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  • 胡皓,孟威,刘安堂,汪汇,朱晓海,江华,钱玉鑫.慢病毒介导的shRNA干扰大鼠施万细胞RSC96 MYH14基因的表达[J].第二军医大学学报,2018,39(10):1132-1137    [点击复制]
  • HU Hao,MENG Wei,LIU An-tang,WANG Hui,ZHU Xiao-hai,JIANG Hua,QIAN Yu-xin.Interference effect of lentiviral mediated shRNA on expression of MYH14 gene in rat Schwann cells RSC96[J].Acad J Sec Mil Med Univ,2018,39(10):1132-1137   [点击复制]
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慢病毒介导的shRNA干扰大鼠施万细胞RSC96 MYH14基因的表达
胡皓1,2,孟威1,刘安堂1,汪汇1,朱晓海1,江华1,钱玉鑫1,3*
0
(1. 海军军医大学(第二军医大学)长征医院整形外科, 上海 200003;
2. 武警总队医院皮肤科, 北京 100600;
3. 上海伊莱美医疗美容医院, 上海 200070
*通信作者)
摘要:
目的 应用RNA干扰技术构建大鼠肌球蛋白重链14(MYH14)基因重组慢病毒载体并鉴定。方法 根据MYH14 mRNA序列设计合成单链引物形成双链寡核苷酸序列,连接入经AgeⅠ和BamHⅠ双酶切线性化的GV298慢病毒质粒载体中,菌液经PCR鉴定并测序验证。取测序正确的菌液提取质粒转染大鼠施万细胞RSC96,利用免疫荧光法观察转染效率,蛋白质印迹法筛选有效敲低MYH14的shRNA质粒,CCK-8法检测转染后RSC96细胞的活力。结果 合成3对MYH14-shRNA序列并将其克隆到GV298载体中,构建了重组质粒MYH14-shRNA1、2、3,经菌液PCR鉴定和测序筛选出测序正确的载体MYH14-shRNA1和MYH14-shRNA2。免疫荧光检测结果显示转染72 h时RSC96细胞荧光表达最强;蛋白质印迹法检测结果显示,与阴性对照(scramble序列)组相比,MYH14-shRNA2转染后RSC96细胞中MYH14蛋白的表达水平降低(0.57±0.15 vs 1.11±0.06,P<0.01),而MYH14-shRNA1转染后MYH14蛋白表达水平差异无统计学意义(P>0.05)。MYH14-shRNA2转染RSC96细胞24 h后的细胞活力与阴性对照组相比差异无统计学意义(1.09±0.16 vs 1.00±0.15,P>0.05)。结论 成功构建大鼠MYH14基因重组慢病毒干扰载体,该载体能有效下调RSC96细胞中MYH14的表达。
关键词:  肌球蛋白重链14  短发夹RNA  慢病毒  施万细胞
DOI:10.16781/j.0258-879x.2018.10.1132
投稿时间:2018-06-12修订日期:2018-09-10
基金项目:国家自然科学基金(31271264).
Interference effect of lentiviral mediated shRNA on expression of MYH14 gene in rat Schwann cells RSC96
HU Hao1,2,MENG Wei1,LIU An-tang1,WANG Hui1,ZHU Xiao-hai1,JIANG Hua1,QIAN Yu-xin1,3*
(1. Department of Plastic Surgery, Changzheng Hospital, Navy Medical University(Second Military Medical University), Shanghai 200003, China;
2. Department of Dermatology, General Hospital of Armed Police Forces, Beijing 100600, China;
3. Shanghai Elikeme Medical Cosmetology Hosipital, Shanghai 200070, China
*Corresponding author)
Abstract:
Objective To construct and identify rat myosin heavy chain 14 (MYH14) gene recombined lentiviral vector by RNA interference technique. Methods Based on the MYH14 mRNA sequence, a single-stranded primer was designed to form a double-stranded oligonucleotide sequence, which was ligated into the GV298 lentiviral vector linearized by AgeⅠ and BamHⅠ double enzymes restriction, and then the bacterial liquid was verified by PCR and sequencing, respectively. The plasmid was extracted in the bacterial liquid with correct sequence and transfected into rat Schwann cells RSC96. The transfection efficiency was observed by immunofluorescence, the shRNA plasmid could effectively knock down MYH14 was screened by Western blotting, and the cell viability of RSC96 cells after transfection was detected by CCK-8. Results Three pairs of MYH14-shRNA sequences were synthesized and cloned into GV298 vector to construct recombinant plasmids MYH14-shRNA1, 2, and 3, and the vector MYH14-shRNA1 and MYH14-shRNA2 were screened by PCR and sequencing. Immunofluorescence showed that the cell fluorescence was the strongest at 72 h after transfection. Western blotting analysis showed that compared with the negative control (scramble sequence) group, the expression level of MYH14 protein in RSC96 cells was significantly decreased after MYH14-shRNA2 transfection (0.57±0.15 vs 1.11±0.06, P<0.01), while there was no significant difference after MYH14-shRNA1 transfection (P>0.05). There was no significant difference in cell viability of RSC96 cells between the negative control and MYH14-shRNA2 groups 24 h after transfection (1.09±0.16 vs 1.00±0.15, P>0.05). Conclusion The rat MYH14 gene recombinant lentiviral vector has been successfully constructed, which can effectively down-regulate the expression of MYH14 in RSC96 cells.
Key words:  myosin heavy chain 14  short hairpin RNA  lentivirus  Schwann cells