大鼠BMSCs、ADSCs、PMSCs体外诱导的成骨样细胞的抗原性差异

发布时间：2018-06-21 18:07

本文选题：同种异体 + 间充质干细胞　；参考：《辽宁医学院》2012年硕士论文

【摘要】：目的探讨大鼠BMSCs、ADSCs、PMSCs诱导成骨后在体外环境下的抗原性差异。方法 8w龄SPF级近交系Wistar大鼠，取材获得三种间充质干细胞：BMSCs、ADSCs、PMSCs，对上述细胞进行分离培养，噻唑蓝（MTT）法记录细胞生长曲线；取第3~5代细胞采用CD分子表面标志物鉴定细胞并定向诱导为成骨样细胞；碱性磷酸酶染色、钙结节染色检测细胞成骨分化能力；应用Transwell共培养体系检测成骨诱导后的三种细胞对同种异体SPF级近交系WKY大鼠脾淋巴细胞增殖的影响；采用流式细胞仪检测淋巴细胞CD4+、CD8+的阳性率；采用免疫酶联反应检测TGF-β1、IL-2的含量。结果 ①细胞形态差别：BMSCs细胞较细长，ADSCs细胞趋向于圆形，PMSCs细胞趋向于多角。②生长曲线分析：PMSCs潜伏期短，于传代后即进入一个相对快速的对数增殖，4-5d后进入平台期；而BMSCs与ADSCs增殖则相对滞后，对数增殖集中于3-5d，，第六日后细胞进入平台期。三种细胞均可通过体外扩增方法进行大量培养。成骨诱导后，三种细胞的生长曲线分析：三种细胞潜伏期为1d， PMSCs于诱导后3-4d进入平台期；BMSCs和ADSCs于诱导后4-5d进入平台期。③P5代（成骨诱导前）CD分子表面标志物鉴定：PMSCs细胞CD29阳性率85%，CD34阳性率为1.33%；BMSCs细胞CD29阳性率为90%，CD34阳性率为0.97%；ADSCs细胞CD29阳性率为80%，CD34阳性率为0.99%。④三种细胞经过茜素红染色后均可见紫红色钙结节，提示三种细胞均可以向成骨样细胞方向诱导；三种细胞经过碱性磷酸酶染色显示深浅不一咖啡色颗粒，Kaplow评级：PMSCs（4分）＞BMSCs（3分）＞ADSCs（2分）。⑤在体外与同种异体淋巴细胞共培养试验中，MTT检测显示三种细胞都表现出抑制淋巴细胞增殖的作用，抑制淋巴细胞增殖的作用与诱导时间正相关。单因素方差分析：三种细胞诱导前后及三者之间横向比较免疫源性均无统计学差异。流式细胞仪检测共培养后的CD4+、CD8+淋巴细胞阳性率均降低。免疫酶联反应检测共培养后上清液中IL-2表达量均降低；TGF-β1表达量均增加。结论体外环境下，三种间充质干细胞成骨诱导前后及三者之间横向比较免疫源性没有差异。
[Abstract]:Objective to investigate the difference of antigenicity of rat BMSCsADP PMSCs induced by osteogenesis in vitro. Methods three kinds of mesenchymal stem cells (BMSCs) were obtained from 8 w SPF inbred strain Wistar rats. The above cells were isolated and cultured. The cell growth curve was recorded by thiazolyl methylene tetrachloride (MTT) method. The cells were identified by CD molecular surface markers and induced into osteoblast-like cells, alkaline phosphatase staining and calcium nodule staining were used to detect the osteogenic differentiation ability of the cells. Transwell co-culture system was used to detect the effect of three kinds of osteoblast induced cells on the proliferation of splenic lymphocytes of SPF class inbred rat, and the positive rate of CD4 / CD8 was detected by flow cytometry. The content of TGF- 尾 _ 1 and IL-2 was detected by immuno-enzyme-linked reaction. Results (1) the differentiation of cell morphology and the tendency of ADSCs to round PMSCs tended to be polygonal. The growth curve analysis showed that the incubation period of BMSCs was shorter than that of BMSCs. After passage, it entered a relatively rapid logarithmic proliferation for 4-5 days, but BMSCs and ADSCs were relatively delayed in proliferation, the logarithmic proliferation was concentrated at 3-5 days, and the cells entered the platform phase after the sixth day. All three kinds of cells can be cultured in large quantities by in vitro amplification. Growth curve analysis of three kinds of cells after osteogenic induction: the incubation period of three kinds of cells was 1 day, PMSCs entered the plateau stage 3-4 days after induction; BMSCs and ADSCs entered the platform.3P5 passage 4 to 5 days after induction. (CD29 positive rate of BMSCs and ADSCs was 1.33 CD34 positive rate of BMSCs cells was 1.33 and CD34 positive rate of BMSCs cells was 0.97% before osteogenesis induction. The CD29 positive rate of ADSCs cells was 80 and CD34 positive rate was 0.99.4 all three kinds of cells could be induced to osteoblast by alizarin red staining. The results of alkaline phosphatase staining showed that the three kinds of cells showed different shades of brown granules: PMSCs (4 points) > BMSCs (3 points) > ADSCs (2 points). In vitro co-culture test with allogeneic lymphocytes, MTT assay showed that all three kinds of cells were present. To inhibit the proliferation of lymphocytes, The inhibition of lymphocyte proliferation was positively correlated with the induction time. Univariate ANOVA: there was no significant difference in immunogenicity between the three kinds of cells before and after induction. The positive rate of CD 4 and CD 8 lymphocytes in co culture was decreased by flow cytometry. The expression of IL-2 in supernatant was decreased by immunoenzyme linked reaction (Elisa) and the expression of TGF- 尾 1 in supernatant was increased. Conclusion in vitro, there is no difference in immunogenicity between the three mesenchymal stem cells before and after osteogenesis.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.12

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