己烯雌酚和胰岛素样因子3对睾丸引带细胞中富含亮氨酸的G蛋白耦联受体8mRNA和蛋白表达水平的影响

摘要

目的 探讨不同水平的己烯雌酚(DES)和胰岛素样因子3(INSL3)对体外培养的睾丸引带细胞中富含亮氨酸的G蛋白耦联受体8(LGR8) mRNA和蛋白表达的影响.方法 将3日龄的雄性昆明小鼠的睾丸引带组织解剖取出,并进行细胞培养.传代后,随机分为正常对照组及实验组(不同浓度DES组:A～E组,不同浓度INSL3组:Ⅰ～Ⅳ组)共11组.其中,实验组加入的DES浓度分别是3.7×10-2 mmol·L-1、3.7×10-3 mmol·L-1、3.7×10-4 mmol·L-1、3.7×10-5 mmol·L-1和二甲基亚砜溶剂对照组;INSL3的浓度分别为3.3×10-3 μmol·L-1、3.3×10-4 μmol·L-1、3.3×10-5 μmol·L-1及3.3×10-6 μmol·L-1.加药持续作用48 h后,应用反转录PCR和流式细胞术分析睾丸引带细胞中LGR8 mRNA及其蛋白的表达情况.结果 A组和Ⅳ组LGR8的mRNA及蛋白水平显著高于正常对照组(P<0.01);与正常对照组相比,B、C、D组LGR8 mRNA及其蛋白表达均显著下降(Pa<0.05),而E组及Ⅰ、Ⅱ、Ⅲ组LGR8 mRNA和其蛋白的表达差异均无统计学意义(Pa>0.05).结论 高剂量的DES和极低剂量的INSL3均能上调睾丸引带细胞中LGR8 mRNA及其蛋白表达水平,推测DES和INSL3可能通过LGR8作用途径来影响睾丸引带的发育.%Objective To study the effect of different concentrations of diethylstilbestrol (DES) and insulin - like factor 3 ( INSL3 ) on the expression of leucine rich repeat containing G protein coupled receptor 8 ( LGR8 ) mRNA and its protein in gubernaculum testis cells in vitro. Methods The gubernaculum testis tissue were dissected from 3 - day - old mice and cultured in Dulbecco's modified Eagle's medium (DMEM). The subcultured cells were randomly divided into 2 normal control groups and 9 experimental groups (DES:from group A to E;INSL3:fromgroup Ⅰ to Ⅳ).The DES groups were given 3.7×10-2 mmol· L -1,3.7×10 -3mmol· L-1,3.7 ×10-4 mmol· L-1,3.7×10 -5 mmol · L-1 of DES dissolved in dimethyl sulfoxide,respectively. And the INSL3 groups were given 3.3 × 10 -3 μmo1 · L-1 ,3.3 × 10-4 μmol · L -1,3.3 × 10-5 μmol · L-1 ,3.3 × 10-6 μmol · L-1 of INSL3 dissolved in double distilled water respectively. Forty - eight hours later, the expression of LGR8 mRNA and protein in gubernacula cells were investigated by reverse transcription -polymerase chain reaction and flow cytometry. Results Compared with the normal control group, the expression of LGR8 mRNA and protein were significantly upregulated in A and Ⅳ groups( Pa ＜ 0.01 ) , significantly reduced in B, C, D groups( Pa ＜ 0.05 ). But there was no significant difference in the Ⅰ,Ⅱ , Ⅲ and E groups(Pu ＞ 0.05 ). Conclusions Both high doses of DES and very low doses of INSI3 can upregulate the expression level of LGR8 mRNA and its protein in gubernaculum testis cells,which may interfere with gubernaeulum testis development by LGR8 pathway.