發(fā)布時(shí)間：2018-03-03 15:50

  本文選題：山羊　切入點(diǎn)：睪丸間質(zhì)細(xì)胞　出處：《西北農(nóng)林科技大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：睪丸間質(zhì)細(xì)胞(interstitial cell)又稱leydig cell(LCs),分布于曲細(xì)精管間的疏松的結(jié)締組織之間,其數(shù)量占睪丸中細(xì)胞總數(shù)的2%~4%,主要功能是合成與分泌雄激素,參與或促進(jìn)精子發(fā)生過程的各種生理功能。很多生理、內(nèi)分泌、藥理或毒理因素等可能干擾睪丸間質(zhì)細(xì)胞的正常功能,導(dǎo)致睪酮分泌異常進(jìn)而導(dǎo)致雄性哺乳動物生殖能力下降或喪失。雖然可以通過原代睪丸間質(zhì)細(xì)胞研究睪丸間質(zhì)細(xì)胞的調(diào)節(jié)及睪酮分泌等功能,但其有限的增殖能力限制了很多研究的開展,特別是各種因素對該細(xì)胞的長期效應(yīng)性研究。本實(shí)驗(yàn)使用屠宰場健康山羊獲得的睪丸組織進(jìn)行間質(zhì)細(xì)胞分離純化培養(yǎng),向細(xì)胞內(nèi)穩(wěn)定轉(zhuǎn)染含有人端粒酶逆轉(zhuǎn)錄酶亞基(human telomerase reverse transcriptase,hTERT)的表達(dá)載體,并進(jìn)行細(xì)胞篩選,以期獲得永生化的山羊睪丸間質(zhì)細(xì)胞系,為后續(xù)的研究奠定基礎(chǔ)。獲得主要結(jié)果如下:1.使用膠原酶消化培養(yǎng)法,2.5 mg/mL膠原酶消化1 h,過濾、低速離心、自然沉降獲取單個(gè)細(xì)胞,培養(yǎng)細(xì)胞為纖維狀,形態(tài)一致,有典型的胞間聯(lián)系,細(xì)胞界限清楚,連接緊密,形態(tài)均勻,增殖旺盛,睪丸間質(zhì)細(xì)胞特異的3β-HSD染色成陽性。2.使用脂質(zhì)體轉(zhuǎn)染法,將質(zhì)粒pCI-neo-hTERT轉(zhuǎn)入第4代的山羊睪丸間質(zhì)細(xì)胞,經(jīng)濃度為500μg/mL的G-418篩選后,挑選出耐藥細(xì)胞克隆擴(kuò)大培養(yǎng),所獲細(xì)胞狀態(tài)良好,生長增殖旺盛,傳至85代。使用RT-PCR和免疫熒光對30代、50代hTERT-GLCs測定,hTERT檢測均呈陽性,而對照的原代睪丸間質(zhì)細(xì)胞呈陰性,證明hTERT在hTERT-GLCs中穩(wěn)定表達(dá),說明外源基因己成功轉(zhuǎn)染到睪丸間質(zhì)細(xì)胞中并表達(dá)。3.對轉(zhuǎn)染后第30代、50代的山羊睪丸間質(zhì)細(xì)胞進(jìn)行RT-PCR鑒定證明hTERT-GLCs可穩(wěn)定表達(dá)其合成睪酮通路上的關(guān)鍵酶和受體:StAR、P450scc、3β-HSD、LH-R,且ELISA檢測證明其睪酮分泌能力和原代睪丸間質(zhì)細(xì)胞無明顯差異,證明其依然具有正常睪酮分泌能力。4.對該細(xì)胞系進(jìn)行生長曲線測定和細(xì)胞周期檢測,相比于原代睪丸間質(zhì)細(xì)胞,該細(xì)胞系具有更快的生長速率和更長的S期;核型分析檢測,其結(jié)果呈現(xiàn)山羊染色體正常的核型(2n=60),表明其染色體正常;端粒長度檢測,其不同代次間端粒長度穩(wěn)定,且長度和Hela細(xì)胞相比沒有明顯差異,表明其端粒酶表達(dá)強(qiáng)度趨于穩(wěn)定;軟瓊脂實(shí)驗(yàn)檢測,該細(xì)胞系軟瓊脂中不能形成細(xì)胞克隆,表明該細(xì)胞依然保留貼壁生長和細(xì)胞通訊依賴性,沒有致瘤性。綜上所述,本實(shí)驗(yàn)分離純化山羊睪丸間質(zhì)細(xì)胞,將人端粒酶逆轉(zhuǎn)錄酶基因hTERT成功導(dǎo)入細(xì)胞基因組中,誘導(dǎo)細(xì)胞端粒酶的激活,阻止端粒縮短并維持足夠且穩(wěn)定的長度,最終使細(xì)胞實(shí)現(xiàn)永生化,成功構(gòu)建山羊睪丸間質(zhì)細(xì)胞系,為后續(xù)研究奠定了基礎(chǔ)。
[Abstract]:Interstitial cell of testis, also known as leydig cell, distributes between loose connective tissue between seminiferous tubules. Its number accounts for 2% of the total number of cells in testis. Its main function is to synthesize and secrete androgen. Participate in or promote the various physiological functions of spermatogenesis. Many physiological, endocrine, pharmacological or toxicological factors may interfere with the normal function of testicular interstitial cells, This leads to abnormal testosterone secretion, which leads to the reduction or loss of reproductive capacity in male mammals. Although the regulation of testicular stromal cells and testosterone secretion can be studied through primary testicular stromal cells, However, its limited proliferative ability limits the development of many studies, especially the long-term effects of various factors on the cells. In this study, the mesenchymal cells were isolated and cultured from the testis of slaughterhouse healthy goats. The expression vector containing human telomerase reverse transcriptasehTERT was stably transfected into the cells and screened to obtain immortalized goat testicular stromal cell lines. The main results are as follows: 1. Using collagenase digestion and culture method to digest for 1 hour, filter, centrifuge at low speed, natural sedimentation to obtain a single cell, the cultured cells are fibrous, and the morphology is the same. There is typical intercellular connection, the cell line is clear, the connection is close, the shape is uniform, the proliferation is exuberant, the specific 3 尾 -HSD staining of testicular interstitial cells is positive .2.Using the liposome transfection method, the plasmid pCI-neo-hTERT is transferred into the 4th generation goat testicular interstitial cells. After screening with G-418 at the concentration of 500 渭 g / mL, the drug-resistant cells were selected for extended culture. The cells were in good condition and proliferative, and passed to 85th generation. RT-PCR and immunofluorescence were used to detect hTERT in hTERT-GLCs of 30 ~ 50 generations. In contrast, the primary stromal cells of testis were negative, which proved that hTERT was stably expressed in hTERT-GLCs. The results showed that exogenous gene had been successfully transfected into testicular stromal cells and expressed .3.The RT-PCR analysis of goat testicular stromal cells at passage 30 and 50 showed that hTERT-GLCs could stably express the key enzyme and receptor on the synthesis of testosterone pathway, the receptor: Starn P450 sccf3 尾 -HSDN LH-R. ELISA showed that there was no significant difference between testosterone secretion and primary testicular stromal cells. The results showed that the cell line still had normal testosterone secretion ability. 4. The cell line had faster growth rate and longer S phase than primary testicular stromal cells, and the karyotype analysis was used to detect the growth curve and cell cycle of the cell line, and the results showed that the cell line had higher growth rate and longer S phase than the primary testicular stromal cells. The results showed that the karyotype of goat chromosome was normal, indicating that the chromosome was normal, the telomere length was stable in different generations, and there was no significant difference between the length of telomere and Hela cells, which indicated that the intensity of telomerase expression tended to be stable. Soft Agar assay showed that the cell clone could not be formed in soft Agar, indicating that the cell remained dependent on adherent growth and cell communication and had no tumorigenicity. In conclusion, goat testicular mesenchymal cells were isolated and purified. The human telomerase reverse transcriptase gene (hTERT) was successfully introduced into the cell genome, which induced the activation of telomerase, prevented telomere shortening and maintained sufficient and stable length, finally immortalized the cells and successfully constructed goat testicular stromal cell line. It lays a foundation for further study.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S827

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本文編號：1561681