自噬对奶山羊雄性生殖干细胞生物学特性的影响
发布时间：2018-08-01 08:01
【摘要】：雄性生殖干细胞(Male germline stem cells,mGSCs),又名精原干细胞(Spermatogonial stem cells,SSCs),是一类未分化的雄性生殖细胞。它定位于曲细精管基底膜,可通过分化为精母细胞最终产生成熟的精子。雄性生殖干细胞是唯一一类可将遗传物质传递给后代的成体干细胞,具有自我更新能力和多向分化的潜能。目前,雄性生殖干细胞的研究主要集中于不同物种的雄性生殖干细胞的体外分离、培养和建系,自我更新机制研究及其去分化和转分化在转化医学中所发挥的作用。而其自我更新机制研究主要集中于细胞因子和转录因子等对雄性生殖干细胞干性维持的调控作用。自噬(Autophagy)为科学家Ashford和Porter在肝细胞中所发现的“自己吃自己”的现象。在细胞正常活动中,如动物的变态发育、老化和分化,自噬负责降解损伤的细胞器和冗余的蛋白以重新组建细胞。目前,对于自噬的研究已经有了很大的进展,但大部分研究集中于其发生机制、肿瘤、衰老等方面,且近年来逐渐有科学工作者着眼于研究自噬对生殖的影响。本试验以自噬为切入点,探讨其对雄性生殖细胞生物学特性的影响,初步研究了自噬对奶山羊雄性生殖干细胞的自我更新及分化的相互关系,为进一步揭示精子发生机理提供理论依据和实验基础。本研究以奶山羊为研究对象,首先在本实验室已建系的永生化雄性生殖干细胞系(Immortal male germlien stem cells of dairy goat,GmGSCs-I-SB)的基础上,转导、筛选稳定表达融合蛋白EGFP-LC3B的自噬示踪雄性生殖干细胞系(GmGSCs-I-SB-EGFP-LC3B),确定自噬现象存在于奶山羊雄性生殖干细胞中,该细胞系可用于实时监测雄性生殖干细胞的自噬发生;进一步通过雷帕霉素和氯喹对GmGSCs-I-SB中自噬的发生进行诱导和抑制,检测到雄性生殖干细胞干性相关基因随自噬发生的增强而表达量升高。另一方面,在处于分化状态中的GmGSCs-I-SB-EGFP-LC3B,也可以观察到自噬体的产生。本试验为深入了解雄性动物生殖干细胞自我更新的机制奠定了基础。1.GmGSCs-I-SB-EGFP-LC3B自噬示踪细胞系的建立首先通过免疫荧光染色,蛋白免疫印迹,透射电镜技术检测GmGSCs-I-SB中内源性的自噬相关标记ATG7,LC3B,P62的表达及自噬体的结构,确定了奶山羊雄性生殖干细胞中有自噬现象的存在。进而根据NCBI公布的pEGFP-C1(U55763.1)和LC3B(XM_011529085.1)序列设计引物,从pEGFP-LC3B-C1载体上扩增出表达融合蛋白EGFP-LC3B的片段,将其插入慢病毒表达载体pTRIP-CAGG-puro上。经EcoRI和BamHI双酶切验证,结果显示pTRIP-CAGG-EGFP-LC3B-puro慢病毒载体构建成功,且测序结果比对正确。然后通过293T细胞包装,转导到奶山羊GmGSCs-I-SB中,经嘌呤霉素筛选得到GmGSCs-I-SB-EGFP-LC3B细胞系。待细胞稳定传至10代后进行流式检测EGFP-LC3B阳性细胞数,并结合免疫荧光染色检测LC3B的表达定位,发现绿色荧光蛋白能够和免疫荧光染色LC3B的红荧光共定位,且蛋白免疫印迹在43KDa处检测到EGFP-LC3B,初步表明我们构建的载体能在奶山羊GmGSCs-I-SB工作。进一步通过无血清饥饿,雷帕霉素刺激和氯喹阻断自噬体的降解等方法分别能观察到GmGSCs-I-SB-EGFP-LC3B中有带绿色荧光标记的自噬体产生,其产生随不同刺激发生相应改变,表明该细胞系能够用来监测自噬的发生。2.自噬对GmGSCs-I-SB生物学特性的影响首先用不同浓度的(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)氯喹处理GmGSCs-I-SB,发现100 nmol/L雷帕霉素和100μmol/L氯喹对GmGSCs-I-SB的活力没有显著影响,但分别对自噬有显著的促进和抑制作用。因此,分别采用此浓度对自噬进行诱导和抑制24 h,检测到自噬抑制组增殖能力降低和凋亡水平增高,且在自噬促进组中,干性基因Plzf等表达量上调。另外,利用GmGSCs-I-SB-EGFP-LC3B细胞系进行类胚体自发分化和定向神经诱导分化试验中,都可明显观察到自噬伴随在整个分化过程中。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827
本文编号：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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827
【共引文献】
相关期刊论文 前2条
1 汤雪明;易静;张蕙心;陈晓群;张岚;;内分泌细胞溶酶体的超微结构研究[J];电子显微学报;1996年Z1期
2 易静,汤雪明;大鼠Leydig细胞中细胞内吞和自体吞噬两条途径的交汇点[J];电子显微学报;2000年03期
相关硕士学位论文 前1条
1 谭俊玲;小鼠生精细胞与支持细胞自噬与自噬诱因的初步研究[D];山东大学;2013年
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