受体源骨髓间充质干细胞对大鼠异位小肠移植慢性排斥反应的影响
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摘要
第一部分大鼠小肠移植慢性排斥反应模型的建立及病理学比较
目的采用F344→Lewis大鼠组合初步建立大鼠小肠移植慢性排斥反应模型,观测小肠组织慢性排斥病理表现。方法利用显微外科技术构建异系异位和异系原位大鼠小肠移植模型,术后0-14天给予皮下注射环孢素。术后90天进行移植物活检,常规病理学检测。乳果糖／甘露醇比值、D-木糖吸收实验检测移植肠功能。
结果(1)异位和原位小肠移植大鼠均能长期存活,大部分超过90天。(2)异位小肠移植在术后90天出现典型的慢性排斥反应的病理学特征,包括粘膜结构改变、间质纤维化、白细胞浸润和动脉内膜增殖。(3)术后90天的原位移植小肠组织学形态相对正常。原位小肠移植大鼠在术后150天左右具有慢性排斥的病理组织学特征,但病变程度轻于术后90天的异位移植小肠。(4)大部分原位小肠移植大鼠在术后150天出现持续的体重下降,且不能被环孢素逆转。当体重下降超过30%时,乳果糖／甘露醇比值和D-木糖吸收实验结果证实移植肠功能下降。结论(1)异位小肠移植在术后90天出现典型的慢性排斥反应的病理学改变,而原位小肠移植在该时间点无慢性排斥反应改变。(2)原位小肠移植于术后150天左右出现慢性排斥改变,以持续性体重下降为临床表现,并伴随肠道吸收功能以及屏障功能的下降。(3)F344→Lewis异位小肠移植手术操作相对简单,经练习后手术成功率可达85%以上。(4)由于异位小肠移植可以出现典型的慢性排斥反应的组织病理学特征,故后继实验采用F344→Lewis异位小肠移植模型,并根据组织病理学检查结果确定术后90天为观察终点。
第二部分大鼠异位小肠移植慢性排斥反应的研究
目的建立大鼠异位小肠移植模型并研究相关慢性排斥反应机理。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、异系治疗组(给予环孢素)。分别在术后7天、14天、30天、60天、90天进行移植物活检,常规病理学检测,建立小肠移植排斥反应组织学评分标准。应用Real time-PCR技术检测移植物内TGFβ1、PDGF、PDGF-C mRNA转录水平,采用免疫组织化学方法检测CD68、TGFβ1、PDGF-C表达情况。结果(1)异系对照组全部大鼠均在术后15天内死亡。(2)异系治疗组自术后14天开始炎细胞浸润程度病理评分显著高于同期同系对照组,术后30天粘膜结构变化以及纤维化程度病理评分显著高于同期对照组,术后60天动脉内膜增殖开始有显著差异(3)CD68检测提示在慢性排斥反应全程CD68的表达持续升高,术后60天达最高水平。(4)异系治疗组的TGF-β1及PDGF-C mRNA转录水平和蛋白表达水平在第60天较同期的同系移植组显著升高,免疫组化可见TGF-β1在间质高表达,PDGF-C主要由组织细胞高表达于胞浆及胞膜。结论(1)术后第60天环孢素组的单核巨噬细胞浸润严重,同时伴TGFβ1、PDGF-C的高表达。(2)异系治疗组术后60天即可出现慢性排斥反应表现,并同时伴有CD68、TGFβ1、PDGF-C分子的显著高表达,提示CD68、TGFβ1、PDGF-C对于判定慢性排斥反应具有指导意义。
第三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间充质干细胞的体外分离、培养、鉴定方法,为进一步开展研究MSCs对小肠移植慢性排斥反应的影响提供细胞来源。方法选用1月龄清洁级Lewis大鼠,处死消毒后取其长骨,用PBS缓冲液冲洗骨髓腔,收集冲洗液,采用密度梯度离心法分离获取细胞,培养后取贴壁细胞进行传代培养。取第3代生长状态良好的细胞,0.25%胰蛋白酶-EDTA消化制成细胞悬液计数并记录生长曲线。取第3、4、5代生长状态良好细胞,以流式细胞术鉴定细胞表型。取第3代MSCs定向诱导分化成骨细胞,采用碱性磷酸酶染色、茜素红染色进行成骨细胞鉴定。CFSE标记第3代MSCs并观察标记效率。结果(1)体外培养的原代MSCs72小时可见贴壁细胞,3周左右可达到90%的融合,2代后即可得到较为纯化的MSC成纤维样细胞。(2)流式细胞仪检测第3代MSCs表面标记物CD29、CD90的阳性率可达96%以上,CD34、CD45的阳性率低于3%。(3)碱性磷酸酶染色和茜素红染色结果证实成骨细胞诱导成功。(4)用CFSE进行细胞标记后,荧光显微镜下见几乎所有MSCs均已被标记绿色荧光,流式细胞仪检测结果显示标记比例达100%,提示CFSE标记效率高。结论(1)MSCs是一类增殖能力旺盛,具有多向分化潜能的干细胞。(2)密度梯度离心联合贴壁培养法是一种简便易行的培养MSCs方法,操作简单,成功率高,可以作为培养大鼠骨髓MSCs的常规方法。(3)CFSE标记可作为标记MSCs的一种有效手段。
第四部分大鼠骨髓间充质干细胞体外免疫抑制功能的实验研究
目的探讨骨髓间充质干细胞(MSCs)在混合淋巴细胞培养反应中对同种异体脾细胞免疫应答反应的影响,并初步探讨其作用机制。方法建立MSCs和同种异体脾细胞共培养体系,反应体系总量300ul,以刀豆蛋白A为刺激因素,CFSE标记部分反应细胞。共分4组：①脾细胞,②脾细胞+conA,③脾细胞+MSCs,④脾细胞+MSCs+conA,每组6复孔,每孔脾细胞数5×105个,MSCs为105个,ConA终浓度为10ug/ml。混合培养72小时后,于倒置荧光显微镜下观察细胞增殖情况,流式细胞仪分析荧光强度以及CD4、CD8比例。结果MSCs和脾细胞共培养组未见明显的增殖子峰,其中CD4、CD8阳性细胞表达明显下降,而CD4/CD8比值无明显变化。结论骨髓MSCs体外能够抑制刀豆蛋白A引起的淋巴细胞增殖反应,对CD4 T细胞、CD8 T细胞均有抑制作用。
第五部分受体源骨髓间充质干细胞对大鼠异位小肠移植慢性排斥反应的影响
目的研究静脉输注受体骨髓间充质干细胞(MSCs)对大鼠异位小肠移植慢性排斥反应的影响及其机制。方法利用显微外科技术构建异系小肠移植模型16只,随机分为2组：对照组和MSCs输注组。小肠移植后24小时,确定手术成功后,MSCs组自阴茎背静脉输注骨髓间充质干细胞107个(溶液为2ml生理盐水),对照组给予同体积的生理盐水,术后0-14天两组均给予环孢素处理。观察两组移植大鼠术后生存状态,于术后第60天全部剖杀获取移植小肠。常规病理学检测,进行慢性排斥反应病理评分。应用Realtime-PCR技术检测移植物内TGFβ1、PDGF-C mRNA转录水平,采用免疫组织化学方法检测CD68、PDGF-C表达情况。另外再建立两只大鼠异位小肠移植模型,给予环孢素处理,术后24小时给予阴茎背静脉输注标记了荧光CFSE的骨髓MSCs,术后7天剖杀获取移植小肠以及自体其他组织器官备冰冻切片检查以观察MSCs在受体内的定位。结果(1)输注的骨髓MSCs可特异的定向迁移至移植小肠。(2)术后60天MSCs组小肠标本的粘膜结构、炎细胞浸润、纤维化程度和动脉内膜增殖程度的评分与环孢素组均有统计学差异。(3)术后60天MSCs组小肠标本表达CD68、TGFβ1、PDGF-C分子水平均低于环孢素组。结论MSCs在一定程度上能移抑制慢性排斥的进程,其作用机制可能是MSCs对淋巴细胞增殖的抑制作用,减少了巨噬细胞浸润以及TGFβ1、PDGF-C分子的表达。
PartⅠEstablishment of Chronic Rejection Model and Pathological Assessment of Small Bowel Transplantation in Rat
Objective:To establish the chronic rejection of rat small bowel transplantation model and to investigate the pathological changes after transplantation. Methods: Allogeneic heterotopic and orthotopic small bowel transplantation model were performed using microsurgery technology, and recipients received cyclosporine through subcutaneous injection at 0-14 days after surgery. Graft samples were harvested at POD90 and examineed by hematoxylin eosin staining and Verhoff's alcoholic hematoxylin ted. The function of transplanted gut was evaluated by ratio of Lactulose and mannitol and D-xylose absorption test. Results:(1)The recipients of heterotopic and orthotopic small bowel transplantation had a survival of more than 90 days. (2)The allografts of heterotopic small bowel transplantation showed histological features of chronic rejection at POD90. Histopathological analysis revealed distinctive abnormalities of allograft including changes of villous architecture, interstitial fibrosis, leukocyte infiltration, and intimal thickening. (3) The above histological changes were not observed in allografts of orthotopic small bowel transplantation. The allografts of orthotopic small bowel transplantation showed histological features of chronic rejection at about POD150, but lesions were mild compared with heterotopic grafts of POD90. (4)Most rat with orthotopic small bowel transplantation rat showed gradual weight loss after POD150, and it couldn't be reversed no matter cyclosporine was applied. When the weight loss more was more than 30%, the absorption and barrier function of orthopotic transplantative gut were decline compared with non-operative rats. Conclusion:(1)Chronic rejection occurred at POD90 in heterotopic small bowel transplantation model.(2)Histological features of chronic rejection in orthotopic small bowel transplantation model were detected after POD 150. (3)The surgical procedure of heterotopic small bowel transplantation is not complicated and surgical success rate is more than 85%.(4)Because typical histopathological features of chronic rejection exsits during the recipients survival time, the model of rat heterotopic small bowel transplantation is applied in the following research. According to pathological results, observative duration were confirm PODO-90 after transplant surgery.
Part II The Research on Chronic Rejection of Heterotopic Small Bowel Transplantation in Rat
Objective:To extablish rat heterotopic small bowel transplantation model and to explore the mechanisms of chronic rejection. Methods:The recipients were divided into 3 groups including isogeneic control group, allogeneic control group and allogeneic transplantation with cyclosporine treatment group(CsA group). Graft samples were harvested at POD7, POD14, POD30, POD60 and POD90 and pathological examination was performed. The CD68 expression indicating infiltration of macrophages was determined by immunohistochemistry. In order to investigate the relationship of TGFβ1 and PDGF-C with chronic rejection, we used real-time quantitative RT-PCR and immunohistochemistry to detect the mRNA transcription and protein expression of TGFβ1 and PDGF-C. Results:(1)All of the recipients in the allogeneic control group died within 15 days after transplantation. (2)The leukocyte infiltration scores of CsA group were higher than those of isogeneic group from POD 14 to POD90. The mucous coat architecture scores and interstitial fibrosis scores of CsA group were higher than those of isogeneic group from POD30 to POD90. Intimal thickening scores of CsA group were significantly higher than those of isogeneic group from the beginning of POD60. (3)The expression of CD68 in CsA group continued to increase throughout the process of chronic rejection, and reached the highest level after POD60. (4)The mRNA and protein expression of TGFβ1 and PDGF-C in CsA group were significantly increased. The immunohistochemical results showed high expression of TGFβ1 in the interstitial and PDGF-C in the cytoplasm and cell membrane. Conclusion:(1)There was extensive infiltration of macrophages in the graft of CsA group at POD60, associated with high expression of TGFpl and PDGF-C. (2) The chronic rejection model of allogeneic heterotopic small bowel transplantation was successfully established at 60 days after surgery. The expression of CD68,TGFβ1 and PDGF-C were significantly increased in CsA group, which could be used to determine chronic rejection.
PartⅢIsolation, Culturing and Identification of Rat MSCs from Bone Marrow in Vitro
Objective:To investigate the methods of the isolation, culture and identification of rat MSCs from bone marrow in vitro.Methods:One month-old male Lewis rats were sacrificed and tibias and femurs tissues were obtained. After the long bones were dissected, the marrows were flushed out with PBS buffer under aseptic condition and purified by percoll gradient centrifugation to get the mononuclear cells. After the mononuclear cells were cultured, the adherent cells were selected for serial subcultivation. The cells of the third generation in good growth condition was harvested and trypsinized to form cell suspension. The cells were counted and the growth curve was made. The cells of the third to five generation were harvested and the cell phenotypes were identified by flow cytometry. The cells of the third generation were induced to differentiate into osteoblasts. The induced cells were identified with alkaline phosphatase staining and alizarin red staining. The cells of the third generation were labeled by fluorescent dye of CFSE. Results:(1)The MSCs were spindle shaped, attached to the culture dish tightly after 72 hours' culture, and proliferated in the culture medium. After 21 days of primary cultivation, MSCs were nearly 100% confluent. (2)The 3th passage MSCs comprised a unique phenotypic population by flow cytometric analysis. The phenotypes were shown to be positive for CD29 and CD90 more than 96% and be negative for CD34 and CD45.(3)The MSCs formed aggregates or nodules and increased their expression of alkaline phosphatase, and calcium accumulation was evident after 3 weeks. Alkaline phosphatase staining and alizarin red staining confirmed the success of induction. (4)CFSE had high efficiency for labeling MSCs, the labeling index was over 100%.
Conclusion:(1)MSCs are stem cells with strong proliferative activity and sustain self-renewal. They are pluripotent and could differentiate to a variety of cell types in some condition. (2)The methods of obtaining rat MSCs with density gradient centrifugation and adherence cultivation were convenient, and could provide immortalized rat MSCs lines for cellular transplant therapy. (3)CFSE staining is a feasible method for labeling MSCs with high efficiency.
PartⅣStudy on the Effect of Bone Marrow Mesenchymal Stem Cells on Immunosuppression in Vitro
Objective:To explore the effect of mesenchymal stem cells derived from bone marrow using mixed lumphocyte culture on the immune response of splenic cells and the corresponding mechanisms.Methods:MSCs and mixed lymphocyte reaction cultures were set up. Splenic lymphocytes from F344 rat were used as reactive cells and labeled with CFSE. The study included 4 groups.Group 1 (the control group) included 5×105 reactive cells cultured. Group 2 were 5×105 reactive cells cultured with conA. Group 3 included 1×105 MSCs from Lewis rats and 5×105reactive cells. Group 4 included 1×105 MSCs from Lewis rats and 5×105 reactive cells cultured with conA as a stimulator. Cells in each group were cultured for 72 hours. The proliferation dynamics of CFSE labeled cells were detected by flow cytometry. In the same groups without CFSE labling, the CD4+ and CD8+ phenotypes of proliferating lymphocytes were determined with monoclone antibodies.Results:Compared with that in group 2, the proliferation of sub-peak in group 4 couldn't be detected. The positive expression of CD4+ and CD8+ were declined compared with that in group 1, but there was no difference in the ratio of CD4+ to CD8+. Conclusion:Bone marrow MSCs demonstrate significant immune regulatory effects on conA-stimulated T-lymphocyte culture. It might provide a remarkable immune suppression in organ transplantation to achieve better outcome in the future.
Part V The Effect of Bone Marrow Mesenchymal Stem Cells Derived from Recipients on the Chronic Rejection in Rat Heterotopic Small Bowel Transplantation
Objective:To observe the effects of MSCs derived from recipients'bone marrow on the chronic rejection of rat heterotopic small bowel transplantation. Methods:Sixteen Lewis rats bearing transplanted small bowel from F344 rats were randomly divided into 2 groups and all of them received cyclosporine A treatment. (1)Control group: 2mL 0.9%NaCl solution was injected through vena dorsalis penis at 24h after small bowel transplantation. (2)Treatment group:1×107MSCs in 2ml 0.9%NaCl solution was injected through vena dorsalis penis at 24h after small bowel transplantation. Graft samples were harvested on POD60 and examined by hematoxylin eosin staining and Verhoffs alcoholic hematoxylin ted. The graft infiltration of macrophages was determined by immunohistochemical staining. mRNA transcription and protein expression of TGFβ1 and PDGF-C were measured by real-time quantitative RT-PCR and immunohistochemistry, respectively. Two other small bowel transplantation models were established with cyclosporine A treatment. At 24h after transplantation, 1×107MSCs labeled with CFSE were injected through vena dorsalis penis, respectively. The two grafts and other self-organ were harvested on 7th day after transplantation to get frozen sections for observation of the fluorescence cells in grafts.Results:(1)Fluorescence cells concentrated in the interstitial of allografts. (2)The scores of mucous coat architecture, leukocyte infiltration, interstitial fibrosis and intimal thickening of treatment group were significantly less than those of control group at POD60.(3)The expression of CD68, TGFβ1 and PDGF-C in treatment group was significantly less than that in control group at POD60. Conclusion:Intravenous infusion with MSCs from recipients could inhibit the process of chronic rejection in rat heterotopic small bowel transplantation to some degree. The mechanisms involve the suppressive effect of MSCs on lymphocytic proliferation and on the expression of CD68,TGFβ1 and PDGF-C.
目的采用F344→Lewis大鼠组合初步建立大鼠小肠移植慢性排斥反应模型,观测小肠组织慢性排斥病理表现。方法利用显微外科技术构建异系异位和异系原位大鼠小肠移植模型,术后0-14天给予皮下注射环孢素。术后90天进行移植物活检,常规病理学检测。乳果糖／甘露醇比值、D-木糖吸收实验检测移植肠功能。
结果(1)异位和原位小肠移植大鼠均能长期存活,大部分超过90天。(2)异位小肠移植在术后90天出现典型的慢性排斥反应的病理学特征,包括粘膜结构改变、间质纤维化、白细胞浸润和动脉内膜增殖。(3)术后90天的原位移植小肠组织学形态相对正常。原位小肠移植大鼠在术后150天左右具有慢性排斥的病理组织学特征,但病变程度轻于术后90天的异位移植小肠。(4)大部分原位小肠移植大鼠在术后150天出现持续的体重下降,且不能被环孢素逆转。当体重下降超过30%时,乳果糖／甘露醇比值和D-木糖吸收实验结果证实移植肠功能下降。结论(1)异位小肠移植在术后90天出现典型的慢性排斥反应的病理学改变,而原位小肠移植在该时间点无慢性排斥反应改变。(2)原位小肠移植于术后150天左右出现慢性排斥改变,以持续性体重下降为临床表现,并伴随肠道吸收功能以及屏障功能的下降。(3)F344→Lewis异位小肠移植手术操作相对简单,经练习后手术成功率可达85%以上。(4)由于异位小肠移植可以出现典型的慢性排斥反应的组织病理学特征,故后继实验采用F344→Lewis异位小肠移植模型,并根据组织病理学检查结果确定术后90天为观察终点。
第二部分大鼠异位小肠移植慢性排斥反应的研究
目的建立大鼠异位小肠移植模型并研究相关慢性排斥反应机理。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、异系治疗组(给予环孢素)。分别在术后7天、14天、30天、60天、90天进行移植物活检,常规病理学检测,建立小肠移植排斥反应组织学评分标准。应用Real time-PCR技术检测移植物内TGFβ1、PDGF、PDGF-C mRNA转录水平,采用免疫组织化学方法检测CD68、TGFβ1、PDGF-C表达情况。结果(1)异系对照组全部大鼠均在术后15天内死亡。(2)异系治疗组自术后14天开始炎细胞浸润程度病理评分显著高于同期同系对照组,术后30天粘膜结构变化以及纤维化程度病理评分显著高于同期对照组,术后60天动脉内膜增殖开始有显著差异(3)CD68检测提示在慢性排斥反应全程CD68的表达持续升高,术后60天达最高水平。(4)异系治疗组的TGF-β1及PDGF-C mRNA转录水平和蛋白表达水平在第60天较同期的同系移植组显著升高,免疫组化可见TGF-β1在间质高表达,PDGF-C主要由组织细胞高表达于胞浆及胞膜。结论(1)术后第60天环孢素组的单核巨噬细胞浸润严重,同时伴TGFβ1、PDGF-C的高表达。(2)异系治疗组术后60天即可出现慢性排斥反应表现,并同时伴有CD68、TGFβ1、PDGF-C分子的显著高表达,提示CD68、TGFβ1、PDGF-C对于判定慢性排斥反应具有指导意义。
第三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间充质干细胞的体外分离、培养、鉴定方法,为进一步开展研究MSCs对小肠移植慢性排斥反应的影响提供细胞来源。方法选用1月龄清洁级Lewis大鼠,处死消毒后取其长骨,用PBS缓冲液冲洗骨髓腔,收集冲洗液,采用密度梯度离心法分离获取细胞,培养后取贴壁细胞进行传代培养。取第3代生长状态良好的细胞,0.25%胰蛋白酶-EDTA消化制成细胞悬液计数并记录生长曲线。取第3、4、5代生长状态良好细胞,以流式细胞术鉴定细胞表型。取第3代MSCs定向诱导分化成骨细胞,采用碱性磷酸酶染色、茜素红染色进行成骨细胞鉴定。CFSE标记第3代MSCs并观察标记效率。结果(1)体外培养的原代MSCs72小时可见贴壁细胞,3周左右可达到90%的融合,2代后即可得到较为纯化的MSC成纤维样细胞。(2)流式细胞仪检测第3代MSCs表面标记物CD29、CD90的阳性率可达96%以上,CD34、CD45的阳性率低于3%。(3)碱性磷酸酶染色和茜素红染色结果证实成骨细胞诱导成功。(4)用CFSE进行细胞标记后,荧光显微镜下见几乎所有MSCs均已被标记绿色荧光,流式细胞仪检测结果显示标记比例达100%,提示CFSE标记效率高。结论(1)MSCs是一类增殖能力旺盛,具有多向分化潜能的干细胞。(2)密度梯度离心联合贴壁培养法是一种简便易行的培养MSCs方法,操作简单,成功率高,可以作为培养大鼠骨髓MSCs的常规方法。(3)CFSE标记可作为标记MSCs的一种有效手段。
第四部分大鼠骨髓间充质干细胞体外免疫抑制功能的实验研究
目的探讨骨髓间充质干细胞(MSCs)在混合淋巴细胞培养反应中对同种异体脾细胞免疫应答反应的影响,并初步探讨其作用机制。方法建立MSCs和同种异体脾细胞共培养体系,反应体系总量300ul,以刀豆蛋白A为刺激因素,CFSE标记部分反应细胞。共分4组：①脾细胞,②脾细胞+conA,③脾细胞+MSCs,④脾细胞+MSCs+conA,每组6复孔,每孔脾细胞数5×105个,MSCs为105个,ConA终浓度为10ug/ml。混合培养72小时后,于倒置荧光显微镜下观察细胞增殖情况,流式细胞仪分析荧光强度以及CD4、CD8比例。结果MSCs和脾细胞共培养组未见明显的增殖子峰,其中CD4、CD8阳性细胞表达明显下降,而CD4/CD8比值无明显变化。结论骨髓MSCs体外能够抑制刀豆蛋白A引起的淋巴细胞增殖反应,对CD4 T细胞、CD8 T细胞均有抑制作用。
第五部分受体源骨髓间充质干细胞对大鼠异位小肠移植慢性排斥反应的影响
目的研究静脉输注受体骨髓间充质干细胞(MSCs)对大鼠异位小肠移植慢性排斥反应的影响及其机制。方法利用显微外科技术构建异系小肠移植模型16只,随机分为2组：对照组和MSCs输注组。小肠移植后24小时,确定手术成功后,MSCs组自阴茎背静脉输注骨髓间充质干细胞107个(溶液为2ml生理盐水),对照组给予同体积的生理盐水,术后0-14天两组均给予环孢素处理。观察两组移植大鼠术后生存状态,于术后第60天全部剖杀获取移植小肠。常规病理学检测,进行慢性排斥反应病理评分。应用Realtime-PCR技术检测移植物内TGFβ1、PDGF-C mRNA转录水平,采用免疫组织化学方法检测CD68、PDGF-C表达情况。另外再建立两只大鼠异位小肠移植模型,给予环孢素处理,术后24小时给予阴茎背静脉输注标记了荧光CFSE的骨髓MSCs,术后7天剖杀获取移植小肠以及自体其他组织器官备冰冻切片检查以观察MSCs在受体内的定位。结果(1)输注的骨髓MSCs可特异的定向迁移至移植小肠。(2)术后60天MSCs组小肠标本的粘膜结构、炎细胞浸润、纤维化程度和动脉内膜增殖程度的评分与环孢素组均有统计学差异。(3)术后60天MSCs组小肠标本表达CD68、TGFβ1、PDGF-C分子水平均低于环孢素组。结论MSCs在一定程度上能移抑制慢性排斥的进程,其作用机制可能是MSCs对淋巴细胞增殖的抑制作用,减少了巨噬细胞浸润以及TGFβ1、PDGF-C分子的表达。
PartⅠEstablishment of Chronic Rejection Model and Pathological Assessment of Small Bowel Transplantation in Rat
Objective:To establish the chronic rejection of rat small bowel transplantation model and to investigate the pathological changes after transplantation. Methods: Allogeneic heterotopic and orthotopic small bowel transplantation model were performed using microsurgery technology, and recipients received cyclosporine through subcutaneous injection at 0-14 days after surgery. Graft samples were harvested at POD90 and examineed by hematoxylin eosin staining and Verhoff's alcoholic hematoxylin ted. The function of transplanted gut was evaluated by ratio of Lactulose and mannitol and D-xylose absorption test. Results:(1)The recipients of heterotopic and orthotopic small bowel transplantation had a survival of more than 90 days. (2)The allografts of heterotopic small bowel transplantation showed histological features of chronic rejection at POD90. Histopathological analysis revealed distinctive abnormalities of allograft including changes of villous architecture, interstitial fibrosis, leukocyte infiltration, and intimal thickening. (3) The above histological changes were not observed in allografts of orthotopic small bowel transplantation. The allografts of orthotopic small bowel transplantation showed histological features of chronic rejection at about POD150, but lesions were mild compared with heterotopic grafts of POD90. (4)Most rat with orthotopic small bowel transplantation rat showed gradual weight loss after POD150, and it couldn't be reversed no matter cyclosporine was applied. When the weight loss more was more than 30%, the absorption and barrier function of orthopotic transplantative gut were decline compared with non-operative rats. Conclusion:(1)Chronic rejection occurred at POD90 in heterotopic small bowel transplantation model.(2)Histological features of chronic rejection in orthotopic small bowel transplantation model were detected after POD 150. (3)The surgical procedure of heterotopic small bowel transplantation is not complicated and surgical success rate is more than 85%.(4)Because typical histopathological features of chronic rejection exsits during the recipients survival time, the model of rat heterotopic small bowel transplantation is applied in the following research. According to pathological results, observative duration were confirm PODO-90 after transplant surgery.
Part II The Research on Chronic Rejection of Heterotopic Small Bowel Transplantation in Rat
Objective:To extablish rat heterotopic small bowel transplantation model and to explore the mechanisms of chronic rejection. Methods:The recipients were divided into 3 groups including isogeneic control group, allogeneic control group and allogeneic transplantation with cyclosporine treatment group(CsA group). Graft samples were harvested at POD7, POD14, POD30, POD60 and POD90 and pathological examination was performed. The CD68 expression indicating infiltration of macrophages was determined by immunohistochemistry. In order to investigate the relationship of TGFβ1 and PDGF-C with chronic rejection, we used real-time quantitative RT-PCR and immunohistochemistry to detect the mRNA transcription and protein expression of TGFβ1 and PDGF-C. Results:(1)All of the recipients in the allogeneic control group died within 15 days after transplantation. (2)The leukocyte infiltration scores of CsA group were higher than those of isogeneic group from POD 14 to POD90. The mucous coat architecture scores and interstitial fibrosis scores of CsA group were higher than those of isogeneic group from POD30 to POD90. Intimal thickening scores of CsA group were significantly higher than those of isogeneic group from the beginning of POD60. (3)The expression of CD68 in CsA group continued to increase throughout the process of chronic rejection, and reached the highest level after POD60. (4)The mRNA and protein expression of TGFβ1 and PDGF-C in CsA group were significantly increased. The immunohistochemical results showed high expression of TGFβ1 in the interstitial and PDGF-C in the cytoplasm and cell membrane. Conclusion:(1)There was extensive infiltration of macrophages in the graft of CsA group at POD60, associated with high expression of TGFpl and PDGF-C. (2) The chronic rejection model of allogeneic heterotopic small bowel transplantation was successfully established at 60 days after surgery. The expression of CD68,TGFβ1 and PDGF-C were significantly increased in CsA group, which could be used to determine chronic rejection.
PartⅢIsolation, Culturing and Identification of Rat MSCs from Bone Marrow in Vitro
Objective:To investigate the methods of the isolation, culture and identification of rat MSCs from bone marrow in vitro.Methods:One month-old male Lewis rats were sacrificed and tibias and femurs tissues were obtained. After the long bones were dissected, the marrows were flushed out with PBS buffer under aseptic condition and purified by percoll gradient centrifugation to get the mononuclear cells. After the mononuclear cells were cultured, the adherent cells were selected for serial subcultivation. The cells of the third generation in good growth condition was harvested and trypsinized to form cell suspension. The cells were counted and the growth curve was made. The cells of the third to five generation were harvested and the cell phenotypes were identified by flow cytometry. The cells of the third generation were induced to differentiate into osteoblasts. The induced cells were identified with alkaline phosphatase staining and alizarin red staining. The cells of the third generation were labeled by fluorescent dye of CFSE. Results:(1)The MSCs were spindle shaped, attached to the culture dish tightly after 72 hours' culture, and proliferated in the culture medium. After 21 days of primary cultivation, MSCs were nearly 100% confluent. (2)The 3th passage MSCs comprised a unique phenotypic population by flow cytometric analysis. The phenotypes were shown to be positive for CD29 and CD90 more than 96% and be negative for CD34 and CD45.(3)The MSCs formed aggregates or nodules and increased their expression of alkaline phosphatase, and calcium accumulation was evident after 3 weeks. Alkaline phosphatase staining and alizarin red staining confirmed the success of induction. (4)CFSE had high efficiency for labeling MSCs, the labeling index was over 100%.
Conclusion:(1)MSCs are stem cells with strong proliferative activity and sustain self-renewal. They are pluripotent and could differentiate to a variety of cell types in some condition. (2)The methods of obtaining rat MSCs with density gradient centrifugation and adherence cultivation were convenient, and could provide immortalized rat MSCs lines for cellular transplant therapy. (3)CFSE staining is a feasible method for labeling MSCs with high efficiency.
PartⅣStudy on the Effect of Bone Marrow Mesenchymal Stem Cells on Immunosuppression in Vitro
Objective:To explore the effect of mesenchymal stem cells derived from bone marrow using mixed lumphocyte culture on the immune response of splenic cells and the corresponding mechanisms.Methods:MSCs and mixed lymphocyte reaction cultures were set up. Splenic lymphocytes from F344 rat were used as reactive cells and labeled with CFSE. The study included 4 groups.Group 1 (the control group) included 5×105 reactive cells cultured. Group 2 were 5×105 reactive cells cultured with conA. Group 3 included 1×105 MSCs from Lewis rats and 5×105reactive cells. Group 4 included 1×105 MSCs from Lewis rats and 5×105 reactive cells cultured with conA as a stimulator. Cells in each group were cultured for 72 hours. The proliferation dynamics of CFSE labeled cells were detected by flow cytometry. In the same groups without CFSE labling, the CD4+ and CD8+ phenotypes of proliferating lymphocytes were determined with monoclone antibodies.Results:Compared with that in group 2, the proliferation of sub-peak in group 4 couldn't be detected. The positive expression of CD4+ and CD8+ were declined compared with that in group 1, but there was no difference in the ratio of CD4+ to CD8+. Conclusion:Bone marrow MSCs demonstrate significant immune regulatory effects on conA-stimulated T-lymphocyte culture. It might provide a remarkable immune suppression in organ transplantation to achieve better outcome in the future.
Part V The Effect of Bone Marrow Mesenchymal Stem Cells Derived from Recipients on the Chronic Rejection in Rat Heterotopic Small Bowel Transplantation
Objective:To observe the effects of MSCs derived from recipients'bone marrow on the chronic rejection of rat heterotopic small bowel transplantation. Methods:Sixteen Lewis rats bearing transplanted small bowel from F344 rats were randomly divided into 2 groups and all of them received cyclosporine A treatment. (1)Control group: 2mL 0.9%NaCl solution was injected through vena dorsalis penis at 24h after small bowel transplantation. (2)Treatment group:1×107MSCs in 2ml 0.9%NaCl solution was injected through vena dorsalis penis at 24h after small bowel transplantation. Graft samples were harvested on POD60 and examined by hematoxylin eosin staining and Verhoffs alcoholic hematoxylin ted. The graft infiltration of macrophages was determined by immunohistochemical staining. mRNA transcription and protein expression of TGFβ1 and PDGF-C were measured by real-time quantitative RT-PCR and immunohistochemistry, respectively. Two other small bowel transplantation models were established with cyclosporine A treatment. At 24h after transplantation, 1×107MSCs labeled with CFSE were injected through vena dorsalis penis, respectively. The two grafts and other self-organ were harvested on 7th day after transplantation to get frozen sections for observation of the fluorescence cells in grafts.Results:(1)Fluorescence cells concentrated in the interstitial of allografts. (2)The scores of mucous coat architecture, leukocyte infiltration, interstitial fibrosis and intimal thickening of treatment group were significantly less than those of control group at POD60.(3)The expression of CD68, TGFβ1 and PDGF-C in treatment group was significantly less than that in control group at POD60. Conclusion:Intravenous infusion with MSCs from recipients could inhibit the process of chronic rejection in rat heterotopic small bowel transplantation to some degree. The mechanisms involve the suppressive effect of MSCs on lymphocytic proliferation and on the expression of CD68,TGFβ1 and PDGF-C.
引文
[1]Jonathan P, Fryer. Intestinal transplantation:an update[J].Curr Opin Gastroenterol,2005,21:162-168.
[2]黎介寿.小肠移植的现状[J].中华器官移植杂志：电子版,2009,3(3)：177-178.
[3]Mazariegos GV, Squires RH,Sindhi RK.Current perspectives on pediatric intestinal transplantation[J].Curr Gastroenterol Rep,2009,11(3):226-233.
[4]Suzuki J, Isobe M, Morishita R, et al.Characteristics of chronic rejection in heart transplantation:important elements of pathogenesis and future treatments[J].Circ J.,2010,74(2):233-239.
[5]Fryer JP. The current status of intestinal transplantation. Curr Opin Organ Transplant,2008,13(3):266-272.
[6]Parizhskaya M, Redondo C, Demetris A, et al.Chronic rejection of small bowel grafts:pediatric and adult study of risk factors and morphologic progression. Pediatr Dev Pathol.,2003,6(3):240-250.
[7]Gokmen MR, Lombardi G, Lechler RI.The importance of the indirect pathway of allorecognition in clinical transplantation[J].Curr Opin Immunol.,2008,20(5):568-74.
[8]Mariafederica U, Ennia D, Maria E, et al.Cytokines and chronic rejection:a study in kidney transplantation long-term survivors[J].Transplantation, 2004,77(4),548-552.
[9]Mizutani K, Terasaki P, Rosen A, et al. Serial ten-year follow-up of HLA and MICA antibody production prior to kidney graft failure[J].Am J Transplant,2005, 5(9):2265-2272.
[10]Mizutani K, Shibata L, Ozawa M,et al.Detection of HLA and MICA antibodies before kidney graft failure[J].Clin Transpl.,2006:255-264.
[11]Stastny P, Lavingia B, Fixler DE,et al. Antibodies against donor human leukocyte antigens and the outcome of cardiac allografts in adults and children[J]. Transplantation,2007,84(6):738-745.
[12]Ma H, Wang J, Wang J, et al.Features of chronic allograft rejection on rat small intestine transplantation[J].Pediatr Transplant,2007,11(2):165-172.
[13]Chen Y, Li X, Tian L,et al.Inhibition of sonic hedgehog signaling reduces chronic rejection and prolongs allograft survival in a rat orthotopic small bowel transplantation model[J].Transplantation,2007,83(10):1351-1357.
[14]靳小石,刘彤,梁晓宇等.中药预防大鼠小肠移植慢性排斥反应的实验研究[R].中国外科周暨第16届亚洲外科年会,2007年.
[15]Zou Z, Zhang Y, Hao L,et al.More insight into mesenchymal stem cells and their effects inside the body[J].Expert Opin Biol Ther.,2010,10(2):215-230.
[16]动物实验方法学.孙敬方主编.北京,人民卫生出版社,2004,150.
[17]Lee RG, Nakamura K, Tsamandas AC, et al.Pathology of human intestinal transplantation[J].Gastroenterology,1996,110:1820-1834.
[18]Jonathan P, Fryer. Intestinal transplantation:an update[J].Curr Opin Gastroenterol,2005,21:162-168.
[19]Goulet O, Lacaille F, Jan D, et al. Intetinal transplantation:indications, results and strategy[J].Current Opinion in Clinical Nutrition and Metabolic Care,2000,3(5):329-338.
[20]Thomas M, Fishbein, Gabriel E, et al.Intestinal transplantation for Gut Failure [J].Gastroenterology,2003,124(6):1615-1628.
[21]Ma H, Wang J, Wang J, et al.Features of chronic allograft rejection on rat small intestine transplantation[J].Pediatr Transplant.,2007,11(2):165-172.
[22]Orloff SL, Yin Q, Corless CL, et al. A rat small bowel transplant model of chronic rejection:histopathologic characteristics [J].Transplantation. 1999,27;68(6):766-779.
[23]Chen Y, Li X, Tian L, Lui VC,et al.Inhibition of sonic hedgehog signaling reduces chronic rejection and prolongs allograft survival in a rat orthotopic small bowel transplantation model.Transplantation.,2007,83(10):1351-1357.
[24]Klaus A, Klima G, Margreiter R,et al.Myoelectric activity during chronic small bowel allograft rejection in rats[J].Dig Dis Sci.,2002,47(11):2506-2511.
[25]Klaus A, Margreiter R, Pernthaler H,et al.Diffuse mesenterial sclerosis:a characteristic feature of chronic small-bowel allograft rejection[J].Virchows Arch. 2003,442(1):48-55.
[26]Gasser M, Otto C, Timmermann W,et al.Putative regulatory function of alloreactive Th2 cells in tolerant recipients after small bowel transplantation. Transplant Proc[J].2001,33(1-2):161-162.
[27]Kouwenhoven EA, Stein-Oakley AN, Jablonski P,et al.EGF and TGF-betal gene expression in chronically rejecting small bowel transplants[J]. Dig Dis Sci. 1999,44(6):1117-1123.
[28]Marco ML.The Fischer-Lewis model of chronic allograft rejection-a summary[J].Nephrol Dial Transplant.2006,21(11):3082-3086.
[29]Guo WH, Tian L, Dallmann MJ,et al.Comparative study of allograft survival of heterotopic and orthotopic small bowel transplantation in rat[J].Transplantation. 2003,75(11):1895-1897.
[30]Bustin S A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.[J].J Mol Endocrinol,2000,25(2): 169-193.
[31]萨姆布鲁克J.分子克隆实验指南第三版[M].科学技术出版社,1998.
[32]刘万里,薛茜,曹明芹等。用SPSS实现完全随机设计多组比较秩和检验的多重比较[J].地方病通报。2007.22(2)：27-29.
[33]Sibley RK. Pathology and immunopathology of solid organ graft rejection. Transplant Proc.1989,21(1 Pt 1):14-17.
[34]Suzuki J, Isobe M, Morishita R, et al.Characteristics of chronic rejection in heart transplantation:important elements of pathogenesis and future treatments[J]. Circ J.2010,74(2):233-239.
[35]Solez K, Colvin RB,Racusen LC, et al.Banff'05 Meeting Report:differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy ('CAN')[J].Am J Transplant.2007,7(3):518-526.
[36]Hruban RH,Beschorner WE, Baumgartner WA,et al.Accelerated arteriosclerosis in heart transplant recipients is associated with a T-lymphocyte-mediated endothelialitis. Am J Pathol.1990,137(4):871-882.
[37]Zou Y, Hernandz F, Burgos E, et al.Organ changes and bacterial translocation in a rat model of chronic rejection after small bowel transplantation[J].Transplantation Proceedings,2006,38(5),1569-1572.
[38]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
[39]Morris PJ, Williams GM, Hume DM, et al.Serotyping for homotransplantation. XII. Occurrence of cytotoxic antibodies following kidney transplantation in man[J]. Transplantation,1968,6(3):392-399.
[40]Wewers MD,Marsh CB.Role of the antibody in the pathogenesis of transplant vascular sclerosis:A hypothesis[J].Transpl Immunol,1997,5(4):283-288.
[41]Herrero-Fresneda I, Torras J, Cruzado JM, et al.Do alloreactivity and prolonged cold ischemia cause different elementary lesions in chronic allograft nephropathy?[J]. Am J Pathol,2003,162(1):127-137.
[42]Azuma H, Nadeau KC, Ishibashi M,et al.Host leukocytes and their products in chronic kidney allograft rejection in rats.Transpl Int.,1994,7Suppl 1:S325-S327.
[43]Heemann UW, Tullius SG, Tamatami T,et al.Infiltration patterns of macrophages and lymphocytes in chronically rejecting rat kidney allografts[J]. Transpl Int.,1994,7(5):349-355.
[44]Russell ME, Wallace AF, Hancock WW, et al. Upregulation of cytokines associated with macrophage activation in the Lewis-to-F344 rat transplantation model of chronic cardiac rejection[J].Transplantation,1995,59(4):572-578.
[45]Pilmore HL, Painter DM, Bishop GA,et al. Early up-regulation of macrophages and myofibroblasts:a new marker for development of chronic renal allograft rejection. Transplantation.2000,69(12):2658-2662.
[46]Kitchens WH, Chase CM, Uehara S, et al. Macrophage depletion suppresses cardiac allograft vasculopathy in mice[J].Am J Transplant.2007,7(12):2675-2682.
[47]Pilmore HL, Painter DM, Bishop GA, et al. Early up-regulation of macrophages and myofibroblasts:a new marker for development of chronic renal allograft rejection[J].Transplantation,2000,69(12):2658-2662.
[48]Yang J, Reutzel-Selke A, Steier C, et al.Targeting of macrophage activity by adenovirus-mediated intragraft overexpression of TNFRp55-Ig, IL-12p40, and vIL-10 ameliorates adenovirus-mediated chronic graft injury, whereas stimulation of macrophages by overexpression of IFNgamma accelerates chronic graft injury in a rat renal allograft model[J].J Am Soc Nephrol,2003,14(1):214-225.
[49]Ye Q, Wu YL, Foley LM, et al.Longitudinal tracking of recipient macrophages in a rat chronic cardiac allograft rejection model with noninvasive magnetic resonance imaging using micrometer-sized paramagnetic iron oxide particles[J]. Circulation.2008,118(2):149-156.
[50]Pilmore H, Painter D, Bishop G,et al. Early up-regulation of macrophages and myofiberoblasts:a new marker for development of chronic renal allograft rejection[J]. Transplantation.2000,69(12):2658-2662.
[51]Savikko J, Taskinen E, Von Willebrand E. Chronic allograft riephropathy is prevented by inhibition of platelet-derived growth factor receptor:tyrosine kinase inhibitors as a potential therapy[J].Transplantation,2003,75(8):1147-1153.
[52]Bissell DM, Roulot D, George J. Transforming growth factor beta and the liver[J].Hepatology,Md,2001,34(5):859-867.
[53]Sharma V, Bologa R, Xu G, et al.Intragraft TGF-beta 1 mRNA:a correlate of interstitial fibrosis and chronic allograft nephropathy[J].Kidney Int. 1996,49(5):1297-1303.
[54]Morris-Stiff G TGFbeta-1 and the development of chronic graft nephropathy: relative roles of gene, mRNA and protein[J].Ann R Coll Surg Engl. 2005,87(5):326-330.
[55]Faust SM, Lu G, Wood SC, et al.TGF beta neutralization within cardiac allografts by decorin gene transfer attenuates chronic rejection[J].J Immunol., 2009,183(11):7307-7313.
[56]Libby P, Warner SJ, Salomon RN,et al.Production of platelet-derived growth factor-like mitogen by smooth-muscle from human atheroma[J].N Eng L J Med,1988,318(23):1493-1498.
[57]Uutela M, Lauren J, Bergsten E, et al.Chromosomal location, exon structure, and vascular expression patterns of the human PDGFC and PDGFC genes[J].Circulation.2001,103(18):2242-2247.
[58]Chen J,Han Y,Lin C,et al.PDGF-D contributes to neointimal hyperplasia in rat model of vessel Injury[J].Biochem Biophys Res Commun.2005,329(3):976-983.
[59]Ponten A, Li X, Thoren P, et al.Transgenic overexpression of platelet-derived growth factor-C in the mouse heart induces cardiac fibrosis, hypertrophy, and dilated cardiomyopathy[J].Am J Pathol.2003,163(2):673-682.
[60]Ponten A, Folestad EB, Pietras K,et al.Platelet-derived growth factor D induces cardiac fibrosis and proliferation of vascular smooth muscle cells in heart-specific transgenic mice[J].Circ Res.2005,97(10):1036-1045.
[61]Lindahl P, Johansson BR, Leveen P,et al. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice[J].Science.1997,277(5323):242-245.
[62]Ding H, Wu X, Bostrom H,et al.A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling[J].Nat Genet.2004,36(10):1111-1116.
[63]Li X, Ponten A, Aase K,et al.PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor[J].Nat Cell Biol,2000,2(5):302-309.
[64]Gilbertson DG, Duff ME, West JW, et al. Platelet-derived growth factor C (PDGF-C), a novel growth factor that binds to PDGF alpha and beta receptor[J].J Biol Chem.2001,276(29):27406-27414.
[65]Campbell JS, Hughes SD, Gilbertson DG, et al. Platelet-derived growth factor C induces liver fibrosis, steatosis,and hepatocellular carcinoma[J].Proc Natl Acad Sci USA,2005,102(9):3389-3394.
[66]Zhuo Y, Hoyle GW, Shan B,et al. Over-expression of PDGF-C using a lung specific promoter results in abnormal lung development[J].Transgenic Res,2006,15(5):543-555.
[67]Grun K, Markova B, Bohmer FD, et al.Elevated expression of PDGF-C in coxsackievirus B3-induced chronic myocarditis[J].Eur Heart J,2005,26(7):728-739.
[68]Zhuo Y, Zhang J, Laboy M,et al. Modulation of PDGF-C and PDGF-D expression during bleomycin-induced lung fibrosis[J].Am JPhysiol Lung Cell Mol Physiol,2004,286(1):L182-L188.
[69]Eitner F, Bucher E, van Roeyen C,et al.PDGF-C is a proinflammatory cytokine that mediates renal interstitial fibrosis[J].J Am Soc Nephrol.2008,19(2):281-289.
[70]Tuuminen R,Nykanen Al,Krebs R,et al.PDGF-A,-C,and-D but not PDGF-B increase TGF-betal and chronic rejection in rat cardiac allografts[J].Arterioscler Thromb Vasc Biol.2009,29(5):691-698.
[71]Haynesworth SE, Goshima J, Goldberg VM,et al.Characterization of cells with osteogenic potential from human marrow[J].Bone.1992,13(1):81-88.
[72]陈嘉薇.环孢素A肾毒性机制的研究进展[J].中华器官移植杂志,2003,4：255-256.
[73]Wekerle T, Sykes M. Mixed chimerism and transplantation tolerance.Annu Rev Med,2001,52:353-370.
[74]Doucet C,Ernoul,ZhangY,et al.Platelet lysates Promote mesenehymal stem cell expansion:a safety substitute for animal serum in cell-based therapy applications. J Cell Physiol.2005,205(2):228-236.
[75]吴凤霞,蔡维君,伍校琼,等.不同年龄大鼠骨髓基质干细胞的增殖特性.神经解剖学杂志,2005,21(5)：531-534.
[76]Ringe J, Kaps C, Schmitt B,et al. Porcine mesenchymnal stem cells.Induction of distinct mesenchymal cell lineages[J].Cell Tissue Res,2002,307(3):321-327.
[77]Lodie TA, Blickarz CE, Devarakonda TJ, et al.Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction[J].Tissue Eng.2002,8(5):739-751.
[78]Jiang XX, Zhang Y, Liu B, et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells[J]. Blood,2005,105(10):4120-4126.
[79]Spaggiari GM, Capobianco A, Becchetti S, et al.Mesenchymal stem cell-natural killer cell interactions:Evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NKcell proliferation[J].Blood 2006,107(4):1484-1490.
[80]Oh JY, Kim MK, Shin MS,et al. The anti-inflammatory and anti-angiogenic role of mesenchymal stem cells in corneal wound healing following chemical injury[J].Stem Cells,2008,26(4):1047-1055.
[81]Maitra B, Szekely E, Gjini K,et al.Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation[J].Bone Marrow Transplant.2004,33(6):597-604.
[82]Deans RJ, Moseley AB.Mesenchymal stem cells:biology and potential clinical uses[J].Exp Hematol.2000,28(8):875-884.
[83]Tse WT, Pendleton JD, Beyer WM,et al.Suppression of allogeneic T-cell proliferation by human marrow stromal cells:implications in transplantation[J]. Transplantation.2003,75(3):389-397.
[84]Le Blanc K, Tammik L, Sundberg B,et al. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex[J].Scand J Immunol.2003,57(1):11-20.
[85]Di Nicola M,Carlo-Stella C,Magni M,et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli[J].Blood.2002,99(10):3838-3843.
[86]Djouad F, Plence P, Bony C,et al.Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals[J].Blood. 2003,102(10):3837-3844.
[87]Waaga AM, Gasser M, Laskowski I, et al.Mechanisms of chronic rejection[J]. Curr Opin Immunol,2000,12(5):517-521.
[1]Mazariegos GV, Squires RH, Sindhi RK.Current perspectives on pediatric intestinal transplantation[J].Curr Gastroenterol Rep.2009,11(3):226-233.
[2]Mitchell RN.Graft vascular disease:immune response meets the vessel wall[J].Annu Rev Pathol.2009,4:19-47.
[3]Chronic Rejection of Small Bowel Grafts:Pediatric and Adult Study of Risk Factors and Morphologic Progression[J].Pediatric and Developmental Pathology 2003,6(3):240-250.
[4]Ma H,Wang J, Wang J, et al.Features of chronic allograft rejection on rat small intestine transplantation[J].Pediatr Transplant.2007,11(2):165-172.
[5]Klaus A, Margreiter R, Pernthaler H,et al.Diffuse mesenterial sclerosis:a characteristic feature of chronic small-bowel allograft rejection[J].Virchows Arch. 2003,442(1):48-55.
[6]Giovanelli M, Gupte GL, Sharif K,et al.Chronic rejection after combined liver and small bowel transplantation in a child with chronic intestinal pseudo-obstruction:a case report[J].Transplant Proc.2008,40(5):1763-1767.
[7]Ramos E, Molina M, Sarria J,et al.Chronic rejection with sclerosing peritonitis following pediatric intestinal transplantation[J].Pediatr Transplant. 2007,11(8):937-941.
[8]Gokmen MR, Lombardi G,Lechler RI.The importance of the indirect pathway of allorecognition in clinical transplantation[J].Curr Opin Immunol. 2008,20(5):568-574.
[9]Mariafederica U, Ennia D, Maria E, et al.Cytokines and chronic rejection:a study in kidney transplantation long-term survivors[J].Transplantation, 2004,77(4):548-552.
[10]Morris PJ, Williams GM, Hume DM, et al. Serotyping for homotransplantation. XII. Occurrence of cytotoxic antibodies following kidney transplantation in man[J].Transplantation,1968,6(3):392-399.
[11]Mizutani K, Terasaki P, Rosen A, et al.Serial ten-year follow-up of HLA and MICA antibody production prior to kidney graft failure[J].Am J Transplant,2005, 5(9):2265-2272.
[12]Mizutani K, Shibata L, Ozawa M,et al. Detection of HLA and MICA antibodies before kidney graft failure[J].Clin Transpl.2006:255-264.
[13]Stastny P, Lavingia B, Fixler DE,et al.Antibodies against donor human leukocyte antigens and the outcome of cardiac allografts in adults and children[J]. Transplantation.2007,84(6):738-745.
[14]Kato T, Mizutani K, Terasaki P,et al.Association of emergence of HLA antibody and acute rejection in intestinal transplant recipients:a possible evidence of acute humoral sensitization[J].Transplant Proc.2006,38(6):1735-1737.
[15]Takahashi H, Kato T, Mizutani K,et al.Simultaneous antibody-mediated rejection of multiple allografts in modified multivisceral transplantation[J].Clin Transpl.2006:529-534.
[16]Nakao A, Toyokawa H,Tsung A, et al.Ex vivo application of carbon monoxide in University of Wisconsin solution to prevent intestinal cold ischemia/reperfusion injury[J].Am J Transplant.2006,6(10):2243-2255.
[17]Solez K, Colvin RB, Racusen LC,et al.Banff'05 Meeting Report:differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy ('CAN')[J].Am J Transplant.2007,7(3):518-526.
[2]黎介寿.小肠移植的现状[J].中华器官移植杂志：电子版,2009,3(3)：177-178.
[3]Mazariegos GV, Squires RH,Sindhi RK.Current perspectives on pediatric intestinal transplantation[J].Curr Gastroenterol Rep,2009,11(3):226-233.
[4]Suzuki J, Isobe M, Morishita R, et al.Characteristics of chronic rejection in heart transplantation:important elements of pathogenesis and future treatments[J].Circ J.,2010,74(2):233-239.
[5]Fryer JP. The current status of intestinal transplantation. Curr Opin Organ Transplant,2008,13(3):266-272.
[6]Parizhskaya M, Redondo C, Demetris A, et al.Chronic rejection of small bowel grafts:pediatric and adult study of risk factors and morphologic progression. Pediatr Dev Pathol.,2003,6(3):240-250.
[7]Gokmen MR, Lombardi G, Lechler RI.The importance of the indirect pathway of allorecognition in clinical transplantation[J].Curr Opin Immunol.,2008,20(5):568-74.
[8]Mariafederica U, Ennia D, Maria E, et al.Cytokines and chronic rejection:a study in kidney transplantation long-term survivors[J].Transplantation, 2004,77(4),548-552.
[9]Mizutani K, Terasaki P, Rosen A, et al. Serial ten-year follow-up of HLA and MICA antibody production prior to kidney graft failure[J].Am J Transplant,2005, 5(9):2265-2272.
[10]Mizutani K, Shibata L, Ozawa M,et al.Detection of HLA and MICA antibodies before kidney graft failure[J].Clin Transpl.,2006:255-264.
[11]Stastny P, Lavingia B, Fixler DE,et al. Antibodies against donor human leukocyte antigens and the outcome of cardiac allografts in adults and children[J]. Transplantation,2007,84(6):738-745.
[12]Ma H, Wang J, Wang J, et al.Features of chronic allograft rejection on rat small intestine transplantation[J].Pediatr Transplant,2007,11(2):165-172.
[13]Chen Y, Li X, Tian L,et al.Inhibition of sonic hedgehog signaling reduces chronic rejection and prolongs allograft survival in a rat orthotopic small bowel transplantation model[J].Transplantation,2007,83(10):1351-1357.
[14]靳小石,刘彤,梁晓宇等.中药预防大鼠小肠移植慢性排斥反应的实验研究[R].中国外科周暨第16届亚洲外科年会,2007年.
[15]Zou Z, Zhang Y, Hao L,et al.More insight into mesenchymal stem cells and their effects inside the body[J].Expert Opin Biol Ther.,2010,10(2):215-230.
[16]动物实验方法学.孙敬方主编.北京,人民卫生出版社,2004,150.
[17]Lee RG, Nakamura K, Tsamandas AC, et al.Pathology of human intestinal transplantation[J].Gastroenterology,1996,110:1820-1834.
[18]Jonathan P, Fryer. Intestinal transplantation:an update[J].Curr Opin Gastroenterol,2005,21:162-168.
[19]Goulet O, Lacaille F, Jan D, et al. Intetinal transplantation:indications, results and strategy[J].Current Opinion in Clinical Nutrition and Metabolic Care,2000,3(5):329-338.
[20]Thomas M, Fishbein, Gabriel E, et al.Intestinal transplantation for Gut Failure [J].Gastroenterology,2003,124(6):1615-1628.
[21]Ma H, Wang J, Wang J, et al.Features of chronic allograft rejection on rat small intestine transplantation[J].Pediatr Transplant.,2007,11(2):165-172.
[22]Orloff SL, Yin Q, Corless CL, et al. A rat small bowel transplant model of chronic rejection:histopathologic characteristics [J].Transplantation. 1999,27;68(6):766-779.
[23]Chen Y, Li X, Tian L, Lui VC,et al.Inhibition of sonic hedgehog signaling reduces chronic rejection and prolongs allograft survival in a rat orthotopic small bowel transplantation model.Transplantation.,2007,83(10):1351-1357.
[24]Klaus A, Klima G, Margreiter R,et al.Myoelectric activity during chronic small bowel allograft rejection in rats[J].Dig Dis Sci.,2002,47(11):2506-2511.
[25]Klaus A, Margreiter R, Pernthaler H,et al.Diffuse mesenterial sclerosis:a characteristic feature of chronic small-bowel allograft rejection[J].Virchows Arch. 2003,442(1):48-55.
[26]Gasser M, Otto C, Timmermann W,et al.Putative regulatory function of alloreactive Th2 cells in tolerant recipients after small bowel transplantation. Transplant Proc[J].2001,33(1-2):161-162.
[27]Kouwenhoven EA, Stein-Oakley AN, Jablonski P,et al.EGF and TGF-betal gene expression in chronically rejecting small bowel transplants[J]. Dig Dis Sci. 1999,44(6):1117-1123.
[28]Marco ML.The Fischer-Lewis model of chronic allograft rejection-a summary[J].Nephrol Dial Transplant.2006,21(11):3082-3086.
[29]Guo WH, Tian L, Dallmann MJ,et al.Comparative study of allograft survival of heterotopic and orthotopic small bowel transplantation in rat[J].Transplantation. 2003,75(11):1895-1897.
[30]Bustin S A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.[J].J Mol Endocrinol,2000,25(2): 169-193.
[31]萨姆布鲁克J.分子克隆实验指南第三版[M].科学技术出版社,1998.
[32]刘万里,薛茜,曹明芹等。用SPSS实现完全随机设计多组比较秩和检验的多重比较[J].地方病通报。2007.22(2)：27-29.
[33]Sibley RK. Pathology and immunopathology of solid organ graft rejection. Transplant Proc.1989,21(1 Pt 1):14-17.
[34]Suzuki J, Isobe M, Morishita R, et al.Characteristics of chronic rejection in heart transplantation:important elements of pathogenesis and future treatments[J]. Circ J.2010,74(2):233-239.
[35]Solez K, Colvin RB,Racusen LC, et al.Banff'05 Meeting Report:differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy ('CAN')[J].Am J Transplant.2007,7(3):518-526.
[36]Hruban RH,Beschorner WE, Baumgartner WA,et al.Accelerated arteriosclerosis in heart transplant recipients is associated with a T-lymphocyte-mediated endothelialitis. Am J Pathol.1990,137(4):871-882.
[37]Zou Y, Hernandz F, Burgos E, et al.Organ changes and bacterial translocation in a rat model of chronic rejection after small bowel transplantation[J].Transplantation Proceedings,2006,38(5),1569-1572.
[38]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
[39]Morris PJ, Williams GM, Hume DM, et al.Serotyping for homotransplantation. XII. Occurrence of cytotoxic antibodies following kidney transplantation in man[J]. Transplantation,1968,6(3):392-399.
[40]Wewers MD,Marsh CB.Role of the antibody in the pathogenesis of transplant vascular sclerosis:A hypothesis[J].Transpl Immunol,1997,5(4):283-288.
[41]Herrero-Fresneda I, Torras J, Cruzado JM, et al.Do alloreactivity and prolonged cold ischemia cause different elementary lesions in chronic allograft nephropathy?[J]. Am J Pathol,2003,162(1):127-137.
[42]Azuma H, Nadeau KC, Ishibashi M,et al.Host leukocytes and their products in chronic kidney allograft rejection in rats.Transpl Int.,1994,7Suppl 1:S325-S327.
[43]Heemann UW, Tullius SG, Tamatami T,et al.Infiltration patterns of macrophages and lymphocytes in chronically rejecting rat kidney allografts[J]. Transpl Int.,1994,7(5):349-355.
[44]Russell ME, Wallace AF, Hancock WW, et al. Upregulation of cytokines associated with macrophage activation in the Lewis-to-F344 rat transplantation model of chronic cardiac rejection[J].Transplantation,1995,59(4):572-578.
[45]Pilmore HL, Painter DM, Bishop GA,et al. Early up-regulation of macrophages and myofibroblasts:a new marker for development of chronic renal allograft rejection. Transplantation.2000,69(12):2658-2662.
[46]Kitchens WH, Chase CM, Uehara S, et al. Macrophage depletion suppresses cardiac allograft vasculopathy in mice[J].Am J Transplant.2007,7(12):2675-2682.
[47]Pilmore HL, Painter DM, Bishop GA, et al. Early up-regulation of macrophages and myofibroblasts:a new marker for development of chronic renal allograft rejection[J].Transplantation,2000,69(12):2658-2662.
[48]Yang J, Reutzel-Selke A, Steier C, et al.Targeting of macrophage activity by adenovirus-mediated intragraft overexpression of TNFRp55-Ig, IL-12p40, and vIL-10 ameliorates adenovirus-mediated chronic graft injury, whereas stimulation of macrophages by overexpression of IFNgamma accelerates chronic graft injury in a rat renal allograft model[J].J Am Soc Nephrol,2003,14(1):214-225.
[49]Ye Q, Wu YL, Foley LM, et al.Longitudinal tracking of recipient macrophages in a rat chronic cardiac allograft rejection model with noninvasive magnetic resonance imaging using micrometer-sized paramagnetic iron oxide particles[J]. Circulation.2008,118(2):149-156.
[50]Pilmore H, Painter D, Bishop G,et al. Early up-regulation of macrophages and myofiberoblasts:a new marker for development of chronic renal allograft rejection[J]. Transplantation.2000,69(12):2658-2662.
[51]Savikko J, Taskinen E, Von Willebrand E. Chronic allograft riephropathy is prevented by inhibition of platelet-derived growth factor receptor:tyrosine kinase inhibitors as a potential therapy[J].Transplantation,2003,75(8):1147-1153.
[52]Bissell DM, Roulot D, George J. Transforming growth factor beta and the liver[J].Hepatology,Md,2001,34(5):859-867.
[53]Sharma V, Bologa R, Xu G, et al.Intragraft TGF-beta 1 mRNA:a correlate of interstitial fibrosis and chronic allograft nephropathy[J].Kidney Int. 1996,49(5):1297-1303.
[54]Morris-Stiff G TGFbeta-1 and the development of chronic graft nephropathy: relative roles of gene, mRNA and protein[J].Ann R Coll Surg Engl. 2005,87(5):326-330.
[55]Faust SM, Lu G, Wood SC, et al.TGF beta neutralization within cardiac allografts by decorin gene transfer attenuates chronic rejection[J].J Immunol., 2009,183(11):7307-7313.
[56]Libby P, Warner SJ, Salomon RN,et al.Production of platelet-derived growth factor-like mitogen by smooth-muscle from human atheroma[J].N Eng L J Med,1988,318(23):1493-1498.
[57]Uutela M, Lauren J, Bergsten E, et al.Chromosomal location, exon structure, and vascular expression patterns of the human PDGFC and PDGFC genes[J].Circulation.2001,103(18):2242-2247.
[58]Chen J,Han Y,Lin C,et al.PDGF-D contributes to neointimal hyperplasia in rat model of vessel Injury[J].Biochem Biophys Res Commun.2005,329(3):976-983.
[59]Ponten A, Li X, Thoren P, et al.Transgenic overexpression of platelet-derived growth factor-C in the mouse heart induces cardiac fibrosis, hypertrophy, and dilated cardiomyopathy[J].Am J Pathol.2003,163(2):673-682.
[60]Ponten A, Folestad EB, Pietras K,et al.Platelet-derived growth factor D induces cardiac fibrosis and proliferation of vascular smooth muscle cells in heart-specific transgenic mice[J].Circ Res.2005,97(10):1036-1045.
[61]Lindahl P, Johansson BR, Leveen P,et al. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice[J].Science.1997,277(5323):242-245.
[62]Ding H, Wu X, Bostrom H,et al.A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling[J].Nat Genet.2004,36(10):1111-1116.
[63]Li X, Ponten A, Aase K,et al.PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor[J].Nat Cell Biol,2000,2(5):302-309.
[64]Gilbertson DG, Duff ME, West JW, et al. Platelet-derived growth factor C (PDGF-C), a novel growth factor that binds to PDGF alpha and beta receptor[J].J Biol Chem.2001,276(29):27406-27414.
[65]Campbell JS, Hughes SD, Gilbertson DG, et al. Platelet-derived growth factor C induces liver fibrosis, steatosis,and hepatocellular carcinoma[J].Proc Natl Acad Sci USA,2005,102(9):3389-3394.
[66]Zhuo Y, Hoyle GW, Shan B,et al. Over-expression of PDGF-C using a lung specific promoter results in abnormal lung development[J].Transgenic Res,2006,15(5):543-555.
[67]Grun K, Markova B, Bohmer FD, et al.Elevated expression of PDGF-C in coxsackievirus B3-induced chronic myocarditis[J].Eur Heart J,2005,26(7):728-739.
[68]Zhuo Y, Zhang J, Laboy M,et al. Modulation of PDGF-C and PDGF-D expression during bleomycin-induced lung fibrosis[J].Am JPhysiol Lung Cell Mol Physiol,2004,286(1):L182-L188.
[69]Eitner F, Bucher E, van Roeyen C,et al.PDGF-C is a proinflammatory cytokine that mediates renal interstitial fibrosis[J].J Am Soc Nephrol.2008,19(2):281-289.
[70]Tuuminen R,Nykanen Al,Krebs R,et al.PDGF-A,-C,and-D but not PDGF-B increase TGF-betal and chronic rejection in rat cardiac allografts[J].Arterioscler Thromb Vasc Biol.2009,29(5):691-698.
[71]Haynesworth SE, Goshima J, Goldberg VM,et al.Characterization of cells with osteogenic potential from human marrow[J].Bone.1992,13(1):81-88.
[72]陈嘉薇.环孢素A肾毒性机制的研究进展[J].中华器官移植杂志,2003,4：255-256.
[73]Wekerle T, Sykes M. Mixed chimerism and transplantation tolerance.Annu Rev Med,2001,52:353-370.
[74]Doucet C,Ernoul,ZhangY,et al.Platelet lysates Promote mesenehymal stem cell expansion:a safety substitute for animal serum in cell-based therapy applications. J Cell Physiol.2005,205(2):228-236.
[75]吴凤霞,蔡维君,伍校琼,等.不同年龄大鼠骨髓基质干细胞的增殖特性.神经解剖学杂志,2005,21(5)：531-534.
[76]Ringe J, Kaps C, Schmitt B,et al. Porcine mesenchymnal stem cells.Induction of distinct mesenchymal cell lineages[J].Cell Tissue Res,2002,307(3):321-327.
[77]Lodie TA, Blickarz CE, Devarakonda TJ, et al.Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction[J].Tissue Eng.2002,8(5):739-751.
[78]Jiang XX, Zhang Y, Liu B, et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells[J]. Blood,2005,105(10):4120-4126.
[79]Spaggiari GM, Capobianco A, Becchetti S, et al.Mesenchymal stem cell-natural killer cell interactions:Evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NKcell proliferation[J].Blood 2006,107(4):1484-1490.
[80]Oh JY, Kim MK, Shin MS,et al. The anti-inflammatory and anti-angiogenic role of mesenchymal stem cells in corneal wound healing following chemical injury[J].Stem Cells,2008,26(4):1047-1055.
[81]Maitra B, Szekely E, Gjini K,et al.Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation[J].Bone Marrow Transplant.2004,33(6):597-604.
[82]Deans RJ, Moseley AB.Mesenchymal stem cells:biology and potential clinical uses[J].Exp Hematol.2000,28(8):875-884.
[83]Tse WT, Pendleton JD, Beyer WM,et al.Suppression of allogeneic T-cell proliferation by human marrow stromal cells:implications in transplantation[J]. Transplantation.2003,75(3):389-397.
[84]Le Blanc K, Tammik L, Sundberg B,et al. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex[J].Scand J Immunol.2003,57(1):11-20.
[85]Di Nicola M,Carlo-Stella C,Magni M,et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli[J].Blood.2002,99(10):3838-3843.
[86]Djouad F, Plence P, Bony C,et al.Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals[J].Blood. 2003,102(10):3837-3844.
[87]Waaga AM, Gasser M, Laskowski I, et al.Mechanisms of chronic rejection[J]. Curr Opin Immunol,2000,12(5):517-521.
[1]Mazariegos GV, Squires RH, Sindhi RK.Current perspectives on pediatric intestinal transplantation[J].Curr Gastroenterol Rep.2009,11(3):226-233.
[2]Mitchell RN.Graft vascular disease:immune response meets the vessel wall[J].Annu Rev Pathol.2009,4:19-47.
[3]Chronic Rejection of Small Bowel Grafts:Pediatric and Adult Study of Risk Factors and Morphologic Progression[J].Pediatric and Developmental Pathology 2003,6(3):240-250.
[4]Ma H,Wang J, Wang J, et al.Features of chronic allograft rejection on rat small intestine transplantation[J].Pediatr Transplant.2007,11(2):165-172.
[5]Klaus A, Margreiter R, Pernthaler H,et al.Diffuse mesenterial sclerosis:a characteristic feature of chronic small-bowel allograft rejection[J].Virchows Arch. 2003,442(1):48-55.
[6]Giovanelli M, Gupte GL, Sharif K,et al.Chronic rejection after combined liver and small bowel transplantation in a child with chronic intestinal pseudo-obstruction:a case report[J].Transplant Proc.2008,40(5):1763-1767.
[7]Ramos E, Molina M, Sarria J,et al.Chronic rejection with sclerosing peritonitis following pediatric intestinal transplantation[J].Pediatr Transplant. 2007,11(8):937-941.
[8]Gokmen MR, Lombardi G,Lechler RI.The importance of the indirect pathway of allorecognition in clinical transplantation[J].Curr Opin Immunol. 2008,20(5):568-574.
[9]Mariafederica U, Ennia D, Maria E, et al.Cytokines and chronic rejection:a study in kidney transplantation long-term survivors[J].Transplantation, 2004,77(4):548-552.
[10]Morris PJ, Williams GM, Hume DM, et al. Serotyping for homotransplantation. XII. Occurrence of cytotoxic antibodies following kidney transplantation in man[J].Transplantation,1968,6(3):392-399.
[11]Mizutani K, Terasaki P, Rosen A, et al.Serial ten-year follow-up of HLA and MICA antibody production prior to kidney graft failure[J].Am J Transplant,2005, 5(9):2265-2272.
[12]Mizutani K, Shibata L, Ozawa M,et al. Detection of HLA and MICA antibodies before kidney graft failure[J].Clin Transpl.2006:255-264.
[13]Stastny P, Lavingia B, Fixler DE,et al.Antibodies against donor human leukocyte antigens and the outcome of cardiac allografts in adults and children[J]. Transplantation.2007,84(6):738-745.
[14]Kato T, Mizutani K, Terasaki P,et al.Association of emergence of HLA antibody and acute rejection in intestinal transplant recipients:a possible evidence of acute humoral sensitization[J].Transplant Proc.2006,38(6):1735-1737.
[15]Takahashi H, Kato T, Mizutani K,et al.Simultaneous antibody-mediated rejection of multiple allografts in modified multivisceral transplantation[J].Clin Transpl.2006:529-534.
[16]Nakao A, Toyokawa H,Tsung A, et al.Ex vivo application of carbon monoxide in University of Wisconsin solution to prevent intestinal cold ischemia/reperfusion injury[J].Am J Transplant.2006,6(10):2243-2255.
[17]Solez K, Colvin RB, Racusen LC,et al.Banff'05 Meeting Report:differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy ('CAN')[J].Am J Transplant.2007,7(3):518-526.