【作者】 刘德忠；

【导师】 石炳毅；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学， 2009， 博士

【摘要】 第一部分大鼠颈部心脏移植模型的建立目的:建立成功率较高的大鼠颈部异位心脏移植模型。方法:采用自制的套管连接血管建立移植心脏血流通路:移植心脏无名动脉连接大鼠右颈总动脉,移植心脏左肺动脉连接大鼠右颈外静脉,建立大鼠颈部心脏移植模型。术后24小时移植心脏正常跳动视为移植成功。结果:建模成功率为88%（30/34）。结论:成功应用套管法建立了大鼠颈部心脏移植模型。第二部分大鼠骨髓间充质干细胞的体外分离、培养与鉴定目的:建立稳定的大鼠骨髓间充质干细胞（Mesenchymal stem cells,MSC）体外分离、培养、鉴定方法。方法:选用1月龄雄性清洁级Wistar大鼠,处死消毒后取其胫骨和股骨,用含15%胎牛血清DMEM冲洗骨髓腔,收集冲洗液,采用密度梯度离心法分离获取细胞,培养后对贴壁细胞进行传代培养。取第3代生长状态良好的细胞,以流式细胞术鉴定细胞表型,并进行冻存复苏实验。结果:MSC在培养瓶中24h后就有细胞贴壁,第3～4d细胞生长明显,经换液去除未贴壁细胞,传代后细胞生长迅速,约6～8d即可长满培养瓶底,达融合状态,三代后即可得到较为纯化的MSC成纤维样细胞,并至少可传代10代以上,冻存对细胞生长无影响。流式细胞术检测细胞表面分子标志,显示CD11B（-）、CD29（+）、CD34（-）、CD45（-）、CD90（+）、CD106（+）。结论:通过密度梯度离心及贴壁筛选培养法获取大鼠骨髓MSC的方法简便易行,可提供稳定的大鼠MSC。第三部分受者同系骨髓间充质干细胞对大鼠心脏移植急性排斥反应的影响目的:观察受者同系骨髓MSC对大鼠心脏移植急性排斥反应的影响,并探讨MSC的免疫调节机制。方法:实验动物随机分为2组,每组10只受体,进行Lewis→Wistar颈部心脏移植:（1）空白对照组（A₃组）:手术后24h,确定手术成功,即经尾静脉注射3mL0.9%氯化钠注射液;（2）MSC处理组（B₃组）:手术后24h,确定手术成功,即经尾静脉注射MSC2×10⁶（悬浮在3mL0.9%氯化钠注射液中）。术后一周,每组随机取4只大鼠,取下腔静脉血及移植物进行组织学及免疫学检查。检测项目:病理学分级,血清IL-2及IL-10,血、移植物CD4⁺、CD8⁺、CD4⁺CD25^high、CD4⁺CD25^highfoxp3⁺T细胞占总淋巴细胞的比例,及CD4⁺/CD8⁺比值。其余6只用以继续观察移植物存活时间。另外再建立2只大鼠心脏移植模型,术后24小时,经尾静脉注射表达绿色荧光蛋白的同系MSC(MSC^GFfP)2×10⁶,术后7天取移植心脏,冰冻切片,荧光显微镜下观察移植物内荧光细胞的分布情况。结果:（1）A₃组大鼠移植心脏存活7.2±1.3d,B₃组大鼠移植心脏存活14.8±2.9d。P＜0.01。（2）血清IL-2:A₃组为233.99±30.19pg/mL,B₃组为249.11±20.34 pg/mL,P＞0.1;血清IL-10:A₃组为81.44±11.01 pg/mL,B₃组为158.39±18.70 pg/mL,P＜0.01。（3）病理学分级,A₃组3B级1例,4级3例;B₃组2级1例,3A级2例,3B级1例,B₃组病理分级总体较A₃组低。（4）大鼠血中CD4⁺/CD8⁺值、CD4⁺CD25^highT细胞占总淋巴细胞的比例、CD4⁺CD25^highFoxp3⁺T细胞占总淋巴细胞的比例,B₃组都明显高于A₃组,差异有统计学意义（P＜0.01）。（5）移植心脏中CD4⁺/CD8⁺值两组之间无显著性差异,CD4⁺CD25^highT细胞占总淋巴细胞的比例、CD4⁺CD25^highFoxp3⁺T细胞占总淋巴细胞的比例,B₃组都明显高于A₃组,差异有统计学意义（P＜0.01）。（6）心脏移植术后24h,经尾静脉向受体大鼠注射同系MSC^GFP后6天,荧光显微镜下观察移植心脏冰冻切片可见荧光细胞在移植心脏表面分布较集中,并向心肌组织浸润,形态及分布类似心肌细胞。结论:静脉注射受者同系骨髓MSC,可以延长大鼠颈部移植心脏的存活时间,减轻急性排斥反应的程度。第四部分受者同系骨髓间充质干细胞对大鼠心脏移植慢性排斥反应的影响目的:观察受者同系骨髓MSC对大鼠心脏移植慢性排斥反应的影响。方法:实验共设4组,每组5只受体大鼠,行颈部心脏移植手术:（1）同系移植组（A₄组）:Wistar→Wistar,CsA2mg/kg·d腹腔注射14天;（2）慢性排斥组（B₄组）:Lewis→Wistar,CsA 2mg/kg·d腹腔注射14天;（3）MSC处理组（C₄组）:Lewis→Wistar,CsA 2mg/kg·d腹腔注射14天,术后第15天、第30天分别经尾静脉注射MSC2×10⁶;（4）单纯CsA组（D₄组）:Lewis→Wistar,CsA 2mg/kg·d腹腔注射至观察终点。观察终点为术后120天。届时,取移植心做病理检查,MASSON染色,分析心脏动脉管壁增厚情况及心肌间质胶原增生情况。结果:所有受体及移植物均良好存活至观察终点。移植心脏内动脉管壁增厚比例（%）:A₄组19.68±2.06,B₄组29.14±1.98,C₄组29.88±2.28,D₄组39.82±1.84。B₄-C₄组之间差异无统计学意义（P＞0.05）。胶原增生比例（%）:A₄组8.76±1.56,B₄组17.98±2.40,C₄组19.50±2.24,D₄组17.64±2.47。A₄组纤维增生较其它各组明显减轻,差异有统计学意义（P＜0.01）;B₄、C₄、D₄各组之间差异无统计学意义（P＞0.05）。结论:早期应用CsA2周后,静脉输注受者同系MSC未能减轻移植心脏慢性排斥反应的程度。更多还原

【Abstract】 PartⅠEstablishment of a Cervical Heterotopic Cardiac Transplantation Model of RatsObjectives:To establish a modified model of rat cervical heterotopic cardiac transplantation for high achievement ratio.Methods:A right cervical heterotopic cardiac transplantation model of rats was established.The allograft blood circulation was established with cuff technique using home-made cannulas.Through the cannulas,the donor innominate artery was anastomosed to the recipient’s right common carotid artery and donor left pulmonary artery to the recipient’s external jugular vein.The successful standard of transplantation was the transplanted heart could beat rhythmically over 24 hours.Results:Successful rate of heterotopic cardic transplantation is 88%（30/34）.Conclusions:A modified model of rat cervical heterotopic cardiac transplantation was established with cuff technique.PartⅡIsolation,Culturing and Identification of Rat MSC From Bone Marrow in vitroObjectives:To establish the methods for the isolation,culturing and identification of rat MSC from bone marrow in vitro. Methods:One month old male Wistar rats were killed to get the tibias and femurs.After the long bones were dissected from rats,the marrows were flushed out with DMEM medium（supplemented with 15%fetal bovine serum） under aseptic condition and purified by Percoll gradient centrifugation to get the mononuclear cells.After the mononuclear cells were cultured,the adherent cells were chosen for serial subcultivation.The third generation cells were harvested and the cell phenotypes were identified by flow cytometry.Results:The mesenchymal stem cells were adhered to plastic surface within 24h.The cell growth was obvious on the 3～4 days.Nonadherent cells were removed by changing the culture medium.After passage,the cells gown rapidly to fill the floor of cell culture flasks and achieved confluence within 6～8 days. Purified MSC fibroblast-like cells could be harvested after 3 generations.There was no obvious influence to cell growth when reserved in frozen condition.The cells could passage for at least 10 generations.The cell phenotypes identified by flow cytometry showed CD29（+）、CD34（-）、CD45（-）、CD11B（-）、CD90 （+）、CD106（+）.Conclusions:The methods of obtaining rat MSC with density gradient centrifugation and adherence cultivation was convenient,and could provide immortalized rat MSC lines.PartⅢEffects of Bone Marrow Mesenchymal Stem Cells Derived from Recipients on the Acute Rejection in Rat Cardiac Transplantation ModelsObjectives:To investigate the effects of MSC derived from recipients’ bone marrow on the acute rejection of rat cardiac transplantation and study the possible underlying mechanisms.Methods:Twenty Wistar rats beating transplanted heart from Lewis rats were divided randomly into 2 groups（10 rats each group ）:（1）Control group （group A3）:3mL 0.9%NaCl solution was injected through tail vein at 24h after heart transplantation.（2） Treatment group（group B₃）:2×10⁶ MSC in 3mL 0.9%NaCl solution was injected through tail vein at 24h after heart transplantation. One week after transplantation,4 rats randomly from each group were anaesthetized to get blood from inferior canal vein and allografts.Grafts pathology was studied.Serum levels of IL-2 and IL-10 was detected by method of ELISA.Flow cytometry was used to detect the ratio of CD4⁺ T cells,CD8⁺ T cells, CD4⁺CD25^high T cells and CD4⁺CD25^highfoxp3⁺ T cells in lymphocytes in the blood and grafts.The rest 6 rats in each group were observed survival time of grafts.Two other cardiac transplantation models were established.At 24h after transplantation,2×10⁶ MSC^GFP were injected throug tail vein,respectively.The two grafts were harvested on 7^th day after transplantation to get frozen sections for observing the fluorescence cells in grafts.Results:（1） The survival time of allografts:group A₃ was 7.2±1.3d,group B₃ was 14.8±2.9d.P＜0.01.（2）Concentrations of serum IL-2:group A₃ was 233.99±30.19pg/mL,group B₃ was 249.11±20.34 pg/mL,P＞0.1;IL-10:group A₃ was 81.44±11.01 pg/mL,group B₃ was 158.39±18.70 pg/mL,P＜0.01. （3）Pathology grade:group A₃:1 case was Grade 3B,3 cases were Grade 4;group B₃:1 case was Grade 2,2 cases were Grade 3A,1 case was Grade 3B.（4） Ratios of CD4⁺/CD8⁺,CD4⁺CD25^high T cells/total lymphocytes and CD4⁺CD25^highFoxp3⁺ T cells/total lymphocytes in blood:group B3 was higher than group A₃,P＜0.01.（5） Ratios of CD4⁺/CD8⁺ in allografts were similar in two groups,P＞0.1;CD4⁺CD25_highT cells/total lymphocytes and CD4⁺CD25^highFoxp3⁺ T cells/total lymphocytes in allografts:group B₃ was higher than group A₃, P＜0.01.（6） Fluorescence cells could be observed to concentrate to the surface of allografts,some cells had infiltrated into cardiac muscle and their shapes and distributions were just like cardiac muscle.Conclusions:Intravenous infusion with MSC from recipients could prolong the survival time of transplanted heart and inhibit acute rejection. PartⅣEffects of Bone Marrow Mesenchymal Stem Cells Derived from Recipients on the Chronic Rejection in Rat Cardiac Transplantation ModelsObjectives:To observe the effects of mesenchymal stem cells derived from recipients’ bone marrow on the chronic rejection of rat cardiac transplantation.Methods:Twenty rats bearing transplanted hearts were divided randomly into 4 groups（5 rats each group）:（1）Homologous transplantation group（group A₄）:Wistar-Wistar,CsA 2mg/kg·d was injected into abdominal cavity for 14 days after transplantation;（2）Chronic rejection group（group B₄）:Lewis-Wistar,CsA 2mg/kg·d was injected into abdominal cavity for 14 days after transplantation; （3）MSC treatment group（group C₄）:Lewis-Wistar,CsA 2mg/kg·d was injected into abdominal cavity for 14 days after transplantation.2×10⁶ MSC was injected through tail vein at the 15^th day and 30^th day after transplantation,respectively;（4） CsA treatment group（group D₄）:Lewis-Wistar,CsA 2mg/kg·d was injected into abdominal cavity from after transplantation to the end of investigation.The investigation time was 120 days after transplantation.On 120^th day after transplantation,the allografts were harvested for pathological examination to analyze the depth of artery wall and the infiltration of collagenous fiber in cardiac muscle by MASSON staining.Results:All recipients and transplanted hearts survived well to the investigating ends.The mean ratio of artery wall depth to artery diameter（%）: group A₄ was 19.68±2.06,group B₄ was 29.14±1.98,group C₄ was 29.88±2.28 and group D₄ was 39.82±1.84.Group B₄ and group C₄ is similar,P＞0.05.The infiltration area of collagenous fiber to cardiac muscle（%）:group A₄ was 8.76±1.56,group B₄ was 17.98±2.40,group C₄ was 19.50±2.24 and group D₄ was 17.64±2.47.Group A₄ was less infiltrated with collagenous fiber than the other 3 groups,P＜0.01.Group B₄,group C₄ and group D₄ were similar in the infiltration of collagenous fiber,P＞0.05.Conclusions:Intravenous infusion with MSC from recipients did not reduce the level of chronic rejection of rat cardiac transplantation.更多还原