基于支持细胞骨架研究补肾填精法治疗男性不育少精子症的效果和机制

发布时间：2018-06-24 01:02

本文选题：补肾填精 + 男性不育　；参考：《北京中医药大学》2017年博士论文

【摘要】：由于生活方式和环境变化、生育年龄推迟等因素,男性不育的发病率不断增高,大部分不育患者无法找到明确病因,仅表现为精子质量下降,其中精子总数减少(少精子症)最容易导致男性不育。少精子症的病因目前尚不明确,研究认为生精细胞和精子凋亡可导致精子生成减少。中医认为男性不育少精子症的主要病机是肾精亏虚,补肾填精法是基本治法。基础实验和临床研究均已经证实五子衍宗丸代表的补肾填精法改善生精功能的机制之一是抑制生精细胞和精子凋亡,文献研究已表明支持细胞骨架破坏可以导致生精细胞凋亡和生精功能受损。笔者前期做过一些研究,把具有生殖毒性的雷公藤多甙与TM4支持细胞体外共培养后,发现支持细胞骨架出现断裂现象,骨架蛋白vimentin表达减少;而动物实验研究发现,五子衍宗丸对雷公藤多甙造成的生精功能损伤有改善作用。基于文献研究和前期研究,提出假设"五子衍宗丸代表的补肾填精法可以通过修复支持细胞骨架,抑制生精细胞和精子凋亡来改善生精",为了验证该假设,设计了以下临床和动物实验研究:1临床部分目的:研究以五子衍宗丸为代表的补肾填精法治疗肾精亏虚型男性不育少精子症的效果与机制。方法:因为男性不育尚缺乏有明确循证医学依据的公认治疗药物,而五子衍宗丸是疗效最被认可的药物,在临床研究中常被用作对照药物,因此本次研究未设立对照组。选择50例肾精亏虚型男性不育少精子症患者,予五子衍宗口服液治疗90天(每日2次,每次10ml),治疗前及治疗90天后分别检测精子质量(一周内连续检测2次,结果均符合少精子症的诊断标准才能入组,选择精子总数较多的检测结果进行记录),并通过Western Blot法分别检测精子凋亡。每30天进行一次随访,治疗前及每次随访时进行肾精亏虚证候评分。观察期间严格记录不良事件,同时详细记录配偶妊娠情况。结果:观察期间,4例患者配偶妊娠。76.7%的患者治疗后精子总数有明显提高。与治疗前相比,治疗后精子浓度、精子总数、精子总活力、前向运动精子百分率及a级精子百分率均有显著的提高,差异有统计学意义(P0.05);而精液量虽有提高,但差异无统计学意义(P0.05)。与治疗前相比,治疗后精子凋亡指标Caspase3的表达下降,差异有统计学意义(P0.05)。肾精亏虚证候评分随着随访次数的增加呈下降趋势,差异有统计学意义(P0.01),其中多数具体症状的评分也整体上呈下降趋势,差异有统计学意义(P0.01)。分组统计显示,20～29岁组、30～39岁组和40～岁组(最大51岁)的精子总数治疗后均有提高,差异有统计学意义(P0.05);体重正常组和过重组的精子总数治疗后均有显著提高,差异有统计学意义(P0.01),而肥胖组的精子总数治疗前后无明显差异;小睾丸组和睾丸体积正常组的精子总数治疗后均有显著提高,差异有统计学意义(P0.01);弱精子症组和前向运动精子百分率正常组的精子总数治疗后均有显著提高,差异有统计学意义(P0.01)。整个观察过程中未见不良反应报告。结论:以五子衍宗丸为代表的补肾填精法能够安全有效的提高肾精亏虚型男性不育少精子症患者的精子质量,提高男性的生育能力,同时可以缓解患者的肾精亏虚症状。补肾填精法提高精子总数的机制之一可能是抑制精子凋亡。2动物实验部分目的:研究五子衍宗丸改善肾精亏虚少精子症模型大鼠精子质量的效果与机制。方法:将25只SD雄性大鼠随机平均分为空白组、模型组和五子衍宗丸低、中、高剂量组,空白组正常喂养,模型组予雷公藤多甙(20mg/kg)灌胃,五子衍宗丸三组除了予雷公藤多甙灌胃,分别予不同剂量五子衍宗丸(0.5,1.0,2.0g/kg)灌胃。30天后,取附睾组织检测大鼠精子总数、前向运动精子百分率、精子总活力等指标,Western Blot法检测睾丸组织波形蛋白(vimentin)、αα微管蛋白的表达,免疫组化法检测睾丸支持细胞中vimentin、αα微管蛋白的表达,免疫组化法和Tunel法分别检测生精细胞的PCNA表达和凋亡。结果:与模型组相比,五子衍宗丸中、高剂量组的精子总数、前向运动精子百分率、精子总活力均显著提高,差异有统计学意义(P0.01);五子衍宗丸高剂量组睾丸组织中vimentin的表达增加,差异有统计学意义(P0.05),五子衍宗丸低、中、高剂量组睾丸组织中αα微管蛋白的表达均增加,差异有统计学意义(P0.05);五子衍宗丸低、中、高剂量组睾丸支持细胞中vimentin的表达均显著增加,差异有统计学意义(P0.01),五子衍宗丸低、中、高剂量组睾丸支持细胞中αα微管蛋白的表达均显著增加,差异有统计学意义(P0.01);五子衍宗丸低、中、高剂量组生精细胞的凋亡率均有下降,差异有统计学意义(p0.05);五子衍宗丸低、中、高剂量组生精细胞的PCNA表达均有所提高,但差异无统计学意义(P0.05)。结论:五子衍宗丸可以提高肾精亏虚少精子症模型大鼠的精子质量,机制之一可能是修复支持细胞骨架蛋白,抑制生精细胞的凋亡。
[Abstract]:The incidence of male infertility is increasing because of the change of life style and environment and the delay of childbearing age. Most infertile patients can not find a clear cause, only the loss of sperm quality, and the decrease of sperm count (oligozoospermia) is the most likely to lead to male infertility. The etiology of oligozoospermia is not yet clear. The apoptosis of spermatozoa and spermatozoa can lead to the decrease of spermatogenesis. The main pathogenesis of male infertility and oligozoospermia is the deficiency of kidney essence and the basic treatment of kidney filling. Basic and clinical studies have confirmed that one of the mechanisms of the five Zi Yan Zong pill to improve the function of spermatogenesis is to inhibit spermatogenic cells and sperm withering. The literature research has shown that the support of cytoskeleton destruction can lead to apoptosis and spermatogenic function of spermatogenic cells. A few studies have been done in the previous study to co culture the glucoside of Tripterygium wilfordii with TM4 support cells in vitro, and find that the support of the cytoskeleton appears, and the expression of skeleton protein vimentin decreases. The study found that the five Zong Zong pill can improve the damage of spermatogenic function caused by Tripterygium wilfordii polysaccharide. Based on literature research and earlier research, the hypothesis that "the kidney filling method of five Zi Yan Zong pill represents the support of the cytoskeleton and inhibition of spermatogenic cell and sperm apoptosis to improve spermatogenesis" is proposed. Clinical and animal experimental studies: 1 clinical purposes: To study the effect and mechanism of tonifying kidney essence with five Zi Yan Zong pill as the representative of the treatment of male sterile oligozoospermia with deficiency of kidney essence. The clinical study was often used as a control drug, so the control group was not set up in this study. 50 cases of male infertility with sperm deficiency were selected for 90 days (2 times a day, each time 10ml). The quality of sperm was detected before and after 90 days of treatment (2 times in a week). The results were consistent with oligozoospermia. The diagnostic criteria could be entered into the group, and the sperm apoptosis was recorded with more total sperm count. The sperm apoptosis was detected by Western Blot method. The renal sperm deficiency syndrome scores were followed up every 30 days. During the period of inspection, the total sperm count of 4 patients with.76.7% was significantly increased after treatment. Compared with before treatment, the sperm concentration, total sperm count, total sperm motility, the percentage of forward motile sperm and the percentage of A-level sperm were significantly improved, the difference was statistically significant (P0.05), while the semen volume increased, but the difference was no common. Study significance (P0.05). Compared with before treatment, the expression of sperm apoptosis index Caspase3 decreased after treatment, the difference was statistically significant (P0.05). The score of kidney deficiency deficiency syndrome was decreased with the increase of follow-up times, the difference was statistically significant (P0.01), and the scores of most specific symptoms were also downward trend, the difference was statistically significant. Statistical significance (P0.01). Group statistics showed that the total sperm count of 20~29 year old group, 30~39 year old group and 40 year old group (maximum 51 years old) were improved after treatment. The difference was statistically significant (P0.05). The difference was statistically significant after the treatment of the normal body weight group and the recombined sperm count (P0.01), and the total sperm count in the obese group was before the treatment. There was no significant difference in the total sperm count in the small testis group and the normal testicular group. The difference was statistically significant (P0.01). The sperm count in the normal group and the asthenospermia group and the forward motile sperm percentage were significantly improved, the difference was statistically significant (P0.01). There was no adverse reaction during the whole observation process. Report. Conclusion: the method of nourishing kidney with five Zong Zong pill can improve the sperm quality of the patients with spermatozoon oligozoospermia, improve the male fertility, and alleviate the symptoms of the deficiency of the sperm. One of the mechanisms of improving the total sperm count by filling the kidney with the method of filling the kidney may be to inhibit the sperm apoptosis.2 Objective: To study the effect and mechanism of five Zong Zong pills to improve the sperm quality of rats with sperm deficiency and oligozoospermia. Methods: 25 SD male rats were randomly divided into blank group, the model group and the five Zong Zong pill were low, middle, high dose, normal feeding in the blank group, and the model group was given 20mg/kg (Tripterygium wilfordii), and five children were derived from the model group. The three groups of Zong pill, except for gavage of Tripterygium wilfordii, were given different doses of five Zong Zong pills (0.5,1.0,2.0g/kg) after.30 after gavage. The total sperm count, the percentage of forward motile sperm, the total motility of spermatozoa were measured by the epididymal tissue, the Western Blot method was used to detect the expression of vimentin, the expression of alpha alpha microtubule, and immunohistochemistry. The expression of vimentin, alpha alpha tubulin in the testis support cells, the expression of PCNA expression and apoptosis in spermatogenic cells were detected by immunohistochemistry and Tunel. Results: compared with the model group, the total sperm count and the percentage of forward motile sperm in the high dose group were significantly improved (P0.0 1): the expression of vimentin in the testis tissue of the high dose group of the five Zong Zong pill increased, the difference was statistically significant (P0.05), the expression of alpha alpha microtubule in the testis tissues of the high dose group increased, and the difference was statistically significant (P0.05); the expression of vimentin in the testis support cells of the high dose group was all obvious. The difference was statistically significant (P0.01). The expression of alpha alpha microtubule in the testis support cells in the high dose group was significantly increased, and the difference was statistically significant (P0.01). The apoptosis rate of the spermatogenic cells in the high dose group was decreased, the difference was statistically significant (P0.05); the five Zong Zong pills were low, middle, The expression of PCNA in the high dose group was improved, but the difference was not statistically significant (P0.05). Conclusion: the five Zi Yan Zong pill can improve the sperm quality of the rat model of oligozoospermia. One of the mechanisms may be to repair the support of the cytoskeleton protein and inhibit the apoptosis of the fine cell.
【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】：2017
【分类号】：R277.5

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