本文選題：Ki67 + SCF��； 參考：《北京中醫(yī)藥大學(xué)》2016年博士論文

【摘要】：近年來,隨著人們生活水平的普遍提高,傳統(tǒng)的生活方式發(fā)生了巨大的改變。諸如精神壓抑、暴飲暴食、長期熬夜等因素直接導(dǎo)致男性精子質(zhì)量水平普遍下降。如何有效提高男性生育能力成為當(dāng)下的熱點(diǎn)話題。但是目前西醫(yī)除去人工授精、試管嬰兒等輔助生殖技術(shù)以及感染、梗阻等病因明確的疾病,臨床上尚無有效解決男性不育癥的治療方法。同時(shí),研究表明輔助生殖技術(shù)雖然可以解決部分患者的生育問題,但是其遠(yuǎn)期影響尚未可知。因此,廣大臨床醫(yī)生將目光轉(zhuǎn)向中醫(yī)藥對(duì)于男性不育癥的診治。本病的中醫(yī)藥治療方法歷史悠久,基礎(chǔ)堅(jiān)實(shí),且臨床療效明顯。這種情況下,如何充分挖掘本病的中醫(yī)藥特色療法已經(jīng)成為一個(gè)重要的課題。本文首先對(duì)左歸丸治療腎陰虧虛型少弱精子癥的臨床療效進(jìn)行系統(tǒng)評(píng)價(jià)；然后探索使用雷公藤多苷制備少弱精子癥大鼠模型,研究其用藥劑量及時(shí)間的最佳組合,制備相對(duì)合理的少弱精子癥模型；之后設(shè)計(jì)保護(hù)實(shí)驗(yàn)和治療實(shí)驗(yàn),每個(gè)實(shí)驗(yàn)包含正常組、模型組以及治療組,綜合評(píng)價(jià)左歸丸對(duì)少弱精子型模型大鼠臟器系數(shù)、精液質(zhì)量、性激素水平、附睪肉毒堿、抑制素B水平的影響,從藥效學(xué)角度對(duì)左歸丸的治療效果進(jìn)行評(píng)價(jià)；最后,采用免疫組織化學(xué)、western blot以及Real-Time PCR檢測技術(shù),對(duì)保護(hù)實(shí)驗(yàn)以及治療實(shí)驗(yàn)中各組睪丸組織Bax、Bal-2、SCF、 Ki67蛋白及mRNA表達(dá)水平進(jìn)行檢測,對(duì)左歸丸可能的抗凋亡及促增殖的作用機(jī)制進(jìn)行定性及定量分析。第一部分左歸丸治療少弱精子型男性不育癥的臨床研究目的：研究左歸丸對(duì)腎陰虧虛型少弱精子癥患者的臨床療效。方法：因本病尚無合適的陽性對(duì)照藥物,故采用自身前后對(duì)照進(jìn)行臨床觀察。本方法的治療周期為90天,每日給予42例入組的腎陰虧虛型少弱精子癥患者左歸丸中藥配方顆粒劑進(jìn)行治療�；颊哂盟幥凹敖Y(jié)束后分別進(jìn)行1次精液常規(guī)檢查；研究開始后,每30天對(duì)入組患者進(jìn)行隨訪一次,隨訪時(shí)對(duì)患者中醫(yī)證候進(jìn)行評(píng)分并嚴(yán)格記錄合并用藥和不良事件,若出現(xiàn)不良反應(yīng)報(bào)告則及時(shí)向上級(jí)醫(yī)師匯報(bào),并進(jìn)行相關(guān)處理。同時(shí)在研究期間詳細(xì)記錄患者配偶懷孕情況。系統(tǒng)評(píng)價(jià)左歸丸對(duì)于腎陰虧虛型少弱精子癥患者精液質(zhì)量各項(xiàng)指標(biāo)以及中醫(yī)證候的改善情況,綜合探討其臨床療效。結(jié)果：與治療前相比,使用左歸丸進(jìn)行治療后精液量、液化狀態(tài)、精子總活動(dòng)力、a級(jí)精子百分率、非前向運(yùn)動(dòng)精子百分率、b級(jí)精子百分率、精子濃度、精子總數(shù)等指標(biāo)均有明顯提高,差異性顯著,具有統(tǒng)計(jì)學(xué)意義(p0.01)；中醫(yī)證候總評(píng)分以及腎陰虧虛相關(guān)中醫(yī)癥候評(píng)分隨著隨訪次數(shù)的增加以及時(shí)間的延長,呈明顯的下降趨勢,差異性顯著,具有明顯的統(tǒng)計(jì)學(xué)意義(p0.01)；左歸丸配方顆粒安全性較高,整個(gè)研究過程中未見不良反應(yīng)報(bào)告。結(jié)論：左歸丸能夠安全有效的提高腎陰虧虛型少弱精子癥患者的精子質(zhì)量各項(xiàng)指標(biāo),提高男性的生育能力,同時(shí)能夠改善患者的中醫(yī)相關(guān)癥狀,特別是對(duì)腎陰虧虛相關(guān)癥狀改善效果顯著。第二部分雷公藤多苷誘導(dǎo)少弱精子型大鼠模型的研究目的：研究多種雷公藤多苷用藥劑量以及造模時(shí)間的組合,并觀察停止用藥后各組大鼠自身恢復(fù)情況,制備少弱精子型大鼠模型。方法：將96只雄性SD大鼠隨機(jī)分為正常組、雷公藤多苷大劑量組、中劑量組以及小劑量組。其中正常組采用去離子水進(jìn)行干預(yù)；雷公藤多苷各劑量組分別采用50 mg/ (Kg·d)、40 mg/(Kg·d)、30mg/(Kg·d)的雷公藤多苷進(jìn)行干預(yù)。給藥第3周及第4周后,每組中選取SD大鼠4只取材；第4周取材后,每組選取4只按照上述實(shí)驗(yàn)方案繼續(xù)喂養(yǎng),并在第5周進(jìn)行取材,剩余的大鼠在第4周給藥完成后就停止進(jìn)行干預(yù),并在實(shí)驗(yàn)開始后的第6、7、8周,每周每組取材4只實(shí)驗(yàn)大鼠,觀察其生殖相關(guān)功能的恢復(fù)情況并進(jìn)行相關(guān)記錄。結(jié)果：造模期間(第3周至第5周),一般狀態(tài)、生殖器官臟器系數(shù)、精液質(zhì)量各項(xiàng)指標(biāo)隨著雷公藤多苷用藥劑量增大以及用藥時(shí)間的延長呈顯著的降低趨勢；睪丸組織病理損傷程度隨著雷公藤多苷用藥劑量增大以及用藥時(shí)間的延長呈顯著的增高趨勢。停止用藥后(第6周至第8周),雷公藤多苷小劑量組上述指標(biāo)逐漸恢復(fù)至正常組水平,未見明顯差異性；中劑量組上述各項(xiàng)指標(biāo)略有恢復(fù),但與正常組相比仍有較大差距；大劑量組生殖功能損傷未見明顯恢復(fù)。結(jié)論：綜合考慮造模時(shí)間、經(jīng)濟(jì)成本以及大鼠的自我恢復(fù)情況,采用40mg/(Kg·d)的雷公藤多苷干預(yù)4周后可以得到少弱精子型大鼠模型。第三部分左歸丸對(duì)雷公藤多苷誘導(dǎo)的少弱精子型模型大鼠的藥效學(xué)研究目的：觀察左歸丸對(duì)少弱精子型模型大鼠一般狀態(tài)、臟器系數(shù)、精液質(zhì)量、睪丸組織病理改變、性激素水平、附睪肉毒堿、抑制素B的影響。方法：將72只雄性SD大鼠隨機(jī)平均分為治療實(shí)驗(yàn)和保護(hù)實(shí)驗(yàn)；每個(gè)實(shí)驗(yàn)又隨機(jī)分為正常組、模型組、左歸丸組,每組12只大鼠。在保護(hù)實(shí)驗(yàn)中,正常組采用去離子水進(jìn)行干預(yù),共計(jì)4周；模型組采用40mg/(Kg·d)的雷公藤多苷進(jìn)行造模,共計(jì)4周；左歸丸治療組采用40mg/(Kg·d)的雷公藤多苷進(jìn)行造模,同時(shí)采用6g/(Kg·d)的左歸丸混懸液(相當(dāng)于人體用藥20倍)進(jìn)行保護(hù)性干預(yù)4周。治療實(shí)驗(yàn)中,正常對(duì)照組采用去離子水進(jìn)行干預(yù)8周；模型組實(shí)驗(yàn)前4周采用40mg/(Kg·d)的雷公藤多苷進(jìn)行造模,實(shí)驗(yàn)第5至8周采用去離子水進(jìn)行干預(yù)；左歸丸治療組實(shí)驗(yàn)前4周采用40mg/ (Kg·d)的雷公藤多苷進(jìn)行造模,實(shí)驗(yàn)第5至8周,采用6g/(Kg·d)的左歸丸混懸液(相當(dāng)于人體用藥20倍)進(jìn)行干預(yù)。結(jié)果：在保護(hù)試驗(yàn)中,左歸丸組與模型組相比,在大鼠睪丸臟器系數(shù)、附睪臟器系數(shù)、精囊腺臟器系數(shù)、精液質(zhì)量各項(xiàng)指標(biāo)、睪酮水平、血清抑制素B方面具有顯著的差異性,具有明顯的統(tǒng)計(jì)學(xué)意義(P0.05或P0.01)；在治療試驗(yàn)中,與雷公藤多苷模型組相比,左歸丸組在大鼠睪丸臟器系數(shù)、附睪臟器系數(shù)、前列腺臟器系數(shù)、精液質(zhì)量各項(xiàng)指標(biāo)、睪酮水平、血清抑制素B具有明顯的差異性,具有明顯的統(tǒng)計(jì)學(xué)意義(P0.05或P0.01)；在保護(hù)實(shí)驗(yàn)與治療實(shí)驗(yàn)兩個(gè)實(shí)驗(yàn)中,左歸丸組大鼠一般狀態(tài)、睪丸組織病理損傷程度與模型組相比都明顯減輕。結(jié)論：左歸丸可以有效改善模型大鼠一般狀態(tài)、生殖器官臟器系數(shù)、精液質(zhì)量、睪酮水平、血清抑制素B水平,具有明顯的保護(hù)及治療作用,效果明顯。第四部分左歸丸對(duì)少弱精子型模型大鼠的抗細(xì)胞凋亡機(jī)制及SCT、Ki67表達(dá)的研究目的：觀察左歸丸對(duì)少弱精子型模型大鼠細(xì)胞凋亡調(diào)控因子Bax、Bcl-2蛋白以及mRNA的影響；同時(shí)觀察左歸丸對(duì)少弱精子型模型大鼠SCF、Ki67蛋白及mRNA表達(dá)的影響方法：分別采用免疫組織化學(xué)、western blot以及Real-Time PCR技術(shù),對(duì)上述左歸丸藥效學(xué)研究中的保護(hù)實(shí)驗(yàn)和治療實(shí)驗(yàn)中全部大鼠睪丸組織進(jìn)行Bax、Bcl-2、SCF、Ki67蛋白及mRNA表達(dá)的檢測。結(jié)果：保護(hù)實(shí)驗(yàn),免疫組織化學(xué)檢測中,與模型組相比較,左歸丸組Bax、Bcl-2、 SCF、Ki67蛋白具有明顯的差異性,具有顯著的統(tǒng)計(jì)學(xué)意義(P0.05或P0.01)；western blot檢測中,左歸丸組與模型組相比較,Bax、Bcl-2、SCF蛋白具有差異性,P0.05,具有統(tǒng)計(jì)學(xué)意義；Real-Time PCR檢測中,與模型組相比較,左歸丸組Bax、Bcl-2、SCF、 Ki67 mRNA具有明顯的差異性,P0.05或P0.01,具有統(tǒng)計(jì)學(xué)意義。治療實(shí)驗(yàn),免疫組織化學(xué)檢測中,與模型組相比較,左歸丸組Bcl-2、SCF、Ki67蛋白具有明顯的差異性,P0.05或P0.01,具有統(tǒng)計(jì)學(xué)意義；western blot檢測中,左歸丸組與模型組相比處于相同水平,未見明顯差異性,但是Bcl-2以及SCF表達(dá)水平明顯高于模型組；Real-Time PCR檢測中,左歸丸組與模型組相比較,SCF、Ki67mRNA表達(dá)水平明顯提高,具有明顯的差異性,P0.05或P0.01,具有統(tǒng)計(jì)學(xué)意義；結(jié)論：左歸丸可以有效調(diào)控模型大鼠Bax、Bcl-2、CF、Ki67蛋白及其mRNA的表達(dá),具有明顯的抑制生精細(xì)胞凋亡、促進(jìn)生精細(xì)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.
【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】：2016
【分類號(hào)】：R277.5
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本文編號(hào)：1952494