人骨髓间充质干细胞与汗腺细胞共同培养诱导细胞表型转化的初步研究  

Cellular phenotype conversion induced by co-culture of human mesenchymal stem cells cocultured with human sweat gland cells

作　　者：李海红[1] 付小兵[1] 周岗[1] 费沛[1] 陈伟[1] 白晓东[1] 蔡存良[1] 孙同柱[1] 

机构地区：[1]解放军总医院三○四临床部全军创伤修复重点实验室

出　　处：《中华医学杂志》2005年第27期1885-1889,共5页National Medical Journal of China

基　　金：国家重点基础研究发展规划资助项目(G1999054204);国家自然科学基金资助项目(30170966;30230370);国家杰出青年基金资助项目(39525024)

摘　　要：目的研究人骨髓间充质干细胞(humanmesenchymalstemcells,hMSCs)在体外与损伤的人汗腺细胞(humansweatglandcells,hSGCs)共培养时的转化情况。方法体外分别分离培养和扩增hMSCs和hSGCs,用二步免疫细胞化学法检测hMSCs和hSGCs抗原表达情况。用5μmol/L的5溴2脱氧尿苷(BrdU)对培养的hMSCs进行连续培养标记。待原代培养的hSGCs达70%融合后,给予47℃高温处理40min造成热损伤体外模型,37℃冷却12h,然后加入(12)×105BrdU标记的hMSCs共同培养,2周后,以抗癌胚抗原(CEA)和抗BrdU单克隆抗体作为一抗,采用免疫细胞化学双染法检测共培养的细胞。结果hMSCs和hSGCs均呈克隆样生长,hMSCs表达CD44和CD105,不表达CD34和CEA;hSGCs表达CK7、CK8、CK19、CEA。用BrdU标记hMSCs阳性率可达90%,hSGCs经高温损伤后,大多数细胞的细胞间连接消失。共培养2周后,部分细胞同时表达CEA和BrdU,并有多核现象。细胞染色示双染细胞可达1%～5%,多核细胞且核染色不同的细胞约为0.01%～0.05%,多核细胞与其他双染细胞相比,细胞明显的宽大扁平。结论在损伤的微环境下,hMSCs可以向hSGCs转化,其机制可能是细胞分化、细胞融合甚至是核融合。Objective To investigate the cellular phenotype conversion during human mesenchymal stem cells (hMSCs)cocultured with injured human sweat gland cells(hSGCs)in vitro. Methods HMSCs and hSGCs were isolated and cultured and expanded respectively. The antigens expression of hMSCs and hSGCs were detected by two-steps immunocytochemistry. HMSCs were labeled with BrdU. The hSGCs were heat-shocked at 47℃ for 40 min when they reached 70% confluency, then cooled for 1-2 h at 37℃ and (1-2)×105 BrdU-labeled hMSCs were added before incubation for up to 2 weeks. The cocultures were observed by phase contrast microscopy and detected by double-staining immunocytochemistry using CEA and BrdU as primary antibodies. Results The cultured hMSCs and hSGCs were clonogenic growth. HMSCs were positive for anti-CD44 and anti-CD105 staining and negative for anti-CD34 and anti-CEA staining. HSGCs express CK7、CK18、CK19 and CEA. The positive rate of BrdU labeled-hMSCs was 90%. The majority of hSGCs lost cell-cell contact after heat-shock. 2 weeks after cocultured, some cocultured cells were positive for both anti-CEA and anti-BrdU staining and some cocultures had more than two nuclei which stained with two different colors by double-staining immunocytochemistry. Statistic results showed 1%-5% of the hMSCs added to the coculture system were recovered as double-staining cells expressing BrdU and CEA while only 0.01%-0.05% cells stained with two different colors in nuclei. The multi-nucleated cells were wide and flatten. Conclusion HMSCs could differentiate into hSGCs in vitro under injured microenvironment. The machnisms of which may be that hMSCs differentiate into hSGCs directly or by cell fusion, even nucleus fusion.

关 键 词：人骨髓间充质干细胞 共同培养 汗腺细胞 细胞表型转化 步研究 癌胚抗原(CEA) HMSCS BRDU标记 免疫细胞化学法 cells 多核细胞 mol/L 单克隆抗体 抗BrdU 培养的细胞 CD105 细胞间连接 表达情况 Cs抗原 连续培养 脱氧尿苷 

分 类 号：R641[医药卫生—外科学]