【作者】 王云炎；

【导师】 温端改； 侯建全；

【作者基本信息】 苏州大学 ， 外科学， 2010， 博士

【摘要】 近几年来,主要组织相容性复合体-I类相关链A(major histocompatibility complex class I-related chain A,MICA)抗体与实体器官移植特别是肾移植的排斥反应和移植肾长期存活率之间的关系成了移植免疫学基础和临床研究的热点。在等待肾移植的患者体内检测到了预存的MICA抗体,说明在移植前患者就接触到了MICA抗原而产生了特异性的抗体;移植后MICA抗体与免疫排斥反应和慢性移植肾失功之间均存在着密切的关系。由于MICA蛋白可以表达在血管内皮细胞表面,但不表达在T淋巴细胞,因而MICA分子可能直接引起移植肾血管的损伤,进而导致移植肾功能损害。有学者研究了MICA抗原对淋巴细胞生物学特性的影响,发现MICA抗原对T细胞和B细胞有促进其增殖的作用。最近对心脏移植的研究中发现,血清中的可溶性MICA(sMICA)分子可以减轻移植物的免疫损伤,对移植物起到保护作用。MICA分子和NKG2D是互为配受体的关系,且NKG2D是NK细胞表面的一种激活性受体。但是MICA抗原对内皮细胞的生物学特性的影响, sMICA在肾移植中的是否有保护作用,以及NK细胞能否被激活而对内皮细胞产生杀伤作用,均未见报道。因此,本课题将分三个部分进行研究:(1)应用MICA重组蛋白对血管内皮细胞进行刺激,观察它对内皮细胞的生物学特性及其分泌功能的影响;(2)外源性抗原对内皮细胞MICA基因表达的调节作用,以及对细胞上清中sMICA的水平的影响;(3)表达MICA分子的内皮细胞与NK细胞共同培养,观察NK细胞对内皮细胞的杀伤作用,并探讨其可能的作用机制。第一部分MICA抗原对内皮细胞生物学功能的影响目的观察主要组织相容性复合体-I类相关链A (MICA)抗原对血管内皮细胞生物学功能的影响。方法将外源性重组MICA蛋白分A5(5ng/ml),A10(10ng/ml),A25(25ng/ml)三个剂量加入培养基中作为实验组,A0组加入等体积的磷酸盐缓冲液(PBS)作为对照组,对人脐静脉内皮细胞(HUVEC)予以培养。用流式细胞仪检测其细胞周期和凋亡情况,采用噻唑蓝(MTT)比色法检测细胞增殖情况,并测定各组细胞上清液中前列环素代谢产物6-酮-前列腺素F1α(6-keto-PGF1α)、内皮素(ET-1)、组织型纤溶酶原激活物(t-PA)及其抑制物(PAI)的水平。结果各实验组(A5,A10,A25 )内皮细胞A570值均高于对照组(A0)。以A5组增殖最为明显,A10组比A5组稍下降,两组之间差异统计学意义(P > 0.05),但明显高于A25组差异有统计学意义(P < 0.05)。在相同剂量下48h时增殖率最高,A570值分别为0.458, 0.446, 0.389。72h和96h呈持续缓慢增殖,随着时间延长增殖率逐渐下降,差异有统计学意义(P < 0.05)。A10, A25组细胞上清中的6-keto-PGF1α水平分别为144.6, 132.8pg/ml,A0, A5组分别为226.5, 232.6pg/ml;A10,A25组ET-1水平分别为23.6, 25.8pg/ml,A0,A5组分别为11.8, 10.4pg/ml。A10,A25组和A0,A5组比较,差异均有统计学意义(P < 0.05);而A10与A25组比较,A0与A5组比较,差异均无统计学意义(P > 0.05)。各实验组(A5,A10,A25 ) t-PA、PAI分别为161.2, 154.2, 157.8ng/ml;221.6, 248.5, 252.4ng/ml;而对照组(A0)分别为233.5ng/ml、137.8ng/ml。实验组和对照组之间比较,差异均有统计学意义(P < 0.05);而各实验组之间比较,差异均无统计学意义(P > 0.05)。各实验组细胞无明显凋亡,细胞周期也无明显改变。结论MICA抗原对内皮细胞的生长周期无明显影响,也不引起其凋亡,但能刺激内皮细胞增殖,以小剂量诱导增殖明显,并呈持续缓慢增殖。同时能够引起内皮细胞功能受损,凝血功能增强,而纤溶功能下降,有助于血栓形成。第二部分外源性抗原对内皮细胞MICA基因表达的影响目的探讨外源性抗原对内皮细胞MICA基因表达的影响。方法将MICA重组蛋白分A5(5ng/ml), A10(10ng/ml), A25(25ng/ml)和热休克蛋白(HSP-70)分B5(5ng/ml), B10(10ng/ml), B25(25ng/ml)各三个剂量的实验组,对内皮细胞(HUVEC)予以诱导培养,对照组A0、B0组分别加等量的磷酸盐缓冲液(PBS)。采用实时荧光定量PCR方法检测内皮细胞MICA mRNA表达、Western blot法检测MICA蛋白和流式细胞仪检测MICA蛋白在细胞膜表面的表达,用ELISA法测定细胞上清液中可溶性MICA (sMICA)的水平。结果用三个剂量MICA重组蛋白诱导48h后,各实验组(A5,A10,A25)内皮细胞MICA mRNA和MICA蛋白的表达量与对照组比较均有显著增加,差异均有统计学意义(P < 0.05)。A5组和A10组MICA mRNA和MICA蛋白均高于A25组,而且差异均有统计学意义(P < 0.05);但A5组与A10组之间比较差异无统计学意义(P > 0.05)。MICA膜蛋白表达以A10组(32.6%)最高,并与A5组(28.2%)、A25组(23.4%)之间有显著性差异(P < 0.05)。各实验组(A5,A10,A25 ) sMICA水平分别为35.8、27.4、21.8pg/ml,较对照组(A0,352.5pg/ml)显著下降,且差异均有统计学意义(P < 0.05),而各实验组之间比较,差异均无统计学意义(P > 0.05)。而HSP蛋白组内皮细胞MICA基因的表达和sMICA水平无明显改变。结论外源性MICA抗原能够诱导内皮细胞自身MICA mRNA、总蛋白和膜蛋白表达上调,尤其是细胞膜蛋白增加明显,但sMICA水平显著下降,而HSP蛋白组无显著变化。第三部分MICA分子介导NK细胞对内皮细胞的杀伤作用目的观察MICA分子在NK细胞杀伤内皮细胞过程中的作用,探讨其可能的机制。方法使用免疫磁珠方法及CD56阳性分选试剂盒分选NK细胞,并将NK细胞与表达MICA膜蛋白的活化内皮细胞,共同培养10h。NK细胞与A0组HUVEC(同第二部分)共培养为A组,与A10组为B组。用荧光素进行染色,在荧光显微镜下观察死亡和存活细胞数量,计算NK细胞对内皮细胞杀伤效率,用ELISA法测定细胞上清中的干扰素-γ(IFN-γ)和穿孔素水平。结果免疫磁珠法可以分选NK细胞,纯度为88.5%。NK细胞对B组内皮细胞的杀伤效率为35.5%,明显高于A组12.6%; B组IFN-γ和穿孔素水平显著高于A组,两组差异均有统计学意义(P < 0.05)。结论经过外源性MICA重组蛋白刺激的内皮细胞表面MICA分子表达增高,能够提高NK细胞对内皮细胞的杀伤效率;IFN-γ和穿孔素可能是发挥细胞毒作用的活性物质。更多还原

【Abstract】 Major histocompatibility complex class I-related chain A (MICA) antibodies are associated with acute rejection and poor renal-allograft survival, which has become focus of basic and clinical research of transplant immunology. Acute rejection was occurred in renal-allograft recipients with good HLA matching and anti-MICA antibodies were detected in serum and rejected allografts. Anti-MICA antibodies also existed in serum of patients waiting for renal transplantation, which indicated that the patients produced specific antibodies against MICA antigen before transplantation. There was close relationship between MICA molecules with chronic renal-allograft dysfunction. MICA protein is expressed on the surface of vascular endothelial cells, but not on T lymphocyte. Therefore, MICA molecule might cause injury of allograft vessles directly and result in renal-allograft dysfunction. It has been reported that soluble MICA (sMICA) can reduce immunologic injury of allograft in heart transplant recipients and protect allograft function. It was demonstrated that MICA antigen can make T and B lymphocytes proliferation.MICA is ligand of NKG2D receptor on NK cell and NKG2D is an activating receptor. But whether MICA antigen can influence endothelial cells, whether sMIICA can protect renal allograft and whether NK cell be activated to kill endothelial cell have not been reported. Therfore, this study will conclude three parts: (1) Observe the impact of biological characteristics and secreting function of endothelial cell after we add MICA antigen to the media for cultivation; (2) Moderating effects of exogenous antigen for MICA expression in endothelial cell and sMICA in superant; (3) Coculture NK cell and endothelial cell expressing MICA protein and observe the cytotoxicity of NK cell against endothelial cell. PartⅠ: Impact of MICA antigen to biological functions of endothelial cellsObjective To investigate the impact of major histocompatibility complex class I-related chain A (MICA) antigen to biological characteristics and secreting function of endothelial cell.Methods HUVECs were divided into three experimental groups(A5,A10,A25 ), which were stimulated with exogenous recombinant MICA protein at 5ng/ml, 10ng/ml, 25ng/ml respectively. Group A0 was added equivalent volume PBS as control. The apoptosis and cell cycle was measured by flow cytometry (FCM). The proliferation of endothelial cell was detected by MTT assay. The level of metabolic product of PGI2 (6-keto-PGF1α)、endothelin (ET-1)、tissue plasminogen activator (t-PA) and its inhibitor (PAI) in supernatant of all groups were detected.Results All experimental groups were higher values of A570 than control group. Both group A5 and A10 were significantly higher than the control group and group A25 (P < 0.05), but there were no significant differences between group A5 and A10 (P > 0.05). The proliferation rate at 48h was the highest in identical dosage. A570 value of experimental groups at 48h was 0.458, 0.446 and 0.389 respectively. The proliferation rate at 72h and 96h were persistently decreased as extending time. There were significant differences (P < 0.05). The level of 6-keto-PGF1αof group A10, A25 was 144.6 and 132.8pg/ml, which was lower than that of group A0 (226.5pg/ml) and A5 (232.6pg/ml). The level of ET-1 of group A10, A25 was 23.6 and 25.8pg/ml while the level of ET-1 of group A0, A5 was 11.8 and 10.4pg/ml. The differences were statistically significant (P < 0.05), but there was no difference between group A0 with A5, group A0 with A5 (P > 0.05). The level of t-PA of experimental groups were 161.2, 154.2 and 157.8ng/ml and the level of PAI were 221.6, 248.5 and 252.4ng/ml. Compared with control group, the level of t-PA in three experimental groups rised markedly and the level of PAI decreased obviously. These differences had statistical significances (P < 0.05). But there was no significant difference among the experimental groups (P > 0.05). There were no cell apoptosis and changes of cell cycle in all experimental groups.Conclusions The small dosage of MICA antigen can stimulate endothelial cell persistent proliferation. Meanwhile, MICA antigen can make endothelial cell injured and cause the coagulation function enhanced, fibrinolytic function declined.PartⅡ: Impact of exogenous antigen for expression of MICA gene in endothelial cellObjective To investigate the expression of MICA gene in endothelial cell stimulated with exogenous antigen.Methods The endothelial cells were divided into control group and three experimental groups, which were stimulated with exogenous recombinant MICA antigen (group A) and heat shock protein (HSP, group B) at 5ng/ml, 10ng/ml and 25ng/ml. The expression of MICA mRNA was detected by real-time fluorescence quantitative PCR. The expression of MICA protein was measured by Western blot. The expression of MICA total protein on the cell surface was detected by using flow cytometry (FCM). The levels of soluble MICA (sMICA) in supernatant of all groups were detected by ELISA.Results The expression of MICA mRNA and MICA protein were increased significantly after MICA stimulation (P < 0.05). The expression of MICA mRNA of group A10 were remarkably higher than the group A25, but had no differences with group A5. The expression of MICA membrane protein of group A10 were higher than that of group A25 (P < 0.05), but had no differences with group A5. The level of sMICA in three experimental groups were 35.8、27.4、21.8pg/ml respectively,which decreased obviously comparing with that of control group( 352.5pg/ml). These differences had statistical significances (P < 0.05). But there was no significant difference among the experimental groups (P > 0.05). However, the expression of MICA gene and sMICA level didn’t change after HSP stimulation.Conclusions The exogenous MICA antigen up-regulate the expression of MICA mRNA and protein, especially the membrane protein on the cell surface significant increase, but sMICA in supernatant decreased.PartⅢ: Killing effect of NK Cell to Endothelial Cell mediated by MICA MoleculeObjective To observe the role of MICA molecule in the process of NK cells killing endothelial cells.Methods NK cells were selected by using immumomagnetic beads (CD56 positive isolation kit) according to the manufacturer’s recommended conditions. It was divided into four groups. Group A was NK cells and HUVECs of group A0 (see PartⅡ), group B was NK cells and HUVECs of group A10. NK cells and HUVECs expressing MICA molecule were cocultured for 10h. All the cells were stained by fluorescein AO/EB and the dead cells were measured under fluorescence microscope. Killing effect of NK cells was calculated by the number of dead cells among total cells. Level of IFN-γand perforin in cell superant were detected by ELISA.Results The purity of NK cells selected by using immumomagnetic beads was 88.5%. The killing effect of NK cells to group Bwas 35.5%, which was higher than that of group A, 12.6%. There were significant differences between two groups (P < 0.05). Level of IFN-γand perforin in group B were higher than that of group A and the differences were statistically significant (P < 0.05).Conclusion Expression of MICA molecule on the surface of HUVECs can raise the cytotoxicity of NK cells to HUVECs. IFN-γand perforin might be bioactive substance playing killing effect.更多还原