中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2013年
32期
5772-5777
,共6页
李华%文峰%漆仲春%周进军%朱亚杰%程朋%魏东%苏晓妹%谭勇%彭晶晶%罗巧丽%李东%张涛
李華%文峰%漆仲春%週進軍%硃亞傑%程朋%魏東%囌曉妹%譚勇%彭晶晶%囉巧麗%李東%張濤
리화%문봉%칠중춘%주진군%주아걸%정붕%위동%소효매%담용%팽정정%라교려%리동%장도
干细胞%脐带脐血干细胞%脐带间充质干细胞%肝细胞%共培养%细胞分化%细胞培养%肝样细胞%甲胎蛋白%白蛋白%人细胞角蛋白19%省级基金%干细胞图片文章
榦細胞%臍帶臍血榦細胞%臍帶間充質榦細胞%肝細胞%共培養%細胞分化%細胞培養%肝樣細胞%甲胎蛋白%白蛋白%人細胞角蛋白19%省級基金%榦細胞圖片文章
간세포%제대제혈간세포%제대간충질간세포%간세포%공배양%세포분화%세포배양%간양세포%갑태단백%백단백%인세포각단백19%성급기금%간세포도편문장
背景：文献报道，从骨髓与脐带中分离获得的间充质干细胞可在体外连续传代培养，仍保持干细胞的特性，并在多种细胞因子的“鸡尾酒式”诱导下分化为肝细胞样细胞。目的：进一步验证人脐带间充质干细胞在体外正常人肝细胞共培养体系下是否可分化为肝细胞并探讨其分化方法。方法：采用贴壁法，从脐带中分离培养间充质干细胞，流式细胞仪检测脐带间充质干细胞表面标志。人肝细胞 LO2细胞与人脐带间充质干细胞建立共培养体系，不添加外源诱导因子，分别于第7，14，21天，通过RT-PCR 法检测肝细胞特异标志物甲胎蛋白、白蛋白、人细胞角蛋白19 mRNA的表达，糖原染色进行功能鉴定。结果与结论：从人脐带中可分离得到贴壁生长的间充质干细胞，其中CD29+细胞比例为96.02%，CD105+细胞比例为96.6%，CD34-细胞比例为99.65%，CD105+CD29+双阳性细胞比例为94.84%。与LO2细胞共培养后第7天仅有甲胎蛋白阳性表达；第14天表达白蛋白、人细胞角蛋白19，第21天时，LO2与人脐带间充质干细胞共培养组未出现甲胎蛋白表达；人细胞角蛋白19和白蛋白的表达比第14天略有增强。共培养21 d后，糖原染色呈阳性。结果证实，无需额外添加外源诱导因子，脐带间充质干细胞可在人正常肝细胞共培养的微环境中，向正常肝细胞分化。
揹景：文獻報道，從骨髓與臍帶中分離穫得的間充質榦細胞可在體外連續傳代培養，仍保持榦細胞的特性，併在多種細胞因子的“鷄尾酒式”誘導下分化為肝細胞樣細胞。目的：進一步驗證人臍帶間充質榦細胞在體外正常人肝細胞共培養體繫下是否可分化為肝細胞併探討其分化方法。方法：採用貼壁法，從臍帶中分離培養間充質榦細胞，流式細胞儀檢測臍帶間充質榦細胞錶麵標誌。人肝細胞 LO2細胞與人臍帶間充質榦細胞建立共培養體繫，不添加外源誘導因子，分彆于第7，14，21天，通過RT-PCR 法檢測肝細胞特異標誌物甲胎蛋白、白蛋白、人細胞角蛋白19 mRNA的錶達，糖原染色進行功能鑒定。結果與結論：從人臍帶中可分離得到貼壁生長的間充質榦細胞，其中CD29+細胞比例為96.02%，CD105+細胞比例為96.6%，CD34-細胞比例為99.65%，CD105+CD29+雙暘性細胞比例為94.84%。與LO2細胞共培養後第7天僅有甲胎蛋白暘性錶達；第14天錶達白蛋白、人細胞角蛋白19，第21天時，LO2與人臍帶間充質榦細胞共培養組未齣現甲胎蛋白錶達；人細胞角蛋白19和白蛋白的錶達比第14天略有增彊。共培養21 d後，糖原染色呈暘性。結果證實，無需額外添加外源誘導因子，臍帶間充質榦細胞可在人正常肝細胞共培養的微環境中，嚮正常肝細胞分化。
배경：문헌보도，종골수여제대중분리획득적간충질간세포가재체외련속전대배양，잉보지간세포적특성，병재다충세포인자적“계미주식”유도하분화위간세포양세포。목적：진일보험증인제대간충질간세포재체외정상인간세포공배양체계하시부가분화위간세포병탐토기분화방법。방법：채용첩벽법，종제대중분리배양간충질간세포，류식세포의검측제대간충질간세포표면표지。인간세포 LO2세포여인제대간충질간세포건립공배양체계，불첨가외원유도인자，분별우제7，14，21천，통과RT-PCR 법검측간세포특이표지물갑태단백、백단백、인세포각단백19 mRNA적표체，당원염색진행공능감정。결과여결론：종인제대중가분리득도첩벽생장적간충질간세포，기중CD29+세포비례위96.02%，CD105+세포비례위96.6%，CD34-세포비례위99.65%，CD105+CD29+쌍양성세포비례위94.84%。여LO2세포공배양후제7천부유갑태단백양성표체；제14천표체백단백、인세포각단백19，제21천시，LO2여인제대간충질간세포공배양조미출현갑태단백표체；인세포각단백19화백단백적표체비제14천략유증강。공배양21 d후，당원염색정양성。결과증실，무수액외첨가외원유도인자，제대간충질간세포가재인정상간세포공배양적미배경중，향정상간세포분화。
BACKGROUND:The studies have shown that the mesenchymal stem cel s derived from bone marrow and umbilical cord can be continuously cultured in vitro, and maintain the characteristics of stem cel s. The mesenchymal stem cel s can differentiate into hepatocyte-like cel s after“cocktail”induction by various cytokines. OBJECTIVE:To further identify whether umbilical cord-derived mesenchymal stem cel s in vitro co-cultured with normal hepatocytes can differentiate into hepatocyte-like cel s, and to investigate the differentiation method. METHODS:Mesenchymal stem cel s were isolated from human umbilical cord with adherent method, and the surface markers of umbilical cord-derived mesenchymal stem cel s were detected with flow cytometry. The umbilical cord-derived mesenchymal stem cel s were co-cultured with liver LO2 cel s without adding exogenous inducers. The expressions of alpha-fetoprotein, albumin and human cytokeratin 19 mRNA of hepatocyte specific markers were detected with reverse transcription PCR at 7, 14 and 21 days after culture, and periodic acid-Schiff staining was used to identify the functions. RESULTS AND CONCLUSION:Mesenchymal stem cel s could isolated from human umbilical cord successful y, showing fibroblastic morphology and adherent cel characterization. Among these cel s, 96.02%cel s were CD29 positive cel s and 96.6%cel s were CD105 positive cel s. The percentage of CD34 negative cel s was 99.65%. The percentage of CD105+CD29+double positive cel s was 94.84%. The mRNA of alpha-fetoprotein was found on the 7th day after co-cultured with LO2 cel s, and the mRNA of albumin and human cytokeratin 19 were found on the 14th day. After co-cultured for 21 days, the alpha-fetoprotein mRNA could not be observed in the co-culture group. The expressions of albumin and human cytokeratin 19 were increased at 14 days. After co-cultured for 21 days, the glycogen staining was positive. Umbilical cord-derived mesenchymal stem cel s can differentiate into hepatocyte-like cel s after co-cultured with normal hepatocytes.
