血红素加氧酶-1基因修饰的骨髓间充质干细胞减轻大鼠小肠移植急性排斥反应的实验研究

发布时间：2018-10-19 14:54

【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。
[Abstract]:Objective: To study the effect of heme oxygenase-1 (HO-1) gene modified bone marrow mesenchymal stem cells (BMMSCs) on acute rejection in rat small intestine transplantation, on the basis of establishing a stable and repeatable model of acute rejection of ectopic small intestine transplantation in rats. To improve the effect of small intestine transplantation on the prognosis of rats. Methods: The BMMSCs in the femur and tibia of Lewis rats were extracted by the method of gradient centrifugation combined with adherent culture. After three generations, BMMMSCs (Ad/ BMMSCs) and HO-1 gene modified BMMSCs (Ad/ HO-1/ BMMSCs) were transfected with adenovirus containing adenovirus (Ad/ BMMSCs) and HO-1 gene. Using BN rats as donor, Lewis rats were used as recipients to establish a model of rejection of small intestine transplantation in ectopic rats. Both the control group and the recipient were Lewis rats. The model of small intestine transplantation rejection was analyzed by the survival and histopathological changes of rats. After the successful modeling, the untreated BMMSCs, Ad/ BMMSCs and Ad/ HO-1/ BMMSCs were treated as basic control. Survival status and survival rate of each group of rats after operation were observed. Histopathology and apoptosis of transplanted intestinal tissues were collected from 1, 5, 7 and 10 days post-operatively, and the expression of HO-1 protein and m RNA was measured by immunohistochemistry, Western blot and PCR. At the same time, the serum samples of each group were kept to detect the levels of various cytokines in the serum. In addition, spleen lymphocytes were extracted to detect the activity of NK cells and the levels of regulatory T cells (Tregs). Results: After culture and identification, BMMSCs extracted from the femur and tibia of rats were easy to be cultured, the quantity was much, the purity was high, the transfection of adenovirus did not change its biological cytologic characteristics. In the model of acute rejection of BN-Lewis's ectopic small intestine transplantation, there was no significant acute rejection within 3 days after operation, and mild to moderate acute rejection occurred in 5 days after operation. All transplant rats showed severe acute rejection. With the aggravation of acute rejection, the transplanted small intestine shows more and more serious inflammatory cell infiltration, glandular epithelium injury, adenocarcinoma of salivary gland, formation of tissue structure, destruction of tissue structure, and mucosal damage. Almost all of the transplanted rats died within 14 days, and the survival time was 10d; BMMSCs treatment significantly improved the survival condition of transplanted rats and significantly improved the survival rate, the histopathological changes were obviously improved, and the rejection index was improved. Apoptosis and NK cell activity decreased significantly. The levels of IL-10, IL-6, IL-17, IL-23, TNF-and IFN-hIL in serum of IL-10, IL-6, IL-17, IL-23, TNF-, and IFN-hIL were significantly decreased. In addition, BMMSCs also induced Treggs formation. However, the above effect of BMMSCs was strong in 7 days and decreased in 10 days. The HO-1 gene modified BMMSCs significantly improved the expression of HO-1 protein in BMMSCs, increased the survival rate of BMMSCs in vivo, and further enhanced the role of BMMSCs in protecting transplanted small intestine. Compared with untreated BMMSCs, the treatment of Ad/ HO-1/ BMMSCs further improved the survival rate, histopathological changes and apoptosis of transplanted rats, and the activity of NK cells decreased significantly. The levels of IL-10, IL-17, IL-23, IL-6, IL-17, IL-23, IL-6, IL-17, IL-23, IL-6, IL-17, IL-23, IL-6, IL-17, IL-23, IL-6, IL-17, IL-23, IL-6, IL-17, IL-23, IL-6, IL-17, IL-23, IL-6, IL-17, IL-23, IL-6, IL-17, IL-23, IL-6, IL-17, IL-23, IL-6, IL-17, IL-23, IL-6, IL-17, IL-23, IL-6, IL-17, IL-23, IL-6, IL-17, IL-23 TNF-IIIa and IFN-IIIa were significantly reduced. Conclusion: The number of BMMSCs extracted from the femur and tibia of rats is high, the purity is high, it is the ideal BBM source of the experiment; the gene modification of BMMSCs can be realized by adenovirus transfection; BMMSCs can reduce the acute rejection response of rat heterotopic small intestine transplantation, but is limited by the low survival rate of BMMSCs in vivo; HO-1 gene modified BMMSCs can improve the survival rate of BMMSCs in vivo, and can further enhance BMMSCs to alleviate acute rejection in rat small intestine transplantation and improve the prognosis of small intestine transplantation rats.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R656.7

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