鸡精原干细胞体外培养及其诱导分化的研究
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摘要
精原干细胞(spermatogonial stem cells, SSCs)是指位于睾丸精曲小管基膜上既能自我更新维持自身群体数量恒定,又能定向分化产生精母细胞的一类干细胞。由于SSCs在体外基因修饰、转基因动物生产等方面比胚胎干细胞更具优势,SSCs逐渐成为干细胞研究领域的一个热点。本研究探索了三种不同的饲养层,即睾丸支持细胞、鸡胚成纤维细胞和鸡胚肝细胞对SSCs体外培养的影响以及培养传代后SSCs保持多能性的潜力;应用特异性化学物质定向诱导鸡胚SSCs向成骨细胞、神经元样细胞分化,并对各诱导剂的组合效果及适合浓度进行了比较。以期能为禽类SSCs的研究和利用提供参考资料,为医学研究提供实验素材。研究结果总结如下几点:
1.分别以睾丸支持细胞、鸡胚成纤维细胞和鸡胚肝细胞作为饲养层,比较三种不同的饲养层对SSCs体外培养的影响。结果显示:以鸡胚成纤维细胞饲养层培养SSCs可传至四代,每代的AKP阳性克隆率分别为45%、40%、36%和21%。以睾丸支持细胞为饲养层进行培养时传至三代,每代的AKP阳性克隆率分别是40%、32%、和22%。而以鸡肝细胞作为饲养层,SSCs未见有克隆形成,不能进行传代培养,表明鸡胚成纤维细胞适合作为SSCs的饲养层,而鸡胚肝细胞不适合作为SSCs饲养层。
2.以地塞米松、β-甘油磷酸钠、维生素C为诱导剂,诱导鸡胚SSCs向成骨细胞分化,比较以上各种诱导剂不同浓度和不同组合形成的诱导液I、II、III、IV,对传代后SSCs的诱导分化效果,钙结节Von Kossa’s法、改良的钙钴法及免疫组化法对成骨细胞进行鉴定,实验结果显示:诱导分化液Ⅱ+Ⅲ的诱导分化效果最好,诱导分化率为85%,与单独使用诱导分化液Ⅱ、Ⅲ、Ⅳ的差异显著(p<0.01),但与单独使用诱导液Ⅰ的差异不显著(P>0.05),其诱导效果略好于单独使用诱导液Ⅰ(诱导分化率为83%)。SSCs被诱导15-21天后分化为成骨细胞,诱导后的细胞Von Kossa’s染色可见细胞间布满黑色颗粒,提示有矿化基质沉积;改良钙钴法碱性磷酸酶染色胞浆呈深棕色或深黑色,免疫组化法染色细胞呈阳性。
3.以维甲酸(RA)、BHA、3-异丁基-1-甲基黄嘌呤(IBMX)为诱导剂,诱导SSCs向神经元样细胞分化,比较以上各种诱导剂不同浓度和不同组合形成的诱导液I、II、III、IV,对传代后SSCs的诱导分化效果,甲苯胺蓝染色和免疫组化进行鉴定。结果显示:诱导分化剂Ⅰ(RA)和诱导分化剂Ⅱ(IBMX)的诱导分化效果最好,诱导分化率分别为83%、81%,两者之间无显著差异(P>0.05),但与其他诱导分化剂之间差异极显著(P<0.01)。SSCs被诱导3-7天后分化为神经元样细胞,甲苯胺蓝染色阳性率为78-85%。免疫组化法检测诱导后细胞的神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF)和胶质纤维酸性蛋白(GFAP)的表达,结果为NSE呈阳性(+)、NF(+)阳性、GFAP呈阴性(-)。
Spermatogonial stem cells were located at seminiferous tubule which not only can maintain the number of themselves but also can differentiate to spermatocyte,are crucial for related stem cells research. This study investigate the vegetative character of spermatogonial stem cells(SSCs) and effect of different feeder when chicken SSCs were cultured in vitro, chicken SSCs were respectively cultured on chicken live cells feeder, chicken CEL feeder and sertoli cells of testicle, explore the capability of SSCs to differentiated into osteoblasts and neuron-like cells under different experimental conditions in vitro. We attempted to prepare the ground for the establishment of chicken SSCs lines and offer cell model for growth and differentiation of cells.
The main results were as follows:
1. In order to investigate the vegetative character of spermatogonial stem cells(SSCs) and effect of different feeder when chicken SSCs were cultured in vitro, chicken SSCs were respectively cultured on chicken live cells feeder, chicken CEL feeder and sertoli cells of testicle. The result showed: SSCs growth well and the vegetative character of them had no any change when they were subcultured on chicken CEL feeder and chicken live cells . the 4 passage rates of AKP positive colony respectively was 45%、40%、36% and 21% when SSCs cultured on chicken CEF and 40%、32% and 22% when SSCs cultured with testicle sertoli . No colony were observed when SSCs were cultured on chicken live cells feeder.
2. SSCs were induced into osteoblasts by desamethason, glycerol 2-phosphate disodium salt hydrate and vitamin C and were examined with Von Kossa’s stain、cytochemistry staining and immunohistochemistry stain. The differentiated cells were positive for ALP, the Von Kossa’s stain was positive and immunohistochemistry stain.The result showed that the inductionⅡcombined withⅢshowed best results, about 85% positive cells formed 15～21d later. Compared to single use of inductionⅠ,Ⅱ,ⅢandⅣ, The results showed that the chicken SSCs could be differentiated into osteoblasts ,which showed the SSCs have the capability of multipotential differentiation in vitro.
3. SSCs were induced into neuron-like cells in the medium containing RA and IBMX, the differentiated cells were identified by toluidine blue stain. The result showed that the inductionⅠandⅡshowds best results and the differentiation rates were 83% and 81% respectively.There was no significant difference between inductionⅠandⅡ(P﹥0.05).SSCs were identified by toluidint blue stain 3～7d later.That showed the SSCs have the capability of multipotential differentiation in vitro.
1.分别以睾丸支持细胞、鸡胚成纤维细胞和鸡胚肝细胞作为饲养层,比较三种不同的饲养层对SSCs体外培养的影响。结果显示:以鸡胚成纤维细胞饲养层培养SSCs可传至四代,每代的AKP阳性克隆率分别为45%、40%、36%和21%。以睾丸支持细胞为饲养层进行培养时传至三代,每代的AKP阳性克隆率分别是40%、32%、和22%。而以鸡肝细胞作为饲养层,SSCs未见有克隆形成,不能进行传代培养,表明鸡胚成纤维细胞适合作为SSCs的饲养层,而鸡胚肝细胞不适合作为SSCs饲养层。
2.以地塞米松、β-甘油磷酸钠、维生素C为诱导剂,诱导鸡胚SSCs向成骨细胞分化,比较以上各种诱导剂不同浓度和不同组合形成的诱导液I、II、III、IV,对传代后SSCs的诱导分化效果,钙结节Von Kossa’s法、改良的钙钴法及免疫组化法对成骨细胞进行鉴定,实验结果显示:诱导分化液Ⅱ+Ⅲ的诱导分化效果最好,诱导分化率为85%,与单独使用诱导分化液Ⅱ、Ⅲ、Ⅳ的差异显著(p<0.01),但与单独使用诱导液Ⅰ的差异不显著(P>0.05),其诱导效果略好于单独使用诱导液Ⅰ(诱导分化率为83%)。SSCs被诱导15-21天后分化为成骨细胞,诱导后的细胞Von Kossa’s染色可见细胞间布满黑色颗粒,提示有矿化基质沉积;改良钙钴法碱性磷酸酶染色胞浆呈深棕色或深黑色,免疫组化法染色细胞呈阳性。
3.以维甲酸(RA)、BHA、3-异丁基-1-甲基黄嘌呤(IBMX)为诱导剂,诱导SSCs向神经元样细胞分化,比较以上各种诱导剂不同浓度和不同组合形成的诱导液I、II、III、IV,对传代后SSCs的诱导分化效果,甲苯胺蓝染色和免疫组化进行鉴定。结果显示:诱导分化剂Ⅰ(RA)和诱导分化剂Ⅱ(IBMX)的诱导分化效果最好,诱导分化率分别为83%、81%,两者之间无显著差异(P>0.05),但与其他诱导分化剂之间差异极显著(P<0.01)。SSCs被诱导3-7天后分化为神经元样细胞,甲苯胺蓝染色阳性率为78-85%。免疫组化法检测诱导后细胞的神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF)和胶质纤维酸性蛋白(GFAP)的表达,结果为NSE呈阳性(+)、NF(+)阳性、GFAP呈阴性(-)。
Spermatogonial stem cells were located at seminiferous tubule which not only can maintain the number of themselves but also can differentiate to spermatocyte,are crucial for related stem cells research. This study investigate the vegetative character of spermatogonial stem cells(SSCs) and effect of different feeder when chicken SSCs were cultured in vitro, chicken SSCs were respectively cultured on chicken live cells feeder, chicken CEL feeder and sertoli cells of testicle, explore the capability of SSCs to differentiated into osteoblasts and neuron-like cells under different experimental conditions in vitro. We attempted to prepare the ground for the establishment of chicken SSCs lines and offer cell model for growth and differentiation of cells.
The main results were as follows:
1. In order to investigate the vegetative character of spermatogonial stem cells(SSCs) and effect of different feeder when chicken SSCs were cultured in vitro, chicken SSCs were respectively cultured on chicken live cells feeder, chicken CEL feeder and sertoli cells of testicle. The result showed: SSCs growth well and the vegetative character of them had no any change when they were subcultured on chicken CEL feeder and chicken live cells . the 4 passage rates of AKP positive colony respectively was 45%、40%、36% and 21% when SSCs cultured on chicken CEF and 40%、32% and 22% when SSCs cultured with testicle sertoli . No colony were observed when SSCs were cultured on chicken live cells feeder.
2. SSCs were induced into osteoblasts by desamethason, glycerol 2-phosphate disodium salt hydrate and vitamin C and were examined with Von Kossa’s stain、cytochemistry staining and immunohistochemistry stain. The differentiated cells were positive for ALP, the Von Kossa’s stain was positive and immunohistochemistry stain.The result showed that the inductionⅡcombined withⅢshowed best results, about 85% positive cells formed 15～21d later. Compared to single use of inductionⅠ,Ⅱ,ⅢandⅣ, The results showed that the chicken SSCs could be differentiated into osteoblasts ,which showed the SSCs have the capability of multipotential differentiation in vitro.
3. SSCs were induced into neuron-like cells in the medium containing RA and IBMX, the differentiated cells were identified by toluidine blue stain. The result showed that the inductionⅠandⅡshowds best results and the differentiation rates were 83% and 81% respectively.There was no significant difference between inductionⅠandⅡ(P﹥0.05).SSCs were identified by toluidint blue stain 3～7d later.That showed the SSCs have the capability of multipotential differentiation in vitro.
引文
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