碱性成纤维细胞生长因子重组腺病毒转染兔骨髓间充质干细胞的表型变化
碱性成纤维细胞生长因子对体外培养的兔骨髓基质细胞生长特性的影响
碱性成纤维细胞生长因子对体外培养的兔骨髓基质细胞生长特性的影响王志勇;陈增海;于胜吉【期刊名称】《山东大学学报：医学版》【年(卷),期】2006(44)11【摘要】目的:研究骨髓基质细胞(BMSC)在体外培养条件下分化为成骨细胞的能力,及碱性成纤维细胞生长因子(bFGF)对其贴附、增殖、分化的影响。
方法:将各组BMSC传代后加入bFGF,其浓度分别为0、5、10、50、1002、00 ng/ml,用倒置相差显微镜、电镜、四唑盐比色法、碱性磷酸酶(ALP)染色、钙染色和电子探针等方法观察其形态、贴附、生长增殖活性、分化成熟和体外钙化的状况。
结果:经10 ng/ml bFGF诱导后,BMSC贴附细胞数量明显增加。
当100 ng/ml时,bFGF对BMSC的促增殖作用达到最大值。
当加入bFGF时,ALP染色阳性率降低,当bFGF 浓度为100μg/L时阳性率最低(P<0.01)。
传代细胞连续培养30 d后钙染色结果呈阳性,电子探针检测矿化区的Ca/P值与羟基磷灰石结晶中Ca/P值相符。
电镜观察可见培养的细胞具有典型的成骨细胞形态特征,并能产生胶原纤维。
结论:BMSC 在体外培养条件下具有分化为成骨细胞的能力,bFGF能够促进细胞的贴壁和增殖,但对其分化成熟具有抑制作用。
【总页数】5页(P1090-1094)【关键词】骨髓基质细胞;成骨细胞;成纤维细胞生长因子2;兔【作者】王志勇;陈增海;于胜吉【作者单位】山东大学齐鲁医院急诊科;山东大学第二医院骨科;中国医学科学院肿瘤医院骨科【正文语种】中文【中图分类】Q954.658【相关文献】1.不同浓度碱性成纤维细胞生长因子对兔骨髓基质细胞的影响 [J], 韦永中;范卫民;陈哲峰;崔维顶2.重组人骨形态发生蛋白2与碱性成纤维细胞生长因子联合促进兔骨髓基质细胞血管内皮细胞生长因子表达及其成骨潜能 [J], 常祺;黄昌林;黄涛3.兔骨髓基质细胞与陶瓷样异种骨的体外培养实验兔骨髓基质细胞形态、生长、附着及增殖性能观察 [J], 李彦林;韩睿;黄河;李世和;曾才铭;王宏邦4.碱性成纤维细胞生长因子对兔骨髓基质细胞生物行为的影响 [J], 刘建;常祺;胡蕴玉;孟国林5.复合骨形态发生蛋白2及碱性成纤维细胞生长因子注射型骨修复材料对兔骨髓基质细胞增殖和超微结构的影响 [J], 崔赓;胡蕴玉;雷伟;孙明林;李洁;靳小兵;汪培铭;杨柳;吕昌伟;吕荣因版权原因，仅展示原文概要，查看原文内容请购买。
重组人碱性成纤维细胞生长因子对兔视网膜缺血再灌注损伤的保护作用
重组人碱性成纤维细胞生长因子对兔视网膜缺血再灌注损伤的保护作用邵宏超;葛嫣然;李利艳;王林洪;马建英;曹凤英;田华【期刊名称】《临床误诊误治》【年(卷),期】2014(000)006【摘要】目的探讨重组人碱性成纤维细胞生长因子( recombinant human basic fibroblast growth factor, rh-bFGF)对兔视网膜缺血再灌注损伤( retinal ischemia reperfusion injury, RIRI)的保护作用。
方法选取健康纯种大耳白兔66只,随机分为假手术组6只,RIRI组30只,rh-bFGF组30只。
通过升高眼压的方法制作兔RIRI模型,RIRI组予0.9%氯化钠注射液,rh-bFGF组予rh-bFGF治疗,假手术组仅用5号头皮针从耳前方角巩膜缘内刺入前房,之后缓慢拔除针头。
RIRI组和rh-bFGF组在RIRI后6 h、12 h、24 h、48 h、72 h常规HE染色观察视网膜组织形态变化,SABC免疫组织化学法检测视网膜组织中凋亡抑制基因Bcl-2和促凋亡基因Bax蛋白表达及Bcl-2/Bax的变化。
结果假手术组视网膜未见凋亡细胞；RIRI组视网膜凋亡细胞主要出现在内核层和神经节细胞层,且在24 h达到高峰；rh-bFGF组与RIRI组比较,观察各时间点细胞损伤情况基本相似。
Bcl-2和Bax在正常视网膜组织中几乎不表达,在RIRI后6 h开始表达,24 h达到高峰,48 h开始下降,72 h后表达明显减弱。
rh-bFGF组与RIRI组比较,Bcl-2表达明显增强,而Bax 表达减弱(P均<0.05)。
结论 Bcl-2和Bax参与了RIRI的调控,rh-bFGF可通过上调Bcl-2表达及下调Bax表达,使Bcl-2/Bax比值升高,起到保护视网膜组织的作用。
%Objective To explore the protective effect of recombinant human basic fibroblast growth factor (rh-bFGF) on retinal ischemia-reperfusioninjury (RIRI) in rabbits. Methods The 66 healthy purebred rabbits were randomly divided into sham-operation group (n=6), ischemia-reperfusion model group (n=30) and rh-bFGF treatment group (n=30). The RIRI models were established by elevating intraocular pressure, the ischemia-reperfusion model group was injected with 0. 9% sodium chloride injec-tion;rh-bFGF drug therapy was performed in rh-bFGF treatment group;the sham-operation group only received the 5th scalp heedle from ear piercing the anterior chamber angle in front of the sclera edge, and then the needle was slowly removed. Retina histopatho-logical changes were observed by routine hematoxylin and eosin stain 6 h, 12 h, 24 h, 48 h and 72 h after RIRI in ischemia-reperfu-sion model and rh-bFGF treatment groups. Expressions of inhibiting gene Bcl-2 and stimulative gene Bax and changes of Bax/Bcl-2 in retinal tissues were detected with SABC immunohistochemical method. Results No apoptotic cell was found in sham-operation group;apoptotic cells mainly existed in the inner nuclear layer and the ganglion cell layer, and apoptotic cells reached the peak 24 h after reperfusion;the rh-bFGF treatment groups had the similar condition with the ischemia-reperfusion model group in cell in-jury at each observational time. No expressions of Bcl-2 and Bax positive cells were found in normal retina tissues;the expressions were found 6 h after reperfusion, and the expressions reached the peak 24 h after reperfusion, and began to descented 48 h after reperfusion, and the decrease was obvious 72 h after reperfusion. Bcl-2 expression was significantly stronger, while Bax expression was significantly reduced in rh-bFGF treatment group,compared with those in ischemia-reperfusion model group (P<0. 05). Con-clusion Bcl-2 and Bax protein are involved in the RIRI regulation. The retina tissue can be protected with Bcl-2 expression of up-regulation and the Bax expression of down-regulation by rh-bFGF, which can increase the Bcl-2/Bax ratio.【总页数】5页(P100-104)【作者】邵宏超;葛嫣然;李利艳;王林洪;马建英;曹凤英;田华【作者单位】063000 河北唐山，河北联合大学附属医院眼科;063000 河北唐山，河北联合大学附属医院眼科;100000 北京，石景山区医院眼科;063000 河北唐山，河北联合大学附属医院眼科;063000 河北唐山，唐山市第九医院门诊部;063000河北唐山，河北联合大学附属医院眼科;063000 河北唐山，河北联合大学附属医院眼科【正文语种】中文【中图分类】R322.91【相关文献】1.神经生长因子联合银杏叶提取物对兔急性青光眼视网膜缺血再灌注损伤的保护作用 [J], 李岳美;李庆和;郑新华2.rh-bFGF 对兔视网膜缺血再灌注损伤的保护作用及其机制 [J], 邵宏超;葛嫣然;刘岩;苏杰;田华3.rh-bFGF对兔视网膜缺血再灌注损伤的保护作用及机制 [J], 邵宏超;葛嫣然;刘岩;苏杰;田华4.热休克蛋白72在兔视网膜缺血再灌注损伤中的表达及重组人碱性成纤维细胞生长因子对其表达的影响 [J], 邵宏超;葛嫣然;刘岩;苏杰;杨馥宇5.活血通络利水方对兔视网膜缺血/再灌注损伤的保护作用 [J], 杨赞章; 张越; 张铭连; 孟兢晶; 李明然因版权原因，仅展示原文概要，查看原文内容请购买。
腺病毒介导的碱性成纤维细胞生长因子对大鼠脊髓损伤腹后外侧索少突胶质细胞的影响
腺病毒介导的碱性成纤维细胞生长因子对大鼠脊髓损伤腹后外侧索少突胶质细胞的影响杨彦玲【摘要】Objective To study the effect of adenovirus-mediated basic fibroblast growth factor ( FGF-2) gene transfer in vivo on oligodendrocyte cell numbers throughout ventrolateral white matter following spinal cord injury in rats. Methods Thirty-two adult female Sprague Dawley rats were injured with the Infinite Horizon Impactor, and then were randomly assigned to four groups; FGF-2-Adts high-titer group (1. 27 × 10 pfu/rat) , FGF-2 -Adts intermediate-titre group (6. 37 × 106 pfu/rat) , FGF-2-Adts low-titer group (3. 18 × 106 pfu/rat) , and green fluorescent protein (GFP) -Adts group (5. 9 × 107 pfu/rat). The transgenic expression in vivo was detected with fluorescence microscopy. The locomotor function of the hindlimbs of rats was evaluated using Riv-lin plate. Slides mounted with tissue sections were processed for immunohistochemical detection and quantification of oligodendrocytes (CC1 + ) in the ventral lateral funiculi (VLF) of injured spinal cords. Results One week after spinal cord injury, GFP showed that many cells had expressed objective gene in vivo and the angles of the occlusal plane of rats in FGF-2 groups were significantly higher than in GFP-Adts group. Also, there was a significant difference among the FGF-2-Adts treatment groups for the volume of gray matter sparing. However, there were no significant differences for total white matter sparing. Stereological quantification of total CC1 * cell numbers inthe spared VLF showed a significant reduction in numbers with GFP controls compared to all other groups 4 weeks after injury. In contrast, the FGF-2 Adts intermediate-titer group had significantly more CC1 + cells when compared to both the FGF-2-Adts high- and low-titer groups. Conclusion Adenovirus-mediated FGF-2 gene transfer can promote the functional recovery of the injured spinal cord by enhancing the proliferation and/or differentiation of oligodendrocytes.%目的观察腺病毒( Adts)介导碱性成纤维细胞生长因子(FGF-2)对大鼠脊髓损伤腹后外侧索少突胶质细胞的影响.方法 32只SD大鼠在T10水平制备脊髓损伤模型,随机分为体内转基因治疗组和损伤对照组,用携带FGF-2和绿色荧光蛋白(GFP)的Adts行直接体内转基因治疗.荧光显微镜观察体内转基因表达,斜板实验检测大鼠运动功能恢复情况,免疫组织化学染色观察腹后外侧索少突胶质细胞(CC1+)数量的变化.结果荧光显微镜观察发现,Adts载体直接引入脊髓后可有效感染脊髓组织并表达报告基因.斜板实验检测结果显示,体内转基因治疗组大鼠的倾斜平面角度明显高于对照组(P＜0.05或P＜0.01).免疫组织化学结果证实,腹后外侧索CCI+数量较对照组明显增加(P＜0.05).结论 Adts介导的FGF-2可促进少突胶质细胞的存活和增殖.【期刊名称】《中国医学科学院学报》【年(卷),期】2012(034)004【总页数】5页(P348-352)【关键词】脊髓损伤;碱性成纤维细胞生长因子;少突胶质细胞;腺病毒载体;基因治疗【作者】杨彦玲【作者单位】延安大学医学院生理教研室,陕西延安716000【正文语种】中文【中图分类】R744脊髓损伤是中枢神经系统的严重创伤，因其高致残率和高病死率一直成为医学研究的热门课题。
转化生长因子β3基因转染兔骨髓间充质干细胞诱导其向软骨表型的分化
中国组织工程研究与临床康复 第15卷 第27期 2011–07–02出版Journal of Clinical Rehabilitative Tissue Engineering Research July 2, 2011 Vol.15, No.27P.O. Box 1200, Shenyang 110004 50481Department of Joint Surgery, 2Department of Rehabilitation, 3Central Laboratory, Affiliated Hospital, Qingdao University Medical College, Qingdao 266000, Shandong Province, ChinaSui Lai-jian ★,Master, Department of Joint Surgery, Affiliated Hospital, Qingdao Univ ersity Medical College, Qingdao 266000, Shandong Province, ChinaCorrespondence to: Wang Ying-zhen, Doctoral supervisor, Department of Joint Surgery, Affiliated Hospital, Qingdao University Medical College, Qingdao 266000, Shandong Province, China wangy ingzhenqd@ Supported by: the Science andTechnology Tackle Key Plan of Qingdao City, No.06-2-2-6- nsh-2*Received: 2011-01-03 Accepted: 2011-02-24转化生长因子ß3基因转染兔骨髓间充质干细胞诱导其向软骨表型的分化*★隋来健1，吕成昱1，张海宁1，徐 浩1，高正玉2，隋爱华3，王英振1Differentiation of rabbit mesenchymal stem cells into cartilage phenotype induced by transforming growth factor beta 3Sui Lai-jian 1, Lü Cheng-yu 1, Zhang Hai-ning 1, Xu Hao 1, Gao Zheng-yu 2, Sui Ai-hua 3, Wang Ying-zhen 1AbstractBACKGROUND: Endogenous induction of cartilage differentiation is to use a certain carrier to integrate the target genes into stem cells to induce self-differentiation by self-induced secretion of cytokines.OBJECTIVE: To observe the ability of rabbit bone marrow mesenchymal stem cells (BMSCs) transfected with transforming growth factor beta 3 (TGF-ß3) to differentiate into cartilage phenotype through adeno-associated virus vector.METHODS: The third passage BMSCs in logarithmic growth phase was transfected by rAAV2-TGF-ß3. Total protein was extracted from cells at 3, 6, 9, 12 days after transfection, and TGF-ß3 was detected through ELISA. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen were determined by RT -PCR and the collagen Ⅱ protein was detected by western blot. The expression of proteoglycan was shown by toluidine blue staining.RESULTS AND CONCLUSION: After transfection, BMSCs can express TGF-ß3 stably, and make a better cartilagetransformation compared to negative control group. TGF-ß3 can be transfected into BMSCs through adeno-associated virus vectors and induce cell differentiation into cartilage phenotype.Sui LJ, Lü CY, Zhang HN, Xu H, Gao ZY, Sui AH, Wang YZ. Differentiation of rabbit mesenchymal stem cells into cartilage phenotype induced by transforming growth factor beta 3.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(27): 5048-5052. [ ]摘要背景：内源性诱导软骨分为就是通过一定的载体将目的基因整合入干细胞内，使其自行分泌诱导因子诱导自身进行分化。
碱性成纤维细胞生长因子重组腺病毒转染兔骨髓间充质干细胞的表型变化
碱性成纤维细胞生长因子重组腺病毒转染兔骨髓间充质干细胞的表型变化蔡弢艺;陈雄生;贾连顺;孙延卿;林斌;陈长青【期刊名称】《中国组织工程研究》【年(卷),期】2014(000)023【摘要】BACKGROUND:Exogenous basic fibroblast growth factor (bFGF) plays an important role in the ligament tissue healing process, and the use of transgenic methods to transfect exogenous genes into cells can promote the secretion of bFGF. OBJECTIVE:To observe phenotypic changes and the bFGF protein expression after bFGF recombinant adenovirus was used to transfect rabbit bone marrow mesenchymal stem cells (BMSCs). METHODS:Passage 2 BMSCs were divided into three groups:Ad.bFGF-eGFP group, Ad.eGFP group and control group. Under a phase contrast microscope we observed the changes in cellmorphology. The expression of bFGF protein in BMSCs was determined by enzyme-linked immunosorbent assay (ELISA). The proliferative curve was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). RESULTS AND CONCLUSION:The transfected cells showed a uniform phenotype of fibroblasts. MTT colorimetric assay revealed that more proliferative activity of transfected BMSCs was shown in the Ad.bFGF-eGFP group than in the Ad.eGFP group and control group. ELISA results showed that expression of bFGF protein was higher in the Ad.bFGF-eGFP group than inthe Ad.eGFP group and control group (P<0.05). BFGF recombinant adenovirus can induce the differentiation of BMSCs into fibroblasts, increase proliferative ability and promote the expression of bFGF protein.%背景：外源性碱性成纤维细胞生长因子在韧带组织的愈合过程中发挥着重要作用，采用转基因方法将外源性基因转入细胞内能促进碱性成纤维细胞生长因子分泌。
碱性成纤维细胞生长因子及内皮细胞生长因子对兔韧带细胞增殖的影
【】 目的 观察碱性成纤维细胞生长因子bFGF和内皮细胞生长因子ECGF对内侧副韧带MCL和前十字韧带ACL细胞增殖行为的影响。方法 培养10周龄新西兰白兔内侧副韧带和前十字韧带细胞，在培养液中分别加入bFGF和ECGF，以XTT方法测定细胞的增殖行为。 结果 bFGF在1 ngml时即对MCL细胞有显著的促增殖作用，5 ngml时开始对ACL细胞有促进作用，其浓度达50 ngml时，对两种细胞的促进作用最大。ECGF在3.125 ngml时即对MCL细胞有显著的促增殖作用，其浓度达12.5 ngml时对ACL细胞有促进作用。bFGF和ECGF在超过其最佳作用浓度后，随浓度升高促进作用不再增强。结论 bFGF和 ECGF可以促进韧带细胞增殖进而促进韧带创伤愈合。 【关键词】 碱性成纤维细胞生长因子;内皮细胞生长因子;韧带;细胞增殖 【Abstract】 Objective To observe the effects of basic fibroblast growth factor bFGF and endothelial cell growth factor ECGF on the proliferation of the cells from medial collateral ligament MCL and anterior cruciate ligament ACLdthods The MCL and ACL cells of tenweekold skeletally immature New Zealand white rabbit were cultured, while bFGF and ECGF were added to the cell culture media, the cellular proliferation was assayed by XTT method.Results The addition of 1 ngml bFGF enhanced the proliferation of MCL cells significantly, 5 ngml bFGF enhanced the proliferation of ACL cells ,this enhancement of proliferation of both MCL cells and ACL cells was maximal in the concentration of 50 ngml. The adition of 3.125 ngml ECGF enhanced the proliferation of MCL cells significantly, 12.5 ngml ECGF enhanced the proliferation of ACL cells.The efficacy of bFGF and ECGF declined gradually when they present at concentrations greater than the dose with the greatest efficacy.Conlusion It is evident that bFGF and ECGF can enhance the proliferation of the ligamentous cells and they can enhance the healing of ligamentous issue 毕业论文网 【Key words】 bFGF, ligament,proliferation,ECGF 韧带损伤是膝部的常见运动创伤，尤以内侧副韧带medial collateral ligament,MCL和前十字韧带anterior cruciate ligament,ACL多见，MCL和ACL是维持膝关节稳定的重要韧带，其损伤可使膝关节丧失稳定性，引起功能障碍，甚至导致骨关节炎[21]。在中央实质部完全断裂后，MCL可自然愈合，有时不需手术介入;而ACL损伤后，即使手术缝合断端，其愈合率也较低。韧带损伤后的愈合过程与一般结缔组织相似，都经过急性炎症期、修复再生期和重塑或成熟期三个阶段[2]。研究表明[3]，在损伤韧带的局部存在着多种生长因子，它们被认为是韧带正常愈合过程的重要调节者。使用生长因子促进韧带愈合是近年来研究的焦点之一。作者通过体外培养MCL和ACL细胞，分别加入碱性成纤维细胞生长因子bFGF和内皮细胞生长因子ECGF，以观察bFGF和ECGF对韧带细胞增殖的影响，为应用bFGF和ECGF促进韧带创伤愈合提供依据。 1 材料与方法 1.1 主要试剂 bFGF暨南大学生物医学工程研究所惠赠，ECGFSigma公司产品，美国，DMEM培养基及胰蛋白酶Gibco公司产品，胶原酶Ⅱ、XTTSigama公司，美国，胎牛血清杭州四季青生物有限公司,酶标仪日本Reader。 毕业论文网 1.2 内侧副韧带和前十字韧带成纤维细胞培养 无菌切取兔龄10周新西兰白兔MCL和ACL，在含100 μgml PG和100 μgml SM双抗Hank’s液中剔除滑膜等外周组织，以眼科剪剪成1 mm3左右的碎片。配制0.25%的胶原酶Ⅱ，将韧带组织移至胶原酶液中，37℃条件下消化1～2 h，组织消化后，无血清DMEM培养液冲洗，离心，以含15%胎牛血清的DMEM培养液悬浮细胞，接种于一次性塑料培养瓶Corning公司，美国中，置于37℃、体积分数为5%CO2培养箱内培养。每3 d换液一次，待细胞汇合后，以胰蛋白酶消化传代。 1.3 XTT法测定韧带细胞增殖 收获第2～5代的MCL和ACL细胞，以5×103孔的浓度，接种于96孔培养板上，待细胞贴壁后，分别加入1、5、10、50、100及500 ngml的bFGF和3.125、6.25、12.5、25、50及100 ngml的ECGF。对照组不加bFGF和ECGF。每组四个复孔。CO2培养箱内培养，每天观察一次。培养3 d后，每孔加入50 μl XTT1 mgml。在37℃、5%CO2培养箱内继续孵育，3 h后，在酶标仪上比色参考波长650 nm,测量波长450 nm，读取OD值。 1.4 统计学处理 实验所得数据以SPSS11.0统计软件进行分析，用方差分析和Dunnett法检验，α=0.05。 2 结果 2.1 膝关节韧带细胞培养 膝关节韧带细胞贴壁时间约5～6 d，体外培养条件下，ACL细胞的增殖速度明显慢于MCL细胞;ACL细胞原代生长约需13～15 d，MCL细胞原代生长约需11～13 d。1∶2传代的细胞不超过6代时，基本上能在5～7 d内汇合成单层。ACL与MCL细胞贴壁生长，形态呈梭形，与典型的成纤维细胞形态相似图1、2。 毕业论文网 图1 原代培养兔ACL细胞×100 图2 原代培养兔MCL细胞×100 2.2 bFGF对兔韧带成纤维细胞增殖的影响 表1可见，MCL和ACL各实验组OD值均大于对照组，经统计学检验，ACL各组OD值方差分析，F=28.02，P各实验组与对照组比较，1 ngml组P0.05，≥5 ngml各组P均0.01。MCL各组OD值方差分析，F=90.66，P各实验组与对照组比较均为P0.01。表明bFGF对MCL和ACL细胞的增殖均有促进作用。1 ngml bFGF 即可促进MCL细胞生长，5 ngml bFGF 即可促进ACL细胞生长。bFGF浓度高至50 ngml时，其促进作用达到最大值;超过其最佳作用浓度后，随浓度升高促进作用不再增强P0.05。 2.3 ECGF对兔韧带成纤维细胞增殖的影响 表2可见，MCL和ACL各实验组OD值均大于对照组，经统计学检验，ACL各组OD值方差分析，F=3.82，P=0.01;各实验组与对照组比较，≥12.5 ngml各组P值均0.05。MCL各组OD值方差分析，F=136.06，P各实验组与对照组比较均为P0.01。表明ECGF 对MCL和ACL细胞的增殖均有促进作用。3.125 ngml ECGF 即可显著促进MCL细胞生长，浓度在12.5 ngml时，其促进作用达到最大值;超过其最佳作用浓度后，随浓度升高促进作用不再增强P0.05。 表1 bFGF对兔韧带细胞增殖的影响表2 ECGF对兔韧带细胞增殖的影响 毕业论文网 3 讨论 bFGF和ECGF是一些具有生物活性的多肽，具有趋化性、促有丝分裂和促进胞外基质合成的作用。它们可与细胞表面特异性受体结合，产生第二信使;第二信使可使细胞核根据特定的基因诱导或抑制基因组DNA向mRNA的转录，改变某些蛋白质的水平，进而促进DNA的复制和有丝分裂。 毕业论文网
碱性成纤维生长因子和人重组骨形态发生蛋白2诱导分化兔骨髓间充质干细胞
碱性成纤维生长因子和人重组骨形态发生蛋白2诱导分化兔骨髓间充质干细胞胡运生;范清宇;马保安;张殿忠;刘云燕【期刊名称】《中国组织工程研究》【年(卷),期】2006(010)001【摘要】背景:骨髓间充质干细胞依赖特定的生长和分化因子能分化为骨、软骨、脂肪等不同组织.人重组骨形态发生蛋白2有明显的骨诱导作用,能诱导未分化的骨髓间充质干细胞不可逆地形成骨与软骨,并最终导致新骨生成;碱性成纤维生长因子亦参与调控骨髓间充质干细胞的软骨与骨的分化.目的:观察碱性成纤维生长因子和人重组骨形态发生蛋白2对兔骨髓间充质干细胞增殖和分化的影响,以便寻求合适的骨形成诱导方法以代替常规的成骨培养体系.设计:随机对照实验.单位:解放军第四军医大学唐都医院骨肿瘤研究所.材料:培养的兔骨髓间充质干细胞.方法:实验于2004-06/12在第四军医大学唐都医院骨肿瘤研究所暨全军骨科中心完成. ①体外培养的兔骨髓间充质干细胞分别经3种不同的生长因子(100μg/L人重组骨形态发生蛋白2,100μg/L碱性成纤维生长因子,100μg/L人重组骨形态发生蛋白2加100μg/L碱性成纤维生长因子)和成骨培养体系处理. ②其增殖和分化通过四甲基偶氮唑盐比色法,碱性磷酸酶、骨钙素检测和钙结节Von Kossa染色观察.主要观察指标:通过检测增殖率和碱性磷酸酶活性比较不同生长因子对兔骨髓间充质干细胞增殖和分化的影响.结果: ①人重组骨形态发生蛋白2能促进兔骨髓间充质干细胞的增殖和分化,特别是其分化. ②碱性成纤维生长因子能刺激兔骨髓间充质干细胞的增殖,与对照组相比,细胞增值率提高近100%;虽然对兔骨髓问充质干细胞分化无明显影响,但其能促进人重组骨形态发生蛋白2对兔骨髓间充质干细胞分化.结论:碱性成纤维生长因子和人重组骨形态发生蛋白2是兔骨髓间充质干细胞有效的分化因子,两者在体外都能促进兔骨髓间充质干细胞的增殖和分化.碱性成纤维生长因子和人重组骨形态发生蛋白2能协同作用加速骨诱导及骨形成,可以用作分化骨组织工程中的种子细胞.%BACKGROUND: Mesenchymal stem cells (MSCs) are capable of differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli, specific growth and differentiation factors.Recombinant human bone morphogenetic protein 2 (rhBMP-2) produced by gene engineering has an obvious osteoinductive activity and can induce undifferentiated mesenchymal cells into cartilage and bone irreversibly, resulting in new bone formation. Basic fibroblast growth factor (bFGF) modulates chondrogenic and osteogenic differentiation of MSCs.OBJECTIVE: To investigate the effect of bFGF and rhBMP-2 on the differentiation and proliferation of cultured rabbit mesenchymal stem cell in order to find out an optimal way of osteogenesis instead of conventional osteogenic supplements (OS).DESIGN: A randomized and controlled trial.SETTING: General Institute of Orthopaedic Oncology, Tangdu Hospital,Fourth Military Medical University of ChinesePLA.MATERIALS: The subjects were rabbit mesenchymal stem cells cultured by the author.METHODS: This experiment was conducted at the Center of Orthopaedic Surgery, General Institute of Orthopaedic Oncology, Tangdu Hospital,Fourth Military Medical University of Chinese PLA from June 2004 to December 2004. ①Rabbit MSCs cultured in vitro were treated with different growth factor (100 μg/L rhBMP-2, 100 μg/L bFGF, 10 μg/L rhBMP-2and 100 μg/L bFGF and OS; ②The proliferation and differentiationof MSCs were observed through activity of MTT, expression of alkaline phosphatase(ALP), and von Kossa staining.MAIN OUTCOME MEASURES: the rate of proliferation and the activity of ALP.RESULTS: ①rhBMP-2 could promote the proliferation and differentiation of MSCs, especially the cell differentiation; ②bFGF could stimulate the proliferation , the cellular proliferation rate increased 100% as compared with control group, and has no effect on differentiation of MSCs , but it could enhance effect on the cell proliferation of rhBMP-2.CONCLUSION: bFGF and rhBMP-2 are effective induction factors for MSCs. Both of them can stimulate the proliferation and differentiation of MSCs in vitro. bFGF and rhBMP-2 exerted a synergetic action in speeding up the pace of osteoinduction and osteogenesis and can be used to differentiate seed cells for tissue engineering bone.【总页数】3页(P163-165)【作者】胡运生;范清宇;马保安;张殿忠;刘云燕【作者单位】解放军第四军医大学唐都医院骨肿瘤研究所暨全军骨科中心,陕西省西安市,710038;解放军第四军医大学唐都医院骨肿瘤研究所暨全军骨科中心,陕西省西安市,710038;解放军第四军医大学唐都医院骨肿瘤研究所暨全军骨科中心,陕西省西安市,710038;解放军第四军医大学唐都医院骨肿瘤研究所暨全军骨科中心,陕西省西安市,710038;解放军第四军医大学唐都医院骨肿瘤研究所暨全军骨科中心,陕西省西安市,710038【正文语种】中文【中图分类】R394.2【相关文献】1.人骨形态发生蛋白2基因重组腺病毒表达载体转染兔骨髓间充质干细胞 [J], 何春耒;姬广林;刘午阳;吴东宝;何澄;王茂源;叶勇军;莫建文;高辉2.人骨形态发生蛋白2基因重组腺病毒表达载体转染兔骨髓间充质干细胞 [J], 何春耒;姬广林;刘午阳;吴东宝;何澄;王茂源;叶勇军;莫建文;高辉3.重组人甲状旁腺素对人骨髓间充质干细胞表达骨形态发生蛋白2的影响 [J], 李丁;王俊芳;张成玉4.骨形态发生蛋白2及碱性成纤维生长因子2对大鼠骨髓间充质干细胞膜片增殖和成骨分化的影响 [J], 张文静; 王佳; 田梦婷; 韩祥祯; 周琦琪; 何惠宇5.骨形态发生蛋白2及碱性成纤维生长因子2对大鼠骨髓间充质干细胞膜片增殖和成骨分化的影响 [J], 张文静; 王佳; 田梦婷; 韩祥祯; 周琦琪; 何惠宇因版权原因，仅展示原文概要，查看原文内容请购买。
兔骨髓间充质干细胞的生物学特征及其对不同生长因子的反应
兔骨髓间充质干细胞的生物学特征及其对不同生长因子的反应【摘要】为了研究兔骨髓间充质干细胞(rBM-MSC)的生物学特征及其对不同生长因子的反应，使用贴壁培养法从兔骨髓单个核细胞中获得间充质干细胞，在光学显微镜和电子显微镜下观察其基本生长行为和生物学特征；使用流式细胞仪检测rBM-MSC的免疫学表型；RT-PCR法检测其胶原表达；诱导rBM-MSC多向分化并使用特异性染色和RT-PCR予以鉴定；最后使用MTT法检测IL-1、3、8和HGF对rBM-MSC生长增殖的影响。
结果显示：贴壁生长的rBM-MSC具有典型的成纤维样细胞形态，可传15代以上，第5代rBM-MSC高表达基质受体CD44，低表达造血细胞标记CD45；RT-PCR显示高表达I型胶原，弱表达II型胶原，不表达X型胶原；在不同的诱导条件下，rBM-MSC 可被诱导分化为成骨细胞、软骨细胞、脂肪细胞和神经元样细胞。
rBM-MSC对IL-3最为敏感，10 ng/ml的低浓度IL-3可显着促进细胞增殖达32％以上，而高浓度IL-3能显着抑制其生长。
结论：分离培养了rBM-MSC，其生物学特征与人和猕猴等BM-MSC相似的生物学特性，低浓度IL-3可有效促进其增殖。
【关键词】骨髓；间充质干细胞；生长因子；兔Biological Characteristics of Rabbit Bone Marrow Mesenchymal Stem Cells and Their Response to Different Growth FactorsAbstractThis study was aimed to analyze the biological characteristics of rabbit bone marrow mesenchymal stem cells (rBM-MSCs) and their response to different growth factors. RabbitBM-MSCs were separated from bone marrow mononuclear cells by using adherent cultivation. Biologicalcharacteristics were investigated by optical and electron microscopy. Immunophenotype of rBM-MSCs was measured by flow cytometry. Theexpression of collagen was detected by RT-PCR. Differentiation potential was identified by specific staining andRT-PCR. The response of rBM-MSCs to IL-1, 3, 8 and HGF with different concentrations were tested by MTT. The results showed that the rBM-MSCs gave rise to a population of adherent cells characterized by the presence of a predominant cell type with a typical fibroblast-like morphology and could be cultured for over 15 passages. CD44 was highly expressed on F5 rBM-MSCs (32%) and CD45 was lowly expressed (%). Type I collagen was highly expressed, while type II collagen was lowly expressed and type X collagen was not detected on rBM-MSCs using RT-PCR method. In various conditions inducting differentiation, rBM-MSCs could differentiate into the osteoblast, chondrocyte, adipocyte and neuron-like cells. The rBM-MSCs weresensitive to IL-3, even low concentration (10 ng/ml) of IL-3 could promote the proliferation of rBM-MSCs effectively (32％, ), whereas high concentration IL-3 inhibited it significantly. It is concluded that rabbit BM-MSCs were successfully isolated and culture-expanded. The biological characteristics of rabbit BM-MSCs are similar to those of human and rhesus BM-MSCs. IL-3 with low concentration can promote the proliferation of rBM-MSCs effectively, but high concentration of IL-3 can inhibit their proliferation.Key wordsbone marrow; mesenchymal stem cell; growth factor; rabbit骨髓间充质干细胞具有高度自我更新和多向分化的潜能，在不同的诱导条件下，可分化为多种组织细胞类型，如骨髓基质细胞、成骨细胞、成软骨细胞和脂肪细胞［1］。
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碱性成纤维细胞生长因子重组腺病毒转染兔骨髓间充质干细胞的表型变化蔡弢艺;陈雄生;贾连顺;孙延卿;林斌;陈长青【摘要】BACKGROUND:Exogenous basic fibroblast growth factor (bFGF) plays an important role in the ligament tissue healing process, and the use of transgenic methods to transfect exogenous genes into cells can promote the secretion of bFGF. OBJECTIVE:To observe phenotypic changes and the bFGF protein expression after bFGF recombinant adenovirus was used to transfect rabbit bone marrow mesenchymal stem cells (BMSCs). METHODS:Passage 2 BMSCs were divided into three groups:Ad.bFGF-eGFP group, Ad.eGFP group and control group. Under a phase contrast microscope we observed the changes in cellmorphology. The expression of bFGF protein in BMSCs was determined by enzyme-linked immunosorbent assay (ELISA). The proliferative curve was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). RESULTS AND CONCLUSION:The transfected cells showed a uniform phenotype of fibroblasts. MTT colorimetric assay revealed that more proliferative activity of transfected BMSCs was shown in the Ad.bFGF-eGFP group than in the Ad.eGFP group and control group. ELISA results showed that expression of bFGF protein was higher in the Ad.bFGF-eGFP group than in the Ad.eGFP group and control group (P<0.05). BFGF recombinant adenovirus can induce the differentiation of BMSCs into fibroblasts, increase proliferative ability and promote the expression of bFGF protein.%背景：外源性碱性成纤维细胞生长因子在韧带组织的愈合过程中发挥着重要作用，采用转基因方法将外源性基因转入细胞内能促进碱性成纤维细胞生长因子分泌。
目的：观察碱性成纤维细胞生长因子重组腺病毒载体转染兔骨髓间充质干细胞后表型变化及碱性成纤维细胞生长因子蛋白的表达。
方法：取 P2代骨髓间充质干细胞，分为3组，正常培养组为对照组，其他2组分别转染重组腺病毒(Ad.bFGF-eGFP)及空病毒(Ad.eGFP)。
相差显微镜观察各组细胞形态变化，MTT法测定细胞增殖曲线，ELISA法分析各组细胞上清液中碱性成纤维细胞生长因子蛋白质量浓度。
结果与结论：转染后细胞呈现均一的成纤维细胞表型；碱性成纤维细胞生长因子重组腺病毒组细胞增殖活性高于空病毒组及对照组；ELISA结果提示碱性成纤维细胞生长因子重组腺病毒组在7 d内上清液中碱性成纤维细胞生长因子蛋白质量浓度较空病毒组及对照组增加，差异有显著性意义(P<0.05)。
提示碱性成纤维细胞生长因子重组腺病毒转染骨髓间充质干细胞可促进其向成纤维细胞分化，增加增殖活力，促进其碱性成纤维细胞生长因子蛋白的表达。
【期刊名称】《中国组织工程研究》【年(卷),期】2014(000)023【总页数】5页(P3727-3731)【关键词】干细胞;骨髓干细胞;碱性成纤维细胞生长因子;骨髓间充质干细胞;成纤维细胞;腺病毒;转染;863项目【作者】蔡弢艺;陈雄生;贾连顺;孙延卿;林斌;陈长青【作者单位】解放军第175医院骨科医院，福建省漳州市 363000;解放军第二军医大学附属长征医院骨科，上海市 200003;解放军第二军医大学附属长征医院骨科，上海市 200003;解放军第175医院骨科医院，福建省漳州市 363000;解放军第175医院骨科医院，福建省漳州市 363000;解放军第二军医大学附属长征医院骨科，上海市 200003【正文语种】中文【中图分类】R394.20 引言 Introduction骨髓间充质干细胞是一类具有自我更新和多向分化潜能的成体干细胞[1]。
骨髓间充质干细胞可以通过骨髓穿刺获得，体外易于分离、纯化、培养、扩增[2]，在体外培养扩增3周可获取2×107个细胞，是组织工程良好的种子细胞来源。
相关生长因子可以促进骨髓间充质干细胞的增殖与分化，不同的细胞因子对骨髓间充质干细胞的功能亦有影响。
碱性成纤维细胞生长因子是重要的有丝分裂促进分子，韧带组织愈合过程中碱性成纤维细胞生长因子对于中胚层及神经外胚层来源的细胞都有强烈的促有丝分裂作用，能有效的促进细胞增殖，自我更新，研究表明其可诱导间充质干细胞向韧带细胞方向分化[3-4]。
肌腱愈合的自然过程中碱性成纤维细胞生长因子的mRNA表达出现于损伤后第1天，于第7天出现高峰，并在随后的2周内下降到较低的水平[5]，在韧带修复过程外膜碱性成纤维细胞生长因子蛋白的高分泌对于韧带早期修复具有促进作用，在损伤韧带的重塑形及提高新生韧带的生物力学强度上也有着重要作用。
外源性碱性成纤维细胞生长因子对细胞增殖促进作用有明显的量效关系，Hankemeier等[6]发现低剂量碱性成纤维细胞生长因子(3 μg/L)添加于培养基中能使骨髓间充质干细胞增加Ⅰ，Ⅲ型胶原、平滑肌肌动蛋白、波形蛋白和纤维粘连蛋白mRNA的表达，其均为韧带的特异性细胞外基质。
在新西兰兔浅屈肌腱修复过程中外源性碱性成纤维细胞生长因子通过皮下注射能减少局部的瘢痕形成并增加肌腱的净质量及生物力学强度，其主要通过促进肌腱细胞成熟及增强胶原纤维直径与密度实现[7]。
实验以腺病毒载体携带碱性成纤维细胞生长因子基因(Ad.bFGF-eGFP)转染骨髓间充质干细胞，观察转染后细胞形态、增殖速率及碱性成纤维细胞生长因子蛋白表达水平的变化，探讨转基因骨髓间充质干细胞作为组织工程韧带种子细胞的可行性。
1 材料和方法 Materials and methods设计：对比观察实验。
材料：实验动物：新西兰大白兔，3月龄，体质量1.0-1.5 kg，由解放军第二军医大学实验动物中心提供。
实验过程中对动物的处置应符合2009年《Ethical issues in animal experimentation》相关动物伦理学标准的条例。
方法：兔骨髓间充质干细胞的体外分离培养方法：在无菌条件下采用髂前上棘穿刺获取兔骨髓2.0-3.0 mL，密度梯度离心及贴壁法采集骨髓间充质干细胞，细胞计数。
以1×108 L-1 (T-25 cm培养瓶)的细胞浓度接种于含体积分数10%胎牛血清的DMEM培养基中，置于37 ℃、体积分数5%CO2的饱和度湿度培养箱中，培养3 d后，全量更换培养基，弃掉未贴壁细胞。
在培养过程中，每3 d全量换液1次。
当原代细胞(P0)生长至瓶底部80%-90%融合时用1∶1的EDTA(2%)和胰蛋白酶(25%)混合液消化传代，在倒置显微镜下观察控制消化时间，以5×107 L-1的浓度接种于T-25cm培养瓶中进行扩增培养。
腺病毒转染：24孔板标记顺序后分成3组，每组接种P2代骨髓间充质干细胞1×104个，培养24 h。
第1，2孔用Ad.bFGF-eGFP病毒液浸染，作为第3，4孔用Ad.eGFP病毒液浸染，前2组MOI值设为50，第5，6孔继续常规培养。
37 ℃体积分数5%CO2孵育箱中孵育24 h后换用体积分数10%胎牛血清DMEM 培养液。
每72 h收集一次培养的上清液，装入15 mL离心管，共收集5次，每次收集后向细胞中重新加入体积分数10%胎牛血清DMEM培养液。
转染后采用流式细胞检测细胞绿色荧光表达。
图1 骨髓间充质干细胞的细胞形态及转染情况(×100)Figure 1 Morphology and transfection of bone marrow mes enchymal stem cells (×100)图注：图中A 为P0代细胞培养24 h细胞贴壁；B培养3 d后开始出现细胞集落；C为P2代骨髓间充质干细胞转染后24 h同一视野相差显微镜照片；D为P2代骨髓间充质干细胞转染后24 h同一视野荧光显微镜照片示，MOI值=50时转染后有较强的绿色荧光表达，细胞生长良好。
图2 各组细胞转染第1天流式细胞仪检测结果Figure 2 Flow cytometry detection of cells at 1 day after transfection in each group图注：重组腺病毒Ad.bFGF-eGFP组转染骨髓间充质干细胞24 h后流式细胞仪显示转染率可达97.8%。
图3 各组细胞转染后增殖速率比较Figure 3 Proliferative ability of transfected cells in different groups图注：重组腺病毒Ad.bFGF-eGFP组细胞浓度在2，3 d显著大于对照组及空病毒组，而空病毒组及对照组间差异无显著性意义。
与对照组及空病毒组比较，aP < 0.05，bP < 0.01。
MTT法检测细胞增殖速率：Ad.bFGF-eGFP转染骨髓间充质干细胞后，与正常第2代骨髓间充质干细胞以1×103/孔的浓度分别接种于96孔培养板，每个培养板每组接种8孔，培养后3 d后在各检测孔吸去培养液，PBS冲洗3遍后加入MTT 100 µL，37 ℃孵育4 h，加DMSO 100 µL，30 min待结晶完全溶解后，以MTT 100 µL+DMSO 100 µL做标准孔，紫外分光光度计450 nm检测吸光度值(A)。