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目的探讨Resistin基因过表达对大鼠胰岛β细胞株RINm5F基础胰岛素分泌的影响。方法体外培养大鼠胰岛β细胞株RINm5F,采用PCR法扩增大鼠Resistin基因全编码区cDNA片段,BamH和Xho双酶切后插入pcDNA3.1B表达载体,经测序验证正确后,采用脂质体转染pcDNA3.1-Resistin真核表达质粒入RINm5F细胞,筛选稳定表达株,并采用RT-PCR验证过表达Resistin的效率。在RINm5F细胞密度达到80%,缓冲液洗3遍,继续于缓冲液中生长2 h,收集上清液,采用ELISA法检测细胞培养上清中的胰岛素水平。同时Trizol一步法提取RINm5F细胞总RNA,采用RT-PCR法检测葡萄糖转运子2(Glut2)mRNA水平,并用紫外吸收剂凝胶密度扫描仪分析条带吸光度值。采用SPSS11.0软件进行统计学分析。结果观察过表达Resistin的RINm5F中ResistinmRNA表达,发现过表达Resistin的细胞株中Resistin mRNA水平为对照组的2.46倍(P<0.001)。过表达Resistin的RINm5F细胞的基础胰岛素分泌低于转染空载质粒的细胞,较对照组下降约32%(P<0.001)。过表达Resistin的RINm5F细胞中Glut2表达明显低于转染空载质粒的细胞,较对照组下降约38%(P<0.05)。结论Resistin基因过表达显著下调胰岛β细胞RINm5F的基础胰岛素分泌,其机制可能与Glut2有关。
Objective To investigate the effect of Resistin gene overexpression on the basal insulin secretion of rat pancreatic β-cell RINm5F. Methods Rat pancreatic islet β cell line RINm5F was cultured in vitro. The full-length cDNA of rat Resistin gene was amplified by PCR. The recombinant plasmid was digested with BamH and Xho and then inserted into pcDNA3.1B expression vector. The correct sequence was verified by liposome The pcDNA3.1-Resistin eukaryotic expression plasmid was transfected into RINm5F cells to screen stable expression strains, and the efficiency of overexpression of Resistin was verified by RT-PCR. RINm5F cell density reached 80%, washed three times in the buffer, continue to grow in the buffer for 2 h, the supernatant was collected, and the insulin level in the cell culture supernatant was measured by ELISA. At the same time, the total RNA of RINm5F cells was extracted by one-step Trizol method. The mRNA expression of Glut2 was detected by RT-PCR and the band absorbance was analyzed by UV absorbance gel density scanner. SPSS11.0 software was used for statistical analysis. Results Resistin mRNA expression in RINm5F over-expressing Resistin was observed. Resistin mRNA level in Resistin-overexpressing cells was 2.46 folds higher than that in control group (P <0.001). The basal insulin secretion of RINm5F cells overexpressing Resistin was lower than that of the transfected cells by about 32% (P <0.001). Glut2 expression in RINm5F cells overexpressing Resistin was significantly lower than that in transfecting empty vector, which was 38% lower than that in control group (P <0.05). Conclusion Resistin overexpression significantly down-regulated the basal insulin secretion of pancreatic β-cell RINm5F, which may be related to Glut2.