【作者】 朱金海；

【导师】 陈良万； 陈燕凌；

【作者基本信息】 福建医科大学 ， 外科学， 2009， 博士

【摘要】 骨髓间充质干细胞(bone marrow mesenchymal stem cells,MSC)是一种高度自我更新能力和多向分化潜能的成体干细胞。研究表明,MSC在体内外具有免疫抑制的能力,能够通过抑制T淋巴细胞的增殖,减轻同种异体移植物的排斥。MSC免疫抑制的功能与其分泌可溶性生长因子有关,包括肝细胞生长因子(HGF)等。而研究证实HGF在器官移植中具有免疫抑制、促增殖、抗凋亡和细胞保护的功能。MSC易于扩增和纯化,易携带外源性基因。利用MSC联合HGF基因转染,可望增强免疫抑制的作用。在本研究中将HGF基因通过慢病毒载体介导稳定转染MSC,对MSC进行修饰,利用MSC归巢效应定位于移植肝,长期分泌HGF,形成一个免疫抑制的微环境,增强MSC的免疫抑制功能,发挥HGF调节免疫和组织修复的功能,取得协同效应,减轻排斥反应,延长移植物存活时间,为肝移植急性排斥提供新的治疗策略。第一部分改良式大鼠同种异体原位肝移植模型的建立目的探讨改良“二袖套法”原位大鼠肝移植模型的建立。方法SD大鼠为供、受体的同基因肝移植70只(SD-SD组),SD、Wistar分别为供、受体的同种异体肝移植60只(SD-Wistar组)。采用改良的“二袖套法”进行大鼠原位肝移植,充分暴露第一肝门,不翻动肝脏先行门静脉灌注。在体一步法离断肝上下腔静脉,不带膈肌环。吻合肝上下腔静脉采用单线连续缝合;双线牵引法安装门静脉袖套。术后充分补液维持大鼠血液动力学稳定。结果供体手术时间(38.2±2.5)min,受体手术时间(45.6±3.5)min,无肝期(15.1±2.2)min,手术成功率93%,1周存活率92%,与传统二袖套法比较差异有统计学意义(P<0.05)。SD-SD组手术成功64只,受体长期存活,术后肝功能很快恢复正常,肝组织病理无明显变化。SD-Wistar组手术成功57只,受体存活时间8-20d,平均10.5d。大鼠于术后3-5d出现急性排斥反应,未经处理后均死亡。结论改良式大鼠原位肝移植操作简便、成功率高、易于复制,是肝移植实验稳定可靠的动物模型。SD-Wistar大鼠组合是一组高排斥的肝移植模型,可发生典型的急性免疫排斥反应。第二部分骨髓间充质干细胞的分离培养鉴定及在移植肝内定居能力的观察目的体外分离培养扩增大鼠骨髓间充质干细胞(MSC),并进行鉴定,观察其在肝移植受体内的定居能力。方法利用直接贴壁培养法培养大鼠MSC,并传代扩增,相差显微镜下观察生长特性。绘制细胞生长曲线。流式细胞仪检测细胞表面标志。定向诱导MSC向成骨和成脂肪分化,鉴定其多向分化潜能。DAPI荧光标记MSC,由门静脉注入同种异体肝移植后受体内,取肝组织冰冻切片,在荧光显微镜下观察其在移植肝内的定居情况。结果直接贴壁法成功分离培养MSC,并在不断的传代培养中得以纯化扩增。细胞生长旺盛,形态呈梭状,均匀分布的漩涡状生长。传代周期5d左右,可在体外传代20代以上,具有强大的增殖能力。流式细胞仪检测第3代的MSC表面标志,CD44、CD90分别为94.81%,99.53%,呈阳性高表达。CD34、CD45、CD11b分别为0.04%、1.94%、1.42%,呈阴性表达,符合MSC表型。在成骨和成脂的诱导剂条件下,MSC可成功分化为成骨和脂肪细胞,具有多向分化的能力。DAPI标记MSC的阳性率达100%。倒置荧光显微镜下观察,可见蓝色荧光标志的MSC定位于移植肝内。结论直接贴壁法分离培养MSC简单易行,获得MSC纯度高,生物学特性稳定。MSC可以在移植肝内存活并定居。第三部分肝细胞生长因子慢病毒的构建及对骨髓间充质干细胞的修饰目的构建携带人肝细胞生长因子(hHGF)基因的慢病毒,感染MSC,观察hHGF基因修饰的MSC细胞(MSC/hHGF)的生物学行为,体外检测MSC/hHGF的免疫抑制作用。方法从含hHGF基因的质粒中,PCR法获取hHGF全长基因,与慢病毒载体分别进行酶切,产物定向连接,转化后行PCR鉴定和测序,构建hHGF慢病毒表达质粒。脂质体介导慢病毒载体三质粒共转染293T细胞。收集病毒超滤离心浓缩,实时定量PCR测定滴度。将hHGF慢病毒感染MSC,荧光显微镜观察荧光表达,流式细胞仪检测慢病毒感染的效率,ELISA法检测细胞培养上清hHGF分泌水平,细胞RT-PCR检测hHGF的mRNA表达。体外混合淋巴细胞反应(MLR)检测MSC和MSC/hHGF两组细胞的免疫抑制作用。结果成功构建携带hHGF基因的慢病毒,滴度达到1x108 TU/ml。hHGF慢病毒高效率感染MSC,最佳MOI值=10。流式细胞仪检测感染效率达98%。荧光表达强,在感染后28d MSC仍稳定表达强烈的绿色荧光。培养上清可检测到hHGF因子,在第5d左右达到高峰,可达40.5ng/ml,这种高水平分泌可持续到感染后第28d。RT-PCR检测出hHGF mRNA表达。MSC组显著抑制淋巴细胞的增殖,抑制率达46.34%。而MSC/hHGF组抑制率可达64.42%,具有更强的抑制效应。结论通过慢病毒载体将hHGF基因稳定转染至MSC细胞,基因整合到MSC基因组中,并正确转录和表达。hHGF基因修饰的MSC获得了更强的免疫抑制作用,说明hHGF与MSC在体外具有免疫抑制协同作用。第四部分慢病毒介导hHGF基因修饰MSC对大鼠同种异体肝移植免疫排斥的影响目的观察慢病毒介导hHGF基因修饰的MSC(MSC/hHGF)在大鼠体内对同种异体肝移植的免疫排斥反应的影响,探讨其诱导免疫耐受的机制。方法建立稳定的同种异体肝移植模型,受体移植成功后,MSC/hHGF组立即经门静脉注入2x106 /ml的MSC/hHGF 1ml。MSC/GFP组注入2x106/ml的MSC/GFP 1ml。MSC组注入2x106/ml的MSC 1ml。对照组注入1ml生理盐水。观察生存时间,移植后7d时检测肝功能,取肝脏组织行病理学检查, ELISA方法检测受体血清中hHGF、IL-2、IL-4、IL-10、IFN-γ等细胞因子的水平,Tunel法检测肝组织内细胞凋亡情况,免疫组化检测TNF-α、NF-κB、Bcl-2、PCNA在肝组织内的表达,RT-PCR检测肝组织内hHGF、TNF-α、NF-κB和Bcl-2 mRNA的表达。结果RT-PCR证实MSC/hHGF组肝组织内有hHGF基因转录,并检测到绿色荧光表达,ELISA检测血清中hHGF水平可达6.2ng/ml。MSC/hHGF组生存时间>100d,MSC/GFP组23.1±3.9d, MSC组23.4±3.1d,均显著长于对照组(10.5±4.2d)。MSC/hHGF组和MSC组均能减轻急性排斥反应,改善受体状况。相比MSC组,MSC/hHGF组疗效更显著,肝功能明显改善,病理学无急性排斥反应或仅轻度,IL-2、IFN-γ水平降低,IL-4、IL-10水平升高,细胞凋亡明显减少,Bcl-2表达和PCNA表达显著升高。TNF-α和NF-κB表达明显减少。结论通过门静脉输注MSC/hHGF到大鼠同种异体移植肝内是有效可行的,成功诱导出免疫耐受。与单纯应用MSC比较,MSC/hHGF在移植肝局部形成分泌高浓度hHGF免疫抑制微环境,能更显著抑制急性排斥反应,延长移植受体的存活时间。hHGF与MSC的免疫抑制协同作用与诱导Th1细胞向Th2细胞偏移、减少致炎因子TNF-α、NF-κB的表达、减少肝细胞凋亡、促进肝细胞增生有关。更多还原

【Abstract】 Bone marrow mesenchymal stem cells(MSC) are adult stem cell of mesodermal origin with abilities of self-renewal and multiple lineage differentiations including osteoblasts,adipocytes, chondrocytes,myoblasts,and early progenitors of neural cells. Researches show that MSC have the immunosuppressive abilities,which inhibit lymphocyte proliferation ,and alleviate allograft rejection. Data have indicated soluble growth factors are relevant to the immunosuppression of MSC, including hepatocyte growth factor(HGF) especially, Which has the functions of promoting cell proliferation, reducing apoptosis,cell protection and immunosuppression. MSC can be purified and cultured easily in vitro,and can take ectogenesis gene easily. In order to study the immune synergistic effect of MSC and HGF,we construct lentiviral vector carrying the human HGF,and then infect the MSC to get hHGF gene-modified MSC(MSC/hHGF). The biological properties and immunoregulatory function in vitro and in vivo of MSC/hHGF were investigated. MSC/hHGF could exhibit the immunosuppression synergetic effect of hHGF and MSC, reduce the acute rejection and prolong the survival time. It can supply a novel strategy and method to induce immune tolerance on liver allograft.PART 1 Modified technique of orthotopic liver transplantation in rats Objective To investigate the surgical technique of establishing a reliable rat model of orthotopic liver transplantation. Methods Syngeneic group SD-SD and allogeneic group SD-Wistar rats liver transplantation were performed in 70 and 60 cases, respectively. 130 cases of orthotopic liver transplantations in rats were performed using modified Kamada’s two-cuff technique.Under the sufficient exposure of the porta hepatis,the liver was perfused through the portal vein with cold perfusion without touching the liver. The anastomosis of the suprahepatic vena cave was sutured end- to-end with 8/0 prolene line. Guided by double line, the continuity of portal vein was established by means of cuff method easily. The fluid was supplemented sufficiently after operation to maintain the stabilization of hemodynamics. Results Orthotopic liver transplantations were performed in 130 rats.The time for donor operation was 38.2±2.5 min,the receptor operative and anhepatic time were 45.6±3.5 min and 15.1±2.2 min respectively.The successful operative rate was 93%.The survival rate after one week was 92%. All the data is better than that of traditional method.There were 64 survivals in SD-SD group and 57 in SD-Wistar group after liver transplantation, and the survival time were long-term and 8～20 days(mean 10.5 days) respectively.The liver function recovered well in SD-SD group ,while in SD-Wistar group we found the liver functional failure and acute rejection in pathology in 3-5 days after liver transplantation which lead to death all without any therapy. Conclusions The modified method is proved to be ideal for its advantages of simple operation,short anhepatic phase and high operative successful rate. The SD to Wistar rat combination is a rejection model and can be used to study immune tolerance in liver transplantation.PART 2 Isolation,culture and identification of the bone marrow mesenchymal stem cells(MSC) in vitro and the distribution of MSC in the rat transplantation liver. Objective To isolate,culture and identify the rat bone marrow mesenchymal stem cells(MSC) .To study the distribution of MSC after injection into portal vein in rats of orthotopic liver transplantation. Methods MSC were collected by flushing the femurs and tibias from 3-4-week-old male SD rat under sterile condition. MSC were separated with direct anchoring method,and then they were amplified and cultured.The characters of the cell,such as morphology,cell growth curve,phenotype were demonstrated.MSC were identified using flow cytometry. The abilities to differentiate along adipocytic and osteoblastic were investigated. MSC were labeled by DAPI and were injected into portal vein in rats after orthotopic liver transplantation. The distribution of MSC was observed by fluorescence after frozen section. Results By direct anchoring method, MSC were gained after purification and proliferation,and passaged every five days.The cell population consisted of spindle-shaped cells with significant renewal capacity,stabilities of the biology as they were cultured until 20 passages. Flow-cytometric analysis showed the 3th passage MSC were positive against CD44 and CD90,with a positive rate of 94.81% and 99.53%,while negative against CD34、CD45 and CD11b with a negative rate of 0.04%、1.94% and 1.42%.MSC can be differentiated into adipocyte and osteoblast cell. The positive rate DAPI reached 100%. MSC labeled with blue fluorescence can be founed in livers of rats. Conclusions Direct anchoring method is simple and feasible when used to separate and culture MSC,so it deserves popularizing.Purified MSC with steady biological characters were gained.MSC can successfully reside in the liver of rats.PART 3 Construction of lentiviral vector recombined by hHGF gene and construction of hHGF gene-modified MSCObjective Construct lentiviral vector carrying the human Hepatocyte growth factor gene,and then infect the MSC to get hHGF gene-modified MSC(MSC/hHGF). The biological properties and immunoregulatory function in vitro of MSC/hHGF were investigated. Methods The hHGF gene was obtained with PCR from pcDNA-hHGF plasmid. The recombination lentiviral vector plasmid hHGF was constructed with digestion and gene recombinant,then identified with PCR and sequencing. The three plasmids system of lentiviral vector were co-transfected to 293T cells to produce hHGF gene. The supernatant was collected and concentrated by ultracentrifugation and the titer of lentivirus was measured by qRT-PCR. The MSC were infected by the constructed lentivirus and were identified with flurescent microscope, flow-cytometric analysis ,ELISA analysis and RT-PCR. The immunosuppression functions of MSC and MSC/hHGF were observed by mixed lymphocyte reaction(MLR). Results Lentiviral vector carrying hHGF gene had been constructed successfully. The titer of lentivirus was 1×108TU/ml. The infection efficiency of MSC by hHGF lentiviral was high and reached up to 98% tested by FCM when the best MOI was 10. A great mount of green fluorescence was observed with the flurescent microscope until 28 day after infection. Peak concentration of hHGF secreted by MSC/hHGF reached to 40.5ng/ml on 5 day. The concentration can maintain in a high level until 28 day after infection. MSC/hHGF could express hHGF mRNA. The MLR test showed that MSC/hHGF could exhibit more inhibitory effect(64.42%) on lymphocyte proliferation than MSC(46.34%) in vitro. Conclusions By Lentiviral vector, hHGF gene was intergrated into MSC genome ,and transcribed and expressed correctly. MSC/hHGF could exhibit more inhibitory effect.MSC and hHGF have immunosuppression synergetic effect in virto. PART 4 The immunosuppression of lentiviral vector mediated hHGF gene-modified MSC on rat liver allograftsObjective TO study the immunosuppression effect of recombinant lentiviral vector mediated hHGF gene-modified MSC on rat liver allografts rejection in vivo, and to reveal the mechanism of immune tolerance. Methods In the rat liver allografts model,after harvest, 1ml MSC/hHGF containing 2x106 cells were implanted to acceptor via portal vein immediately in the MSC/hHGF group. As controls,1ml MSC/GFP and 1ml MSC containing 2x106 cells were implanted in MSC/GFP group and MSC group respectively by the same method ,and 1ml physiological saline in control group. Then we observed the survival time,examined the liver function and pathology, measured the level of cytokine including hHGF,IL-2,IL-4,IL-10 and IFN-γby ELISA analysis,examined the level of apoptosis of gragts by Tunel method and the expression level of TNF-α,NF-κB,Bcl-2 and PCNA by immunohistology and RT-PCR. Results RT-PCR demonstrated the transcription of hHGF gene in the grafts. Green fluorescence could been observed with the flurescent microscope.The serum cytokine hHGF reached to 6.2ng/ml. The survival time was repectively >100 days, 23.1±3.9 days,23.4±3.1 days in MSC/hHGF group,MSC/GFP group and MSC group,which was significantly higher than control group(10.5±4.2 days). Both of group MSC/hHGF group and MSC group could reduce the histologic rejection Banff scores and improve liver function. Compared with MSC group, MSC/hHGF group exhibit more inhibitory effect, liver function significantly improved,level of acute rejection alleviated,level of cytokine IL-2 and IFN-γdecreased, level of cytokine IL-4 and IL-10 increased,level of apoptosis reduced,the expression level of Bcl-2 and PCNA increased,but the expression of TNF-αand NF-κB descended. Conclusions These data in vivo demonstrate planting MSC/hHGF via portal to rat liver allograft is efficient and effectively and can induce immune tolerance. Compared with injection of MSC alone, MSC/hHGF treatment could alleviate acute rejection and prolong the survival time more significantly.MSC/hHGF could exhibit the immunosuppression synergetic effect of hHGF and MSC ,which made the Th1 cell toward Th2 cell, suppressed IL-2 and IFN-γ,and hence enhanced the expression of Th2 cytokines such as IL-4 and IL-10,suppressed production of proinflammatory factor such TNF-αand NF-κB,reduced the level of apoptosis,promoted liver regeneration. It can supply a novel strategy and method to induce immune tolerance on liver allograft.更多还原