趋化因子受体CXCR7通过分化抑制因子-1促进间充质干细胞旁分泌肝细胞生长因子的研究

Chemokine receptor 7 promotes the paracrine of hepatocyte growth factor by mesenchymal stem cells via inhibitor of differentiation-1

目的探讨间充质干细胞(MSC)旁分泌肝细胞生长因子(HGF)的可能机制。方法①体外培养C57BL/6小鼠间充质干细胞(mMSC),并构建慢病毒质粒稳转的高表达趋化因子受体7(CXCR7)的mMSC,同时设置空白对照组和空载体对照组。待细胞传代培养20代后,采用荧光显微镜和流式细胞仪鉴定转染效率;采用实时荧光定量反转录-聚合酶链反应(RT-PCR)检测mMSC中CXCR7 mRNA表达水平。②另取传至4~6代的mMSC,分为MSC对照组〔MSC-blank组,野生型mMSC加入100μg/L脂多糖(LPS)〕、高表达CXCR7组(MSC-OE-CXCR7组,慢病毒质粒稳转高表达CXCR7 mMSC加入100μg/L的LPS)、高表达CXCR7对照组(MSC-OENC-CXCR7组,慢病毒空载体质粒稳转mMSC加入100μg/L的LPS)、CXCR4抑制剂组(MSC-IE-CXCR4组,mMSC经0.1 mg/L CXCR4抑制剂TC14012预处理24 h后加入100μg/L的LPS)和CXCR4抑制剂对照组(MSC-IENC-CXCR4组,mMSC加入与TC14012等量的DMEM培养液预处理24 h后再加入100μg/L的LPS)。经LPS处理6 h后收集各组细胞,采用RT-PCR法检测分化抑制因子-1(ID-1)的mRNA表达;收集细胞上清液,采用酶联免疫吸附试验(ELISA)检测HGF水平。结果①经荧光显微镜及流式细胞仪检测鉴定慢病毒质粒介导的高表达CXCR7的mMSC构建成功。与空白对照组比较,高表达CXCR7慢病毒载体组mMSC中CXCR7 mRNA表达水平明显升高(2^(-ΔΔCt):5.81±0.97比1.02±0.12,P<0.05);而空载体对照组mMSC中CXCR7 mRNA表达水平与空白对照组差异无统计学意义(2^(-ΔΔCt):0.95±0.22比1.02±0.12,P>0.05)。②与MSC-blank组相比,MSC-OE-CXCR7组高表达CXCR7或者MSC-IE-CXCR4组抑制CXCR4均可引起mMSC中ID-1 mRNA高表达(2^(-ΔΔCt):5.56±0.66、2.47±0.58比1.00±0.10,均P<0.05),同时可增加HGF外分泌水平(ng/L:632.02±149.98、217.21±40.53比108.53±24.62,均P<0.05);但MSC-OENC-CXCR7组和MSC-IENC-CXCR4组mMSC中ID-1 mRNA表达水平和HGF外分泌水平与MSC-blank组比较差异均无统计学意义ID-1 mRNA(2^(-ΔΔCt)):1.01±0.27、1.21±0.32比1.00±0.10,HGF(ng/L):133.56±25.19、107.11±25.30比108.53±24.62,均P>0.05。结论诱导MSC中CXCR7高表达或者抑制CXCR4表达均可增加MSC中ID-1表达,并促进HGF分泌,从而促进肺微血管内皮修复。

Objective To investigate the possible mechanism of mesenchymal stem cells(MSC)secreting hepatocyte growth factor(HGF).Methods①C57BL/6 mouse mesenchymal stem cells(mMSC)were cultured in vitro,and mMSC with high expression of chemokine receptor 7(CXCR7)were transduced by lentivirus plasmid.Blank control group and empty carrier control group were set at the same time.After 20 generations of cell culture,the transfection efficiency was identified by fluorescence microscopy and flow cytometry.The mRNA expression levels of CXCR7 in mMSC were detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction(RT-PCR).②mMSC with passage number 4-6 were divided into MSC control group[MSC-blank group,100μg/L lipopolysaccharide(LPS)was added to wild-type MSC],highly expressed CXCR7 group(MSC-OE-CXCR7 group,100μg/L LPS was added to mMSC transduced by lentivirus plasmid with high expression of CXCR7),highly expressed CXCR7 control group(MSC-OENC-CXCR7 group,100μg/L LPS was added to mMSC transduced by no load lentivirus plasmid),CXCR4 inhibitor group(MSC-IE-CXCR4 group,100μg/L LPS was added to mMSC after 0.1 mg/L CXCR4 inhibitor TC14012 pretreatment for 24 hours),and CXCR4 inhibitor control group(MSC-IENC-CXCR4 group,100μg/L LPS was added to mMSC after DMEM culture medium with equal amount of TC14012 pretreatment for 24 hours).Cells in each group were collected after treatment with LPS,and mRNA expression of inhibitor of differentiation-1(ID-1)was detected by RT-PCR.The cell supernatant was collected,and the levels of HGF were detected by enzyme linked immunosorbent assay(ELISA).Results①The high expression of CXCR7 for mMSC which were transduced through lentivirus plasmid were successfully constructed detected by fluorescence microscope and flow cytometry.Compared with the blank control group,the expression of CXCR7 mRNA in the lentivirus with high expression of CXCR7 group was significantly increased(2^(-ΔΔCt):5.81±0.97 vs.1.02±0.12,P<0.05).There was no significant difference in CXCR7 mRNA expression between the empty carrier control group and the blank control group(2^(-ΔΔCt):0.95±0.22 vs.1.02±0.12,P>0.05).②Compared with the MSC-blank group,high expression of CXCR7 in MSC-OE-CXCR7 group or inhibition of CXCR4 in MSC-IE-CXCR4 group could induce high expression of ID-1 mRNA in mMSC(2^(-ΔΔCt):5.56±0.66,2.47±0.58 vs.1.00±0.10,both P<0.05)and increase HGF exocrine level(ng/L:632.02±149.98,217.21±40.53 vs.108.53±24.62,both P<0.05).However,there were no significant differences in ID-1 mRNA expression and HGF exocrine level of mMSC among MSC-OENC-CXCR7 group,MSC-IENC-CXCR4 group and MSC-blank group[ID-1 mRNA(2^(-ΔΔCt)):1.01±0.27,1.21±0.32 vs.1.00±0.10,HGF(ng/L):133.56±25.19,107.11±25.30 vs.108.53±24.62,both P>0.05].Conclusion High expression of CXCR7 or inhibition of CXCR4 in MSC can increase the expression of ID-1 and promote the secretion of HGF,thus promoting pulmonary microvascular endothelial repair.