【作者】 孟庆友；

【导师】 李晓强；

【作者基本信息】 苏州大学 ， 胸心血管外科， 2014， 博士

【摘要】 目的:研究miR-126调控内皮祖细胞及对静脉血栓溶解、机化和再通的影响，为慢性深静脉血栓的治疗提供一种新的治疗思路。方法：首先分离、培养及鉴定骨髓源性内皮祖细胞，将miR-126模拟物、抑制剂或阴性对照物用电转的方法转染到内皮祖细胞中，采用MTT、流式细胞术检测miR-126对内皮祖细胞增殖及细胞周期的影响；采用划痕实验、穿膜实验检测miR-126对内皮祖细胞运动迁移能力的影响；采用matrigel管腔形成实验检测miR-126对内皮祖细胞成小管能力的影响。进一步预测miR-126靶基因PIK3R2，并构建包含野生型PIK3R23’UTR和突变体PIK3R23’UTR的Luciferase报告基因载体，检测miR-126对PIK3R2的直接调控作用，Western blot分析PIK3R2蛋白以及PI3K/AKT信号通路中PI3K、Akt和p-Akt蛋白的变化。再构建miR-126慢病毒表达载体（pLVX-IRES-ZsGreenl-miR-126），HEK293T细胞包装、重组、扩增慢病毒颗粒，荧光显微镜观察绿色荧光表达， PCR、双酶切、基因测序进行鉴定。pLVX-IRES-ZsGreenl-miR-126体外转染内皮祖细胞，用实时荧光定量PCR(qPCR)方法检测转染后的内皮祖细胞中miR-126的表达。然后结扎大鼠肾下段下腔静脉建立深静脉血栓模型，将慢病毒转染的内皮祖细胞移植到静脉血栓模型中。分为三组：A组(12只)，空白对照组，经尾静脉注射1ml PBS；B组(12只)，EPCs/pLVX-IRES-ZsGreenVector (EPCs/vector)，经尾静脉注入含有1.0×106EPCs/vector的PBS细胞悬液。C组(12只)，EPCs/pLVX-IRES-ZsGreen-miR-126(EPCs/miR-126)，经尾静脉注入含有1.0×106EPCs/miR-126的PBS细胞悬液。移植后7天、14天取出血栓段下腔静脉及血栓，通过称重评价血栓溶解情况，荧光示踪观察内皮祖细胞归巢到静脉血栓情况，HE染色评价静脉血栓机化及CD34免疫组化观察血栓再通变化。采用SPSS15.0软件进行分析，P<0.05为差异，有统计学意义。结果:成功培养、鉴定了大鼠骨髓源性内皮祖细胞。电转成功后，miR-126对内皮祖细胞早期具有促进增殖及改变细胞周期的作用，后期没有影响。划痕实验及穿膜实验都证实了miR-126模拟物能够显著促进内皮祖细胞的迁移运动能力；miR-126抑制剂则能抑制内皮祖细胞的迁移运动能力。miR-126模拟物可以增强内皮祖细胞的成小管能力，miR-126抑制剂能抑制内皮祖细胞的成小管能力。野生型PIK3R23’UTR和突变体PIK3R23’UTR的Luciferase报告基因载体构建及鉴定成功，共转染miR-126模拟物和pMIR/PIK3R2报告基因载体后，萤光素酶活性明显降低，而共转染miR-126抑制剂和pMIR/PIK3R2报告基因载体后，萤光素酶活性变化不大，共转染miR-126模拟物或抑制剂和pMIR/PIK3R2/mut报告基因载体后则不影响信号强度。上调内皮祖细胞中miR-126表达水平后，其PIK3R2蛋白表达水平明显下降，PI3K和磷酸化的Akt（p-Akt）蛋白表达上调，而下调内皮祖细胞中miR-126表达水平后，其PIK3R2蛋白表达水平明显增加，PI3K和磷酸化的Akt（p-Akt）蛋白表达下调。经酶切及测序结果提示慢病毒表达载体pLVX-IRES-ZsGreenl-miR-126构建正确，能有效转染内皮祖细胞，转染miR-126后在内皮祖细胞的表达较对照组高216倍。内皮祖细胞被移植到静脉血栓后，在7天和14天两个时间段内，EPCs/miR-126组中静脉血栓的重量最低，萤光阳性细胞数目最多，HE染色和CD34免疫组化提示更能促进血栓的机化和再通，均有统计学意义。结论: miR-126能够促进内皮祖细胞成血管能力，包括增殖、迁移和成小管能力，miR-126能够负性调控其靶基因PIK3R2，激活PI3K/AKT信号通路。上调内皮祖细胞的miR-126表达后能够促进静脉血栓溶解、机化和再通。这可能是内皮祖细胞介导的治疗深静脉血栓的一种新思路。更多还原

【Abstract】 Objective: This research is to study of miR-126regulating endothelial progenitor cells（EPCs）and promoting venous thrombosis resolution,organization and recanalization.And these findings indicate a potential therapeutic intervention of deep venous thrombosis.Methods: Firstly, rat bone marrow-derived EPCs were isolated, cultivated andidentificated. Secondly,EPCs were transfected with control oligoes and miR-126mimicsor inhibitor by electroporation. Proliferation capacity and cell cycle were analyzed by MTTand flow cytometry.Cell migration analysis was done by wound healing and transwellassay.And tubulogenic activity test was performed by matrigel tube formation assay.Thirdly,by gain-of-function examination, its putative target genes were searched for usingonline search tool. PIK3R2was selected as the candidate target gene of rno-miR-126inEPCs.And further Luciferase reporters were constructed containing either a wild-typePIK3R23’UTR sequence (pMIR/PIK3R2/wt), or a mutated PIK3R23’UTR(pMIR/PIK3R2/mut). Luciferase activity was assessed by co-transfecting the luciferasereporter vectors with the miR-126mimics, inhibitor or NC. The luciferase activity ofreporter was observed. PIK3R2expression was detected by Western blot assays,and PIK3,Akt and p-Akt proteins in PI3K/AKT signal channel by Western blot assays,too.Fourthly,to stably express miR-126in EPCs, the lentiviral expression vectorpLVX-IRES-ZsGreen-miR-126was constructed.They was then transfected into293Tcells. The supernatant containing the lentivirus was harvested at72h. EPCs were infectedwith1ml lentivirus suspension. Green fluorescence was observed to indicate thetransduction efficiency at48h post transduction. miR-126expression in EPCs was detected by real-time quantitative PCR (qPCR).Forthermore, Experimental rat models ofdeep vein thrombosis were obtained by complete ligation of inferior vena cava below therenal veins. When deep venous thrombosis was formed after IVC ligation, the alive ratswere divided as3groups for cells transplantion via tail intravenous injection: A (n=12),blank control group (blank control), which received1ml PBS; B (n=12),EPCs/pLVX-IRES-ZsGreen Vector group (EPCs/vector), which received1.0×106EPCstransfected with lentivirus particle of pLVX-IRES-ZsGreen vector; C (n=12),EPCs/pLVX-IRES-ZsGreen-miR-126group (EPCs/miR-126), which received1.0×106EPCs transfected with lentivirus particle of pLVX-IRES-ZsGreen-miR-126. On the7th and14th day post operation, the rats were sacrificed and the thrombus segment of inferior venacava and thrombus were acquired,then were weighed. The homing of EPCs was shown byGFP expression and observed using fluorescence microscope. And the samples weretreated by dimethybenzene and embedded in paraffin. In order to observe thrombusorganization and recanalization, hematoxylin and eosin (HE) staining andimmunohistochemical staining for CD34and were performed. All statistical analyses wereperformed using SPSS15.0software. A two-tailed value of P <0.05was consideredstatistically significant.Results: Rat bone marrow-derived EPCs were isolated, cultivated and identificatedsuccessfully.After EPCs were transfected by electroporation successfully, proliferationcapacity of EPCs was enhanced and cell cycle was changed in early stage when transfectedwith miR-126mimics.The relative wound size of EPCs transfected with NC or miR-126oligonucleotides was analyzed at0,24,48and72h post wounding. Compared with NCmimics, miR-126mimics significantly enhanced the migration of EPCs across the woundspace at24,48and72h. In contrast, miR-126inhibitor significantly suppressed themigration of EPCs at the same time. Consistent with the wound healing assay, migrationchamber assay also showed that miR-126mimics significantly promoted while miR-126inhibitor significantly inhibited EPCs migration into the chamber membrane comparedwith control group. EPCs transfected with miR-126mimics showed a significant improvement of capillary tube formation compared with NC by matrigel tube formationassay. In contrast, miR-126inhibitor-infected cells showed a significant impairment ofcapillary tube formation compared with NC. After an extensive review of onlinemicroRNA database (that is, TargetScan, Microrna.org and miRanda), we selected PIK3R2as the candidate target gene of rno-miR-126in EPCs. Luciferase reporter assays firstperformed to verify a direct interaction between miR-126and the3’UTR of PIK3R2.Luciferase reporters were constructed containing either a wild-type PIK3R23’UTRsequence (pMIR/PIK3R2/wt), or a mutated PIK3R23’UTR (pMIR/PIK3R2/mut).Luciferase activity was assessed by co-transfecting the luciferase reporter vectors with themiR-126mimics, inhibitor or NC. Luciferase activity of pMIR/PIK3R2/wt was markedlydecreased in cells transfected with miR-126mimics, compared to luciferase activity ofpMIR/PIK3R2/mut. Conversely, the luciferase activity of reporter plasmid was notinterfered after transfection with miR-126inhibitor. miR-126mimics decreased thePIK3R2expression compared to NC mimics, while miR-126inhibitor increased PIK3R2protein levels. PI3K and phospho-Akt (p-Akt) levels were significantly enhanced in theEPCs transfected with miR-126mimics compared to negative control mimics. A reverseresult was observed when miR-126was knocked down in EPCs transfected with miR-126inhibitor compared to negative control inhibitor.Restriction enzyme digestion andsequencing results suggest that lentiviral vector pLVX-IRES-ZsGreenl-miR-126wasconstructed correctly.And EPCs were effectively transfected by them.Aftertransfection,the miR-126expression in EPCs was higher216times than the control group.On the7thand14thday after EPCs transplantation, Compared with control group, theweight of thrombus decreased in EPCs/vector and EPCs/miR-126groups. Furthermore, thethrombus weight of EPCs/miR-126group was even lower than that of EPCs/vectorgroup.EPCs homing were observed in section by using fluorescence microscope. Andtransplanted EPCs were recruited into the thrombus in both EPCs/vector andEPCs/miR-126group. In contrast, No GFP was observed in control group. We can alsofind that the larger number of GFP positive cells was appeared in EPCs/miR-126group compared with EPCs/vector group. HE staining indicated there were more degree oforganization in EPCs/vector and EPCs/miR-126group,even higher in EPCs/miR-126group. Compared with control group, more neovascularition were formed in EPCs/vectorand EPCs/miR-126group,and were maximum in EPCs/miR-126group.All werestatistically significant.Conclusions: MiR-126enhances the ability of proangiogenic properties of EPCs,including proliferation, migration and tubulogenic activity, by targeting PIK3R2directly.The mechanism of miR-126effecting on EPCs is through PI3K/Akt signal pathway. EPCsvia upregulation of miR-126expression can promote venous thrombosisresolution,organization and recanalization. Based on the results in this research,regulationof miR-126in EPCs may represent a potential therapeutic intervention in EPCs-mediatedtherapy for deep venous thrombosis.更多还原