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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3.7×10-2mmol.L-1、3.7×10-3mmol.L-1、3.7×10-4mmol.L-1、3.7×10-5mmol.L-1和二甲基亚砜溶剂对照组;INSL3的浓度分别为3.3×10-3μmol.L-1、3.3×10-4μmol.L-1、3.3×10-5μmol.L-1及3.3×10-6μmol.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 investigate the effects of different levels of diethylstilbestrol (DES) and insulin-like factor 3 (INSL3) on the mRNA and protein expression of leucine-rich G protein-coupled receptor 8 (LGR8) in cultured testicular cells. Methods Testicular tapes of 3-day-old male Kunming mice were dissected out and cultured in vitro. After passage, they were randomly divided into normal control group and experimental group (different concentrations of DES group: A ~ E group, different concentrations of INSL3 group: Ⅰ ~ Ⅳ group) a total of 11 groups. The concentrations of DES in experimental group were 3.7 × 10-2mmol.L-1, 3.7 × 10-3mmol.L-1, 3.7 × 10-4mmol.L-1, 3.7 × 10-5mmol.L-1and Dimethylsulfoxide solvent control group; INSL3 concentrations were 3.3 × 10-3μmol.L-1, 3 × 10-4μmol.L-1, 3 × 10-5μmol.L-1 and 3.3 × 10-6μmol, respectively. L-1. After 48 h of continuous dosing, the expression of LGR8 mRNA and its protein in testicular tape cells was analyzed by reverse transcription PCR and flow cytometry. Results The mRNA and protein levels of LGR8 in group A and group Ⅳ were significantly higher than those in normal control group (P <0.01). Compared with normal control group, LGR8 mRNA and protein expression in group B, C and D were significantly decreased (P <0.05 ), While there was no significant difference in the expression of LGR8 mRNA and protein between group E and groups Ⅰ, Ⅱ and Ⅲ (P> 0.05). Conclusions Both high-dose DES and very low dose INSL3 can up-regulate the expression of LGR8 mRNA and protein in testicular cells, suggesting that DES and INSL3 may affect the development of testis through LGR8 pathway.