重组人脂肪因子chemerin对巨噬细胞凋亡影响及其机制的研究

发布时间：2018-04-03 05:00

本文选题：重组人chemerin　切入点：脂肪因子　出处：《首都医科大学》2016年硕士论文

【摘要】：目的趋化因子chemerin是新发现的脂肪因子。本课题前期研究显示冠心病患者心外膜脂肪组织中chemerin的m RNA及蛋白表达明显增高,且与冠状动脉粥样硬化严重程度正相关。脂肪因子chemerin被发现可以促进单核巨噬细胞黏附及泡沫细胞形成,而这两个病理过程在As发生、发展过程中发挥着重要作用。巨噬细胞凋亡贯穿于As的各个阶段,其在As病变晚期,可引起继发性细胞坏死和炎性反应,促进斑块脂质核心的形成及扩大,而且巨噬细胞凋亡后释放出的蛋白酶等可损伤纤维帽,最终导致不稳定斑块(即易损斑块)的形成。本研究旨在探讨chemerin对巨噬细胞凋亡的影响及其作用机制,从而为As及冠心病的预防和治疗提供新的干预靶点。方法(一)人单核细胞培养及向巨噬细胞分化10%胎牛血清的RPMI1640培养基培养人单核细胞株THP-1,使用CD14-FITC进行鉴定;加入100nmol/L PMA孵育72h,将THP-1人单核细胞诱导分化为巨噬细胞,获得THP-1巨噬细胞。(二)制备促进游离胆固醇聚集(FC-loading)模型按文献使用含1%胎牛血清的DMEM培养基加入100μg/ml乙酰低密度脂蛋白及10μg/ml胆固醇乙酰转移酶抑制剂-58035促进FC在巨噬细胞内聚集,诱导巨噬细胞凋亡。(三)凋亡检测以Annexin-FITC和PI双标法经流式细胞仪检测。将经PBS液洗涤过的上述巨噬细胞制成细胞悬液,分别加入5μl Annexin-V和5μl PI,室温避光15min,然后加入200μl 1×Buffer缓冲液上流式细胞仪检测。每个样本计数5000个细胞,以激发光波长488nm,发射光波长515nm、560nm标准。从流式细胞仪上的细胞散点图进行分析,Annexin-V+/PI-细胞为凋亡细胞(绿色荧光);Annexin-V+/PI+细胞为坏死细胞(红色-橙色荧光);Annex-in-V-/PI-为活细胞;以凋亡的巨噬细胞占巨噬细胞总数的百分比为凋亡率。(四)不同浓度重组人chemerin对FC-loading诱导巨噬细胞凋亡的影响按浓度梯度0.1μg/l、10μg/l、500μg/l加入重组人chemerin,同时设立空白对照组、中和chemerin组,中和chemerin组预先使chemerin及chemerin中和抗体反应30min,37℃5%CO2培养箱孵育8h;上述各组分别加入巨噬细胞培养液孵育18h,然后在FC聚集巨噬细胞的状态下再孵育12h。按上述方法检测巨噬细胞凋亡率。(五)明确JNK及P38MAPK信号通路的作用根据以上实验确定重组人chemerin的实验浓度,设立重组chemerin组和中和chemerin组;重组chemerin组和中和chemerin组分别应用10μmol/l JNK阻断剂(SP600125)和10μmol/l P38MAPK阻断剂(SB203580)进行干预,再进行上述实验。结果(一)不同浓度重组人chemerin对巨噬细胞凋亡的影响重组人chemerin可促进FC诱导的巨噬细胞凋亡,并且随着chemerin浓度的增加巨噬细胞凋亡增强。巨噬细胞凋亡率对照组为19.04%±0.39%、重组chemerin0.1μg/l组为22.77%±0.86%、10μg/l组为26.37%±1.06%、500μg/l组为33.44%±0.87%(见图3)。各chemerin处理组巨噬细胞凋亡率同对照组相比,差异均有统计学意义(P(27)0.05)。使用chemerin中和抗体干预后,各组巨噬细胞凋亡率同对照组相比差异无统计学意义(P0.05)。(二)JNK与P38MAPK信号通路在chemerin促巨噬细胞凋亡过程中的作用巨噬细胞凋亡率重组chemerin组为23.81%±0.83%、中和chemerin组为17.75%±1.23%、重组chemerin+JNK阻断剂组为13.96%±0.68%、中和chemerin+JNK阻断剂组为11.62%±0.36%、重组chemerin+P38MAPK阻断剂组为11.99%±0.23%、中和chemerin+P38MAPK阻断剂组为10.10%±0.43%。重组chemerin组和中和chemerin组应用JNK阻断剂前后的差值分别为9.85%±0.34%和6.13%±1.09%,前者明显高于后者,差异有统计学意义(P(27)0.05)。由此可知,阻断JNK信号通路可明显削弱chemerin的促巨噬细胞凋亡作用。但应用JNK阻断剂后的重组chemerin组和中和chemerin组仍然有差异(P(27)0.05),则提示尚有其它通路参与chemerin的促巨噬细胞凋亡过程。进一步阻断P38MAPK通路,重组chemerin组和中和chemerin组在应用P38MAPK抑制剂前后的差值分别为11.82%±0.60%和7.65%±0.83%,前者明显高于后者,差异有统计学意义(P(27)0.05)。由此推断,P38MAPK信号通路亦参与了chemerin的促巨噬细胞凋亡过程。结论重组人chemerin可促进FC诱导的巨噬细胞凋亡,并且JNK及P38MAPK信号通路参与此凋亡过程。
[Abstract]:The purpose of chemokine Chemerin is adipokines newly discovered. In our previous study showed that the expression of M RNA and protein Chemerin in patients with coronary heart disease were significantly higher in epicardial adipose tissue, and positively correlated with the severity of coronary atherosclerosis. The fat factor Chemerin was found to promote monocyte adhesion and formation of foam cells, and these two pathological process in As, plays an important role in the process of development. Each stage of macrophage apoptosis throughout the As, the As advanced lesions, can cause secondary cell necrosis and inflammatory reaction, promote plaque formation and expansion of the lipid core, and apoptosis of macrophages after the release of protease can damage the fibrous cap, resulting in no stable plaques (i.e. plaque formation). This study aimed to investigate the effects of Chemerin on macrophage apoptosis and its mechanism of action, and thus to As Provide new targets for prevention and treatment of coronary heart disease. Methods: (a) human monocyte to macrophage differentiation and RPMI1640 culture medium with 10% fetal bovine serum culture medium of human monocytic cell line THP-1, CD14-FITC joined 100nmol/L PMA identification; incubating for 72h, THP-1 human monocyte differentiation into macrophages. THP-1 macrophages. (two) preparation to promote free cholesterol aggregation (FC-loading) containing 1% fetal bovine serum DMEM added 100 g/ml acetyl low density lipoprotein and 10 g/ml cholesterol acyltransferase inhibitor -58035 promote FC aggregation in the macrophage culture medium model according to the literature, apoptosis of macrophages induced by apoptosis (three). Detection of Annexin-FITC and PI double staining by flow cytometry. The macrophages were washed with PBS solution were added into cell suspension, 5 L Annexin-V and 5 L PI, 15min and dark at room temperature. Add 200 L 1 x Buffer buffer flow cytometry. Each sample count of 5000 cells with excitation wavelength 488nm and emission wavelength of 515nm, 560nm standard. From the analysis of flow cytometry on cell scatter, Annexin-V+/PI- cell apoptosis cells (green fluorescence); Annexin-V+/PI+ cell necrosis cells (red orange fluorescence); Annex-in-V-/PI- living cells; apoptosis of macrophage percentage of macrophages apoptosis rate. (four) different concentrations of recombinant human Chemerin effect on FC-loading induced apoptosis of macrophages by concentration gradient of 0.1 g/l, 10 g/l, 500 g/l with recombinant human Chemerin, and blank control group, and Chemerin group, and Chemerin group to make Chemerin and Chemerin neutralizing antibody reaction 30min box incubated with 8h 5%CO2 37 degrees of the culture; groups were added to macrophage was cultured in 18h incubation, however In the FC under the state of aggregation of macrophages incubated 12h. by the method of macrophage apoptosis detection rate. (five) of JNK and P38MAPK pathway according to the experimental concentrations of recombinant human Chemerin to determine the above experiments, the establishment of recombinant Chemerin group and Chemerin group and Chemerin group; recombinant and neutralizing Chemerin group respectively using 10 mol/l JNK blocker (SP600125) and 10 mol/l P38MAPK blockers (SB203580) intervention, and the experimental results. (a) of different concentrations of recombinant human Chemerin Chemerin on the effect of recombinant human macrophage apoptosis can promote the apoptosis of macrophages induced by FC, and with the increase of the concentration of Chemerin increased. Apoptosis of macrophages apoptosis ratio in control group 19.04% + 0.39% u g/l, recombinant chemerin0.1 group is 22.77% + 0.86%, 10 + 1.06% and 26.37% in g/l group, 500 g/l group is 33.44% + 0.87% (see Figure 3). The Chemerin Group of macrophage apoptosis rate compared with the control group, the differences were statistically significant (P (27) 0.05). The use of Chemerin neutralizing antibody intervention, the apoptosis rate of macrophages were compared with the control group, the difference was not statistically significant (P0.05). (two) JNK and P38MAPK signal pathway in apoptosis of macrophage macrophage apoptosis rate of recombinant Chemerin group is 23.81% + 0.83% in Chemerin pro, and group Chemerin was 17.75% + 1.23%, recombinant chemerin+JNK blocker group is 13.96% + 0.68%, and chemerin+JNK blocker group is 11.62% + 0.36%, the recombinant chemerin+ P38MAPK blocker group is 11.99% + 0.23%, and the chemerin+P38MAPK blocker group was 10.10% + 0.43%. group and Chemerin group and recombinant Chemerin the application of JNK blocking agent before and after the difference were 9.85% + 0.34% and 6.13% + 1.09%, the former was significantly higher than that of the latter, the difference was statistically significant (P (27) 0.05). Thus, resistance Off the JNK signaling pathway can markedly reduce apoptosis of macrophage Chemerin. But the application of JNK blocking agent after the recombinant Chemerin group and Chemerin group and there are still differences (P (27) 0.05), suggest that there are other pathways involved in Chemerin on macrophage apoptosis process. Further blocking the P38MAPK pathway, recombinant Chemerin group and Chemerin group and the difference between before and after the application of P38MAPK inhibitors were 11.82% + 0.60% and 7.65% + 0.83%, the former was significantly higher than that of the latter, the difference was statistically significant (P (27) 0.05). Thus, the P38MAPK signaling pathway also participates in Chemerin on macrophage apoptosis. Conclusion recombinant human Chemerin can promote the apoptosis of macrophages induced by FC and JNK. And the P38MAPK signaling pathway is involved in this apoptotic process.

【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R541.4

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