本文關鍵詞： 趨化因子CXC的配體基因 短發(fā)夾RNA 骨髓間充質干細胞 慢病毒 趨化效應　出處：《第三軍醫(yī)大學學報》2017年01期 　論文類型：期刊論文

【摘要】：目的構建大鼠趨化因子CX3C的配體1(chemokine CX3C ligand 1,CX3CL1)基因慢病毒RNA干擾(RNA interference,RNAi)載體,觀測其沉默CX3CL1基因對骨髓間充質干細胞(bone marrow derived mesenchymal stem cells,b MSCs)生長及其趨化效應的影響。方法首先分離、培養(yǎng)大鼠骨髓b MSCs,并予以流式細胞術檢測鑒定。針對CX3CL1基因mRNA序列,篩選3個小干擾RNA(small interfering RNA,siRNA)靶點并予以合成。把合成的siRNA導入b MSCs,Western blot檢測其對靶基因編碼蛋白的抑制效應,以此明確最佳siRNA。設計與合成針對最佳siRNA序列的短發(fā)夾RNA(short hair RNA,shRNA),連入CD513B-1慢病毒載體,構建CD513B-1/CX3CL1 shRNA慢病毒,并行測序鑒定。測序正確者用293T細胞包裝成具有高效感染力的CD513B-1/CX3CL1 shRNA重組慢病毒,該重組病毒用于感染b MSCs。分離培養(yǎng)大鼠脾巨噬細胞,將其與被感染的b MSCs共培養(yǎng)。倒置顯微鏡下觀測被感染b MSCs的綠色熒光蛋白(GFP)的表達情況;CCK-8檢測被感染b MSCs的生長變化;Real-time PCR檢測被感染b MSCs的CX3CL1與核抗原PCNA基因的表達變化;Western blot檢測被感染b MSCs的CX3CL1和PCNA蛋白,與被感染的b MSCs共培養(yǎng)的脾巨噬細胞的趨化因子M-CSF和IL-8的表達變化。結果大鼠b MSCs得以分離、培養(yǎng)和鑒定。篩檢得CX3CL1基因的最佳干擾序列:CTCTATGAGCAATTATTTA;測序證實,成功構建重組慢病毒載體CD513B-1/CX3CL1 shRNA。熒光觀察表明,被CD513B-1/CX3CL1shRNA病毒感染的b MSCs明顯表達GFP。CCK-8檢測結果顯示,與對照細胞比較,沉默CX3CL1的b MSCs的生長減慢,48 h開始更為明顯(P0.01);Real-time PCR、Western blot檢測結果證實,CX3CL1基因的shRNA慢病毒能有效沉默b MSCs的CX3CL1,并下調(diào)其核抗原基因PCNA及其蛋白的表達,沉默CX3CL1的b MSCs能下調(diào)與其共培養(yǎng)的脾巨噬細胞的趨化因子M-CSF、IL-8的表達。結論成功構建CX3CL1基因RNAi重組慢病毒載體。該病毒載體能有效沉默b MSCs的CX3CL1基因,使b MSCs的生長減慢并下調(diào)其核抗原PCNA基因及其編碼蛋白的表達。沉默CX3CL1的b MSCs能下調(diào)與其共培養(yǎng)的脾巨噬細胞的趨化因子M-CSF、IL-8的表達。
[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.
【作者單位】： 解放軍第324醫(yī)院腦血管病中心;
【基金】：重慶市基礎與前沿研究計劃項目(CSTC2014jcyjA10077)~~
【分類號】：R743.3
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本文編號：1511372