| [Objective]As one of the main reasons of the old die, Atherosclerosis (As) was great harmful to people’s health, with its incidence was Higher and higher, it had been the focus of attention in society.Generally first,Atherosclerosis had lipid and compound carbohydrate accumulation, bleeding and thrombosis, fibre hyperplasia and calcinosis, later,Artery become transformation and calcification gradually, which often involved the elastic of muscular artery,once it had development enough to block the artery cavity, tissue or organs which these arteries supplyed would ischemia or necrosis.As the pathology base of cerebrovascular disease,atherosclerosis (As) was a kind of progressing inflammatory disease,there were abundance inflammatory mediators and mononuclear cells in the atheromatous plaque, it demonstrated that mononuclear cells played the key role in the inflammatory process.In Classical theories of Atherosclerosis, Interleukin was considered as playing an important role in chronic vascular inflammation.Interleukin8was secreted primarily by monocytes macrophages、endothelial cells, it had chemotaxis function for mononuclear cells,urged it adhere to vascular endothelial, attracted neutrophils and lymphatic cells into the subendothelial clearance, promoted smooth muscle cell proliferation and increased the instability of plaques,involved in the formation of atherosclerosis.ox-LDL played an important role in the process of inducing macrophages apoptosis,increasing the instability of atherosclerotic plaque.Series research had showed that OX-LDL had important cytotoxic effects to macrophages, endothelial cells in atherosclerotic plaque.In AS, ox-LDL played a role of Starting and enlarging, promoting Macrophages change into foam cells, regulating cell proliferation and induce apoptosis.Atherosclerosis (AS) was a kind of chronic inflammatory disease, the inflammatory mediators were found in a wide variety of cells which were associated with atherosclerosis,such as CD14、 CD36、CD86.With the help of ox-LDL,they Can induce the expression of matrix metalloproteinases, increase the coagutant activity of endothelial cell,promote the releasion of inflammatory factor and instability of plaque, play a key role in the process of atherosclerosis pathology.The prophase study has found that there were various kinds of apoptosis and necrosis cells in AS plaque,including endothelial cells, smooth muscle cells and macrophage cell. Compared with smooth muscle cells and endothelial cells, macrophages were more prone to apoptosis,so Macrophages played the leading role in apoptosis.The apoptosis of macrophages was important factor in instability atherosclerotic plaque, In a sense, the balance of apoptosis and cell proliferation decided the Occurrence and development of atherosclerotic plaque.Although the role of CD14、CD34、 CD86in monocyte/macrophage surface has reported respectively, what the relation ship among them and the role of early and late apoptosis when IL-8impact on ox-LDL-induced RAW264.7cells. This research was very few, it was worth discussing.Therefore, it was of great significance to explore the relationship of CD14、 CD34、 CD86、 early/terminar apoptosis and circulation system disease for the treatment of diseases and the research of new drug.it also made it become a new hotspot research of coronary heart disease.They could form a complex network between inflammation factors in the living, maybe, there were many positive and negative feedback loop formation. Promoted inflammation factors and anti-inflammatory factor both appear in chronic inflammation of the atherosclerosis,They sinked into the imbalances and balance contradictory unity.The cytokines which Participated in these inflammatory reaction had TNF-a, TNF-βIL-1, IL-10, γ-interferon etc, however,TNF-a, TNF-β, IL-1β, IL-8belonged to promote inflammation cell factors among them, and IL-10belonged to anti-inflammation cell factor.Although it had clearly that ox-LDL could stimulate macrophages to secrete IL-1β and the role of IL-10,IL-1β, IL-8had been reported in AS, what would happen when IL-8impacted on ox-LDL-induced RAW264.7cells and what the interaction between them,there was little research, it should be deserves further study.As the index which reflected the state of inflammation,it should be furher explicated between their relations and the effect of the development in AS,this provided the new ideas for prevention and cure of atherosclerosis.The cell model of ox-LDL-induced RAW264.7cells was often used to simulate human mononuclear macrophages in role of AS, to explore the pathology and pathogenesis of atherosclerosis.Accorded to explore the role of IL-8in ox-LDL-induced RAW264.7cells, researched the key role of IL-8in the pathology and pathogenesis of atherosclerosis,foud out the pathogenic targets and interverie,it would bringed the new treatment strategies hope for atherosclerosis in the future.This topic was divided into three parts:The first part:RAW264.7cells were stimulated in different groups for48h, and later,the cells were collected.The cells were detected for IL-8mRNA expression level by PCR, then,the cells were detected for IL-8expression level by WESTERN BLOT. The aim was to study whether IL-8were related to macrophages, whether IL-8mRNA messenger transduction were consistent heightly with protein translation in RAW264.7, the further deduction maybe make between the IL-8level and serious degree of AS.The second part: RAW264.7cells were stimulated in different groups for48h, the cells were collected and detected for CD14、CD34、 CD86、early apoptosis and terminal apoptosis by flow cytometry.The change of CD14、 CD34、 CD86in macrophages and its role of early apoptosis/terminal apoptosis in AS had been reported, inflammation factors were more or less related to them in Induing Apoptosis; recruited more inflammatory factor, promoted the stability of atherosclerotic plaque, participated in the occurrence and development of atherosclerosis.However,what the role of IL-8in OX-LDL induced RAW264.7cells and what the function relation was between them,all of them was few researched,it should deserves further study.The third part RAW264.7cells were stimulated in different groups for48h, the liquid supernatant were collected and detected for the level of IL-1β,IL-10by ELISA.In chronic inflammatory reaction of AS, inflammation factors and anti-inflammatory factor in the imbalances and balance the contradictory unity.It had reported that IL-1β could promote inflammation and IL-10had adverse effect, but what the relationship was between IL-8and them, what its role was, there had been no reported. It would be provided the new ideas for prophylaxis and treatment to further research the change rule between promote inflammation factors and anti-inflammatory factor in AS.[Methods]RAW264.7was cultivated and divided into four groups,A group (control group):the cells were chltivated in the tissue Culture Plate for48hours without intervene; B group:the cells were stimulated by IL-8(100μmol/L)for48hours; C group:the cells were stimulated by ox-DL(200μ g/ml)for48hours;Dgroup:the cells were stimulated by IL-8(100umol/L)and ox-LD(200μg/ml)for48hours.Cells and supernatants were collected in48hours, the expression of IL-8mRNA and protein was examined by PCR and WESTERN BLOT,respectively;CD14、CD34、CD86and the early/later apoptotic rate of U264.7cells was examined by flow cytometry (FCM), The levels of IL-1β, IL-10in the supernatants were determined by ELISA.Statistic analysisAll results were analysed by SPSS13.0software package. The level of IL-8mRNA in different RAW264.7cells groups were detected by polymerase chain reaction (PCR) and IL-8protein was detected by WESTTERN-BLOT respectively.The positive rate of CD14、 CD34、CD86, early apoptosis/terminal apoptosis were detected by flow cytometry and the level of IL-1β、IL-10in different RAW264.7cells groups were detectes by ELISA. Sample mean was expressed by mean±standard deviation. If the measurement data was satisfy homogeneity of variance,it would be analysised by one-way ANOVA,then difference between groups was of statistic significance (P<0.05) and it would be analysised by LSD; If the measurement data was not satisfy homogeneity of variance,it would be analysised by Welch,then difference between groups was of statistic significance (P<0.05) and it would be analysised by Dunnettf’s T3.[Results]1、The data that the level of IL-8mRNA was analyzed by Welch, difference between groups was of statistic significance (P<0.05.Comparing with A group by Dunnettf’s T3,B、 C、 D group were significant difference (P=0.000),and there was no significant difference between B group and C group (P=0.949).It indicated that there was positive feedback for IL-8to promote the level of IL-8mRNA in ox-LDL-induced RAW264.7cells.2、 The data that the level of IL-8was analyzed by Welch, difference between groups was of statistic significance (P<0.05).Compared with A group by Dunnettf’s T3,B、 C、 D group were significant difference (P=0.000),and there was no significant difference between B group and C group (P=0.182).It indicated that there was positive feedback for IL-8to promote IL-8protein Synthesis in ox-LDL-induced RAW264.7cells.3、 The data that CD14、 CD34、 CD86、 early apoptosis and terminal apoptosis were analyzed by Welch, the P value of CD86、early apoptosis were0.857、0.952.(P>0.05),but difference between groups in CD14、 CD34、terminal apoptosis all were significant difference (P<0.05)4、 The level of IL-1β in liquid supernatant which cultivated RAW264.7cells were analyzed by Welch, difference between groups were significant difference (P <0.05).Compared with A group by Dunnettf’s T3,the level of IL-1β in B、D groups were promoted (P<0.05), B group was higher than C group (P<0.05)5、 The level of IL-10in liquid supernatant which cultivated RAW264.7cells were analyzed by Welch, difference between groups were significant difference (P <0.05). Compared with A group by Dunnettf’s T3,the level of IL-10in B、C、 D groups were inhibited Significantly (P<0.05), B group was higher than C group (P<0.05)。and it was no significant difference between B group and C group (P=0.886)[Conclusions]The following conclusions are make through above three part of experiments:1、 IL-8Can promote ox-LDL-induced RAW264.7cells over expressed IL-8mRNA, and It is consistent Highly in messenger transduction and Protein translation. It presentes that IL-8participate in AS and accelerate the development of the AS by positive feedback to promote their own secretion.2、 IL-8Can increase the level of CD14、 CD34in ox-LDL-induced RAW264.7cells, induce the late apoptosis of macrophages, promote the instability of atherosclerotic plaque.3、 IL-8Can increase the level of IL-1β and inhibit the level of IL-10in ox-LDL-induced RAW264.7cells,induce more macrophages and lymphatic cell activate to take part in the inflammation of atheromatous plaque. |
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