發(fā)布時(shí)間：2018-06-24 01:02

  本文選題：補(bǔ)腎填精 + 男性不育　； 參考：《北京中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：由于生活方式和環(huán)境變化、生育年齡推遲等因素,男性不育的發(fā)病率不斷增高,大部分不育患者無(wú)法找到明確病因,僅表現(xiàn)為精子質(zhì)量下降,其中精子總數(shù)減少(少精子癥)最容易導(dǎo)致男性不育。少精子癥的病因目前尚不明確,研究認(rèn)為生精細(xì)胞和精子凋亡可導(dǎo)致精子生成減少。中醫(yī)認(rèn)為男性不育少精子癥的主要病機(jī)是腎精虧虛,補(bǔ)腎填精法是基本治法�；A(chǔ)實(shí)驗(yàn)和臨床研究均已經(jīng)證實(shí)五子衍宗丸代表的補(bǔ)腎填精法改善生精功能的機(jī)制之一是抑制生精細(xì)胞和精子凋亡,文獻(xiàn)研究已表明支持細(xì)胞骨架破壞可以導(dǎo)致生精細(xì)胞凋亡和生精功能受損。筆者前期做過(guò)一些研究,把具有生殖毒性的雷公藤多甙與TM4支持細(xì)胞體外共培養(yǎng)后,發(fā)現(xiàn)支持細(xì)胞骨架出現(xiàn)斷裂現(xiàn)象,骨架蛋白vimentin表達(dá)減少;而動(dòng)物實(shí)驗(yàn)研究發(fā)現(xiàn),五子衍宗丸對(duì)雷公藤多甙造成的生精功能損傷有改善作用。基于文獻(xiàn)研究和前期研究,提出假設(shè)"五子衍宗丸代表的補(bǔ)腎填精法可以通過(guò)修復(fù)支持細(xì)胞骨架,抑制生精細(xì)胞和精子凋亡來(lái)改善生精",為了驗(yàn)證該假設(shè),設(shè)計(jì)了以下臨床和動(dòng)物實(shí)驗(yàn)研究:1臨床部分目的:研究以五子衍宗丸為代表的補(bǔ)腎填精法治療腎精虧虛型男性不育少精子癥的效果與機(jī)制。方法:因?yàn)槟行圆挥腥狈τ忻鞔_循證醫(yī)學(xué)依據(jù)的公認(rèn)治療藥物,而五子衍宗丸是療效最被認(rèn)可的藥物,在臨床研究中常被用作對(duì)照藥物,因此本次研究未設(shè)立對(duì)照組。選擇50例腎精虧虛型男性不育少精子癥患者,予五子衍宗口服液治療90天(每日2次,每次10ml),治療前及治療90天后分別檢測(cè)精子質(zhì)量(一周內(nèi)連續(xù)檢測(cè)2次,結(jié)果均符合少精子癥的診斷標(biāo)準(zhǔn)才能入組,選擇精子總數(shù)較多的檢測(cè)結(jié)果進(jìn)行記錄),并通過(guò)Western Blot法分別檢測(cè)精子凋亡。每30天進(jìn)行一次隨訪(fǎng),治療前及每次隨訪(fǎng)時(shí)進(jìn)行腎精虧虛證候評(píng)分。觀(guān)察期間嚴(yán)格記錄不良事件,同時(shí)詳細(xì)記錄配偶妊娠情況。結(jié)果:觀(guān)察期間,4例患者配偶妊娠。76.7%的患者治療后精子總數(shù)有明顯提高。與治療前相比,治療后精子濃度、精子總數(shù)、精子總活力、前向運(yùn)動(dòng)精子百分率及a級(jí)精子百分率均有顯著的提高,差異有統(tǒng)計(jì)學(xué)意義(P0.05);而精液量雖有提高,但差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。與治療前相比,治療后精子凋亡指標(biāo)Caspase3的表達(dá)下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。腎精虧虛證候評(píng)分隨著隨訪(fǎng)次數(shù)的增加呈下降趨勢(shì),差異有統(tǒng)計(jì)學(xué)意義(P0.01),其中多數(shù)具體癥狀的評(píng)分也整體上呈下降趨勢(shì),差異有統(tǒng)計(jì)學(xué)意義(P0.01)。分組統(tǒng)計(jì)顯示,20～29歲組、30～39歲組和40～歲組(最大51歲)的精子總數(shù)治療后均有提高,差異有統(tǒng)計(jì)學(xué)意義(P0.05);體重正常組和過(guò)重組的精子總數(shù)治療后均有顯著提高,差異有統(tǒng)計(jì)學(xué)意義(P0.01),而肥胖組的精子總數(shù)治療前后無(wú)明顯差異;小睪丸組和睪丸體積正常組的精子總數(shù)治療后均有顯著提高,差異有統(tǒng)計(jì)學(xué)意義(P0.01);弱精子癥組和前向運(yùn)動(dòng)精子百分率正常組的精子總數(shù)治療后均有顯著提高,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。整個(gè)觀(guān)察過(guò)程中未見(jiàn)不良反應(yīng)報(bào)告。結(jié)論:以五子衍宗丸為代表的補(bǔ)腎填精法能夠安全有效的提高腎精虧虛型男性不育少精子癥患者的精子質(zhì)量,提高男性的生育能力,同時(shí)可以緩解患者的腎精虧虛癥狀。補(bǔ)腎填精法提高精子總數(shù)的機(jī)制之一可能是抑制精子凋亡。2動(dòng)物實(shí)驗(yàn)部分目的:研究五子衍宗丸改善腎精虧虛少精子癥模型大鼠精子質(zhì)量的效果與機(jī)制。方法:將25只SD雄性大鼠隨機(jī)平均分為空白組、模型組和五子衍宗丸低、中、高劑量組,空白組正常喂養(yǎng),模型組予雷公藤多甙(20mg/kg)灌胃,五子衍宗丸三組除了予雷公藤多甙灌胃,分別予不同劑量五子衍宗丸(0.5,1.0,2.0g/kg)灌胃。30天后,取附睪組織檢測(cè)大鼠精子總數(shù)、前向運(yùn)動(dòng)精子百分率、精子總活力等指標(biāo),Western Blot法檢測(cè)睪丸組織波形蛋白(vimentin)、αα微管蛋白的表達(dá),免疫組化法檢測(cè)睪丸支持細(xì)胞中vimentin、αα微管蛋白的表達(dá),免疫組化法和Tunel法分別檢測(cè)生精細(xì)胞的PCNA表達(dá)和凋亡。結(jié)果:與模型組相比,五子衍宗丸中、高劑量組的精子總數(shù)、前向運(yùn)動(dòng)精子百分率、精子總活力均顯著提高,差異有統(tǒng)計(jì)學(xué)意義(P0.01);五子衍宗丸高劑量組睪丸組織中vimentin的表達(dá)增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05),五子衍宗丸低、中、高劑量組睪丸組織中αα微管蛋白的表達(dá)均增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05);五子衍宗丸低、中、高劑量組睪丸支持細(xì)胞中vimentin的表達(dá)均顯著增加,差異有統(tǒng)計(jì)學(xué)意義(P0.01),五子衍宗丸低、中、高劑量組睪丸支持細(xì)胞中αα微管蛋白的表達(dá)均顯著增加,差異有統(tǒng)計(jì)學(xué)意義(P0.01);五子衍宗丸低、中、高劑量組生精細(xì)胞的凋亡率均有下降,差異有統(tǒng)計(jì)學(xué)意義(p0.05);五子衍宗丸低、中、高劑量組生精細(xì)胞的PCNA表達(dá)均有所提高,但差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:五子衍宗丸可以提高腎精虧虛少精子癥模型大鼠的精子質(zhì)量,機(jī)制之一可能是修復(fù)支持細(xì)胞骨架蛋白,抑制生精細(xì)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.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R277.5

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