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新生小鼠Lin⁻ Sca-1⁺心脏干细胞分离培养方案的优化

李端端, 石山慧, 周密, 马秀霞, 高建忠, 张雅琼, 杨丽文, 左琳

山西医科大学1基础医学院生理系；第一临床医学院病理科；第一临床医学院；第二临床医学院，太原 030001

摘要

心脏原位干细胞(cardiac stem cells, CSCs)治疗心肌梗死具有公认的疗效，但是其分离及培养技术尚不完善，这是制约其临床应用的关键技术问题。为解决这一问题，本研究旨在优化Lin− (lineage-negative) Sca-1+ (stem cell antigen-1-positive) CSCs分离方案。用组织块酶解法(混合酶液)分离C57BL/6J新生(出生0~3天)小鼠Lin− Sca-1+ CSCs。在已有的研究基础上，严格控制消化时间、次数、温度、搅拌速度、离心时间和转速等多重因素，结合免疫磁珠分选获得纯度较高的Lin− Sca-1+ CSCs。此后，对分离纯化的原代细胞进行培养，优化培养基成分、换液时间和方式。采用流式细胞术及免疫荧光染色技术，检测分选细胞的纯度及传代培养细胞中Sca-1+细胞的百分比。结果显示：(1) 本法分离获得的Lin− Sca-1+ CSCs纯度高达(85.03 ± 5.60)%；(2) 离体培养过程中，细胞生长状态良好，原代培养5天开始有干细胞克隆球生成，培养7天即可铺满皿底，生长曲线显示，离体培养第3天细胞进入对数增长期；(3) 免疫组化染色结果显示：干细胞特异性标志Sca-1的表达随着传代的进行有一定的衰减。流式细胞术检测结果显示，第一代、第三代和第五代培养细胞的Sca-1阳性率分别为(71.82 ± 2.63)%、(58.38 ± 3.70)%、(46.19 ± 4.72)%。以上结果表明，本研究所建立的组织块酶解法结合免疫磁珠分选法可获得纯度较高的Lin− Sca-1+ CSCs，同时干细胞离体培养体系相对稳定。本研究所建立的分离及培养方法简单稳定、可靠有效，为进一步研究Sca-1+ CSCs治疗心肌梗死奠定了良好的方法学基础。

关键词： 干细胞; 分离和纯化 ; 原代培养 ; 鉴定

分类号：R329

Optimization of isolation and culture of Lin⁻ Sca-1⁺ cardiac stem cells from newborn mice

LI Duan-Duan, SHI Shan-Hui, ZHOU Mi, MA Xiu-Xia, GAO Jian-Zhong, ZHANG Ya-Qiong, YANG Li-Wen, ZUO Lin

Department of Physiology, Basic Medical College； Department of Pathology, the First Affiliated Hospital； The First Affiliated Hospital； 4The Second Affiliated Hospital, Shanxi Medical University, Taiyuan 030001, China

Abstract

Cardiac stem cells (CSCs) transplantation has been recognized to be effective on the treatment of myocardial infarction (MI), but some techniques still need to be developed in the isolation and culture of CSCs, which is the key problem restricting the clinical application of CSCs. This study was focused on the isolation of Lin− (lineage-negative) Sca-1+ (stem cell antigen-1-positive) CSCs from newborn C57BL/6J mice (0–3 d) by mixed enzymatic-explant isolation in combination with immunomagnetic separation. The digesting time, digesting frequency, incubation temperature, stirring speed, centrifugation time and rotational speed were strictly controlled in the experiment. In order to increase the survival rate of CSCs, the medium changing time and manner were optimized in primary CSCs culture. The percentages of Sca-1+ cells in primary and passage cells were detected by flow cytometry and immunofluorescence staining. The results showed that: (1) the proportion of Lin− Sca-1+ cells within the collected cells could be as high as (85.03 ± 5.60)% after isolation and purification; (2) In vitro culture of Lin− Sca-1+ CSCs grew into spheres on the 5th day, and over the whole bottom of the dish on the 7th day. The growth curve showed that the cells were in logarithmic growth phase on the 3rd day; (3) Immuno- fluorescence staining data showed that the expression of Sca-1, the CSCs membrane-specific marker, was decreased after subculture, and flow cytometry data showed that the percentages of Sca-1+ cells were (71.82 ± 2.63)%, (58.38 ± 3.70)% and (46.19 ± 4.72)% in passage 1 (P1), P3, and P5 CSCs, respectively. The above results suggest that high purity of Lin− Sca-1+ CSCs can be obtained by enzymolysis combined with immunomagnetic separation method. Moreover, the CSCs culture system is stable. In our experiment, the Sca-1+ CSCs isolation and culture method has been successfully established, and it is simple, stable, effective and reliable. The method can provide a stable methodological basis for the treatment of MI by Lin− Sca-1+ CSCs transplantation.

Key words: Stem cells; isolation and purification ; primary cell culture ; identification

收稿日期：2016-12-11　　录用日期：2017-02-28

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引用本文：

李端端, 石山慧, 周密, 马秀霞, 高建忠, 张雅琼, 杨丽文, 左琳. 新生小鼠Lin⁻ Sca-1⁺心脏干细胞分离培养方案的优化[J]. 生理学报 2017; 69 (4): 477-484.

LI Duan-Duan, SHI Shan-Hui, ZHOU Mi, MA Xiu-Xia, GAO Jian-Zhong, ZHANG Ya-Qiong, YANG Li-Wen, ZUO Lin. Optimization of isolation and culture of Lin⁻ Sca-1⁺ cardiac stem cells from newborn mice. Acta Physiol Sin 2017; 69 (4): 477-484 (in Chinese with English abstract).