西门塔尔牛胰腺间充质干细胞原代培养及其多向分化潜能研究

Primary Culture of Simmental Bovine Pancreatic Mesenchymal Stem Cells and Their Multi-directional Differentiation Potential

旨在对西门塔尔牛胰腺间充质干细胞(pancreatic mesenchymal stem cells,PMSCs)进行原代培养并研究其体外分化潜能,为细胞疗法和组织工程学方面提供新的种子细胞。本研究从3月龄的西门塔尔牛胚胎中无菌分离胰腺组织,分别采用胶原酶消化法和组织块贴壁法分离PMSCs,进行原代培养;绘制第3代、第9代、第15代细胞生长曲线并测定群体倍增时间及克隆形成能力,采用免疫荧光检测干细胞表面标志物(CD29、CD44、CD73、CD90、CD34和CD45),RT-PCR检测干细胞细胞表面标志物(CD29、CD44、CD73、CD90、CD106、CD166、CD34和CD45);染色体核型分析检测其基因稳定性,通过向成脂、成骨、成软骨和成肝样细胞诱导分化,检测其多向分化潜能。结果表明,两种方法都可成功分离出PMSCs,细胞贴壁后形态均为长梭形,漩涡状生长,生长趋势呈典型的S形;第9代细胞群体倍增时间显著低于第15代而高于第3代(P<0.01);第9代PMSCs克隆形成率显著低于第3代而显著高于第15代(P<0.05);免疫荧光结果表明,PMSCs特异性表达CD29、CD44、CD73和CD90,RT-PCR结果显示PMSCs特异性表达CD29、CD44、CD73、CD90、CD106和CD166,未表达造血细胞表面标志物CD34和CD45,与国际细胞治疗学会组织干细胞委员会指定的MSCs表面标记物相对应;核型分析表明,PMSCs为正常二倍体(2n=60,XY),染色体基因组未发生变异;特异性染色和RT-PCR结果表明,从西门塔尔牛体内获得的PMSCs可分化为脂肪细胞、骨细胞、软骨细胞和肝样细胞。本试验证实两种方法均可成功建立西门塔尔牛PMSCs体外分离培养体系,PMSCs具有活性好、增殖速度快的特点,与MSCs有相似的生物学特性和多项分化的潜能。可为组织工程学提供新的种子细胞。

The purpose of the experiment was to primary culture and study the in vitro differentiation potential of Simmental bovine pancreatic mesenchymal stem cells(PMSCs),and to provide new seed cells for cell therapy and tissue engineering.Pancreatic tissue was aseptically isolated from 3-month-old Simmental cattle embryos,and PMSCs were isolated by collagenase digestion and tissue block adherence,respectively,and then primary cultured.The growth curves of 3 rd,9 th and 15 th passage cells were drawn and the population doubling time and clone formation ability were determined.Stem cell surface markers(CD29,CD44,CD73,CD90,CD34 and CD45) were detected by immunofluorescence,and stem cell surface markers(CD29,CD44,CD73,CD90,CD106,CD166,CD34 and CD45) were detected by RT-PCR.Its gene stability was detected by karyotype analysis,the multi-directional differentiation potential was detected by inducing adipogenic,osteogenic,chondrogenic and hepatocyte-like cells.The results showed that PMSCs could be successfully isolated by both methods.After the cells adhered,they were all long spindle-shaped,swirling growth,and the growth trend was typical S-shaped.The doubling time of the cell population in the 9 th passage was significantly lower than that in the 15 th passage and higher than that in the 3 rd passage(P<0.05);The clone formation rate of the 9 th passage of PMSCs was significantly lower than that of the 3 rd passage and significantly higher than that of the 15 th passage(P<0.05);Immunofluorescence results showed that CD29,CD44,CD73 and CD90 specifically expressed in PMSCs,and RT-PCR results showed that CD29,CD44,CD73,CD90,CD106 and CD166 specifically expressed in PMSCs,but the hematopoietic cell surface markers CD34 and CD45 did not express,which were consistent with international corresponding to the surface markers of MSCs designated by the Tissue Stem Cell Committee of the Society for Cell Therapy;Karyotype analysis showed that PMSCs were normal diploid(2 n=60,XY),and there was no mutation in the chromosomal genome;Specific staining and RT-PCR results showed that PMSCs obtained from Simmental cattle could differentiate into adipocytes,osteocytes,chondrocytes and hepatocyte-like cells.This experiment confirmed that both methods could successfully establish the in vitro separation and culture system of Simmental cattle PMSCs.PMSCs showed good proliferation activity,similar biological properties and multiple differentiation potentials with MSCs.The results could provide new seed cells for tissue engineering.