【摘要】：支氣管肺發(fā)育不良（BPD）是早產(chǎn)兒常見的慢性肺部疾病，目前發(fā)病機(jī)制尚未明確，缺乏有效的防治措施，死亡率和致殘率較高。BPD的主要病理機(jī)制為肺泡化阻滯和肺微血管損傷。本研究從BPD存在明顯的微血管形成阻滯入手，通過分別構(gòu)建Ang1和VEGF質(zhì)粒，慢病毒組裝轉(zhuǎn)染間充質(zhì)干細(xì)胞后，輸注到新生小鼠支氣管肺發(fā)育不良模型體內(nèi)，采用形態(tài)學(xué)和免疫組化、westernblotting、Q-PCR、電鏡等手段觀察移植后肺泡結(jié)構(gòu)和微血管生成以及VEGF、Ang1、肺灌洗液炎性細(xì)胞計(jì)數(shù)指標(biāo)的變化，旨在探討攜帶Ang1和VEGF基因的MSC對于BPD血管新生和肺泡化的影響及作用機(jī)制，闡明肺損傷中循環(huán)受損與肺泡阻滯的關(guān)聯(lián)，為今后BPD基于細(xì)胞基礎(chǔ)的基因干預(yù)治療提供實(shí)驗(yàn)依據(jù)。本研究分為以下四部分： 研究內(nèi)容一：血管內(nèi)皮生長因子及血管生成素1載體的構(gòu)建 本部分研究我們成功采用Gateway技術(shù)構(gòu)建了PLV.Ex2d.P/neo-CMVVEGF/IRES/EGFP、 PLV.Ex3d.P/puro-CMVAng-1IRES/DsRed-Express2和空白質(zhì)粒PLV.EX2D.p/puro-CMVDsRed express2、PLV.EX2D.neo-CMVEGFP。所得質(zhì)粒經(jīng)PCR及測序證實(shí)完全正確，為所需目的質(zhì)粒。為更好的實(shí)現(xiàn)示蹤，本研究將EGFP綠色熒光質(zhì)粒和DsRed紅色熒光蛋白構(gòu)建入載體質(zhì)粒中。EGFP表達(dá)綠色熒光，而DsRed表達(dá)紅色熒光，其激發(fā)和發(fā)射波長較長，其發(fā)射峰位于培養(yǎng)基、組織培養(yǎng)器材及細(xì)胞成分等產(chǎn)生的熒光背景范圍之外，具有較高的信噪比，而且在細(xì)胞內(nèi)熒光轉(zhuǎn)換效率高，更易檢測，可在熒光顯微鏡下直觀觀察。不同顏色光有利于在體實(shí)驗(yàn)中同時定位不同的細(xì)胞。為下一步病毒包裝、細(xì)胞感染及體內(nèi)示蹤打下了基礎(chǔ)。 研究內(nèi)容二：VEGF、Ang1質(zhì)粒慢病毒包裝及MSCs穩(wěn)轉(zhuǎn)細(xì)胞株建立 本部分研究我們通過將目的質(zhì)粒與輔助質(zhì)粒pLV/helper-SL3、pLV/helper-SL4及pLV/helper-SL5混合采用脂質(zhì)體法制備DNA-Lipofectamine2000復(fù)合物，并共同轉(zhuǎn)染293FT細(xì)胞進(jìn)行慢病毒的包裝，產(chǎn)生相應(yīng)的慢病毒顆粒，通過熒光表達(dá)情況來測定病毒滴度和感染效率，成功包裝出具高效感染力的攜帶VEGF和Ang1的慢病毒顆粒。其中，CMV-VEGF滴度為1×108TU/ml，Ang1-DsRed(puro)為5×108TU/ml，均達(dá)到了下一步感染MSCs要求。本研究通過慢病毒介導(dǎo)，成功獲得分別攜帶VEGF基因和GFP基因及Ang1和DsRed基因的小鼠骨髓間充質(zhì)干細(xì)胞穩(wěn)轉(zhuǎn)細(xì)胞株。轉(zhuǎn)導(dǎo)48小時后觀察發(fā)現(xiàn)VEGF轉(zhuǎn)導(dǎo)率約為60%， Ang-1的轉(zhuǎn)染率5%左右，且熒光很弱，而GFP和DsRed的轉(zhuǎn)染率則高達(dá)80%以上，且熒光相對較強(qiáng)。經(jīng)單克隆法篩選純化均獲得穩(wěn)定轉(zhuǎn)染細(xì)胞株。經(jīng)多次傳代及凍存復(fù)蘇，，活力良好。經(jīng)Q-PCR及Westernblot檢測，證實(shí)可穩(wěn)定表達(dá)VEGF或Ang-1，且熒光顯微鏡下觀察熒光表達(dá)良好。顯示成功構(gòu)建了慢病毒介導(dǎo)的攜帶VEGF基因和GFP基因及攜帶Ang1和DsRed基因的小鼠骨髓間充質(zhì)干細(xì)胞穩(wěn)轉(zhuǎn)細(xì)胞株。經(jīng)雞胚尿囊膜血管新生實(shí)驗(yàn)證實(shí)，MSCs VEGF及MSCs Ang-1細(xì)胞培養(yǎng)上清液均可促進(jìn)雞胚尿囊膜新生血管生成，但前者生成的血管結(jié)構(gòu)異常，存在出血現(xiàn)象。而兩者一起應(yīng)用，則無此現(xiàn)象，表明MSCs VEGF及MSCs Ang-1細(xì)胞培養(yǎng)上清液含有促血管因子的表達(dá)，而且VEGF和Ang1在血管生成方面存在一定的協(xié)同作用。 研究內(nèi)容三：高氧誘導(dǎo)新生鼠BPD模型的建立及其肺血管損傷觀察 采用高氧（60-70%）持續(xù)吸入的方法，我們成功制作了新生昆明小鼠BPD模型，并通過HE染色、免疫組化、血管計(jì)數(shù)和投射電鏡等觀察了肺泡結(jié)構(gòu)和微血管發(fā)育情況。研究發(fā)現(xiàn)BPD組小鼠肺部病理呈現(xiàn)BPD典型的肺泡簡化特征，與空氣組比較，BPD組生后7d時RAC即明顯降低，至生后21d時差異更加顯著。同時，我們采用CD34免疫組化方法，對高氧吸入致肺微血管的影響進(jìn)行了初步的觀察。結(jié)果顯示高氧吸入組小鼠生后3d和7d時，MVD略低于空氣組，但兩者差異無統(tǒng)計(jì)學(xué)意義，生后14d時MVD低于正�？諝饨M，至生后21d差異更加顯著，顯示高氧持續(xù)吸入可導(dǎo)致微血管生成障礙，血管內(nèi)皮細(xì)胞數(shù)量減少。本研究還對Ang1、VEGF及EphrinB2這三個重要的促血管生成因子在模型組和對照組小鼠肺中的表達(dá)情況進(jìn)行了觀察。免疫組化結(jié)果顯示高氧持續(xù)暴露可導(dǎo)致Ang1、VEGF及EphrinB2表達(dá)的持續(xù)下調(diào)。我們還對BPD模型的透射電鏡下肺超微結(jié)構(gòu)進(jìn)行了觀察，發(fā)現(xiàn)與正�？諝饨M對比，高氧模型組肺泡II型上皮細(xì)胞腫脹，電子密度降低。胞質(zhì)內(nèi)板層小體較少或消失，結(jié)構(gòu)松散，有排空現(xiàn)象。線粒體腫脹，體積增大。毛細(xì)血管內(nèi)皮細(xì)胞腫脹明顯；微血管結(jié)構(gòu)紊亂，有出血，肺泡腔內(nèi)可見出血的變形紅細(xì)胞。血管內(nèi)壁毛糙，并有較多黑色顆粒狀凝聚，管腔內(nèi)未見正常紅細(xì)胞。證實(shí)BPD模型鼠不僅肺泡結(jié)構(gòu)和肺泡上皮細(xì)胞存在異常，肺微血管結(jié)構(gòu)和氣血屏障亦受損。以上結(jié)果從不同層面進(jìn)一步證實(shí)了我們的最初猜想，即高氧暴露損傷了肺循環(huán)的正常發(fā)育，并導(dǎo)致了BPD的形成，顯示肺循環(huán)異常在BPD發(fā)病中起重要作用。 研究內(nèi)容四：攜帶VEGF及Ang1基因的MSCs對BPD模型鼠干預(yù) 本部分研究內(nèi)容通過模型鼠的在體實(shí)驗(yàn)，顯示MSCs、MSCs-Ang-1及MSCsV+A可減輕BPD模型鼠肺損傷和肺循環(huán)損害，其中以MSCs V+A效果最為明顯，可有效改善BPD模型鼠的體重，提高RAC，增強(qiáng)肺血管生成，減少肺部因高氧導(dǎo)致的炎癥反應(yīng)，減輕肺膠原沉積和纖維化，顯示Ang-1和VEGF在修復(fù)肺血管損傷方面具有協(xié)同效應(yīng)。經(jīng)免疫熒光、GFP免疫組化及共聚焦顯微鏡觀察顯示，MSCsAng-1和MSCs VEGF腹腔注射后，均可遷移至肺部定植，但植入數(shù)量較少。Westernblotting及Q-PCR檢測顯示MSCs Ang-1和MSCs VEGF腹腔注射后，均可增加肺部Ang1或VEGF的表達(dá)。通過墨汁灌注方法，觀察各組肺血管情況�？梢娕c空氣鹽水組致密呈網(wǎng)狀的血管結(jié)構(gòu)相比，高氧鹽水組血管網(wǎng)明顯稀疏，未能形成立體完整的血管網(wǎng)體系。而MSCs組與高氧鹽水組比較，血管網(wǎng)稍有改善。而MSCs-VEGF組和MSCs-Ang-1組及MSCs-V+A組與MSCs組比較，血管網(wǎng)進(jìn)一步明顯致密，提示血管數(shù)量有所改善。但本研究亦發(fā)現(xiàn)單純使用MSCsVEGF并不能有效減輕BPD的肺損傷，反而在一定程度上導(dǎo)致了肺發(fā)育的惡化，這可能與單純的VEGF誘導(dǎo)生成的血管結(jié)構(gòu)不成熟，通透性高，容易導(dǎo)致出血和炎性細(xì)胞滲出，從而導(dǎo)致肺功能惡化有關(guān)。 總之，以上結(jié)果證實(shí)了本課題的最初設(shè)想，采取VEGF和Ang1雙基因通過MSCs導(dǎo)入，可發(fā)揮協(xié)同效應(yīng)，有效的修復(fù)受損肺循環(huán)，減輕BPD肺損傷，這為BPD的有效干預(yù)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R722.6

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