两种月龄近交系五指山小型猪脂肪间充质干细胞生物学特性的比较  

Comparison of biological characteristics of adipose derived mesenchymal stem cells in Wuzhishan inbreed miniature pigs aged two different months

作　　者：高元慧[1] 向杨[2] 曹卉[1] 王顺兰[1] 郑琳麟 何浩伟 张应爱[1] 张淑芳[1] 黄邓高[1] Gao Yuanhui;Xiang Yang;Cao Hui;Wang Shunlan;Zheng Linlin;He Haowei;Zhang Yingai;Zhang Shufang;Huang Denggao(Central Laboratory,Affiliated Haikou Hospital of Xiangya School of Medicine,Central South University,Haikou 570208,Hainan Province,China;Department of Urology,Affiliated Haikou Hospital of Xiangya School of Medicine,Central South University,Haikou 570208,Hainan Province,China)

机构地区：[1]中南大学湘雅医学院附属海口医院,中心实验室,海南省海口市570208 [2]中南大学湘雅医学院附属海口医院,泌尿外科,海南省海口市570208

出　　处：《中国组织工程研究》2021年第19期2988-2993,共6页Chinese Journal of Tissue Engineering Research

基　　金：海南省自然青年基金(819QN390),项目负责人:高元慧;海南省自然青年基金(819QN386),项目负责人:黄邓高;海南省重大科技计划项目(ZDKJ2017007),项目负责人:张淑芳。

摘　　要：背景:干细胞是再生医学研究的热点,影响干细胞生物学特性的因素很多,其中供体年龄是重要的影响因素之一。目的:观察两种月龄近交系五指山小型猪脂肪间充质干细胞生物学特性的变化,筛选最佳种子细胞来源。方法:以4月龄和12月龄近交系五指山小型猪脂肪间充质干细胞为研究对象,分别选取第3代细胞和第10代细胞,观察细胞形态,绘制细胞增殖曲线,检测细胞表面抗原、细胞周期、细胞凋亡情况,然后进行成骨诱导分化茜素红染色和成脂诱导分化油红O染色以及细胞衰老β-半乳糖苷酶染色等。结果与结论:①4月龄和12月龄细胞均呈漩涡状贴壁生长,表面标志物CD29、CD44和CD90表达均为阳性,CD8a、CD45和HLA-DR表达均为阴性;②4月龄和12月龄第3代细胞增殖能力均较强;第10代细胞,与4月龄相比,12月龄G0/G1期百分比增高(P<0.001),早期凋亡增加(P<0.001),而S期和G2/M期百分比显著下降(P<0.001);③4月龄和12月龄第3代细胞的体外诱导分化能力无显著性差异;12月龄第10代细胞的钙化结节率和脂滴率与4月龄相比显著降低(P<0.001);④4月龄和12月龄第3代细胞β-半乳糖苷酶几乎不表达;12月龄第10代细胞β-半乳糖苷酶阳性细胞与4月龄相比显著增加(P<0.001),且细胞形态变得更加扁平和肥大、折光性弱;⑤结果表明,4月龄近交系五指山小型猪脂肪间充质干细胞具有更强的增殖和分化能力,且细胞衰老活性更低,提示4月龄更适合用于自体移植的种子细胞。BACKGROUND: Stem cells have been the focus of regenerative medicine research. Many factors affect the biological characteristics of stem cells, among which donor age is one of the critical factors.OBJECTIVE: To observe biological characteristics of two types of inbreed line miniature pigs of Wuzhishan adipose-derived mesenchymal stem cells so as toscreen the optimal seed cell source.METHODS: Inbreed line miniature pigs of Wuzhishan adipose-derived mesenchymal stem cells aged 4 and 12 months were selected as materials. The passage3 and passage 10 of cells were selected to observe their cell morphology, draw cell proliferation curve, and detect cell surface antigen, cell cycle and apoptosis.Afterwards, osteogenic differentiation was performed with Alizarin red staining, followed by adipogenic differentiation with Oil Red O staining and cellsenescence β-galactosidase staining.RESULTS AND CONCLUSION: (1) The morphology of cells at 4 and 12 months both showed vortex-shaped adherent growth. The expression of surface markersCD29, CD44 and CD90 was positive, while that of CD8a, CD45 and HLA-DR was negative. (2) The proliferation of cells at 4 and 12 months was stronger in passage 3.In passage 10, the percentage of cells in the G0/G1 phase (P < 0.001) and early apoptosis (P < 0.001) increased, while in the S and G2/M phases, it decreased at 12months compared with 4 months (P < 0.001). (3) There was no significant difference in the differentiation ability of cells at passage 3 in vitro between 4 and 12months. The calcification nodule rate and lipid droplet rate of cells at 12 months of passage 10 were significantly lower than at 4 months of passage 10 (P < 0.001).(4) The β-galactosidase of cells was almost not expressed at 4 and 12 months of passage 3. However, in the passage 10, the positive rate of cells at 12 monthswas significantly higher than that at 4 months (P < 0.001);and the cell morphology became more flattened, hypertrophic and less refractive. (5) It is concludedthat adipose-derived mesenchymal stem cells of

关 键 词：干细胞 脂肪间充质干细胞 近交系五指山小型猪 年龄 增殖 分化 衰老 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]