转染人热休克蛋白70对同种异体移植心脏移植物血管病的影响

Effect of transforming heat shock protein70 gene transfer on angionosis of cardiac allogeneil graft

目的探讨经冠状动脉内灌注重组腺病毒载体介导HSP70（Ad．HSP70）转基因治疗对同种异体心脏移植物血管病的影响。方法以套管技术建立颈部异位异种心脏移植模型。基因转移组在体外经冠状动脉缓慢灌注含3．8×10^10VP／mlAd—HSP70的4℃HTK液，持续45min；空白载体组灌注含3．8×10^10VP／ml不携带基因腺病毒空白载体（Ad）的HTK液（B组）；对照组仅灌注无病毒的HTK液（A组）持续同样时间。术后1个月收集移植物组织做检测。组织内目的基因表达产物通过免疫分析、RT—PCR进行半定量分析，免疫组化染色进行定性和定位分析。结果RT—PCR证实，移植物内外源性HSP70基因转录，免疫组化检测证实Ad—HSPT0转染的移植物（C组）HsP70成功转到血管周围的心肌细胞和冠状动脉微血管，HSP70表达水平明显高于其他两组（P〈0．01）。HSP70过量表达减少NF—KB的活化及VCAM-1的表达，HSP70过量表达的移植物内炎症细胞浸润数明显减少，巨噬细胞和NK细胞浸润减少，移植物血管内皮损伤轻。结论经冠状动脉灌注重组腺病毒载体介导保护性自稳蛋白一人热休克蛋白70基因转移到心脏移植物是相当有效可行的，并可减轻移植物冠脉内皮损伤，预防移植物血管病。经冠状动脉灌注重组腺病毒载体介导基因转移在移植物内局部表达有潜力的治疗制剂，此方法具有广阔的临床应用前景。

Objective To study the effect of recombinant adenovirus vector-mediated HSP70 （Ad. HSP70） gene transfer on cardiac allogeneil graft. Methods In cervical heterotopic cardiac transplantation modelsby cuff technique, after harvest, wistar rat donor hearts＇ coronary arteries were perfused ex vivo with HTK solution containing 3.8×10^10 VP/ml of donor heart of Ad.HSP70 at 4 ℃ for 45 minute （group C）, then implanted in the necks of immunosuppressed rat recipients. In control group, the hearts were perfused with HTK Solution containing 3.8×10^10 VP/ml of donor heart adenoviral blank-vector （Ad） not delivered that gene （group B ） or with virus-free HTK solution （group A） for the same period by the same method. The allografts were collected after one month. Gene product expression in tissue was quantified by immunoassay, reverse transcription-polymerase chain reations （RT-PCR） and visualized and localized by i staining. Results RT-PCR demonstrated the transcription of exogenous HSP70 gene in the allografts. Immunohistochemical examination determination of Ad. HSP70-infected grafts （group 3） confirmed successful HSP70 gene transfer expression in perivascular myocardial cell and coronary capillaries. Expression level of foreign gene significantly higher in Ad.HSP70-infected grafts compared with other two （P〈0.05）. The number of inflammatory cell infiltration in cardiac allografts of Ad. HSP70ted group was less than that of other groups （P〈0.01）. Over expression of exogenous HSP70 gene the expression of VCAM-1 and NF-KB attenuated macrophage and nature killer cell infiltration in the ts and protected allograft endothelial cell. Conclusion Intracoronary gene transfer of recombinant adenovirus vector encoding HSP70 to cardiac allogeneil graft is efficient and effective and protects allograft endothelial cell. Recombinant adenovirus vectors can induce localized expression of such potential therapeutic agent within donor allografts that would make this approach clinically applicable.