外源性Smad7基因小鼠肺内定向表达的研究

Expression of exogenous Smad7 gene in lung:in vivo study with mice

目的探讨放射线诱导Egr-1基因启动子调控腺病毒介导的Smad7基因在小鼠肺内表达的规律性、其表达的细胞内定位及小鼠耐受性。方法将Egr-1基因启动子的放射敏感元件和Smad7 cDNA包装到复制缺陷型腺病毒内,制备成AD．Egr-Smad7。120只C57BL／6J小鼠随机分组进入实验,每组6只。小鼠气管内给药,24 h后单次全胸放射。(1)66只分为11组:生理盐水组、AD．Egr-Smad7及未包装Egr-Smad7腺病毒(各分5个不同病毒滴度)组,观察给药后呼吸困难、紫绀情况及72 h内死亡情况,研究重组腺病毒滴度与小鼠急性耐受性关系。(2)36只分为6组:空白对照(C)组、单纯放射(R)组、AD．Egr-Smad7未放射(RA)组、AD．Egr-Smad7放射(RAR)组、未包装Egr-Smad7病毒未放射(AV)组及未包装Egr-Smad7病毒放射(AVR)组,放射8 Gy,5 h后免疫组织化学法检测外源性Smad7基因肺内表达的细胞内定位。(3)168只分为28组:C组、R组、RA组、BAR组、AV组及AVR组(各组按照腺病毒滴度又分为1×108、5×108、1×109 pfu／0．1 ml组,按照放射后时间又分为6 h及9 h组),放射8 Gy,研究重组腺病毒滴度与外源性Smad7基因肺内表达的关系。(4)324只分为54组:C组、R组、RA组、RAR组、AV组及AVR组(各组按照射后时间又分为放射后0、1、2、3、5、7、9、12及15 h组),放射8 Gy,研究照射后不同时间Smad7基因肺内表达的时效关系。(5)126只分为21组:C组、R组、RA组、RAR组、AV组及AVR组(各放射组按放射剂量又分为5、8、10、12、15及20 Gy组),照射后5 h研究Smad7基因肺内表达与放射剂量的关系。采用Western印迹法检测小鼠肺组织Smad7蛋白表达。结果重组腺病毒滴度在109pfu及以下时,小鼠可安全耐受,各组6只均存活,而5×109pfu及以上组小鼠5只以上死亡。AVR组外源性Smad7基因在肺泡上皮细胞、血管内皮细胞及细支气管上皮细胞胞浆内均有明显表达,而各对照组未见表达。外源性Smad7基因表达随重组腺病毒滴度升高而增加(P<0．01);单次放射0-12 Gy间其表达随放射剂量升高而增加,15 Gy时下降;放射后2-9 h其表达量最高(P<0．05),而后降低,15 h降至接近正常水平。结论重组腺病毒滴度在109pfu及以下时,小鼠可安全耐受。以腺病毒为载体,辐射诱导Egr-1启动子能够调控外源性Smad7基因在小鼠肺组织各种细胞胞质内广泛表达,外源性Smad7基因表达随重组病毒滴度升高而增加,并与放射剂量及放射后间隔时间有关。

Objective To investigate the regular pattern of expression, and expression localization in lung of Smad7 gene mediated by adenovirus and regulated by irradiation via early growth response （Egr） fector-1 promoter and the safe dose of the recombinant adenovirus. Methods The radio-inducible element of Egr-1 gene promoter and cDNA encoding Smad7 were enclosed into the replication-defective adenovirus, thus establishing the recombinant adenovims AD. Egr-Smad7. 720 C57BL/6J mice were randomly divided into 6 different groups： Group C （blank control group, receiving normal saline only）, Group R （receiving radiation with the dose of 8 Gy at the whole chest only）, Group AR （undergoing intratracheal instillation of AD. Egr-Smad7 without radiation）, Group RAR （ undergoing intratracheal instillation of AD. Egr-Smad7 and then radiation 24 hours later）, Group AV （receiving the replication-defective adenovims without radiation）, and Group AVR （receiving both the replication-defective adenovims and radiation）. The foUowing studies were conducted. （1） Tolerance to the adenovims. Sixty mice were divided into 11 equal subgroups： Subgroups C, and Subgroups RA and AV receiving AD. Egr-Smad7 at 5 different concentrations： 10^8, 5 ×10^8, 10^9, 5 × 10^9, and 10^10 pfu/0. 1 ml respectively. The mice were observed for the symptoms of dyspnea and cyanosis and death within 72 hours. （2） Localization of the expression of exogenous Smad7 in the lungs. 36 mice were divided into 6 equal Subgroups： C, R, RA, RAR, AV, and AVR, the adenovirus titre being 10^9 pfu/0. 1 ml. Five hours after the irradiation all mice were killed to take out their lungs. （3） Relationship between the titre of AD. Egr-Smad7 and expression of Smad7 in lung. 168 mice were divided into 28 Subgroups： C, R, RA, RAR, AV, and AVR, the titre of virus being 10^8, 5 × 10^8, or 10^9 respectively, and the time after irradiation being 6 hours or 9 hours. The mice were killed at specific time points and their lungs were taken out. （4） Time-effect on Smad7 expression in lung. 134 mice were divided into 54 equal Subgroups： R, RA, RAR, AV, and AVR. The mice were killed 0, 1, 2, 3, 5, 7, 9, 12, and 15 hours after irradiation and their lungs were taken out. （5） Dose-effect of irradiation on the Smad7 expression in lung. 126 mice were divided into 21 Subgroups： C, R, RA, RAR, AV, and AVR, the irradiation dose being 5, 8, 10, 12, 15, or 20 Gy. Five hours after the irradiation the mice were killed and their lungs were taken out. Western blotting was used to detect the mRNA expression of Smad7 in lung and the protein expression of Smad7 in lung was examined by immunohistocbemistry. Results （1） The concentration of 10^9 pfu and lower was safe, no matter the adenovirus contained Egf-Smad7 or not. （2） mRNA expression of exogenous SmaD7 was found in the cytoplasm of bronchial epithelium cells, alveolar epithelial cells, and blood vessel endothelial cells in Subgroup RAR but not in the other subgroups. （3） Western blotting showed that the protein expression of Smad7 was obvious in the lung tissues of Subgroup RAR, being more obvious 6 hours after irradiation than 9 hours later （ P 〈 0. 01 ）, AD. Egr-Smad7 dosedependently increased the protein expression of exogenous Smad7 in Subgroup RAR （ P 〈 0.01 ）, and that the protein expression of Smad7 was very weak in the other subgroups in comparison with Subgroup RAR （ all P 〈 0.01 ）. （4） The protein expression of Smad7 in the lung tissues of Subgroup RAR began to increase 1 hour after irradiation, peaked 5 hours later, then gradually decreased, returned to the level 1 hour later by the 12 th hour after irradiation, and almost disappeared 15 hours later. （5） The protein expression of Smad7 in Subgroup RAR increased after irradiation in a dose-dependent manner （P 〈0.01 ）, with a peak when the dose was 12 Gy. Conclusion γ irradiation markedly induces the expression of exogenous Smad7 gene mediated by recombinant adenovirus in the cells of lung tissues dose-dependently that is correlated with time interval as well. It is important to look for a safe titre of the recombinant adenovirus. Blocking the signal transduction of transforming growth factor-β may become a novel strategy for gene therapy aiming at preventing irradiation-induced lung fibrosis.