人脂肪干细胞基本生物学特性、表面抗原及成骨、成脂分化潜能的研究

发布时间：2018-03-07 01:26

本文选题：脂肪　切入点：干细胞　出处：《安徽医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的研究人脂肪干细胞的基本生物学特性、表面抗原特点及其向成骨细胞和脂肪细胞诱导分化潜能。方法①取健康人腹部皮下脂肪组织,胶原酶消化法分离出脂肪干细胞,进行体外培养,观察其形态特征。②cck—8比色法检测1、3、6、10代脂肪干细胞活性,并绘制细胞生长曲线,计算群体倍增时间。③流式细胞仪检测第1、3、6代脂肪干细胞的细胞周期及表面抗原,SPSS11.0软件分析数据。④取第3代人脂肪干细胞,分别用成骨和成脂诱导培养液诱导其向成骨细胞和脂肪细胞分化,cck—8比色法检测诱导后的细胞活性,并绘制细胞生长曲线,计算群体倍增时间;RT-PCR检测人脂肪干细胞诱导后成骨细胞特异性骨钙蛋白(osteocalcin,OCN)、骨桥蛋白(osteopontin,OPN)及骨保护素(Osteoprotegerin,OPG)基因的表达和脂肪细胞特异性过氧化物酶体增殖物激活受体γ(PPARγ)基因与CCAAT增强子结合蛋白(C/EBPa)基因的表达,Western-blot进一步检测OPG蛋白的表达;碱性磷酸酶(ALP)染色、冯库萨(Von Kossa)染色、油红O染色定性鉴定其成骨和成脂分化潜能。结果①形态特征:原代及传代的人脂肪干细胞形态均类似成纤维细胞,生长能力旺盛。②生长曲线及倍增时间:1、3、6、10代人脂肪干细胞的生长曲线大致相同,呈近似“S”形,群体倍增时间约为36h。③细胞周期分析:第1、3、6代人脂肪干细胞中,超过80%的细胞都处于G0/G1期。④表面抗原:第1、3、6代脂肪干细胞表面抗原表达无明显区别,P＞0.05。其中,CD10、CD13、CD55、CD59及CD71表达阳性,CD11b、CD14、CD18、CD34、CD45、CD56及HLA-DR表达阴性。⑤诱导分化:人脂肪干细胞经成骨诱导培养后,细胞增殖速度变慢,群体倍增时间延长至约66h,诱导14d后,RT-PCR检测到OCN、OPN基因的表达,同时,RT-PCR、Western-blot检测到OPG基因与蛋白的表达,ALP染色和Von Kossa染色阳性;人脂肪干细胞经成脂诱导后,细胞增殖速度同样变慢,群体倍增时间延长至约70h,诱导14d后,RT-PCR检测到PPARγ与C/EBPa基因的表达,油红O染色阳性。结论人腹部皮下脂肪组织可分离培养出脂肪干细胞,大多细胞处于静止期,表面抗原CD10、CD13、CD55、CD59、CD71表达呈阳性,CD11b、CD14、CD18、CD34、CD45、CD56、HLA-DR表达呈阴性,体外培养和传代对其表面抗原的表达无明显影响,能向成骨细胞和脂肪细胞诱导分化,有望成为组织工程理想的种子细胞来源之一。
[Abstract]:Objective to study the basic biological characteristics, surface antigen characteristics and differentiation potential of human adipose stem cells into osteoblasts and adipocytes. Methods 1 adipose stem cells were isolated from healthy human abdominal subcutaneous adipose tissue and cultured in vitro by collagenase digestion. The morphological characteristics of adipose stem cells were measured by 2cck-8 colorimetry, and the growth curve of adipose stem cells was plotted. The cell cycle and surface antigen of adipose stem cells of passage 1 and 3 were analyzed by flow cytometry with population doubling time 3. 3. The third generation of human adipose stem cells were analyzed by SPSS 11.0 software. Osteoblasts and adipocytes were induced to differentiate into osteoblasts and adipocytes by cck-8 colorimetry, and cell growth curves were plotted. Expression of osteocalcinin-specific osteocalcin OCNN, osteopontinin Osteoprotegerin Osteoprotegerin OPGG gene and adipocyte specific peroxisome proliferator activated receptor 纬 (PPAR 纬) in human adipose stem cells induced by human adipose stem cells were determined by RT-PCR. The expression of C / EBPA gene and CCAAT enhancer binding protein was further detected by Western-blot. Alkaline phosphatase (ALP) staining, Von Kossa staining and oil red O staining were used to identify their osteogenic and adipogenic potential. Results 1Morphologic characteristics: the morphology of primary and passage human adipose stem cells was similar to that of fibroblasts, and the growth curve of human adipose stem cells was similar to that of fibroblasts, and the growth curve of human adipose stem cells was similar to that of "S". The population doubling time was about 36h.3 cell cycle analysis: in the first and third generation of human adipose stem cells, More than 80% cells were in G _ 0 / G _ 1 phase .4 surface antigen: there was no significant difference in the expression of surface antigen of adipose stem cells in the 1st and 6th generation of adipose stem cells (P > 0.05). Among them, the positive expression of CD10, CD13, CD55, CD59, and CD71 expression were induced by CD11bP14, CD18, CD34, CD45, CD56 and HLA-DR expression negative. 5. The differentiation of human adipose stem cells was induced by osteogenesis. After 14 days of induction, the expression of OCN-OPN gene was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the expression of OPG gene and protein was detected by RT-PCR and Von Kossa staining. The cell proliferation rate also slowed down, the population doubling time was prolonged to about 70 h. After 14 days of induction, the expression of PPAR 纬 and C% EBPA gene was detected by RT-PCR, and the oil red O staining was positive. Conclusion ASCs can be isolated and cultured from human abdominal subcutaneous adipose tissue. Most of the cells are in the stationary phase. The positive expression of CD10, CD13, CD5, CD59, CD71, and CD11bt4, CD14, CD34, CD34, CD45, CD56, and HLA-DR are negative, but the expression of surface antigen is not affected by culture and passage in vitro. It can induce differentiation into osteoblasts and adipocytes, which is expected to be one of the ideal seed cells for tissue engineering.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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