沉默CX3CL1基因对骨髓间充质干细胞生长及其趋化效应的影响  

Effect of lentivirus-mediated silencing of CX3CL1 gene on growth and chemotaxis effect in bone marrow derived mesenchymal stem cells

作　　者：郝磊[1] 田洪[1] 牟长河[1] 张玉波[1] 周虎传[1] 宋川[1] 刘磊[1] Hao Lei Tian Hong Mou Changhe Zhang Yubo Hou Huzhuan Song Chuan Liu Lei(Center of Cerebrovascular Diseases, No. 324 Hospital of PLA, Chongqing, 400020, Chin)

机构地区：[1]解放军第324医院脑血管病中心,重庆400020

出　　处：《第三军医大学学报》2017年第1期34-41,共8页Journal of Third Military Medical University

基　　金：重庆市基础与前沿研究计划项目(CSTC2014jcyjA10077)~~

摘　　要：目的构建大鼠趋化因子CX3C的配体1(chemokine CX3C ligand 1,CX3CL1)基因慢病毒RNA干扰(RNA interference,RNAi)载体,观测其沉默CX3CL1基因对骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,b MSCs)生长及其趋化效应的影响。方法首先分离、培养大鼠骨髓b MSCs,并予以流式细胞术检测鉴定。针对CX3CL1基因mRNA序列,筛选3个小干扰RNA(small interfering RNA,siRNA)靶点并予以合成。把合成的siRNA导入b MSCs,Western blot检测其对靶基因编码蛋白的抑制效应,以此明确最佳siRNA。设计与合成针对最佳siRNA序列的短发夹RNA(short hair RNA,shRNA),连入CD513B-1慢病毒载体,构建CD513B-1/CX3CL1 shRNA慢病毒,并行测序鉴定。测序正确者用293T细胞包装成具有高效感染力的CD513B-1/CX3CL1 shRNA重组慢病毒,该重组病毒用于感染b MSCs。分离培养大鼠脾巨噬细胞,将其与被感染的b MSCs共培养。倒置显微镜下观测被感染b MSCs的绿色荧光蛋白(GFP)的表达情况;CCK-8检测被感染b MSCs的生长变化;Real-time PCR检测被感染b MSCs的CX3CL1与核抗原PCNA基因的表达变化;Western blot检测被感染b MSCs的CX3CL1和PCNA蛋白,与被感染的b MSCs共培养的脾巨噬细胞的趋化因子M-CSF和IL-8的表达变化。结果大鼠b MSCs得以分离、培养和鉴定。筛检得CX3CL1基因的最佳干扰序列:CTCTATGAGCAATTATTTA;测序证实,成功构建重组慢病毒载体CD513B-1/CX3CL1 shRNA。荧光观察表明,被CD513B-1/CX3CL1shRNA病毒感染的b MSCs明显表达GFP。CCK-8检测结果显示,与对照细胞比较,沉默CX3CL1的b MSCs的生长减慢,48 h开始更为明显(P<0.01);Real-time PCR、Western blot检测结果证实,CX3CL1基因的shRNA慢病毒能有效沉默b MSCs的CX3CL1,并下调其核抗原基因PCNA及其蛋白的表达,沉默CX3CL1的b MSCs能下调与其共培养的脾巨噬细胞的趋化因子M-CSF、IL-8的表达。结论成功构建CX3CL1基因RNAi重组慢病毒载体。该病毒载体能有效沉默b MSCs的CX3CL1基Objective To construct lentiviral vector-medicated RNA interference （RNAi） targeting rat chemokine CX3C ligand 1 （CX3CL1） and investigate its effect on the growth and chemotaxis effect of bone marrow derived mesenchymal stem cells （bMSCs）. Methods bMSCs were isolated from rat bone marrow, cultured and identified with flow cytometry. Three potential RNAi sites were screened based on the mRNA sequence of CX3CL1 gene and then synthesized. The 3 synthesized siRNAs were transferred into bMSCs, Western blotting was used to analyze the inhibitory effect of target gene so as to identify the optimal siRNA sequence. Then the short hair RNA （shRNA） oligonucleotide targeting the optimal siRNA sequence was designed, synthesized and inserted into CD513B-1 plasmid to form CD513B-1/CX3CL1 shRNA lentiviral vector. After identified by sequencing, CD513B-1/CX3CL1 shRNA plasmid was transfected into the 293T cells to produce lentiviral particles. The bMSCs were transfected with the lentiviral particles. After rat splenic macrophages were isolated and cultured, and the cells were cocultured with the transfected bMSCs. The expression of green fluorescent protein （GFP） in the transfected bMSCs was observed by inverted microscopy. Cell count kit-8 （CCK-8） assay was employed to test the growth of transfected bMSCs. The expression of CX3CL1 and proliferating cell nuclear antigen （PCNA） in the transfected bMSCs was detected by real-time PCR. The levels of the proteins in the transfected bMSCs and those of M-CSF and IL-8 in the splenic macrophages were detected by Western blotting respectively. Results Rat bMSCs were successfully isolated from bone marrow, cultured and identified. The optimal siRNA sequence CTCTATGAGCAATTATTTA was confirmed. The result of sequencing revealed that lentiviral vector CD513B-1/CX3CL1 shRNA was constructed successfully. The transfected bMSCs significantly expressed GFP. The results of CCK8 assay showed that the growth of bMSCs were slower after CX3CL1 silencing, especially in 48

关 键 词：趋化因子CX3C的配体1基因 短发夹RNA 骨髓间充质干细胞 慢病毒 趋化效应 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R394-33[医药卫生—基础医学]