摘要
生殖干细胞在有性繁殖的生物中肩负着将遗传信息在世代中传递的重要任务,即通过精卵结合产生新的个体。生殖细胞的种系传递特性使得它成为动物胚胎学、发育生物学和细胞生物学等学科关注的研究对象之一。生殖细胞的可塑性使其能代替胚胎干细胞(Embryonic stem cell, ESC)用于治疗和遗传修饰,而不涉及伦理和免疫排斥问题,故引起生物学界极大关注。因此,如何迅速开展诱导来源于其他组织的干细胞分化成为雄性生殖细胞的研究显得很有必要和迫切。目前将ES细胞诱导为生殖细胞的研究已经开展,但诱导方法较多,效率较低。
相对于人、鼠的ES细胞,使用鸡胚ES细胞可以克服伦理方面的限制,且取材方便,模拟临床治疗实验具有十分明显优势。因此本研究以家鸡做为实验动物模型,在比较ES和SSCs异同点的基础上,筛选适宜视黄酸(RA)浓度,比较不同基质蛋白、支持细胞、不同时期睾丸提取液和睾丸细胞条件培养液诱导ES向雄性生殖细胞方向分化的效果,为建立适宜的诱导体系和后来的临床研究提供参考。同时将诱变后有抗病毒活性的MMx蛋白通过SSCs体内介导法制备抗病转基因鸡,为制备转基因动物和培育新品种提供新的途径和思路。研究结果如下：
(1)采用免疫荧光和碱性磷酸酶联合检测法鉴定鸡ES细胞和SSCs细胞的干细胞特性,RT-PCR方法检测两种细胞在Cvh、C-kit、DAZL、GDF3、integrin α6、integrin β1、 Nanog、Oct-4、Sox2、Stra8基因表达上的异同性,并通过免疫细胞化学和Western blot检测TRA-1-60、TRA-1-81、C-kit、DAZL、integrin a6、integrin β1、Sox2蛋白在两细胞上的表达差异情况。结果表明：ESC和SSCs基因表达存在差异,即未分化的ES细胞表达GDF3、Nanog、Sox2基因；SSCs表达C-kit、integrin α6、integrin β1、Cvh、Stra8、 Dazl基因,两种细胞mRNA均检测到Oct-4、β-actin基因的表达。蛋白水平上,免疫细胞化学方法和western blot方法检测的5个蛋白中ES细胞表达Sox2蛋白,SSCs表达C-kit、 integrina6、integrinβ1、Dazl蛋白。这两细胞的特异性标记物,可为ES与SSCs细胞的鉴定提供依据。
(2)在鉴定鸡胚ES细胞性别的基础上,将雄性ES细胞接种到24孔板中,分别采用10-3mol/L、10-4mol/L、10-5mol/L、10-6mol/LRA浓度进行诱导,通过细胞形态变化和上章获得的差异基因表达量变化,筛选适宜RA诱导浓度,同时探讨RA促进鸡ES向生殖细胞方向分化的潜能。结果显示：根据半定量PCR和诱导过程中细胞形态观察,确定了RA的适宜诱导浓度为10-5mol/L。在适宜浓度诱导过程中,诱导4天时类胚体出现,随后类胚体逐渐增大,8d时类胚体开始解体,随后变为单个小的圆形细胞,但10天时未观察到生殖细胞样细胞。诱导过程中相应基因表达量也发生变化：ES细胞特异基因Nanog、Sox2表达量下降,生殖细胞特异基因Stra8、Dazl、integrin α6、integrin β1、C-kit表达量上升。特别是Stra8基因,与自分化相比,显著升高。结果证明RA可启动生殖细胞相应基因的表达,对鸡胚ES细胞向雄性生殖细胞方向分化具有积极作用。
(3)ES细胞在体内特定小生境中发育,其内各种基质蛋白与其他细胞相互作用影响ES细胞的增殖分化。本实验将ES细胞分别接种于胶原蛋白(collagen)、纤维连接蛋白(fibronectin)和层粘连蛋白(laminin)包被的培养皿中,在适宜RA浓度作用下研究ES细胞向生殖细胞方向发育的可能性。结果显示：三组诱导实验中,细胞水平上RA+fibronectin诱导4天时最早观察到类胚体的出现,随后细胞体积增大,在10天时观察到精原样细胞。层粘连蛋白和胶原蛋白组在诱导6天时观察到类胚体形态,而层粘连蛋白组在10天时观察到SSCs样细胞形态。相关基因表达量变化也较明显。三组中ES细胞特异基因Nanog和Sox2基因表达量均迅速下降,而纤维连接蛋白实验组中下降程度最高。Dazl表达量均有变化,RA+collagen组中在诱导2天时表达量开始升高,到第8d表达量最高,10天时表达量下降；RA+laminin组中在诱导4天时表达量为最高,随后表达量下降,到第10天表达量降到最低,显著低于SSCs本身表达量。RA+fibronectin组自第4天开始表达量一直维持高表达状态。Stra8、integrin β1、integrina6基因在三组中规律较一致,随着诱导时间的延长,表达量持续升高,但诱导10天时表达量未达到SSCs本身表达量。C-kit表达量规律不明显,RA+collagen组诱导4天时表达量最高,随后降低。RA+fibronectin组表达量变化不大。结论：ES特异基因表达量为下降趋势,C-kit、Stra8、integrinβ1、 integrina6表达量总体为上升趋势,其中RA+fibronectin和诱导组中基因变化明显。根据细胞形态变化情况和特异基因变化情况比较,三组试验诱导效果均比RA单独诱导效果好,且三种基质蛋白中RA+fibronectin诱导组效果较好。
(4)研究表明支持细胞是精原细胞小境的关键因子,可能会通过分泌一些生长因子或者通过与细胞接触来调节生殖细胞发育分化过程。本实验在适宜RA浓度条件下,不同时期睾丸提取液和睾丸组织条件培养基、鸡胚睾丸支持细胞共培养后及综合因素诱导,探讨不同方法对ES向雄性生殖细胞方向分化的影响。结果显示：RA+支持细胞实验组在诱导2天时类胚体出现,随后其体积增大,诱导10天观察到精原样细胞。基因变化过程中,Nanog基因表达量降低,而Sox2基因高表达。Dazl在诱导2天时表达量达到最高,随后逐渐降低。而Stra8、integrinβ1、integrina6基因表达量在诱导4天开始一直维持高水平表达,且在诱导10天时检测到Sox2、Daz1、integrinβ1、integrina6蛋白表达。在不同时期睾丸提取液和睾丸条件培养液实验组中,细胞形态变化差异较小,在诱导4天时类胚体出现,诱导10天时SSCs样细胞出现。在性成熟期睾丸提取液和条件培养液试验中8d出现SSCs样细胞。基因表达量上Nanog表达量逐渐上升,而Sox2表达量下降。性成熟睾丸提取液和条件培养液中,Dazl、Stra8、integrinβ1和integrina6表达量逐渐升高。而在RA+Fibronectin+支持细胞综合因素诱导过程中,诱导第2天即出现类胚体,同时ES特异基因Nanog、Sox2表达量下降明显。Dazl, Stra8,integrin(31, integrina6表达量逐渐升高。C-kit在诱导4天时表达量升高,随后保持相同水平,诱导10天时,检测到Dazl、C-kit蛋白的表达。结论：不同时期睾丸提取液和睾丸组织培养条件培养基诱导下,性成熟期睾丸提取液和睾丸组织条件培养液诱导效果较好。与单因素和双因素诱导相比,综合因素诱导效果并没有明显变化。几组实验中RA与支持细胞共诱导组效果最好。
(5)利用精原干细胞介导的方法体内转染融合基因EGFP-MMx,制备抗病毒性疾病转基因鸡。采用脂质体包埋法,睾丸内注射EGFP-MMx融合基因,术后采集30d、40d、50d、60d、70d、80d睾丸组织进行冰冻切片、基因组PCR、点杂交实验,转染后60d公鸡精液人工授精于正常母鸡,对后代血液采用PCR检测技术、RT-PCR和Western blot方法检测外源基因EGFP-MMx的整合表达情况。结果显示：睾丸冰冻切片结果显示大部分实验鸡有绿色荧光,且在60d荧光数及亮度达到最高水平。提取18只实验公鸡睾丸组织基因组,分别以EFGP和MMx基因序列设计引物进行PCR检测和点杂交,阳性率分别为77.8%和72.2%。提取4只实验公鸡7个不同时期的精液基因组进行PCR检测,阳性率为25%。对于38只F1代进行血液DNA.RNA和蛋白水平检测,阳性率分别为10.53%(4/38)、10.53%(4/38)、7.89%(3/38)。结果证明外源基因通过睾丸注射法可整合到基因组上,可作为培育抗病转基因鸡的新途径。
综上,本实验探索了不同方法对家鸡ES细胞向雄性生殖细胞的诱导效果及SSCs细胞体内介导制备转基因鸡的可行性,得出以下结论：RA作用下,将ES细胞和支持细胞共培养效果较好,可较好的诱导ES向雄性生殖细胞方向分化；通过SSCs体内介导法,获得3只携带抗病基因的转基因鸡。这些结果一方面能为雄性生殖细胞的临床应用提供理论基础,另一方面可为培育新品种提供新的思路。
In the sexual reproduction of organisms, germ cells undertake the genetic information passed in generations and generate new individuals by sperm-egg binding. The characteristic of the germ cells make it become one of the research objects of animal embryology, developmental biology and cell biology. Male germ cells could be used to replace embryonic stem cells for disease therapy and genetic modification since it has the merits of pluripotency and do not involve in ethical and immune rejection problem, therefore, it caused great concern in the biological community. It's necessary and urgent to begin the research of how to induce stem cells from other tissues differentiated into spermatogonial stem cells. Recently, the research on ESCs differentiated into male germ cells has already been done. But the efficiency is still low.
Poultry embryonic stem cells has no restricted within ethics as to human embryonic stem cells(hES) and mouse embryonic stem cells(mES), and can easily be obtained, holding an dominant advantage in simulation clinical treatment. Hence, in this study, poultry species were used as animal model in this study. On the basis of the difference between ES and SSCs, retinoic acid(RA) concentration wes initially screened, in combination with three matrix proteins, sertoli cells, testicular abstracts and testicular cell conditioned medium obtained from different periods, to identify the efficiency of germ cell differentiation derived from chicken ESCs. The study provides conference for build appropriate induction system and clinical research. Meanwhile used in vivo SSCs-mediated way transfer active-antiviral MMx protein to generate antiviral transgenic chicken, and provided new ways and thinking for prepared transgenic animals and cultivated new species. The results addressed as follows:
(1)To identify the two stem cells, the similarities and differences between the chicken embryonic stem cell (ESC) and spermatogonial stem cell (SSCs) were studied. The characteristics of ESC and SSCs were analyzed with the expression of alkaline phosphatase (AKP), immunologic markers and pluripotency markers. Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence and western blotting were used on the two cells. Of the ten genes we detected, Oct4was detected in both of the two cells, while GDF3、Nanog、 Sox2genes only expressed in undifferentiated ES and Dazl, Cvh, Stra8, integrina6, integrinβ1and C-kit genes were found in SSCs, which showed there are differences in ES and SSCs at the level of mRNA. Immunofluorescence and western blotting showed that Sox2protein expressed in ES while C-kit, integrina6, integrinβ1and Dazl proteins were found in SSCs. Our result showed chicken ES and SSCs had common characteristics of stem cell, but exhibited significant discrepancy at the level of mRNA and protein. The different specific markers between the two cells not only suggested the differentiation potential of the two cells but also offered theoretical and practical basis for identifying the two cells.
(2) In this part, On the basis of identifying the chicken ES cells sex, the ZZ ES cells were transferred onto24-well plates. For screening the suitable concentration of RA, both cell morphologic and genes expression were detected under different concentration (10-3mol/L、10-4mol/L、10-5mol/L、10-6mol/L). The results showed10-5mol/L was the best. Under this concentration, embryonic body was observed on induced4days. With the prolongation of induction time, EB became large gradually. Until induced8d, EB began to disintegrate and became single round cells. SSCs-like cells did not be observed on the induced10days. On this induction process, expression of many genes changed gradually. ES specific genes Nanog and Sox2expression descended while germ cell specific genes Stra8、Dazl、integrina6、intrgrinβ1、C-kit ascended especially Stra8. we supposed RA was in charge of notable heighten of Stra8expression. In conclusion, RA can promote ES differentiation into germ cell.
(3)ES cells developed in the specific niche. The proliferation and differentiation of ES cells are affected by the interaction between variety of matrix proteins and other cells. This experiment studied the possibility of ES cells differentiated to germ cells when ES directly contacted with collagen, fibronectin and laminin under the RA medium. The results showed:on the RA+fibronectin group, embryonic body was observed at induced4days while on the other two groups were induced6days. On RA+fibronectin and RA+laminin groups, SSCs-like cells were seen on induced10days while RA+collagen group did not been observed on induced10days. Related gene expression changes were more significantly. The expression of Nanog and Sox2genes gradually descended especially RA+fibronectin group. We found distinctive difference on Dazl expression between the three groups. On the RA+collagen group, the expression of Dazl gene rising from induced2days, and reached the peak at8days, then descended on10days. On RA+laminin group, Dazl expression reached the peak at induced4days, then gradually descent. On RA+fibronectin group, the expression of Dazl gene began to express from the2st day and expressed significantly at the4th day, then maintained a higher level at the6th,8th and10th. The expression of Stra8、integrinβ1、integrina6genes increased as induced time prolonging. Conclusion:as the induced time prolonging, the expression of Nanog and Sox2genes descent and C-kit, Stra8, integrin(31, integrina6ascend. Compared on cell morphologic change and specific genes expression, the RA+marix protein groups had better induce effection than RA especially RA+fibronectin.
(4) Research suggested sertoli cells are the key factor on SSCs niche, which can secrete some growth factors to regulate the progress of germ cell development. In this experiment, different methods were used to induce ES differentiation into germ cell, which were testes abstract and testes medium from different periods, chichen embryonic sertoli cells co-culture with ES and comprehend factors.The results were:On the chicken embryonic sertoli cells group, embryonic body was observed on the induced2days and SSCs-like cells on the induced10days less. Nanog gene expressed lower as time going induced by RA. The expression of Dazl was seen at the peak on induced2days and gradually descent following. Stra8, integrin(31and integrina6genes began to express from the2st day and expressed significantly at the4th day, then maintained a higher level at the6th,8th and10th. On the groups of RA+testes abstract and testes medium from different periods, embryoid was observed on the induced4days and SSCs-like cells on the8d at sexual maturity testes abstract and testes medium groups while on the10days on the other groups. Nanog gene expressed higher as time going induced by RA while Sox2is lower. Dazl、Stra8、integrinβ1、and integrina6expression were gradually ascend. On the comprehend group, embryoid was seen on induced2days and SSCs-like cells on the8d. Nanog and Sox2genes expressed lower than RA group as time going and Dazl, Stra8, integrinp1, integrina6and C-kit higher. Conclusion:under RA+testes abstract and testes medium from different periods, sexual maturity testes abstract and testes medium induced effect was better than the other groups. The induction effect of comprehend group (RA+fibronectin+sertoli cells) was almost same as the sertoli cells group. Between those groups, RA+sertoli group is the best induced methods.
(5)In order to generate anti-viral transgenic chickens through transfected spermatogonial stem cell with fusion gene EGFP-MMx. After injecting fusion gene EGFP-MMx into testes, tissues frozen section, PCR and dot blot of testes was performed at the30d,40d,50d,60d,70d and80d, a few of normal hen were inseminated artificially with cock semen of60d. Finally PCR, reverse transcription-PCR and Western Blot were used to detect the integration of exogenous gene EGFP-MMx of the offspring. Tissues frozen section results showed18experimental chickens have the green fluorescence and the number and brightness of fluorescence was at the highest level. DNA was extracted from18experimental chicken and PCR and dot blot were preformed to detect the EGFP and MMx gene. The positive rate was77.8%and72.2%respectively. The four experimental chickens' sperm were collected and DNA was abstracted for PCR performing. The PCR positive rate was25%(1/4). PCR and RT-PCR positive rate was10.53%(438) in F1blood genome and RNA respectively, while Western Blot positive rate of F1blood was7.89%(3/38). Taken together, it was proved the possibility of cultivating anti-viral transgenic chickens by the method of testes injection method which could integrate foreign genes into the genome
In conclusion, we compared different methods effect on chicken ES differentiation to male germ cell and explored the possibility of cultivating anti-viral transgenic chickens by the method of testes injection method:under the RA medium, the group of ES cells cocultured with sertoli cells group has the best effect on chicken ES differentiation to male germ cell; by SSCs-mediated in vivo, we obtained3transgenic chickens with antiviral gene. The results can provide the theoretical basis for SSCs clinical application and provide an idea for breeding new variety
引文
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