肾阳虚小鼠睾丸生殖细胞凋亡的实验研究
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摘要
目的：探讨“房室不节、劳倦过度”所致肾阳虚小鼠生殖细胞的凋亡机制，同时，研究金匮肾气丸抑制生殖细胞凋亡的作用机理。
方法：以已经建立的“房室不节、劳倦过度”肾小虚小鼠为实验动物，对小鼠睾丸组织的形态学进行观察，并分析小鼠睾丸内FSH和睾酮含量，同时利用RT-PCR和免疫组织化学技术对凋亡相关基因进行分析，利用TUNEL对小鼠睾丸生殖细胞的凋亡率进行测定。
结果：“劳倦过度、房室不节”导致小鼠的睾丸内生殖细胞凋亡率上升，睾丸内睾酮含量、Fas、FasL及Caspase3表达和分布异常，金匮肾气丸和甲基睾酮对这种异常有一定反向调节作用。此外，模型小鼠的Bcl-XL mRNA水平也低于正常水平。
结论：“劳倦过度、房室不节”导致小鼠睾丸内睾酮水平下降，通过Fas、FasL的外源性死亡通路引起生殖细胞凋亡，在这一过程中，有Bcl-XL的参与。金匮肾气丸可以通过提高睾丸内睾酮含量，调节Fas、Fas L表达，可以降低“劳倦过度、房室不节”造成的小鼠睾丸生殖细胞凋亡。
Objective:This experimental study focused on the mechanism of germ cell apoptosisin the testis of Kidney-Yang deficiency syndrome mice model.Meanwhile,the effects ofJinguishenqi bolus on decreasing the germ cell apoptotic index were explored.
Methods:In this study,a mice model of Kidney-yang deficiency syndrome was usedas experimental animal which had been established by us before.The method used todevelop Kidney-yang deficiency syndrome mice model based on principle of Chinesemedicine classical theory.The mice model was caused by overfatigue and sexualoverstrain.The morphological changes of mice testis tissue were observed after HEstaining.Ultramicrostructure were observed with transmission electron microscopy aswell.The concentrations of follicle-stimulating hormone and testosterone in the testis ofmice were measured with radioimmunoassay method.Then the mRNA levels of certainapoptosis related genes including Bcl-2,Bax,Bcl-XL,Fas,FasL and P53 were assessed byRT-PCR.Furthermore,the expression of Fas、FasL and Caspase3 protein in mice testiswere determined by immunohistochemical method.In addition,the germ cell apoptosisratios were detected by TUNEL.
Results:The testicular germ cell apoptotic index of Kidney-Yang deficiencysyndrome mice model caused by overfatigue and sexual overstrain increased significantly.And typical ultrastructural changes of apoptosis cell such as cell shrinkage,roughmembrane,nuclei shrinkage and margination of chromatin in model mice testis could beseen under ultramicroscope.While intratesticular testosterone concentration in mice modelwas much higher than normal mice,the content of follicle-stimulating hormone in mice model showed no change compared with normal mice.RT-PCR results showed thatmRNA levels of Fas and FasL in model mice testis were much higher than that in normalmodel.And abnormalities of location and expression of Fas and FasL protein in modelmice testis were detected by immunohistochemical method.And there was a higherexpression of Fas activated apoptopic signal pathway downsteam enzyme Caspase3 inmodel mice testis.When the model mice were treated with Jinguishenqi bolus ormethyltestosterone,the content of intratesticular testosterone in mice testis increasedobviously.And Fas,FasL and Caspase3 expression were downregulated by Jinguishenqibolus or methyltestosterone too,and so the apoptopic index.And there was also mRNAexpression change of one of Bcl-2 family protein named Bcl-XL in model mice testis.Itseemed that Jinguishenqi bolus or methyltestosterone had no any effect on this diversity.
Conclusion:Intratesticul testosterone plays an important roles on regulatingapoptosis of male germ cell.Kidney-yang deficiency syndrome model mice caused byoverfatigue and sexual overstrain have a low level of intratesticul testosteroneconcentration.The low content of intratesticul testosterone may upregulate theexpression Fas and FasL and then initiate a Fas dependent apoptotic pathway.Caspase3which has also been regulated works as a major executer in the Fas dependent apoptoticpathway.Jinguishenqi bolus can induce the apoptopic index of germ cells of model malemice.The mechanism of Jinguishenqi bolus on germ cell apoptosis may mainly dependon increasing intratesticul testosterone.In this way,the Fas and FasL expression havebeen depressed and so does Caspase3.Furhermore,Bcl-XL is also involved in theapoptosis process of the Kidney-Yand deficiency model mice.
方法：以已经建立的“房室不节、劳倦过度”肾小虚小鼠为实验动物，对小鼠睾丸组织的形态学进行观察，并分析小鼠睾丸内FSH和睾酮含量，同时利用RT-PCR和免疫组织化学技术对凋亡相关基因进行分析，利用TUNEL对小鼠睾丸生殖细胞的凋亡率进行测定。
结果：“劳倦过度、房室不节”导致小鼠的睾丸内生殖细胞凋亡率上升，睾丸内睾酮含量、Fas、FasL及Caspase3表达和分布异常，金匮肾气丸和甲基睾酮对这种异常有一定反向调节作用。此外，模型小鼠的Bcl-XL mRNA水平也低于正常水平。
结论：“劳倦过度、房室不节”导致小鼠睾丸内睾酮水平下降，通过Fas、FasL的外源性死亡通路引起生殖细胞凋亡，在这一过程中，有Bcl-XL的参与。金匮肾气丸可以通过提高睾丸内睾酮含量，调节Fas、Fas L表达，可以降低“劳倦过度、房室不节”造成的小鼠睾丸生殖细胞凋亡。
Objective:This experimental study focused on the mechanism of germ cell apoptosisin the testis of Kidney-Yang deficiency syndrome mice model.Meanwhile,the effects ofJinguishenqi bolus on decreasing the germ cell apoptotic index were explored.
Methods:In this study,a mice model of Kidney-yang deficiency syndrome was usedas experimental animal which had been established by us before.The method used todevelop Kidney-yang deficiency syndrome mice model based on principle of Chinesemedicine classical theory.The mice model was caused by overfatigue and sexualoverstrain.The morphological changes of mice testis tissue were observed after HEstaining.Ultramicrostructure were observed with transmission electron microscopy aswell.The concentrations of follicle-stimulating hormone and testosterone in the testis ofmice were measured with radioimmunoassay method.Then the mRNA levels of certainapoptosis related genes including Bcl-2,Bax,Bcl-XL,Fas,FasL and P53 were assessed byRT-PCR.Furthermore,the expression of Fas、FasL and Caspase3 protein in mice testiswere determined by immunohistochemical method.In addition,the germ cell apoptosisratios were detected by TUNEL.
Results:The testicular germ cell apoptotic index of Kidney-Yang deficiencysyndrome mice model caused by overfatigue and sexual overstrain increased significantly.And typical ultrastructural changes of apoptosis cell such as cell shrinkage,roughmembrane,nuclei shrinkage and margination of chromatin in model mice testis could beseen under ultramicroscope.While intratesticular testosterone concentration in mice modelwas much higher than normal mice,the content of follicle-stimulating hormone in mice model showed no change compared with normal mice.RT-PCR results showed thatmRNA levels of Fas and FasL in model mice testis were much higher than that in normalmodel.And abnormalities of location and expression of Fas and FasL protein in modelmice testis were detected by immunohistochemical method.And there was a higherexpression of Fas activated apoptopic signal pathway downsteam enzyme Caspase3 inmodel mice testis.When the model mice were treated with Jinguishenqi bolus ormethyltestosterone,the content of intratesticular testosterone in mice testis increasedobviously.And Fas,FasL and Caspase3 expression were downregulated by Jinguishenqibolus or methyltestosterone too,and so the apoptopic index.And there was also mRNAexpression change of one of Bcl-2 family protein named Bcl-XL in model mice testis.Itseemed that Jinguishenqi bolus or methyltestosterone had no any effect on this diversity.
Conclusion:Intratesticul testosterone plays an important roles on regulatingapoptosis of male germ cell.Kidney-yang deficiency syndrome model mice caused byoverfatigue and sexual overstrain have a low level of intratesticul testosteroneconcentration.The low content of intratesticul testosterone may upregulate theexpression Fas and FasL and then initiate a Fas dependent apoptotic pathway.Caspase3which has also been regulated works as a major executer in the Fas dependent apoptoticpathway.Jinguishenqi bolus can induce the apoptopic index of germ cells of model malemice.The mechanism of Jinguishenqi bolus on germ cell apoptosis may mainly dependon increasing intratesticul testosterone.In this way,the Fas and FasL expression havebeen depressed and so does Caspase3.Furhermore,Bcl-XL is also involved in theapoptosis process of the Kidney-Yand deficiency model mice.
引文
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