TLR4介导的P38MAPK信号通路对阻塞性睡眠呼吸暂停综合征伴高血压大鼠脂肪因子的影响

发布时间：2018-05-07 04:35

本文选题：阻塞性睡眠呼吸暂停综合征 + 高血压　；参考：《桂林医学院》2013年硕士论文

【摘要】：目的：研究TLR4介导的p38MAPK信号通路在阻塞性睡眠呼吸暂停综合征（Obstractive SleepApnea Symptom，OSAS）伴高血压大鼠脂肪组织、脂肪细胞中的表达，和对脂肪细胞因子leptin的影响，探讨OSAS伴高血压可能的发病机制。方法：45只健康雄性SD大鼠随机均分为间歇低氧4周组、间歇低氧6周组和正常对照组，间歇低氧4周组和6周组大鼠放入实验舱中，循环充入氮气和氧气,每次循环90s,每天间歇性低氧8h,正常对照组大鼠暴露于空气中不予任何特殊处理。分别测定实验前和每周试验后各组大鼠尾动脉血压。于间歇低氧28、42d后分别处死间歇低氧4周组和6周组大鼠，HE染色观察各组大鼠心肌、肾动脉的病理变化，ELISA法检测各组大鼠血浆中leptin含量的变化，RT-PCR、Western-Blot法检测脂肪组织中TLR4、p38MAPK mRNA和蛋白的表达；取各模型组大鼠脂肪组织进行原代培养脂肪细胞，显微镜下观察细胞形态改变，流式细胞仪检测细胞增殖活性;油红O染色法进行细胞鉴定；各组原代脂肪细胞分为12组:正常对照组，间歇低氧4周对照组，间歇低氧6周对照组，脂多糖诱导正常组，脂多糖诱导间歇低氧4周组，脂多糖诱导间歇低氧6周组，TLR4阻滞正常组，TLR4阻滞间歇低氧4周组，TLR4阻滞间歇低氧6周组， p38MAPK阻滞正常组， P38MAPK阻滞间歇低氧4周组，p38MAPK阻滞间歇低氧6周组。收集上述各组脂肪细胞，利用RT-PCR法和Western-blot法分别检测TLR4、p38MAPK mRNA和蛋白的表达，另收集上述各组脂肪细胞培养液，利用ELISA法测定培养液中leptin的含量。结果：1模型大鼠血压逐渐升高，且出现以心肌细胞肿胀、肾动脉管壁增厚为特征的相关OSAS病理改变，从而成功建立OSAS合并高血压大鼠模型。 2与正常对照组相比，间歇性低氧4周组、间歇性低氧6周组血清leptin含量升高（P0.05，P0.01），且间歇性低氧6周组升高更显著（P0.05）。 3与正常对照组相比，间歇低氧4周组、间歇低氧6周组脂肪组织中TLR4、p38MAPK mRNA和蛋白表达水平均显著升高(P0.05，P0.01)。 4成功培养原代大鼠脂肪细胞。细胞24小时贴壁时为类圆形，3d后渐成梭形或多角梭形，6d左右进入指数增长期；流式细胞仪检测证明细胞具有增殖分化能力；经诱导分化为成熟脂肪细胞，油红O染色可见细胞内出现红染颗粒. 5TLR4介导p38MAPK信号通路在脂肪细胞中的表达：与对照组相比，，脂多糖可以上调TLR4、p38MAPK mRNA和蛋白的表达（P0.01, P0.05）；TLR4阻滞剂可以下调TLR4、p38MAPK mRNA和蛋白的表达（P0.01，P0.05）；p38MAPK阻滞剂仅下调p38MAPK mRNA和蛋白的表达（P0.05），对TLR4表达无意义。 6大鼠脂肪细胞中TLR4介导的p38MAPK信号通路对leptin分泌的调控：与对照组相比，脂多糖预处理组分泌leptin增加（P0.01），TLR4阻滞剂组分泌leptin减少(P0.05），P38MAPK阻滞剂组分泌leptin无意义（P0.05）。结论： TLR4介导的p38MAPK信号通路在脂肪组织和脂肪细胞中表达，并通过对脂肪因子leptin的调控影响OSAS合并高血压病情进展，为进一步寻找更有效的治疗方法打下基础。
[Abstract]:Objective: To investigate the possible pathogenesis of OSAS associated hypertension by TLR4 mediated p38MAPK signaling pathway in Obstractive SleepApnea Symptom (OSAS) with adipose tissue, adipocyte expression, and the effect on Adipocyte Factor leptin in rats with obstructive sleep apnea syndrome (OSAS).
Methods: 45 healthy male SD rats were randomly divided into intermittent hypoxic 4 weeks group, intermittent hypoxia 6 week group and normal control group, intermittent hypoxic 4 week group and 6 week group of rats were put into the experimental cabin and circulate into nitrogen and oxygen, each cycle was 90s, intermittent hypoxia 8h, the normal control rats were exposed to air in no special treatment. The blood pressure of the tail artery of rats in each group before and after the experiment was measured. After intermittent hypoxic 28,42d, the rats were killed 4 weeks and 6 weeks respectively. The pathological changes of the myocardium and renal artery were observed by HE staining. The changes of leptin content in the plasma of the rats were detected by ELISA, and TLR4 and p38 in the adipose tissue were detected by RT-PCR and Western-Blot method. The expression of MAPK mRNA and protein;
The adipose tissue of the rat model group was taken for primary culture of adipocytes. The morphological changes of the cells were observed under the microscope and the cell proliferation activity was detected by flow cytometry. The oil red O staining method was used to identify the cells. The primary adipocytes in each group were divided into 12 groups: normal control group, intermittent hypoxia 4 week control group, intermittent hypoxia 6 week control group and lipopolysaccharide induction group. In normal group, lipopolysaccharide induced intermittent hypoxia for 4 weeks, LPS induced intermittent hypoxia for 6 weeks, TLR4 block in normal group, TLR4 block intermittent hypoxia for 4 weeks, TLR4 block intermittent hypoxia for 6 weeks, p38MAPK block in normal group, P38MAPK block intermittent hypoxia for 4 weeks and p38MAPK block intermittent hypoxia for 6 weeks. The above adipocytes were collected and RT-PCR method was collected. The expression of TLR4, p38MAPK mRNA and protein were detected by the Western-blot method, and the culture fluid of the adipocyte was collected, and the content of leptin in the medium was measured by ELISA.
Results: the blood pressure of the 1 model rats increased gradually, and the pathological changes of OSAS were characterized by the swelling of the cardiac myocytes and the thickening of the renal artery wall, so that the OSAS combined with the hypertensive rat model was successfully established.
2 compared with the normal control group, the serum leptin content in the intermittent hypoxic group was increased in the intermittent hypoxic group for the 4 week group (P0.05, P0.01), and the intermittent hypoxic group increased significantly in the 6 week group (P0.05).
3 compared with the normal control group, the expression of TLR4, p38MAPK mRNA and protein in the adipose tissue of the intermittent hypoxia group for 4 weeks and the intermittent hypoxia group for 6 weeks increased significantly (P0.05, P0.01).
4 the primary rat adipocytes were successfully cultured. The cells were round on the wall for 24 hours. After 3D, the cells became fusiform or multi angle, and 6D entered the exponential growth period. Flow cytometry showed that the cells had the ability to proliferate and differentiate; the cells were differentiated into mature adipocytes, and red O staining could be found in the cells.
5TLR4 mediates the expression of p38MAPK signaling pathway in adipocytes: compared with the control group, lipopolysaccharide can up regulate the expression of TLR4, p38MAPK mRNA and protein (P0.01, P0.05); TLR4 blockers can down regulate the expression of TLR4, p38MAPK mRNA and protein. It is meaningless to express.
The regulation of leptin secretion by the TLR4 mediated p38MAPK signaling pathway in 6 rat adipocytes: compared with the control group, the secretion of leptin in the lipopolysaccharide preconditioning group increased (P0.01), the TLR4 blocker group secreted the leptin decrease (P0.05), and the P38MAPK blocker group secreted leptin (P0.05).
Conclusion: TLR4 mediated p38MAPK signaling pathway is expressed in adipose tissue and adipocytes, and the regulation of adipose factor leptin affects the progression of OSAS combined with hypertension, so as to lay a foundation for further finding more effective treatment methods.

【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R766;R544.1

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