左归丸治疗少弱精子型男性不育症的临床和实验研究

发布时间：2018-05-29 21:40

  本文选题：Ki67 + SCF　； 参考：《北京中医药大学》2016年博士论文

【摘要】：近年来,随着人们生活水平的普遍提高,传统的生活方式发生了巨大的改变。诸如精神压抑、暴饮暴食、长期熬夜等因素直接导致男性精子质量水平普遍下降。如何有效提高男性生育能力成为当下的热点话题。但是目前西医除去人工授精、试管婴儿等辅助生殖技术以及感染、梗阻等病因明确的疾病,临床上尚无有效解决男性不育症的治疗方法。同时,研究表明辅助生殖技术虽然可以解决部分患者的生育问题,但是其远期影响尚未可知。因此,广大临床医生将目光转向中医药对于男性不育症的诊治。本病的中医药治疗方法历史悠久,基础坚实,且临床疗效明显。这种情况下,如何充分挖掘本病的中医药特色疗法已经成为一个重要的课题。本文首先对左归丸治疗肾阴亏虚型少弱精子症的临床疗效进行系统评价；然后探索使用雷公藤多苷制备少弱精子症大鼠模型,研究其用药剂量及时间的最佳组合,制备相对合理的少弱精子症模型；之后设计保护实验和治疗实验,每个实验包含正常组、模型组以及治疗组,综合评价左归丸对少弱精子型模型大鼠脏器系数、精液质量、性激素水平、附睾肉毒碱、抑制素B水平的影响,从药效学角度对左归丸的治疗效果进行评价；最后,采用免疫组织化学、western blot以及Real-Time PCR检测技术,对保护实验以及治疗实验中各组睾丸组织Bax、Bal-2、SCF、 Ki67蛋白及mRNA表达水平进行检测,对左归丸可能的抗凋亡及促增殖的作用机制进行定性及定量分析。第一部分左归丸治疗少弱精子型男性不育症的临床研究目的：研究左归丸对肾阴亏虚型少弱精子症患者的临床疗效。方法：因本病尚无合适的阳性对照药物,故采用自身前后对照进行临床观察。本方法的治疗周期为90天,每日给予42例入组的肾阴亏虚型少弱精子症患者左归丸中药配方颗粒剂进行治疗。患者用药前及结束后分别进行1次精液常规检查；研究开始后,每30天对入组患者进行随访一次,随访时对患者中医证候进行评分并严格记录合并用药和不良事件,若出现不良反应报告则及时向上级医师汇报,并进行相关处理。同时在研究期间详细记录患者配偶怀孕情况。系统评价左归丸对于肾阴亏虚型少弱精子症患者精液质量各项指标以及中医证候的改善情况,综合探讨其临床疗效。结果：与治疗前相比,使用左归丸进行治疗后精液量、液化状态、精子总活动力、a级精子百分率、非前向运动精子百分率、b级精子百分率、精子浓度、精子总数等指标均有明显提高,差异性显著,具有统计学意义(p0.01)；中医证候总评分以及肾阴亏虚相关中医症候评分随着随访次数的增加以及时间的延长,呈明显的下降趋势,差异性显著,具有明显的统计学意义(p0.01)；左归丸配方颗粒安全性较高,整个研究过程中未见不良反应报告。结论：左归丸能够安全有效的提高肾阴亏虚型少弱精子症患者的精子质量各项指标,提高男性的生育能力,同时能够改善患者的中医相关症状,特别是对肾阴亏虚相关症状改善效果显著。第二部分雷公藤多苷诱导少弱精子型大鼠模型的研究目的：研究多种雷公藤多苷用药剂量以及造模时间的组合,并观察停止用药后各组大鼠自身恢复情况,制备少弱精子型大鼠模型。方法：将96只雄性SD大鼠随机分为正常组、雷公藤多苷大剂量组、中剂量组以及小剂量组。其中正常组采用去离子水进行干预；雷公藤多苷各剂量组分别采用50 mg/ (Kg·d)、40 mg/(Kg·d)、30mg/(Kg·d)的雷公藤多苷进行干预。给药第3周及第4周后,每组中选取SD大鼠4只取材；第4周取材后,每组选取4只按照上述实验方案继续喂养,并在第5周进行取材,剩余的大鼠在第4周给药完成后就停止进行干预,并在实验开始后的第6、7、8周,每周每组取材4只实验大鼠,观察其生殖相关功能的恢复情况并进行相关记录。结果：造模期间(第3周至第5周),一般状态、生殖器官脏器系数、精液质量各项指标随着雷公藤多苷用药剂量增大以及用药时间的延长呈显著的降低趋势；睾丸组织病理损伤程度随着雷公藤多苷用药剂量增大以及用药时间的延长呈显著的增高趋势。停止用药后(第6周至第8周),雷公藤多苷小剂量组上述指标逐渐恢复至正常组水平,未见明显差异性；中剂量组上述各项指标略有恢复,但与正常组相比仍有较大差距；大剂量组生殖功能损伤未见明显恢复。结论：综合考虑造模时间、经济成本以及大鼠的自我恢复情况,采用40mg/(Kg·d)的雷公藤多苷干预4周后可以得到少弱精子型大鼠模型。第三部分左归丸对雷公藤多苷诱导的少弱精子型模型大鼠的药效学研究目的：观察左归丸对少弱精子型模型大鼠一般状态、脏器系数、精液质量、睾丸组织病理改变、性激素水平、附睾肉毒碱、抑制素B的影响。方法：将72只雄性SD大鼠随机平均分为治疗实验和保护实验；每个实验又随机分为正常组、模型组、左归丸组,每组12只大鼠。在保护实验中,正常组采用去离子水进行干预,共计4周；模型组采用40mg/(Kg·d)的雷公藤多苷进行造模,共计4周；左归丸治疗组采用40mg/(Kg·d)的雷公藤多苷进行造模,同时采用6g/(Kg·d)的左归丸混悬液(相当于人体用药20倍)进行保护性干预4周。治疗实验中,正常对照组采用去离子水进行干预8周；模型组实验前4周采用40mg/(Kg·d)的雷公藤多苷进行造模,实验第5至8周采用去离子水进行干预；左归丸治疗组实验前4周采用40mg/ (Kg·d)的雷公藤多苷进行造模,实验第5至8周,采用6g/(Kg·d)的左归丸混悬液(相当于人体用药20倍)进行干预。结果：在保护试验中,左归丸组与模型组相比,在大鼠睾丸脏器系数、附睾脏器系数、精囊腺脏器系数、精液质量各项指标、睾酮水平、血清抑制素B方面具有显著的差异性,具有明显的统计学意义(P0.05或P0.01)；在治疗试验中,与雷公藤多苷模型组相比,左归丸组在大鼠睾丸脏器系数、附睾脏器系数、前列腺脏器系数、精液质量各项指标、睾酮水平、血清抑制素B具有明显的差异性,具有明显的统计学意义(P0.05或P0.01)；在保护实验与治疗实验两个实验中,左归丸组大鼠一般状态、睾丸组织病理损伤程度与模型组相比都明显减轻。结论：左归丸可以有效改善模型大鼠一般状态、生殖器官脏器系数、精液质量、睾酮水平、血清抑制素B水平,具有明显的保护及治疗作用,效果明显。第四部分左归丸对少弱精子型模型大鼠的抗细胞凋亡机制及SCT、Ki67表达的研究目的：观察左归丸对少弱精子型模型大鼠细胞凋亡调控因子Bax、Bcl-2蛋白以及mRNA的影响；同时观察左归丸对少弱精子型模型大鼠SCF、Ki67蛋白及mRNA表达的影响方法：分别采用免疫组织化学、western blot以及Real-Time PCR技术,对上述左归丸药效学研究中的保护实验和治疗实验中全部大鼠睾丸组织进行Bax、Bcl-2、SCF、Ki67蛋白及mRNA表达的检测。结果：保护实验,免疫组织化学检测中,与模型组相比较,左归丸组Bax、Bcl-2、 SCF、Ki67蛋白具有明显的差异性,具有显著的统计学意义(P0.05或P0.01)；western blot检测中,左归丸组与模型组相比较,Bax、Bcl-2、SCF蛋白具有差异性,P0.05,具有统计学意义；Real-Time PCR检测中,与模型组相比较,左归丸组Bax、Bcl-2、SCF、 Ki67 mRNA具有明显的差异性,P0.05或P0.01,具有统计学意义。治疗实验,免疫组织化学检测中,与模型组相比较,左归丸组Bcl-2、SCF、Ki67蛋白具有明显的差异性,P0.05或P0.01,具有统计学意义；western blot检测中,左归丸组与模型组相比处于相同水平,未见明显差异性,但是Bcl-2以及SCF表达水平明显高于模型组；Real-Time PCR检测中,左归丸组与模型组相比较,SCF、Ki67mRNA表达水平明显提高,具有明显的差异性,P0.05或P0.01,具有统计学意义；结论：左归丸可以有效调控模型大鼠Bax、Bcl-2、CF、Ki67蛋白及其mRNA的表达,具有明显的抑制生精细胞凋亡、促进生精细胞增殖的作用。
[Abstract]:In recent years, with the general improvement of people's living standards, great changes have taken place in the traditional way of life. Such as mental depression, binge eating, long stay in the night and so on directly lead to the decline of male sperm quality. How to effectively improve male fertility has become a hot topic at present. But western medicine is now removing artificial insemination, At the same time, there is no effective solution to the treatment of male infertility. At the same time, the research shows that although the assisted reproductive technology can solve the problems of some patients, the long-term effect has not been known. Therefore, the general clinicians will turn their eyes to the traditional Chinese medicine. The Chinese medicine has a long history, solid foundation and obvious clinical effect. In this case, how to fully excavate the characteristic therapy of traditional Chinese medicine has become an important subject. Firstly, the clinical efficacy of Zuogui Pill in the treatment of deficiency asthenospermia with kidney yin deficiency syndrome is systematically studied in this paper. Then explore the use of Tripterygium wilfordii polysaccharide to prepare the oligozoospermia rat model, study the best combination of dosage and time, prepare a relatively reasonable oligozoospermia model, and then design protection experiment and treatment experiment, each experiment includes normal group, model group and treatment group, comprehensive evaluation of Zuogui Pill on oligozoospermia type The effect of the viscera coefficient, semen quality, sex hormone level, epididymal carnitine and inhibin B level in the model rats was evaluated. The therapeutic effect of Zuogui pill was evaluated from the pharmacodynamic point of view. Finally, immunohistochemistry, Western blot and Real-Time PCR were used to detect Bax, Bal-2, SCF in each group of testis in the protection experiment and the treatment experiment. Ki67 protein and mRNA expression level were detected and the mechanism of the possible anti apoptosis and proliferation promoting mechanism of Zuogui pill was qualitatively and quantitatively analyzed. The clinical purpose of the first part of Zuogui Pill in the treatment of male infertility with oligozoospermia type: the clinical effect of Zuogui Pill on asthenospermia with kidney yin deficiency type. There were no suitable positive control drugs, so the clinical observation was carried out before and after the control. The treatment period of this method was 90 days, and 42 cases of kidney yin deficiency asthenia asthenia asthenia with Zuogui pill were treated with traditional Chinese medicine granules. The patients were examined before and after 1 times of the semen routine examination. After the study, the study began, The patients were followed up every 30 days, the TCM syndromes were scored and the combined medication and adverse events were strictly recorded. If the adverse reaction report was reported to the superior physicians in time, and the related treatment was carried out in the period of study. The quality of semen of asthenospermia patients with deficiency asthenospermia and the improvement of TCM syndrome were studied. Results: compared with before treatment, the amount of semen, the state of liquefaction, the total activity of sperm, the percentage of a sperm, the percentage of non forward motile sperm, the percentage of B sperm, the concentration of sperm, and the sperm concentration compared with the treatment before treatment. The total sperm count and other indexes were significantly improved, with significant difference, with statistical significance (P0.01). The total score of TCM syndrome and the syndrome score of TCM syndrome related to kidney yin deficiency were obviously decreased with the increase of the number of follow-up times and the prolongation of time. The difference was significant (P0.01), and the formula of Zuo GUI pill was the formula. There is no adverse reaction report in the whole study. Conclusion: Zuogui pill can improve the sperm quality indexes of the patients with deficiency asthenospermia with kidney yin deficiency, improve the male fertility, and improve the symptoms of Chinese medicine, especially the improvement of kidney yin deficiency related symptoms. The second part of Tripterygium wilfordii induced oligozoospermia model in rats: To study the combination of the dosage of multiple Tripterygium Wilfordii and the time of modeling, and to observe the self recovery of rats in each group after cessation of drugs. Methods: 96 male SD rats were randomly divided into normal group, thunder male rats In the large dose group, medium dose group and small dose group, the normal group was treated with deionized water, and Tripterygium wilfordii was treated with 50 mg/ (Kg. D), 40 mg/ (Kg. D), 30mg/ (Kg. D) by Tripterygium wilfordii polysaccharide. After third and 4 weeks, 4 samples of SD rats were selected in each group; each group was selected for fourth weeks. 4 rats were fed in accordance with the above experimental scheme, and the materials were taken for fifth weeks. The remaining rats stopped intervening after fourth weeks of administration, and after the first week of the experiment, 4 rats in each group were harvested every week to observe the recovery of their reproductive function and record the related records. Results: during the model period (third weeks). To fifth weeks), the general state, the organ coefficient of reproductive organs, the quality of the semen with the increase of the dosage of Tripterygium Wilfordii and the prolongation of the time, the degree of pathological injury of the testicle increased with the increase of the dosage of Tripterygium Wilfordii and the prolongation of the time of drug use. After the medicine (sixth weeks to eighth weeks), the above indexes of the small dose group of Tripterygium wilfordii gradually recovered to the normal group level, and no obvious difference was found. The above indexes in the middle dose group were slightly restored, but there was a big gap between the normal group and the normal group. The self recovery of the rat and the 40mg/ (Kg. D) interference of Tripterygium wilfordii was used for 4 weeks. The third part of Zuogui pill was used to study the pharmacodynamics of the oligozoospermia model rats induced by Tripterygium wilfordii polysaccharide. The effect of semen quality, pathological changes of testis tissue, sex hormone level, epididymis carnitine and inhibin B. Methods: 72 male SD rats were randomly divided into treatment experiment and protective experiment. Each experiment was randomly divided into normal group, model group, Zuogui pill group, 12 rats in each group. In the protection experiment, the normal group was used deionized water. Intervention, for a total of 4 weeks, the model group used 40mg/ (Kg d) of Tripterygium wilfordii poly glycoside for 4 weeks. The treatment group of Zuogui pill used 40mg/ (Kg. D) of Tripterygium wilfordii polysaccharide to build the model, and 6g/ (Kg. D) left GUI pill suspension (equivalent to 20 times the human medication) for 4 weeks. In the treatment experiment, the normal control group was removed from the control group. Subwater intervention for 8 weeks; 4 weeks before the model group, 40mg/ (Kg. D) of Tripterygium wilfordii was used to make the model, the experiment fifth to 8 weeks to use deionized water to intervene, and 4 weeks before the experimental group of Zuogui pill, 40mg/ (Kg. D) of Tripterygium wilfordii was used to make the model, the experiment was fifth to 8 weeks, and the 6g/ (Kg. D) left GUI pill suspension (equivalent to the human body) Results: in the protection test, in the protection test, the left GUI pill group was significantly different in the rat testicular organ coefficient, the epididymal organ coefficient, the seminal vesicle organ coefficient, the seminal fluid quality index, the testosterone level and the serum inhibin B, and had significant statistical significance (P0.05 or P0.01) in the treatment test. Compared with the Tripterygium wilfordii multi glycoside model group, Zuogui pill group was significantly different in the rat testicular organ coefficient, epididymal organ coefficient, prostate organ coefficient, semen quality index, testosterone level and serum inhibin B, with significant statistical significance (P0.05 or P0.01); in the two experiments of protection experiment and treatment experiment, left Conclusion: Zuogui pill can effectively improve the general state of the model rats, the organ coefficient of the genital organs, the quality of the semen, the level of testosterone and the level of serum inhibin B, which has obvious protective and therapeutic effect. The effect is obvious in the fourth part of Zuogui pill. Study on the mechanism of anti apoptosis and the expression of SCT and Ki67 in the rats with oligozoospermia model: To observe the effect of Zuo GUI Pill on the apoptosis regulator Bax, Bcl-2 protein and mRNA in the rat model of oligozoospermia model, and observe the influence methods of Zuo GUI Pill on the expression of SCF, Ki67 protein and mRNA in the model rats of oligozoospermia model: respectively Immunohistochemistry, Western blot and Real-Time PCR technique were used to detect the Bax, Bcl-2, SCF, Ki67 protein and mRNA expression of all rat testis in the protective experiment and treatment experiment of the pharmacodynamic study of Zuo GUI pill. Results: protection experiment, immunohistochemistry test, compared with model group, Bax, Bc in Zuo GUI pill group L-2, SCF, Ki67 protein has significant difference, and has significant statistical significance (P0.05 or P0.01); in Western blot detection, Zuo GUI pill group is compared with model group, Bax, Bcl-2, SCF protein have difference, P0.05, with statistical significance. There was significant difference, P0.05 or P0.01, with statistical significance. In the treatment experiment and immunohistochemistry, the Bcl-2, SCF, and Ki67 protein in Zuo GUI pill group had obvious difference, P0.05 or P0.01, with statistical significance. In Western blot detection, the left GUI pill group was at the same level as the model group, and no obvious difference was found. Difference, but the expression level of Bcl-2 and SCF was significantly higher than that of the model group; in Real-Time PCR detection, the expression level of SCF, Ki67mRNA in Zuo GUI pill group was significantly higher than that in model group, with significant difference, P0.05 or P0.01, with statistical significance. Conclusion: Zuo GUI pill can effectively regulate the model rats Bax, Bcl-2, CF, Ki67 protein and The expression of mRNA can inhibit spermatogenic cell apoptosis and promote the proliferation of spermatogenic cells.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R277.5
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本文编号：1952494