牛雄性生殖干细胞分离培养特性及移植研究
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摘要
雄性生殖干细胞(male germ stem cells, mGSCs)是性腺分化开始一直存在于睾丸曲细精管中、唯一能将遗传物质传递给后代的干细胞。在mGSCs的细胞学特性、分离纯化方法、体外长期培养系统、体外诱导mGSCs形成单倍体细胞和精子、mGSCs的基因修饰和同种异种间的移植等方面,许多研究者做了许多工作,新的成就不断涌现,但是关于哺乳动物mGSCs的研究,仍然处于起步探索阶段。本试验在前人研究的基础上,分析牛性腺发育和曲细精管中mGSCs的分布规律;以新生牛睾丸为材料,比较不同mGSCs分离纯化的方法及效果;不同饲养层细胞和细胞因子对体外培养新生牛mGSCs行为的影响;新生牛ES-mGSCs的形成及体外分化潜能;定向诱导新生牛mGSCs体外形成精子样细胞;新生牛mGSCs移植无内源性生殖细胞模型兔睾丸,探讨新生牛mGSCs异种移植的可行性。
mGSCs来源于PGCs,观察不同发育阶段牛性腺组织结构显示,6~10 w牛胎儿,性腺开始分化,雄性生殖细胞开始克隆聚集;15 w胎牛形成曲细精管;20 w胎牛生殖细胞增殖较快;出生时,曲细精管完全形成,曲细精管中只有支持细胞和雄性生殖干细胞,并且间质组织少,有利于生殖细胞的分离。
采用不同解离方法,对53例新生牛睾丸处理结果显示,以0.1%的胶原酶+5 ng/mL DNaseⅠ消化30 min和0.25%胰酶+0.04% EDTA+5 ng/mL DNaseⅠ消化5 min,分段处理能有效离解睾丸组织,获取单细胞或细胞团。
对5例上述离解的睾丸细胞的mGSCs进行纯化,结果显示,3次贴壁差法比percoll密度梯度离心获得的mGSCs细胞活力强;mGSCs活性和纯度分别达到76.7%和98.2%;3次贴壁差法纯化的新生牛mGSCs根据大小可分为2类,一类细胞直径超过25μm,数量较少,占总细胞数量的15%左右,细胞质的流动性是其显著特点;另一类细胞直径小于15μm,数量较大,占总细胞数量的85%左右。两类细胞中CD117(c-kit)阳性细胞为18.91%。
分离新生牛的生殖细胞中,透射电镜可观察到典型As~Aal以及A1~A4型精原细胞,其主要区分为核仁形态、常染色质和异染色质的分布不同,未见胞质桥结构,所有观察的细胞均显示细胞质结构简单,含有包囊体,局部区域分布有简单的线粒体和核糖体。表明新生牛睾丸中已有多类型的精原细胞;支持细胞与生殖细胞之间存在桥粒和空隙连接。
在DMEM+10%NBS基础培养液中,支持细胞在传至12代,细胞形态从类似铺路石样细胞转变成短梭状细胞。mGSCs-BSCs共培养系统中,BSCs可以促进前3代mGSCs
Male germ stem cells(mGSCs)settle in testicular seminiferous tubules after the differentiation of genital gland and is the sole kind of adult stem cells which can transfer genetic matter to offspring. Some investigators had researched the cytology characters, separation and purifying, the in vitro cultural system, in vitro induction into haploid cells or spermatozoa, gene modification and transplantation of the mGSCs into homogeneous and heterogeneous testis and reached new achievements. But few results of investigation about neonatal bovine mGSCs were reported.
According to previous investigations, the following contents were studied in the thesis: development of neonatal bovine testes and mGSCs, different methods and effects of separation and purifying on mGSCs, establishment of cultural systems of mGSCs and observation of in vitro behaviors of mGSCs, directional induction and differentiation of mGSCs into spermatozoa-like cells, analysis of formation and in vitro differentiation potential of ES-like cells from neonatal bovine mGSCs,and finally,the assay of tissues difference between testis tissues of model rabbits deficient in endogenic germ cells and those of model rabbits transplanted with neonatal bovine mGSCs. The conclusion showed as the follows:
Bovine testis tissues were observed from 6-week fetuses to neonatal calf. The results demonstrated that male germ cells began to form clone at 6~10 w; and seminiferous tubules appeared at 15 w; and more germ cells located at center of seminiferous tubules at 20 w; and sertoli cells and germ cells located at sub-center of seminiferous tubules and leydig cells was less among seminiferous tubules in neonatal testis.
Neonatal testis tissues were procedurally treated through 0.1% collagenase 30 min and 0.25% trypsin +0.04% EDTA+5 ng/mL DNaseI 5 min, and single cells and small cells masses were acquired. Activity and purification degree of mGSCs is better through 3 times isolation with adhesion than through percoll density gradient centrifugation,reaching an activity and purifying of 76.7% and 98.2% respectively. Through 3 times adhering isolation, 2 kinds of mGSCs were acquired,including cells diameter less than 15μm and over 25μm. The remarkable character of the former was cytoplasm fluidity were about 85% of total cells and
mGSCs来源于PGCs,观察不同发育阶段牛性腺组织结构显示,6~10 w牛胎儿,性腺开始分化,雄性生殖细胞开始克隆聚集;15 w胎牛形成曲细精管;20 w胎牛生殖细胞增殖较快;出生时,曲细精管完全形成,曲细精管中只有支持细胞和雄性生殖干细胞,并且间质组织少,有利于生殖细胞的分离。
采用不同解离方法,对53例新生牛睾丸处理结果显示,以0.1%的胶原酶+5 ng/mL DNaseⅠ消化30 min和0.25%胰酶+0.04% EDTA+5 ng/mL DNaseⅠ消化5 min,分段处理能有效离解睾丸组织,获取单细胞或细胞团。
对5例上述离解的睾丸细胞的mGSCs进行纯化,结果显示,3次贴壁差法比percoll密度梯度离心获得的mGSCs细胞活力强;mGSCs活性和纯度分别达到76.7%和98.2%;3次贴壁差法纯化的新生牛mGSCs根据大小可分为2类,一类细胞直径超过25μm,数量较少,占总细胞数量的15%左右,细胞质的流动性是其显著特点;另一类细胞直径小于15μm,数量较大,占总细胞数量的85%左右。两类细胞中CD117(c-kit)阳性细胞为18.91%。
分离新生牛的生殖细胞中,透射电镜可观察到典型As~Aal以及A1~A4型精原细胞,其主要区分为核仁形态、常染色质和异染色质的分布不同,未见胞质桥结构,所有观察的细胞均显示细胞质结构简单,含有包囊体,局部区域分布有简单的线粒体和核糖体。表明新生牛睾丸中已有多类型的精原细胞;支持细胞与生殖细胞之间存在桥粒和空隙连接。
在DMEM+10%NBS基础培养液中,支持细胞在传至12代,细胞形态从类似铺路石样细胞转变成短梭状细胞。mGSCs-BSCs共培养系统中,BSCs可以促进前3代mGSCs
Male germ stem cells(mGSCs)settle in testicular seminiferous tubules after the differentiation of genital gland and is the sole kind of adult stem cells which can transfer genetic matter to offspring. Some investigators had researched the cytology characters, separation and purifying, the in vitro cultural system, in vitro induction into haploid cells or spermatozoa, gene modification and transplantation of the mGSCs into homogeneous and heterogeneous testis and reached new achievements. But few results of investigation about neonatal bovine mGSCs were reported.
According to previous investigations, the following contents were studied in the thesis: development of neonatal bovine testes and mGSCs, different methods and effects of separation and purifying on mGSCs, establishment of cultural systems of mGSCs and observation of in vitro behaviors of mGSCs, directional induction and differentiation of mGSCs into spermatozoa-like cells, analysis of formation and in vitro differentiation potential of ES-like cells from neonatal bovine mGSCs,and finally,the assay of tissues difference between testis tissues of model rabbits deficient in endogenic germ cells and those of model rabbits transplanted with neonatal bovine mGSCs. The conclusion showed as the follows:
Bovine testis tissues were observed from 6-week fetuses to neonatal calf. The results demonstrated that male germ cells began to form clone at 6~10 w; and seminiferous tubules appeared at 15 w; and more germ cells located at center of seminiferous tubules at 20 w; and sertoli cells and germ cells located at sub-center of seminiferous tubules and leydig cells was less among seminiferous tubules in neonatal testis.
Neonatal testis tissues were procedurally treated through 0.1% collagenase 30 min and 0.25% trypsin +0.04% EDTA+5 ng/mL DNaseI 5 min, and single cells and small cells masses were acquired. Activity and purification degree of mGSCs is better through 3 times isolation with adhesion than through percoll density gradient centrifugation,reaching an activity and purifying of 76.7% and 98.2% respectively. Through 3 times adhering isolation, 2 kinds of mGSCs were acquired,including cells diameter less than 15μm and over 25μm. The remarkable character of the former was cytoplasm fluidity were about 85% of total cells and
引文
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