TERT和BDNF基因共修饰脐血间充质干细胞移植治疗新生鼠缺氧缺血性脑损伤的实验研究

Neonatal hypoxic-ischemic brain damage seriously threatens newborn's health and even their lives. Cell transplantation replacement therapy is one of the effective treatments for it. Compared with MSC of the other tissues, umbilical cord blood mesenchymal stem cell (UCB-MSC) has more advantages, in addition to the potential of differentiation to neural cell. However, the application of UCB-MSC in cell transplantation therapy is hindered due to its limited life. TERT can make cell immortalization. In the preliminary work, we found that intracerebroventricular application of TERT could alleviate hypoxic-ischemic brain damage. Therefore, we intend to transfect UCB-MSC with TERT not only to extend UCB-MSC's lifespan but also to increase the expression of endogenous TERT. Researches show that brain-derived neurotrophic factor (BDNF) is effective for hypoxic-ischemic brain damage, and that endogenous expression of BDNF works best. Therefore, we propose to introduce BDNF into telomerase reconstituted UCB-MSC. Concerning that UCB-MSC selectively migrates to the ischemic area in the brain, UCB-MSC can increase BDNF recruitment to the cortical lesion site and amplify the theraputic effect of BDNF. This piece of work provides new idea for effective treatment for hypoxic-ischemic brain damage in the newborns.

新生儿缺氧缺血性脑损伤严重威胁新生儿健康甚至生命，细胞移植替代治疗是其有效治疗措施之一。脐血间充质干细胞不仅有向神经细胞分化潜能，且具有其他组织来源的干细胞无可比拟的优点。但脐血间充质干细胞寿命有限，这一特点极大地限制了它在细胞治疗中的应用。TERT可使细胞永生化。前期工作中，我们发现侧脑室注射TERT可减轻缺氧缺血性诱导的脑损伤。因此，我们拟在脐血间充质干细胞中转染TERT基因，既延长细胞寿命，又增加TERT的内源性表达。研究表明，脑源性神经生长因子(BDNF)对缺氧缺血性脑损伤具有良好的治疗作用，BDNF的内源性表达能达到最佳治疗效果。因此，我们拟将BDNF导入端粒酶重建的脐血间充质干细胞表达，利用脐血间充质干细胞对神经损伤部位的趋化作用，使脐血间充质干细胞分泌的BDNF更好地发挥其神经修复功能。为寻找新生儿缺氧缺血性脑损伤的有效治疗措施提供了新思路。

新生儿缺氧缺血性脑损伤严重威胁新生儿健康甚至生命，细胞移植替代治疗是其有效治疗措施之一。脐血间充质干细胞寿命有限，这一特点极大地限制了它在细胞治疗中的应用。端粒酶逆转录酶（teolomerase reverse transcriptase, TERT） 可使细胞永生化。脑源性神经生长因子（brain derived neurotrophic factor, BDNF）对缺氧缺血性脑损伤具有良好的治疗作用，BDNF 的内源性表达能达到最佳治疗效果。本课题将 BDNF 导入端粒酶重建的脐血间充质干细胞，通过体内体外实验研究TERT和BDNF基因共修饰脐血间充质干细胞（TERT-BDNF-UCB）对缺氧缺血脑损伤后细胞凋亡和神经修复的影响。采用RT-PCR和Western blot检测TERT和BDNF mRNA和蛋白的表达，采用TUNEL法检测细胞凋亡，采用双盲法进行改良神经功能行为学评分（modified neurological severity score，mNSS）进行行为学检查，采用Morris水迷宫进行神经行为学评价。RT-PCR和Western blot结果证实构建TERT-BDNF-UCB成功。体外实验结果显示，TERT-BDNF-UCB可显著减少氧糖剥夺（oxygen-glucose deprivation, OGD）诱导的神经元凋亡。与未治疗组和UCB组、TERT-UCB和BDNF-UCB组相比，TERT-BDNF-UCB组大鼠的神经损伤评分有显著的改善，学习记忆能力明显提高。因此，TERT-BDNF-UCB较单纯脐血间充质干细胞具有更好的应用前景。