烟草烟雾暴露对小鼠骨骼肌炎症的影响

发布时间：2018-09-19 21:00

【摘要】：目的烟草烟雾暴露可诱发肺部异常炎症反应导致肺气肿,同时可以引起其他部位的炎症,本文着重探讨烟草烟雾对骨骼肌炎症的影响。方法48只雄性6个月龄大小的昆明小鼠随机分为4组(每组12只)：12周正常对照组(A组),24周正常对照组(B组),12周烟雾暴露组(C组)；24周烟雾暴露组(D组)。通过单纯长期香烟烟雾被动吸入方式建立实验模型,观察： (1)小鼠大体情况的变化；解剖后肺脏病理形态变化；小鼠烟雾暴露前后体重的变化； (2)骨骼肌的变化：①取肺组织及骨骼肌组织制作病理切片,观察形态学改变；②取小鼠骨骼肌,通过蛋白印迹法(Western blot WB)检测胞浆中肌球蛋白重链亚型Ⅱβ链(Myosin heavy chain isoforms-Ⅱ β MHC-Ⅱ β)的改变,来显示肌纤维的变化。 (3)骨骼肌炎症介质的含量：应用酶联免疫吸附试验(Enzyme-linked immunosorbent assay ELISA)检测白细胞介素-8(Interleukin-8IL-8)、肿瘤坏死因子-α(Tumor necrosis factor-α TNF-α)的浓度。 (4)骨骼肌炎症调控基因的表达：①通过Western blot方法检测组蛋白去乙酰化酶2(Histone deacetylase2HDAC2)蛋白的表达；②应用逆转录聚合酶链反应(Reverse transcriptase polymerase chain reaction RT-PCR)法检测HDAC2的mRNA表达；③通过Western blot方法检测核因子-κB/P65(Nuclear factor-κ b/P65)蛋白的表达。结果(1)观察大体情况,C、D组小鼠对比A、B组,烟雾暴露组均显得毛发稀疏,无光泽,部分竖毛。吸烟12周后,C、D组小鼠体重偏低,与A、B组相比(P0.05),差异具有统计学意义；吸烟24周后,D组小鼠与B组比较,烟雾暴露组体重更明显偏低。解剖后观察肺脏大体病理形态发现：烟雾暴露组肺体积比正常组膨大,边缘钝圆,表面可见多个大小不等的大泡,肺脏回缩较差。 (2)观察香烟烟雾暴露小鼠肺组织病理切片发现：肺泡腔扩大、部分肺泡间隔断裂、肺泡腔融合、肺组织肺大泡形成伴周围炎症细胞浸润,表明已形成肺气肿模型。进一步观察小鼠骨骼肌组织病理切片发现：烟雾暴露组骨骼肌组织中有周围炎症细胞浸润伴随肌细胞形态结构改变,排列紊乱,局部性坏死病灶产生,表明肺气肿小鼠骨骼肌炎症模型已经建立。Western blot法检测A、B、C、D组小鼠骨骼肌组织中MHC-Ⅱβ链蛋白表达量分别为：1.2103±0.2197、1.1782±0.2046、1.1151±0.1956、0.6775±0.1645,正常对照组(A、B)、12周烟雾暴露组(C)与24周烟雾暴露组(D)之间的表达差异具有统计学意义(P0.05),说明MHC-Ⅱβ链蛋白的表达量在烟雾暴露组要低于正常对照组。 (3) ELISA法检测B、D组骨骼肌匀浆上清液中IL-8的平均浓度分别为：195.49和233.88,既工L-8浓度在烟雾暴露组大于正常对照组；检测炎症因子TNF-α的平均浓度为124.6651以及150.7417,既TNF-α浓度在烟雾暴露组大于正常对照组。 (4) RT-PCR法检测B、D组HDAC2的mRNA表达水平,通过测定CT值,用Paff1法计算：烟雾暴露组／正常对照组(Median)=0.64,证实HDAC2的mRNA表达水平在烟雾暴露组要低于正常对照组。Western blot法检测A、B、C、D组小鼠骨骼肌组织中HDAC2表达量分别为0.5942±0.1078、0.5366±0.2179、0.2978±0.1135、0.2290±0.0772,正常对照组(A、B)与12周烟雾暴露组和24周烟雾暴露组之间的表达差异具有统计学意义(p=0.000),显示HDAC2在烟雾暴露组明显低于正常对照组；检测NF-κB/P65蛋白表达量分别为：0.4901±0.2591、0.5166±0.2319、0.5263±0.2448、1.1230±0.3725,正常对照组(A、B)、12周烟雾暴露组与24周烟雾暴露组之间表达差异具有统计学意义(P(0.05),说明NF-κB/P65蛋白表达量在烟雾暴露组要高于正常对照组。结论：1、长期烟草烟雾暴露可以导致小鼠骨骼肌细胞空泡样变、萎缩及溶解,肌纤维中MHC-Ⅱ β链蛋白含量减少,显示可以导致骨骼肌结构和功能的改变 2、长期烟草烟雾暴露可以导致骨骼肌炎症因子(IL-8、TNF-α)及炎症调控基因NF-κB活性明显增强、HDAC2表达明显减弱,显示骨骼肌中存在着明显的炎症反应。
[Abstract]:Objective Tobacco smoke exposure can induce abnormal pulmonary inflammation and induce emphysema in other parts of the lung. This article focuses on the effect of tobacco smoke on skeletal muscle inflammation.
Methods 48 6-month-old male Kunming mice were randomly divided into 4 groups: 12-week normal control group (group A), 24-week normal control group (group B), 12-week smoke exposure group (group C) and 24-week smoke exposure group (group D).
(1) The changes of the general condition of the mice; the pathological changes of the lungs after dissection; the changes of the weight of the mice before and after exposure to smoke;
(2) Changes of skeletal muscle: 1) The lung and skeletal muscle tissues were taken to make pathological sections to observe the morphological changes; 2) The skeletal muscle of mice was taken to detect the changes of myosin heavy chain isoforms - II beta MHC - II beta in the cytoplasm by Western blot WB.
(3) Skeletal muscle inflammatory mediators: Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha).
(4) Expression of inflammation-regulating genes in skeletal muscle: 1. Histone deacetylase 2 (HDAC 2) protein was detected by Western blot; 2. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of HDAC 2 mRNA; 3. Western blot was used to detect the expression of HDAC 2 protein. The expression of nuclear factor kappa B/P65 (Nuclear factor- kappa b/P65) protein.
Results (1) After 12 weeks of smoking, the weight of mice in group C and D was lower than that in group A and B (P 0.05). After 24 weeks of smoking, the weight of mice in group D was lower than that in group B. The pulmonary gross pathological morphology showed that the lung volume of smoke exposure group was larger than that of normal group, with blunt edges, multiple bullae of different sizes on the surface and poor retraction of the lungs.
(2) Observing the pathological section of lung tissue of smoke exposed mice, it was found that alveolar cavity enlarged, part of alveolar septum ruptured, alveolar cavity fused, alveoli formed in lung tissue accompanied by infiltration of peripheral inflammatory cells, indicating that the model of emphysema had been formed. Peripheral inflammatory cell infiltration was accompanied by morphological and structural changes of myocytes, disordered arrangement, and localized necrosis, indicating that the model of skeletal muscle inflammation in emphysema mice had been established. The expression of MHC-II beta chain protein in skeletal muscle tissues of A, B, C, D mice was detected by Western blot: 1.2103 [0.2197], 1.1782 [0.2046], 1.1151 [0.1956], 0.6775 [0] respectively. 1645, normal control group (A, B), 12-week smoke exposure group (C) and 24-week smoke exposure group (D) were significantly different (P 0.05), indicating that the expression of MHC-II beta chain protein in the smoke exposure group was lower than the normal control group.
(3) The average concentrations of IL-8 in the supernatant of skeletal muscle homogenate of B and D groups were 195.49 and 233.88, respectively, which were higher in smoke exposure group than in normal control group, and 124.6651 and 150.7417, respectively, which were higher in smoke exposure group than in normal control group.
(4) RT-PCR was used to detect the expression of HDAC2 mRNA in the skeletal muscle tissues of mice in groups B, D and B. The expression of HDAC2 mRNA in the skeletal muscle tissues of mice in groups A, B, C and D was 0.5942 (+ 0.107) respectively. The expression of HDAC2 in smoke exposed group was significantly lower than that in normal control group (p=0.000), and the expression of NF-kappa B/P65 protein was 0.4901+0.2591, 0.5166+0.2319 and 0.5166+0.2319 respectively. The expression of NF-kappa B/P65 protein in smoke exposure group was higher than that in normal control group (A, B), 12-week smoke exposure group and 24-week smoke exposure group (P (0.05).
CONCLUSION: 1. Long-term tobacco smoke exposure can induce vacuole-like degeneration, atrophy and dissolution of skeletal muscle cells in mice, and decrease the content of MHC-II beta chain protein in muscle fibers, indicating that it can lead to changes of skeletal muscle structure and function.
2. Long-term tobacco smoke exposure can lead to increased activity of inflammatory factors (IL-8, TNF-a) and inflammation-regulating gene NF-kappa B in skeletal muscle, and decreased expression of HDAC2, indicating that there is an obvious inflammatory reaction in skeletal muscle.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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