【作者】 李桂琼；

【导师】 陈庆伟；

【作者基本信息】 重庆医科大学 ， 老年医学， 2012， 博士

【摘要】 目的：在体外分离、诱导培养和鉴定大鼠骨髓来源的EPCs，5-BrdU进行标记。方法：采用Percoll密度梯度离心法和差速贴壁培养法，分离并抽取SD大鼠股骨和胫骨骨髓单个核细胞(MNCs)，在给予碱性成纤维细胞生长因子(Basic fibroblastgrowth factor，bFGF)等生长因子的条件下诱导培养14天。通过普通光学显微镜观察细胞的形态学特征。免疫细胞化学染色以鉴定其表面抗原表达情况。用双荧光染色检测Dil标记的乙酰化低密度脂蛋白(Acetyl low density lipoprotein，acLDL)和FITC标记的荆豆凝集素l(Ulex europaeus agglutinin，UEA-1)进行鉴定。分离培养的EPCs用不同浓度的5-溴脱氧尿嘧啶（5-Brdu）进行标记，MTT观察5-Brdu对其增殖活性的影响。5-BrdU标记EPCs备用。结果：贴壁细胞起初呈铺路石样，随后呈现梭形样排列生长。CD34、CDl33、Flk-1(VEGFR)和vWF(Ⅷ因子)在贴壁细胞中呈不同时段的阳性表达。EPCs摄取Dil-acLDL，且结合FITC-UEA-1。5-BrdU可以标记EPCs，且不会影响细胞增殖活性。结论：采用Percoll密度梯度离心法和差速贴壁结合法，同时给予VEGF、bFGF及EGF诱导，可以获得较高纯度的EPCs，且该细胞具备了成熟的ECs的部分功能学特性，5-Brdu标记EPCs可行。目的：通过化学偶联法制备抗肌凝蛋白轻链/CD34双特异性抗体（BsAb），并装配在内皮祖细胞（EPCs）表面。方法：抽取、分离和培养自体EPCs。通过化学偶联法构建BsAb以及FITC标记的BsAb。分别将10ng、25ng、50ng和100ng的FITC标记的BsAb与1×10~6个EPCs混合，在荧光显微镜及流式细胞仪下观察抗体与细胞的结合情况，以筛选出抗体装配的最佳浓度。使用上述实验所筛选出的条件，装配BsAb和EPCs备用。结果：通过化学偶联法成功制备抗肌凝蛋白轻链/CD34双特异性抗体（BsAb），并能够装配在内皮祖细胞（EPCs）表面。BsAb在EPCs表面装配的最佳浓度为50ng/106细胞。结论：BsAb构建成功，并在EPCs表面成功装配BsAb。目的：本部分我们探讨了超声破坏微泡对促进BsAb标记的EPCs向缺血心肌定向移植的可行性和治疗效果。方法：使用前两部分中所制备的BsAb标记的EPCs。选取42只6周龄健康的SD大鼠制备大鼠急性心肌梗死模型，划分为①正常组（Normal），②心梗组（MI），③单纯双抗组（BsAb），④单纯超声+微泡组（US+MB），⑤单纯细胞组（EPCs）；⑥细胞+抗体组（EPCs+BsAb），⑦细胞+超声微泡+抗体组（EPCs+BsAb+US+MB）。建模8小时后，给予超声微泡和细胞移植处理，最后一组经尾静脉注射1ml经BsAb装配的EPCs，并注射脂质超声微泡溶液1ml，超声波辐照大鼠心前区。移植30天后，取大鼠心肌组织，切片进行免疫组织化学染色5-BrdU以检测EPCs的迁移和分布情况。利用超声心动图检测各组大鼠心功能变化情况，计算左心室射血分数(LVEF)及左心室短轴缩短率(FS)等以评价心功能。采用免疫组织化学法检测大鼠在细胞移植后30天后心脏中Ⅷ因子的表达情况。利用Real time PCR和Westernblot检测细胞移植后各组大鼠VEGF及SDF-1基因的mRNA表达和蛋白表达水平。结果： EPCs+BsAb+US+MB组大鼠心肌5-BrdU免疫染色阳性细胞明显高于MI组，P<0.01。超声心电图检测大鼠左心室射血分数(LVEF)及左心室短轴缩短率(FS)等，EPCs+BsAb+US+MB组左心室收缩功能明显优于MI组，基本恢复至Normal组水平。Ⅷ因子免疫组化染色显示，EPCs+BsAb+US+MB组毛细血管数较其他各组多。RT-PCR和Western blot检测VEGF及SDF-1的表达水平，EPCs+BsAb+US+MB组VEGF和SDF-1的mRNA水平和蛋白表达水平均高于MI组，P<0.01，与Normal组没有显著差异。结论：BsAb与超声破坏微泡技术联合使用，可以促进BsAb标记的EPCs归巢至大鼠缺血心肌，促进血管新生，为干细胞移植治疗心肌梗死提供了新的思路。目的：探讨FTO基因多态性与癌症风险。方法：收集Pubmed和Embase数据库的文献。使用Meta分析，计算固定效应模型95%置信区间(CI)的合并比值比(OR)。13个研究中心的16277名患者以及31153名对照人员被纳入研究。结果：除了胰腺癌外(OR=1.10,95%CI1.03–1.19)，FTO多态性与癌症风险增加相关性无显著性差异。(OR=1.01,95%CI0.98–1.04)。研究中没有文献偏差(Begg’stest: P=0.760; Egger’s test: P=0.553)。结论：Meta分析表明尽管FTO基因多态性和胰腺癌有微弱关联，但是FTO基因多态性和癌症风险增加没有明显关联。但是这个结论仍然需要谨慎，因为这些研究中都没有把BMI/肥胖考虑进去。更多还原

【Abstract】 Objective: To extract, culture and identify rat bone marrow-derived EPCs in vitro,labeled cells with5-BrdU.Methods: The SD rat femur and tibia bone marrow mononuclear cells (MNCs) wereisolated and extracted by Percoll density gradient centrifugation and speed differenceadhesion culture. MNCs were cultured with administration of basic fibroblast growthfactor (bFGF), vascular endothelialcell growth factor (VEGF) and epidermal growthfactor (EGF) for14days. The morphological features were further observed withoptical microscope. Immunocytochemistry was used to identify their surface antigenexpression. Double fluorescence staining was used to detect Di-labeled Acetyl lowdensity lipoprotein (Di-acLDL) and FITC-labeled Ulex europaeus agglutinin (UEA-1).Identified EPCs were incubated with5-BrdU. EPCs were incubated with5-BrdU atdifferent concentrations, cells proliferation was assayed by MTT. EPCs Labeled with5-BrdU were for subsequent use.Results: Adherent cells grew showing an initial cobblestone, and then a spindle-like arrangement style. CD34, CDl33, Flk-1(VEGFR) and vWF (VIII of factor) werepositively expressed at different stages in the adherent cells. The EPCs uptookDi-acLDL and bound FITC-UEA-1.5-BrdU can be used to lable EPCs，did not affectcells proliferation.Conclusions: Percoll density gradient centrifugation and speed difference cultivationcombined withVEGF, bFGF, and EGF could obtain highly purified EPCs, and thesecells showed some of the features of mature ECs.5-BrdU can be used to lable EPCs. Objective: To prepare anti-myosin light chain/CD34bispecific antibody (BsAb) bychemical heteroconjugation, and to assemble them on the surface of EPCs.Methods: EPCs were extracted, isolated and cultured as described above. BsAb andFITC-BsAb were generated by chemical heteroconjugation. EPCs were mixed withFITC-BsAb at10ng,20ng,50ng and100ng, the binding activity was detected byfluorescence and flow cytomety to screen out the optimal concentration of the antibody.EPCs were assembled with these BsAbs for further purpose.Results: BsAb was successfully generated and assembled on the surface of EPCs. Themost optimal concentration was50ng/106cells.Conclusions: BsAb was successfully generated and further assembled on the surfaceof EPCs. Objective: To study the feasibility of ultrasound microbubble destruction promotingBsAb-labeled EPCs to transplant to the ischemic myocardium.Methods: BsAb-labeled EPCs were obtained as previously discribed.426-week,healthy SD rats were used as acute myocardial infarction model. They were divided asfollowing:1. Normal;2. MI;3. Antibody;4. US and MB;5. EPCs;6EPCs andantibody;7. EPCs, US, MB and antibody. Rats were treated with ultrasound andtransplanation8hours after modeling. The last group was injected1ml BsAb-labeled ofEPCs, and was further injected with lipid ultrasound microbubble solution1ml. Theprecordium of the rats were irradiation. After30days, the myocardial tissues werestained with5-BrdU to detect the migration and distribution of EPCs.Echocardiography was used to detect the heart function. The function were detected byleft ventricular ejection fraction (LVEF) and left ventricular fractional shortening (FS).VIIIwas detected by immunochemistry after30days. Real time PCR and western blotwere equipped to detect the expression of VEGF and SDF-1.Results: Group7(EPCs, UM and antibody) was stained with more5-BrdUsignificantly. Group7showed high LVEF and FS compared with MI groups. Group7had more positive capillaries. The mRNA and protein level of VEGF and SDF-1ingroup7rats increased compared with MI groups through real time PCR and Westernblot. There was no difference between group7and Normal group of the mRNA andprotein level of VEGF and SDF-1through real time PCR and Western blot.Conclusions: Combined with UM, BsAb could promote the target-directed migrationof EPCs and capillaries genesis. The work provided new strategy for the treatment of myocardial infarction. Objective: to clarify the association between FTO polymorphism and cancer risk.Methods: Published literature from PubMed and Embase databases had been retrieved.Pooled odds ratio (OR) with95%confidence interval (CI) was calculated byfixed-effects model.13studies involving16,277cases and31,153controls had beenidentified.Results: FTO polymorphism was not significantly associated with the increased riskof cancer (OR=1.01,95%CI0.98–1.04), except that a statistically significantassociation with pancreatic cancer (OR=1.10,95%CI1.03–1.19). No publication biaswas detected (Begg’s test: P=0.760; Egger’s test: P=0.553).Conclusions: Our meta-analysis implicated that no association between FTOpolymorphism and the increased risk of cancer existed, although it was marginallyassociated with pancreatic cancer. However, the conclusion should be made withcaution for most included studies had not take BMI/obesity into account.更多还原