【作者】 李庚；

【导师】 董念国；

【作者基本信息】 华中科技大学 ， 心血管外科， 2014， 博士

【摘要】 第一部分：巨噬细胞极化与主动脉瓣膜钙化的相关性研究目的：收集人体瓣膜标本,鉴定M1、M2型巨噬细胞,比较钙化瓣膜与正常瓣膜之间M1、M2差异。RT-PCR检测TNF-α、IL-6、IL-12α、TGF-β1、IL-10和AMAC-1细胞因子表达情况。方法：根据《赫尔辛基宣言》和知情同意原则收集人体主动脉瓣膜标本。依瓣膜病变程度的轻重拟定瓣膜钙化分级,0、1、2、3共四级。对照组瓣膜为0级,取自无瓣膜病变的急性主动脉夹层者、无瓣膜病变的心脏移植受体心以及配型失败的供心。实验组瓣膜钙化为1-2级,取自因主动脉瓣退行性病变行瓣膜置换者,瓣膜明显增厚、钙化。一部分瓣膜标本固定、包埋、切片后行免疫组化染色鉴定；另一部分标本直接液氮保存。结果：免疫组化染色结果显示,实验组和对照组主动脉瓣膜中：①巨噬细胞浸润密度(细胞数/mm2)分别为(52.6±29.6,N=20),(22.5±16.0,N=14),P<0.05,具有统计学意义。②M1型巨噬细胞浸润密度分别为(48±30.0,N=20),(5.43±6.70,N=14),P<0.05,具有统计学意义。③M2型巨噬细胞浸润密度分别为(10.75±13.2,N=20),(20.79-±：14.5,N=20)P=0.0567,结果不具有统计学意义。④M1所占总巨噬细胞比例分别为0.941±0.246,0.297±0.301,P<0.05,具有统计学意义。⑤M2所占总巨噬细胞比例分别为0.251±0.254,0.945±0.112,且P<0.05,具有统计学意义。RT-PCR检测M1型细胞因子TNF-α、IL-6、IL-12α与M2型细胞因子TGF-β1、IL-10和AMAC-1。实验组表达水平均较对照组高,其中TGF-β1、IL-12α、IL-10、AMAC-1,P<0.05,具有统计学意义。TNF-α和IL-6的P值大于0.05,不具统计学意义。结论：增厚钙化主动脉瓣膜中,巨噬细胞含量增高。实验组以M1型巨噬细胞浸润为主,对照组以M2型巨噬细胞为主。提示M1型巨噬细胞同主动脉瓣膜钙化可能存在相关性。细胞因子结果表明,实验组TGF-β1、IL-12α、IL-10、AMAC-1等细胞因子较对照组均增高,说明瓣膜钙化可能是多种炎症因子参与作用的结果。第二部分：巨噬细胞诱导VICs钙化及机制研究第一节：体外诱导巨噬细胞极化和VICs钙化目的：刺激U937单核细胞为巨噬细胞,并分别诱导其向M1、M2极化。收集人主动脉瓣膜临床标本,体外分离培养VICs,使用钙化培养基诱导其钙化。方法：PMA刺激U937单核细胞分化为巨噬细胞。不同浓度LPS和IL-4分别刺激巨噬细胞,WB和RT-PCR检测M1、M2型相关标志性蛋白。从临床瓣膜标本中分离培养瓣膜间质细胞(VICs),钙化培养基培养7天后检测BMP2, ALP等钙化相关蛋白,14天后检测钙化斑块形成。结果：U937人单核细胞经PMA刺激后分化为巨噬细胞。经LPS刺激后,iNOS表达上调,经IL-4刺激后,Arg-1和TGF-β表达上调。分离并鉴定人主动脉瓣膜VICs,表型鉴定结果为a-SMA (+)> vimentin (+)。钙化培养基培养7天后检测钙化相关因子ALP、BMP2等明显增高,与正常培养基相比,ALP升高5.1-0.23倍,BMP2升高3.51±0.24倍,P<0.05,具有统计学意义。14天后行茜素红染色,见明显钙结节染色,证明钙化培养基诱导VICs钙化效果可靠。结论：U937细胞人单核细胞分化的巨噬细胞,经LPS/IL-4刺激建立M1/M2细胞模型。分离培养VICs,钙化培养基诱导建立VICs钙化模型。第二节：SOCS蛋白与巨噬细胞极化的联系目的：探讨SOCS3和SOCS2是否参与人巨噬细胞极化。方法：将U937单核细胞诱导为巨噬细胞,给予不同浓度LPS刺激,检测SOCS3表达。给予不同浓度IL-4刺激,检测SOCS2表达。SOCS3病毒,转染巨噬细胞后,给予LPS刺激,观察M1型巨噬细胞相关细胞因子变化。结果：LPS可上调巨噬细胞iNOS表达,诱导其M1型分化。同时,LPS可增加巨噬细胞SOCS3蛋白表达,且SOCS3表达趋势与iNOS高度一致。IL-4可上调巨噬细胞Arg-1表达,诱导其向M2型分化。同时,IL-4可增加SOCS2蛋白表达。SOCS3转染实验结果表明：巨噬细胞转染SOCS3病毒后,相同浓度LPS刺激可明显上调iNOS和TNF-α表达。结论：M1型、M2型巨噬细胞的极化与SOCS3、SOCS2蛋白密切相关。上调SOCS3蛋白可诱导巨噬细胞M1型极化。第三节：M1型巨噬细胞促进VICs钙化的研究目的：探讨巨噬细胞M1型极化是否促进VICs钙化及相关机制。方法：用PMA诱导U937单核细胞分化巨噬细胞。LPS500ng/ml刺激巨噬细胞,12小时。流式细胞仪检测M1型巨噬细胞比例。重复上述实验,留取细胞上清液,深低温冰箱冻存,备用。选取第3-5代VICs,种植于六孔板,分组如下：A组为对照组,1640培养基lm1+钙化培养基1血；B组为PMA刺激的巨噬细胞上清液1m1+钙化培养基1ml；C组为经LPS和PMA联合刺激后的巨噬细胞上清液1m1+钙化培养基lml。7天后行RT-PCR以及Westen Blot检测相关钙化指标ALP、BMP2、OPN和TNF-α。结果：经LPS刺激后可明显上调M1型巨噬细胞比例。经PMA和LPS刺激的巨噬细胞上清液与VICs共培养,可明显上调ALP、BMP2、OPN表达。结论：M1型巨噬细胞极化可促进VICs钙化。更多还原

【Abstract】 Part Ⅰ:The correlation of macrophage polarization and aortic valve calcificationObjective:Collect the human valve specimens, and identify M1and M2macrophages, compare the differences between M1and M2of calcified valves and normal valves. Detect the expression of TNF-a, IL-6, IL-12a, TGF-β1, IL-10and AMAC-1by RT-PCR.Methods:According to the "Declaration of Helsinki" and the principle of informed consent to collect specimens of human aortic valves. Propose grading valvular calcification,0,1,2,3of four stage. Control valve calcification is stage zero. The valves of the control group were from the acute aortic dissection patients with no valve disease, heart transplant recipients without heart valve disease, and the donor heart of matching defeat. Valvular calcification in the experimental group stage1-2. The valves of the experiment group were from the cardiac valve replacement patients caused of aortic degeneration, the valves were obviously thickened or calcification; Part of these valve specimens were fixed, embedded, sliced underwent immunohistochemical staining for identification. The rest part of the specimen directly liquid nitrogen preservation.Results:Immunohistochemical staining showed that the experimental group and the control group in the aortic valve:①macrophage infiltration density (cells/mm2), respectively (52.6±29.6, N=20),(22.5±16.0, N=14), the difference was statistically significant (P<0.05).②Ml macrophages infiltration density was (48±30.0, N=20),(5.43±6.70, N=14), P<0.05, statistically significant.③M2macrophage infiltration density was (10.75±13.2, N=20),(20.79±14.5, N=20) P=0.0567, the result was not statistically significant. The proportion of the total④Ml macrophages were0.941±0.246,0.297±0.301, P<0.05, statistically significant.⑤M2macrophages proportion of the total cells were0.251±0.254,0.945±0.112, and P<0.05, statistically significant. RT-PCR detection of M1-type cytokines TNF-a, IL-6, IL-12a and M2-type cytokine TGF-β1, IL-10and AMAC-1. The expression levels of the experimental groups were higher than the control group, in which TGF-β1, IL-12a, IL-10, AMAC-1, P<0.05, statistically significant. TNF-α and IL-6P values greater than0.05, not statistically significant.Conclusion:Calcified aortic valve thickened, the macrophage content increased. The experimental group M1macrophages infiltration based, control group M2macrophages based. Tip M1macrophages with aortic valve calcification may be correlated. Cytokine results showed that the experimental group TGF-β1, IL-12α, IL-10, AMAC-1and other cytokines were significantly higher than the control group, indicating that the results of valvular calcification may be a variety of inflammatory cytokines involved in the role. Part Ⅱ:Mechanisms of macrophage-induced VICs calcificationSection Ⅰ:macrophage polarization and VICs calcification in vitroObjective:Stimulate U937monocytes into macrophages, and induce them to M1, M2macrophage. Human aortic valve clinical specimens were collected, isolated and cultured VICs in vitro, using the calcification medium to induce calcification.Methods:PMA stimulated U937monocytes differentiate into macrophages. Different concentrations of LPS and IL-4were stimulated to macrophages, WB and RT-PCR detection of M1, M2type associated logos protein. Isolated and cultured valvular interstitial cells (VICs) from clinical specimens, valvular calcification medium for7days then detection of BMP2, ALP and other calcification-related protein,14days after the detection of calcified plaque.Results:U937human monocytic differentiation into macrophages after PMA stimulation. After LPS stimulation, iNOS upregulation, and after IL-4stimulation, Arg-1and TGF-β expression increased. Separation and identification of human aortic valve VICs, phenotypic identification results for a-SMA (+), vimentin (+). Calcification medium for7days after the detection of calcifications associated factor ALP, BMP2, such as significantly increased, compared with the normal medium, ALP increased5.10±0.23fold, BMP2increased3.51±0.24fold, P<0.05, statistically significant.14days after Alizarin red staining, dyeing come out significant calcium nodules proved reliable VICs calcification calcification medium induced effects.Conclusion:U937human monocytic cells differentiated macrophages stimulated by LPS/IL-4establish M1/M2cell model. Isolation and culture VICs, calcification medium induced VICs calcification model. Section Ⅱ:relationship between SOCS protein and macrophage polarizationObjective:To investigate whether SOCS3and SOCS2involved in human macrophage polarization.Methods:U937monocytes were induced into macrophages, LPS stimulation at different concentrations to detect SOCS3expression. Different concentrations of IL-4given stimulation SOCS2expression detected. After transfection of SOCS3virus to macrophages, giving LPS stimulation and observed Ml macrophages associated cytokines.Results:LPS can upregulate the expression of iNOS, and induce macrophages differentiation to Ml. Meanwhile, LPS increases the expression of macrophage SOCS3, and SOCS3expression was highly consistent trend of iNOS. IL-4can upregulate macrophage Arg-1expression and induce differentiation of M2. Meanwhile, IL-4can increase SOCS2expression. SOCS3transfection results showed that:the macrophages transfected SOCS3virus, the same concentration of LPS stimulation significantly increased iNOS and TNF-a expression.Conclusion:Ml and M2macrophages is closely related to SOCS3, SOCS2proteins. Upregulated SOCS3protein can induce macrophage M1polarization. Section Ⅲ:Research about Ml macrophages promote VICs calcificationObjective:To investigate whether the Ml macrophage polarization promotes VICs calcification and related mechanisms.Methods:PMA-induced U937monocyte differentiate into macrophages. Stimulated macrophages by LPS500ng/ml,12hours. Flow cytometry test M1macrophages ratio. Repeat the above experiments, specimens from cell supernatant, deep hypothermia freezer frozen and set aside. Select the3-5generations VICs, grown in six-well plates, grouped as follows:A group was the control group,1640medium lml+calcification medium1ml, group B, PMA-stimulated macrophage culture supernatants lml+calcification medium1ml; group C LPS and PMA combined stimulated macrophage culture supernatants lml+lml calcification medium.7days later, using RT-PCR and Westen Blot detect calcifications associated indicators ALP, BMP2and OPN.Results:After LPS stimulation significantly increased the proportion of M1macrophages. By PMA and LPS-stimulated macrophages were cultured supernatant VICs can be significantly increased ALP, BMP2, OPN expression.Conclusion:M1macrophages polarization can promote VICs calcification.更多还原