鸡卵清提取液诱导293T细胞高表达tRFs&tiRNAs分子及细胞功能验证  

Chicken Egg White Extract Induces High Expression of t RFs&ti RNAs Molecules in 293T Cells and Its Cell Function Verification

作　　者：阮光萍 姚翔 何洁 莫平 王凯 杨再玲 庞荣清 朱向情 潘兴华 RUAN Guangping;YAO Xiang;HE Jie;MO Ping;WANG Kai;YANG Zailing;PANG Rongqing;ZHU Xiangqing;PAN Xinghua(Basic Medical Laboratory of 920th Hospital of Joint Logistics Support Force of PLA,Kunming 650032,China;The Integrated Engineering laboratory of Cell Biological Medicine of State and Regions,Kunming 650032,China;The Transfer Medicine Key Laboratory of Cell Therapy Technology of Yunnan Province,Kunming 650032,China)

机构地区：[1]解放军联勤保障部队第920医院基础医学实验室,昆明650032 [2]干细胞与免疫细胞生物医药技术国家地方联合工程实验室,昆明650032 [3]云南省细胞治疗技术转化医学重点实验室,昆明650032

出　　处：《中国细胞生物学学报》2021年第11期2158-2168,共11页Chinese Journal of Cell Biology

基　　金：云南省科技计划项目重大科技专项(批准号:2018ZF007);云南省重点项目(批准号:2018FA041、202101AS070039);云南省面上项目(批准号:202101AT070212);国家自然科学基金(批准号:31970515);中国人民解放军联勤保障部队第920医院院管课题(批准号:2019YGB17、2019YGA05)资助的课题。

摘　　要：tRNA源性片段(tRNA-derived fragments,tRFs)和tRNA源性应激诱导RNAs(tRNA-derived stress-induced RNAs,tiRNAs)是tRNAs的衍生片段,属于短的非编码RNA家族,通过转录、翻译、信号通路等途径参与复杂的生物反应。该文旨在验证鸡卵清提取液诱导293T细胞后升高的3个tRFs&tiRNAs分子的细胞功能。将293T细胞加于6孔板中,3个孔加普通培养基,3个孔加50%鸡卵清提取液的培养基,共培养3天。对照组3个样本和诱导组3个样本进行高通量测序检测tRFs&tiRNAs分子在两组中的差异表达。经检测验证诱导后的细胞有3个tRFs&tiRNAs分子稳定升高。这3个分子上调表达有统计学意义。合成这3个分子转染293T细胞,WB检测多能因子OCT4和NANOG的变化,定量PCR检测多能基因OCT4和NANOG的变化和端粒的相对表达量,流式检测多能因子OCT4和NANOG的变化。同时检测这3个分子转染293T细胞后细胞增殖、细胞凋亡和细胞周期的变化情况。结果表明,3个分子转染293T细胞后,WB检测到多能因子OCT4和NANOG表达对比未转染细胞明显升高,定量PCR检测多能基因OCT4和NANOG相对表达量对比未转染细胞明显升高,端粒对比未转染细胞明显增长。流式检测到多能因子OCT4和NANOG阳性表达细胞对比未转染细胞明显增多。这3个分子转染293T细胞后细胞活性增强,细胞凋亡减少,细胞周期也发生了一定的改变。该研究证明了这3个分子过表达可促进293T细胞多能因子OCT4和NANOG表达升高,促进端粒增长,使细胞年轻化。同时这3个分子过表达可使细胞活性增强,细胞凋亡减少。tRFs (tRNA-derived fragments) and tiRNAs (tRNA-derived stress-induced RNAs) are derived fragments of tRNAs,which belong to the short non-coding RNA family and participate in complex biological reactions by transcription,translation and signaling pathways.To verify the cell function of the three tRFs&tiRNAs molecules raised after the chicken egg white extract induces 293T cells.293T cells were added to a 6-well plate,among which 3 wells added with ordinary medium,and 3 wells added with 50% chicken egg white extract medium,and cultured for 3 days.Three samples in the control group and three samples in the induction group were subjected to high-throughput sequencing to detect the differential expression of tRFs&tiRNAs molecules in the two groups.It was verified by testing that the induced cells had 3 tRFs&tiRNAs molecules steadily increasing.The up-regulated expression of these 3 molecules is statistically significant.These three molecules were synthesized and transfected into 293T cells.The changes of pluripotency factors OCT4 and NANOG were detected by WB.The changes of pluripotency genes OCT4 and NANOG and the relative expression of telomeres were detected by quantitative PCR,and the expression variety of pluripotency factors OCT4 and NANOG were detected by flow cytometry.Simultaneously detect the changes of cell proliferation,apoptosis and cell cycle after transfection of these three molecules into 293T cells.The results indicated after the 3 molecules were transfected into 293T cells;the expression of pluripotent factors OCT4 and NANOG was significantly higher than that of untransfected cells detected by WB,and the relative expression levels of pluripotent genes OCT4 and NANOG were significantly higher than that of untransfected cells detected by quantitative PCR.Telomeres are significantly increased compared with untransfected cells.Flow cytometry detected a significant increase in cells expressing pluripotency factors OCT4 and NANOG compared with untransfected cells.After these 3 molecules were transfected in

关 键 词：tRFs&tiRNAs分子 293T细胞 差异表达 转染 细胞功能 验证 

分 类 号：Q343[生物学—遗传学]