左归丸对不育症SD大鼠Ki67蛋白及mRNA表达的影响

Effect of Zuogui Pill on Ki67 Protein and mRNA Expression in Infertility SD Rats

目的:观察左归丸对雷公藤多苷(tripterygium wilfordii,GTW)构建的少弱精子症模型大鼠睾丸组织中组织细胞增殖核抗原(Ki67)蛋白及mRNA表达的影响,初步探讨左归丸改善大鼠模型生殖功能的作用机制。方法:将36只雄性SD大鼠随机分为正常组、模型组、左归丸组,每组12只,共干预8周。正常组全程给予去离子水灌胃;模型组前4周给予40 mg·(kg·d)^(-1)的GTW进行造模,实验5～8周给予去离子水灌胃干预。左归丸组前4周用40 mg·(kg·d)^(-1)的GTW造模,实验5～8周,给予6 g·(kg·d)^(-1)的左归丸混悬液灌胃。8周后将所有大鼠处死,无菌取睾丸组织,固定、包埋并切片。免疫组化法检测各组大鼠睾丸Ki67蛋白的表达,比较各组平均光密度值。采用RT-PCR方法检测各组大鼠睾丸组织Ki67mRNA的表达情况。结果:模型组大鼠睾丸组织各级生精细胞核中Ki67较正常组表达明显减弱(P<0.05);左归丸组睾丸组织中各级生精细胞核中Ki67蛋白表达较模型组明显增多(P<0.05)。模型组ki67 mRNA表达水平较正常组明显降低(P<0.05);正常组与左归丸组ki67 mRNA表达水平相当;左归丸组ki67 mRNA表达水平明显高于模型组(P<0.05)。结论:左归丸能够上调Ki67蛋白及mRNA的表达,促进生精细胞增殖,提高各级生精细胞的数量并改善其功能,最终提高精子质量。

Objective: To observe the effect of Zuogui Pill on the expression of proliferating cell nuclear antigen( Ki 67) protein and mRNA in the testis of rats with oligozoospermia model constructed by Tripterygium wilfordii( GTW),and to explore the mechanism of Zuogui Pill in improving the reproductive function of rat model. Methods: 36 male rats were randomly divided into normal group,model group and Zuogui Pill group,with 12 rats in each group for a total of 8 weeks. The normal group was given deionized water throughout the whole process;40 mg·( kg·d)-1 of GTW was given in the model group for the first 4 weeks,and deionized water was administered to the stomach for 5 to 8 weeks. In the first 4 weeks of the Zuogui pill group,40 mg·( kg·d)-1 of GTW was used for modeling,and 5 to 8 weeks after the experiment,6 g·( kg·d)-1 of Zuogui Pill suspension was administered. All rats were sacrificed after 8 weeks,and testicular tissue was aseptically taken,fixed,embedded and sectioned. The expression of Ki67 protein in the testis of each group was measured by immunohistochemistry,and the average optical density values of different groups were compared. RT-PCR was used to detect and compare the expression of Ki67 mRNA in testis of each group. Results: The expression of Ki67 in the nucleus of spermatogenic cells in the testis of the model group was significantly lower than that in the normal group( P < 0. 05). The expression of Ki67 protein in the nucleus of spermatogenic cells in the testis of Zuogui Pill group wassignificantly higher than that in the model group( P < 0. 05). The expression level of ki67 mRNA in the model group was significantly lower than that in the normal group( P < 0. 05). The expression level of ki67 mRNA in the normal group and Zuogui Pill group was similar;the expression level of ki67 mRNA in Zuoguiwan group was significantly higher than that in the model group( P < 0. 05). Conclusion: Zuogui Pill can up-regulate the expression of Ki67 protein and mRNA,promote the proliferation of spermatogenic cells,increase the number of spermatogenic cells at all levels and improve their function,and ultimately improve sperm quality.