本文關(guān)鍵詞： 非免疫缺陷受體 精原干細(xì)胞 生殖細(xì)胞移植 精子發(fā)生 轉(zhuǎn)基因技術(shù)　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：雄性生殖細(xì)胞是指通過有性生殖繁衍后代的雄性生物生殖系統(tǒng)中能將遺傳信息傳遞給后代的一類細(xì)胞,其中精原干細(xì)胞是整個機(jī)體內(nèi)唯一貫穿一生,既可通過增殖維持自身數(shù)目穩(wěn)定又能定向分化為精子的成體干細(xì)胞;同時,精原干細(xì)胞在自發(fā)或誘導(dǎo)條件下,具有分化為三胚層的潛能,具有類似胚胎干細(xì)胞的特征而備受生物醫(yī)學(xué)、轉(zhuǎn)化醫(yī)學(xué)和畜牧業(yè)廣泛關(guān)注。目前對精原干細(xì)胞的研究主要集中在分子標(biāo)記的篩選、體外培養(yǎng)體系建立及維持、經(jīng)典及表觀遺傳修飾相關(guān)信號通路的研究、基于精原干細(xì)胞修飾的轉(zhuǎn)基因動物制作以及睪丸組織塊移植等領(lǐng)域,而這些研究中采用的免疫缺陷受體模型大大限制了精原干細(xì)胞的科研與應(yīng)用,特別是實際生產(chǎn)及其在畜牧行業(yè)的推廣;同時,基于精原干細(xì)胞顯微移植的轉(zhuǎn)基因動物制作具有一定的技術(shù)難度。因此,精原干細(xì)胞介導(dǎo)的簡單易行的轉(zhuǎn)基因動物制作策略亟需探討。本研究主要著力于探究睪丸組織及精原干細(xì)胞非免疫缺陷移植受體模型制作的方法,探索體內(nèi)簡單易行的雄性生殖細(xì)胞介導(dǎo)的轉(zhuǎn)基因技術(shù)的新方法。本研究獲得如下結(jié)果:1,本研究探究了不同濃度的腺嘌呤(200mg/kg,260mg/kg,320mg/kg和380mg/kg)對非免疫缺陷大鼠(Sprague-Dawley,SD大鼠)睪丸的損傷情況,通過相關(guān)生理指標(biāo)和睪丸切片觀察,候選得到精原干細(xì)胞移植受體在SD大鼠體內(nèi)的最佳的作用濃度(320mg/kg)。同時作為陽性對照,摸索了白消安(一種廣泛使用精原干細(xì)胞受體模型制備的藥物)在SD大鼠受體制作的合適作用濃度;從存活率、體重、睪丸指數(shù)及睪丸切片確定了腺嘌呤處理優(yōu)于白消安處理,可作為替代方案阻滯內(nèi)源性精子發(fā)生,為精原干細(xì)胞移植提供合適的微環(huán)境。同時,使用酶消化、差異貼壁及免疫磁珠分選等方法富集高純度小鼠精原干細(xì)胞(Lin28陽性率達(dá)到84%,THY-1陽性率達(dá)到92%),同時利用體外慢病毒感染修飾的方法標(biāo)記小鼠的精原干細(xì)胞,并移植到受體大鼠曲細(xì)精管內(nèi),首次檢測到非免疫缺陷受體內(nèi)異種移植完整的精子發(fā)生。本實驗也使用細(xì)胞系和分離的原代生殖細(xì)胞及支持細(xì)胞作為研究對象,研究腺嘌呤對其體外作用機(jī)理;使用TUNEL證實染色體中DNA發(fā)生斷裂,使用蛋白免疫印跡實驗檢測到了凋亡早期相關(guān)蛋白Caspase3和凋亡執(zhí)行蛋白PARP濃度依賴性表達(dá),初步探明了腺嘌呤通過誘導(dǎo)細(xì)胞凋亡對生殖細(xì)胞產(chǎn)生了特異的生殖毒性。2,本研究使用慢病毒體內(nèi)感染雄性生殖細(xì)胞的策略,獲得了超過67%的轉(zhuǎn)基因后裔,生殖能力正常,外源轉(zhuǎn)基因可穩(wěn)定遺傳給后代;同時也揭示了該策略與親本年齡及慢病毒注射部位沒有相關(guān)性(P0.05),這提示我們可以在大家畜中嘗試使用慢病毒注射睪丸間質(zhì)部位獲得轉(zhuǎn)基因后裔,簡化大家畜生產(chǎn)及育種中的繁瑣環(huán)節(jié)和技術(shù)難題。同時,也比較了不同慢病毒系統(tǒng)對睪丸內(nèi)精原干細(xì)胞感染能力,與慢病毒p CD513B-CMV-MCS-EF1相比,慢病毒p Lenti V-H1-MCS-CMV感染精原干細(xì)胞之后獲得的轉(zhuǎn)基因后代能正常表達(dá)外源轉(zhuǎn)基因片段;進(jìn)一步研究揭示不同慢病毒整合到子代基因組后,外源基因啟動子序列甲基化水平存在顯著差異:前者外源轉(zhuǎn)基因片段啟動子甲基化水平高達(dá)87.8%,而后者僅為45.5%。本研究為以后使用慢病毒載體制作轉(zhuǎn)基因動物特別是對于曲細(xì)精管移植難于實現(xiàn)的大家畜提供了新思路。3,本研究探索了使用非免疫缺陷小鼠(成年昆明小鼠)作為睪丸組織塊移植的受體,既可以完成同種不同品系間睪丸移植的精子發(fā)生,也首次檢測到異種睪丸組織塊移植后(大鼠睪丸移植到小鼠皮下),具有完整的異源精子發(fā)生。本研究同時發(fā)現(xiàn),使用大量的睪丸組織塊移植到非免疫缺陷受體小鼠皮下檢測到移植組織塊經(jīng)歷了破碎、重塑進(jìn)而完成精子發(fā)生的過程;同時也發(fā)現(xiàn)在非免疫缺陷小鼠使用免疫抑制劑可提高異源組織塊存活和完成精子發(fā)生的成功率,將完整精子發(fā)生的成功率從2-5%提高到了8.3%,并且重現(xiàn)了移植組織塊經(jīng)歷破碎、重塑進(jìn)入完成精子發(fā)生的過程,為精子發(fā)生及睪丸組織發(fā)育的研究提供了新模型,可為轉(zhuǎn)基因動物制作提供新途徑。綜上所述,本研究著力探索了非免疫缺陷受體模型下精原干細(xì)胞研究的兩大策略:其一是基于精原干細(xì)胞移植的相關(guān)研究,另一個是睪丸組織塊移植的研究。探究了腺嘌呤作用于SD大鼠(免疫正常大鼠)睪丸組織生殖毒性,獲得一種新型的可用于精原干細(xì)胞移植受體模型,經(jīng)過輸出小管移植獲得了完整的異種移植精子發(fā)生;同時優(yōu)化了基于慢病毒載體的依賴精原干細(xì)胞轉(zhuǎn)基因動物制作的途徑,從甲基化水平揭示了不同載體不同啟動子在體內(nèi)表達(dá)外源基因差異的原因。同時也對非免疫缺陷受體皮下睪丸組織塊移植進(jìn)行了探索,配合使用免疫抑制劑提高了異源組織塊存活率和精子發(fā)生率。本研究對于精原干細(xì)胞功能、精子發(fā)生及睪丸組織發(fā)育提供了新見解,對于轉(zhuǎn)基因動物制作提供了簡單易行的策略,對于大家畜種和珍稀動物在生物學(xué)研究及實際應(yīng)用具有重要意義。
[Abstract]:Male germ cells refers to the male reproductive system by sexual reproduction biological offspring can transmit genetic information to a class of cell progeny, which spermatogonial stem cells are the body only throughout life, can maintain its stability and the number of proliferation to differentiate into sperm of adult stem cells; at the same time, fine the original stem cells in spontaneous or induced conditions, can differentiate into three germ layers with similar potential of embryonic stem cells has attracted extensive attention in biomedical characteristics, translational medicine and animal husbandry. The spermatogonial stem cell research mainly concentrated in the screening of molecular markers, system establishment and maintenance of in vitro epigenetic research modification of related signaling pathways and classic spermatogonial stem cells modified by transgenic animal production and testicular tissue transplantation field based on immune defects in these studies used by Model greatly limits the spermatogonial stem cell research and application, especially the actual production and promotion in the livestock industry; at the same time, there is a certain difficulty in transgenic animal spermatogonial stem cell transplantation based on production. Therefore, to explore the strategies to transgenic animal spermatogonial stem cells mediated by simple production. The research mainly focuses on exploring testis and spermatogonial stem cells transplantation model making method of non immune deficiency, a new method of transgenic technology of male germ cells mediated in vivo to explore simple. This study as follows: 1, the research results of different concentrations of adenine (200mg/kg, 260mg/kg, 320mg/kg and 380mg/kg) the non immunodeficient rat (Sprague-Dawley SD rats) injury of testis, the related physiological indexes and testicular sections were obtained, the candidate spermatogonial stem cell transplantation in the receptor The optimal concentration of SD rats (320mg/kg). At the same time as a positive control, groping busulfan (a widely used spermatogonial stem cell drug receptor model preparation) in the appropriate concentration of production of receptor SD in rats; from the survival rate, weight, testis index and testis were determined adenine treatment is superior to busulfan treatment, can be used as alternatives to block endogenous spermatogenesis, provide suitable microenvironment for spermatogonial stem cell transplantation. At the same time, the use of enzyme digestion and differential attachment and immunomagnetic separation methods such as enrichment of high purity of mouse spermatogonial stem cells (Lin28 positive rate reached 84%, the positive rate of THY-1 reached 92% at the same time), in vitro using lentivirus infection modified method labeling of mouse spermatogonial stem cells, and transplanted into recipient rat seminiferous tube, first detected by immunocompetent xenograft in vivo intact sperm. This experiment also makes Primary germ cells and Sertoli cells and isolated cell lines as the research object, the research on the mechanism of adenine in vitro; TUNEL chromosome DNA confirmed fracture, using Western blot to detect early apoptosis and apoptosis related protein Caspase3. PARP protein concentration dependent expression, preliminarily adenine produced the reproductive toxicity of specific.2 on germ cells by inducing apoptosis, this study strategy using lentiviral infection of male germ cells, transgenic descendants of more than 67% of the normal reproductive capacity, transgene can be stably inherited; at the same time also revealed no correlation between the strategies and parental age and lentivirus injection site (P0.05), suggesting that we can try to use in all animal lentivirus injected testicular interstitial site to obtain transgenic offspring, we simplify. The tedious aspects and technical problems in production and breeding. At the same time, also compared the different lentiviral system on testicular spermatogonial stem cells to infection, compared with CD513B-CMV-MCS-EF1 lentiviral P, lentivirus P Lenti V-H1-MCS-CMV infection of spermatogonial stem cells of transgenic progeny obtained after the normal expression of transgene fragments; further study revealed different slow the virus integrated into the genome of offspring, exogenous gene promoter sequence there are significant differences in methylation level: the former transgene fragment promoter methylation levels up to 87.8%, while the latter is only 45.5%. in this study for later use lentiviral vector for making transgenic animal especially provides a new idea for.3 seminiferous tube transplantation is difficult to achieve all animals, this study explored the use of non immunodeficient mice (adult Kunming mice) as testis tissue transplantation receptor, can complete the same Different strains of the testicular transplantation spermatogenesis, also first detected in testicular tissue block transplantation (xenogeneic transplantation of rat testis to mice), with complete heterologous sperm occurrence. The study also found that the use of a large number of testicular tissue transplanted to immunodeficient mice subcutaneous non receptor detected tissue after transplantation broken, and then complete the remodeling process of spermatogenesis; also found that the use of immunosuppressive agents in immunodeficient mice can improve the heterologous tissue survived and complete spermatogenesis success rate, the success rate of complete spermatogenesis increased from 2-5% to 8.3%, and re transplantation tissue through crushing, complete the process of spermatogenesis in remodeling that provides a new model for the study of the development and organization of spermatogenesis, which can provide a new way for the production of transgenic animal. In summary, this study explores the non Immunodeficiency receptor model two strategies under the spermatogonial stem cell research: one is the related research of spermatogonial stem cell transplantation based on a study of testis tissue transplantation. To explore the role of adenine in SD rats (Immune normal rats) reproductive toxicity in testis, a model can be used to spermatogonial stem cell transplantation receptor model, after efferent duct transplantation has achieved complete xenograft sperm; optimize the sperm dependent lentiviral vector based on the original way of making stem cells of transgenic animal, the level of methylation reveals why different carriers from different promoters of exogenous gene expression in vivo. The difference is also carried out exploration on the testis non receptor block with immunodeficiency subcutaneous transplantation, immunosuppression increases the incidence of heterologous tissue and sperm survival rate. This study for spermatogonial stem cell function Organization, and spermatogenesis provides new insights into the development, provides a simple strategy for the production of transgenic animal, for all livestock and rare animal plays an important role in biological research and practical application.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q78;Q954.4
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本文編號：1549993