慢病毒介导转染VEGF和Ang1基因的小鼠骨髓间充质干细胞对氧诱导新生鼠BPD模型的干预研究

发布时间：2018-08-26 19:22

【摘要】：支气管肺发育不良（BPD）是早产儿常见的慢性肺部疾病，目前发病机制尚未明确，缺乏有效的防治措施，死亡率和致残率较高。BPD的主要病理机制为肺泡化阻滞和肺微血管损伤。本研究从BPD存在明显的微血管形成阻滞入手，通过分别构建Ang1和VEGF质粒，慢病毒组装转染间充质干细胞后，输注到新生小鼠支气管肺发育不良模型体内，采用形态学和免疫组化、westernblotting、Q-PCR、电镜等手段观察移植后肺泡结构和微血管生成以及VEGF、Ang1、肺灌洗液炎性细胞计数指标的变化，旨在探讨携带Ang1和VEGF基因的MSC对于BPD血管新生和肺泡化的影响及作用机制，阐明肺损伤中循环受损与肺泡阻滞的关联，为今后BPD基于细胞基础的基因干预治疗提供实验依据。本研究分为以下四部分：研究内容一：血管内皮生长因子及血管生成素1载体的构建本部分研究我们成功采用Gateway技术构建了PLV.Ex2d.P/neo-CMVVEGF/IRES/EGFP、 PLV.Ex3d.P/puro-CMVAng-1IRES/DsRed-Express2和空白质粒PLV.EX2D.p/puro-CMVDsRed express2、PLV.EX2D.neo-CMVEGFP。所得质粒经PCR及测序证实完全正确，为所需目的质粒。为更好的实现示踪，本研究将EGFP绿色荧光质粒和DsRed红色荧光蛋白构建入载体质粒中。EGFP表达绿色荧光，而DsRed表达红色荧光，其激发和发射波长较长，其发射峰位于培养基、组织培养器材及细胞成分等产生的荧光背景范围之外，具有较高的信噪比，而且在细胞内荧光转换效率高，更易检测，可在荧光显微镜下直观观察。不同颜色光有利于在体实验中同时定位不同的细胞。为下一步病毒包装、细胞感染及体内示踪打下了基础。研究内容二：VEGF、Ang1质粒慢病毒包装及MSCs稳转细胞株建立本部分研究我们通过将目的质粒与辅助质粒pLV/helper-SL3、pLV/helper-SL4及pLV/helper-SL5混合采用脂质体法制备DNA-Lipofectamine2000复合物，并共同转染293FT细胞进行慢病毒的包装，产生相应的慢病毒颗粒，通过荧光表达情况来测定病毒滴度和感染效率，成功包装出具高效感染力的携带VEGF和Ang1的慢病毒颗粒。其中，CMV-VEGF滴度为1×108TU/ml，Ang1-DsRed(puro)为5×108TU/ml，均达到了下一步感染MSCs要求。本研究通过慢病毒介导，成功获得分别携带VEGF基因和GFP基因及Ang1和DsRed基因的小鼠骨髓间充质干细胞稳转细胞株。转导48小时后观察发现VEGF转导率约为60%， Ang-1的转染率5%左右，且荧光很弱，而GFP和DsRed的转染率则高达80%以上，且荧光相对较强。经单克隆法筛选纯化均获得稳定转染细胞株。经多次传代及冻存复苏，，活力良好。经Q-PCR及Westernblot检测，证实可稳定表达VEGF或Ang-1，且荧光显微镜下观察荧光表达良好。显示成功构建了慢病毒介导的携带VEGF基因和GFP基因及携带Ang1和DsRed基因的小鼠骨髓间充质干细胞稳转细胞株。经鸡胚尿囊膜血管新生实验证实，MSCs VEGF及MSCs Ang-1细胞培养上清液均可促进鸡胚尿囊膜新生血管生成，但前者生成的血管结构异常，存在出血现象。而两者一起应用，则无此现象，表明MSCs VEGF及MSCs Ang-1细胞培养上清液含有促血管因子的表达，而且VEGF和Ang1在血管生成方面存在一定的协同作用。研究内容三：高氧诱导新生鼠BPD模型的建立及其肺血管损伤观察采用高氧（60-70%）持续吸入的方法，我们成功制作了新生昆明小鼠BPD模型，并通过HE染色、免疫组化、血管计数和投射电镜等观察了肺泡结构和微血管发育情况。研究发现BPD组小鼠肺部病理呈现BPD典型的肺泡简化特征，与空气组比较，BPD组生后7d时RAC即明显降低，至生后21d时差异更加显著。同时，我们采用CD34免疫组化方法，对高氧吸入致肺微血管的影响进行了初步的观察。结果显示高氧吸入组小鼠生后3d和7d时，MVD略低于空气组，但两者差异无统计学意义，生后14d时MVD低于正常空气组，至生后21d差异更加显著，显示高氧持续吸入可导致微血管生成障碍，血管内皮细胞数量减少。本研究还对Ang1、VEGF及EphrinB2这三个重要的促血管生成因子在模型组和对照组小鼠肺中的表达情况进行了观察。免疫组化结果显示高氧持续暴露可导致Ang1、VEGF及EphrinB2表达的持续下调。我们还对BPD模型的透射电镜下肺超微结构进行了观察，发现与正常空气组对比，高氧模型组肺泡II型上皮细胞肿胀，电子密度降低。胞质内板层小体较少或消失，结构松散，有排空现象。线粒体肿胀，体积增大。毛细血管内皮细胞肿胀明显；微血管结构紊乱，有出血，肺泡腔内可见出血的变形红细胞。血管内壁毛糙，并有较多黑色颗粒状凝聚，管腔内未见正常红细胞。证实BPD模型鼠不仅肺泡结构和肺泡上皮细胞存在异常，肺微血管结构和气血屏障亦受损。以上结果从不同层面进一步证实了我们的最初猜想，即高氧暴露损伤了肺循环的正常发育，并导致了BPD的形成，显示肺循环异常在BPD发病中起重要作用。研究内容四：携带VEGF及Ang1基因的MSCs对BPD模型鼠干预本部分研究内容通过模型鼠的在体实验，显示MSCs、MSCs-Ang-1及MSCsV+A可减轻BPD模型鼠肺损伤和肺循环损害，其中以MSCs V+A效果最为明显，可有效改善BPD模型鼠的体重，提高RAC，增强肺血管生成，减少肺部因高氧导致的炎症反应，减轻肺胶原沉积和纤维化，显示Ang-1和VEGF在修复肺血管损伤方面具有协同效应。经免疫荧光、GFP免疫组化及共聚焦显微镜观察显示，MSCsAng-1和MSCs VEGF腹腔注射后，均可迁移至肺部定植，但植入数量较少。Westernblotting及Q-PCR检测显示MSCs Ang-1和MSCs VEGF腹腔注射后，均可增加肺部Ang1或VEGF的表达。通过墨汁灌注方法，观察各组肺血管情况。可见与空气盐水组致密呈网状的血管结构相比，高氧盐水组血管网明显稀疏，未能形成立体完整的血管网体系。而MSCs组与高氧盐水组比较，血管网稍有改善。而MSCs-VEGF组和MSCs-Ang-1组及MSCs-V+A组与MSCs组比较，血管网进一步明显致密，提示血管数量有所改善。但本研究亦发现单纯使用MSCsVEGF并不能有效减轻BPD的肺损伤，反而在一定程度上导致了肺发育的恶化，这可能与单纯的VEGF诱导生成的血管结构不成熟，通透性高，容易导致出血和炎性细胞渗出，从而导致肺功能恶化有关。总之，以上结果证实了本课题的最初设想，采取VEGF和Ang1双基因通过MSCs导入，可发挥协同效应，有效的修复受损肺循环，减轻BPD肺损伤，这为BPD的有效干预提供了新的思路。
[Abstract]:Bronchopulmonary dysplasia (BPD) is a common chronic pulmonary disease in premature infants. At present, the pathogenesis of BPD is still unclear, effective prevention and treatment measures are lacking, mortality and disability rate are high. The main pathological mechanisms of BPD are alveolarization block and pulmonary microvascular injury. Mesenchymal stem cells (MSCs) were transfused with plasmids of 1 and VEGF and lentiviruses. The changes of alveolar structure and microangiogenesis, VEGF, Ang1 and inflammatory cell count in lung lavage fluid were observed by morphology and immunohistochemistry, Western blotting, Q-PCR and electron microscopy. To investigate the effects and mechanism of MSC carrying Ang1 and VEGF genes on angiogenesis and alveolarization in BPD, and to clarify the relationship between circulatory impairment and alveolar block in lung injury.
Contents 1: Construction of vascular endothelial growth factor and angiopoietin 1 vector
In this part, we successfully constructed PLV.Ex2d.P/neo-CMVVEGF/IRES/EGFP, PLV.Ex3d.P/puro-CMVAng-1IRES/DsRed-Express2 and blank plasmids PLV.EX2D.p/puro-CMVDsRed express2, PLV.EX2D.neo-CMVEGFP by Gateway technique. The plasmids obtained were confirmed to be completely correct by PCR and sequencing, and were used for the purpose of better tracing. EGFP green fluorescent plasmid and DsRed fluorescent protein were constructed into vector plasmid. EGFP expressed green fluorescence, while DsRed expressed red fluorescence. The excitation and emission wavelengths of EGFP green fluorescent plasmid and DsRed fluorescent protein were longer. The emission peaks of EGFP green fluorescent plasmid and DsRed fluorescent protein were located outside the fluorescence background of culture medium, tissue culture apparatus and cell components, and had higher signal-to-noise ratio and were fine. Intracellular fluorescence conversion efficiency is high, easy to detect, and can be directly observed under fluorescence microscope. Different colored light is conducive to the simultaneous localization of different cells in vivo experiments.
Research contents two: VEGF, Ang1 plasmid lentivirus packaging and MSCs stable transfection cell line establishment
In this part, DNA-Lipofectamine2000 complex was prepared by mixing the target plasmid with the auxiliary plasmid pLV/helper-SL3, pLV/helper-SL4 and pLV/helper-SL5. The DNA-Lipofectamine2000 complex was co-transfected into 293FT cells to package lentiviruses, and the lentiviral particles were produced. The virus titer and sensitivity were determined by fluorescence expression. The CMV-VEGF titer was 1 *108TU/ml, Ang1-DsRed (puro) was 5 *108TU/ml, which met the requirements of MSCs infection in the next step. In this study, mice with VEGF gene, GFP gene and Ang1 and DsRed gene were successfully obtained by lentivirus mediation. After 48 hours of transduction, the transfection rate of VEGF was about 60%, Ang-1 was about 5%, and the fluorescence was very weak. The transfection rate of GFP and DsRed was over 80%, and the fluorescence was relatively strong. The stable transfected cells were obtained by monoclonal screening and purification. Good. The stable expression of VEGF or Ang-1 was confirmed by Q-PCR and Western blot, and the fluorescent expression was observed under fluorescence microscope. Lentivirus-mediated stable transfection of mouse bone marrow mesenchymal stem cells carrying VEGF and GFP genes and Ang 1 and DsRed genes was successfully constructed. Both MSCs VEGF and MSCs Ang-1 cell culture supernatant could promote angiogenesis of chicken embryo allantoic membrane, but the angiogenesis of the former was abnormal and hemorrhagic. However, the combination of MSCs VEGF and MSCs Ang-1 cell culture supernatant had no such phenomenon, indicating that MSCs VEGF and MSCs Ang-1 cell culture supernatant contained the expression of angiogenic factors, and that VEGF and Ang 1 were in angiogenesis prescription. There are some synergistic effects.
Research contents three: establishment of hyperoxia induced neonatal rat BPD model and observation of pulmonary vascular injury
The BPD model of neonatal Kunming mice was successfully established by continuous inhalation of hyperoxia (60-70%). The alveolar structure and microvascular development were observed by HE staining, immunohistochemistry, vascular counting and electron microscopy. It was found that the lung pathology of BPD mice showed typical simplified alveolar features, and BPD mice were born in BPD group compared with air group. RAC decreased significantly at the 7th day after birth, and the difference was more significant at the 21st day after birth. At the same time, we used CD34 immunohistochemical method to observe the effect of hyperoxia inhalation on pulmonary microvasculature. The difference between normal air group and control group was more significant at 21 days after birth, indicating that inhalation of hyperoxia could lead to microangiogenesis disorder and decrease of the number of vascular endothelial cells. The expression of Ang1, VEGF and EphrinB2 was continuously down-regulated by oxygen exposure. The ultrastructure of lung in BPD model was observed by transmission electron microscopy. Compared with normal air group, the alveolar type II epithelial cells in hyperoxia model group were swollen and the electron density was decreased. Mitochondria were swollen and enlarged in size.The endothelial cells of capillaries were swollen obviously.The structure of microvessels was disordered and hemorrhage was observed in the alveolar cavity.The inner wall of blood vessels was rough and there were many black granular aggregates.Normal red blood cells were not found in the lumen.It was confirmed that the alveolar structure and alveolar epithelial cells were abnormal in BPD model rats. These results further confirm our initial conjecture that hyperoxia exposure impairs the normal development of pulmonary circulation and leads to the formation of BPD, suggesting that abnormal pulmonary circulation plays an important role in the pathogenesis of BPD.
Research contents four: MSCs carrying VEGF and Ang1 genes interfere with BPD model rats.
This part of the study showed that MSCs, MSCs-Ang-1 and MSCsV+A can alleviate the lung injury and pulmonary circulation damage in BPD model rats through in vivo experiments. MSCs V+A has the most obvious effect, which can effectively improve the weight of BPD model rats, increase RAC, enhance pulmonary angiogenesis, reduce lung inflammation caused by hyperoxia, and reduce lung collagen deposition. Immunofluorescence, GFP immunohistochemistry and confocal microscopy showed that MSCs Ang-1 and MSCs VEGF could migrate to the lungs after intraperitoneal injection, but the number of implants was small. Western blotting and Q-PCR showed that MSCs Ang-1 and MSCs VEGF could be transplanted into the abdominal cavity. After injection, the expression of Ang1 or VEGF in the lung was increased. The pulmonary vessels were observed by ink perfusion method. Compared with the air saline group, the vascular network in the hyperoxic saline group was obviously thinner and could not form a three-dimensional intact vascular network system. The vascular network in the MSCs group was slightly improved compared with the hyperoxic saline group. Compared with MSCs group, MSCs-VEGF group, MSCs-Ang-1 group, MSCs-V+A group and MSCs-V+A group, the vascular network was further dense, suggesting an improvement in the number of blood vessels. Tube structure is immature, high permeability, easy to lead to bleeding and inflammatory cell exudation, resulting in deterioration of lung function.
In conclusion, the above results confirm the original assumption of this topic. The introduction of VEGF and Ang1 genes through MSCs can play a synergistic effect, effectively repair damaged pulmonary circulation and reduce BPD lung injury, which provides a new idea for the effective intervention of BPD.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R722.6

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