结缔组织生长因子对体外培养肌腱细胞增殖和量效关系的实验研究

发布时间：2018-11-13 10:03

【摘要】： 目的：研究不同浓度的CTGF对体外培养肌腱细胞增殖的影响，探讨CTGF参与肌腱损伤修复的可能机制。方法：本实验以成年兔后肢趾屈肌腱为材料，，用酶消化后，将分离的细胞用含10％胎牛血清的培养基培养，贴壁达80％后传代。观察细胞的形态及生长规律。取第二代细胞，通过免疫组化的方法进行肌腱细胞鉴定。取第三代肌腱细胞，按细胞数约为4×10~3／孔接种在96孔培养板的48孔，每6孔为一组，共分为8组，根据每组CTGF浓度不同，分为5ng／ml CTGF组、10ng／ml CTGF组、20ng／ml CTGF组、50ng／ml CTGF组、100ng／ml CTGF组、200ng／ml CTGF组、500ng／ml CTGF组及对照组(不加CTGF)。显微镜下观察各组细胞均进入对数生长期时，用噻唑蓝(methyl thiazolyl tetrazolium，MTT)法染色，在酶联免疫检测仪上测出各孔吸光度(optical density，OD)值，并计算各组OD均值进行比较。结果：MTT实验通过比较各组OD值发现：5ng／mlCTGF组肌腱细胞增殖与对照组无显著性差异；5ng／ml、10ng／ml、20ng／ml、50ng／mlCTGF组肌腱细胞增殖有随CTGF浓度增加而上升的趋势，且各组之间的差异均有显著性(P＜0.05)；50ng／ml、100ng／ml、200ng／ml CTGF、500ng／ml CTGF组肌腱细胞增殖有随浓度增加而稍呈下降的趋势，但各组之间的差异均无显著性(P＞0.05)。结论：10ng／mlCTGF、20ng／mlCTGF、50ng／mlCTGF、100ng／mlCTGF、200ng／mlCTGF、500ng／mlCTGF有促进肌腱细胞增殖作用；5ng／mlCTGF对肌腱细胞增殖无明显作用。CTGF浓度在0ng／ml～50ng／ml之间时，肌腱细胞的增殖随浓度增加呈上升趋势；在50ng／ml～500ng／ml之间时，肌腱细胞的增殖随浓度增加稍呈下降趋势。50ng／ml为促肌腱细胞增殖的最佳CTGF浓度。
[Abstract]:Aim: to investigate the effects of different concentrations of CTGF on the proliferation of cultured tendon cells in vitro and to explore the possible mechanism of CTGF involved in tendon repair. Methods: after digested with enzyme, the isolated cells were cultured on the culture medium containing 10% fetal bovine serum. The morphology and growth of cells were observed. The second generation cells were identified by immunohistochemical method. The third passage of tendon cells were divided into 8 groups according to the number of cells (4 脳 10 ~ 3 / well) inoculated in the 96-well culture plate. According to the concentration of CTGF in each group, they were divided into 5ng/ml CTGF group, 10ng/ml CTGF group and 20ng/ml CTGF group. 50ng/ml CTGF group, 100ng/ml CTGF group, 200ng/ml CTGF group, 500ng/ml CTGF group and control group (without CTGF).) When the cells in each group entered logarithmic growth period, the absorbance (optical density,OD) of each hole was measured by using the enzyme linked immunosorbent assay (Elisa) with thiazolyl blue (methyl thiazolyl tetrazolium,MTT method, and the mean value of OD in each group was calculated and compared. Results: MTT test showed that there was no significant difference in the proliferation of tendon cells between the 5ng/mlCTGF group and the control group by comparing the OD values of each group. The proliferation of tendon cells in the 10ng / ml 10 ng / ml CTGF group increased with the increase of CTGF concentration, and there were significant differences among the groups (P < 0. 05). The proliferation of tendon cells in the 50ng / ml CTGF,500ng/ml CTGF group decreased slightly with the increase of the concentration, but there was no significant difference between the two groups (P > 0. 05). Conclusion: 10ng / ml CTGF1 20ng / ml CTGFF 50ng / ml CTGFG / 100ng / ml CTGFF 200ng / ml CTGF500ng / ml CTGF can promote the proliferation of tendon cells. 5ng/mlCTGF had no obvious effect on the proliferation of tendon cells. When the concentration of CTGF was between 0ng/ml~50ng/ml, the proliferation of tendon cells increased with the increase of 0ng/ml~50ng/ml concentration. Between 50ng/ml~500ng/ml and 50ng/ml~500ng/ml, the proliferation of tendon cells decreased slightly with the increase of CTGF concentration. 50ng/ml was the best CTGF concentration to promote the proliferation of tendon cells.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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