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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 regularity of expression of adenovirus-mediated Smad7 gene in lungs induced by radiation-induced Egr-1 gene promoter, its intracellular localization and tolerance in mice. Methods The Egr-1 gene promoter and Smad7 cDNA were packaged into replication-deficient adenovirus to prepare AD. Egr-Smad7.120 C57BL / 6J mice were randomized into groups of 6 mice per group. Intratracheal administration of mice, 24 h after a single full chest radiation. (1) 66 were divided into 11 groups: saline group, AD. Egr-Smad7 and Egr-Smad7 adenovirus (each with 5 different virus titer) were used to observe the dyspnoea, cyanosis and the death within 72 h after administration. The titer of recombinant adenovirus was compared with that of mice Sexual relations. (2) Thirty-six mice were divided into 6 groups: blank control group (C), radiotherapy alone group (R), AD Egr-Smad7 non-radioactive (RA) group, AD. Egr-Smad7 radioactive (RAR) group, untreated Egr-Smad7 non-radioactive (AV) group and uncoated Egr-Smad7 viral radioactive (AVR) group were irradiated with 8 Gy for 5 h, then the expression of exogenous Smad7 Intracellular localization of genes in the lungs. (3) 168 were divided into 28 groups: C group, R group, RA group, BAR group, AV group and AVR group (each group was divided into 1 × 108, 5 × 108, 1 × 109 pfu /0.1 ml group, which were divided into 6 h and 9 h group according to the time after radiation. The radiation dose was 8 Gy. The relationship between recombinant adenovirus titers and the expression of exogenous Smad7 gene in lungs was studied. (4) 324 were divided into 54 groups: C group, R group, RA group, RAR group, AV group and AVR group (each group was divided into 0,1,2,3,5,7 , 9, 12 and 15 h groups), 8 Gy were irradiated to study the time-dependent expression of Smad7 gene in lungs at different time after irradiation. (5) 126 were divided into 21 groups: C group, R group, RA group, RAR group, AV group and AVR group (radiation group was divided into 5,8,10,12,15 and 20 Gy group ), And the relationship between the expression of Smad7 gene in lungs and the radiation dose was studied 5 hours after irradiation. Western blotting was used to detect the expression of Smad7 protein in mouse lung tissue. Results The recombinant adenovirus titer of 109pfu and below, the mice can be safely tolerated, each group of six survived, and 5 × 109pfu and above mice died of more than 5. The exogenous Smad7 gene in AVR group was significantly expressed in the cytoplasm of alveolar epithelial cells, vascular endothelial cells and bronchiolar epithelial cells, but not in each control group. The expression of exogenous Smad7 gene increased with the titer of recombinant adenovirus (P <0.01). The expression of Smad7 gene increased with the increase of radiation dose from 0-12 Gy in single radiation and decreased at 15 Gy. The expression level was highest at -9 h (P <0.05), and then decreased at 15 h, which was close to the normal level. Conclusion The recombinant adenovirus titer of 109pfu and below, the mice can be safely tolerated. Using adenovirus as a carrier, radiation-induced Egr-1 promoter can regulate the expression of exogenous Smad7 gene in the cytoplasm of various lung tissues of mice. The expression of exogenous Smad7 gene increases with the increase of recombinant virus titer, And with the radiation dose and the time interval after radiation.