HO-1修饰间充质干细胞对急性肺损伤的作用及机制研究

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinical critical illness. At present, the mortality rate among ALI/ARDS patients remains high. Bone marrow mesenchymal stem cells (BMSC) are characterized with multi-lineage differentiation and tissue reparative ability, which can restore traumatic tissues by the means of differentiation or fusion. Meanwhile, BMSC have the immunomodulatory effects and can serve as a favorable gene-carrier. Heme oxygenase-1( HO-1) is a rate-limiting enzyme in the metabolism of hemoglobin. HO-1 can protect cells from injuring by its anti-oxidation, anti-apoptosis and anti-inflammation when in stress condition. Recent research demonstrated that HO-1 and its metabolites could supress the Toll like receptor signaling pathway. By combining the stem cell transplantation technique and the genetic engineering technique, the modified BMSC (BMSC-HO-1) may be more helpful for the recovery of ALI/ARDS. In this study, we will plane to choose lentivirus vector as the gene transfer tool for the gene modification of BMSC, then we will compare the differences of the phenotype and differentiation in BMSC after gene modification. And, we will evaluate the protective effect and the differentiation capacity of the HO-1 gene modified BMSC, the expression and activities of HO-1 and the damage degree of lung in ALI rat model after the BMSC-HO-1 was transfused. At last, we will use Transwell co-culture system to observe the changes of inflammatory condition, the balance of oxidation- anti-oxidation and the apoptosis of BMSC, AEC and PVEC after HO-1 gene modification, which may help to elucidate the potential therapeutic mechanisms of BMSC-HO-1 in ALI/ARDS rat model.

目前急性肺损伤(ALI)/急性呼吸窘迫综合征(ARDS)的临床病死率仍高。骨髓间充质干细胞(BMSC)具有多向分化潜能、组织修复及免疫调节作用。此外，BMSC还是良好的基因运载工具。HO-1是血红素代谢中的限速酶，能从抗氧化，抗凋亡，抗炎等方面发挥细胞保护作用，研究发现HO-1及其代谢产物对TLRs致炎通路起负调控作用。本课题以含HO-1基因慢病毒载体为转染工具，对BMSC行基因修饰(BMSC-HO-1)，观察修饰前后BMSC细胞表型及分化功能有无变化；建立大鼠ALI模型，将BMSC或BMSC-HO-1回输到大鼠体内观察BMSC转化为肺组织细胞的指标，HO-1体内表达和生物学活性以及肺损伤指标；利用Transwell体系，在炎症微环境中将BMSC或BMSC-HO-1与大鼠AECⅡ和PVEC共培养，从氧化失衡、细胞凋亡及炎症失控多个方面探讨BMSC-HO-1对急性肺损伤的保护机制。

目前急性肺损伤（ALI）/急性呼吸窘迫综合征（ARDS）的临床病死率仍高。骨髓间充质干细胞(BMSC)具有多向分化潜能、组织修复及免疫调节作用。此外，BMSC还是良好的基因运载工具。HO-1是血红素代谢中的限速酶，能从抗氧化，抗凋亡，抗炎等方面发挥细胞保护作用，研究发现HO-1及其代谢产物对TLRs致炎通路起负调控作用。本课题以含HO-1基因慢病毒载体为转染工具，对BMSC行基因修饰(BMSC-HO-1)，观察修饰前后BMSC细胞表型及分化功能有无变化；建立大鼠ALI模型，将BMSC或BMSC-HO-1回输到大鼠体内观察BMSC转化为肺组织细胞的指标，HO-1体内表达和生物学活性以及肺损伤指标；利用Transwell体系，在炎症微环境中将BMSC或BMSC-HO-1与AECⅡ和PVEC共培养，从氧化失衡、细胞凋亡及炎症失控多个方面探讨BMSC-HO-1对急性肺损伤的保护机制。实验结果显示经HO-1修饰后BMSC的表型及多胚层分化能力无变化，BMSC-HO-1对肺损伤大鼠的保护作用较BMSC更为显著，且BMSC-HO-1可成功归巢至肺损伤大鼠肺组织内并成功分化为肺组织细胞；BMSC-HO-1能从氧化失衡、细胞凋亡及炎症失控多个方面对AECⅡ和PVEC产生保护作用，且对LPS-TLR4信号通路存在调控作用：减少LPS诱导的TLR4表达上调，减少TLR4/MyD88，TLR4/TRIF及TLR4/IRAK-1复合物形成。本研究为ALI及ARDS的防治提供了新思路，后期研究成果的转化将有利于提高急性肺损伤及急性呼吸窘迫综合征的救治成功率。项目资助已发表中文统计源期刊论文16篇。培养硕士研究生3名，博士研究生1名，已有2名硕士研究生顺利毕业。项目批准经费23万元，目前统计支出17.4万元，各项支出基本与预算相符。剩余5.6万元，剩余经费计划用于继续完善部分实验及论文发表相关费用。