【作者】 郭军华；

【导师】 何清湖；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2014， 博士

【摘要】 本研究分为三个部分：第一部分UU感染对精液质量的影响及知柏地黄丸的干预作用目的：本研究探讨UU感染对大鼠精液质量的影响以及知柏地黄丸的调节作用,采用彩色精子动态检测系统进行精子运动参数测定,并进一步证实UU感染会导致精子质量的下降以及知柏地黄丸的治疗作用,为下一步揭示UU感染致男性不育相关发病机制奠定理论基础。方法：4-5月龄SD大鼠40只,随机抽取30只,经膀胱接种UU悬液,建立UU睾丸感染动物模型。剩余10只同步注射生理盐水作为正常对照组,于接种后的第7天取睾丸穿刺液运用培养法检测UU,判断造模成功否。造模成功后,UU感染模型大鼠再随机分成模型组、阿奇霉素组、知柏地黄丸组。知柏地黄丸组给药量为1g/kg.d (即1ml/d),阿奇霉素组给药量为0.105mg/kg.d(即1ml/d),正常组、模型组给予相同剂量的生理盐水。以上各组均灌胃给药,每日一次,连续给药21d。采用颈椎脱臼法处死大鼠,取附睾组织,用眼科手术剪刀剔除附睾上的脂肪组织,小心去掉小血管,采用机械分离法分离精子细胞,彩色精子动态检测系统进行精子运动参数测定。结果：模型组大鼠A级精子、B级精子、直线速度、平均速度均明显低于正常组。经治疗后,中药组和西药组A级精子、B级精子、直线速度、曲线速度、平均速度水平提高,与模型组比较,差异有统计学意义(P<0.05,P<0.01),而两组均不能提高直线运动水平。中药组在提高A级精子、B级精子、曲线速度、平均速度方面效果优于西药组,差异有统计学意义(P<0.05,P<0.01)结论：UU感染能够降低A级精子、B级精子、直线速度、平均速度水平,知柏地黄丸和阿奇霉素对其都有治疗作用,且知柏地黄丸的效果优于阿奇霉素。第二部分UU感染对大鼠生精细胞线粒体超微结构及膜电位的影响及知柏地黄丸的干预作用目的：本研究探讨UU感染对大鼠生精细胞线粒体超微结构与膜电位的影响以及知柏地黄丸的干预作用,采用电镜进行生精细胞线粒体超微结构的观察,采用流式细胞仪对线粒体膜电位的检测,试图揭示UU感染致精子活力下降可能作用机制及知柏地黄丸干预机制。方法：采用颈椎脱臼法处死大鼠,取睾丸组织,用眼科手术剪刀和镊子剥去睾丸被膜和脂肪垫,小心去掉小血管,截取睾丸片段,采用机械分离法分离生精细胞,流式细胞仪检测线粒体膜电位；取睾丸片段,戊二醛固定,电镜观察生精细胞线粒体结构。结果：模型大鼠生精细胞线粒体膜电位水平显著低于正常组。经治疗后,中药组和西药组均能显著提高生精细胞线粒体膜电位,与模型组比较,差异有统计学意义(P<0.01)；且中药组的效果明显优于西药组,差异有统计学意义(P<0.01)。结论：UU感染能够降低生精细胞线粒体膜电位,知柏地黄丸和阿奇霉素都能提高线粒体膜电位,且知柏地黄丸效果更优。第三部分UU感染对大鼠生精细胞线粒体呼吸代谢功能的影响及知柏地黄丸的干预作用目的：本研究探讨UU感染对大鼠生精细胞线粒体呼吸代谢功能的影响以及知柏地黄丸的干预作用,采用EL ISA方法检测SDH的活性及含量,采用免疫组化法(DAB法)检测COX活性,采用RT-PCR法检测SDH、COXⅠ及Ⅱ的mRNA表达,试图揭示UU感染对线粒体呼吸产能功能的影响机制,为知柏地黄丸提高精子活力提供理论基础。方法：如上法取睾丸组织,采用EL ISA方法检测SDH的活性及含量,采用免疫组化法检测COX活性,采用RT-PCR法检测SDH、COXⅠ及Ⅱ的mRNA表达。结果：模型组大鼠睾丸组织中SDH酶活性和含量,均显著低于正常组,差异有统计学意义(P<0.01)。经治疗后,中药组和西药组均能显著提高SDH酶含量,与模型组比较,差异有统计学意义(P<0.01),而西药组不能提高SDH酶活性,但是两组均未能使SDH酶活性和含量达到正常,与正常组比较,P<0.01。模型组大鼠睾丸组织COX酶活性,与其他组比较,差异无统计学意义(P>0.05)。模型组大鼠睾丸组织SDH酶、COX酶Ⅰ、Ⅱ亚基mRNA表达均低于正常组。经治疗后,仅西药组能够提高睾丸组织SDH酶mRNA表达,中药组和西药组都能提高COX l mRNA表达,与模型组比较,差异有统计学意义(P<0.05),但两者效果相当,差异无统计学意义(P>0.05)。结论：UU感染能够降低SDH酶活性和含量,SDH酶及COX l、ll亚基mRNA表达,不能降低COX酶活性。治疗后,中药组和西药组均能显著提高SDH酶含量,COX l mRNA表达,均不能提高COX ll mRNA表达；中药组能提高SDH酶活性,西药组能够提高睾丸组织SDH酶mRNA表达。总结：1.UU感染能够明显降低大鼠A级精子、B级精子、直线速度、平均速度水平。知柏地黄丸和阿奇霉素能够提高A级精子、B级精子、曲线速度、平均速度水平,且知柏地黄丸效果优于阿奇霉素。2.UU感染能够显著降低线粒体膜电位,引起线粒体超微结构的改变。知柏地黄丸可能通过提高线粒体膜电位,改善线粒体结构的破坏,对线粒体起到修复和保护作用,而提高精子活力。3.UU感染能够降低大鼠睾丸组织中SDH酶活性和含量,对COX酶活性没有影响,说明UU感染所致精子活力下降与COX酶无关,可能主要与SDH酶活性和含量有关。4.UU感染能够使SDH酶mRNA表达降低。治疗后SDH酶mRNA表达提高,而SDH酶活性不提高,说明SDHmRNA表达的提高可能和SDH酶活性无关或会降低SDH酶的活性,可能和SDH酶的含量有关。UU感染能够使COX酶Ⅰ、Ⅱ亚基mRNA表达降低。治疗后COX I mRNA表达提高,COX ll mRNA表达和COX酶活性没有提高,说明COX酶的活性可能和COX ll mRNA有关,和COX I mRNA表达无关。更多还原

【Abstract】 There are three parts in this study:Part1Impacts on the quality of semen by UU infected and interve-ntion of Zhibai DihuangDecoctionObjective:This study explores the effects of semen quality of rats and regulatory role of Zhibai Dihuang Decoction after UU infected. The sperm motility parameters are measured by color sperm dynamic detection system. And further confirm that UU infection will lead to a decline of sperm quality and Zhibai DihuangDecoction can treat it, for further revealing the related theoretical basis for the pathogenesis of male infertility by UU infected.Methods:From40SD rats of4-5months age, randomly selecte30and inoculate the bladder with UU suspension, to establish the animal model of infected testis by UU. The remaining10rats are synchronously injected normal saline as the control group.Seventh days after inoculation, detect UU of the testis puncture fluid by the culture method,to judge the model.If the model is successful,they are randomly divided into the model group,azithromycin group, Zhibai Dihuang Decoction group.the dosage of ZhibaiDihuang Decoction group was1g/kg·D (1ml/d), azithromycin group0.105g/kg·D (1ml/d).the control groupandthe model group are given the same dosage of normal saline.every group is administered for21d orally, once a day.The rats are sacrificed by cervical dislocation, take the epididymal tissue, remove epididymal fat tissue and the small blood vessels carefully by the ophthalmology operation scissor.isolating sperm cells by the mechanical method and determinating sperm motion parameters by the color sperm dynamic detection system.Results:A grade sperm,B grade sperm, linear velocity, mean velocity of the model group, are significantly lower than the control group.After treatment, A grade sperm, B grade sperm, linear velocity, curvilinear velocity, average velocity of Zhibai Dihuang Decoction group and azithromycin group improved comparing with the model group and the difference has statistical significance (P<0.05, P<0.01),but the linear velocity of two group not improve.A grade sperm, B grade sperm, curvilinear velocity, average velocity of Zhibai Dihuang Decoction group is better than azithromycin group and the difference has statistical significance (P<0.05, P<0.01)Conclusions:UU can reduce the level of A grade sperm, B grade sperm, linear velocity and average velocity.Zhibai Dihuang Decoction and azithromycin can treat it and Zhibai Dihuang Decoction better than azithromycin.Part2Effects of rat spermatogenic cell mitochondrial ultrastructure and mitochondria membrane potential after UU infected and intervention of Zhibai Dihuang DecoctionObjective:This study to explore the effects of rat spermatogenic cells ultrastructure and mitochondria membrane potential by UU infected and intervention effect of Zhibai Dihuang Decoction, using electron microscopy to observe the ultrastructure of mitochondria of spermatogenic cells,using flow cytometry to detecte the mitochondrial membrane potential,to reveal the mechanism of UU infection inducing sperm motility and intervention mechanism of Zhibai Dihuang DecoctionMethods:The rats are sacrificed by cervical dislocation, take the testis tissue, remove testis fat tissue and the small blood vessels carefully by the ophthalmology operation scissor, intercepte testicular fragments,isolate sperm cells by the mechanical method and measure mitochondrial membrane potential by Flow cytometric detection.Testis fragments were fixed in glutaraldehyde, and observate the spermatogenic cells mitochondrial structure by electron microscope.Results:The spermatogenic cell mitochondrial membrane potential of the model group is Significantly lower than the control group.After treatment, the spermatogenic cell mitochondrial membrane potential of Zhibai Dihuang Decoction group and azithromycin group significantly improve compared with the model group, and the difference has statistical significance (P<0.01).Even Zhibai Dihuang Decoction group is better than azithromycin group, and the difference was statistically significant (P<0.01).Conclusion:UU can reduce the spermatogenic cell mitochondrial membrane potential.Zhibai Dihuang Decoction and azithromycin can improve it, but Zhibaidihuang Decoction is better.Part3Effects of the rat spermatogenic cell mitochondrial respir-atory metabolism function by UU infected and intervention of Zhibai Dihuang DecoctionObjective:This study to explore the Effects of the rat spermatogenic cell mitochondrial respiratory metabolism function by UU infected and intervention of Zhibai Dihuang Decoction,using ELISA to detect the activity and content of SDH,Using immunohistochemical method (DAB method) to detect the activity of COX,using RT-PCR to detect the expression of SDH, COX I and II mRNA,to reveal the mechanism of mitochondrial respiratory capacity function by UU infected,to provide the theoretical basis for the Zhibai Dihuang Decoction improve sperm motility.Methods:The above method to take the testis,using ELISA to detect the activity and content of SDH,Using immunohistochemical method (DAB method) to detect the activity of COX,using RT-PCR to detect the expression of SDH, COX I and II mRNA.Results:The activity and content of SDH in the model group rats’testicular tissue were significantly lower than the control group and the difference has statistical significance(P<0.01). After the treatment,the content of SDH of Zhibai Dihuang Decoction group and azithromycin group both significantly increase, compared with the model group, the difference was statistically significant (P<0.01),but the activity of SDH of the azithromycin group does not enhance. And the activity and content of SDH in the two groups failed to be normal, compared with the normal group,P<0.01. The activity of COX in the model group rats’testis did not decline, compared with other groups and the difference was not statistically significant (P>0.05).The expression of SDH,COX I, COX II mRNA in the model group rats’testicular tissue was lower than the normal group.After treatment, only the expression of SDH mRNA of the azithromycin group improve.the expression of COX I mRNA of, Zhibai Dihuang Decoction group and azithromycin group increase, compared with the model group, the difference was statistically significant (P<0.05),But compared with each othe,the difference was no significant (P>0.05).Conclusion:UU can reduce the activity and content of SDH and the expression of SDH,COXI,COX ⅡmRNA, but can not reduce the activity of COX.After treatment, the content of SDH and the expression of COX I mRNA in both Zhibai Dihuang Decoction group and azithromycin group significantly increase, but the expression of COX Ⅱ mRNA not increase.Zhibai Dihuang Decoction can improve the activity of SDH, azithromycin can improve the expression of SDH mRNA in the testis tissue.Summary1.UU can reduce the level of A grade sperm, B grade sperm, linear velocity and average velocity.Zhibai Dihuang Decoction and azithromycin can treat it and Zhibai Dihuang Decoction better than azithromycin.2. UU can reduce the spermatogenic cell mitochondrial membrane potential and cause mitochondrial ultrastructure changes.Zhibai Dihuang Decoction can improve sperm motility by means of improving the mitochondrial membrane potential,reducing the structure of mitochondria damage, and repairing or protectiving the mitochondria.3. UU can reduce the activity and content of SDH, but can not reduce the activity of COX.It indicate that the decline in sperm motility caused by UU has nothing to do with the COX, may be mainly related to the activity and content of SDH.4. UU can reduce the expression of SDHmRNA.After treatment,the expression of SDH enzyme mRNA increased, while the activity of SDH did not improve,which revealed that the expression of SDHmRNA increasing may be independent of the activity of SDH or reduce the activity of SDH, may be related to the content of SDH.UU can reduce the expression of COXI,COX II mRNA.After treatment,the expression of COX I mRNA increased, the expression of COX II mRNA and the activity of COX did not increase, which reveal that the activity of COX may be related to the expression of COX II mRNA, and not to the expression of COX I mRNA.更多还原