本文選題：重組人chemerin　切入點(diǎn)：脂肪因子　出處：《首都醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的趨化因子chemerin是新發(fā)現(xiàn)的脂肪因子。本課題前期研究顯示冠心病患者心外膜脂肪組織中chemerin的m RNA及蛋白表達(dá)明顯增高,且與冠狀動(dòng)脈粥樣硬化嚴(yán)重程度正相關(guān)。脂肪因子chemerin被發(fā)現(xiàn)可以促進(jìn)單核巨噬細(xì)胞黏附及泡沫細(xì)胞形成,而這兩個(gè)病理過(guò)程在As發(fā)生、發(fā)展過(guò)程中發(fā)揮著重要作用。巨噬細(xì)胞凋亡貫穿于As的各個(gè)階段,其在As病變晚期,可引起繼發(fā)性細(xì)胞壞死和炎性反應(yīng),促進(jìn)斑塊脂質(zhì)核心的形成及擴(kuò)大,而且巨噬細(xì)胞凋亡后釋放出的蛋白酶等可損傷纖維帽,最終導(dǎo)致不穩(wěn)定斑塊(即易損斑塊)的形成。本研究旨在探討chemerin對(duì)巨噬細(xì)胞凋亡的影響及其作用機(jī)制,從而為As及冠心病的預(yù)防和治療提供新的干預(yù)靶點(diǎn)。方法(一)人單核細(xì)胞培養(yǎng)及向巨噬細(xì)胞分化10%胎牛血清的RPMI1640培養(yǎng)基培養(yǎng)人單核細(xì)胞株THP-1,使用CD14-FITC進(jìn)行鑒定;加入100nmol/L PMA孵育72h,將THP-1人單核細(xì)胞誘導(dǎo)分化為巨噬細(xì)胞,獲得THP-1巨噬細(xì)胞。(二)制備促進(jìn)游離膽固醇聚集(FC-loading)模型按文獻(xiàn)使用含1%胎牛血清的DMEM培養(yǎng)基加入100μg/ml乙酰低密度脂蛋白及10μg/ml膽固醇乙酰轉(zhuǎn)移酶抑制劑-58035促進(jìn)FC在巨噬細(xì)胞內(nèi)聚集,誘導(dǎo)巨噬細(xì)胞凋亡。(三)凋亡檢測(cè)以Annexin-FITC和PI雙標(biāo)法經(jīng)流式細(xì)胞儀檢測(cè)。將經(jīng)PBS液洗滌過(guò)的上述巨噬細(xì)胞制成細(xì)胞懸液,分別加入5μl Annexin-V和5μl PI,室溫避光15min,然后加入200μl 1×Buffer緩沖液上流式細(xì)胞儀檢測(cè)。每個(gè)樣本計(jì)數(shù)5000個(gè)細(xì)胞,以激發(fā)光波長(zhǎng)488nm,發(fā)射光波長(zhǎng)515nm、560nm標(biāo)準(zhǔn)。從流式細(xì)胞儀上的細(xì)胞散點(diǎn)圖進(jìn)行分析,Annexin-V+/PI-細(xì)胞為凋亡細(xì)胞(綠色熒光);Annexin-V+/PI+細(xì)胞為壞死細(xì)胞(紅色-橙色熒光);Annex-in-V-/PI-為活細(xì)胞;以凋亡的巨噬細(xì)胞占巨噬細(xì)胞總數(shù)的百分比為凋亡率。(四)不同濃度重組人chemerin對(duì)FC-loading誘導(dǎo)巨噬細(xì)胞凋亡的影響按濃度梯度0.1μg/l、10μg/l、500μg/l加入重組人chemerin,同時(shí)設(shè)立空白對(duì)照組、中和chemerin組,中和chemerin組預(yù)先使chemerin及chemerin中和抗體反應(yīng)30min,37℃5%CO2培養(yǎng)箱孵育8h;上述各組分別加入巨噬細(xì)胞培養(yǎng)液孵育18h,然后在FC聚集巨噬細(xì)胞的狀態(tài)下再孵育12h。按上述方法檢測(cè)巨噬細(xì)胞凋亡率。(五)明確JNK及P38MAPK信號(hào)通路的作用根據(jù)以上實(shí)驗(yàn)確定重組人chemerin的實(shí)驗(yàn)濃度,設(shè)立重組chemerin組和中和chemerin組;重組chemerin組和中和chemerin組分別應(yīng)用10μmol/l JNK阻斷劑(SP600125)和10μmol/l P38MAPK阻斷劑(SB203580)進(jìn)行干預(yù),再進(jìn)行上述實(shí)驗(yàn)。結(jié)果(一)不同濃度重組人chemerin對(duì)巨噬細(xì)胞凋亡的影響重組人chemerin可促進(jìn)FC誘導(dǎo)的巨噬細(xì)胞凋亡,并且隨著chemerin濃度的增加巨噬細(xì)胞凋亡增強(qiáng)。巨噬細(xì)胞凋亡率對(duì)照組為19.04%±0.39%、重組chemerin0.1μg/l組為22.77%±0.86%、10μg/l組為26.37%±1.06%、500μg/l組為33.44%±0.87%(見(jiàn)圖3)。各chemerin處理組巨噬細(xì)胞凋亡率同對(duì)照組相比,差異均有統(tǒng)計(jì)學(xué)意義(P(27)0.05)。使用chemerin中和抗體干預(yù)后,各組巨噬細(xì)胞凋亡率同對(duì)照組相比差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。(二)JNK與P38MAPK信號(hào)通路在chemerin促巨噬細(xì)胞凋亡過(guò)程中的作用巨噬細(xì)胞凋亡率重組chemerin組為23.81%±0.83%、中和chemerin組為17.75%±1.23%、重組chemerin+JNK阻斷劑組為13.96%±0.68%、中和chemerin+JNK阻斷劑組為11.62%±0.36%、重組chemerin+P38MAPK阻斷劑組為11.99%±0.23%、中和chemerin+P38MAPK阻斷劑組為10.10%±0.43%。重組chemerin組和中和chemerin組應(yīng)用JNK阻斷劑前后的差值分別為9.85%±0.34%和6.13%±1.09%,前者明顯高于后者,差異有統(tǒng)計(jì)學(xué)意義(P(27)0.05)。由此可知,阻斷JNK信號(hào)通路可明顯削弱chemerin的促巨噬細(xì)胞凋亡作用。但應(yīng)用JNK阻斷劑后的重組chemerin組和中和chemerin組仍然有差異(P(27)0.05),則提示尚有其它通路參與chemerin的促巨噬細(xì)胞凋亡過(guò)程。進(jìn)一步阻斷P38MAPK通路,重組chemerin組和中和chemerin組在應(yīng)用P38MAPK抑制劑前后的差值分別為11.82%±0.60%和7.65%±0.83%,前者明顯高于后者,差異有統(tǒng)計(jì)學(xué)意義(P(27)0.05)。由此推斷,P38MAPK信號(hào)通路亦參與了chemerin的促巨噬細(xì)胞凋亡過(guò)程。結(jié)論重組人chemerin可促進(jìn)FC誘導(dǎo)的巨噬細(xì)胞凋亡,并且JNK及P38MAPK信號(hào)通路參與此凋亡過(guò)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.

【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R541.4

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