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

发布时间：2018-02-14 18:53

  本文关键词： 趋化因子CXC的配体基因 短发夹RNA 骨髓间充质干细胞 慢病毒 趋化效应　出处：《第三军医大学学报》2017年01期 　论文类型：期刊论文

【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(P0.01);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基因,使b MSCs的生长减慢并下调其核抗原PCNA基因及其编码蛋白的表达。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。
[Abstract]:Objective to construct the vector of rat chemokine CX3C ligand 1 (CX3CL1) lentivirus RNA interference RNAi, and to observe the effect of CX3CL1 gene silencing on the growth and chemotaxis of bone marrow derived mesenchymal stem cells (MSCs) and its chemoattractant effect. Methods to investigate the effect of CX3CL1 gene silencing on the growth and chemotaxis of bone marrow derived mesenchymal stem cells in rat bone marrow mesenchymal stem cells (BMSCs), and to investigate the effects of CX3CL1 gene silencing on the growth and chemotaxis of marrow derived mesenchymal stem cells. Rat bone marrow bMSCs were cultured and identified by flow cytometry. Three small interfering RNA(small interfering siRNAs were screened and synthesized according to the mRNA sequence of the CX3CL1 gene. The synthesized siRNA was introduced into bMSCs interfering to detect its inhibitory effect on the target gene encoding protein. In order to identify the best siRNA, we designed and synthesized the short hairpin RNA(short hair hair shRNAs for the best siRNA sequence, and connected them into the CD513B-1 lentivirus vector to construct CD513B-1/CX3CL1 shRNA lentivirus. Sequencing was performed. The correctly sequenced cells were packaged with 293T cells as highly infectious CD513B-1/CX3CL1 shRNA recombinant lentivirus. The recombinant virus was used to infect bMSCs.Rat splenic macrophages were isolated and cultured. The expression of green fluorescent protein (GFP) of infected b MSCs was observed under inverted microscope. CCK-8 was used to detect the growth change of infected b MSCs. Real-time PCR was used to detect CX3CL1 and nuclear antigen PCNA gene of infected b MSCs. Western blot was used to detect the CX3CL1 and PCNA proteins of the infected b MSCs. The expression of chemokines M-CSF and IL-8 in splenic macrophages co-cultured with infected b MSCs was changed. Results Rat b MSCs was isolated, cultured and identified. The recombinant lentivirus vector CD513B-1/CX3CL1shRNA was successfully constructed. Fluorescence observation showed that b MSCs infected by CD513B-1/CX3CL1shRNA virus significantly expressed GFP.CCK-8, which was compared with that of control cells. The results of Western blot analysis showed that the shRNA lentivirus containing CX3CL1 gene could effectively silence the expression of CX3CL1 and down-regulate the expression of its nuclear antigen PCNA and its protein. Silencing b MSCs of CX3CL1 can down-regulate the expression of chemokine M-CSF- IL-8 in splenic macrophages co-cultured with it. Conclusion the recombinant lentivirus vector of CX3CL1 gene RNAi was successfully constructed, which can effectively silence the CX3CL1 gene of b MSCs. The growth of b MSCs was slowed down and the expression of nuclear antigen PCNA gene and its encoded protein was down-regulated, while b MSCs silencing CX3CL1 could down-regulate the expression of M-CSF IL-8, a chemokine of splenic macrophages co-cultured with b MSCs.
【作者单位】： 解放军第324医院脑血管病中心;
【基金】：重庆市基础与前沿研究计划项目(CSTC2014jcyjA10077)~~
【分类号】：R743.3
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本文编号：1511372