兔脂肪干细胞BrdU标记情况的初步研究

发布时间：2018-03-04 07:06

本文选题：干细胞　切入点：细胞分化　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5-溴脱氧尿嘧啶核苷(BrdU)标记脂肪间充质干细胞(ADSCs)的效果,确定体外标记的最佳浓度和时间,评价BrdU标记的ADSCs的适用性。方法:取6月龄新西兰白兔颈后皮下脂肪,采用Ⅰ型胶原酶消化方法体外分离培养脂肪干细胞,利用成骨、成脂诱导培养基定向向成脂、成骨细胞诱导并用组织化学方法鉴定其诱导结果,从而鉴定其具有多向分化的干细胞潜能。选取第3代ADSCs,分别采用5、10、15、20μmol/L浓度的BrdU体外标记干细胞24、48、72小时,利用免疫组化实验计算不同浓度和标记时间下脂肪干细胞的标记率,确定BrdU对ADSCs的最佳标记方法。通过台盼蓝染色和细胞倍增时间的测量确定最佳标记方法的安全性。对第3代脂肪干细胞采用最佳标记方法标记后更换为普通培养液继续培养,适时传代,连续检测第5、7、9、11代脂肪干细胞的BrdU标记率,了解该标记方法在脂肪干细胞体外扩增培养过程中的衰减情况。同时将标记后的第3代脂肪干细胞作为实验组,未标记细胞作为对照组,分别种植于乳酸-羟基乙酸共聚物(PLGA)支架材料上,体外培养3天后自体回植于动物皮下,4周后取出支架材料,制作石蜡切片采用免疫组化法鉴定BrdU标记的脂肪干细胞在体内标记的效果。结果:兔脂肪干细胞体外培养具有成纤维细胞样外形,增埴迅速,连续传代14代细胞形态无明显改变,无自发性向脂肪细胞分化现象。成脂诱导12天后油红O染色胞浆内发现红染的油滴,成骨诱导14天后Von kossa染色阳性,由此证明在体外定向诱导后脂肪干细胞可以向脂肪细胞和成骨细胞分化。BrdU可标记脂肪干细胞的核,采用10μmol/L的BrdU体外标记48小时是适宜的标记方法,该方法对脂肪干细胞的增殖无明显影响。初测标记率达95%,标记率随传代次数而降低,传代8次(约体外培养4周)后标记率仍达41%。同期进行的体内实验证实,BrdU标记后的脂肪干细胞移植入体内4周后免疫组化检测有BrdU阳性细胞存在,从而证明移植的脂肪干细胞可以在体内存活。结论:兔脂肪干细胞易于体外分离培养,具有较强的自我更新能力及多向分化的潜能.BrdU标记脂肪干细胞的操作简单易行,10μmol/L的BrdU标记脂肪干细胞48h后,体外标记效果满意.标记细胞移植体内1月后BrdU仍显示较好的示踪作用,因此BrdU可以用于脂肪干细胞的标记。
[Abstract]:Aim: to study the effect of 5-bromodeoxyuridine BrdU (BrdU) on the labeling of adipose mesenchymal stem cells (ADSCs), determine the optimal concentration and time of in vitro labeling, and evaluate the applicability of BrdU labeled ADSCs. Methods: adipose stem cells were isolated and cultured in vitro from 6-month-old New Zealand white rabbits with posterior subcutaneous fat. Adipose stem cells were isolated and cultured by type I collagenase digestion. Adipogenic medium was used to induce adipogenesis. Osteoblasts were induced and identified by histochemical method, so as to identify their multidirectional stem cell potential. The third generation of ADSCs was labeled with BrdU at the concentration of 5 10 ~ 1520 渭 mol/L for 24872 hours in vitro. The labeling rate of adipose stem cells at different concentrations and labeling time was calculated by immunohistochemistry. The best labeling method for ADSCs by BrdU was determined. The safety of the best labeling method was determined by trypan blue staining and cell doubling time measurement. The third generation adipose stem cells were labeled with the best labeling method and then replaced with normal culture medium. After timely passage, the BrdU labeling rate of adipose stem cells (ASCs) of generation 5, 7, 9 and 11 was continuously detected, and the attenuation of the labeling method in the process of proliferation and culture of adipose stem cells in vitro was investigated. At the same time, the third generation of labeled adipose stem cells were used as experimental group. The unlabeled cells were used as the control group and planted on the lactic acid-glycolic acid copolymer (PLGA) scaffold. After 3 days of culture in vitro, autologous cells were implanted subcutaneously in the animals for 4 weeks, then the scaffolds were removed. Paraffin sections were made to evaluate the effect of BrdU labeled adipose stem cells in vivo by immunohistochemical method. Results: rabbit adipose stem cells cultured in vitro showed fibroblast-like appearance, rapid growth, and no obvious changes in cell morphology in successive passage 14 passages. There was no spontaneous differentiation into adipocytes. After 12 days of lipogenesis, red oil droplets were found in the cytoplasm of oil red O staining, and Von kossa staining was positive after 14 days of osteogenic induction. The results showed that adipose stem cells could differentiate into adipocytes and osteoblasts after directional induction in vitro. BrdU could label the nucleus of adipose stem cells. It was suitable to label adipose stem cells with 10 渭 mol/L BrdU for 48 hours in vitro. This method had no significant effect on the proliferation of adipose stem cells. The initial labeling rate was 95%, and the labeling rate decreased with the passage times. After 8 passages (about 4 weeks in vitro culture), the labeling rate was still 41%. The results of in vivo experiments confirmed that there were BrdU positive cells in adipose stem cells after transplantation with BrdU in vivo for 4 weeks. This proves that transplanted adipose stem cells can survive in vivo. Conclusion: rabbit adipose stem cells are easy to be isolated and cultured in vitro, and have strong self-renewal ability and multidirectional differentiation potential. BrdU labeling of adipose stem cells is simple and easy to be used to label adipose stem cells with 10 渭 mol/L BrdU for 48 h. The labeling effect in vitro was satisfactory. BrdU still showed a good tracer effect after in vivo transplantation of labeled cells, so BrdU could be used as a marker of adipose stem cells.
【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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