缺血后适应基础上HGF修饰的间充质干细胞移植治疗肺缺血再灌注损伤的实验研究

Lung ischemia-reperfusion (I/R) injury after pulmonary transplantation can contribute significantly to postoperative pulmonary dysfunction. To date,the mechanisms underlying lung I/R injury remain unclear,and effective treatments for its prevention are lacking. Increasing evidence has suggested that the therapeuticpotential of bone marrow mesenchymal stem cell （BMSC） could be applied to I/R injured tissue such as the heart, liver, lung, and kidneys.However, the quantity and quality of engrafted BMSC are critical for the benefits of BMSC therapy. Hepatocyte growth factor (HGF), a multifunctional growth factor known to affect angiogenesis, apoptosis, inflammation, and stem cell homing processes. Several studies have showed that ischemic postconditioning (I-postC) could cause a significant reduction in the systemic inflammatory response, inhibit the expression of apoptotic molecules, and activate endogenous protective molecules. Thus, in the present study, we will use HGF gene tranfection to enhance BMSC"quantity "and combine I-postC to increase BMSC "quality"in the grafted lung tissue. To test the proposal, we will determine the effect of the secreted HGF on BMSC differention, survival and proliferation in vitro.Using rat model of lung IRI, we will invesigate the synergetic therapeutic effect of transplantations of BMSC based on I-postC via determination of lung function parameters and graft pathologic histology.Furthermore, we will explore the underlying mechanisms of the combined treatment strategy by investigating the effect of HGF and I-postC on BMSC engraft, differentiation, survival, and microvessel formation, and inflammation cytokine and immune cell changes. This study may provide a new strategy for treatment of lung IRI, even othe organs.

肺缺血再灌注损伤(IRI)是肺移植术后早期移植肺功能不全和移植失败的主要原因。近年来骨髓间充质干细胞（BMSC）已经在IRI的治疗中取得重要的进展，但如何提高干细胞的"质"和"量"是治疗的关键。因此，本研究拟将携带肝细胞生长因子（HGF）的慢病毒载体转染大鼠BMSC,利用HGF促进血管生成、抑制细胞凋亡、吸引干细胞归巢等功能，改善BMSC的"质"；进一步应用缺血后适应（I-postC）改善移植物微环境，从而提高BMSC在肺组织的"量"。研究包括体外考察HGF对BMSC生物学功能的影响；建立大鼠移植肺IRI模型，观察I-postC基础上HGF修饰的BMSC移植对IRI的协同治疗作用；同时研究HGF和I-postC对BMSC在肺组织中的定植、分化、存活以及微血管的生成，炎症因子表达以及免疫细胞情况，探讨该联合治疗的保护机制。本研究可能为临床肺及其他器官移植术后IRI治疗提供一条全新的思路。

背景：我们以前研究表明间充质干细胞移植可以有效的保护肺缺血再灌注损伤。大量研究表明肝细胞生长因子在细胞和组织修复和再生中发挥重要的作用。在本课题中，我们研究HGF修饰的间充质干细胞在肺缺血再灌注损伤中的作用。 .方法：分离大鼠骨髓间充质干细胞，构建HGF表达的腺病毒载体。建立大鼠肺缺血再灌注损伤模型，评价动脉血气、肺湿/干重比，细胞因子表达和肺损伤病理改变。.结果：我们成功分离培养获得大鼠骨髓间充质干细胞，纯度达95%以上。重组腺病毒构建rAV-HGF表达载体，转染间充质干细胞(MSC-HGF)。体外实验表明，转染HGF基因对MSC的形态、增殖以及分化能力无明显影响，但可以促进MSC的存活。在缺血再灌注肺损伤模型中，相比较单纯给予MSC组，体内给予MSC-HGF可以有效的降低肺湿/干比，PaCO2水平，改善PaO2和SOD水平，抑制MDA和MPO产生，同时更显著地降低TNF水平，增加IL-10水平。病理组织学显示，MSC-HGF可以有效的改善肺缺血再灌注损伤，减轻肺水肿、微血管出血和炎性浸润；抑制肺上皮细胞凋亡。进一步 研究发现，HGF修饰可以有效的促进MSC在肺组织中的定植和存活，体外实验也表明HGF修饰可以有效的抑制H2O2诱导的MSC凋亡。.结论：我们的研究表明HGF修饰的MSC能够有效的增强MSC的抗缺血再灌注功能，可能为器官的缺血再灌注损伤提供一种新的策略和手段。.完成指标情况：已经全部完成本课题的研究计划，完成论文撰写，1篇SCI论文被移植权威杂志修回（Transplantation)。完成1名研究生的培养。