血管内皮生长因子基因转染骨髓间充质干细胞对支气管肺发育不良大鼠肺发育的影响

Effects of bone marrow mesenchymal stem cells transfected by vascular endothelial growth factor gene on lung development of rats with bronchopulmonary dysplasia

目的研究血管内皮生长因子（VEGF）基因转染骨髓问充质干细胞（MSCs）移植对支气管肺发育不良大鼠肺泡结构和肺部微血管的作用。方法采用密度梯度离心法分离sD大鼠骨髓MSCs，贴壁培养传代并鉴定，应用DNA重组技术构建真核表达载体pcDNA3．1（-）／VEGF164，然后转染至MSCs。将新生SD大鼠置于950mI／L氧箱中14d后，随机分为转染组（MSCs/VEGF组）、对照组（MSCs组）、空白组（无血清培养基组），每组10只，分别于气管内注入1×10^5个转染VEGF的MSCs、单纯MSCs和等量无血清培养基。分别于移植后1、4周取大鼠肺组织行HE染色观察肺组织结构和放射状肺泡计数（RAC），免疫组织化学染色法评价VEGF蛋白表达和新生血管密度情况，Western blot方法检测肺组织中VEGF164蛋白表达情况。结果免疫组织化学结果显示移植后1、4周转染组大鼠肺组织RAC、VEGF蛋白表达、再生血管密度较对照组和空白组均明显增加（P均〈0．05）。Westernblot检测结果显示移植后1、4周转染组VEGFl64蛋白表达较对照组和空白组均明显增加（P均〈0．05）。结论VEGF基因转染MSCs移植能显著促进肺泡发育以及微血管再生，VEGF与新生大鼠肺发育有密切关系。

Objective To explore the influence of vascular endothelial growth factor（VEGF） gene-transfected bone marrow mesenchymal stem cells（MSCs） on pulmonary alveolar structure and microvascular in rats with broncho- pulmonary dysplasia. Methods Bone marrow MSCs were isolated from SD rats by way of density gradient centrifugation ,purified,and transfected with peDNA3.1 （ - ）/VEGF164 and blank plasmid pcDNA3.1 respectively using the li- posome mediated method. After placing newborn SD rats in the oxygen box of 950 mL/L for 14 days, they were randomly divided into the transfected group（ MSCs/VEGF group）, the control group（ MSCs group）, and the blank group（ serumfree medium group） ,with 10 rats in each group respectively, and they were injected respectively with 1× 10^5 MSCs transfected by VEGF,MSCs and the same amount of simple serum-free medium by airway. After transplantation for 1 week and 4 weeks,lung tissue was observed by means of hematoxylin eosin staining to study lung structure and radial alveolar counts（ RAC ）, and VEGF protein expression and angiogenesis densities were evaluated by immunohistochemistry, and finally VEGF164 protein was detected using Western blot. Results After transplantation for 1 week,4 weeks, the RAC, VEGF expression, vascular density by immunohistochemistry in transfected group were significantly more than those in the control group and the blank group（ all P 〈0.05 ）. After transplantation for 1 week ,4 weeks,the VEGFI64 protein level in transfected group was significantly more than that in the control group and the blank group （ all P 〈 0. 05）. Conclusions Eukaryotic expression vector pcDNA3.1 （ - ）/VEGF164 can effectively be expressed in MSCs. Transplantation of vascular endothelial growth factor gene by means of transfected MSCs brings better improvement in pulmonary alveolar structure and microvascular regeneration. VEGF is closely related to lung development in newborn rats.