人可溶型CD155分子产生机制的研究
发布时间：2019-06-29 10:56
【摘要】： 人CD155分子最先由Bernhardt等人于1989年发现,该分子能够介导脊髓灰质炎病毒(poliovirus,PV)入侵机体,引起脊髓灰质炎,因此命名为PVR(poliovirus receptor)。免疫球蛋白超家族成员(IgSF)CD155属于黏附分子中的Nectin家族,参与细胞的黏附和黏附连接(AJs)的形成,并且在介导恶性肿瘤细胞的转移中发挥重要作用。因CD155同Nectins的差别,2003年,Takai等人将CD155命名为Necl-5(Nectin like molecule-5),随后的研究发现该分子还与胚胎期神经系统的发育有关。CD155在人多种正常细胞上低水平表达,而在多种肿瘤细胞上高表达。最近研究表明,CD155为CD226的配体,CD155与CD226分子的相互作用介导了NK细胞杀伤肿瘤的作用,并且成为NK细胞杀伤肿瘤的研究热点之一,备受关注。作为黏附分子和免疫调节膜分子,该分子存在着可溶型形式(sCD155),可能在调节病毒入侵、细胞黏附和肿瘤的免疫监视中起到重要作用。以往研究发现许多可溶型膜分子与一些疾病的发生和发展有关,产生机制可以是通过细胞直接分泌,也可以由一些蛋白酶水解跨膜分子的胞膜外区,使其脱落形成。而sCD155的产生机制和功能至今尚无确切定论。 本实验首先采用间接免疫荧光染色和FCM分析了人多种细胞系的CD155表达。CD155分子在细胞系上的表达分布有着一定的规律性:不表达于人B细胞系、NK细胞系和单核细胞系;而在T细胞系和巨核细胞系有表达,在内皮细胞系、上皮细胞系、上皮细胞来源的癌细胞系、胶质瘤细胞系和黑色素瘤细胞系上均有高水平表达。 采用本室制备的10株抗CD155胞膜外区单克隆抗体(mAb),纯化后标记辣根过氧化物酶(HRP),通过竞争ELISA法确定了这10株mAbs识别CD155胞膜外区的3组表位,筛选出夹心ELISA最佳包被和检测抗体分别为FMU-CD155.2和HRP/FMU-CD155.10,建立了检测sCD155的夹心ELISA方法,敏感性为200ng/L,具有良好的稳定性。利用此夹心ELISA方法对正常人和肿瘤病人血清中的sCD155进行了检测,正常人的血清sCD155浓度范围为1.426～6.549μg/L(中位数2.529μg/L),部分肿瘤(白血病、淋巴瘤、胰腺肿瘤、乳腺癌和肺癌)病人血清中的sCD155水平低于正常人,其余肿瘤病人血清中的sCD155水平与正常人无显著差异。 采用K562细胞为探讨sCD155产生机制的细胞模型,以不同蛋白酶抑制剂处理K562细胞,用PMA刺激K562细胞不同时间,有的实验加入丝氨酸蛋白酶抑制剂AEBSF和半胱氨酸蛋白酶抑制剂NEM,用间接免疫荧光染色及FCM分析膜型CD155(mCD155)表达,同时用夹心ELISA法检测细胞培养上清中sCD155水平。结果表明静止的K562细胞mCD155表达的阳性率约为15.7%。5mM AEBSF或0.2mM NEM处理细胞6h,mCD155表达阳性率均显著增加,分别上升到96.1%和54.1%,并以时间和剂量依赖方式调节静止细胞mCD155的表达。PMA刺激K562细胞可同时增加mCD155和sCD155的表达,而加入AEBSF和NEM的细胞mCD155表达均有增加,PMA+AEBSF组较PMA+NEM组mCD155上升幅度大;同时加入蛋白酶抑制剂AEBSF或NEM后,细胞培养上清中sCD155的浓度均明显下降。而其他蛋白酶抑制剂对K562细胞mCD155表达无影响。 总之,本文首次证实了酶切mCD155产生sCD155的蛋白酶为丝氨酸蛋白酶和半胱氨酸蛋白酶。成功建立了检测sCD155的夹心ELISA方法,为进一步探讨sCD155产生机制和功能研究打下实验基础。
[Abstract]:Human CD155 is first discovered by Bernhardt et al. in 1989, which can mediate the invasion of poliovirus (PV) into the body and cause poliomyelitis, so it is named PVR. The immunoglobulin superfamily member (IgSF) CD155 belongs to the Nectin family in the adhesion molecule, participates in the formation of the cell adhesion and adhesion connection (AJs), and plays an important role in mediating the metastasis of malignant tumor cells. In 2003, Takai et al., named Necl-5 (Nectin like molecule-5), found that the molecule was also associated with the development of the embryonic-phase nervous system due to the difference between CD155 and Necins. CD155 is expressed at a low level on a variety of normal cells and is highly expressed on a variety of tumor cells. Recent studies have shown that CD155 is a ligand of CD226, and the interaction of CD155 with CD226 molecules mediate the killing of NK cells and become one of the hot spots of NK cell killing. As an adhesion molecule and an immunomodulatory membrane molecule, the molecule has a soluble form (sCD155), which may play an important role in the regulation of viral invasion, cell adhesion, and immunosurveillance of tumors. In the past, many soluble membrane molecules have been found to be related to the occurrence and development of some diseases, and the production mechanism can be directly secreted by the cells, and the extracellular region of the transmembrane molecule can be hydrolyzed by some proteases to form the membrane. The mechanism and function of sCD155 are not conclusive yet. In this experiment, indirect immunofluorescence staining and FCM were used to analyze the CD of human cell lines. The expression profile of CD155 in the cell line has a certain regularity: it is not expressed in the human B cell line, the NK cell line and the monocyte line, and is expressed in the T cell line and the megakaryocyte, and is in the endothelial cell line, the epithelial cell line and the epithelial cell source. Both the cancer cell line, the glioma cell line, and the melanoma cell line High-level expression was carried out.10 strains of anti-CD155 extracellular domain monoclonal antibody (mAb) prepared in this room were used to purify and label the horseradish peroxidase (HRP), and the three epitopes of the outer region of the CD155 cell membrane were identified by competitive ELISA. The best coating and detection antibodies for sandwich ELISA were FMU-CD155.2 and HRP/ F, respectively. MU-CD155.10, a sandwich ELISA for the detection of sCD155 was established, with a sensitivity of 200 ng. The serum sCD155 concentration in the normal and tumor patients was 1.426-6.549. m u.g/ L (median 2.529. mu.g/ L) and partial tumor (white). sCD155 level in serum of patients with blood disease, lymphoma, pancreatic tumor, breast cancer and lung cancer is lower than normal, and sCD155 in the serum of the remaining tumor patients There was no significant difference between the level and the normal. The cell model of the mechanism of sCD155 was studied by K562 cells. The K562 cells were treated with different protease inhibitors. The different time of the K562 cells was stimulated with PMA, and a serine protease inhibitor AE was added. BSF and cysteine protease inhibitor NEM, using indirect immunofluorescence staining and FCM to analyze the expression of membrane CD155 (mCD155) and sandwich ELISA. The results showed that the positive rate of mCD155 expression in the quiescent K562 cells was about 15.7%. The positive rate of mCD155 expression in 5 mM AEBSF or 0.2 mM NEM treated cells increased significantly, which increased to 96.1% and 54.1%, respectively, and depended on time and dose. The expression of mCD155 and sCD155 was regulated by PMA-stimulated K562 cells, and the expression of mCD155 and sCD155 was increased. The expression of mCD155 in both AEBSF and NEM was increased, and the expression of mCD155 in PMA + AEBSF group was greater than that of PMA + NEM group. The concentration of sCD155 in supernatant was significantly reduced, while other protease inhibitors were The expression of mCD155 in K562 cells was not affected. In conclusion, the expression of sC in mCD155 was confirmed for the first time in this paper. The protease of D155 is a serine protease and a cysteine protease. A sandwich ELISA method for detecting sCD155 is successfully established to furthe
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2507761
[Abstract]:Human CD155 is first discovered by Bernhardt et al. in 1989, which can mediate the invasion of poliovirus (PV) into the body and cause poliomyelitis, so it is named PVR. The immunoglobulin superfamily member (IgSF) CD155 belongs to the Nectin family in the adhesion molecule, participates in the formation of the cell adhesion and adhesion connection (AJs), and plays an important role in mediating the metastasis of malignant tumor cells. In 2003, Takai et al., named Necl-5 (Nectin like molecule-5), found that the molecule was also associated with the development of the embryonic-phase nervous system due to the difference between CD155 and Necins. CD155 is expressed at a low level on a variety of normal cells and is highly expressed on a variety of tumor cells. Recent studies have shown that CD155 is a ligand of CD226, and the interaction of CD155 with CD226 molecules mediate the killing of NK cells and become one of the hot spots of NK cell killing. As an adhesion molecule and an immunomodulatory membrane molecule, the molecule has a soluble form (sCD155), which may play an important role in the regulation of viral invasion, cell adhesion, and immunosurveillance of tumors. In the past, many soluble membrane molecules have been found to be related to the occurrence and development of some diseases, and the production mechanism can be directly secreted by the cells, and the extracellular region of the transmembrane molecule can be hydrolyzed by some proteases to form the membrane. The mechanism and function of sCD155 are not conclusive yet. In this experiment, indirect immunofluorescence staining and FCM were used to analyze the CD of human cell lines. The expression profile of CD155 in the cell line has a certain regularity: it is not expressed in the human B cell line, the NK cell line and the monocyte line, and is expressed in the T cell line and the megakaryocyte, and is in the endothelial cell line, the epithelial cell line and the epithelial cell source. Both the cancer cell line, the glioma cell line, and the melanoma cell line High-level expression was carried out.10 strains of anti-CD155 extracellular domain monoclonal antibody (mAb) prepared in this room were used to purify and label the horseradish peroxidase (HRP), and the three epitopes of the outer region of the CD155 cell membrane were identified by competitive ELISA. The best coating and detection antibodies for sandwich ELISA were FMU-CD155.2 and HRP/ F, respectively. MU-CD155.10, a sandwich ELISA for the detection of sCD155 was established, with a sensitivity of 200 ng. The serum sCD155 concentration in the normal and tumor patients was 1.426-6.549. m u.g/ L (median 2.529. mu.g/ L) and partial tumor (white). sCD155 level in serum of patients with blood disease, lymphoma, pancreatic tumor, breast cancer and lung cancer is lower than normal, and sCD155 in the serum of the remaining tumor patients There was no significant difference between the level and the normal. The cell model of the mechanism of sCD155 was studied by K562 cells. The K562 cells were treated with different protease inhibitors. The different time of the K562 cells was stimulated with PMA, and a serine protease inhibitor AE was added. BSF and cysteine protease inhibitor NEM, using indirect immunofluorescence staining and FCM to analyze the expression of membrane CD155 (mCD155) and sandwich ELISA. The results showed that the positive rate of mCD155 expression in the quiescent K562 cells was about 15.7%. The positive rate of mCD155 expression in 5 mM AEBSF or 0.2 mM NEM treated cells increased significantly, which increased to 96.1% and 54.1%, respectively, and depended on time and dose. The expression of mCD155 and sCD155 was regulated by PMA-stimulated K562 cells, and the expression of mCD155 and sCD155 was increased. The expression of mCD155 in both AEBSF and NEM was increased, and the expression of mCD155 in PMA + AEBSF group was greater than that of PMA + NEM group. The concentration of sCD155 in supernatant was significantly reduced, while other protease inhibitors were The expression of mCD155 in K562 cells was not affected. In conclusion, the expression of sC in mCD155 was confirmed for the first time in this paper. The protease of D155 is a serine protease and a cysteine protease. A sandwich ELISA method for detecting sCD155 is successfully established to furthe
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
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1 邓虎平;人可溶型CD155分子产生机制的研究[D];第四军医大学;2007年
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