【作者】 杨洋；

【导师】 沈中阳；

【作者基本信息】 天津医科大学 ， 外科学 普外， 2016， 博士

【摘要】 目的:在建立稳定的、可重复性强的大鼠异位小肠移植急性排斥反应模型的基础上,探讨血红素加氧酶-1(heme oxygenase,HO-1)基因修饰的骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMMSCs)在减轻大鼠小肠移植急性排斥反应,改善小肠移植大鼠预后中的作用。方法:本研究首先采用梯度离心联合贴壁培养的方法提取Lewis大鼠股骨及胫骨中的BMMSCs,培养至3代以后,通过转染空腺病毒(adenovirus,Ad)以及携带有HO-1基因的腺病毒制备腺病毒空载的BMMSCs(Ad/BMMSCs)和HO-1基因修饰的BMMSCs(Ad/HO-1/BMMSCs);以BN大鼠为供体,以Lewis大鼠为受体,建立异位大鼠小肠移植排斥反应模型。对照组供受体均为Lewis大鼠。通过大鼠存活情况以及组织病理学变化分析小肠移植排斥反应模型建模是否成功;在成功建模的基础上,术后经阴茎背静脉分别给予未处理的BMMSCs、Ad/BMMSCs以及Ad/HO-1/BMMSCs各1×107个,等量的生理盐水注射作为基础对照。观察术后各组大鼠的生存状况并分析生存率。分别于术后1、5、7、10d时搜集移植肠组织检测组织病理学以及细胞凋亡情况,并通过免疫组织化学、Western Blot、PCR测定移植肠组织中HO-1蛋白以及m RNA的表达。同时,保留各组血清样本检测血清中各种细胞因子的水平。此外,提取脾脏淋巴细胞检测其中NK细胞(natural killer cell,NK cell)的活性以及调节性T细胞(regulatory T cells,Tregs)的水平。结果:经培养鉴定,由大鼠股骨及胫骨提取的BMMSCs易培养、数量多、纯度高,腺病毒转染不会改变其生物细胞学特性;在反复预实验练习后熟练掌握小肠移植技术。在BN至Lewis的异位小肠移植急性排斥反应模型中,术后3 d内无明显的急性排斥反应发生,术后5 d时会发生轻至中度的急性排斥反应,术后7 d时会发生中至重度的急性排斥反应,而至术后第10 d,所有的移植大鼠均表现为重度的急性排斥反应。随着急性排斥反应的加重,移植小肠表现为越来越严重的炎性细胞浸润、腺窝上皮损伤、腺窝凋亡小体形成、组织结构破坏以及粘膜溃疡等。几乎所有的移植大鼠均于14 d天之内死亡,中位生存时间为10 d;BMMSCs治疗显著改善了移植大鼠地生存状况并显著提高了其术后生存率,组织病理学改变明显改善,排斥指数、细胞凋亡及NK细胞活性显著下降。血清IL-10、TGF-β等与抗炎或Tregs分化相关的细胞因子表达水平显著升高,而与促炎或Th17分化相关的细胞因子IL-2、IL-6、IL-17、IL-23、TNF-α及IFN-γ的表达水平则明显降低。此外,BMMSCs还诱导了Tregs的生成。但BMMSCs的以上效应一般7 d内较强,在10 d时便呈下降趋势;HO-1基因修饰BMMSCs,显著提高了BMMSCs中HO-1蛋白的表达,提高了BMMSCs在体内的存活率,并进一步增强了BMMSCs保护移植小肠的作用。与未处理的BMMSCs治疗相比,Ad/HO-1/BMMSCs治疗进一步提高了移植大鼠术后生存率,组织病理学变化及细胞凋亡进一步好转,NK细胞活性显著下降而Tregs水平显著升高,细胞因子IL-10、TGF-β显著上升而IL-2、IL-6、IL-17、IL-23、TNF-α及IFN-γ则显著降低。结论:大鼠股骨、胫骨提取的BMMSCs数量多、纯度高,是实验理想的BMMSCs来源;以腺病毒转染来实现BMMSCs的基因修饰是可行的;BMMSCs治疗可减轻大鼠异位小肠移植的急性排斥反应,但受限于BMMSCs在体内较低的存活率;HO-1基因修饰BMMSCs可提高BMMSCs在体内的存活率,并可进一步增强BMMSCs减轻大鼠小肠移植急性排斥反应,改善小肠移植大鼠预后的作用。更多还原

【Abstract】 Objective: To establish a stable and repeatable acute rejection model of rat heterotopic small bowel transplantation(SBTx) and determine whether heme oxygenase-1(HO-1) gene modified bone marrow mesenchymal stem cell(BMMSCs)could reduce acute rejection and improve outcomes for SBTx in rat.Methods:Firstly, BMMSCs were isolated from the femur and tibia of Lewis rats and cultured in a cell culture incubator by gradient centrifuge and adhesive culture methods. The third-passage BMMSCs were transfected with empty adenovirus vector or adenovirus carrying HO-1 gene to form Ad/BMMSCs or Ad/HO-1/BMMSCs,respectively. Then, acute rejection model of heterotopic SBTx was performed allogeneically from BN rats to Lewis rats. Lewis to Lewis SBTx served as control.Living condition and histopathology change of the recipients were assessed to determine whether the acute rejection model was estabished successfully. On the basic of successful modeling, recipient rats in the Ad/HO-1/BMMSCs treatment group were treated with 1 × 107 Ad/HO-1/BMMSCs through the superficial dorsal veins of the penis immediately after surgery. Control rats were treated with 1 × 107Ad/BMMSCs or 1 × 107 BMMSCs. Isopycnic saline infusions served as the basic control. The animals were sacrificed after 1, 5, 7 or 10 days for specimen collection.Small bowel histology and apoptosis were detected, HO-1 protein and m RNA were analysed by immunohistochemical staining, Western Blot and PCR. In addition,serum samples were collected to measure cytokine concentrations in serum,lymphocytes were isolated from the recipient spleens to assesse NK cell activity and numbers of T regulatory(Treg) cells at each time point.Results: Our result showed that BMMSCs isolated from rat femur and tibia have the characteristics of easy cultivation, large quantity and high purity. BMMSCs transfected with recombinant adenovirus did not changed in terms of biological property. We finally estabilished the acute rejection model of SBTx by repetitious practice, and we found there was no obvious acute rejection within 3 days post operation. While, mild or moderate acute rejection was observed on day 5 and moderate or severe acute rejection was observed on day 7. All recipient suffered severe acute rejection on day 10. The intestine graft presented heavier cellular infiltration into the muscle layer and mucosa, more apoptotic cells and severer architectural distortion and mucosal ulceration, with development of the acute rejection. All recipient died within 2 weeks and the median survival time was 10 days.BMMSCs treatment significantly improved animal living condition and changes of histopathology. Acute rejection grading scores, apoptosis and NK cell activity significantly reduced. Serum IL-10 and TGF-β concentrations, which related to anti-inflammatory or Tregs differentiation, significantly increased. While, Serum IL-2,IL-6, IL-17, IL-23, TNF-α and IFN-γ concentrations, which related to proinflammatory or Th17 differentiation, significantly decreased. Besides, BMMSCs induced the production of Tregs. However, these effects usually maintained within 7days and decreased on day 10. HO-1 gene modification significantly improved the survival rate of BMMSCs in vivo and inhanced the protective effect in SBTx. The recipient survival rate, changes of histopathology and ultrastructure and apoptosis further improved, NK cell activity significantly reduced while Tregs level significantly increased, Serum IL-10 and TGF-β concentrations significantly increased while serum IL-2, IL-6, IL-17, IL-23, TNF-α and IFN-γ concentrations significantly decreased, compare to that in the BMMSCs treated group.Conclusion: Rat femur and tibia are the ideal source of BMMSCs as the characteristics of easy cultivation, large quantity and high purity. It’s feasible to modify BMMSCs by adenovirus transfection. BMMSCs treatment reduces acute rejection for heterotopic SBTx in rat, but the effect may be limited by the low survival rate in vivo. HO-1 gene modification inhances the effect of BMMSCs on reducing acute rejection and improving the outcomes of SBTx in rat.更多还原