【作者】 林慧庆；

【导师】 王建军；

【作者基本信息】 华中科技大学 ， 外科学， 2008， 博士

【摘要】 第一部分大鼠同种异体原位肺移植模型的建立目的:通过大鼠套管法吻合技术建立大鼠左侧原位肺移植模型。方法:将60只SPF级SD大鼠随机分为供、受体两组,采用三套管吻合技术完成大鼠肺移植模型。结果:30例手术中,供肺灌注到摘取时间(10±1)分钟,供肺完成套管时间(10±4)分钟,供体动静脉、支气管吻合时间(15±2)分钟,移植肺热缺血时间(16±2)分钟,冷缺血时间(30±5)分钟,手术平均时间(80±10)分钟,术后15天生存率达86%,其中两例死于急性肺水肿,一例死于肺静脉栓塞,一例死于对侧气胸。结论:三套管大鼠同种异体肺移植模有效、可靠,为临床肺移植提供了较好的实验基础,但是模型操作复杂,需要长时间的学习和反复操作来提高肺移植模型的稳定性。第二部分落新妇甙对大鼠肺移植排斥反应的抑制作用目的:运用排斥反应分级及观察落新妇甙对大鼠移植肺中支气管肺泡灌洗液诱导肺成纤维细胞胶原增生的抑制作用实验,探讨落新妇甙对大鼠肺移植排斥反应的抑制作用。方法:采用大鼠左侧原位肺移植模型。随机将肺移植后的60只大鼠分为4组:A组肺移植后用生理盐水(1ml/天)灌胃,B组肺移植后用环孢素A (5mg/kg/天)灌胃,C组肺移植后用落新妇甙(1mg/kg/天)灌胃,D组肺移植后用环孢素A (2.5mg/kg/天)及落新妇甙(1mg/kg/天)灌胃。术后第30天(每组抽取5只大鼠)切取移植肺观察肺急性排斥反应分级情况;另外每组10只大鼠,取受体支气管肺泡灌洗液。用收集的支气管肺泡灌洗液在体外刺激人胚胎成纤维细胞,通过MTT比色法和酶联免疫(ELISA)法分别检测人胚肺成纤维细胞的增殖及Ⅰ型胶原的合成,并对检测结果进行分析。结果:(1)B组、C组及D组移植肺排斥反应分级均低于A组(P<0.01);D组移植肺急性排斥反应最轻,优于C组(P<0.05)和B组(P<0.01)。(2)受者移植肺中支气管肺泡灌洗液显著促进了人胚肺成纤维细胞的增殖和胶原合成;(3)B、C、D组的支气管肺泡灌洗液诱导的人胚肺成纤维细胞Ⅰ型胶原合成有显著的抑制作用(P<0.05)。结论:落新妇甙可抑制大鼠移植肺中支气管肺泡灌洗液诱导的人胚肺成纤维细胞的增殖及胶原合成,对大鼠肺移植后的闭塞性细支气管炎具有抑制作用;对肺移植排斥反应有较强的抑制作用,并与环孢素A具有协同作用。第三部分落新妇甙对大鼠肺移植急性排斥反应抑制作用的机制研究实验一落新妇甙对肺移植大鼠活化T细胞的动态影响目的:观察落新妇甙对大鼠移植肺活化T细胞的影响及动态变化,探讨落新妇甙对大鼠移植肺活化T细胞的作用。方法:采用大鼠左侧单肺原位移植模型。大鼠10只不做移植,抽取外周血作为空白对照;随机将肺移植后的40只大鼠分为2组:A组肺移植后用生理盐水(1ml/天)灌胃,B组肺移植后落新妇甙(1mg/kg/天)灌胃。移植前(空白对照)、术后第2天、术后第5天,分别用荧光标记抗体双染色结合流式细胞技术检测各阶段大鼠T细胞表达的活化抗原CD69、CD25和CD71的表达。结果:术后第2天,各组检测的CD69、CD25和CD71抗原均显著高于对照组(P<0.01),且B组和A组有统计学差异(P<0.05),显示术后第2天,活化T细胞增加,但落新妇甙能显著抑制T细胞的活化。术后第5天,B组CD69、CD25和CD71抗原基本降至正常水平,显著低于A组。结论:落新妇甙能显著抑制急性排斥反应诱导的T细胞活化,对肺移植排斥反应有较强的抑制作用。实验二落新妇甙诱导移植肺活化T细胞凋亡减轻急性排斥反应目的:探讨落新妇甙对大鼠肺移植后机体急性排斥反应的影响和机制。方法:建立大鼠左肺原位移植模型,术后将受体大鼠随机分为两组:对照组和实验组。对照组术后生理盐水1ml/天灌胃,实验组术后落新妇甙1mg/kg/天灌胃,观察肺移植的存活时间、大鼠脾细胞T淋巴细胞转化率和脾淋巴细胞IL-2活性,以及外周血中活化T细胞凋亡。结果:与对照组相比,实验组脾细胞T淋巴细胞转化率明显降低,两组差异有显著意义(P<0.05)。移植大鼠脾淋巴细胞IL-2活性在实验组中为4.25±2.65UI/ml,对照组IL-2活性为23.46±1.82UI/ml,两组有显著差异(P<0.01)。实验组能有效的诱导急性排斥反应中活化T细胞凋亡。结论落新妇甙通过下调IL-2产生和抑制体内IFN-γmRNA,诱导活化T细胞凋亡,抑制T淋巴细胞增殖分化,广泛抑制了以T细胞为主的肺移植术后急性排斥反应,从而延长大鼠肺移植的存活时间。实验三活化T细胞信号凋亡传导通路在落新妇甙抑制肺移植排斥反应中作用目的:用大鼠左肺原位移植模型,探讨落新妇甙对大鼠肺移植排斥反应中活化T细胞p38丝裂原激活蛋白激酶(p38MAPK)和PI3K/Akt信号传导通路的影响。方法:建立大鼠左肺原位移植模型,在第30天提取大鼠外周血分离活化T细胞进行体外培养,并进行实验分组,A~D组。A组(对照组),行细胞培养;B组(落新妇甙组),培养液中加入落新妇甙15mg/L;C组,落新妇甙(15mg/L)加p38MAPK抑制剂SB203580(5μmol/L);D组,术后每天落新妇甙(15mg/L)加PI3K/Akt抑制剂LY294002(10μmol/L)。TUNEL法检测T淋巴细胞凋亡情况。RT-PCR和Western blot法检测T细胞p38MAPK和PI3K及Akt下游表达的情况。结果:B组(落新妇甙组)T细胞凋亡指数和p38MAPK表达明显高于对照组,而PI3K/Akt明显低于对照组。C组(落新妇甙加p38MAPK抑制剂组)T细胞凋亡和p38MAPK表达均明显低于B组(落新妇甙组)(P<0.01),PI3K/Akt明显高于对照组,但均与对照组无显著差异(P>0.05)。D组(落新妇甙加PI3K/Akt抑制剂LY294002组)T细胞凋亡和PIK3/Akt表达明显高于对照组,p38MAPK明显低于对照组。结论:落新妇甙诱导大鼠肺移植排斥反应中活化T细胞凋亡与其激活p38MAPK信号传导通路、及抑制PI3K/Akt信号传导通路有关。更多还原

【Abstract】 SECTION ONE The Establishment of Orthotopic Left Pulmonary Allograft Model in RatsObjective: To establish rat orthotopic left lung allograft transplantation model by the“cuff-like”vessel and bronchial anastomosis technique. Methods: Sixty male Sprague-Dawley rats were randomly allocated to donors and recipients. Thirty left lung transplantation were performed by using the“cuff-like”vessel and bronchial anastomosis technique. Results: All rats were successfully performed orthotopic left lung allograft transplantation. The mean time for the donor lung (from perfusion to the harvest) was (10±1)mins; the mean time for cuff the vessel and bronchus was (10±4)mins; the mean time for anastomoses was (15±2)mins. The mean operative time was (80±10)mins. The 15d survival rates of rats who received transplants were 86%. Among the rats who received transplants, 2 died of pulmonary edema, 1 died of pulmonary thrombosis, the rest one dies of pneumothorax of the right side. Conclusion: Rat orthotopic left lung transplantation with successive anastomosis in cavity is a valid, reproducible, single and cheap model for studying donor lung preservation, ischemia-reperfusion, acute rejection and chronic rejection about lung transplantation.SECTION TWO Suppression effect of Astilbin on lung allograft rejection in ratsObjective: Using histopathological rejection grade and observeing the local fibrogenesis to study the suppression effect of Astilbin on lung allograft rejection in rats. Methods: The model of rat left lung transplantation was set up.Sixty lung transplanted rats were divided in to 4 groups randomly: group A were fed with normal saline 1ml per day, group B were fed with CsA 5mg/kg per day, group C were fed with Astilbin 1mg/kg per day, and group D were fed with Astilbin 1mg/kg per day aud CsA 2.5mg/kg per day. Histopathological rejection grade of the lung graft were analyzed in after 30 days (5 rats of each group were chosen). BALF(10 rats of each group were chosen) collected from recipients rats treated with drugs while undergoing rat orthotopic left lung transplantation model was used to stimulate human lung fibroblast cells in vitro. Fibroblast proliferation and typeⅠcollagen synthesis was dectected respectively by MTT and ELISA to evaluate the effect of astilbin. Results: (1) In group B , C and D, the histopathological rejection grade of the lung graft was significantly lower as compared with group A. The histopathological rejection grad of the grafts in group D was significantly lower as compared with group C (P<0.05) and group B (P<0.01). (2) In vitro, the BALF induced fibroblast proliferation and typeⅠcollagen synthesis; (3) Group B, C and D significantly decreased human lung fibroblast cells profliferation for stimulation of the BLAF, especially in Group D (P<0.05) Conclusions: Astilbin significantly decreased human lung fibroblast cells proliferation and typeⅠcollagen synthesis for stimulation of BLAF collected from recipients rats treated with drugs while undergoing lung transplantation, suggesting astilbin may inhibit obliterating bronchiolitis after lung transplantation. Astilbin can suppress lung allograft rejection, and has synergistic reaction with CsA.SECTION THREE The Mechanism of Astilbin on the Suppression of Lung Allograft Rejection PART ONEExpression and Implication of CD69, CD25 and CD71 on CD3+ lymphocytes in peripheral blood in the rats with lung transplantation using astilbinObjectives: To investigate the expression and implication of CD69, CD25 and CD71 on CD3+ lymphocytes in peripheral blood in the rats with lung transplantation using astilbin. Methods: The model of rat left lung transplantation was set up. Ten rats were taking peripheral blood as the control group. Forty lung transplanted rats were divided in to 2 groups randomly: group A were fed with normal saline 1ml per day, group B were fed with Astilbin 1mg/kg per day The lymphocytes from the peripheral blood in rats in different periods and were immunologically labeled by CD3·PE and CD69/CD25/CD71·FIFC. CD3/ CD69, CD3 /CD25 and CD3/CD71 were determined by flow cytometry (FCM). Results: The levels of CD3/ CD69, CD3 /CD25 and CD3/CD71 were markedly higher in the 2 days after the lung transplantation, which means the activated T cells were related to the lung allograft acute rejection. On the 5 days after the lung transplantation, the levels of CD3/ CD69 in group B were significant lower in the group A,(P<0.01), which means the astilbin act on the activated T cells and protect the allograft. Conclusion: Astilbin can act on the activated T cells and suppress lung allograft rejection maybe partially related to its effect in activated T cells.PART TWO Effect of astilbin on the inducement of apoptosis in activated T cells to suppress acute rejection on rat lung transplantation model.Objective:To study the suppression effect of Astilbin on lung allograft rejection in rats. Methods: The model of rat left lung transplantation was set up. sixty lung transplanted rats were divided in to 2 groups randomly: control group were fed with normal saline 1ml per day, astilbin group were fed with Astilbin 1mg/kg per day. Survival time of each group, activity of IL-2 in spleen lymph cells , transforming rate of T cells in spleen and apoptosis of T cells were observed. Results: Transforming rate of T cells in spleen was significant lower in the astilbin group than the control group. Activity of IL-2 in spleen lymph cells was 4.25±2.65UI/ml and 23.46±1.82UI/ml respectively in the astilbin group and the control group. There were significant difference between two groups (P<0.05). The astilbin group can effectively derive apoptosis of activated T cells in acute rejection. Conclusions: Astilbin can suppress lung allograft rejection maybe partially related to its lower the IL-2 concentration and IFN-γmRNA of activated T cells , which inducement of apoptosis in activated T cells.PART THREE Effect of astilbin on activation of p38MAPK and suppression of PI3K/Akt signal pathway in activated T cells of rat lung transplantation model with acute rejection.Objective: To investigate the effect of astilbin on activation of p38 mitogen activated protein kinase (p38MAPK) and suppression of PI3K/Akt in activated T cells of rat lung transplantation model with acute rejection. Methods: The model of rat left lung transplantation was established. At the day30, activated T cells were taken and cultured. The cultured activated T cells were divided into four groups: Group A: control group, were cultured with PBS; Group B: astilbin group, add astilbin (15mg/L); Group C: add Astilbin(15mg/L) and SB203580(5μmol/L); Group D, Astilbin+LY294002(10μmol/L). Apoptosis of T cells were observed by TUNEL. The expression of p38MAPK and PI3K/Akt were measured by RT-PCR and Western blot. Results: Apoptosis indexes and the expression of p38MAPK in activated T cells in Group B were found significant higher than those of the Group A, while PI3K/Akt was significant lower than Group A.(P<0.01). Apoptosis indexes and the expression of p38MAPK in activated T cells in Group B were found significant higher than those of the Group C, while PI3K/Akt was significant lower than Group C.(P<0.01). Apoptosis indexed and the expression of PI3K/Akt in Group D was significant higher than Group A, while the p38MAPK was significant lower than Group A.(P<0.01).Conclusion: Astilbin induces apoptosis of activated T cells of lung transplantation maybe partially related to its activation expression of p38MAPK and suppression of PI3K/Akt signal pathway. Key words: lung transplantation; astilbin, p38MAPK, PI3K/Akt更多还原