Chinese Journal of Tissue Engineering Research
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Zhang Yang, Li Xiu-lan
Received:
2013-03-21
Revised:
2013-05-04
Online:
2013-07-02
Published:
2013-07-02
Contact:
Li Xiu-lan, Master, Investigator, Doctoral supervisor, Cell Engineering Laboratory of Tianjin Orthopaedic Hospital, Tianjin 300211, China
lixiulan1954@sina
About author:
comZhang Yang★, Master, Associate investigator, Cell Engineering Laboratory of Tianjin Orthopaedic Hospital, Tianjin 300211, China
Supported by:
Tianjin Applied Basis and Cutting-Edge Technology Research Plan, No. 11JCYBJC10200*
CLC Number:
Zhang Yang, Li Xiu-lan. Safety and clinical application of induced pluripotent stem cells[J]. Chinese Journal of Tissue Engineering Research, doi: 10.3969/j.issn.2095-4344.2013.27.021.
2.1 提高诱导性多潜能干细胞安全性的措施 2.1.1 避免c-Myc基因的使用 在Yamanaka经典方法中,c-Myc基因是4个主要的转录因子之一,同时也是公认的癌基因,外源c-Myc基因的导入具有引发癌变的可能。 Aasen等[5]、Wernig等[6]和Nakagawa等[7]均报道了在不使用c-Myc基因的情况下,仅采用Oct-4、Sox2和Klf4这3个转录因子,也能高质量的诱导小鼠胚胎成纤维细胞和人皮肤成纤维细胞重编程为诱导性多潜能干细胞。Feng等[8]不使用c-Myc和Klf4这两个因子,用Essrb与Oct4和Sox2共同作用把胎鼠成纤维细胞重编程为诱导性多潜能干细胞。Thomson研究小组[9]不使用c-Myc和Klf4,用Oct-4、Sox2、Nanog和Lin28可以诱导人的成纤维细胞为诱导性多潜能干细胞,避开致癌性。去除c-Myc基因后的诱导效率明显低于4个转录因子共同作用,同时会延迟诱导性多潜能干细胞克隆出现的时间,提示c-Myc基因并不是体细胞重编程为诱导性多潜能干细胞所必需的,但细胞重编程中c-Myc基因的参与有助于提高诱导效率。 2.1.2 以其它介导方式代替反转录病毒的使用 诱导性多潜能干细胞前期研究中普遍使用反转录病毒载体,只能感染分裂期细胞而不能感染静止期细胞,目前建立诱导性多潜能干细胞系的传统方法所使用的慢病毒载体属于反转录病毒的一种,可以高效感染分裂细胞和非分裂细胞。不管是采用慢病毒还是反转录病毒基因载体均可能导致载体序列及外源因子序列永久地整合入细胞的基因组DNA中,引起插入突变,影响诱导性多潜能干细胞功能及分化,甚至导致肿瘤发生,限制了诱导性多潜能干细胞在基础研究和临床研究中的应用。 腺病毒介导:由反转录病毒载体介导的基因组插入突变并不是诱导诱导性多潜能干细胞所必需的,利用瞬时转染的方式亦可能获得诱导性多潜能干细胞。Stadtfeld等[10]采用腺病毒载体代替反转录病毒载体,通过外源转录基因的瞬时表达代替基因的永久性整合,诱导诱导性多潜能干细胞均获成功,见图1。该方法一般不会把病毒载体整合进宿主基因组中,但诱导效率低,且仍有外源基因整合进宿主基因组的可能。"
Jia等[15]建立了一个携带POU5Fl、Sox-2、Lin28、Nanog四个重组因子及2A肽段和IRES的质粒,可以诱导人脂肪干细胞重编程为诱导性多潜能干细胞,同时进一步利用PhiC31分子内重组系统使质粒骨架在细菌里被清除和被降解。微环状载体外源沉默机制的低活化作用使其具有更长的异位表达,因此具有较高转染效率,重编程效率低于病毒载体[16-17],高于其他质粒转染重编程方法[11, 14]。该载体仅有1个质粒,无需随后的药物筛选或载体清除,也不包含癌基因。采用质粒转染方法获得的这些诱导性多潜能干细胞没有外源性DNA,从而避免了癌变问题,更适合未来的临床应用。 RNA转染:Yakubov等[18]尝试采用RNA转染方法将人包皮成纤维细胞重编程为诱导性多潜能干细胞,用质粒pTMA作为平台通过体外转录方法分别使用Oct-4、lin-28、Sox-2、Nanog 4个转录因子的cDNA体外合成mRNA,RNA转染人包皮成纤维细胞,成功获得了RNA诱导诱导性多潜能干细胞,见图5。这种方法避免了破坏DNA整合和相关基因组,有可能代替DNA载体诱导诱导性多潜能干细胞。"
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