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
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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。
Objective: To study the effect of the connective tissue growthfactor of different density on proliferation oftendon cells in vitro, and toinvestigate the possible mechanism of CTGF in repairing the tendoninjury.
Methods: We got the tendon from the posterior limbs of rats,tendoncells were obtained from the tendon segments after enzymatic digestion,then we cultured the cells with 10% fetal calf serum(FCS), the cells werepassaged when 80% were adherenced.we observed the morphous andgrowth regularity of the cells.The second passage celles were identifiedby immunohistochemistry to make sure that they were tendon cells. Thethird passage tendon cells of rabbit in vitro were planted in 48 wells of a96-well culture plate and every well contained 4×10~3 cells. They weredivided into 8 groups and every group contained 6 wells. According toCTGF concentration, 5ng/ml CTGF group、10ng/ml CTGF group、20ng/ml CTGF group、50ng/ml CTGF group、100ng/ml CTGF group、200ng/ml CTGF group、500ng/ml CTGF group, control group(no BFGF).When all the groups being in exponential phase of growth undermicroscope, they were dyed by MTF method and OD value of every wellwas detected by enzyme linked immunodetection meter.
Results: We got the results after the MTT test by contrast to OD mean of each group.There was no difference in the proliferation of tendoncells between 5ng/ml CTGF group and control group; 5ng/ml、10ng/ml、20ng/ml、50ng/mlCTGF groups had significant difference(P＜0.05),andthe proliferation of tendon cells got faster with the higher concentrationof CTGF in this term.There were no significant difference(P＞0.05)amongthe 50ng/ml、100ng/ml、200ng/ml、500ng/ml CTGF group.
Conclusions: 10ng/ml、20ng/ml、50ng/ml、100ng/ml、200ng/ml、500ng/ml、CTGF could promote proliferation of tendon cells; 5ng/mlCTGF has no significant effect on proliferation of tendon cells.theproliferation of tendon cells got faster with the higher concentration ofCTGF when CTGF concentration varying from 0ng/ml to 50ng/ml; butthe proliferation of tendon cells got a bit slower with the higherconcentration of CTGF when CTGF concentration varying from 50ng/mlto 500ng/ml.CTGF at 50ng/ml is found to be the most effectiveconcentration.
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。
Objective: To study the effect of the connective tissue growthfactor of different density on proliferation oftendon cells in vitro, and toinvestigate the possible mechanism of CTGF in repairing the tendoninjury.
Methods: We got the tendon from the posterior limbs of rats,tendoncells were obtained from the tendon segments after enzymatic digestion,then we cultured the cells with 10% fetal calf serum(FCS), the cells werepassaged when 80% were adherenced.we observed the morphous andgrowth regularity of the cells.The second passage celles were identifiedby immunohistochemistry to make sure that they were tendon cells. Thethird passage tendon cells of rabbit in vitro were planted in 48 wells of a96-well culture plate and every well contained 4×10~3 cells. They weredivided into 8 groups and every group contained 6 wells. According toCTGF concentration, 5ng/ml CTGF group、10ng/ml CTGF group、20ng/ml CTGF group、50ng/ml CTGF group、100ng/ml CTGF group、200ng/ml CTGF group、500ng/ml CTGF group, control group(no BFGF).When all the groups being in exponential phase of growth undermicroscope, they were dyed by MTF method and OD value of every wellwas detected by enzyme linked immunodetection meter.
Results: We got the results after the MTT test by contrast to OD mean of each group.There was no difference in the proliferation of tendoncells between 5ng/ml CTGF group and control group; 5ng/ml、10ng/ml、20ng/ml、50ng/mlCTGF groups had significant difference(P＜0.05),andthe proliferation of tendon cells got faster with the higher concentrationof CTGF in this term.There were no significant difference(P＞0.05)amongthe 50ng/ml、100ng/ml、200ng/ml、500ng/ml CTGF group.
Conclusions: 10ng/ml、20ng/ml、50ng/ml、100ng/ml、200ng/ml、500ng/ml、CTGF could promote proliferation of tendon cells; 5ng/mlCTGF has no significant effect on proliferation of tendon cells.theproliferation of tendon cells got faster with the higher concentration ofCTGF when CTGF concentration varying from 0ng/ml to 50ng/ml; butthe proliferation of tendon cells got a bit slower with the higherconcentration of CTGF when CTGF concentration varying from 50ng/mlto 500ng/ml.CTGF at 50ng/ml is found to be the most effectiveconcentration.
引文
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