摘要
目的观察肝细胞生长因子(hepatocyte growth factor,HGF)修饰的BMSCs对大鼠同种异体肝移植后免疫排斥反应的影响,探讨其诱导免疫耐受的机制。方法 3～4周龄清洁级雄性SD大鼠8只,体重75～85 g,用于分离培养BMSCs;成年清洁级雄性SD大鼠64只,体重200～250 g,作为供体;成年清洁级雄性Wistar大鼠64只,体重230～280 g,作为受体。建立稳定的同种异体肝移植动物模型并随机分为4组(n=16),A、B、C、D组分别立即经门静脉注入1 mL生理盐水、1 mL细胞密度为2×106个/mL的BMSCs、1 mL细胞密度为2×106个/mL的BMSCs/绿色荧光蛋白、1 mL细胞密度为2×106个/mL的BMSCs/hHGF。术后作实验动物生存曲线分析;术后7 d检测实验动物肝功能,并取肝脏组织行病理组织学观察,RT-PCR检测肝组织内hHGF mRNA表达,ELISA法检测血清中hHGF、IL-2、IL-4、IL-10、IFN-γ细胞因子的表达,TUNEL法检测肝组织内细胞凋亡情况,免疫组织化学方法检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)在肝组织内的表达。结果 D组生存时间显著高于A、B、C组(P<0.01);B、C组生存时间明显高于A组(P<0.01),但B、C组间生存时间差异无统计学意义(P>0.05)。RT-PCR示检测D组肝组织内有hHGF mRNA表达,ELISA检测血清中hHGF水平达(6.2±1.0)ng/mL。相比B、C组,D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为轻度;IL-2、IFN-γ水平降低,IL-4、IL-10水平升高,细胞凋亡明显减少,PCNA表达显著升高,除IL-4水平比较差异无统计学意义(P>0.05)外,其余指标比较差异均有统计学意义(P<0.05)。结论通过门静脉输注BMSCs/hHGF至大鼠同种异体肝移植模型能成功诱导免疫耐受。与单纯应用BMSCs相比,BMSCs/hHGF在移植肝局部形成高浓度hHGF免疫抑制微环境,能更显著抑制急性排斥反应,延长移植受体存活时间。BMSCs/hHGF的免疫抑制作用与诱导Th1细胞向Th2细胞偏移、减少肝细胞凋亡、促进肝细胞增殖有关。
Objective To study the effect of recombinant lentiviral vector mediated human hepatocyte growth factor （hHGF） gene-modified bone marrow mesenchymal stem cells （BMSCs） on the immunological rejection after allograft liver transplantation in rats, and to reveal the mechanism of immune tolerance. Methods Eight male Sprague Dawley （SD） rats of clean grade （aged 3 to 4 weeks, weighing 75-85 g） were selected for the isolation and culture of BMSCs; 64 adult male SD rats of clean grade （weighing 200-250 g） were used as donors; and 64 adult male Wistar rats of clean grade （weighing 230-280 g） were used as receptors. After establishing a stable model of rat allogeneic liver transplantation, 1 mL saline, 2×l0^6/mL of BMSCs 1 mL, 2 × 10^6/mL of BMSCs/green fluorescent protein 1 mL, and 2 × 10^6/mL of BMSCs/hHGF 1 mL were injected via the portal vein in groups A, B, C, and D respectively. Then the survival time of the rats was observed. The hepatic function was determined and the histological observation of the liver was performed. The hHGF mRNA expression was detected by RT-PCR, the level of cytokine including hHGF, interleukin 2 （IL-2）, IL-4, IL-10, and interferon y （IFN-T） by ELISA assay, the level of apoptosis by TUNEL method, and the expression level of proliferating cell nuclear antigen （PCNA） by immunohistochemical method. Results The survival time of group D was significantly higher than that of groups A, B, and C （P 〈 0.01）; the survival time of groups B and C was significantly higher than that of group A （P 〈 0.01）, but there was no significant difference between group B and group C （P 〉 0.05）. RT-PCR demonstrated the transcription of hHGF mRNA in the grafts of group D; the serum cytokine hHGF reached to （6.2 ± 1.0） ng/mL. Compared with groups B and C, group D exhibited significant inhibitory effect, significantly improved liver function, and showed mild acute rejection. In addition, the levels of cytokine IL-2 and IFN-7 decreased; the levels of cytokine IL-4 and IL-10 increased; the level of apoptosis reduced; and the expression level of PCNA increased. Except for the expression of IL-4 （P 〉 0.05）, there were significant differences in the other indexes between group D and groups B, C （P 〈 0.05）. Conclusion BMSCs/hHGF implanting to rat liver allograft via portal vein can induce immune tolerance. Compared with injection of BMSCs alone, BMSCs/hHGF treatment can alleviate acute rejection and prolong the survival time significantly. The immunosuppressive effect of BMSCs/hHGF is correlated with Th2 shifts up of Thl/Th2 shift, reduced apoptosis, promoted liver regeneration.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2011年第7期871-876,共6页
Chinese Journal of Reparative and Reconstructive Surgery
基金
福建省教育厅科研基金资助项目(JA09110)
