小鼠急性病毒性心肌炎心肌HO-1mRNA及其蛋白表达

Expression of oxygenase-1 protein and mRNA on mouse acute viral myocarditis caused by coxsackie viruses B3

目的:探讨小鼠急性病毒性心肌炎(viral myocarditis,VMC)心肌细胞血红素氧合酶-1(heme oxygenase-1,HO-1)基因及其蛋白表达的动态变化。方法:72只清洁级近交系4-6周龄雄性BALB/c小鼠随机分为2组:心肌炎组(V组)40只(腹腔注射CVB3病毒),实验对照组(C组)32只。于病毒接种后第4d、8d、15d、21d分别采血后处死小鼠并留取心脏标本。采用免疫组化法和原位杂交法检测心肌HO-1蛋白及mRNA表达,分光光度计法分间接测定COHb含量,光镜及透射电镜观察心肌组织病理及细胞超微结构改变。结果:(1)心肌炎组可见炎症细胞浸润,心肌细胞可见大面积坏死,晚期可见钙化灶形成;电镜下可见肌原纤维溶解断裂,线粒体膜消失,含溶酶体丰富的单核巨噬细胞浸润,对照组小鼠心肌未见上述变化。(2)血COHb含量变化:心肌炎组小鼠血COHb含量在第8d和第15d均较对照组明显增高,2组差异显著(0.047±0.005和0.031±0.004;0.076±0.006和0.030±0.005,P<0.01)。(3)血清cTnI含量变化:V组小鼠血清cTnI含量与C组比较除第21d无明显差别外,其余各时点均高于C组,差异有统计学意义,P<0.01。(4)HO-1免疫组化结果:心肌炎组HO-1蛋白均成阳性表达,各时点心肌HO-1平均吸光度值均高于对照组(P<0.01)。(5)HO-1mRNA原位杂交结果:心肌炎组HO-1mRNA原位杂交染色各时点均呈阳性表达,各时点吸光度值均高于对照组(P<0.01)。结论:病毒性心肌炎可诱导心肌组织HO-1mRNA及其蛋白的表达,可能是受损心肌发挥自我保护作用机制之一。

AIM： To observe the dynamic variation of oxygenase - 1 protein and mRNA on mouse acute viral myocarditis caused by coxsackie viruses B3. METHODS- A total 72 inbred male BALB/c mice of 4 - 6 weeks were divid- ed randomly into 2 groups as follows： 32 mice were inoculated intraperitoneally （ip） with virus free 1640 culture solution 0.1 mL on day 0 as blank group （C） ; 40 mice were ip 0.1 mL tissue culture infectious dose 50 （TCID50 is 10-436/mL） coxsackie viruses 133 （CVB3） on day 0 as VMC group （V）, then each mouse in both groups was ip0. 1 mL NS every day. 8 mice in each of C group and V group were sacrificed on 4, 8, 15, and 21 d respectively after infections. The blood speci- mens gathered by taking out the eyeballs of mice were tested for the content of carboxyhemoglobin （COHb） using spectrophotometer method. The heart tissue slides were also stained by immunohistochemistry （IHC） for HO - 1 and in situ hybridization （ISH） for HO - 1 mRNA. The histological and ultrastructure changes were observed under light microscope and electron microscope. RESULTS ： （ 1 ） The histopathological changes of myocardial cells ： many inflammatory cells were found in the heart and large area myocardial cells necrosis was observed under light microscope. The inflammatory area was reduced at late stage in the heart of mouse in group V, while the myocardium in group C was normal. （2） The myocardial observation by electron microscopy： the myofibril in group V was dissolved and mitochondrial membrane disappeared, mononuclear cell infiltration was also observed under electron microscopy, which contained many lysosomes. The myocardial cells in group C were normal. （3） The changes of blood COHb level ： compared with group C, the group V COHb level showed significantly higher on the day 8 and day 15 after CVB3 innoculation （0.047 ±0. 005 vs 0. 031 ± 0. 004 ; 0. 076±0. 006 vs 0. 030 ±0.005, P 〈0. 01）. No obvious change in group C was observed. （4） The result of HO- 1 IHC staining： myocardial cells had positive expression in group V, group C was negative. The absorbance （A） values in group V was significantly higher than that in group C （P 〈0. 01 ） at different time points. （5） The result of HO - 1 ISH was similar to HO- 1 IHC. The A values in group V was all higher than that in group C （P 〈0. 01 ）. CONCLUSION： Viral myocarditis caused by coxsackie viruses B3 induces the expression of HO - 1 mRNA and protein, and these expressions may play selfprotection in inflammation injury in myocardial cells.