重组6型腺相关病毒介导的绿色荧光蛋白在野百合碱诱导肺动脉高压大鼠肺组织中的表达

Expression of green fluorescent protein in lung tissue of monocrotaline-induced pulmonary arterial hypertension rat transduced by recombinant adeno-associated virus type 6

目的探讨重组6型腺相关病毒(r AAV6)介导的绿色荧光蛋白Zs Green在野百合碱诱导的动脉型肺动脉高压(PAH)大鼠中的转染效果及表达,为进一步利用此病毒载体进行PAH机制研究提供实验依据。方法将24只雄性Sprague-Dawley(SD)大鼠随机分为正常对照组、PAH模型组和PAH病毒组,每组8只。PAH模型组和PAH病毒组大鼠颈背部皮下单次注射野百合碱60 mg/kg建立PAH模型,造模后1d PAH病毒组大鼠气管内注射100μl含1×1011vg r AAV6-Zs Green液,PAH模型组大鼠气管内注射等体积PBS,正常对照组大鼠颈背部皮下注射等体积生理盐水,且气管内注射等体积PBS。造模3周后各组大鼠称重,采用右心导管法检测右室收缩压(RVSP)和平均肺动脉压(m PAP)。取大鼠新鲜肺组织制备冰冻切片,采用苏木精伊红染色检查大鼠肺组织病理学改变并计算肺动脉中膜厚度(WT)百分比,采用免疫荧光法观察大鼠肺组织中Zs Green荧光强度变化。收集游离心脏标本测定右心室肥厚指数(RVHI)。采用Western blot法检测各组大鼠肺组织中Zs Green蛋白的表达。结果 PAH模型鼠体重明显低于正常对照组,造模大鼠RVSP、m PAP、RVHI和WT百分比均明显高于正常对照组大鼠,提示野百合碱诱导PAH大鼠造模成功。荧光显微镜下可见PAH病毒组大鼠肺组织中Zs Green平均荧光强度(A值)为33.471±5.147,明显高于正常对照组的4.581±1.871和PAH模型组的4.167±1.249,差异均有统计学意义(均P<0.05),并在肺组织中呈广泛分布,包括肺血管内中膜。Western blot检测显示,正常对照组、PAH模型组和PAH病毒组大鼠肺组织中Zs Green蛋白相对表达量(灰度值)分别为0.072±0.017、0.067±0.019和0.317±0.062,PAH病毒组大鼠组肺组织Zs Green蛋白相对表达量明显高于正常对照组和PAH模型组,差异有统计学意义(均P<0.05)。苏木精伊红染色显示,PAH病毒组大鼠肺组织炎性细胞浸润及肺组织结构紊乱并未比PAH模型组大鼠明显增加。结论 r AAV6可介导Zs Green转染野百合碱诱导的PAH大鼠,提示r AAV6-Zs Green可能成为PAH基因治疗的有效载体。

Objective To explore the transfection efficiency and expression of green fluorescent protein in lung tissue of monocrotaline（ MCT） induced pulmonary arterial hypertension（ PAH） rat transduced by recombinant adeno-associated virus type 6（ r AAV6）. Methods Twenty-four male Sprague-Dawley rats were randomly divided into three groups（ n = 8 for each group）： control group,PAH model group,and PAH virusgroup. Rats from PAH model group and PAH virus group received single subcutaneous injections of monocrotaline（MCT,60 mg/kg）,while isovolumetric saline was injected subcutaneously into the rats of the control group. One day later,1×1011 virus genomes rA AV6 carrying report gene ZsG reen（ green fluorescent protein） were intratracheally administrated of PAH virus group. Meanwhile,isovolumetric sterile phosphate buffered solution（ PBS）was given to the rats of PAH model group and control groups. After 21 days,all rats were anaesthetized and right heart catheterization was conducted to measure right ventricular systolic pressure（ RVSP） and mean pulmonary arterial pressure（ mP AP）. Lung tissues and heart were obtained for further Western blot detection and histopathology analysis. Results The weight was significantly lower in monocrotaline stimulated rats compared to normal control rats. Conversely,RVSP and mP AP detected by right heart catheterization,and right ventricular hypertrophy index were elevated obviously in the PAH model and PAH virus group rats,which represented the successful establishment of MCT-PAH rat model. The spontaneous green fluorescence was widely observed in the lung tissue of the PAH virus groups rats（33. 471±5. 147）,and rarely in the rats of the PAH model（ 4. 167± 1. 249） and control groups（4. 581± 1. 871）. In addition,the level of green fluorescent protein（ ZsG reen） transduced by rA AV6 was found to be significantly higher in PAH model group rats（ 0. 317± 0. 062 vs. 0. 067± 0. 019）（ P 0. 05）. There was no specific histologic finding for more inflammatory cells infiltration and structure destruction in PAH virus rat lung tissue than in PAH model rats. Conclusions Recombinant AAV serotype 6 vector is demonstrated to be efficient and low-toxicity in ZsG reen transgene expression in the lung tissue of MCT induced PAH rats,and rA AV6-ZsG reen may act as a potential good vector for gene therapy of PAH in the future.