摘要
目的探讨体外诱导人脂肪间充质干细胞（hADSCs）向胰岛β样细胞分化的方法。方法手术获取人脂肪组织，分离培养hADSCs，流式细胞术鉴定其分子表面标志。应用含胰十二指肠同源框因子1（PDX1）与神经元素3（Ngn3）的慢病毒转染hADSCs，并联合乙基咔唑（NECA）及多种细胞因子进行诱导分化后，采用实时定量聚合酶链反应（Real—time PCR）检测胰岛素分泌细胞特异基因肌腱膜的纤维肉瘤肿瘤基因同系物A（Mafa）、胰岛素1（Insulin1）、葡萄糖转运体2（Glut2）及叉头蛋白转录因子01（Fox01）的表达，采用免疫荧光法鉴定诱导后细胞内胰岛素及C肽的表达，采用化学发光法检测诱导后细胞的胰岛素分泌水平。结果hADSCs、CD105、CD44、CD29及CD90表达阳性，CD45及CD34表达阴性。Real-time PCR结果显示诱导后的细胞高表达PDX1、Ngn3、Insulin1及Glut2；免疫荧光结果显示诱导后的细胞胞质内表达胰岛素及C肽；化学发光法测得诱导后的细胞胰岛素分泌量为（156．23±30．97）mIU／L，高糖刺激后分泌量为（836．50±231．22）mlU／L。结论人脂肪间充质干细胞在体外可以通过转染含PDX1和Ngn3慢病毒并联合NECA及多种细胞因子的方法诱导分化为胰岛B样细胞。
Objective This study was to investigate the feasibility of human adipose - derived stem cells to differentiate into islet β-like cells. Methods Human adipose derived stem cells （hADSCs） from human abdominal adipose tissue by surgery were isolated and identified by Fluorescence activated cell sorting （FACS）, and hADSCs transfected by lentivirus harboring Pancreatic and duodenal homeobox 1 （ PDX1 ） and neurogenin 3 （ Ngn3 ） were differentiated into islet β- like cells induced by 5＇ - N - ethylcarbo - xamidoad - enosine （NECA） and multiple cytokines. The expressions of Mus musculus v - maf musculoaponeurotic fibrosarcoma oncogene family protein A （ Mafa）, Insulin1, Glucose transporter 2 （Glut2） and Forkhead box O1 （FoxO1） were examined by Real- time polymerase chain reaction （PCR） and insulin and C - peptide were examined by Immunofluorescence cytochemistry. The levels of insulin secretion were examinde by electrochemiluminescence test. Results hADSCs expressed CD105, CD44, CD29 and cDg0, but did not express CIM5 and CD34. PDX1, Ngn3, Insulinl and Glut2 were expressed in the transduced cells analyzed by Real - time PCR. Immunofluorcscence cytochemistry demonstrated the transduced cells were insulin - positive and C - peptide - positive. And the levels of insulin in the transduced cells culture supernatant were （ 156. 23 ± 30. 97 ） mIU/L and the levels of insulin secertion were （836. 50 ± 231.22） mIU/L with high glucose stimulation. Conclusion Adipose - derived stem cells can differentiate into islet β- ceils in vitro. These islet β- cells can synthesize and store insulin.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2016年第1期46-50,共5页
Chinese Journal of Experimental Surgery
基金
南京市医学科技发展重点项目（ZKX10016）
