高胰岛素促单核/巨噬源性泡沫细胞ACAT-1表达及漏芦对其影响

【作者】 李宏霞；

【导师】 张壮；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2012， 硕士

【摘要】 心脑血管疾病严重危害着人类健康,动脉粥样硬化是心脑血管疾病共同的病理基础。全世界每年死于心脑血管疾病的比率接近总死亡人数的三分之一；我国每年死于心、脑血管疾病的人数占总死亡人数的41%,近年来心脑血管疾病的年轻化趋势日益渐重,故早期预防动脉粥样硬化的发生具有十分重要的社会意义。目的：高胰岛素血症是致动脉粥样硬化发生的独立危险因素,单核巨噬细胞源性泡沫细胞是动脉粥样硬化早期病变的基础,研究高浓度胰岛素与动脉粥样硬化的形成机制及寻找早期防治动脉粥样硬化的药物,将为预防并治疗高胰岛素血症并发症开辟新出路。以往研究表明,漏芦可减轻动脉粥样硬化的病变程度,抑制巨噬细胞摄取ox-LDL,对单核巨噬细胞源性泡沫细胞的形成有显著抑制作用。本实验通过佛波酯诱导人单核细胞成巨噬细胞后,再与ox-LDL孵育,同时给予高胰岛素处理,在其基础上再给予不同浓度的漏芦提取物,进而探索漏芦提取物抑制高胰岛素促进巨噬细胞摄入ox-LDL的机制,从而探索漏芦对于早期动脉粥样硬化的预防与治疗效果。本实验研究对于漏芦预防动脉粥样硬化早期病变有一定的意义。方法：1.MTT实验确定诱导单核细胞的最佳PMA浓度：人单核细胞与PMA孵育,观察PMA对人单核细胞转化成巨噬细胞的影响。2.MTT实验确定胰岛素、氟伐他汀及漏芦的实验浓度：人巨噬细胞与不同浓度胰岛素、氟伐他汀及漏芦作用48h后,通过检测细胞活性观察三者的细胞毒性。3.油红0染色观察泡沫细胞转化率、酶法测定泡沫细胞胆固醇酯含量、实时荧光定量PCR实验检测ACAT-lmRNA的表达量及Western blot实验检测ACAT-1蛋白的表达量：PMA诱导人单核细胞成巨噬细胞后,再与ox-LDL孵育,并且同时分别给予胰岛素、氟伐他汀及漏芦培养48h。结果：1.MTT实验确定诱导单核细胞的最佳PMA浓度：PMA作用单核细胞24h及48h后,在40-160nmol/L范围内,随PMA浓度的升高巨噬细胞转化率升高,在160-640nmol/L范围内,随PMA浓度的升高巨噬细胞转化率降低；PMA作用24h后,160nmol/L浓度组与单核细胞组比较差异最大,有明显统计学意义(P<0.01),作用48h后,160nmol/L浓度组与单核细胞组比较差异最大,有统计学意义(Y<0.05),且PMA诱导24h较48h巨噬细胞转化率高,故选择160nmol/LPMA诱导单核细胞24h。2.MTT实验确定胰岛素、氟伐他汀及漏芦的实验浓度：不同浓度胰岛素作用于人巨噬细胞48h后,胰岛素浓度在0.005-0.1u／L范围内时,与巨噬细胞组比较差异均无统计学意义(P>O.05)。氟伐他汀10-5mol/L浓度组与巨噬细胞组相比差异有统计学意义(P<O.05),有细胞毒性,浓度低于10-5mol/L时,与巨噬细胞组比较差异均无统计学意义(P>O.05),故本实验采用10-6mol/L作为药物作用浓度。漏芦浓度在0.005-0.5g／L浓度范围时与巨噬细胞组比较差异均无统计学意义(P>0.05),1g／L浓度组与巨噬细胞组比较差异有统计学意义(P<0.05),出现细胞毒性,故选用O.5、0.05、0.005g／L分别作为漏芦高、中、低药物浓度进行实验。3.油红0染色观察泡沫细胞转化率：ox-LDL+0.02U/L胰岛素浓度组与ox-LDL组相比差异有统计学意义(P<0.05),泡沫细胞转化率升高,并且随着胰岛素浓度升高,转化率升高。正常人空腹胰岛素水平为0.005-0.015U／L,胰岛素水平高于0.015U／L即为高胰岛素血症,故本实验选取0.02U/L的胰岛素浓度作为研究浓度。ox-LDL+胰岛素组与ox-LDL组相比差异有统计学意义(P<0.05),泡沫细胞转化率升高：ox-LDL+胰岛素+氟伐他汀组、ox-LDL+胰岛素+高浓度漏芦组、ox-LDL+胰岛素+中浓度漏芦组、ox-LDL+胰岛素+低浓度漏芦组与ox-LDL+胰岛素组相比差异均有统计学意义(P<0.05),泡沫细胞转化率降低,且随漏芦浓度升高,泡沫细胞转化率降低越明显。4.酶法测定巨噬细胞源性泡沫细胞胆固醇酯含量：ox-LDL组较巨噬细胞组差异有统计学意义(P<0.05),胆固醇酯含量增高；0.02U/L胰岛素组、0.04U／L胰岛素组、0.06U／L胰岛素组均较ox-LDL组差异有统计学意义(P<0.05),胆固醇酯含量均增高,随着胰岛素浓度升高,胆固醇酯含量升高。ox-LDL组较巨噬细胞组差异有统计学意义(K0.05),胆固醇酯含量增高；ox-LDL+胰岛素组较ox-LDL组差异有统计学意义(P<0.05),胆固醇酯含量增高：ox-LDL+胰岛素+氟伐他汀组、ox-LDL+胰岛素+高浓度漏芦组、ox-LDL+胰岛素十中浓度漏芦组、ox-LDL+胰岛素+低浓度漏芦组均较ox-LDL+胰岛素组差异有统计学意义(P<0.05),胆固醇酯含量降低,且随漏芦浓度升高,胆固醇酯含量降低越明显。5.实时荧光定量PCR实验检测ACAT-lmRNA的表达量：ox-LDL组较巨噬细胞组差异有统计学意义(P<0.05), ACAT-lmRNA的表达量升高；ox-LDL+胰岛素组较ox-LDL组差异有统计学意义(P<0.05), ACAT-lmRNA的表达量升高；ox-LDL+胰岛素+氟伐他汀组、ox-LDL+胰岛素+高浓度漏芦组、ox-LDL+胰岛素+中浓度漏芦组、ox-LDL+胰岛素+低浓度漏芦组均较ox-LDL+胰岛素组差异有统计学意义(P<0.05), ACAT-lmRNA的表达量降低,且随漏芦浓度升高,ACAT-lmRNA的表达量降低越明显。6. Western blot实验观察ACAT-1蛋白的表达量：ox-LDL组较巨噬细胞组差异有统计学意义(P<0.05), ACAT-1蛋白的表达量升高；ox-LDL+胰岛素组较ox-LDL组差异有统计学意义(P<0.05), ACAT-1蛋白的表达量升高；ox-LDL+胰岛素+氟伐他汀组、ox-LDL+胰岛素+高浓度漏芦组、ox-LDL+胰岛素+中浓度漏芦组、ox-LDL+胰岛素+低浓度漏芦组均较ox-LDL+胰岛素组差异有统计学意义(P<0.05), ACAT-1蛋白的表达量降低,且随漏芦浓度升高,ACAT-1蛋白的表达量降低越明显。结论：1.PMA诱导单核细胞转化成巨噬细胞后,再与ox-LDL孵育,能够模拟单核巨噬细胞源性泡沫细胞的形成过程。2.油红0染色及酶法测定泡沫细胞胆固醇酯含量发现高浓度胰岛素具有促进巨噬细胞源性泡沫细胞形成的作用,漏芦提取物可以抑制高浓度胰岛素促进泡沫细胞形成过程的作用。3. Western blot实验及实时荧光定量PCR实验均发现：高浓度胰岛素促进泡沫细胞形成的作用与促进ACAT-1的表达关系密切,漏芦提取物能够抑制高浓度胰岛素促进ACAT-1表达过程的作用。4.由此可见高浓度胰岛素促进泡沫细胞形成与漏芦提取物抑制泡沫细胞形成的作用均与ACAT-1有密切的关系。本实验为漏芦预防动脉粥样硬化提供了初步实验依据。更多还原

【Abstract】 Atherosclerosis is the common pathological basis of cardiovascular and cerebrovascular diseases which is very harmful to human health. The pepole dying of cardiovascular and cerebrovascular diseases in the world reaches one third of total deaths; in China, the pepole dying of cardiovascular and cerebrovascular diseases reaches41%oftotal deaths.In recent years there is a growing number of young people suffering from cardiovascular and cerebrovascular diseases, and therefore early prevention of atherosclerosis has a very important social significance.The hyperinsulinnemia is an independent risk factor of the atherosclerosis. The foam cells from mononuclear-macrophagocyte are the foundation of the atherosclerosis inearly lesion phase.The study about the formation mechanism of the hyperinsulinnemiaand atherosclerosis and looking for the drugs of early prevention and treatment of atherosclerosis will open up a new way to provide prevention and treatment for complications of hyperinsulinemia.The previous studies show that Loulu could reduce the lesion of atherosclerosis and inhibit macrophage uptake of ox-LDL. The foam cells from mononuclear-macrophagocyte could be significantly inhibited by Loulu.Objective:Tn this study, monocyte has been induced to macrophage by PMA, incubating withox-LDL,at the same time,high insulin and Loulu have been added to the cells,finally we can explore the mechanism that the extract of Loulu inhibits macrophage uptake of ox-LDL which is promoted by hyperinsulinemia. We can explore the prevention and treatment effect of the Loulu on early atherosclerosis.lt has an very important meaning to prevention of atherosclerosis.Methods:1. Detecting the optimal PMA concentration of inducing monocytes by MTT assay:After THP-1cells being incubated with PMA, to observe the influence of monocyte from the different concentrations of PMA.2. Determining the concentration of experimental drugs such as insulin, Fluorine simvastatin and Loulu by MTT:After macrophages being incubated with insulin, Fluorine simvastatin and Loulu48h, to observe cytotoxicity of insulin, Fluorine simvastatin and Loulu by detecting cells activity.3. Detecting the rate of foam cell transformation by Oil red O staining, detectingthe cholesterol ester content of foam cells by enzymatic, observing the expression of ACAT-lmRNA by real time PCR and observing the expression of AC AT-1protein by Western blot:after monocyte has been induced to macrophage by PMA,incubating with ox-LDL48h, at the same time, high insulin, Fluorine simvastatin and Loulu have been added to the cells.Results:1. Detecting the optimal PMA concentration of inducing monocytes by MTT assay:After incubating with PMA24h and48h, we could see that the conversion of macrophagesincreases following increasing of PMA concentration between40nmoL/L and160nmoL/L, and the conversion of macrophages reduces following increasing of PMAconcentration between160nmoL/L and480nmoL/L.There is statistically significant difference between160noml/L group and monocyte group (P<0.05). Compared with48h, the conversion of macrophages rate of24h is higher, so we choose the160noml/L PMA to induce THP-1cells24h.2. Determining the concentration of experimental drugs such as insulin, Fluorine simvastatin and Loulu by MTT:After cultivating by different concentrations of insulin48h,compared with the control group,there is no statistically significant difference between0.005U/L and0.1U/L (P>0.05). After cultivating by different concentrations Fluorine simvastatin of48h,compared with the control group,there is no statistically significant difference below10-5mol/L (P>0.05), and there is statistically significant difference exceed10-5mol/L (P<0.05).Therefore, we choose the10-6mol/L as the action concentration of drug. After cultivated by different concentrations of Loulu48h, compared with the control group,there is no statistically significant difference between0.005g/l and0.5g/L (P>0.05),there is statistically significant in lg/L(P<0.05),which appears cytotoxicity.3. Detecting the rate of foam cell transformation by Oil red O staining:There is statistically significant difference between group of ox-LDL and group of ox-LDL and0.02U/L insulin(P<0.05); foam cell transformation rate increases with insulin concentrations. Normal fasting insulin level is0.005-0.015U/L, and definitionof the hyperinsulinemia is insulin level which is higher than the0.015U/L, so we select0.02U/L insulin concentration as the research concentration.There is statistically significant between group of ox-LDL and group of ox-LDL and insulin (P<0.05); foam cell transformation rate increases with insulin concentrations. Compared with the ox-LDL+insulin group by ox-LDL-+insulin+Fluorine simvastatin group,ox-LDL+insulin+high concentrations of Loulu group, ox-LDL+insulin+concentration Loulu group, ox-LDL+insulin+low concentrations of the Loulu group,the difference is statistically significant (P<0.05), foam cell transformation rate reduces following increasing of Loulu concentration.4. Detecting the cholesterol ester content of foam cells by enzymatic:There is statistically significant between group of ox-LDL and macrophage group (P<0.05),the cholesterol ester content of foam cells increased. Compared with ox-LDLgroup,0.02U/L insulin group,0.04U/L insulin group and0.06U/L insulin group have statistical difference(P<0.05), cholesterol ester content increased with insulinconcentrations increasing.There is statistically significant difference between group of ox-LDL and macrophage group (P<0.05), the cholesterol ester content of foam cells increased. Compared with the ox-LDL+insulin group by ox-LDL-+insulin+Fluorine simvastatin group,ox-LDL+insulin+high concentrations of Loulu group, ox-LDL+insulin+concentration Loulu group ox-LDL+insulin+low concentrations of the Loulu group,thedifference is statistically significant (P<0.05), the cholesterol ester content of foam cells reduces following increasing of Loulu concentration.5. Observing the expression of ACAT-1mRNA by real time PCR:There is statistically significant between group of ox-LDL and macrophage group (P<0.05),the ACAT-lmRNA increased. Compared with the ox-LDL+insulin group by ox-LDL-H-insulin+Fluorine simvastatin group,ox-LDL+insulin+high concentrations of Loulu group, ox-LDL+insulin+concentration Loulu group, ox-LDL+insulin+low concentrations of the Loulu group,the differences is statistically significant (P<0.05), the ACAT-1mRNA reduces following increasing of Loulu concentration.6.Observing the expression of ACAT-1protein by Westren blot:There is statistically significant between group of ox-LDL and macrophage group (P<0.05),the ACAT-1protein increased. Compared with the ox-LDL+insulin group by ox-LDL-+insulin+Fluorine simvastatin group,ox-LDL+insulin+high concentrations of Loulu group, ox-LDL+insulin+concentration Loulu group, ox-LDL+insulin+low concentrations of the Loulu group,the differences is statistically significant (P<0.05), the ACAT-1protein reduces following increasing of Loulu concentration.Conclusions:1. Induced by PMA,macrophages were incubated with ox-LDL,which can simulate the formation of foam cells.2. Detecting the rate of foam cell transformation by Oil red O staining and detecting the cholesterol ester content of foam cells by enzymatic:high concentrations of insulin can promote foam cell formation, which can be inhibited by Loulu.3. By Western blot and real time PCR:the function which high concentrations of insulin can promote foam cell formation is related to the exprssion of ACAT-1which is promoted by insulin, which can be inhibited by Loulu.4. So the function that insulin of high concentrations promoting foam cell formation and Loulu inhibiting foam cell formation is close related with the expression of ACAT-1. This study provides preliminary experimental evidence for Loulu preventing atherosclerosis.更多还原