补肾生精汤对房劳小鼠生精障碍影响的实验研究

【作者】 腰向颖；

【导师】 董连荣；

【作者基本信息】 北京中医药大学 ， 中医基础理论， 2002， 硕士

【摘要】 本课题在中医“肾主生殖”理论指导下，通过“房劳伤肾”的中医病因学说，造成房劳肾虚生精障碍模型，并观察了补肾方药对模型的影响，以揭示房劳肾虚在生精细胞调亡水平的表现，提供补肾方药在生精细胞凋亡方面的实验依据，从而为“肾主生殖”的实验研究提供一些生精细胞凋亡方面的实验依据。本论文分为文献综述和实验研究两部分。1文献综述1.1中医临床运用“肾主生殖”理论治疗男性不育的概况目前中医临床治疗男性不育症的有关资料，大体可分为三类：（1）按纯中医理论进行辨证论治；（2）按现代实验检查结果施治；（3）实验检查结果与中医辨证互参施治。由于肾在中医生殖理论中的主导地位，中医临床对男性不育症的治疗，无论是按中医理论辨证论治，还是按实验检查结果用药或是两者互参施治，仍以补益肾中精气、调和肾阴肾阳为主。当前中医临床已经突破了传统的“望、闻、问、切”四诊的范围，吸取了现代科技成果，使中医在诊治本病方面正向量化发展。1.2 “肾主生殖”与现代医学理论相关的研究概况“肾主生殖”与现代医学理论相关的研究内容，包括与生殖内分泌的关系、与精液的关系、与睾丸组织形态的关系、与免疫系统的关系、与微量元素的关系等五个方面，可知中医“肾主生殖”与现代医学理论的许多层面相关，因此必须全面地认识中医“肾主生殖”在现代医学理论中的内涵。1.3生精细胞凋亡的研究概况在男性生殖领域对生精细胞凋亡的研究是一个较为热门也较为前沿的课题。近20年的有关资料显示，生精细胞凋亡具有年龄性、阶段性、种族性的特点，以及各种生殖激素的变化可以影响生精细胞的凋亡，并且由于动物年龄的不同，同样的生殖激素变化，其生精细胞凋亡的情况不同，另外支持细胞和间质细胞的改变也可以影响生精细胞的凋亡。对其调节机制的研究发现：一些细胞因子、生长因子以及细胞浆蛋白等通常以调节凋亡过程或在凋亡细胞中表达两种方式参与凋亡发生；另外，光照、温度、超声波、锌、镉、输精管结扎、化学物质、电离辐射等因素对生精细胞凋亡均有影响。中医药与生精细胞凋亡的研究才刚刚起步,但随着现代分子生物学技术的发展所提供的更科学、更现代化的研究手段和方法，生精细胞凋亡对阐明中医“肾主生殖”理论的实质，促进中医药诊治男性不育症的现代化，无疑提供了新的思路，新的契入点。2实验研究2.1房劳法导致雄性小鼠生精障碍模型的建立参照朱传义、李震等方法，将健康雌性小鼠皮下注射复方己酸孕酮0.05ml，3天左右以阴道涂片检查角化细胞程度，证实其进入动情期，用来诱导房事过度。每只房劳组雄鼠与5只雌鼠合笼，每日检查笼中阴道栓，每隔5日依次互相更换雌鼠。正常对照组除不与雌鼠合笼外，其余条件相同。持续8周后，将各组雄鼠处死取材测定指标。本实验主要是观察房劳生精障碍小鼠睾丸生精细胞的凋亡情况,采用了流式细胞测定仪检测睾丸组织亚<WP=5>单倍体DNA含量和琼脂糖凝胶电泳两种方法进行测定。流式法参照聂振英等的方法:迅速将左侧睾丸的1/2切下放入平皿，加入少量生理盐水，用剪刀将睾丸组织剪至匀浆状，用吸管吸取组织匀浆滴入5ml试管内，加入5%胃蛋白酶消化液1ml，恒温（37℃）水浴消化40分钟，消化期间要间断振荡或吹打，然后用50目尼龙网过滤，离心沉淀1500prm5分钟，再用盐水洗三次，每次以短时低速离心沉淀（500-800prm）5分钟，去除细胞碎片，以300目尼龙网过滤去细胞团块，70%乙醇固定，置0-4℃冰箱12小时，离心弃去固定液，加入3mlPBS液重悬5分钟，400目尼龙网过滤1次，500-1000r/min，弃去PBS液；用1mlPI染液，4℃避光保存30分钟；然后通过FACS420型流式细胞仪进行检测。琼脂糖凝胶电泳法分为睾丸组织DNA的提取和电泳两步,睾丸组织DNA的提取使用组织DNA提取试剂盒,将DNA提取后,便可进行琼脂糖凝胶电泳及溴化乙锭染色观察各组电泳情况.结果显示房劳组生精细胞凋亡率明显高于对照组、琼脂糖凝胶电泳呈特殊的梯形状，说明房劳生可导致生精细胞凋亡增加，其发生机制与房劳后T水平降低从而引发生精细胞凋亡的异常升高有关。2.2 补肾生精汤对房劳小鼠生精障碍的影响观察根据房劳伤肾耗精及“男子以精为主”、“种子之法，男必先养其精”的中医理论，结合导师董连荣教授多年临床及实验研究成果，观察补肾生精汤对房劳肾虚生精障碍模型的影响。补肾生精汤系由“七宝美髯丹”加减化裁而成，“七宝美髯丹”据说由明代邵应节创制，《医方集解》载有“明嘉靖间，方士邵应节进此方，世宗服之，连生皇子，遂盛行于世。”刘艳芳等认为精子的生成依赖于肾阴的滋润和肾阳的温煦，而本方有益肾填精、平补阴阳之效，故临床常被加减治疗男性不育。方法：将房劳模型组小鼠16只，随机分成房劳治疗组和房劳自然恢复组，共两组，每组8只。治疗组给予补肾生精汤26mg/kg/d灌胃。自然恢复组给予蒸馏水26mg/kg/d 灌胃。共30天，然后处死动物、测定指标。结果显示房劳治疗组用药后睾丸间质细胞基本接近正常、T明显升高、生精细胞凋亡得到抑制，说明本方能够提高T含量、抑制生精细胞凋亡，从而改变睾丸生精状况。本实验结果表明，中医“肾主生殖”理论在雄更多还原

【Abstract】 "The Shen (kidney) dominating reproduction" is an important viewpoint in modern medicine territory and traditional medicine territory. Based on and guided by this theory, We carried out this study "The study of effect of BSSJT (Bu Shen Sheng Jing Tang) on Fang Lao male rat sperm cells obstruction" Our study is also guided by the Chinese traditional medicine theory "Fang Lao hurting Shen(kidney)". We made two models: NOMAL group and FANG LAO group, We also observe the effect of BSSJT treating the Latter group.This paper divided into two parts: theoretical part and experimental part.In the former, We collected and analyzed a large of materials between "The Shen(kidney) dominating reproduction" and modern medicine. Including the relation of sperm, morphology, minimum element, environment of the sperm and so on. Through this means, We find our study relate to modern medicine and modern technology. At the same time, we find the study had already broke the traditional method, and related to modern medicine in different positions and different levels. So we must treat it with developing and overall view. As we know, in the sum, there are three kinds method to treat the males sterility: one is according to Chinese Traditional medicine; the second is according to the result of the modern experimental; the last combined with the two former. Based on this reason, we should recognize the importance of the experiment.In order to release the essence of "The Shen(kidney)dominating reproduction" we engage in experimental study, the method and results as follows:Experiments 1 made two groups model, one is NOMAL group; the other is FANG LAO group the male rats have excessive sexual intercourse with sexual aroused female rats for 8 weeks in order to make the apoptosis model. Comparing two models, we find that FANG LAO group is obviously worse than the NOMAL group.Experiments 2: the study of effect of BSSJT, we made the FANG LAO model group into two groups: FANG LAO TREATMENT group and FANG LAO BEING RENEWED NATRUALY group. The former group’s male rats are pumped BSSJT 26mg/kg/d into stomach for 30 days; the latter group are given water 26mg/kg/d into stomach for the same days, then killed the animals. Comparing two models, we find that TREATMENT group is better than the BEING RENEWED NATRUALY group.A great variety of chemicals and environmental conditions can effect the result of the experimental. The more further of the study the more deeper of the recognize to the theory of "The Shen(kidney )dominating the reproduction".更多还原