靶向抑制Molt-4 T细胞增殖的Smo-siRNA:筛选和评价  

Screening and evaluation of Smo-siRNA targeted to inhibition of Molt-4 cell proliferation

作　　者：朱华民 王旭[3] 徐艳[3] 杨力建[2] 陈少华[2] 吴秀丽[2] 李扬秋[2,3] Zhu Hua-min;Wang Xu;Xu Yan;Yang Li-jian;Chen Shao-hua;Wu Xiu-li;Li Yang-qiu(Shenzhen Hospital, Southern Medical University, Shenzhen 518102, Guangdong Province, China;Institute of Hematology, Medical College of Jinan University, Guangzhou 510632, Guangdong Province, China;Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou 510632, Guangdong Province, China)

机构地区：[1]南方医科大学深圳医院,广东省深圳市518102 [2]暨南大学医学院血液病研究所,广东省广州市510632 [3]暨南大学再生医学教育部重点实验室,广东省广州市510632

出　　处：《中国组织工程研究》2018年第13期2104-2108,共5页Chinese Journal of Tissue Engineering Research

摘　　要：背景:研究表明,T淋巴细胞白血病的发生发展与Hedgehog通路的异常有关。Smo基因是该信号通路中的关键基因,控制着Hedgehog信号向细胞膜内的传递。目的:筛选一种可以高效抑制Molt-4细胞株增殖和诱导凋亡的小干扰RNA(Smo-siRNA)。方法:(1)根据siRNA设计原理,设计并化学合成Smo-siRNA 1,2,3以及无关干扰序列的阴性对照siRNA;(2)利用Nuclefector^(TM)核转染仪将以上Smo-siRNA分别转入人T淋巴细胞白血病细胞株(Molt-4细胞),分别在转染24,48,72 h采用qRT-PCR检测Smo mRNA相对表达水平,CCK-8法检测细胞生长抑制率,Hoechst33258染色观察细胞凋亡形态,流式细胞仪(Annexin V/PI法)检测细胞凋亡率。结果与结论:(1)利用Nuclefector^(TM)核转染仪成功将Smo-siRNA转入Molt-4细胞,Smo-siRNA 1沉默效果最佳,有效降低Molt-4细胞Smo mRNA表达水平(P<0.05),并且以48 h作用效果最明显;(2)转染后24 h,Smo-siRNA可明显抑制Molt-4细胞生长(P<0.05);(3)Hoechst染色证实Molt-4细胞符合凋亡的细胞形态学变化;(4)与对照组比较,Smo-siRNA1组细胞的凋亡率明显增加(P<0.05);(5)结果表明,小干扰RNA下调Molt-4细胞Smo基因表达可明显抑制Molt-4细胞增殖,并促进凋亡,提示Smo-siRNA具有作为T细胞白血病靶向基因治疗或者协同治疗的潜能。BACKGROUND： Studies have shown that the occurrence and development of T lymphocytic leukemia is related to the abnormality of Hedgehog pathway. The Smo gene is a key gene in this signaling pathway and controls the transmission of Hedgehog signaling into the cell membrane. OBJECTIVE： To design and screen a highly efficient and specific Smo-siRNA which is able to downregulate the Smo gene expression in Molt-4 cells, thereby inhibiting the Molt-4 cells proliferation and inducing apoptosis. METHODS： （1） Smo-siRNAs numbered 1, 2 or 3, and the scrambled non-siRNA control （SC） were obtained by chemosynthesis. Untreated and sc-treated cells were used as controls. （2） Smo expression levels in Molt-4 cells were analyzed using qRT-PCR at 24, 48, 72 hours after siRNAs delivered by NuclefectorTM.Cell proliferation in vitro was assayed by the cell counting kit-8.The morphology and percentage of apoptotic cells were revealed by Hoechst33258 staining and flow cytometry, respectively. RESULTS AND CONCLUSION： （1） Smo-siRNAs were successfully transferred into Molt-4 cells, and exhibited best silencing results. After transfection with Smo-siRNA1, the mRNA level of Smo was significantly reduced （P 〈 0.05）, and the lowest level was at 48 hours after transfection. （2） Cell proliferation of Molt-4 cells was significantly inhibited by Smo-siRNA at 24 hours after transfection. （3） Hoechst staining results showed morphological changes of Molt-4 were in accordance with those of apoptotic cells. （4） The apoptotic rate was significantly increased in the Smo-siRNA group compared with the control group （P 〈 0.05）. Findings from this study showed that suppression of Smo by RNA interference could effectively inhibit proliferation and induce apoptosis in Molt-4 cells, indicating that Smo-siRNA as gene targeted therapy or synergistic treatment has therapeutic potential in T-cell malignancies.

关 键 词：干细胞 小干扰RNA Smo基因 MOLT-4细胞 细胞凋亡 细胞增殖 

分 类 号：R394.2[医药卫生—医学遗传学]