透明质酸和其受体CD44在慢性哮喘气道重塑中的作用机制研究

发布时间：2018-05-19 06:22

本文选题：气道平滑肌细胞 + 透明质酸　；参考：《华中科技大学》2012年硕士论文

【摘要】：第一部分透明质酸与其受体CD44在慢性哮喘ASMCs中的表达变化研究目的 1、研究SD大鼠在慢性哮喘状态下，气道平滑肌细胞分泌的透明质酸（hyaluronicacid）的表达，以及其受体CD44基因和蛋白水平的表达。 2、研究SD大鼠在慢性哮喘状态下，气道平滑肌细胞中透明质酸合成酶（HAS）和透明质酸酶（HYAL）基因水平的表达。材料与方法建立SD大鼠慢性哮喘模型，处死后培养原代气道平滑肌细胞，使用2~5代ASMCs。分组：正常对照组（C组）和慢性哮喘组（A组）。应用realtime PCR检测ASMCs中CD44,透明质酸合酶1、2、3和透明质酸酶1、 2基因水平变化；Elisa方法检测ASMCs细胞培养上清液中HA的含量；Western-blot方法检测ASMCs中CD44蛋白水平的表达。实验结果 1、慢性哮喘组大鼠ASMCs中CD44mRNA和蛋白水平表达较正常对照组明显升高（p＜0.05）。 2、慢性哮喘组大鼠ASMCs分泌的HA较正常对照组明显升高（p＜0.001）。 3、相对于正常对照组，慢性哮喘组大鼠ASMCs中透明质酸合酶1、2及透明质酸酶1、2mRNA表达均升高（p＜0.05）。结论与正常对照组相比，慢性哮喘气道平滑肌细胞分泌的HA以及细胞膜上其受体CD44表达明显增高，透明质酸合酶以及透明质酸酶mRNA水平表达明显增高。第二部分透明质酸对SD大鼠慢性哮喘模型气道平滑肌细胞增殖和凋亡的影响研究目的 1、研究SD大鼠在慢性哮喘状态下，气道平滑肌细胞增殖和凋亡的表达。 2、研究SD大鼠气道平滑肌细胞在低分子量透明质酸（LMH-HA）干预下，其细胞学活性的表达变化。 3、研究SD大鼠在慢性哮喘状态下，气道平滑肌细胞中细胞外信号调节激酶1/2（extracellular signal-regulated kinase, ERK1/2）与磷酸化细胞外信号调节激酶1/2（p-ERK1/2）蛋白表达和活化情况。 4、研究SD大鼠气道平滑肌细胞在低分子量透明质酸（LMH-HA）干预下，气道平滑肌细胞中细胞外信号调节激酶1/2（extracellular signal-regulated kinase,ERK1/2）与磷酸化细胞外信号调节激酶1/2（p-ERK1/2）蛋白表达和活化情况。材料与方法慢性哮喘模型制备，原代ASMCs培养与第一部分相同。实验分组：（1）正常对照组（C组）：即为正常对照组大鼠ASMCs，无任何刺激干预；（2）对照+低分子量透明质酸组（C+HA组）：正常对照组大鼠ASMCs加入1mg/L低分子量透明质酸刺激24小时；（3）对照+低分子量透明质酸+PD98059组（C+HA+PD98059组）：正常对照组大鼠ASMCs先加入10umol/L PD98059干预1小时，再给予1mg/L低分子量透明质酸刺激24小时；（4）慢性哮喘组（A组）：即为慢性哮喘组大鼠ASMCs，不加任何干预刺激；（5）慢性哮喘+低分子量透明质酸组（A+HA组）：慢性哮喘组大鼠ASMCs加入1mg/L低分子量透明质酸刺激24小时；（6）慢性哮喘+低分子量透明质酸+PD98059组（A+HA+PD98059组）：慢性哮喘组大鼠ASMCs先加入10umol/L PD98059干预1小时，再给予1mg/L低分子量透明质酸刺激24小时。应用Western-blot方法检测各组ASMCs中p-ERK1/2、ERK1/2蛋白水平的表达；流式细胞术、MTT法检测各组ASMCs增殖和凋亡改变。实验结果 1、对比正常对照组，慢性哮喘组大鼠ASMCs增殖水平升高，凋亡水平降低，差异具有统计学意义（p＜0.05）。 2、正常对照组和哮喘对照组大鼠ASMCs经LMW-HA干预后，细胞增殖均较对照组明显增加（p＜0.01）；并且ASMCs经PD98059阻断后加入LMW-HA干预，细胞增殖较对照组以及LMW-HA干预组降低，且差异有统计学意义（p＜0.01）。 3、正常对照组和哮喘对照组大鼠ASMCs经LMW-HA干预后，，细胞凋亡较对照组均减少（p＜0.05）；并且ASMCs经PD98059阻断后加入LMW-HA干预，细胞凋亡较对照组以及LMW-HA干预组增加，且差异有统计学意义（p＜0.05）。 4、对照组ASMCs经LMW-HA干预后，p-ERK1/2、ERK1/2蛋白水平以及p-ERK1/2、ERK1/2活性比值较对照组升高（p＜0.05）。对照组ASMCs经PD98059阻断再加入LMW-HA干预后，p-ERK1/2、ERK1/2活性比值较对照组以及LMW-HA干预组显著降低（p＜0.05）。结论在慢性哮喘状态下，ASMCs增殖较正常对照组显著增加，同时凋亡水平明显降低；ASMCs在低分子量透明质酸（LMH-HA）干预下，增殖活性显著增高，凋亡减少，并且这一过程是通过ERK1/2信号转导通路实现。以上结果提示，在慢性哮喘状态下，气道平滑肌细胞向中细胞外基质中分泌的HA升高，而ECM中升高的HA和CD44又通过ERK1/2信号转导通路促进ASMCs的异常增殖，从而参与慢性哮喘气道重塑过程。
[Abstract]:Part one
Expression of hyaluronic acid and its receptor CD44 in chronic asthma ASMCs
research objective
1, to investigate the expression of hyaluronic acid (hyaluronicacid) secreted by airway smooth muscle cells in SD rats and the expression of CD44 gene and protein level of its receptor.
2, we studied the expression of hyaluronan synthase (HAS) and hyaluronidase (HYAL) gene in airway smooth muscle cells of SD rats in chronic asthma.
Materials and methods
A SD rat model of chronic asthma was established, and the primary airway smooth muscle cells were cultured after death. The 2~5 generation ASMCs. was used.
Group: normal control group (group C) and chronic asthma group (group A).
Realtime PCR was used to detect CD44, hyaluronan synthase 1,2,3 and hyaluronidase 1 in ASMCs.
2 changes in gene level; Elisa method was used to detect the content of HA in ASMCs cell culture supernatant; Western-blot method was used to detect the expression of CD44 protein in ASMCs.
experimental result
1, the expression of CD44mRNA and protein in ASMCs of chronic asthma group was significantly higher than that of normal control group (P < 0.05).
2, the HA secretion of ASMCs in chronic asthma group was significantly higher than that in normal control group (P < 0.001).
3, compared with normal control group, the expression of hyaluronan synthase 1,2 and hyaluronidase 1,2mRNA in ASMCs of chronic asthma group increased (P < 0.05).
conclusion
Compared with the normal control group, the expression of HA and the expression of CD44 on the cell membrane of the airway smooth muscle cells increased significantly, and the expression of hyaluronidase and hyaluronidase mRNA increased significantly.
The second part is the effect of hyaluronic acid on the proliferation and apoptosis of airway smooth muscle cells in chronic asthmatic model of SD rats.
research objective
1, we studied the expression of airway smooth muscle cell proliferation and apoptosis in SD rats under chronic asthma.
2, we studied the changes of cellular activity of SD rat airway smooth muscle cells under the intervention of low molecular weight hyaluronic acid (LMH-HA).
3, the expression and activation of extracellular signal regulated kinase 1/2 (extracellular signal-regulated kinase, ERK1/2) and phosphorylated extracellular signal regulated kinase 1/2 (p-ERK1/2) protein in the airway smooth muscle cells of SD rats were studied in the condition of chronic asthma.
4, the expression and activation of the extracellular signal regulated kinase 1/2 (extracellular signal-regulated kinase, ERK1/2) and phosphorylated extracellular signal regulated kinase 1/2 (p-ERK1/2) in the airway smooth muscle cells in the airway smooth muscle cells of SD rats were studied under the intervention of low molecular weight hyaluronic acid (LMH-HA).
Materials and methods
The chronic asthma model was prepared, and the primary ASMCs culture was the same as that in the first part.
Group of experiments:
(1) normal control group (group C): the normal control group rats were ASMCs without any stimulation intervention.
(2) control + low molecular weight hyaluronic acid group (group C+HA): in normal control rats, ASMCs was added to 1mg/L low molecular weight hyaluronic acid for 24 hours.
(3) control + low molecular weight hyaluronic acid +PD98059 group (group C+HA+PD98059): normal control group rats ASMCs first added 10umol/L PD98059 intervention for 1 hours, and then give 1mg/L low molecular weight hyaluronic acid stimulation for 24 hours.
(4) chronic asthma group (group A): chronic asthma group ASMCs, without any intervention stimulation;
(5) chronic asthma + low molecular weight hyaluronic acid group (group A+HA): in chronic asthma group, ASMCs was added to 1mg/L low molecular weight hyaluronic acid for 24 hours.
(6) chronic asthma + low molecular weight hyaluronic acid +PD98059 group (group A+HA+PD98059): chronic asthma group rats ASMCs first added 10umol/L PD98059 intervention for 1 hours, and then give 1mg/L low molecular weight hyaluronic acid stimulation for 24 hours.
Western-blot method was used to detect the expression of p-ERK1/2 and ERK1/2 protein in ASMCs of each group, and the proliferation and apoptosis of ASMCs in each group were detected by flow cytometry and MTT.
experimental result
1, compared with the normal control group, the level of ASMCs proliferation and the level of apoptosis were decreased in chronic asthma group, and the difference was statistically significant (P < 0.05).
2, the proliferation of ASMCs in the normal control group and the asthma control group was significantly higher than that in the control group (P < 0.01) after the intervention of LMW-HA (P < 0.01), and the proliferation of ASMCs was lower than that of the control group and the LMW-HA intervention group after PD98059 blockage, and the difference was statistically significant (P < 0.01).
3, in the normal control group and the asthma control group, the apoptosis of ASMCs was reduced by LMW-HA (P < 0.05) after the intervention of the control group (P < 0.05), and the apoptosis was increased after PD98059 blockage, and the apoptosis was increased compared with the control group and the LMW-HA intervention group, and the difference was statistically significant (P < 0.05).
4, compared with the control group, the ratio of p-ERK1/2, ERK1/2 protein and ERK1/2 activity was higher in the control group than in the control group (P < 0.05). The ratio of ASMCs to the control group was significantly lower than that of the control group (P < 0.05). The ratio of the activity of ASMCs to the control group was significantly lower than that of the control group and the intervention group (P < 0.05). The ratio of ASMCs to the control group was significantly lower than that of the control group (P < 0.05). (P < 0.05).
conclusion
In the state of chronic asthma, the proliferation of ASMCs was significantly higher than that in the normal control group, and the level of apoptosis was significantly reduced, and the proliferation activity of ASMCs was significantly increased and apoptosis decreased under the intervention of low molecular weight hyaluronic acid (LMH-HA). The process was realized through the ERK1/2 signal transduction pathway. The secreted HA of the smooth muscle cells increased to the medium extracellular matrix, while the increased HA and CD44 in ECM promoted the abnormal proliferation of ASMCs through the ERK1/2 signal transduction pathway, thus participating in the process of airway remodeling in chronic asthma.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R562.25

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