Bcl-2基因转染对高糖低氧诱导的移植内皮祖细胞损伤的保护作用及机制研究

Lower limb ischemia is common and frequently-occurring disease in the elderly nowadays, the patients with end-stage lower limb ischemia who have lost a good opportunity for surgical and interventional treatment have to face the outcome of amputation. Since the first case of endotheliar progenitor cell (EPC) transplantation for end-stage lower limb ischemia was achieved successfully in 2002, this treatment has been rapidly carried out in the world, we also confirmed the efficacy of the treatment in clinic and experiment. However, some domestic and international literature indicates that the efficacy of EPC transplantation in treating critical limb ischemia remains to be improved, especially for diabetic critical limb ischemia, recent studies have shown that high glucose hypoxia environment will reduce the number and function of transplanted EPC, maybe this is the key to the point. Therefore, how to protect transplanted EPC from high glucose hypoxia induced injury is current research focus. This proposal is expected to transfect apoptosis regulatory gene Bcl-2 in rat bone marrow-derived EPC, then measuring the anti-apoptotic ability and cell functions of genitically modified EPC in vitro high glucose hypoxia environment and in ischemic limb of diabetic rats, proving Bcl-2 gene modification has a protective effect in high glucose hypoxia induced transplanted EPC injury, in order to provide new ideas to improve the therapeutic efficacy of EPC transplantation in diabetic critical limb ischemia.

下肢缺血性疾病已成为危害中老年人的常见病和多发病，已失去手术和介入治疗机会的终末期患者面临截肢的严重后果。自2002年首例内皮祖细胞（EPC）移植治疗终末期下肢缺血性疾病获得成功以来，该治疗在世界范围内得到迅速开展，我们在实验和临床中也均验证了该治疗的有效性。但同时国内外大量文献表明该治疗的疗效有待提高，尤其对糖尿病性严重肢体缺血性疾病的疗效不佳，最新研究证明移植的EPC在高糖低氧环境下数量减少、功能减弱，是影响疗效的关键所在。因此,如何保护高糖低氧导致的移植EPC损伤是当前的研究热点。本课题预期通过凋亡调控基因Bcl-2转染大鼠骨髓源EPC的方法，进行转基因EPC在体外高糖低氧环境下和糖尿病大鼠缺血肢体内的抗凋亡和促新生血管生成能力的研究，从机制上证明Bcl-2基因转染对高糖低氧诱导的移植EPC损伤有保护作用，为临床上改善EPC移植治疗糖尿病性严重肢体缺血性疾病的疗效提供新思路。

下肢缺血性疾病已成为危害中老年人的常见病和多发病，已失去手术和介入治疗机会的终末期患者面临截肢的严重后果。自2002 年首例内皮祖细胞（EPC）移植治疗终末期下肢缺血性疾病获得成功以来，该治疗在世界范围内得到迅速开展，我们在实验和临床中也均验证了该治疗的有效性。但同时国内外大量文献表明该治疗的疗效有待提高，尤其对糖尿病性严重肢体缺血性疾病的疗效不佳，最新研究证明移植的EPC 在高糖低氧环境下数量减少、功能减弱，是影响疗效的关键所在。因此,如何保护高糖低氧导致的移植EPC 损伤是当前的研究热点。本课题通过凋亡调控基因Bcl-2 转染大鼠骨髓源EPC 的方法，进行转基因EPC 在体外高糖低氧环境下和糖尿病大鼠缺血肢体内的抗凋亡和促新生血管生成能力的研究。体外实验部分，结果发现EPC在高糖低氧环境下培养24小时，EPC的凋亡明显增加，细胞的NF-kBp65，Tie-2，JNK，Notch-1和p38MAPK基因表达增强，而Bcl-2和VEGF-R2基因表达受到抑制。而Bcl-2基因转染可以明显降低高糖低氧环境对EPC的上述作用。动物实验部分，我们将Bcl-2基因转染的EPC注射到大鼠缺血后肢的肌肉内，发现大鼠血浆的VEGF水平明显升高，另外缺血肢体的毛细血管密度增高。研究表明Bcl-2基因转染能够通过影响EPC新生血管生成和氧化应激信号通路，抑制高糖低氧环境对EPC细胞的损害，并且提高EPC的促新生血管生成能力。从机制上证明Bcl-2 基因转染对高糖低氧诱导的移植EPC 损伤有保护作用，为临床上改善EPC 移植治疗糖尿病性严重肢体缺血性疾病的疗效提供新思路。