转sTNFR基因处理对供心缺血/再灌注损伤的保护作用

Protective effect of sTNFR gene transduction on ischemia-reperfusion injury of donor′s heart

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2×10^12pfu/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）×10^6/孔,明显低于对照组和AdmLacZ组的（5.0±2.2）×10^6/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 investigate the transfection effects of sTNFR gene directly perfused to donor hearts in vitro, and to explore the protective mechanism of sTNFR gene transduction to donor hearts against ischemia - reperfusion injury using the model of heterotopic heart transplantation in mice. Methods The experiment was carried out in three groups, with each group containing 10 donor mice and 10 recipient mice. The donor hearts were infused with iced saline containing replication - defective recombinant adenovirus encoding murine sTNFR - p55 （for AdmLacZ sTNFR gene group）, replication-defective adenovirus vector AdHCVsplLaeZ （for AdmLacZ vector group） and PBS （for control group） respectively, for 1 h, before the donor hearts were transplanted to the recipients using cervical heterotopic heart transplantation method. The sTNFR - p55 level in serum and the graft infiltrating cell （ GIC ） count in each recipient group were examined, with the allogeneic MLR in GIC analyzed as well. Results High level of sTNFR - p55 was detected in the serum of the AdmsTNFR gene group 12 hours after the transfection at MOI 2 × 10^12pfu/L, which reached its peak （59.5±6.5） μg/L 24 hours after the transfection. In contrast, the sTNFR -p55 level in AdmLacZ vector group was staying at a much lower level （ 1.5 ±0.56） μg/L （P 〈 0.05） 24 hours after the transfection. The sTNFR expression could still be kept at high level in the serum 14 days after transplantation in AdmsTNFR gene group. The GIC count in AdmsTNFR gene groups was （0.5 ±0.12） × 10^6/well, much lower than that in AdmLacZ vector group （5.0±2.2） ×10^6/well （P〈0.05）. When GIC were cocultured with the γ- irradiation- inactivated allogeneic lymphocytes from donor mice for 72 h MLR, the proliferation of GIC was found obviously inhibited in the AdmsTNFR gene group, as compared with that in the other two groups. Conclusion These data indicate that adenovirus - mediated sTNFR - p55 gene can be transferred successfully to cardiac allografts by direct peffusion under hypothermic conditions （0 -4℃ ） in vitro, which can bring about sustained high local concentration of sTNFR - p55, decrease of GIC count in the graft heart and inhibition of the proliferation of GIC.