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目的研究可溶性肿瘤坏死因子受体(sTNFR)基因直接体外灌注转染供者器官的效果,利用小鼠异位心脏移植模型探讨sTNFR基因转染对供心缺血/再灌注损伤的保护机制。方法实验分3组,每组C57/BL6(H-2Kb)供体鼠和BalB/c(H-2Kd)受体鼠各10只,在0~4℃溶液中对3组C57/BL6(H-2Kb)小鼠的供心分别经主动脉缓慢灌注含编码小鼠sTNFR-p55基因的复制缺陷重组腺病毒载体(Adm sTNFR组)、复制缺陷重组腺病毒载体AdHCVsp1LacZ(AdmLacZ组)的PBS和单纯PBS(对照组),灌注1 h后,将供心移植给BalB/c(H-2Kd)受体鼠。检测各组移植受体鼠外周血血清sTNFR的表达水平和移植物浸润细胞(G IC)的数量,并分析Adm-sTNFR基因修饰后G IC的同种反应活性(MLR)。结果以2×1012pfu/L的滴度转染移植心脏后,12 h即可在Adm sTNFR组小鼠的血清中检测到高水平的sTNFR-p55表达,24 h达到分泌高峰,为(59.5±6.5)μg/L;而AdmLacZ组24 h的sTNFR-p55水平为(1.5±0.56)μg/L(P<0.05),对照组更低;术后14天,Adm sTNFR组仍持续表达有效高水平的sTNFR-p55〔(20.1±3.7)μg/L〕。Adm sTNFR组移植心脏的G IC数目为(0.5±0.12)×106/孔,明显低于对照组和AdmLacZ组的(5.0±2.2)×106/孔(P<0.05),其比例为1∶10~1∶20。将不同组的G IC与γ射线(20 Gy)灭活的供体鼠来源的T细胞作72 h MLR,Adm sTNFR组的G IC增殖受到了明显的抑制,AdmLacZ组和对照组的G IC则显示较强的对同种刺激的增殖活性。结论在低温(0~4℃)的条件下可以将编码sTNFR基因的腺病毒成功地转入移植心脏,通过基因表达可长时间维持有效的sTNFR-p55的局部浓度,减轻了受体鼠移植器官内的炎性细胞浸润,并抑制移植器官内浸润细胞的同种反应活性。
Objective To study the effect of sTNFR gene transfection on donor organs in vitro and to explore the protective mechanism of sTNFR gene transfection on donor heart ischemia / reperfusion injury using mouse heterotopic heart transplantation model. Methods The experiment was divided into three groups. Each group of 10 C57 / BL6 (H-2Kb) donor and BalB / c (H-2Kd) -2Kb) mice were slowly perfused with replication-deficient recombinant adenovirus vector (Adm sTNFR group) encoding mouse sTNFR-p55 gene, PBS containing recombinant adenovirus vector AdHCVsp1LacZ (AdmLacZ group) and pure PBS (control group). After 1 h of perfusion, donor hearts were transplanted to BalB / c (H-2Kd) recipient mice. The expression of sTNFR and the number of grafted infiltrating cells (GIC) in the peripheral blood of the transplanted recipients of each group were detected, and the alloreactivity (MLR) of GIC after Adm-sTNFR gene modification was analyzed. Results High transcripts of sTNFR-p55 were detected in sera of Adm sTNFR mice 12 h after transfected with 2 × 1012 pfu / L of titers and reached the peak of secretion at 24 h (59.5 ± 6.5 ) μg / L, while the level of sTNFR-p55 in AdmLacZ group was (1.5 ± 0.56) μg / L at 24 h and was lower in the control group; Adm sTNFR group continued to express high level of sTNFR-p55 [(20.1 ± 3.7) μg / L]. The number of G ICs in the hearts transplanted with Adm sTNFR was (0.5 ± 0.12) × 106 / well, which was significantly lower than that of the control and AdmLacZ groups (5.0 ± 2.2) × 106 / well (P <0.05) ~ 1:20. T lymphocytes derived from different groups of G ICs and γ-rays (20 Gy) inactivated donor mice were used for 72 h MLR. G IC proliferation in Adm sTNFR group was significantly inhibited. G ICs in AdmLacZ group and control group Shows a strong stimulation of proliferation of the same kind of activity. Conclusions The adenovirus encoding sTNFR gene can be successfully transplanted into the heart under low temperature (0 ~ 4 ℃), and the local concentration of sTNFR-p55 can be maintained for a long time by gene expression, and the transplanted organ of recipient mice Inflammatory cell infiltration within the transplanted organ and inhibit alloreactivity of infiltrating cells within the transplanted organ.