腺病毒介导的p21基因转染肺动脉高压大鼠模型的研究  

Effect of p21 gene transfection mediated by replication deficient adenovirus on the pulmonary hypertensive rat model

作　　者：陈少杰[1] 王一彪[1] 陈欧[1] 朱晓波[1] 马宇[1] 

机构地区：[1]山东大学第二医院儿科,济南250033

出　　处：《中华儿科杂志》2008年第2期139-142,共4页Chinese Journal of Pediatrics

基　　金：山东省医药卫生科研项目（2005HZ041）

摘　　要：目的探讨腺病毒介导的抗增殖性p21基因转染对左向右分流肺动脉高压大鼠模型的肺动脉高压形成的影响。方法将携带目的基因的重组腺病毒AdCMV—p21采用脂质体转染法包装、扩增，并稀释成滴度为1．67×10^8pfu／L的溶液。将大鼠随机分为空白对照组（n=10）、模型组（n=15）、实验组（n=10）、实验对照组（n=10）。模型组和实验组采用套管连接法建立左向右分流肺动脉高压模型，实验组和实验对照组均采用气管吸入法转染AdCMV—p21。测量各组大鼠平均肺动脉压（mPAP）、右心室平均压（mRVP）、右心肥厚指数（RVHI），比较肺高压程度，左肺行免疫组化染色，观察转染效果，右肺HE染色观察肺动脉形态学改变，计算管壁厚度与血管外径比值（WT％）。结果模型组大鼠的mPAP、mRVP、RVHI、WT％较空白对照组高，差异有统计学意义（P〈0．05），表示肺动脉高压模型建立成功；实验组和实验对照组经免疫组化染色可见大鼠肺血管壁平滑肌细胞核内含棕黄色颗粒，表示转染重组腺病毒成功，实验组转基因阳性细胞率为（42．8±11．6）％，与实验对照组相比差异无统计学意义（P〉0．05）；实验组大鼠mPAP为（20．06±3．40）mmHg（1mmHg=0．133kPa），mRVP为（22．53±2．53）mmHg，管壁厚度为（9．64±2．14）μm，WT％为（30．8±3．5）％，与模型组各指标相比水平较低（P〈0．05），与实验对照组和空白对照组相比水平偏高（P〈0．05）。结论腺病毒介导的p21基因可通过气管吸入法成功转染至肺高压大鼠模型的肺动脉平滑肌细胞内，并可在一定程度上抑制肺动脉高压的发展。Objective To investigate the feasibility of transferring p21 gene into lung tissue with recombinant adenovirus with full-length cDNA of p21 inserted in the Wistar rat model of pulmonary hypertension （PAH） induced by left-to-right shunt, study the expression of the desired gene in vivo, find if overexpression of desired gene can inhibit pulmonary hypertension. Methods With full-length cDNA of p21 transfected HEK293 cells with clonfectin, and was packed, amplified in order to obtain the high-titer recombinant adenovirus （AdCMV-p21）. The infection titer was determined by TCID50 method and was diluted into 1.67 × 10^8 pfu/L Wistar rats were randomly allocated to control group （n = 10）, model group （n = 15）, test group （n = 10） and test control group （n = 10）. In model group and test group left-to-right shunt pulmonary hypertension was developed by using cuff technique. AdCMV-p21 was transfected into test group and test control group using tracheal inhalation. The mPAP, mRVP and RVHI were measured and compared between every two groups. The left lung was immunohistochemically stained to observe the result of transfection. The right lung was HE stained to observe morphological changes in arteria pulmonalis and calculate WT%, Results The mRVP, mPAP and WT% in model group and test group were significantly higher than those in control （ P 〈 0. 05 ）, which suggested that the rat model of PAH was established successfully. Brown spots in the nucleus of VSMCs of pulmonary artery were seen in test group and test control group, which indicated that AdCMV-p21 was transfected successfully. The rate of transfected cells in test group was （42.8 ±11.6）% ,which was equal to that of test control group （P〉0.05）. In test group, the mPAP was （20. 06 ± 3.40） mm Hg （ 1 mm Hg = 0. 133 kPa）, mRVP was （22.53 ± 2. 53 ） mm Hg,WT% was （30. 8±3.5 ） %, which were significantly lower than those in model group （ P 〈 0. 05 ）, but higher than those in control group and test control grou

关 键 词：细胞周期蛋白质类 腺病毒科 高血压 肺性 基因疗法 疾病模型 动物 

分 类 号：R543.205[医药卫生—心血管疾病] R735.202[医药卫生—内科学]