CAJ | 학술논문

目的探讨慢病毒表达载体介导的小鼠CXC型趋化因子受体4(CXCR4)基因体外对小鼠骨髓间充质干细胞(MSC)生物学特性的影响.方法克隆小鼠CXCR4基因,构建携带CXCR4基因与增强型绿色荧光蛋白(EGFP)双顺反子结构的重组慢病毒表达载体LV-CXCR4-IRES-EGFP,同时构建对照载体LV-IRES-EGFP.脂质体法包装病毒,并测定滴度.通过优化慢病毒感染MSC条件,建立高表达CXCR4的MSC,荧光显微镜观察EGFP表达,流式细胞术检测细胞表面CXCR4表达,CCK-8法检测MSC对单向混合淋巴细胞培养体系中淋巴细胞增殖的影响,划痕实验和Transwell实验检测MSC体外迁移能力.结果成功克隆小鼠CXCR4基因,并构建重组慢病毒载体LV-CXCR4-IRES-EGFP和对照载体LV-IRES-EGFP.制备共表达CXCR4和报告基因EGFP的高滴度慢病毒颗粒.优化感染条件后,LV-CXCR4-IRES-EGFP感染组MSC表面CXCR4的表达明显高于LV-IRES-EGFP对照组(P＜0.05),分别为(90.3±3.37)％和(1.53±0.34)％.过表达CXCR4基因不影响MSC对T淋巴细胞增殖的抑制作用(P＞0.05).划痕实验和Transwell实验结果表明,过表达CXCR4可明显提高MSC对划痕损伤修复能力和向高浓度基质细胞衍生因子-1(SDF-1)的迁移能力,并且具有剂量依赖性.结论成功构建重组慢病毒载体LV-CXCR4-IRES-EGFP,慢病毒载体系统可高效介导CXCR4基因在小鼠MSC表达,并具有生物学活性.
목적 탐토만병독표체재체개도적소서CXC형추화인자수체4(CXCR4)기인체외대소서골수간충질간세포(MSC)생물학특성적영향.방법 극륭소서CXCR4기인,구건휴대CXCR4기인여증강형록색형광단백(EGFP)쌍순반자결구적중조만병독표체재체LV-CXCR4-IRES-EGFP,동시구건대조재체LV-IRES-EGFP.지질체법포장병독,병측정적도.통과우화만병독감염MSC조건,건립고표체CXCR4적MSC,형광현미경관찰EGFP표체,류식세포술검측세포표면CXCR4표체,CCK-8법검측MSC대단향혼합림파세포배양체계중림파세포증식적영향,화흔실험화Transwell실험검측MSC체외천이능력.결과 성공극륭소서CXCR4기인,병구건중조만병독재체LV-CXCR4-IRES-EGFP화대조재체LV-IRES-EGFP.제비공표체CXCR4화보고기인EGFP적고적도만병독과립.우화감염조건후,LV-CXCR4-IRES-EGFP감염조MSC표면CXCR4적표체명현고우LV-IRES-EGFP대조조(P＜0.05),분별위(90.3±3.37)％화(1.53±0.34)％.과표체CXCR4기인불영향MSC대T림파세포증식적억제작용(P＞0.05).화흔실험화Transwell실험결과표명,과표체CXCR4가명현제고MSC대화흔손상수복능력화향고농도기질세포연생인자-1(SDF-1)적천이능력,병차구유제량의뢰성.결론 성공구건중조만병독재체LV-CXCR4-IRES-EGFP,만병독재체계통가고효개도CXCR4기인재소서MSC표체,병구유생물학활성.
Objective To construct mouse CXC chemokine receptor type 4 (Cxcr4) gene overexpressing lentiviral vector and to evaluate its biological effect on mouse mesenchymal stem cells (MSCs).Methods Cxcr4 gene was amplified and subcloned into pCR-Blunt vector.Cxcr4 gene and enhanced green fluorescent protein (EGFP) gene expressed bicistronic recombinant lentiviral vector LV-CXCR4-IRES-EGFP and control vector LV-IRES-EGFP were constructed,respectively.Both plasmids were co-transfected into 293FT packaging cell line with packaging plasmid pSPAX2 and enveloping plasmid pMD.2G using Lipofectamine 2000 to produce lentiviral virus,respectively.The recombinant viruses were harvested and the virus titer was determined by limiting dilution.Mouse MSCs were infected with viral supernatant.EGFP expression was visualized using fluorescence microscope and efficiency of infection was determined by flow cytometry (FCM).Cell counting kit-8 (CCK-8) was applied in mixed lymphocyte reaction (MLR) to evaluate the suppressive effect of MSCs on mice splenocyte proliferation in vitro.Wound healing ability of MSCs was measured by scratch experiment and migration capacity by a chemotaxis assay using a transwell assay.Results The Cxcr4 fragment was amplified by reverse transcription polymerase chain reaction (RT-PCR) and verified by DNA sequencing.The restriction enzyme digestion experiment demonstrated that the recombinant lentiviral vector LV-CXCR4-IRES-EGFP and the control vector LV-IRES-EGFP were successfully constructed.Expression of CXCR4 was detected by fluorescence microscopy,which indicated that the lentiviral particles expressing CXCR4 were packaged.Furthermore,expression of EGFP were detected by fluorescence microscopy in MSCs after infection and the expression of CXCR4 protein on MSCs surface in CXCR4-MSC group was significantly increased comparing to those in the control group(P ＜ 0.05).CXCR4-MSCs group and the control group were (90.3 ± 3.37) ％ and (1.53 ± 0.34) ％,respectively.Meanwhile,overexpression of CXCR4 had no effect on their capacity of immune regulation when co-cultured with splenocyte (P ＞ 0.05).Moreover,overexpression of CXCR4 can not only accelerated the wound healing after scratch,but also enhanced the migration ability of cells in the transwell induced by high concentration of SDF-1 in a dose-dependent manner compared with the EGFP control group.Conclusion The CXCR4 expressing lentiviral vector LV-CXCR4-IRES-EGFP was successfully constructed.The lentiviral vector can not only efficiently infect mouse MSCs,but also stably express CXCR4 in MSCs.The MSCs modified with CXCR4 have biological characteristic in vitro.