“续断种子方”系列实验研究2：对精子发生发育的影响

续断种子方对无精子症模型小鼠精子发生c-kit/CFTR的调控

王权胜¹,唐振宇²，唐乾利³，代波¹庞秋华¹陆海旺¹

（1广西中医药大学第一附属医院男科 广西南宁 530023

2广西中医药大学广西南宁 530001

3右江民族医学院 广西百色 533000）

基金项目：广西自然科学基金项目：“续断种子方”对大鼠精子发生发育过程中c-kit/CFTR的调控，项目编号2013GXNSFAA019130

1作者简介：王权胜(1975年5月)，男，汉族，广西玉林人，副主任医师，博士，研究方向：男性不育，E-Mail：415811600@qq.com，联系电话15296543026

2 通讯作者 唐振宇 510437052@qq.com

[摘要]:目的 观察续断种子方对无精子症模型小鼠睾丸精子发生发育过程中c-kit/CFTR表达的影响，探索中药干预精子发生发育的可能起作用的路径。方法按照随机数字表法分为对照组，模型组，麒麟丸组，续断种子方高、中、低剂量组。采用白消安和环磷酰胺建立无精子症小鼠模型。各治疗组给予相应药物灌胃,对照组和模型组小鼠给予等体积生理盐水,每日1 次,连续8周。检测小鼠附睾的精子质量、睾丸组织显微超微结构、睾丸组织c-kit/CFTR蛋白的表达。结果 模型组偶见散在精子，睾丸生精上皮不同程度破坏，精原细胞、精母细胞线粒体空化明显，部分线粒体脊消失，核膜局部破损，c-kit/CFTR免疫组化棕黄色阳性表达的细胞散在；续断种子方高剂量组、麒麟丸组、对照组小鼠附睾精子浓度较高，睾丸生精上皮见精子、精子细胞、精母细胞，线粒体空化少，可见丰富的高尔基体、内质网结构，c-kit/CFTR免疫组化棕黄色阳性表达的细胞辉度大。结论续断种子方可以有效促进睾丸生精上皮的精子发生发育，增加精子的数量，达到“以通为用，和营生精”之效。

[关键词]：精子发生；模型小鼠；续断种子方；c-kit；CFTR；和营生精

Regulation ofXuduanzhongzifangon c-kit/CFTRDuring the Generating and Developing Processes of rats’ Sperm

Wang Quansheng1, Tang Zhenyu2, Tang Qianli3, Dai Bo1, Pang Qiuhua1, Lu Haiwang1

(1 Department of andrology of First Affiliated Hospital of Guangxi University of Chinese medicine, Nanning, Guangxi, 530023

2 Guangxi university of Chinese medicine, Nanning, Guangxi, 530001

3 Youjiang ethnic medical university, Baise, guangxi 533000)

[Abstract]: Objectiveto observe the impact of Xuduanzhongzifangon c-kit during the generating and developing processes of sperm of azoospermia rat models. Method Randomly divided the azoospermia rat models into control group, model group, qilinwangroup, high, middle and low dosage of Xuduanzhongzifanggroups. CBN and cyclophosphamide were used to establish the rat models of azoospermia. Each group was administered with corresponding drugs, the control group and model group were given equal volume of saline once a day for eight weeks. Indicators such as Sperm quality, microscopic ultrastructures and expression of c-kit protein of testicular tissues were tested. Results scattered sperms were occasionally seen in Model group, testicular seminiferous epithelium were damaged to varying degrees, obvious cavitation can be seen at the mitochondrias of spermatogonia and spermatocytes, some of the mitochondria ridges were disappeared, some of the nuclear membrane were damaged, scattered c-kit/CFTRbrown positive immunohistochemical expression cells were detected. Compared to the model group, a higher concentration of sperms were tested at the control group, qilinwangroup, high, middle and low dosage of Xuduanzhongzifanggroups; Sperm, sperm cells and spermatocytes were seen at testicular seminiferous epithelium, fewer cavitation were seen at the mitochondrias; Extensive Golgi apparatus and endoplasmic reticulum structure were detected; Greater cell luminance of c-kit /CFTR brown positive immunohistochemical expression cells were detected. Conclusion Xuduanzhongzifangcan effectively promote the generating and developing processes of sperms of testicular seminiferous epithelium and increase the sperm counts.Xuduanzhongzifanghas the effect of “tonifying the Spleen and Kidney, invigorating the circulation of blood and promote the spermatogenic process”.

Keywords:spermatogenesis; rat model;Xuduanzhongzifang; c-kit; invigorating the circulation of blood and promote the spermatogenic process

中医对男性不育症的治疗源远流长，积累了丰富的经验，但其治疗的作用机制尚未阐述清楚。通过文献研究和临床经验总结,我们认为“营气不从，肾精亏虚”是男性不育症的主要病因病机，治以续断种子方，前期临床观察确有一定疗效^[1]。本文通过建立无精子症小鼠模型，以续断种子方为被试因素，以模型小鼠左侧睾丸组织为受试对象，以麒麟丸为阳性参照，研究续断种子方对无精子症模型小鼠睾丸精子发生发育过程中c-kit/CFTR表达的影响，探索中药干预精子发生发育的可能起作用的路径。

1材料与方法

1.1 主要仪器及试剂

MACRO精子计数板和WLJY-9000型伟力彩色精子质量检测系统（北京伟力新世纪科技发展有限公司）；752紫外可见分光光度计（上海菁华科技仪器有限公司）；TDL-4ZB型低速自动平衡离心机（湖南星科科学仪器有限公司）；Biocell2010奥地利酶标仪。SP免疫组化试剂盒，一抗为兔抗小鼠c-kit多克隆抗体(Santa Cruz公司)，二抗为生物素标记的羊抗兔IgG(H+L)(Pierce公司)

1.2 实验动物及分组

120只清洁级 C5BL/6小鼠，6周-8周龄，24g～28g，由广西医科大学实验动物中心提供，动物许可证号SCXK(桂)2009-0002。适应性喂养一周。饲养过程中，小鼠均自由采食、饮水。每天换水，隔日换1次笼中的木屑垫料以保持清洁。环境温度20～25℃。按照随机数字表法分为正常对照组，模型组，续断种子方高、中、低剂量组，麒麟丸组，每组各20只。

1.3无精子症小鼠模型的建立

参考文献^[2]的造模方法，建立小鼠无精子症动物模型，造模方法：除正常对照组小鼠外，其余小鼠均采用白消安(Sigma产品)和环磷酰胺粉针剂(上海华联制药有限公司)，以0.9％生理盐水5ml稀释后，分别按白消安10 mg／kg和环磷酰胺120 mg／kg一次性腹腔内注射，同时正常对照组注射等量生理盐水后，各组小鼠正常喂养4周，4周后随机抽取模型组小鼠4只做病检，光学显微镜下见睾丸生精小管内精子、精子细胞以及精母细胞消失，仅见精原细胞，提示无精子症动物模型造模成功。

1.4给药方法

续断种子方（经验方，药物组成为：续断15g、骨碎补15g、杜仲15g、牛膝15g、菟丝子15g、枸杞子10g、女贞子10g、党参15g、白术10g、山药15g），采用单味中药配方颗粒（免煎中药，由江苏省江阴市天江药业有限公司生产，产品批号1203002）；麒麟丸（汕头市麒麟药业有限公司生产，批准文号：国药准字z10930034，批号：20130607）。