摘要
目的本试验模拟糖尿病病程不同阶段患者体内葡萄糖和胰岛素浓度的变化,研究不同浓度的葡萄糖和胰岛素对Colon 26肿瘤细胞TGF-β1 mRNA表达的影响。方法体外常规培养Colon 26肿瘤细胞。根据所用培养液外加葡萄糖和胰岛素浓度的不同,将实验所用细胞共分12组:A组(对照组)RPMI 1640培养液;B组加5 mmol/L葡萄糖;C组加10 mmol/L葡萄糖;D组加25 mmol/L葡萄糖;E组加5μIU/ml胰岛素;F组加25μIU/ml胰岛素;G组加125μIU/ml胰岛素;H组加5 mmol/L葡萄糖+5μIU/ml胰岛素;K组加5 mmol/L葡萄糖+125μIU/ml胰岛素;L组加25 mmol/L葡萄糖+125μIU/ml胰岛素;M组加25 mmol/L葡萄糖+5μIU/ml胰岛素;N组加25 mmol/L葡萄糖+1.25μIU/ml胰岛素。每组设3个复孔,置37℃5%CO2饱和湿度的培养箱培养48 h,收集细胞后抽提总RNA,RT-PCR同时扩增目的片段TGF-β1和内参照β-actin,琼脂糖凝胶电泳,扩增产物在凝胶成像系统上扫描分析,计算TGF-β1 mRNA相对表达量。整个实验过程均重复3次。结果D组TGF-β1 mRNA的表达(0.768±0.017)高于A组(0.540±0.012),差异有显著性(P<0.01);而B、C组TGF-β1 mRNA的表达(0.545±0.033、0.558±0.035)与A组相比均无显著性差别(P>0.05)。E、F、G组对Colon 26肿瘤细胞TGF-β1 mRNA的表达(分别为0.566±0.039、0.549±0.030、0.531±0.022)与A组相比无显著性差别(P>0.05)。L、M、N组的TGF-β1 mRNA表达(分别为0.889±0.028、0.764±0.027、0.752±0.035)均高于A组(0.540±0.012),差异有显著性(P<0.01);而H、K组的TGF-β1 mRNA表达(0.550±0.037、0.537±0.025)与A组相比均无显著性差别(P>0.05)。结论高浓度的葡萄糖能够上调Colon 26肿瘤细胞TGF-β1 mRNA的表达,不同浓度的胰岛素不影响Colon 26肿瘤细胞TGF-β1 mRNA的表达,高浓度的葡萄糖和胰岛素同时作用能够上调Colon 26肿瘤细胞TGF-β1 mRNA的表达。
Objective To study the effect of glucose and insulin on the expression of TGF-β1 mRNA in Colon 26 cells by mimicing the changes of glucose and insulin levels in different courses of diabetes mellitus.Methods Colon 26 tumor cells were cultured in vitro.According to the concentrations of glucose and insulin in culture medium,cells were divided into 12 groups： A group cultured with RPMI 1640 as control,B、C、D groups cultured with 5 mmol/L,10 mmol/L,25 mmol/L glucose respectively,E、F、G groups cultured with 5 μIU/ml、25 μIU/ml、125 μIU/ml insulin resprctively,while H group cultured with 5 mmol/L glucose and 5 μIU/ml insulin,K group cultured with 5 mmol/l glucose and 125 μIU/ml insulin,L group cultured with 25 mmol/L glucose and 125 μIU/ml insulin,M group cultured with 25 mmol/L glucose and 5 μIU/ml insulin,N group cultured with 25 mmol/l glucose and 1.25 μIU/ml insulin.There were 3 repeated holes in each group.After added to different culture media,cells were cultured for 48 h in saturated humidity incubator with 5%CO2 at 37 ℃.Expressions of TGF-β1 were detected by RT-PCR and were repeated three times.Results In D group,the level of TGF-β1 mRNA（0.768±0.017）was higher significantly than A（0.540±0.012,P〈0.01）.However,expression of TGF-β1 in B and C group had no differences（0.545±0.033、0.558±0.035） compared with that in A group respectively（P〉0.05）.Compared with group A,there was no statistical difference about the expression of TGF-β1 mRNA in Colon 26 cells in E,F and G groups（0.566±0.039、0.549±0.030、0.531±0.022,P〉0.05）.In L,M and N groups,the level of TGF-β1 mRNA was higher（0.889±0.028、0.764±0.027、0.752±0.035） significantly than that of group A（P〈0.01）.No differences were found in H and K groups（0.550±0.037、0.537±0.025） compared with A group（P〉0.05）.Conclusion High concentration of glucose may up-regulate the expression of TGF-β1 mRNA in Colon 26 tumor cells.Different concentrations of insulin have no effects on the expression of TGF-β1 mRNA in Colon 26 tumor cells.High concentration of glucose and insulin may up-regulate the expression of TGF-β1 mRNA in Colon 26 tumor cells.
出处
《同济大学学报（医学版）》
CAS
2009年第2期32-37,共6页
Journal of Tongji University(Medical Science)
基金
河北省重大疾病专项资助项目(07276103D)
