南方医科大学学报
南方醫科大學學報
남방의과대학학보
JOURNAL OF SOUTHERN MEDICAL UNIVERSITY
2014年
11期
1621-1626,1631
,共7页
祝旭龙%颜谭%姚维杰%王永恒%程冲%向俊西%吕毅%高庆东%李建辉%GAO Qingdong%LI Jian-hui
祝旭龍%顏譚%姚維傑%王永恆%程遲%嚮俊西%呂毅%高慶東%李建輝%GAO Qingdong%LI Jian-hui
축욱룡%안담%요유걸%왕영항%정충%향준서%려의%고경동%리건휘%GAO Qingdong%LI Jian-hui
骨髓%间充质干细胞%分离与培养%肝细胞%胆管上皮细胞%大鼠
骨髓%間充質榦細胞%分離與培養%肝細胞%膽管上皮細胞%大鼠
골수%간충질간세포%분리여배양%간세포%담관상피세포%대서
bone marrow%mesenchymal stem cell%isolation and culture%hepatocytes%biliry epithelial cells
目的：建立一个分离、培养、纯化大鼠的骨髓间充质干细胞的方法。方法使用贴壁法分离大鼠的骨髓间充质干细胞，通过培养初期的频繁换液和传代过程中减少胰蛋白酶的暴露时间，达到骨髓间充质干细胞的纯化。流式细胞仪检测细胞表面标志物，MTT法检测细胞生长状况，暴露于不同的诱导培养基使细胞成骨、成脂，并向肝胆系分化。结果分离所得的细胞阳性表达CD29、CD44、CD90，而对于造血细胞标志物CD45呈阴性。在成骨、成脂培养基的诱导下，细胞ALP、茜素红、油红O染色均呈阳性，并发现传至10代的细胞仍保持分化功能。将骨髓间充质干细胞贯续暴露于不同的细胞因子和激素，得到了表达肝细胞、胆管上皮细胞特异标志的子代细胞。结论通过改良后的方法可以高效、简洁地分离出形态均一的大鼠骨髓间充质干细胞，并能够向多胚层的细胞分化。
目的：建立一箇分離、培養、純化大鼠的骨髓間充質榦細胞的方法。方法使用貼壁法分離大鼠的骨髓間充質榦細胞，通過培養初期的頻繁換液和傳代過程中減少胰蛋白酶的暴露時間，達到骨髓間充質榦細胞的純化。流式細胞儀檢測細胞錶麵標誌物，MTT法檢測細胞生長狀況，暴露于不同的誘導培養基使細胞成骨、成脂，併嚮肝膽繫分化。結果分離所得的細胞暘性錶達CD29、CD44、CD90，而對于造血細胞標誌物CD45呈陰性。在成骨、成脂培養基的誘導下，細胞ALP、茜素紅、油紅O染色均呈暘性，併髮現傳至10代的細胞仍保持分化功能。將骨髓間充質榦細胞貫續暴露于不同的細胞因子和激素，得到瞭錶達肝細胞、膽管上皮細胞特異標誌的子代細胞。結論通過改良後的方法可以高效、簡潔地分離齣形態均一的大鼠骨髓間充質榦細胞，併能夠嚮多胚層的細胞分化。
목적：건립일개분리、배양、순화대서적골수간충질간세포적방법。방법사용첩벽법분리대서적골수간충질간세포，통과배양초기적빈번환액화전대과정중감소이단백매적폭로시간，체도골수간충질간세포적순화。류식세포의검측세포표면표지물，MTT법검측세포생장상황，폭로우불동적유도배양기사세포성골、성지，병향간담계분화。결과분리소득적세포양성표체CD29、CD44、CD90，이대우조혈세포표지물CD45정음성。재성골、성지배양기적유도하，세포ALP、천소홍、유홍O염색균정양성，병발현전지10대적세포잉보지분화공능。장골수간충질간세포관속폭로우불동적세포인자화격소，득도료표체간세포、담관상피세포특이표지적자대세포。결론통과개량후적방법가이고효、간길지분리출형태균일적대서골수간충질간세포，병능구향다배층적세포분화。
Objective To optimize the protocols for isolation and culture of mesenchymal stem cells from rat bone marrow (BMSCs). Methods BMSCs were isolated by adherence to plastic with frequent medium change and reduced trypsinization time. The cell growth curves were drawn and the surface markers of BMSCs were detected by flow cytometry. The cells were induced to differentiate into osteogenic, adipogenic, hepatic and cholic lineages. Results The cells isolated using this method were positive for CD29, CD44, and CD90 and negative for the hematopoietic surface markers CD45. The osteogenic and adipogenic differentiation of the BMSCs was verified by alkaline phosphatase staining, Alizarin red staining and Oil red staining. The cell subcultures up to passage 10 maintained capacities of differentiation into osteogenic and adipogenic lineages. The BMSCs induced with sequential addition of growth factors, cytokines and hormones differentiated into cells expressing hepatocyte-and cholangiocyte-specific markers. Conclusion The optimized method allows efficient isolation of homogenous populations of MSCs from rat bone marrow, which can be induced into multiple cell lineages.
