建立稳定表达猪LIF蛋白的小鼠STO细胞系

Establishment of STO cell lines expressing porcine leukemia inhibitory factor

目的:在STO小鼠成纤维细胞中表达猪白血病抑制因子(leukemia inhibitory factor,LIF),并尝试使用其作为饲养层培养大鼠诱导多能性干细胞(i PS细胞)。方法:通过脂质体转染的技术,将已构建的猪LIF基因真核表达载体p CAG-p LIF转染至小鼠STO细胞中,获得能够稳定表达猪LIF的转基因STO细胞(STO-p LIF细胞)。通过RT-PCR、q PCR、Western blot等方法检测STO-p LIF细胞中猪LIF基因的表达量,选择高效表达猪LIF的STO-p LIF细胞作为饲养层,培养大鼠i PS细胞。通过生长曲线检测、碱性磷酸酶染色、干细胞多能因子的RT-PCR和免疫荧光染色等方法,检验STO-p LIF细胞饲养层在大鼠i PS细胞培养方面的功能。结果:STO-p LIF细胞中LIF RNA和蛋白表达水平均上调(P<0.05),而该细胞系作为饲养层所培养的大鼠i PS细胞在形态、多能性因子表达方面更接近于传统培养的大鼠i PS细胞,与无外源添加LIF所培养的大鼠i PS存在差异(P<0.05)。结论:成功获得了稳定表达猪LIF基因的STO-p LIF细胞,并证明该细胞作为饲养层在大鼠i PS细胞培养中具有一定优势。

Objective：To establish the STO cell lines expressing the recombinant porcine leukemia inhibitory factor（LIF）and try to culture the rat induced pluripotency stem cells（ri PSCs）with the established STO-p LIF cells as the feeder layer. Methods：We established the stable STO cell lines（STO-p LIF cells） which express LIF gene effectively by lipofectin transfection with the pig LIF eukaryotic expression vector p CAG-p LIF. The expression level of the inserted exogenous LIF gene was tested by RT-PCR,q PCR and Western blot. The cell lines which expressed pig LIF efficiently were used as the feeder cells to culture the ri PSCs. Besides the RTPCR testing the pluripotency factors,we also undertook the growth curve and the alkaline phosphates expressions detection as well as the immunocytofluorescent to identify the pluripotency of the ri PSCs. Results： The expression level of the inserted exogenous LIF gene of the STO-p LIF cell lines was higher than that of the STO（P〈0.05）, and the ri PSCs cultured with the STO-p LIF were close to the traditional ri PSCs, and had difference with the ri PSCs cultured with the N2B27 without LIF（P〈0.05）.Conclusion：The stable STO cell lines effectively expressing porcine recombinant porcine leukemia inhibitory factor were established successfully and these cells can maintain the pluripotency and self renew of ri PSCs as the feeder cells.