目的探讨CRISPR/CAS9靶向敲除Oct3/4基因后化疗药物索拉菲尼(Sorafenib)对肝癌细胞的作用影响。方法通过实时荧光定量PCR和免疫印迹检测人肝癌细胞Li-7、HepG2、Huh7、BEL-7405和人肝正常细胞LX-2中Oct3/4的表达水平。通过CRISPR设计工...目的探讨CRISPR/CAS9靶向敲除Oct3/4基因后化疗药物索拉菲尼(Sorafenib)对肝癌细胞的作用影响。方法通过实时荧光定量PCR和免疫印迹检测人肝癌细胞Li-7、HepG2、Huh7、BEL-7405和人肝正常细胞LX-2中Oct3/4的表达水平。通过CRISPR设计工具(http://crispr.mit.edu/)设计靶向Oct3/4的gRNA,并通过CRISPR/CAS9技术敲除肝癌细胞HepG2的Oct3/4,评估Oct3/4缺失情况下,化疗药物Sorafenib对肝癌细胞HepG2的凋亡水平和DNA损伤水平的影响。结果Oct3/4在人肝癌细胞Li-7、HepG2、Huh7、BEL-7405中的mRNA表达水平显著高于人肝正常细胞LX-2(P<0.05);同时,Oct3/4在人肝癌细胞Li-7、HepG2、Huh7、BEL-7405中的蛋白表达水平也显著高于人肝正常细胞LX-2。使用CRISPR/CAS9对HepG2细胞进行基因编辑后,挑取嘌呤霉素筛选出的2个单克隆细胞株进行Oct3/4表达水平的检测,1#号单克隆细胞株Oct3/4表达水平低于野生型WT,2#号单克隆细胞株没有表达Oct3/4。测序后发现1#号基因组上的Oct3/4第1个外显子只有1条染色体缺失了2个碱基,另一条染色体并未缺失;2#号基因组上的Oct3/4第1个外显子2条染色体均存在不同程度的缺失,一条缺失2个碱基,另一条缺失4个碱基。使用Sorafenib处理野生型HepG2细胞和2#Oct3/4 KO HepG2细胞后,Oct3/4 KO细胞株的凋亡水平显著上升(P<0.05),DNA损伤水平显著上升(P<0.05)。结论Oct3/4基因的敲除能够显著提高化疗药物Sorafenib促肝癌细胞凋亡和DNA损伤的作用。展开更多
Embryonic stem cells (ES cells) are derived from the inner cell mass (ICM) of blastocysts. ES cells can divide and produce identical copies of them over and over again (self-renewal) in vitro for a long time, and reta...Embryonic stem cells (ES cells) are derived from the inner cell mass (ICM) of blastocysts. ES cells can divide and produce identical copies of them over and over again (self-renewal) in vitro for a long time, and retain the capability of differentiating into all cell types when induced by appropriate signals. Their capability of multilineage dif- ferentiation might be exploited for cell-based therapies. Therefore, ES cells have a broad prospect in many clinical applications. To achieve success in the clinical applications, we have to understand how ES cells propagate and differen- tiate into specific cell types. The cytokine LIF can sustain the self-renewal of certain mouse ES cells (mES cells) through activation of the signal transduction pathway LIF/gp130/ STAT3. In this pathway the transcription factor STAT3 is a crucial factor. Furthermore, Oct-3/4 plays a very important role in maintaining the ES cell pluripotency. Oct-3/4 regu- lates embryo development through its co-factor Sox2 and Rox-1. Recently nanog, a new homeodomain gene, was found and it has been shown to be crucial for the renewal and pluripotency of ES cells. Three other signals BMP, Wnt and ERK also can influence differentiation and propagation of ES cells. This review article summarizes recent progress in this area, mainly focusing on the LIF signaling pathway and the transcription factors Oct-3/4 and Nanog. Although it is still unclear how these components cooperate, a model is presented here to provide a design for solving this problem.展开更多
文摘目的探讨CRISPR/CAS9靶向敲除Oct3/4基因后化疗药物索拉菲尼(Sorafenib)对肝癌细胞的作用影响。方法通过实时荧光定量PCR和免疫印迹检测人肝癌细胞Li-7、HepG2、Huh7、BEL-7405和人肝正常细胞LX-2中Oct3/4的表达水平。通过CRISPR设计工具(http://crispr.mit.edu/)设计靶向Oct3/4的gRNA,并通过CRISPR/CAS9技术敲除肝癌细胞HepG2的Oct3/4,评估Oct3/4缺失情况下,化疗药物Sorafenib对肝癌细胞HepG2的凋亡水平和DNA损伤水平的影响。结果Oct3/4在人肝癌细胞Li-7、HepG2、Huh7、BEL-7405中的mRNA表达水平显著高于人肝正常细胞LX-2(P<0.05);同时,Oct3/4在人肝癌细胞Li-7、HepG2、Huh7、BEL-7405中的蛋白表达水平也显著高于人肝正常细胞LX-2。使用CRISPR/CAS9对HepG2细胞进行基因编辑后,挑取嘌呤霉素筛选出的2个单克隆细胞株进行Oct3/4表达水平的检测,1#号单克隆细胞株Oct3/4表达水平低于野生型WT,2#号单克隆细胞株没有表达Oct3/4。测序后发现1#号基因组上的Oct3/4第1个外显子只有1条染色体缺失了2个碱基,另一条染色体并未缺失;2#号基因组上的Oct3/4第1个外显子2条染色体均存在不同程度的缺失,一条缺失2个碱基,另一条缺失4个碱基。使用Sorafenib处理野生型HepG2细胞和2#Oct3/4 KO HepG2细胞后,Oct3/4 KO细胞株的凋亡水平显著上升(P<0.05),DNA损伤水平显著上升(P<0.05)。结论Oct3/4基因的敲除能够显著提高化疗药物Sorafenib促肝癌细胞凋亡和DNA损伤的作用。
文摘Embryonic stem cells (ES cells) are derived from the inner cell mass (ICM) of blastocysts. ES cells can divide and produce identical copies of them over and over again (self-renewal) in vitro for a long time, and retain the capability of differentiating into all cell types when induced by appropriate signals. Their capability of multilineage dif- ferentiation might be exploited for cell-based therapies. Therefore, ES cells have a broad prospect in many clinical applications. To achieve success in the clinical applications, we have to understand how ES cells propagate and differen- tiate into specific cell types. The cytokine LIF can sustain the self-renewal of certain mouse ES cells (mES cells) through activation of the signal transduction pathway LIF/gp130/ STAT3. In this pathway the transcription factor STAT3 is a crucial factor. Furthermore, Oct-3/4 plays a very important role in maintaining the ES cell pluripotency. Oct-3/4 regu- lates embryo development through its co-factor Sox2 and Rox-1. Recently nanog, a new homeodomain gene, was found and it has been shown to be crucial for the renewal and pluripotency of ES cells. Three other signals BMP, Wnt and ERK also can influence differentiation and propagation of ES cells. This review article summarizes recent progress in this area, mainly focusing on the LIF signaling pathway and the transcription factors Oct-3/4 and Nanog. Although it is still unclear how these components cooperate, a model is presented here to provide a design for solving this problem.
