利用限定因子诱导牛iPS细胞
摘要
iPS细胞在家畜新品种培育及细胞重编程机制的研究中具有重要意义。本研究采用慢病毒感染法以融合有绿色荧光蛋白的pSox2、hOct4、pMyc和pKlf4四种限定因子诱导牛胎儿皮肤成纤维细胞为iPS细胞,并对形成的iPS细胞克隆进行鉴定。试验结果如下:
牛胎儿成纤维细胞经过慢病毒感染后的第15 d开始发生形态改变,由长梭形变成边缘不规则的圆球形。接到饲养层后细胞贴附生长,形态改变的细胞不但数量增多,而且聚集一起呈扁平克隆状致密生长,边界清楚。高倍镜下观察牛iPS克隆细胞核较大,核质比率较高,荧光下观察牛iPS克隆中有部分细胞能够表达绿色荧光蛋白。进一步传代培养,牛iPS细胞克隆数不断增多,而且传至十几代后仍保持稳定增长。冻存-复苏后的细胞继续保持稳定的传代培养性能。待细胞接到饲养层后随着iPS细胞克隆的增殖和稳定传代,绿色荧光表达强度逐渐减弱,而且表达绿色荧光蛋白的克隆也逐渐减少。
秋水仙素处理牛iPS细胞克隆进行核型分析检测,结果显示诱导出的牛iPS细胞染色体条数为60,与正常的牛细胞核型相吻合;碱性磷酸酶染色呈阳性。RT-PCR扩增鉴定iPS细胞克隆中多潜能相关基因Oct4、Sox2、Nanog、c-Myc和Klf4的表达情况,结果Oct4、Nanog、c-Myc和Klf4均呈阳性,Sox2为阴性,而作为对照的牛胎儿成纤维细胞中均无上述5种基因的表达。免疫荧光染色法对牛iPS细胞克隆中多能性相关基因的蛋白进行检测,Oct4和Nanog蛋白呈阳性,但其余蛋白表达均呈现阴性。牛iPS细胞克隆在体外悬浮培养的第10 d,可观察到拟胚体的形成,并且拟胚体易于聚集融合。将牛iPS细胞多点多次注射到裸鼠背侧皮下,6周后在裸鼠注射部位成功长出大小不等的多个畸胎瘤,取出的畸胎瘤形态呈不规则的圆球状,被一层白膜包裹,质地柔软,侵袭性强,畸胎瘤表面分布很多血管与裸鼠皮下组织相连。经组织切片和HE染色可观察到裸鼠皮下生长的畸胎瘤中存在由内、中、外三胚层分化而来的组织。
结论:利用限定因子pSox2、hOct4、pc-Myc和pKlf4成功将牛胎儿成纤维细胞诱导为牛iPS细胞。
Induced pluripotent stem cell (iPSC)by defined factors is of great importance to producing transgenic animals and studying the reprogramming mechanism of cell. So, in this study, we attempted to derive induced pluripotent cells from bovine fetal skin fibroblast cells (bFFC) with four defined factors.
Four lentivirus plasmids carried pSox2, hOct4, pMyc and pKlf4 gene respectively were transfected into 293T cells, and the produced lentivirus that could generate the defined factors were colleced and mixed, then the mixture was added into cell culture medium to infect bFFCs. We continued to observe the cell morphology and GFP protein expression. Once the shape of fibroblasts cells began to change, they would be seeded onto mouse embryonic fibroblast feeders. After several passages, clonies with clear-edege were selected and identified. Results as follow:
After infected with lentivirus for 15 days, some bFF cells began to exhibit a progressive morphological change from spindle-shaped to globular. When seeded onto feeder, some flat colonies with cutting edege formed, Clonies cells had big caryon and exhibited a high nucleus/cytoplasm ratio. After a dozen passages, the intensity of green fluorescence gradually decreased in those clonies cells, and the number of clonies which express green fluorescent protein gradually decreased.
Most clonies cells exhibited normal karyotype, and were positive for alkaline phosphatase test. When detected the mRNA by RT-PCR, all selected clonies were positive for Oct4, Nanog, c-Myc and Klf4 but negtive for Sox2. By contrast, bFF cells were negative for all these genes mRNA detection. As a positive control, the mouse embryonic stem cells transcripted all these five pluripotent-related genes mRNA. Immunocytoche- mical analysis showed that Oct4 and Nanog protein could be expressed in those induced clonies cells, but the other pluripotency markers ( Sox2,SSEA-1,SSEA-3,SSEA-4,TRA-1-60 and TRA-1-80 ) could not be expressed. When suspended and cultured in ordinary culture medium, some embryoid bodies formed 10 days later, and these embryoid bodies were apt to aggregation. When injected the induced clonies cells bovine iPS cells into the dorsal skin of nude mice, a lot of teratomas containning tissues from ectoderm, mesoderm, and endoderm formed 6 weeks later. The teratoma was irregular spherical shape, soft, strong and aggressive. The surface of teratomas distribute many blood vessels which iconnected with nude mice subcutaneous tissue.
Conclusion: We successfully induced the bovine iPS cells from fetal bovine primitive fibroblast cell by four defined factors pSox2, hOct4, pMyc and pKlf4, and founded the bovine iPS cell line.
牛胎儿成纤维细胞经过慢病毒感染后的第15 d开始发生形态改变,由长梭形变成边缘不规则的圆球形。接到饲养层后细胞贴附生长,形态改变的细胞不但数量增多,而且聚集一起呈扁平克隆状致密生长,边界清楚。高倍镜下观察牛iPS克隆细胞核较大,核质比率较高,荧光下观察牛iPS克隆中有部分细胞能够表达绿色荧光蛋白。进一步传代培养,牛iPS细胞克隆数不断增多,而且传至十几代后仍保持稳定增长。冻存-复苏后的细胞继续保持稳定的传代培养性能。待细胞接到饲养层后随着iPS细胞克隆的增殖和稳定传代,绿色荧光表达强度逐渐减弱,而且表达绿色荧光蛋白的克隆也逐渐减少。
秋水仙素处理牛iPS细胞克隆进行核型分析检测,结果显示诱导出的牛iPS细胞染色体条数为60,与正常的牛细胞核型相吻合;碱性磷酸酶染色呈阳性。RT-PCR扩增鉴定iPS细胞克隆中多潜能相关基因Oct4、Sox2、Nanog、c-Myc和Klf4的表达情况,结果Oct4、Nanog、c-Myc和Klf4均呈阳性,Sox2为阴性,而作为对照的牛胎儿成纤维细胞中均无上述5种基因的表达。免疫荧光染色法对牛iPS细胞克隆中多能性相关基因的蛋白进行检测,Oct4和Nanog蛋白呈阳性,但其余蛋白表达均呈现阴性。牛iPS细胞克隆在体外悬浮培养的第10 d,可观察到拟胚体的形成,并且拟胚体易于聚集融合。将牛iPS细胞多点多次注射到裸鼠背侧皮下,6周后在裸鼠注射部位成功长出大小不等的多个畸胎瘤,取出的畸胎瘤形态呈不规则的圆球状,被一层白膜包裹,质地柔软,侵袭性强,畸胎瘤表面分布很多血管与裸鼠皮下组织相连。经组织切片和HE染色可观察到裸鼠皮下生长的畸胎瘤中存在由内、中、外三胚层分化而来的组织。
结论:利用限定因子pSox2、hOct4、pc-Myc和pKlf4成功将牛胎儿成纤维细胞诱导为牛iPS细胞。
Induced pluripotent stem cell (iPSC)by defined factors is of great importance to producing transgenic animals and studying the reprogramming mechanism of cell. So, in this study, we attempted to derive induced pluripotent cells from bovine fetal skin fibroblast cells (bFFC) with four defined factors.
Four lentivirus plasmids carried pSox2, hOct4, pMyc and pKlf4 gene respectively were transfected into 293T cells, and the produced lentivirus that could generate the defined factors were colleced and mixed, then the mixture was added into cell culture medium to infect bFFCs. We continued to observe the cell morphology and GFP protein expression. Once the shape of fibroblasts cells began to change, they would be seeded onto mouse embryonic fibroblast feeders. After several passages, clonies with clear-edege were selected and identified. Results as follow:
After infected with lentivirus for 15 days, some bFF cells began to exhibit a progressive morphological change from spindle-shaped to globular. When seeded onto feeder, some flat colonies with cutting edege formed, Clonies cells had big caryon and exhibited a high nucleus/cytoplasm ratio. After a dozen passages, the intensity of green fluorescence gradually decreased in those clonies cells, and the number of clonies which express green fluorescent protein gradually decreased.
Most clonies cells exhibited normal karyotype, and were positive for alkaline phosphatase test. When detected the mRNA by RT-PCR, all selected clonies were positive for Oct4, Nanog, c-Myc and Klf4 but negtive for Sox2. By contrast, bFF cells were negative for all these genes mRNA detection. As a positive control, the mouse embryonic stem cells transcripted all these five pluripotent-related genes mRNA. Immunocytoche- mical analysis showed that Oct4 and Nanog protein could be expressed in those induced clonies cells, but the other pluripotency markers ( Sox2,SSEA-1,SSEA-3,SSEA-4,TRA-1-60 and TRA-1-80 ) could not be expressed. When suspended and cultured in ordinary culture medium, some embryoid bodies formed 10 days later, and these embryoid bodies were apt to aggregation. When injected the induced clonies cells bovine iPS cells into the dorsal skin of nude mice, a lot of teratomas containning tissues from ectoderm, mesoderm, and endoderm formed 6 weeks later. The teratoma was irregular spherical shape, soft, strong and aggressive. The surface of teratomas distribute many blood vessels which iconnected with nude mice subcutaneous tissue.
Conclusion: We successfully induced the bovine iPS cells from fetal bovine primitive fibroblast cell by four defined factors pSox2, hOct4, pMyc and pKlf4, and founded the bovine iPS cell line.
引文
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