摘要
多能性干细胞是一类具有无限自我更新能力及分化为功能细胞的多潜能特殊细胞。科学家们通过各种方法希望获得多能性干细胞,然而目前方法都存在一定问题。通过4种转录因子诱导将已分化成体细胞重编程为多能性干细胞(iPS细胞),很好的解决了目前存在于多能性干细胞获得中的问题。但是iPS细胞的诱导方法并不完善,需要解决其安全性和诱导效率方面的问题。本课题通过原核表达人类Oct4蛋白与细胞穿膜肽融合蛋白,利用人神经干细胞对单个蛋白诱导iPS细胞进行了探索,主要结果如下:
(1)利用pET系统构建了带有人类Oct4转录因子、11-Arginine穿膜肽和6×His纯化标签的原核表达载体。将重组质粒转化入含大肠杆菌稀有密码子的Rostta2(DE3)工程菌以终浓度为1mM的IPTG,28℃12h诱导表达,获得了43kDa左右的hOct4-11R-His融合蛋白,蛋白处于细菌裂解上清中。经Ni+纯化柱纯化,经Western blotting检测表明融合蛋白表达正确。
(2)利用EGFP-11R-His和罗丹明标记的hOct4-11R-His蛋白加入BJ和人神经干细胞观察穿膜肽进入细胞情况。证明蛋白加入量为10ug/ml时穿膜效率高且细胞状态好。hOct4-11R-His蛋白对神经干细胞具有高效的穿膜效率和细胞核趋向性。
(3)利用hOct4-11R-His诱导人神经干细胞ReNcell NSC,以终浓度为10ug/ml加入细胞,12h后换液培养4天为一轮,4轮后细胞出现克隆样生长。经AP染色后部分细胞呈阳性,定量PCR检测Oct4和Nanog基因明显上调。
蛋白直接诱导iPS细胞避免了病毒和其他以DNA为基础重编程方法所产生的潜在隐患,不涉及任何的遗传修饰,是目前最安全的诱导方法,我们的研究证明通过单个蛋白诱导多能性干细胞是可行的,为进一步研究奠定了基础。
Pluripotent stem cells have both the abilities to unlimitedly self-renew and to differentiate into various functional lineages. Scientists have been making efforts to find effective ways to obtain pluripotent stem cells. In the past five years, generation of pluripotent stem cells using 4 transcriptional factors provided a promising strategy to solve the problems that hamper the acquisition of pluripotent stem cells. However, this strategy needs to be further improved mainly due to low efficiency and safety. To overcome these weakness, we used a fusion protein between a single transcriptional factor, hOct4, and cell penetrating peptides, which is produced using prokaryotic expression systems, to induce human neural stem cells into pluripotent stem cells. The results are as follows:
1. We successfully constructed a pET-based prokaryotic expression system consisting of hOct4 cDNA, 11 Arg cell penetrating peptide and His purification tag (hOct4-11R-His). Subsequently, the recombinant plasmid was transferred into Rosetta2(DE3)E.coli. The expression of 43 KDa fusion protein was induced by IPTG (1mM) at 28℃. Finally, hOct4-11R-His from the supernatant part of bacterial lysate was purified by Ni affinity chromatography and identified by Western Blotting.
2. The penetrating capacity of Rhodamine-labelled hOct4-11R-His and EGFP-11R-His was investigated using BJ cells and human neural stem cells. The result showed that recombinant proteins effectively enter the tested cells without cytotoxicity at the concentration of 10μg/ml. hOct4-11R-His was demonstrated to have an effective cell penetrating capacity to human neural stem cells and a tropism to nucleus.
3. The NSC were treated 12h with the recombinant protein hOct4-11R-His at 10ug/ml in the ReNcell NSC maintain medium supplemented with 1mM valproic acid (VPA),followed by changing to the same media without the recombinant protein, and culturing for 96h before the next cycle of the treatment. After six rounds of treatment, several colonies with iPS-like morphology were observed. Quantitative Real Time-PCR showed increased expression of endogenous Oct4 and Nanog in hOct4-11R-His-treated neural stem cells. Moreover, AP positive cells also appeared in this cell population.
The strategy of protein-mediated induced pluripotent stem (iPS) cells production, which is not involved in any genetic modification, avoids the potential tumor-inducing risk resulting from usage of viral vector and is currently the safest way to acquire iPS cells. Our works showed that single transcriptional factors-mediated iPS production is feasible and provided a research platform for further investigation.
引文
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