【作者】 陈军；

【导师】 张凯伦；

【作者基本信息】 华中科技大学 ， 心胸外科， 2009， 博士

【摘要】 目的探讨成年大鼠骨髓间充质干细胞(BMSC)在体外分离、培养的方法；观察BMSC的生物学特性；第2、3、4、5代BMSC的SDF-1受体CXCR4基因的表达。方法提取成年大鼠骨髓，全骨髓培养法通过多次传代获取纯化的BMSC。通过相差显微镜观察细胞形态学变化，免疫组化检测第2、3、4、5代BMSC的CD34、CD44、CXCR4的表达。RT-PCR检测第2、3、4、5代BMSC的CXCR4的表达变化。结果全骨髓培养法培养的BMSC细胞增殖能力强、细胞均一性较好。免疫组化检测显示BMSC高表达CD44，基本不表达CD34，CXCR4有较强表达，RT-PCR示BMSC的CXCR4 mRNA表达呈阳性，各代BMSC的CXCR4的表达无明显变化。结论全骨髓培养法是一种简便易行、有效分离BMSC的方法。BMSC表达CXCR4。目的探讨成功制备大鼠心肌梗死模型的方法。方法结扎SD大鼠左前降支制作急性心肌梗死模型，分析80只大鼠术中术后并发症的成因和防治方法，并经心电图、组织学和超声心动图等检查确认模型成功建立。结果急性心肌梗死大鼠术后早期心电图出现ST段抬高、室壁发白；晚期心电图出现病理性Q波，室壁瘢痕形成，心功能明显下降。手术中的主要并发症和死因有心律失常、出血、呼吸道阻塞和肺损伤等。手术后的主要并发症和死因有感染、肺不张、心心衰等。结论本实验通过减少肺损伤、心律失常的发生，及采取抗感染等措施成功建立SD大鼠心肌梗死模型。目的：研究基质细胞衍生因子-1(SDF-1)／CXCR4轴在骨髓间充质干细胞(BMSC)迁移到梗死心肌中的作用。方法全骨髓贴壁法培养BMSC，结扎SD大鼠左前降支制作急性心肌梗死模型并制备正常和梗死心肌组织提取液，利用Boyden小室体外迁移体系观察不同浓度SDF-1和心肌组织提取液对BMSC的趋化作用；心肌梗死后不同时期的梗死组织提取液对BMSC的体外趋化作用的差异；AMD3 100处理BMSC对心肌组织提取液趋化BMSC的影响。结果SDF-1对BMSC有剂量依赖性的趋化作用；梗死后24h至1w的心肌组织提取液对BMSC有明显的趋化作用，72h至1w作用最明显，而这种作用可部分被SDF-1受体CXCR4的阻断剂AMD3100抑制。结论梗死心肌组织提取液对BMSC有明显的趋化作用，SDF-1／CXCR4可能是梗死心肌组织提取液中趋化BMSC的主要趋化因子／受体对。目的探讨SDF-1对大鼠骨髓间充质干细胞(BMSC)增殖的影响；SDF-1预处理对大鼠BMSC在无氧无血清条件下凋亡的影响。方法本实验采用全骨髓贴壁法培养BMSC。不同剂量SDF-1(1ng／mL、10ng／mL、100ng／mL)与BMSC共培养48h，MTT法分析BMSC增殖的增殖情况。将细胞分别与10ng／mL、100ng／mLSDF-1作用24h后在无氧无血清条件下培养6h，流式细胞仪和TUNEL法检测细胞凋亡变化。结果MTT结果表明，SDF-1呈剂量依赖性促进BMSC增殖，以100ng／mL SDF-1促BMSC增殖作用最明显。在无氧无血清条件下，SDF-1预处理组细胞的凋亡率与对照组的凋亡率相比降低(P＜0．05)，100ng／mL SDF-1预处理组凋亡率最低。结论SDF-1呈剂量依赖性促进BMSC增殖。SDF-1预处理BMSC能抑制其无氧无血清条件下的凋亡。目的探讨基质细胞衍生因子-1(SDF-1)预处理的大鼠骨髓间充质干细胞(BMSC)移植对大鼠急性心肌梗死(AMI)的治疗效果。方法全骨髓贴壁法培养BMSC；将BMSC与100ng／mL SDF-1作用24h。建立大鼠AMI模型，将经100ng／mL SDF-1预处理的BMSC和未经处理的BMSC植入大鼠梗死心肌周边，2W后超声观察心脏功能的变化。结果SDF-1预处理BMSC移植组对大鼠急性心肌梗死后心功能改善最为明显。结论SDF-1预处理BMSC是一种能增强BMSC移植治疗心肌梗死效果的新方法。更多还原

【Abstract】 Experiment 1Experimental study on isolation and culture of bone mesenchymalstem cells and its CXCR4 expressionObjective To investigate the method of the isolation and culture of bone marrow-derivedmesenchymal stem cells(BMSC);To observe the biological characterization of BMSC andthe expression of Stromal cell-derived factor-1 (SDF-1) receptor-CXCR4 in the2th,3th,4th,Sth generation of BMSC.Methods Bone marrow mesenchymal stem cells were isolated from adult SD mice by thewhole bone marrow culture and were purified through several passages.The morphology ofBMSC was observed under inverted micro scope.Immunohistochemistry was used todetermine the expression of CD34,CD44,and CXCR4 in the 2nd,3rd,4th,5th generationof BMSC respectively.RT-PCR was used to measure the expression of CXCR4 in the 2nd,3rd,4th,5th generation of BMSC respectively.Results BMSC obtained by the whole bone marrow culture had good performances ofpurity,uniform and ability of proliferation in the process of culture in vitro.Immunohistochemical analysis revealed that part of BMSCS were CXCR4 positive andmost BMSCS were CD44 positive,while most BMSCS were CD34 negative.The mRNAof CXCR4 was successfully reversely transcribed by RT-PCR in every generation ofBMSC,and their quantity has no difference.Conclusions The whole bone marrow culture method is a helpful method that can obtainBMSC conveniently and effectively.BMSC exhibit CXCR4 expression. Experiment 2Study on rat model of acute myocardial infarctionObjective To find out how rat model of acute myocardial infarction can be establishedsuccessfully.Methods Left anterior descending coronary arteries were ligated to produce acutemyocardial infarction in SD rats.The cause of complications in and after operation and itsprevention and cure approaches were analyzed in 80 rats.Credible rat models wereevidenced by electrocardiogram,histology examination and ultrasonic cardiogram.Results Rats established model of acute myocardial infarction had elevation of ST segmentin ECG in early stage,patho-Q wave in late stage、scarring in ventricular wall and worseheart function.Intraoperative main complications and death causes were arrhythmia,hemorrhage,airway obstruction,injury of lungs,an so on;postoperative maincomplications and death causes were infection,pulmonary atelectasis and congestive heartfailure,an so on.Conclusion Our methods include decreasing lung injury,injecting anti infective and so on.They give a guarantee for making a successful rat AMI model. Experiment 3In vitro study on the role of SDF-1/CXCR4 axis in the migration ofbone mesenchymal stem cells into heart after acutemyocardial infarctionObjective To study the role of stromal cell derived factor 1/CXC chemokine receptor 4axis (SDF-1/CXCR4 axis) in the migration of bone mesenchymal stem cells into the heartafter acute myocardial infarction.Methods Bone mesenchymal stem cells culture were performed with the wholemarrow-adherence way,Acute myocardial infarction (AMI) model was built by ligation ofleft anterior descending coronary in SD rats,cardiac tissular extract were made fromnormal heart and infracted heart.By using Boyden chamber in vitro migration assaysystem,the effects of different concentrations of SDF-1,cardiac tissular extract fromnormal heart and from infracted myocardium at different time point after acute infractionon bone mesenchymal stem cells migration were observed,the influence of AMD3100treatment on bone mesenchymal stem cells’ migration induced by cardiac tissular extractwere also observed.Results SDF-1 had a concentration-dependent chemotaxis effect on bone mesenchymalstem cells.Cardiac tissular extract made from hearts one week after AMI can induce themigration of bone mesenchymal stem cells obviously,and this effect can be suppressed byAMD3100 which is a blocker of CXCR4.Conclusion Cardiac tissular extract from hearts one week after AMI can induce themigration of marrow stromal cells,SDF-1/CXCR4 axis play an important role in thisprocess. Experiment 4Effects of stromal-derived factor 1 on proliferation and apoptosis ofrat bone mesenchymal stem cells in vitroObjective To detemrine the effect of SDF-1 on in-vitro proliferation of bone mesenchymalstem cells (BMSC);To investigate the effects of stromal-derived factor 1 preconditioning(PC) on apoptosis of bone mesenchymal stem cell (BMSC) treated with hypoxia plusserum deprivation.Methods BMSC culture was performed with the whole marrow-adherence way;BMSCwere incubated with different doses of SDF-l(lng/mL,10ng/mL,100ng/mL)for 48h,thenproliferation of BMSC were measured by MTT assays.BMSC were incubated in mediumfor 24 hours with 10ng/mL、100ng/mL SDF-1 respectively,then they were treated withhypoxia plus serum deprivation for 6 hour.Apoptic rate were detected by flow cytometerand TUNEL method.Results MTT assay showed that SDF-1 could enhance BMSC proliferation in dose dependmanner compared with the control group and the effect on proliferation of BMSC washighest in 100ng/ml group among all groups.The number of apoptotic cells wassignificantly reduced in SDF-1 PC group compared with the control group,and 100ng/mLSDF-1 PC group has the lowest level of apoptosis.Conclusion SDF-1 could enhance BMSC proliferation in dose depend manner.SDF-1preconditioning suppresses the apoptosis of BMSC treated with hypoxia plus serumdeprivation. Experiment 5Study about cellular transplant applied with rat bone mesenchymalstem cells preconditioned with stromal-derived factor 1 totreat acute myocardial infarctionObjective To observe therapeutic efficacy of cellular transplant with bone mesenchymalstem cells (BMSC) preconditioned with SDF-1 in rats with acute myocardial infarction.Methods BMSC cultures were performed with the whole marrow-adherence way;BMSCwere incubated in medium for 24 hours with 100ng/mL SDF-1.Acute myocardial infarction(AMI) model was built in SD rats,and BMSC preconditioned or non-preconditioned withSDF-1 were transplanted into border zone around infarct area,then heart function wasdetermined after 2 weeks by ultrasonography.Results We established AMI model successfully.Two weeks after BMSC transplant,significant improvement in cardiac function was observed in 100ng/mL SDF-1preconditioned group compared with the non-preconditioned group.Conclusion We conclude that SDF-1 PC is a novel approach for enhancing therapeuticefficacy of cellular transplant in rats with acute myocardial infarction.更多还原