【作者】 魏来；

【导师】 赵强； 王克强； 周光华；

【作者基本信息】 复旦大学 ， 外科学， 2006， 博士

【摘要】 第一部分人内皮型一氧化氮合成酶基因重组腺病毒载体(Ad．CMV-heNOS)的构建【目的】构建携带人内皮型一氧化氮合成酶基因的重组腺病毒载体Ad．CMV-heNOS，同步构建携带报告基因——增强型绿色荧光蛋白基因的重组腺病毒载体Ad．CMV-EGFP。【方法】将heNOS基因插入载体PSUCMV中构建重组质粒PSUCMV-heNOS，后者经酶切及测序鉴定正确后与5型腺病毒右臂的质粒Pbhge3通过Lipofectamine2000共转染至293细胞，制备携带人内皮型一氧化氮合成酶基因的重组腺病毒载体Ad．CMV-heNOS，鉴定正确后体外扩增、纯化并测定滴度。同法构建重组腺病毒载体Ad．CMV-EGFP并测定滴度。【结果】重组质粒PSUCMV-heNOS经酶切及测序鉴定正确。PCR检测证实PSUCMV-heNOS与Pbhge3共转染293细胞所得腺病毒载体Ad．CMV-heNOS构建成功，经扩增纯化后测定病毒滴度为5.15×10~9pfu／ml。同法构建的Ad．CMV-EGFP滴度为3.8×10~9pfu／ml。【结论】成功构建并纯化得到了高滴度重组复制缺陷型腺病毒载体Ad．CMV-heNOS和Ad．CMV-EGFP，为后续实验中高效基因转染做好准备。第二部分大鼠骨髓内皮祖细胞分离、培养和鉴定的实验研究【目的】研究大鼠骨髓内皮祖细胞(EPC)分离、培养并向内皮细胞方向诱导分化的方法和条件。【方法】从大鼠骨髓中通过密度梯度离心法分离单个核细胞，经差速贴壁后取二次贴壁细胞选择性诱导培养2w。以Dil-ac-LDL、FITC-UEA-1双荧光染色法以及vWF、Flk-1免疫荧光染色法鉴定EPC；流式细胞术(FACS)检测EPC纯度；细胞培养液一氧化氮(NO)含量测定分析EPC功能。【结果】二次贴壁细胞经诱导培养后3d开始伸展，5d形成集落，7-10d增殖加速并出现条索状结构，2w大部分细胞呈多角形。Dil-ac-LDL、FITC-UEA-1双染，vWF及Flk-1免疫荧光染色阳性率均大于70％。FACS检测其中vWF阳性细胞占77.93％，Flk-1阳性细胞占81.50％。二次贴壁并经诱导培养的细胞培养液中NO含量明显高于普通培养的细胞，但低于成熟内皮细胞(P＜0.05)。【结论】通过差速贴壁并经选择性诱导培养可以从大鼠骨髓中分离培养出纯度较高的具备内皮细胞部分特征的EPC。第三部分大鼠动力型肺动脉高压模型的建立【目的】研究不同比例肺组织切除后对大鼠体、肺动脉压、右心室肥厚程度和肺血管重构的影响，以期建立一种新的大鼠动力型肺动脉高压模型。【方法】68只8w龄Wister大鼠分为右肺全切除组(组Ⅰ)、左肺全切除组(组Ⅱ)、右肺上中二叶切除组(组Ⅲ)、假手术组(组Ⅳ)和阴性对照组(组Ⅴ)。手术后4w测定各组大鼠体、肺动脉收缩压，检测右心室肥厚指标(RV／(LV+S))以及肺血管显微形态学指标——肺肌型小动脉相对中膜面积(RMA)及其占肺小血管的百分比(SMA％)。【结果】组Ⅰ大鼠4w后存活率66.7％(12／18)，存活大鼠体重较组Ⅳ、Ⅴ减轻(P＜0.05)。组Ⅰ肺动脉收缩压、RV／(LV+S)、RMA及SMA％均明显高于其它四组(P＜0.01)；组Ⅱ、组Ⅲ之间各指标无显著性差异(P＞0.05)；组Ⅳ、Ⅴ之间各指标也无显著性差异(P＞0.05)；组ⅡRMA明显高于组Ⅳ、Ⅴ(P＜0.05)；组Ⅲ肺动脉收缩压和RMA均明显高于组Ⅳ、Ⅴ(P＜0.05)；五组大鼠体动脉收缩压无显著性差异(P＞0.05)。【结论】大鼠右肺全切除术后4w可形成明显肺动脉高压、右心室肥厚和肺小血管重构，而对体动脉压影响不大。与其它动力型肺动脉高压模型相比，该法操作简便，死亡率较低，实验周期较短，具有一定的应用价值。而左肺全切除和右肺上中二叶切除术后4w肺动脉压和肺血管结构虽有一定的变化，但相对不明显。第四部分Ad．CMV-heNOS体外转染内皮祖细胞，并抑制离体平滑肌细胞增殖的实验研究【目的】了解重组腺病毒载体对大鼠骨髓来源EPC的转染效率，观察Ad．CMV-heNOS转染EPC后heNOS基因的表达情况以及对离体平滑肌细胞(SMC)增殖的影响。【方法】以不同MOI值的腺病毒载体Ad．CMV-EGFP体外转染EPC，流式细胞仪(FACS)检测转染阳性率。Ad．CMV-heNOS以MOI=50转染EPC后，RT-PCR检测细胞中heNOS mRNA的转录水平，免疫组化染色法、Western-Blot法检测heNOS蛋白的表达水平，硝酸还原酶法检测细胞培养液中NO水平。将EPC按干预方式不同分为Ad．CMV-hcNOS转染组、空载体Ad．CMV转染组以及PBS“转染”组，各组EPC与大鼠SMC(EGFP标记)混合培养72h后，FACS检测混合细胞中SMC所占百分比(SMC％)；将各组EPC与SMC非混合性共培养24h后，FACS检测SMC细胞周期变化及细胞凋亡情况。【结果】随着MOI值逐渐增大，Ad．CMV-EGFP体外转染EPC的转染阳性率也逐渐增高，MOI=50时阳性率可达85％以上，MOI再增大，阳性率增高有限。RT-PCR、免疫组化、Western-blot等方法证明：MOI=50时Ad．CMV-heNOS可成功将heNOS基因转入EPC中并有正确的表达；而且与对照组相比，Ad．CMV-heNOS转染组的EPC培养液中NO含量更高。EPC经不同方式干预后与SMC以1：1混合培养72h，在细胞总数相似的情况下，Ad．CMV-heNOS转染组的SMC％较其它两组明显降低(P＜0.05)。EPC与SMC经非混合性共培养24h后，与阴性对照相比，Ad．CMV-heNOS转染组更多SMC仍处于G1期(P＜0.01)；空载体转染组和PBS“转染”组SMC细胞周期相似，处于G1期的比例低于Ad．CMV-heNOS转染组(P＜0.05)而高于阴性对照(P＜0.01)；同时还发现Ad．CMV-heNOS转染组有少量SMC(1％左右)出现凋亡现象。【结论】MOI=50时重组腺病毒载体转染EPC可获最佳量效比。Ad．CMV-heNOS能成功将heNOS基因转入EPC中并正确表达，且能催化更多NO生成。在体外共培养体系中，EPC可干扰SMC细胞周期的运行，抑制SMC的增殖，而转染heNOS基因的EPC这种抑制作用更强。第五部分转染heNOS基因的内皮祖细胞靶向移植治疗大鼠动力型肺动脉高压的实验研究【目的】研究转基因EPC经颈静脉注射途径移植入动力型肺动脉高压(HPH)大鼠体内后的存活及分布规律：转染heNOS基因的EPC能否在大鼠体内正确表达heNOS、并起到缓解肺动脉高压病理改变、降低肺动脉压的作用。【方法】建立大鼠HPH模型，将转染EGFP基因的EPC经颈静脉注射途径移植入HPH大鼠体内，在不同时间点(1d、3d、1w、2w)处死动物并取出肺、心、肝、肾行快速冰冻切片，观察绿色荧光细胞在各器官中的分布规律。将40只HPH大鼠按干预方式随机分为：转染heNOS基因的EPC移植组(组Ⅰ)、转染空载体的EPC移植组(组Ⅱ)、单纯EPC移植组(组Ⅲ)和PBS液移植组(组Ⅳ)，干预后2w测定各组大鼠体、肺动脉收缩压，检测RV／(LV+S)以及肺肌型小动脉RMA和SMA％；通过RT-PCR和Western Blot检测各组大鼠肺组织中heNOS表达；同时以WesternBlot比较组Ⅰ大鼠肺与心、肝、肾中heNOS表达水平的差异。【结果】EGFP标记的EPC移植后1d和3d肺组织切片中可发现较多荧光细胞，1w以后荧光强度逐渐减弱，2w时仍可见荧光细胞分布于肺小血管壁上，有的已成为血管内皮的一部分；而各时间点心、肝、肾组织切片中仅在个别视野偶见荧光细胞。各组大鼠经不同方式干预2w后，组Ⅰ肺动脉收缩压、RV／(LV+S)、RMA和SMA％均显著低于组Ⅳ(P＜0.01)；组Ⅱ、Ⅲ之间各指标差异不显著(P＞0.05)，但均明显低于组Ⅳ(P＜0.05)而高于组Ⅰ(P＜0.05)(SMA％除外)；四组大鼠体动脉收缩压无显著性差异(P＞0.05)。RT-PCR和Western Blot证实组Ⅰ大鼠肺组织中有heNOS的正确表达，而Western Blot同时发现：与肺组织相比，组Ⅰ大鼠心、肝、肾中仅有微量heNOS表达。【结论】转基因EPC经颈静脉注射途径移植入HPH大鼠体内后具有明显的肺组织靶向性，且在肺组织中可以较长时间存活(＞2w)并正确表达目的基因。单纯EPC移植可以有效缓解动力型肺动脉高压的进展；而heNOS基因转染结合EPC移植则进一步加强了这种作用，甚至可能部分逆转肺动脉高压的病理改变。更多还原

【Abstract】 Part 1 Construction of recombinate adenovirus vector carrying gene heNOSObjective To construct a recombinate adenovirus vector carrying gene heNOS as wellas a recombinate adenovirus vector carrying gene EGFP as a reporter.Methods heNOS gene were cloned into adenovirus shuttle vector PSUCMV bystandard procedure to produce recombinate plasmid PSUCMV-heNOS. AfterPSUCMV-heNOS was identified, it was transferred into adenoviral packaging cell （293cell） with adenovirus Pbhge3 by lipofectamine 2000 to construct vectorAd.CMV-heNOS. Then the virus were amplified and purified. At the same time,recombinate adenovirus vector Ad.CMV-EGFP carrying gene EGFP was established inthe same way.Results The recombinate PSUCMV-heNOS was correctly constructed and confirmedby restriction endonucleas analysis and DNA sequencing analysis. The result of PCRdemonstrated the vector Ad.CMV-heNOS was successfully constructed. And theconcentration of Ad.CMV-heNOS and Ad.CMV-EGFP reached to 5.15×l0⁹pfu/ml and3.8×l0⁹pfu/ml respectively.Conclusion The high-titer recombinate replication-defective adenovirus vectorcarrying gene heNOS and EGFP were successfully constructed and purified.Part 2 Isolation, culture and identification of endothelial progenitor cells from rat bone marrowObjective To investigate the methods of isolating and culturing endothelial progenitor cells（EPCs） from rat bone marrow, as well as their ability of differentiating into endothelial cells.Methods The mononuclear cells were isolated from rat bone marrow by density gradient centrifugation and cultured for 48h. Then the suspending cells were cultured in the selective culture medium（with the supplement of VEGF, bFGF, IFG-1 and EGF） for2w. The attached cells were stained with Dil-ac-LDL and FITC-UEA-1, and the expression of vWF and Flk-1 was also assessed by immunofluorescence. The percentage of EPCs was analyzed by fluorescence-activated cell sorting（FACS）. Their function of producing nitric oxide（NO） was evaluated by quantification of NO level in culture medium.Results The secondarily attached cells stretched and exhibited the clonal morphology after 3 and 5 days’ inducing culture respectively. The cells proliferated faster after 7-10 days’ incubation when cord-like structure was observed. After 2 weeks’ induction, most of the cells exhibited multangular morphology. More than 70% attached cells took up Dil-ac-LDL, bound FITC-UEA-1 （double positive fluorescence）, and expressed vWF and Flk-1 （immunofluorescence）. According to the results from FACS, the percentage of vWF+ and Flk-1+ cells were 77.93% and 81.50% respectively. The level of NO in the selective culture medium of secondarily attached cells was higher than that of the primarily attached cells but lower than that of the mature endothelial cells （P＜0.05）. Conclusion EPCs were enriched in rat bone marrow and may exhibit some of the characteristics endothelial cells owned after inducing culture in vitro. Part 3 Establishment of hemodynamic pulmonary hypertension（HPH） in ratsObjective To explore the influence on the systemic and pulmonary artery pressure, right ventricular hypertrophy and pulmonary vascular structural remodeling in rats after removal of different proportion of lungs. So as to establish a new model of hemodynamic pulmonary hypertension in rats.Methods Sixty-eight Wistar rats were randomly divided into group I （total resection of right lung）, group II （total resection of left lung）, group III （resection of the upper and middle lobe of right lung）, group IV （sham operated） and group V （control）. Four weeks later, the systolic systemic artery pressure（sSAP） and systolic pulmonary artery pressure（sPAP） were measured. The ratio of right ventricular mass to left ventricular plus septal mass [RV/（LV+S）] was detected. The relative medial area（RMA） of small pulmonary muscularized arteries（SMA） and the percentage of SMA to all the small pulmonary vessels （SMA%） were measured and calculated.Results There were 66.7% rats survived 4 weeks after the operation（12/18） in group I. And the weight of group I is less than that of group IV and V（P<0.05）. Group I hadobviously higher sPAP, RV/（LV+s）, RMA and SMA%（P<0.01）. There was no difference in all the indexes between group II and III（P>0.05） and between group IV and V（P>0.05）. But group II had higher RMA than group IV and V（P<0.05） while group III had higher sPAP and RMA than group IV and V（P<0.05）. There was no difference in sSAP among the five groups（P>0.05）.Conclusion Four weeks after the total resection of right lung, pulmonary artery hypertension, right ventricular hypertrophy and small pulmonary artery structural remodeling were emerged. But sSAP was not influenced. Compared with other HPH models, this method was easily performed, the mortality of rats was relatively low and the pulmonary hypertension was quickly induced.Part 4 Transfection of Ad.CMV-heNOS to EPCs and its inhibition effects on the proliferation of smooth muscle cells （SMCs） in vitroObjective To explore the transfection efficiency of adenovirus vector to EPCs from rat bone marrow, the expression of heNOS after the transfection of Ad.CMV-heNOS to EPCs, and their inhibition effects on SMCs in vitro.Methods After the transfection of Ad.CMV-EGFP to EPCs with different MOI, we used FACS to evaluate the transfection efficiency. Then we transfected Ad.CMV-heNOS to EPCs with MOI 50:1. After transfection, heNOS mRNA transcription was detected by RT-PCR, heNOS expression was detected by immunohistochemical staining and Western-blot. The level of NO in culture medium was also measured. Then we divided EPCs into 3 groups including Ad.CMV-heNOS transfected group, Ad.CMV transfected group and PBS group. All the groups of EPCs were mixedly co-cultured with EGFP-labeled SMCs for 72 hours. Then SMCs% were detected by FACS. We used "inserts" containing PET membrane to establish a co-culture system in which EPCs and SMCs can influence each other without mixing. After 24 hours’ co-culture, FACS measured the alteration of SMCs cell cycle and apoptosis.Results The transfection efficiency of Ad.CMV-EGFP to EPCs increased with MOI. When MOI reached 50, the transfection efficiency increased to more than 85%. From then on, as MOI increased, the transfection efficiency increased limitedly. So we choose50:1 as the most suitable MOI. Using RT-PCR, immunohistochemical staining and Western-blot, we found that heNOS gene could be correctly transfected into EPCs. After the transfection, the level of NO in the culture medium was also higher than control group. After the mixed co-culture of EPCs with SMCs for 72h, the SMC% of the Ad.CMV-heNOS transfected group was lower than that of the other two groups（P<0.05）. After non-mixed co-culture of EPCs with SMCs for 24h, there were obviously more SMCs of Ad.CMV-heNOS transfected group still in G₁ stage（P＜0.01） than the other groups. What’s more, about 1% SMCs showed up apoptosis in Ad.CMV-heNOS transfected group.Conclusion The most suitable MOI for transfection in the research is 50:1. heNOS gene could be successfully transfected into EPCs by Ad.CMV-heNOS, followed by correct expression and effects of catalyzing more NO. In the co-culture system in vitro, EPSc themselves could interfere with the cell cycle of SMCs and inhibit their proliferation. Furthermore, the transfection of gene heNOS could enhance this effects. Part 5 Targeted transplantation of heNOS-transfected EPCs into rats with hemodynamic pulmonary hypertensionObjective To explore the survival, disposition of EPCs and expression level of heNOS in vivo after injection of heNOS-transfected EPCs to HPH rats through jugular veins. To investigate if heNOS-transfected EPCs could relieve the pathologic changes of HPH and decrease the pulmonary arterial pressure.Methods After the HPH model was established, we transplanted EGFP-labeled EPCs to the rats through jugular vein. The rats were put to death and the organs such as lung, heart, liver and kidney were taken out and made into frozen section on 1 day, 3 days, 1 week and 3 weeks after the operation respectively. Then we observed the disposition of green fluorescence cells. Forty HPH rats were randomly divided into group I （Ad.CMV-heNOS transfected EPCs were transplanted）, group II （Ad.CMV transfected EPCs were transplanted）, group III （EPCs were transplanted） and group IV （only PBS was transplanted）. Two weeks later, we measured sSAP, sPAP, RV/（LV+s）, RMA and SMA%. We detected the heNOS expression by RT-PCR and Western-blot. For group I, we even compared the heNOS expression in lung with in heart, liver and kidney by Western-blot.Results 1 day and 3 days after the EGFP-labeled EPCs transplanted, a lot of fluorescence cells could be seen in the frozen sections of lung. One week later, the fluorescence intensity decreased. Two weeks after the transplantation, there were still a few fluorescence cells locating on the wall of small pulmonary arteries. And some of them had become a part of the vessel endothelium. However, there were very few fluorescence cells in heart, liver and kidney all the time. Two weeks after the intervention, group I displayed obviously lower sSAP, RV/（LV+s）, RMA and SMA% （P＜0.01）. There was no difference between group II and group III（P>0.05）. And the index of group II and III were lower than that of group IV but higher than group I（P<0.05） with the exception of SMA%. There was no difference in sSAP among the four groups. The expression of heNOS in lungs was successfully proved by RT-PCR and Western-blot. At the same time, there was very little expression of heNOS in heart, liver and kidney.Conclusion After injecion of heNOS-transfected EPCs to HPH rats through jugular vein, EPCs could be targeted into lung, correctly express heNOS and survive for more than 2 weeks. Transplantation of untransfected EPCs could relieve the progression of pulmonary hypertension obviously. And heNOS-transfected EPCs could strengthen this effect. In addition, it could even partially reverse pathologic change of hemodynamic pulmonary hypertension.更多还原