中药预防大鼠小肠移植慢性排斥反应的实验研究
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摘要
第一部分大鼠小肠移植模型的建立和鉴定
目的建立大鼠小肠移植模型并研究排斥反应机理。方法利用显微外科技术构建同系和异系大鼠小肠移植模型，分为3组：同系移植组、异系移植组、异系移植使用CsA组(环孢素组)。分别在术后7天、14天、28天、60天进行移植物活检，常规病理学检测，应用Realtime-PCR技术检测移植物内IL-4、INF-γ分子mRNA转录水平以观察急性排斥反应，采用免疫组织化学方法检测CD68以观察单核巨噬细胞浸润情况，使用Realtime-PCR技术和Western blot技术检测移植物内TGFβ1、PDGF mRNA转录水平及蛋白表达水平，并应用免疫组化方法对上述两种蛋白的表达进行定位，以明确其与排斥反应的关系。结果(1)异系移植组全部受体大鼠均在术后11天内死亡。(2)环孢素组IL-4、INF-γmRNA分子转录水平在术后第7天较同期的同系移植组有显著升高，第14天、28天、60天标本的检测结果较同期的同系移植组无显著差异。(3)坏孢素组在术后第60天的慢性排斥反应评分显著高于同期的同系移植组。(4)CD68检测提示在术后第60天环孢素组较同期的同系移植组显著升高。(5)坏孢素组的TGF-β1及PDGFmRNA转录水平和蛋白表达水平在第60天也较同期的同系移植组显著升高，免疫组化可见TGF-β1及PDGF在间质内高表达，表明环孢素组术后第60天开始出现慢性排斥反应，并同时伴有TGF-β1及PDGF分子的表达水平升高。结论(1)移植术后的早期存在急性排斥反应。(2)术后第60天环孢素组的单核巨噬细胞浸涧严重。(3)异系大鼠小肠移植经环孢素治疗组术后60天即可出现慢性排斥反应表现，并同时伴有CD68、TGFβ1、PDGF因子的显著高表达，提示CD68、TGFβ1、PDGF列于判定慢性排斥反应具有指导意义。
第二部分单味中药对慢性排斥反应的预防作用
目的观察单味中药对于慢性排斥反应的预防作用并筛选有效药物。方法利用显微外科技术构建异系小肠移植模型，受体大鼠应用环孢素1个月后按照实验要求随机分组，分别给予雷公藤、粉防己碱、青藤碱，同时设立同系对照组及异系对照组。术后7天、14天、28天、60天、90天行移植物活检，常规病理学检测，应用免疫组织化学方法检测CD68以观察单核巨噬细胞浸润情况，使用Realtime-PCR技术及Western blot技术和免疫组织化学方法检测TGFβ1、PDGF mRNA转录水平和蛋白表达水平，于实验终点应用流式细胞技术检测移植物和外周血T细胞亚群以观察受体和移植物的免疫功能。结果(1)慢性排斥反应病理评分在第90天各组间比较中出现显著差异，其中粉防己碱组、青藤碱组和异系对照组评分显著高于同期雷公藤组和同系对照组。(2)CD68的检测结果在术后第90天组间比较中有显著差异，粉防已碱组、青滕碱组、异系对照组中表达显著高于雷公藤组和同系对照组。(3)TGFβ1和PDGF mRNA转录水平在术后第60天和第90天的组间比较中有显著差异，粉防己碱组、青藤碱组、异系对照组的转录水平显著高于雷公藤组和同系对照组，TGFβ1和PDGF蛋白定量及免疫组化结果与mRNA转录水平一致。(4)实验终止时，外周血T细胞亚群检测表明雷公藤组和粉防己碱组CD4+／CD8+显著降低，移植物T细胞亚群检测表明异系对照组CD4+／CD8+显著低于其他各组。(5)肝肾功能检测表明粉防己碱组和青藤碱组AST值显著升高。结论(1)粉防己碱组、青藤碱组和异系对照组的组织破坏程度和单核巨噬细胞浸润程度重于雷公藤组和同系对照组。(2)术后第60天和第90天，粉防己碱组、青藤碱组、异系对照组的TGFβ1和PDGF因子较同时点各组处于较高水平。(3)雷公藤和粉防己碱对于免疫功能有一定的抑制作用。(4)异系列照组移植物CD8+淋巴细胞浸润增多。(5)粉防己碱和青藤碱对于肝功能有一定影响。(6)雷公藤组与其它中药治疗组相比可更为有效地减低TGFβ1、PDGF因子的表达，减轻和延缓慢性排斥反应的发生和发展。
第三部分复方中药对慢性排斥反应的预防作用
目的观察复方中药对慢性排斥反应的预防作用并进行筛选有效复方中药。方法利用显微外科技术构建异系小肠移植模型，应用环孢素1个月后，按照实验要求随机分组，在予以雷公藤的基础上分别给予黄芪、丹参、黄芪-丹参，同时设立同系对照组及异系对照组。分别于术后术后7天、14天、28天、60、90天行移植物活检，常规病理学检测，应用免疫组织化学方法检测CD68以观察单核巨噬细胞浸润情况，使用Realtime-PCR技术和Western blot技术及免疫组织化学方法检测TGFβ1、PDGF mRNA转录水平及蛋白表达水平，以明确各移植组TGFβ1、PDGF水平的变化，应用流式细胞技术检测实验终止时移植物和外周血T细胞亚群以观察受体和移植物的免疫功能。结果(1)慢性排斥反应病理评分在第90天各组间比较中出现显著差异，其中丹参组、黄芪-丹参组、异系对照组的病理评分显著高于同期黄芪组和同系对照组。(2)CD68的检测结果显示术后第90天组间比较中有显著差异，其中异系对照组CD68表达显著高于同期各实验组和同系对照组。(3)TGFpl和PDGF mRNA转录水平、蛋白表达水平检测结果显示在第60天和第90天组间比较有显著差异。在第60天组间比较，异系对照组的转录水平显著高于同期各组，第90天组间比较表明异系对照组、丹参组、黄芪-丹参组的转录水平显著高于同期的黄芪组和同系对照组。(4)实验终止时移植物T细胞亚群检测表明，异系对照组移植物中CD4+／CD8+显著减低，外周血T细胞亚群检测中各组之间无显著差异。结论(1)丹参组、黄芪-丹参组、异系对照组的组织损伤程度要重于黄芪组和同系对照组。(2)异系对照组单核巨噬细胞浸润严重。(3)在第60天和第90天异系对照组、丹参组、黄芪-丹参组的TGFβ1和PDGF因子处于较高水平。(4)异系对照组移植物中CD8+淋巴细胞浸润增多。(5)雷公藤-黄芪配伍与其它配伍组相比可更为有效地抑制TGFβ1、PDGF的表达，减轻移植后期移植物中单核巨噬细胞的浸润，显著延缓小肠移植术后排斥反应的进程。
SectionⅠThe Establishment and Characterization of the Rat Small BowelTransplantation Model
Objective: To establish the rat small bowel transplantation model and investigatethe mechanism of rejection. Methods: Isogeneic and allogeneic small boweltransplantation were performed in rats with microsurgical technology. Recipients weredivided into three groups: isogeneic control, allogeneic control, and allogeneictransplantation with CsA treatment (CsA group). Graft tissue were harvested on POD7,POD14, POD28 and POD60 and routine pathological examination was carried out.To monitor the acute rejection, the mRNA of IL-4 and IFN-γ, were quantified byreal-time quantitative RT-PCR. The infiltration of macrophages was determined bydetecting CD68 with immunohistochemical staining. In order to investigate therelation of TGFβ1 and PDGF with graft rejection, mRNA transcription and proteinexpression of TGFβ1 and PDGF were measured by real-time quantitative RT-PCR andWestern blot analysis. Immunohistochemical staining was also performed to detect thelocation of TGFβ1 and PDGF expression. Results: (1)All recipients in allogeneiccontrol group were died within 11 days after transplantation. (2)There were significantincreasing of IL-4 and IFN-γmRNA transcription in CsA group in POD7 comparedwith isogeneic group, but no significant differences were detected in POD14, POD28and POD60. (3)The chronic rejection scores of CsA group in POD60 were higher thanthat in isogeneic group. (4)CD68 expression in CsA group increasing significantly inPOD60. (5)Both the mRNA and protein expression of TGFβ1 and PDGF wereincreased significantly. The immunohistochemical results showed a high expression ofTGFβ1 and PDGF in the matrix. Conclusion: (1)Acute rejection occurred in the early stage of CsA group. (2)There was extensive infusion of macrophages in the graftof CsA group in POD60. (3)Chronic rejection was occurred in CsA group in POD60,with the increased levels of TGFβ1 and PDGF. (4)The chronic rejection model ofallogeneic small bowel transplantation could be established successfully within 60days after operation. The expression of CD68, TGFβ1 and PDGF were significantlyincreased, which could be used to determine chronic rejection.
SectionⅡThe Research on Prophylaxis of Chronic Rejection with Single Chinese Herbin Rat Small Bowel Transplantation
Objectives To demonstrate the prophylaxis of chronic rejection with single Chineseherb and select the best one. Methods Isogeneic and Allogeneic small boweltransplantation were performed in rats with microsurgical technology. After CsAtreatment of allogeneic recipients for one month, all experimental recipients weretreated with Tripterygii Wilfordii, Tetrandrine and Sinomenine, respectively. At thesame time, allogeneic control and isogeneic control were designed. Graft tissue wereharvested in POD7, POD14, POD28 and POD60. Routine pathological examinationwas carried out. The infiltration of macrophages was determined by detecting CD68with immunohistochemical staining. The mRNA transcription and expression ofTGFβ1 and PDGF were measured by real-time quantitative RT-PCR, Western blotanalysis and Immunohistochemical staining. T cell subsets in peripheral blood andgraft in the end of research were analyzed by flow cytometry measure. Results (1)The pathological scores of Tetrandrine group, Sinomenine group and allogeneiccontrol in POD90 increased significantly than the other groups of the same stage. (2)CD68 detected by immunohistochemical staining showed higher expression in thegroups of Sinomenine, Tetrandrine and allogeneic control in POD90. (3)There werealso significant increase of mRNA transcription and expression of TGFβ1 and PDGF in the groups of Sinomenine, Tetrandrine and allogeneic control in POD60 andPOD90. (4) Flow cytometry measure showed decrease of CD4+/CDS+ in peripheralblood in Tripterygii Wilfordii group and Tetrandrine group, and CD4+/CDS+ in thegrafts of allogeneic control decreased either. (5)AST were increased meaningfully inTetrandrine and Sinomenine groups. Conclusion (1)Serious damages andmacrophages infiltration were existed in Tetrandrine group, Sinomenine group andallogeneic control. (2)TGFβ1 and PDGF were significantly increased in the groups ofSinomenine, Tetrandrine and allogeneic control in POD60 and POD90. (3) Thedecrease of CD4+/CD8+ in peripheral blood and graft in Tripterygii Wilfordii,Tetrandrine group and allogeneic control showed the effect of immunoimpression ofthese herbs, and the damages of the immune function in grafts of allogeneic group. (4)Tetrandrine and Sinolnenine showed side-effects to liver function. (5)Compared withthe other herbs, Tripterygii Wilfordii could be the best one to control the cytokines ofTGFβ1 and PDGF, and thus prophylaxes the chronic rejection.
SectionⅢThe Research on Prophylaxis of Chronic Rejection with Compound Chinese Herbfollowing Small Bowel Transplantation
Objectives To demonstrate the prophylaxis of chronic rejection with compoundChinese herbs and choose the effective mixtures. Methods Isogeneic and allogeneicsmall bowel transplantation were performed in rats with microsurgical technology.After CsA treatment of allogeneic recipients for one month, all experimentalrecipients were treated with Tripterygii Wilfordii-Radix Astragali mixture, TripterygiiWilfordii-tanshinone mixture and Tripterygii Wilfordii- Radix Astragali- tanshinonemixture, respectively. At the same time, allogeneic control and isogeneic control weredesigned. Graft tissue were harvested in POD7, POD14, POD28 and POD60.Routine pathological examination was carried out. CD68 was detected by immunohistochemical staining. The mRNA transcription and protein expression ofTGFβ1 and PDGF were measured by real-time quantitative RT-PCR, Western blotanalysis and Immunohistochemical staining. The T cell subsets in peripheral bloodand graft in the end of research were analyzed by flow cytometry measure. Results(1)The pathological scores of Tanshinone group, Radix Astragali-tanshinone group,and altogeneic control in POD90 increased significantly than the other groups of thesame stage. (2)Compared with the other groups, CD68 expression in allogeneiccontrol increased significantly in POD90. (3)There were significant increase ofmRNA transcription and expression of TGFβ1 and PDGF in POD60 and POD90. Thecomparison in POD60 showed the higher level of mRNA transcription and expressionof TGFβ1 and PDGF in allogeneic control, and in POD90, the groups of allogeneiccontrol, Tanshinone, and Radix Astragali-tanshinone showed significant increasethan the other groups. (4)The CD4+/CDS+ in the grafts of allogeneic control werelower than the other groups, and no difference of CD4+/CD8+ in peripheral blooddetected. Conelusion (1)Serious damages were existed in Tanshinone group, RadixAstragali- tanshinone group, and allogeneic control in POD90. (2)There wereintensive infiltration of macrophages in group of allogeneic control. (3)TGFβ1 andPDGF were significantly increased in allogeneic control in POD60, and groups ofallogeneic control, Tanshinone, and Radix Astragali-tanshinone in POD90. (4) therewas damages of the immune function exist in grafts of allogeneic group. (5)Comparedwith the other groups, Tripterygii Wilfordii- tanshinone mixture could be the bestcompound inhibiting the expression of TGF-β1 and PDGF , and alleviating theinfusion of macrophages, which could be used for prophylaxis of chronic rejection.
目的建立大鼠小肠移植模型并研究排斥反应机理。方法利用显微外科技术构建同系和异系大鼠小肠移植模型，分为3组：同系移植组、异系移植组、异系移植使用CsA组(环孢素组)。分别在术后7天、14天、28天、60天进行移植物活检，常规病理学检测，应用Realtime-PCR技术检测移植物内IL-4、INF-γ分子mRNA转录水平以观察急性排斥反应，采用免疫组织化学方法检测CD68以观察单核巨噬细胞浸润情况，使用Realtime-PCR技术和Western blot技术检测移植物内TGFβ1、PDGF mRNA转录水平及蛋白表达水平，并应用免疫组化方法对上述两种蛋白的表达进行定位，以明确其与排斥反应的关系。结果(1)异系移植组全部受体大鼠均在术后11天内死亡。(2)环孢素组IL-4、INF-γmRNA分子转录水平在术后第7天较同期的同系移植组有显著升高，第14天、28天、60天标本的检测结果较同期的同系移植组无显著差异。(3)坏孢素组在术后第60天的慢性排斥反应评分显著高于同期的同系移植组。(4)CD68检测提示在术后第60天环孢素组较同期的同系移植组显著升高。(5)坏孢素组的TGF-β1及PDGFmRNA转录水平和蛋白表达水平在第60天也较同期的同系移植组显著升高，免疫组化可见TGF-β1及PDGF在间质内高表达，表明环孢素组术后第60天开始出现慢性排斥反应，并同时伴有TGF-β1及PDGF分子的表达水平升高。结论(1)移植术后的早期存在急性排斥反应。(2)术后第60天环孢素组的单核巨噬细胞浸涧严重。(3)异系大鼠小肠移植经环孢素治疗组术后60天即可出现慢性排斥反应表现，并同时伴有CD68、TGFβ1、PDGF因子的显著高表达，提示CD68、TGFβ1、PDGF列于判定慢性排斥反应具有指导意义。
第二部分单味中药对慢性排斥反应的预防作用
目的观察单味中药对于慢性排斥反应的预防作用并筛选有效药物。方法利用显微外科技术构建异系小肠移植模型，受体大鼠应用环孢素1个月后按照实验要求随机分组，分别给予雷公藤、粉防己碱、青藤碱，同时设立同系对照组及异系对照组。术后7天、14天、28天、60天、90天行移植物活检，常规病理学检测，应用免疫组织化学方法检测CD68以观察单核巨噬细胞浸润情况，使用Realtime-PCR技术及Western blot技术和免疫组织化学方法检测TGFβ1、PDGF mRNA转录水平和蛋白表达水平，于实验终点应用流式细胞技术检测移植物和外周血T细胞亚群以观察受体和移植物的免疫功能。结果(1)慢性排斥反应病理评分在第90天各组间比较中出现显著差异，其中粉防己碱组、青藤碱组和异系对照组评分显著高于同期雷公藤组和同系对照组。(2)CD68的检测结果在术后第90天组间比较中有显著差异，粉防已碱组、青滕碱组、异系对照组中表达显著高于雷公藤组和同系对照组。(3)TGFβ1和PDGF mRNA转录水平在术后第60天和第90天的组间比较中有显著差异，粉防己碱组、青藤碱组、异系对照组的转录水平显著高于雷公藤组和同系对照组，TGFβ1和PDGF蛋白定量及免疫组化结果与mRNA转录水平一致。(4)实验终止时，外周血T细胞亚群检测表明雷公藤组和粉防己碱组CD4+／CD8+显著降低，移植物T细胞亚群检测表明异系对照组CD4+／CD8+显著低于其他各组。(5)肝肾功能检测表明粉防己碱组和青藤碱组AST值显著升高。结论(1)粉防己碱组、青藤碱组和异系对照组的组织破坏程度和单核巨噬细胞浸润程度重于雷公藤组和同系对照组。(2)术后第60天和第90天，粉防己碱组、青藤碱组、异系对照组的TGFβ1和PDGF因子较同时点各组处于较高水平。(3)雷公藤和粉防己碱对于免疫功能有一定的抑制作用。(4)异系列照组移植物CD8+淋巴细胞浸润增多。(5)粉防己碱和青藤碱对于肝功能有一定影响。(6)雷公藤组与其它中药治疗组相比可更为有效地减低TGFβ1、PDGF因子的表达，减轻和延缓慢性排斥反应的发生和发展。
第三部分复方中药对慢性排斥反应的预防作用
目的观察复方中药对慢性排斥反应的预防作用并进行筛选有效复方中药。方法利用显微外科技术构建异系小肠移植模型，应用环孢素1个月后，按照实验要求随机分组，在予以雷公藤的基础上分别给予黄芪、丹参、黄芪-丹参，同时设立同系对照组及异系对照组。分别于术后术后7天、14天、28天、60、90天行移植物活检，常规病理学检测，应用免疫组织化学方法检测CD68以观察单核巨噬细胞浸润情况，使用Realtime-PCR技术和Western blot技术及免疫组织化学方法检测TGFβ1、PDGF mRNA转录水平及蛋白表达水平，以明确各移植组TGFβ1、PDGF水平的变化，应用流式细胞技术检测实验终止时移植物和外周血T细胞亚群以观察受体和移植物的免疫功能。结果(1)慢性排斥反应病理评分在第90天各组间比较中出现显著差异，其中丹参组、黄芪-丹参组、异系对照组的病理评分显著高于同期黄芪组和同系对照组。(2)CD68的检测结果显示术后第90天组间比较中有显著差异，其中异系对照组CD68表达显著高于同期各实验组和同系对照组。(3)TGFpl和PDGF mRNA转录水平、蛋白表达水平检测结果显示在第60天和第90天组间比较有显著差异。在第60天组间比较，异系对照组的转录水平显著高于同期各组，第90天组间比较表明异系对照组、丹参组、黄芪-丹参组的转录水平显著高于同期的黄芪组和同系对照组。(4)实验终止时移植物T细胞亚群检测表明，异系对照组移植物中CD4+／CD8+显著减低，外周血T细胞亚群检测中各组之间无显著差异。结论(1)丹参组、黄芪-丹参组、异系对照组的组织损伤程度要重于黄芪组和同系对照组。(2)异系对照组单核巨噬细胞浸润严重。(3)在第60天和第90天异系对照组、丹参组、黄芪-丹参组的TGFβ1和PDGF因子处于较高水平。(4)异系对照组移植物中CD8+淋巴细胞浸润增多。(5)雷公藤-黄芪配伍与其它配伍组相比可更为有效地抑制TGFβ1、PDGF的表达，减轻移植后期移植物中单核巨噬细胞的浸润，显著延缓小肠移植术后排斥反应的进程。
SectionⅠThe Establishment and Characterization of the Rat Small BowelTransplantation Model
Objective: To establish the rat small bowel transplantation model and investigatethe mechanism of rejection. Methods: Isogeneic and allogeneic small boweltransplantation were performed in rats with microsurgical technology. Recipients weredivided into three groups: isogeneic control, allogeneic control, and allogeneictransplantation with CsA treatment (CsA group). Graft tissue were harvested on POD7,POD14, POD28 and POD60 and routine pathological examination was carried out.To monitor the acute rejection, the mRNA of IL-4 and IFN-γ, were quantified byreal-time quantitative RT-PCR. The infiltration of macrophages was determined bydetecting CD68 with immunohistochemical staining. In order to investigate therelation of TGFβ1 and PDGF with graft rejection, mRNA transcription and proteinexpression of TGFβ1 and PDGF were measured by real-time quantitative RT-PCR andWestern blot analysis. Immunohistochemical staining was also performed to detect thelocation of TGFβ1 and PDGF expression. Results: (1)All recipients in allogeneiccontrol group were died within 11 days after transplantation. (2)There were significantincreasing of IL-4 and IFN-γmRNA transcription in CsA group in POD7 comparedwith isogeneic group, but no significant differences were detected in POD14, POD28and POD60. (3)The chronic rejection scores of CsA group in POD60 were higher thanthat in isogeneic group. (4)CD68 expression in CsA group increasing significantly inPOD60. (5)Both the mRNA and protein expression of TGFβ1 and PDGF wereincreased significantly. The immunohistochemical results showed a high expression ofTGFβ1 and PDGF in the matrix. Conclusion: (1)Acute rejection occurred in the early stage of CsA group. (2)There was extensive infusion of macrophages in the graftof CsA group in POD60. (3)Chronic rejection was occurred in CsA group in POD60,with the increased levels of TGFβ1 and PDGF. (4)The chronic rejection model ofallogeneic small bowel transplantation could be established successfully within 60days after operation. The expression of CD68, TGFβ1 and PDGF were significantlyincreased, which could be used to determine chronic rejection.
SectionⅡThe Research on Prophylaxis of Chronic Rejection with Single Chinese Herbin Rat Small Bowel Transplantation
Objectives To demonstrate the prophylaxis of chronic rejection with single Chineseherb and select the best one. Methods Isogeneic and Allogeneic small boweltransplantation were performed in rats with microsurgical technology. After CsAtreatment of allogeneic recipients for one month, all experimental recipients weretreated with Tripterygii Wilfordii, Tetrandrine and Sinomenine, respectively. At thesame time, allogeneic control and isogeneic control were designed. Graft tissue wereharvested in POD7, POD14, POD28 and POD60. Routine pathological examinationwas carried out. The infiltration of macrophages was determined by detecting CD68with immunohistochemical staining. The mRNA transcription and expression ofTGFβ1 and PDGF were measured by real-time quantitative RT-PCR, Western blotanalysis and Immunohistochemical staining. T cell subsets in peripheral blood andgraft in the end of research were analyzed by flow cytometry measure. Results (1)The pathological scores of Tetrandrine group, Sinomenine group and allogeneiccontrol in POD90 increased significantly than the other groups of the same stage. (2)CD68 detected by immunohistochemical staining showed higher expression in thegroups of Sinomenine, Tetrandrine and allogeneic control in POD90. (3)There werealso significant increase of mRNA transcription and expression of TGFβ1 and PDGF in the groups of Sinomenine, Tetrandrine and allogeneic control in POD60 andPOD90. (4) Flow cytometry measure showed decrease of CD4+/CDS+ in peripheralblood in Tripterygii Wilfordii group and Tetrandrine group, and CD4+/CDS+ in thegrafts of allogeneic control decreased either. (5)AST were increased meaningfully inTetrandrine and Sinomenine groups. Conclusion (1)Serious damages andmacrophages infiltration were existed in Tetrandrine group, Sinomenine group andallogeneic control. (2)TGFβ1 and PDGF were significantly increased in the groups ofSinomenine, Tetrandrine and allogeneic control in POD60 and POD90. (3) Thedecrease of CD4+/CD8+ in peripheral blood and graft in Tripterygii Wilfordii,Tetrandrine group and allogeneic control showed the effect of immunoimpression ofthese herbs, and the damages of the immune function in grafts of allogeneic group. (4)Tetrandrine and Sinolnenine showed side-effects to liver function. (5)Compared withthe other herbs, Tripterygii Wilfordii could be the best one to control the cytokines ofTGFβ1 and PDGF, and thus prophylaxes the chronic rejection.
SectionⅢThe Research on Prophylaxis of Chronic Rejection with Compound Chinese Herbfollowing Small Bowel Transplantation
Objectives To demonstrate the prophylaxis of chronic rejection with compoundChinese herbs and choose the effective mixtures. Methods Isogeneic and allogeneicsmall bowel transplantation were performed in rats with microsurgical technology.After CsA treatment of allogeneic recipients for one month, all experimentalrecipients were treated with Tripterygii Wilfordii-Radix Astragali mixture, TripterygiiWilfordii-tanshinone mixture and Tripterygii Wilfordii- Radix Astragali- tanshinonemixture, respectively. At the same time, allogeneic control and isogeneic control weredesigned. Graft tissue were harvested in POD7, POD14, POD28 and POD60.Routine pathological examination was carried out. CD68 was detected by immunohistochemical staining. The mRNA transcription and protein expression ofTGFβ1 and PDGF were measured by real-time quantitative RT-PCR, Western blotanalysis and Immunohistochemical staining. The T cell subsets in peripheral bloodand graft in the end of research were analyzed by flow cytometry measure. Results(1)The pathological scores of Tanshinone group, Radix Astragali-tanshinone group,and altogeneic control in POD90 increased significantly than the other groups of thesame stage. (2)Compared with the other groups, CD68 expression in allogeneiccontrol increased significantly in POD90. (3)There were significant increase ofmRNA transcription and expression of TGFβ1 and PDGF in POD60 and POD90. Thecomparison in POD60 showed the higher level of mRNA transcription and expressionof TGFβ1 and PDGF in allogeneic control, and in POD90, the groups of allogeneiccontrol, Tanshinone, and Radix Astragali-tanshinone showed significant increasethan the other groups. (4)The CD4+/CDS+ in the grafts of allogeneic control werelower than the other groups, and no difference of CD4+/CD8+ in peripheral blooddetected. Conelusion (1)Serious damages were existed in Tanshinone group, RadixAstragali- tanshinone group, and allogeneic control in POD90. (2)There wereintensive infiltration of macrophages in group of allogeneic control. (3)TGFβ1 andPDGF were significantly increased in allogeneic control in POD60, and groups ofallogeneic control, Tanshinone, and Radix Astragali-tanshinone in POD90. (4) therewas damages of the immune function exist in grafts of allogeneic group. (5)Comparedwith the other groups, Tripterygii Wilfordii- tanshinone mixture could be the bestcompound inhibiting the expression of TGF-β1 and PDGF , and alleviating theinfusion of macrophages, which could be used for prophylaxis of chronic rejection.
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65 原春辉，刘永锋，李桂臣，等．黄芪对犬移植胰腺缺血再灌注损伤的保护作用[J]．中华普通外科杂志，2005，20(11)：739
66 Gao OT, Cheung JK, Li J, et al. A Chinese herbal decoction, Danggui Buxue Tang, prepared from Radix Astragali and Radix Angelicae Sinensis stimulates the immune responses [J]. Planta Med. 2006 Oct; 72(13): 1227
67 Kang H, Ahn KS, Cho C, et al. Immunomodulatory effect of Astragali Radix extract on murine TH1/TH2 cell lineage development[J]. Biol Pharm Bull. 2004 Dec; 27(12): 1946-50.
68 Kajimura K, Takagi Y, Miyano k, et al. Polysaccharide of Astragali radix enhances IgM antibody production in aged mice[J]. Biol Pharm Bull. t997 Nov; 20(11): 1178-82
69 李春霞，侯桂华，宋静，等．黄芪在同种移植排斥中对CD4+CD25+T细胞及Foxp3的影响[J]．山东大学学报，2006，44(8)：760
70 赵柏，陈照彦．黄芪一虫草合剂对肾损伤转化生长因子β1的影响[J]．哈尔滨医科大学学报，2004，38(4)：323
71 Bedossa P, Paradis V. Transforming growth factor-beta (TGF-beta): a key-role in liver fibrogenesis[J]. J Hepatol. 1995, 22(s2): 37
72 arnovsky MJ, Russell ME, Hancock W, et al. Chronic rejection in experimental cardiac transplantation in a rat model [J]. Clin Transplant. 1994, 8(3): 308
73 动物实验方法学．孙敬方 主编．北京，人民卫生出版社，2004，150
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2. Debra Sudan. Cost and quality of life after intestinal transplantation[J]. Gastroenterology,2006,130:s158
3. Kareem M. Intestinal transplantation for short bowel syndrome and gastrointestinal failure: current consensus,rewarding outcomes,and practical guideline [J]. Gastroenterology,2006,130: s132
4. Hosenpud JD, Bennett LE, Keck BM, et al. The registry of the international society for heart and lung transplantation: eighteenth official report-2001 [J]. J Heart Lung Transplant[J], 2001, 20: 805
5. James S, Joren C. Recent advances in the immunology of chronic rejection [J]. Current Opinion in Nephrology and Hypertension,2002,11:315
6. Yousem SA, Berry GJ, Cagle PT, et al. Revision of the 1990 working formulation for the classification of pulmonary allografi rejection: Lung Rejection Study Group [J]. J Heart Lung Transplant, 1996, 15: 1-15
7. Maria P, Clara R, Anthony D, et al. Chronic rejection of small bowel grafts: pediatric and adult study of risk factors and morphologic progression[J]. Pediatric and developmental pathology, 2003, 6: 240
8．王庆文．移植肾慢性排斥反应得发病机制和治疗[J]．肾脏病与透析肾移植杂志，2000，9(2)：181
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23. Bedossa P, Paradis V. Transforming growth factor-beta (TGF-beta): a key-role in liver fibrogenesis[J]. J Hepatol. 1995, 22(s2): 37
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24 Mariafederica U, Ennia D, Maria E, et al. Cytokines and chronic rejection: a study in kidney transplantation long-term survivors[J]. Transplantation, 2004, 77, 548
25 Shirwan H. Chronic allograft rejection .Do the Th2 cells preferentitally induced by indirect alloantigen recognition play a dominant role[J]. Transplantation, 1999, 68: 715
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63 He X, Li C, Yu S. Protective effects of radix astragali against anoxic damages to in vitro cultured neurons
64 Zhang X, Feng J, Mu K, et al. Effects of single herbal drugs on adhesion and migration ofmelanocytes[J]. J Tradit Chin Med. 2005 Sep; 25(3): 219
65 原春辉，刘永锋，李桂臣，等．黄芪对犬移植胰腺缺血再灌注损伤的保护作用[J]．中华普通外科杂志，2005，20(11)：739
66 Gao OT, Cheung JK, Li J, et al. A Chinese herbal decoction, Danggui Buxue Tang, prepared from Radix Astragali and Radix Angelicae Sinensis stimulates the immune responses [J]. Planta Med. 2006 Oct; 72(13): 1227
67 Kang H, Ahn KS, Cho C, et al. Immunomodulatory effect of Astragali Radix extract on murine TH1/TH2 cell lineage development[J]. Biol Pharm Bull. 2004 Dec; 27(12): 1946-50.
68 Kajimura K, Takagi Y, Miyano k, et al. Polysaccharide of Astragali radix enhances IgM antibody production in aged mice[J]. Biol Pharm Bull. t997 Nov; 20(11): 1178-82
69 李春霞，侯桂华，宋静，等．黄芪在同种移植排斥中对CD4+CD25+T细胞及Foxp3的影响[J]．山东大学学报，2006，44(8)：760
70 赵柏，陈照彦．黄芪一虫草合剂对肾损伤转化生长因子β1的影响[J]．哈尔滨医科大学学报，2004，38(4)：323
71 Bedossa P, Paradis V. Transforming growth factor-beta (TGF-beta): a key-role in liver fibrogenesis[J]. J Hepatol. 1995, 22(s2): 37
72 arnovsky MJ, Russell ME, Hancock W, et al. Chronic rejection in experimental cardiac transplantation in a rat model [J]. Clin Transplant. 1994, 8(3): 308
73 动物实验方法学．孙敬方 主编．北京，人民卫生出版社，2004，150
1. Jonathan P. Fryer. Intestinal transplantation: an update[J]. Curr Opin Gastroenterol, 2005, 21: 162-168
2. Debra Sudan. Cost and quality of life after intestinal transplantation[J]. Gastroenterology,2006,130:s158
3. Kareem M. Intestinal transplantation for short bowel syndrome and gastrointestinal failure: current consensus,rewarding outcomes,and practical guideline [J]. Gastroenterology,2006,130: s132
4. Hosenpud JD, Bennett LE, Keck BM, et al. The registry of the international society for heart and lung transplantation: eighteenth official report-2001 [J]. J Heart Lung Transplant[J], 2001, 20: 805
5. James S, Joren C. Recent advances in the immunology of chronic rejection [J]. Current Opinion in Nephrology and Hypertension,2002,11:315
6. Yousem SA, Berry GJ, Cagle PT, et al. Revision of the 1990 working formulation for the classification of pulmonary allografi rejection: Lung Rejection Study Group [J]. J Heart Lung Transplant, 1996, 15: 1-15
7. Maria P, Clara R, Anthony D, et al. Chronic rejection of small bowel grafts: pediatric and adult study of risk factors and morphologic progression[J]. Pediatric and developmental pathology, 2003, 6: 240
8．王庆文．移植肾慢性排斥反应得发病机制和治疗[J]．肾脏病与透析肾移植杂志，2000，9(2)：181
9. Hayry P, Isoniemi H, Yilmaz S, et al. Chronic atlografi rejection[J], Immunol Rev, 1993, 134
10. Simone AJ, Cees van K, Leendert C. Pathogenesis of chronic allografi rejection[J]. Transpl Int ,2003, 16: 137
11．孙启全．器官移植物慢性排斥反应的免疫发病机制[J]．J Nephrol Dialy Transplant，2003，12(2)：186
12. Mariafederica U, Ennia D, Maria E, et al. Cytokines and chronic rejection: a study in kidney transplantation long-term survivors[J]. Transplantation, 2004, 77, 548
13. Alexander K, Raimund M, Heinz P, et al. Diffuse mesenterial sclerosis: a characteristic feature of chronic small bowel allograft rejection[J]. Virchows Arch, 2003, 442: 48
14. Shirwan H. Chronic allograft rejection. Do the Th2 cells preferentitally induced by indirect alloantigen recognition play a dominant role[J] Transplantation, 1999, 68: 715
15. Le Moine A, Goldman M, Abramowicz D. Multiple pathway to allograft rejection[J]. Transplantation, 73: 1373
16. Waaga AM, Gasser M, Laskowski I, et al. Mechanism of chronic rejection[J]. Curr Opin Immunol, 2000, 12: 517
17. Allan JS, Madsen JC. Recent advance in the immunology of chronic rejection[J]. Curr Opin Nephrol Hypertens, 2002, 11: 315
18. Bradley JA, Mowat AM, Bolton EM. Processed MHC class Ⅰ alloantigen as the stimulus for CD4+T cell dependent antibody-mediated graft rejection[J]. Immunol Today, 1992, 13: 434
19. Game DS, Lechler RI. Pathways of allorecongnition: implications for transplantation tolerance [J]. Transpl Immunol, 2002, 10: 101
20. Sayegh MH. Why do we reject a graft? Role of indirect allorecognition in graft rejection[J]. Kidney Int, 1999, 56: 1967
21. Shiwan H. Chronic allograft rejection[J].Transplantation, 1999, 68: 715
22. Womer KL, Stone JR, Murphy B, et al. Indirect allorecogntion of donor class Ⅰ and class Ⅱ major histocompatibility complex peptides promotes the development of transplant vasculopathy[J]. J AM Soc Nephrol, 2001, 12: 250
23. Bedossa P, Paradis V. Transforming growth factor-beta (TGF-beta): a key-role in liver fibrogenesis[J]. J Hepatol. 1995, 22(s2): 37
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