自噬對(duì)奶山羊雄性生殖干細(xì)胞生物學(xué)特性的影響
發(fā)布時(shí)間：2018-08-01 08:01
【摘要】：雄性生殖干細(xì)胞(Male germline stem cells,mGSCs),又名精原干細(xì)胞(Spermatogonial stem cells,SSCs),是一類未分化的雄性生殖細(xì)胞。它定位于曲細(xì)精管基底膜,可通過(guò)分化為精母細(xì)胞最終產(chǎn)生成熟的精子。雄性生殖干細(xì)胞是唯一一類可將遺傳物質(zhì)傳遞給后代的成體干細(xì)胞,具有自我更新能力和多向分化的潛能。目前,雄性生殖干細(xì)胞的研究主要集中于不同物種的雄性生殖干細(xì)胞的體外分離、培養(yǎng)和建系,自我更新機(jī)制研究及其去分化和轉(zhuǎn)分化在轉(zhuǎn)化醫(yī)學(xué)中所發(fā)揮的作用。而其自我更新機(jī)制研究主要集中于細(xì)胞因子和轉(zhuǎn)錄因子等對(duì)雄性生殖干細(xì)胞干性維持的調(diào)控作用。自噬(Autophagy)為科學(xué)家Ashford和Porter在肝細(xì)胞中所發(fā)現(xiàn)的“自己吃自己”的現(xiàn)象。在細(xì)胞正常活動(dòng)中,如動(dòng)物的變態(tài)發(fā)育、老化和分化,自噬負(fù)責(zé)降解損傷的細(xì)胞器和冗余的蛋白以重新組建細(xì)胞。目前,對(duì)于自噬的研究已經(jīng)有了很大的進(jìn)展,但大部分研究集中于其發(fā)生機(jī)制、腫瘤、衰老等方面,且近年來(lái)逐漸有科學(xué)工作者著眼于研究自噬對(duì)生殖的影響。本試驗(yàn)以自噬為切入點(diǎn),探討其對(duì)雄性生殖細(xì)胞生物學(xué)特性的影響,初步研究了自噬對(duì)奶山羊雄性生殖干細(xì)胞的自我更新及分化的相互關(guān)系,為進(jìn)一步揭示精子發(fā)生機(jī)理提供理論依據(jù)和實(shí)驗(yàn)基礎(chǔ)。本研究以奶山羊?yàn)檠芯繉?duì)象,首先在本實(shí)驗(yàn)室已建系的永生化雄性生殖干細(xì)胞系(Immortal male germlien stem cells of dairy goat,GmGSCs-I-SB)的基礎(chǔ)上,轉(zhuǎn)導(dǎo)、篩選穩(wěn)定表達(dá)融合蛋白EGFP-LC3B的自噬示蹤雄性生殖干細(xì)胞系(GmGSCs-I-SB-EGFP-LC3B),確定自噬現(xiàn)象存在于奶山羊雄性生殖干細(xì)胞中,該細(xì)胞系可用于實(shí)時(shí)監(jiān)測(cè)雄性生殖干細(xì)胞的自噬發(fā)生;進(jìn)一步通過(guò)雷帕霉素和氯喹對(duì)GmGSCs-I-SB中自噬的發(fā)生進(jìn)行誘導(dǎo)和抑制,檢測(cè)到雄性生殖干細(xì)胞干性相關(guān)基因隨自噬發(fā)生的增強(qiáng)而表達(dá)量升高。另一方面,在處于分化狀態(tài)中的GmGSCs-I-SB-EGFP-LC3B,也可以觀察到自噬體的產(chǎn)生。本試驗(yàn)為深入了解雄性動(dòng)物生殖干細(xì)胞自我更新的機(jī)制奠定了基礎(chǔ)。1.GmGSCs-I-SB-EGFP-LC3B自噬示蹤細(xì)胞系的建立首先通過(guò)免疫熒光染色,蛋白免疫印跡,透射電鏡技術(shù)檢測(cè)GmGSCs-I-SB中內(nèi)源性的自噬相關(guān)標(biāo)記ATG7,LC3B,P62的表達(dá)及自噬體的結(jié)構(gòu),確定了奶山羊雄性生殖干細(xì)胞中有自噬現(xiàn)象的存在。進(jìn)而根據(jù)NCBI公布的pEGFP-C1(U55763.1)和LC3B(XM_011529085.1)序列設(shè)計(jì)引物,從pEGFP-LC3B-C1載體上擴(kuò)增出表達(dá)融合蛋白EGFP-LC3B的片段,將其插入慢病毒表達(dá)載體pTRIP-CAGG-puro上。經(jīng)EcoRI和BamHI雙酶切驗(yàn)證,結(jié)果顯示pTRIP-CAGG-EGFP-LC3B-puro慢病毒載體構(gòu)建成功,且測(cè)序結(jié)果比對(duì)正確。然后通過(guò)293T細(xì)胞包裝,轉(zhuǎn)導(dǎo)到奶山羊GmGSCs-I-SB中,經(jīng)嘌呤霉素篩選得到GmGSCs-I-SB-EGFP-LC3B細(xì)胞系。待細(xì)胞穩(wěn)定傳至10代后進(jìn)行流式檢測(cè)EGFP-LC3B陽(yáng)性細(xì)胞數(shù),并結(jié)合免疫熒光染色檢測(cè)LC3B的表達(dá)定位,發(fā)現(xiàn)綠色熒光蛋白能夠和免疫熒光染色LC3B的紅熒光共定位,且蛋白免疫印跡在43KDa處檢測(cè)到EGFP-LC3B,初步表明我們構(gòu)建的載體能在奶山羊GmGSCs-I-SB工作。進(jìn)一步通過(guò)無(wú)血清饑餓,雷帕霉素刺激和氯喹阻斷自噬體的降解等方法分別能觀察到GmGSCs-I-SB-EGFP-LC3B中有帶綠色熒光標(biāo)記的自噬體產(chǎn)生,其產(chǎn)生隨不同刺激發(fā)生相應(yīng)改變,表明該細(xì)胞系能夠用來(lái)監(jiān)測(cè)自噬的發(fā)生。2.自噬對(duì)GmGSCs-I-SB生物學(xué)特性的影響首先用不同濃度的(0、50、100、200、300、400、500、600、700、800、900、1000nmol/L)雷帕霉素和(0、50、100、200、300、400、500、600、700、800、900、1000μmol/L)氯喹處理GmGSCs-I-SB,發(fā)現(xiàn)100 nmol/L雷帕霉素和100μmol/L氯喹對(duì)GmGSCs-I-SB的活力沒(méi)有顯著影響,但分別對(duì)自噬有顯著的促進(jìn)和抑制作用。因此,分別采用此濃度對(duì)自噬進(jìn)行誘導(dǎo)和抑制24 h,檢測(cè)到自噬抑制組增殖能力降低和凋亡水平增高,且在自噬促進(jìn)組中,干性基因Plzf等表達(dá)量上調(diào)。另外,利用GmGSCs-I-SB-EGFP-LC3B細(xì)胞系進(jìn)行類胚體自發(fā)分化和定向神經(jīng)誘導(dǎo)分化試驗(yàn)中,都可明顯觀察到自噬伴隨在整個(gè)分化過(guò)程中。
[Abstract]:Male reproductive stem cells (Male germline stem cells, mGSCs), also known as spermatogonial stem cells (Spermatogonial stem cells, SSCs), are undifferentiated male reproductive cells. They are located in the basilar membrane of the fine tubule and can be differentiated into spermatocytes to produce mature spermatogones. Male reproductive stem cells are the only kind of genetic material that can be passed on. Adult stem cells, which are delivered to offspring, have the potential for self renewal and multidifferentiation. At present, the research of male reproductive stem cells mainly focuses on the isolation, culture and construction of male reproductive stem cells of different species, the study of self renewal mechanism and the role of dedifferentiation and transdifferentiation in transformation medicine. The study of my update mechanism focuses on the regulatory role of cytokines and transcription factors on the dry maintenance of male reproductive stem cells. Autophagy (Autophagy) is the "self eating" phenomenon found by scientists Ashford and Porter in hepatocytes. In normal cellular activities, such as animal metamorphosis, aging and differentiation, autophagy is responsible. Degradation of damaged organelles and redundant proteins to reorganize cells. Currently, a lot of progress has been made in the study of autophagy, but most of the studies have focused on its pathogenesis, tumor, aging and so on. In recent years, scientists have gradually focused on the study of the effect of autophagy on reproduction. The relationship between autophagy and the self renewal and differentiation of male reproductive stem cells in dairy goats was preliminarily studied. This study provided a theoretical basis and experimental basis for further revealing the mechanism of spermatogenesis. On the basis of the reproductive stem cell line (Immortal male germlien stem cells of dairy goat, GmGSCs-I-SB), it transduced and screened the autophagy tracer male reproductive stem cell line (GmGSCs-I-SB-EGFP-LC3B), which stably expressed the fusion protein EGFP-LC3B, and determined that the autophagy existed in the male reproductive stem cells of milk goats. The cell line could be used for real-time monitoring of male reproductive stem cells. Autophagy occurs in sexual reproductive stem cells and is further induced and inhibited by rapamycin and chloroquine in the occurrence of autophagy in GmGSCs-I-SB. The expression of the stem related genes in male reproductive stem cells increases with the enhancement of autophagy. On the other hand, the GmGSCs-I-SB-EGFP-LC3B in the differentiated state can also be observed from the autophagy. In order to understand the mechanism of the self renewal of the male reproductive stem cells, this experiment establishes the basis for the establishment of the.1.GmGSCs-I-SB-EGFP-LC3B autophagy tracer cell line, first by immunofluorescence staining, protein immunoblotting, and transmission electron microscopy to detect the expression of autophagy related markers ATG7, LC3B, P62 in the GmGSCs-I-SB. The autophagic structure was used to determine the existence of autophagy in the male reproductive stem cells of dairy goats, and then the primers were designed based on the pEGFP-C1 (U55763.1) and LC3B (XM_011529085.1) sequence published by NCBI. The fragments expressing the fusion protein EGFP-LC3B were amplified from the pEGFP-LC3B-C1 vector and inserted into the Lentivirus Expression Vector pTRIP-CAGG-puro. By EcoRI and BamHI double enzyme digestion, the results showed that the pTRIP-CAGG-EGFP-LC3B-puro lentivirus vector was successfully constructed and the sequencing results were correct. Then, the GmGSCs-I-SB-EGFP-LC3B cell line was screened by purinamycin through 293T cell packaging and transduced to the milk goat GmGSCs-I-SB. After the cell was stabilized to 10 generations, the flow cytometry was carried out to detect EGFP-LC The number of 3B positive cells, combined with immunofluorescence staining to detect the expression of LC3B, found that the green fluorescent protein could co locate with the red fluorescence of the immunofluorescent staining LC3B, and the protein immunoblotting was detected at EGFP-LC3B at 43KDa. It was preliminarily indicated that the carrier we constructed could work in the dairy goat GmGSCs-I-SB. Further through the serum-free starvation, thunder Paramictin stimulation and chloroquine blocking autophagic degradation can be used to observe the production of autophagic with green fluorescent markers in GmGSCs-I-SB-EGFP-LC3B, and its production varies with different stimuli. It shows that the cell line can be used to monitor autophagy and the effect of autophagy on the biological characteristics of GmGSCs-I-SB is different. The concentration of (0,501002003004005006007008009001000nmol/L) rapamycin and (0,501002003004005006007008009001000 mol/L) chloroquine treated GmGSCs-I-SB, and found that 100 nmol/L rapamycin and 100 micron mol/L chloroquine had no significant effect on the viability of GmGSCs-I-SB, but had significant promotion and inhibition on autophagy. This concentration was used to induce and inhibit autophagy by 24 h, respectively, to detect the decrease of proliferation and increase in the level of apoptosis in the autophagy inhibition group, and the expression of Plzf and so on in the autophagy promotion group. In addition, the GmGSCs-I-SB-EGFP-LC3B cell lines were used in the self differentiation and directed differentiation test of the embryoid body. It is obvious that autophagy is involved in the whole differentiation process.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S827
本文編號(hào)：2156835
[Abstract]:Male reproductive stem cells (Male germline stem cells, mGSCs), also known as spermatogonial stem cells (Spermatogonial stem cells, SSCs), are undifferentiated male reproductive cells. They are located in the basilar membrane of the fine tubule and can be differentiated into spermatocytes to produce mature spermatogones. Male reproductive stem cells are the only kind of genetic material that can be passed on. Adult stem cells, which are delivered to offspring, have the potential for self renewal and multidifferentiation. At present, the research of male reproductive stem cells mainly focuses on the isolation, culture and construction of male reproductive stem cells of different species, the study of self renewal mechanism and the role of dedifferentiation and transdifferentiation in transformation medicine. The study of my update mechanism focuses on the regulatory role of cytokines and transcription factors on the dry maintenance of male reproductive stem cells. Autophagy (Autophagy) is the "self eating" phenomenon found by scientists Ashford and Porter in hepatocytes. In normal cellular activities, such as animal metamorphosis, aging and differentiation, autophagy is responsible. Degradation of damaged organelles and redundant proteins to reorganize cells. Currently, a lot of progress has been made in the study of autophagy, but most of the studies have focused on its pathogenesis, tumor, aging and so on. In recent years, scientists have gradually focused on the study of the effect of autophagy on reproduction. The relationship between autophagy and the self renewal and differentiation of male reproductive stem cells in dairy goats was preliminarily studied. This study provided a theoretical basis and experimental basis for further revealing the mechanism of spermatogenesis. On the basis of the reproductive stem cell line (Immortal male germlien stem cells of dairy goat, GmGSCs-I-SB), it transduced and screened the autophagy tracer male reproductive stem cell line (GmGSCs-I-SB-EGFP-LC3B), which stably expressed the fusion protein EGFP-LC3B, and determined that the autophagy existed in the male reproductive stem cells of milk goats. The cell line could be used for real-time monitoring of male reproductive stem cells. Autophagy occurs in sexual reproductive stem cells and is further induced and inhibited by rapamycin and chloroquine in the occurrence of autophagy in GmGSCs-I-SB. The expression of the stem related genes in male reproductive stem cells increases with the enhancement of autophagy. On the other hand, the GmGSCs-I-SB-EGFP-LC3B in the differentiated state can also be observed from the autophagy. In order to understand the mechanism of the self renewal of the male reproductive stem cells, this experiment establishes the basis for the establishment of the.1.GmGSCs-I-SB-EGFP-LC3B autophagy tracer cell line, first by immunofluorescence staining, protein immunoblotting, and transmission electron microscopy to detect the expression of autophagy related markers ATG7, LC3B, P62 in the GmGSCs-I-SB. The autophagic structure was used to determine the existence of autophagy in the male reproductive stem cells of dairy goats, and then the primers were designed based on the pEGFP-C1 (U55763.1) and LC3B (XM_011529085.1) sequence published by NCBI. The fragments expressing the fusion protein EGFP-LC3B were amplified from the pEGFP-LC3B-C1 vector and inserted into the Lentivirus Expression Vector pTRIP-CAGG-puro. By EcoRI and BamHI double enzyme digestion, the results showed that the pTRIP-CAGG-EGFP-LC3B-puro lentivirus vector was successfully constructed and the sequencing results were correct. Then, the GmGSCs-I-SB-EGFP-LC3B cell line was screened by purinamycin through 293T cell packaging and transduced to the milk goat GmGSCs-I-SB. After the cell was stabilized to 10 generations, the flow cytometry was carried out to detect EGFP-LC The number of 3B positive cells, combined with immunofluorescence staining to detect the expression of LC3B, found that the green fluorescent protein could co locate with the red fluorescence of the immunofluorescent staining LC3B, and the protein immunoblotting was detected at EGFP-LC3B at 43KDa. It was preliminarily indicated that the carrier we constructed could work in the dairy goat GmGSCs-I-SB. Further through the serum-free starvation, thunder Paramictin stimulation and chloroquine blocking autophagic degradation can be used to observe the production of autophagic with green fluorescent markers in GmGSCs-I-SB-EGFP-LC3B, and its production varies with different stimuli. It shows that the cell line can be used to monitor autophagy and the effect of autophagy on the biological characteristics of GmGSCs-I-SB is different. The concentration of (0,501002003004005006007008009001000nmol/L) rapamycin and (0,501002003004005006007008009001000 mol/L) chloroquine treated GmGSCs-I-SB, and found that 100 nmol/L rapamycin and 100 micron mol/L chloroquine had no significant effect on the viability of GmGSCs-I-SB, but had significant promotion and inhibition on autophagy. This concentration was used to induce and inhibit autophagy by 24 h, respectively, to detect the decrease of proliferation and increase in the level of apoptosis in the autophagy inhibition group, and the expression of Plzf and so on in the autophagy promotion group. In addition, the GmGSCs-I-SB-EGFP-LC3B cell lines were used in the self differentiation and directed differentiation test of the embryoid body. It is obvious that autophagy is involved in the whole differentiation process.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S827
【共引文獻(xiàn)】
相關(guān)期刊論文 前2條
1 湯雪明;易靜;張蕙心;陳曉群;張嵐;;內(nèi)分泌細(xì)胞溶酶體的超微結(jié)構(gòu)研究[J];電子顯微學(xué)報(bào);1996年Z1期
2 易靜,湯雪明;大鼠Leydig細(xì)胞中細(xì)胞內(nèi)吞和自體吞噬兩條途徑的交匯點(diǎn)[J];電子顯微學(xué)報(bào);2000年03期
相關(guān)碩士學(xué)位論文 前1條
1 譚俊玲;小鼠生精細(xì)胞與支持細(xì)胞自噬與自噬誘因的初步研究[D];山東大學(xué);2013年
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