氯化铵裂解法与非裂解法提取人脂肪干细胞生物学特性的差异

《中国组织工程研究》 Chinese Journal of Tissue Engineering Research 45 ·研究原著·

李梓菲，女，1990年生，吉林省长春市人，汉族，中国医学科学院北京协和医学院整形外科学在读博士，主要从事乳房整形美容相关临床研究、脂肪干细胞相关基础研究。

通讯作者：栾杰，博士，主任医师，中国医学科学院北京协和医学院整形外科医院，北京市 100144

文献标识码:A 来稿日期：2019-01-07 送审日期：2019-01-17 采用日期：2019-03-12 在线日期：2019-09-26

Li Zifei, MD candidate, Plastic Surgery Hospital, chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100144, China

Corresponding author: Luan Jie, MD, Chief physician, Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100144, China

氯化铵裂解法与非裂解法提取人脂肪干细胞生物学特性的差异 李梓菲，王 黔，栾 杰，穆大力，刘春军，辛敏强，付 苏，徐伯扬，刘温悦，陈 琳，程 浩，王成龙，康德旎，李尚善，祁 珺 (中国医学科学院北京协和医学院整形外科医院，北京市 100144) DOI:10.3969/j.issn.2095-4344.1869 ORCID: 0000-0002-8958-4975(李梓菲)

文章快速阅读：

文题释义： 氯化铵红细胞裂解液：含有氯化铵成分的红细胞裂解液，此种裂解液被广泛应用于骨髓干细胞及脂肪干细胞的提取，其目的是去除组织内的红细胞，使目的细胞群进一步纯化。其作用机制为：红细胞内有大量碳酸酐酶，可自发将NH3转化为NH4+，CO2转化为HCO3-，促使胞外NH3和CO2连续内流造成细胞裂解。 基质血管成分：脂肪组织中除了含有成熟脂肪细胞外，还包含一组混杂的细胞群，即基质血管成分。基质血管成分包括血管内皮细胞、脂肪祖细胞、成纤维细胞、周细胞、脂肪干细胞，造血干细胞、红细胞等，其具有多向分化潜能及促进组织血管再生等功能，在组织及器官的再生、修复中具有极大的应用前景。

摘要 背景：脂肪干细胞的提取及纯化流程尚未建立统一标准。最常用的纯化脂肪干细胞的方法是利用红细胞裂解液处理基质血管成分。但这一步骤是否对脂肪干细胞存在不良影响仍缺乏证据，是否有利于未来临床应用仍有待探讨。 目的：比较氯化铵红细胞裂解液法和非裂解法提取脂肪干细胞的效率，并进一步比较两种方法提取脂肪干细胞的生物学特性。 方法：收集吸脂手术患者脂肪组织，经Ⅰ型胶原酶消化后，利用或不用氯化铵红细胞裂解液对基质血管成分进行纯化，MuseTM细胞状态分析仪对活细胞计数及活细胞比例评估；将基质血管成分接种后培养人脂肪干细胞至第2代，光学显微镜观察脂肪干细胞形态，流式细胞学分析脂肪干细胞表型，CCK-8法绘制细胞增殖曲线，成脂及成骨培养基诱导后油红O及茜素红染色法分别评估成脂、成骨分化能力。该实验经中国医学科学院整形外科医院伦理委员会批准，并与患者签署相关知情同意书。 结果与结论：①与裂解组相比，非裂解组提取即刻的基质血管成分中非红活细胞数更多，活细胞百分比更大；②两组脂肪干细胞均呈梭形、鱼群状排列；③两组细胞均高表达CD90、CD73、CD105等细胞表面抗原，低表达或不表达CD11b、CD34、CD19、CD45、HLA-DR等细胞表面抗原；④非裂解组细胞增殖速度更快，成脂及成骨能力两组间无明显区别；⑤结果表明，利用氯化铵红细胞裂解液法提取基质血管成分降低了脂肪干细胞提取效率，抑制了脂肪干细胞增殖能力。非裂解过程不影响脂肪干细胞表型及成脂、成骨分化能力。因此不建议在人脂肪干细胞提取过程中进行氯化铵法红细胞裂解。 关键词： 红细胞裂解液；细胞提取；脂肪干细胞；细胞活性；细胞表型；细胞增殖 中图分类号：R459.9；R394.2；R622 基金资助： 中国医学科学院医学与健康科技创新工程：脂肪干细胞应用于组织再生与修复的关键问题研究(2017-I2M-3-006)，项目负责人：栾杰

获取人脂肪抽吸物 Ⅰ型胶原酶消化 基质血管成分 (1)红细胞裂解法处理； (2)非红细胞裂解法处理 即刻细胞计数 培养至第2代 (1)光镜下观察细

胞形态； (2)流式细胞仪检测细胞表型； (3)CCK-8检测细胞增殖能力； (4)成脂分化能力； (5)成骨分化能力

结论： (1)非裂解法提取人脂肪干细胞是一种更有效的方法； (2)其可以更好地保留细胞增殖能力，并不影响细胞表型及成脂、成骨分化能力

文章特点— (1)对脂肪干细胞的提取过程进行优化，使之更有效和安全，以适用于脂肪干细胞的临床应用是研究的热点之一； (2)该研究比较了氯化铵红细胞裂解液法和非裂解法提取脂肪干细胞的效率，以及2种方法提取脂肪干细胞的生物学特性。 李梓菲，王黔，栾杰，穆大力，刘春军，辛敏强，付苏，徐伯扬，刘温悦，陈琳，程浩，王成龙，康德旎，李尚善，祁珺. 氯化铵裂解法与非裂解法提取人脂肪干细胞生物学特性的差异[J]. 中国组织工程研究，2020，24(1):45-50. DOI:10.3969/j.issn.2095-4344.1869

46 Ammonium chloride lysis method versus non-lysis method for isolation of human adipose-derived stem cells

Li Zifei, Wang Qian, Luan Jie, Mu Dali, Liu Chunjun, Xin Minqiang, Fu Su, Xu Boyang, Liu Wenyue, Chen Lin, Cheng Hao, Wang Chenglong, Kang Deni, Li Shangshan, Qi Jun (Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100144, China)

Abstract BACKGROUND: A general standard has not been established for the extraction and purification of adipose-derived stem cells (ADSCs). An erythrocyte lysis step for stromal vascular fraction is the commonly used method in the procedure for ADSCs isolation. However, there is a lack of evidence on whether this step will have adverse effects on human ADSCs (hADSCs). OBJECTIVE: To test the efficiency of two hADSCs isolation methods, which are erythrocyte-lysis method based on ammonium chloride and non-lysis method. Moreover, the biological characteristics of the hADSCs isolated by the two methods were also compared. METHODS: After collagenase digestion of lipoaspirate, erythrocyte lysis buffer was used to purify stromal vascular fraction in erythrocyte-lysis method, while in non-lysis method the buffer was not used. A MuseTM cell analyzer was used to assess living cell counting and proportion of stromal vascular fraction in both methods. Then hADSCs were cultured to the second passage for next testing. Cell morphology was observed under light microscope. Cell phenotype was detected by flow cytometry. Cell counting kit-8 was used to evaluate cell proliferation. Oil red O staining and alizarin staining were used to evaluate adipogenic and osteogenic ability of hADSCs after adipogenic and osteogenic induction. This study was approved by the Ethics Committee of the Plastic Surgery Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, and informed consents were signed by all participants. RESULTS AND CONCLUSION: (1) Compared with the erythrocyte lysis group, hADSCs obtained in the non-lysis group contained a larger number and a larger percentage of non-erythrocyte living cells. (2) The two groups of hADSCs were spindle-shaped and arranged as a fish shape. (3) The cell phenotypes of both groups met the phenotypic requirements for human mesenchymal stem cells. (4) The cell proliferation in the non-lysis group was faster than that in the erythrocyte lysis group, while there was no difference in the adipogenic and osteogenic abilities between the two groups. In conclusion, the use of erythrocyte lysis buffer reduces the isolation efficiency of hADSCs and inhibits cell proliferation. The non-lysis isolation method does not affect phenotypes, adipogenic and osteogenic ability of hADSCs. Therefore, it is not recommended to implement erythrocyte lysis during the isolation of hADSCs. Key words: erythrocyte lysis buffer; cell isolation; adipose-derived stem cells; cell viability; cell phenotype; cell proliferation Funding: Innovation Fund for Medical Science (CIFMS), No. 2017-I2M-3-006 (to LJ)