for their activities. 生理学简介 介绍 生理学是研究生物体功能的科学。它研究生物体如何进行各种活动，如何饮食，如何运动，如何适应不断改变的环境，如何繁殖后代。这门学科包罗万象，涵盖了生物体整个生命过程。生理学成功地解释了生物体如何进行日常活动，基于的观点是生物体好比是结构复杂而灵巧的机器，其操作受物理和化学规律控制。 尽管从生物学整个范畴看，生物体某些活—动m过a程ny是ar相e 似的——如基因编码的复制——但许多过程还是某些生物体群组特 有的。鉴于此有必要将这门学科分成不同部分研究，如细菌生理学、植物生理学和动物生理学。 要研究一种动物如何活动，首先需要了解它的构成。要充分了解一个生物体的生理学活动就必须掌握全面的解剖学知识。一个生物体的各部分起着什么作用可通过实验观察得知。尽管我们对志愿者进行了许多重要的生理调查，但是实验条件需要精确控制，所以我们当前大多生理知识还是源于对其它动物如青蛙，兔子，猫和狗等的研究。当我们明确大多数动物物种的特定生理过程存在共同之处时，相同的生理原理适用于人类也是合理的。通过这种方法，我们获得了大量的知识，从而让我们对人类生理学有了更深入的了解，为我们有效治疗许多疾病提供了一个坚实的基础。 机体的基本组成物质是细胞，细胞结 合在一起形成组织。组织的基本类型有上皮组织，结缔组织，神经组织和肌组织，每类组织都有各自的特征。许多结缔组织中细胞量相对较少，但是有大量的细胞外基质。相比而言，光滑的肌组织由大量密密麻麻的肌细胞通过特定的细胞连接组成。各种器官如脑，心脏，肺，小肠和肝等由不同种类的组织聚集而成。这些器官是不同生理系统的组成部分。心脏和血管组成心血管系统；肺，器官，支气管，胸壁和膈肌组成呼吸系统；骨骼和骨骼肌组成骨骼肌系统；大脑，脊髓，自主神经和神经中枢以及周围躯体神经组成神经系统等等。 细胞在形体和功能上差异很大，但是它们有某些共同的特征。第一，它们由限制
药学英语课文翻译 课后翻译节选 中英双语对照 第四版
本篇包括人卫第四版Unit 3B，Unit4A，5A，8A，10A，12AB， 13A等七篇课文 Unit 3 Text B The Other Side of Antibiotics 抗生素的另一面 Antibiotics have eliminated or controlled so many infectious diseases that virtually everyone has benefited from their use at one time or another. Even without such personal experience, however, one would have to be isolated indeed to be unaware of the virtues, real and speculative, of these “miracle” drugs1. The American press, radio, and television have done a good job of reporting the truly remarkable story of successes in the chemical war on germs. What′s more, any shortcomings on their part have been more than made up for by the aggressive public relations activity of the pharmaceutical companies which manufacture and sell antibiotics. 抗生素可以消除或控制很多种感染疾病，以致几乎每人生病时都习惯于使用它而受益，但是如果一个人没有这样的亲身经历，他必定是离群索居才会不知道这些“特效药物”或真实或推测的优点。美国的出版物、电台或电视台用大量的篇幅报道了有关对细菌的化学战中获得的这些显著功绩。而它的缺点却被生产和销售抗生素的制药公司通过公关活动掩藏了。 In comparison, the inadequacies and potential dangers of these remarkable drugs are much less widely known. And the lack of such knowledge can be bad, especially if it leads patients to pressure their doctors into prescribing antibiotics when such medication isn’t really needed, or leads them to switch doctors until they find one who is, so to speak, antibiotics-minded2. 相比而言，使用这些药物的危险性并不广为人知。对这种知识的缺乏将更糟糕，特别是当患者要求医生开处方用抗生素而事实并不需要，或患者频繁地更换医生直至找到一个同意开抗生素处方的医生。 Because the good side of the antibiotics story is so very well-known, there seems more point here to a review of some of the immediate and long-range problems that can come from today’s casual use of these drugs. It should be made clear in advance that calamities from the use of antibiotics are rare in relation to the enormous amounts of the drugs administered. But the potential hazards, so little touched on generally, do need a clear statement. 因为抗生素的好的一面已广为人知，今天抗生素的滥用导致短期或长期问题。我们预先应该知道与抗生素的巨大的使用量相比,它产生危害的例子是少见的。但是，尽管十分少见，需要对这种潜在的危险作一个清楚的说明。 The antibiotics are not, strictly speaking, exclusively prescription drugs. A number of them are permitted in such over-the-counter products as nasal sprays, lozenges, troches, creams, and ointments. Even if these products do no harm there is no point whatsoever in using them. If you have an infection serious enough to warrant the launching of chemical warfare, you need much bigger doses of the antibiotics than any of the non-prescription products are allowed to contain. 严格来讲，抗生素并不全是处方药。许多抗生素被允许作为非处方药(如鼻喷雾剂、键剂、片剂、软膏和乳膏),尽管它们没有危害，也不能随意地使用。如果你患了严重的感染，你就得需要比非处方药所允许最大剂量更大剂量的抗生素了。 Over-the-counter products, however, account for only a small percentage of total antibiotics production. It is the prescription dosages that give people trouble. 然而，非处方药品只是整个抗生素类产品的一小部分，正是处方药物给人类带来了麻烦。
药学专业英语文章及翻译
Expression of livin in gastric cancer and induction of apoptosis in SGC-7901 cells by shRNA-mediated silencing of livin gene Background-Because of increased resistance to apoptosis in tumor cells, inhibition of specific antiapoptotic factors may provide a rational approach for the development of novel therapeutic strategies.Livin, a novel inhibitor of apoptosis protein family, has been found to be expressed in various malignancies and is suggested to have poorly prognostic significance. However, no data are available concerning the significance of livin in gastric cancer. In this study, we detected the expression of livin in human gastric carcinoma and investigated the apoptotic susceptibility of SGC-7901 cell by shRNAmediated silencing of the livin gene. Methods-The mRNA and protein expression of livin were analyzed by RT-PCR and western blot assay.The relationship between livin expression and clinical pathologic parameters was investigated. The small interfering RNA eukaryotic expression vector specific to livin was constructed by gene recombination, and the nucleic acid was sequenced. Then it was transfected into SGC-7901 cells by Lipofectamin 2000. RT-PCR and Western blot assay were used to validate gene-silencing efficiency of livin in SGC-7901 cells. Stable clones were obtained by G418 screening. The cell apoptosis was assessed by flow cytometry (FCM). Cell growth state and 50 % inhibition concentration (IC50) of 5-FU and cisplatin was determined by MTT method. Results-The expression of livin mRNA and protein were detected in 19 of 40 gastric carcinoma cases (47.5%) and SGC-7901 cells. No expression of livin was detected in tumor adjacent tissues and benign gastric lesion. The positive correlation was found between livin expression and poor differentiation of tumors as well as lymph node metastases (P < 0.05). Four small interfering RNA eukaryotic expression vector specific to livin were constructed by gene recombination. And one of them can efficiently decrease the expression of livin, the inhibition of the gene was not less than 70% (P < 0.01). The recombinated plasmids were extracted and transfected gastric cancer cells. The stable clones were obtained by G418 screening, and were amplified and cultured. When livin gene was silenced, the reproductive activity of the gastric cancer cells was significantly lower than the control groups(P < 0.05). The study also showed that IC50 of 5-Fu and cisplatin on gastric cancer cells treated by shRNA was
药学英语课文翻译
药学英语课文翻译 Unit1药品 根据他们的产品或来源药物可以被分为三种： Ⅰ、全合成 Ⅱ、天然产物和 Ⅲ、由部分合成产物（半合成产物） 本书的重点是关于最重要化合物Ⅰ和Ⅲ——这类药物合成。然而，这并不意味天然产物和其他的药物就不重要。它们可以被用作有价值的先导化合物，并且它们通常被用作起始原料或作为重要合成产物的中间体。 合成而更加经济的。 在过去的几年里发酵，即微生物工程，已经变得极其重要。通过现代技术和基因的选择结果，导致了微生物高突变体演变的产生，发酵已经变成了对物质广泛围的选择方式。真核细胞（酵母和霉菌）和原核细胞（单细菌细胞和放线菌）都被用作微生物。以下为可获得的生产形式: 1.细胞原料（单细胞蛋白质） 2.酶 3.初级的降解产物（初级酶代物） 4.次级的降解产物（次级的代物） 在次级代物中，必先提起的是抗生素，以下五种药代表了每年世界围价值170亿美元的药物： 青霉素，头孢菌素，四环素，红霉素，氨基糖苷类。 大约有5000种抗生素已经从微生物中分离出来了，但在这些中仅有那些少于100种用于治疗使用。然而，一定知道，那些衍生物通过部分合成被改进用于治疗。在过去十年中，单单从β-酰胺半合成的就有五万种药物。发酵在容积大于400m3的不锈钠发酵罐中进行，避免了微生物噬菌体的污染等等，整个过程必须在无菌条件下进行。 （倒数第五段开始） 大量使用的试剂不仅仅是酸（盐酸、硫酸、硝酸、醋酸），还有无机和有机碱（氢氧化钠、氢氧化钾、碳酸钾、重碳酸钾、铵碱、三乙胺、吡啶）。还有辅助化学物质包括活性炭和催化剂。所有这些补充的化学物质（比如中间体）在最终产物中可能是杂志的来源。 在1969年，世界卫生组织出版了关于“药品安全质量保护”的论述。目录2是有关“药品赔偿和安全保护质量的规定”（世界卫生组织，1969年第418号技术报告，目录2；
药学英语课文3翻译
Foods That Fight Cancer 抗癌症的食物
Foods That Fight Cancer Diet is now considered a major weapon(武器) against cancer. The National Cancer Institute （协会）estimates that about one-third of all cancers are linked to diet, and recent research indicated（指出）that what you eat may help to significantly reduce your risk. Cancer develops over a long time, which means that you have years-typically decades（数十年）-in which to hinder（阻碍）or promote it. Researchers are finding that what you eat may interfere（妨碍）with cancer growth at various stages. For example, certain foods can block (v.阻碍) the chemicals that initiate（开始）cancer. Antioxidants（抗氧化剂）, found in some vitamins and minerals（矿物）, can snuff（消灭）out oxygen free radicals（基础；原子团）, substances that are thought to make cells more susceptible（易受影响的）to cancer, and they can even repair some of the cellular damage that has been done. And some food-wheat bran in particular-has been shown to shrink（收缩）precancerous（癌症前期的）cells. A recent review（评论）of 17 studies from 17 nations reveals that people who eat the most fruits and vegetables have about half the cancer rates of those who eat the least. That includes cancers of the lung, colon（结肠）, breast, cervix（宫颈）, esophagus（食道）, oral cavity（口腔）, stomach, bladder（膀胱）, pancreas（胰腺）and ovary（卵巢）. In fact, some research suggests that frequent consumption（肺痨;消耗）of fruits and vegetables can cut the risk of lung cancer even in smokers. “It is almost mind-boggling（难以理解的）,” says Tim Byers, an epidemiologist （流行病学家）with the U. S. Centers for Disease Control and Prevention, “that ordinary fruits and vegetables can be so effective against such a potent（有效的）carcinogen（致癌物质）as cigarette smoke.” One of the most studied antioxidants in vegetables and fruits thought to protect against cancer is beta-carotene（B-胡萝卜素）, concentrated（聚集）in deep green, yellow and orange vegetables such as carrots, sweet potatoes and spinach（菠菜）. Fruits high in beta-carotene include apricots （杏）and cantaloupes（哈密瓜）. In test-tube（试管）studies at Harvard University, beta-carotene had a direct toxic effect on cells taken from malignant（有害的）tumors（肿瘤）. It also reduced the growth of lung-cancer cells and altered（改变）the proteins needed for tumors to grow. Research also shows that beta-carotene can change in the body to retinoid acid（视黄酸）, a substance used in clinical trials to treat certain cancers. Here are some of the foods that contain cancer-fighting chemicals. Tomatoes. One of the compounds in tomatoes that is thought to reduce the risk of cancer is lycopene（番茄红素）, the pigment（色素）that makes tomatoes red. Lycopene, an antioxidant that is also found in watermelons and apricots, quenches（结束）certain cancer- triggering oxygen free radicals. Having little lycopene in your blood is associated with a higher risk of pancreatic cancer, according in a Johns Hopkins University study. People with pancreatic cancer showed lower levels of lycopene compared with healthy individuals. Those with the least blood lycopene had over five times the risk of pancreatic cancer as healthy people with the most blood lycopene. Lycopene is present in tomato products, including sauces（酱油）, tomato paste and even ketchup（番茄酱）. Green Vegetables. A recent Italian study showed that dark-green leafy vegetables lower the risk of many cancers. Spinach, broccoli（西兰花）, kale（甘兰）and dark-green lettuces（油麦菜）are chock-full of antioxidants, including beta-carotene, folate and lutein，. A good rule of