人可溶型CD155分子產(chǎn)生機(jī)制的研究
發(fā)布時(shí)間：2019-06-29 10:56
【摘要】： 人CD155分子最先由Bernhardt等人于1989年發(fā)現(xiàn),該分子能夠介導(dǎo)脊髓灰質(zhì)炎病毒(poliovirus,PV)入侵機(jī)體,引起脊髓灰質(zhì)炎,因此命名為PVR(poliovirus receptor)。免疫球蛋白超家族成員(IgSF)CD155屬于黏附分子中的Nectin家族,參與細(xì)胞的黏附和黏附連接(AJs)的形成,并且在介導(dǎo)惡性腫瘤細(xì)胞的轉(zhuǎn)移中發(fā)揮重要作用。因CD155同Nectins的差別,2003年,Takai等人將CD155命名為Necl-5(Nectin like molecule-5),隨后的研究發(fā)現(xiàn)該分子還與胚胎期神經(jīng)系統(tǒng)的發(fā)育有關(guān)。CD155在人多種正常細(xì)胞上低水平表達(dá),而在多種腫瘤細(xì)胞上高表達(dá)。最近研究表明,CD155為CD226的配體,CD155與CD226分子的相互作用介導(dǎo)了NK細(xì)胞殺傷腫瘤的作用,并且成為NK細(xì)胞殺傷腫瘤的研究熱點(diǎn)之一,備受關(guān)注。作為黏附分子和免疫調(diào)節(jié)膜分子,該分子存在著可溶型形式(sCD155),可能在調(diào)節(jié)病毒入侵、細(xì)胞黏附和腫瘤的免疫監(jiān)視中起到重要作用。以往研究發(fā)現(xiàn)許多可溶型膜分子與一些疾病的發(fā)生和發(fā)展有關(guān),產(chǎn)生機(jī)制可以是通過細(xì)胞直接分泌,也可以由一些蛋白酶水解跨膜分子的胞膜外區(qū),使其脫落形成。而sCD155的產(chǎn)生機(jī)制和功能至今尚無確切定論。 本實(shí)驗(yàn)首先采用間接免疫熒光染色和FCM分析了人多種細(xì)胞系的CD155表達(dá)。CD155分子在細(xì)胞系上的表達(dá)分布有著一定的規(guī)律性:不表達(dá)于人B細(xì)胞系、NK細(xì)胞系和單核細(xì)胞系;而在T細(xì)胞系和巨核細(xì)胞系有表達(dá),在內(nèi)皮細(xì)胞系、上皮細(xì)胞系、上皮細(xì)胞來源的癌細(xì)胞系、膠質(zhì)瘤細(xì)胞系和黑色素瘤細(xì)胞系上均有高水平表達(dá)。 采用本室制備的10株抗CD155胞膜外區(qū)單克隆抗體(mAb),純化后標(biāo)記辣根過氧化物酶(HRP),通過競爭ELISA法確定了這10株mAbs識別CD155胞膜外區(qū)的3組表位,篩選出夾心ELISA最佳包被和檢測抗體分別為FMU-CD155.2和HRP/FMU-CD155.10,建立了檢測sCD155的夾心ELISA方法,敏感性為200ng/L,具有良好的穩(wěn)定性。利用此夾心ELISA方法對正常人和腫瘤病人血清中的sCD155進(jìn)行了檢測,正常人的血清sCD155濃度范圍為1.426～6.549μg/L(中位數(shù)2.529μg/L),部分腫瘤(白血病、淋巴瘤、胰腺腫瘤、乳腺癌和肺癌)病人血清中的sCD155水平低于正常人,其余腫瘤病人血清中的sCD155水平與正常人無顯著差異。 采用K562細(xì)胞為探討sCD155產(chǎn)生機(jī)制的細(xì)胞模型,以不同蛋白酶抑制劑處理K562細(xì)胞,用PMA刺激K562細(xì)胞不同時(shí)間,有的實(shí)驗(yàn)加入絲氨酸蛋白酶抑制劑AEBSF和半胱氨酸蛋白酶抑制劑NEM,用間接免疫熒光染色及FCM分析膜型CD155(mCD155)表達(dá),同時(shí)用夾心ELISA法檢測細(xì)胞培養(yǎng)上清中sCD155水平。結(jié)果表明靜止的K562細(xì)胞mCD155表達(dá)的陽性率約為15.7%。5mM AEBSF或0.2mM NEM處理細(xì)胞6h,mCD155表達(dá)陽性率均顯著增加,分別上升到96.1%和54.1%,并以時(shí)間和劑量依賴方式調(diào)節(jié)靜止細(xì)胞mCD155的表達(dá)。PMA刺激K562細(xì)胞可同時(shí)增加mCD155和sCD155的表達(dá),而加入AEBSF和NEM的細(xì)胞mCD155表達(dá)均有增加,PMA+AEBSF組較PMA+NEM組mCD155上升幅度大;同時(shí)加入蛋白酶抑制劑AEBSF或NEM后,細(xì)胞培養(yǎng)上清中sCD155的濃度均明顯下降。而其他蛋白酶抑制劑對K562細(xì)胞mCD155表達(dá)無影響。 總之,本文首次證實(shí)了酶切mCD155產(chǎn)生sCD155的蛋白酶為絲氨酸蛋白酶和半胱氨酸蛋白酶。成功建立了檢測sCD155的夾心ELISA方法,為進(jìn)一步探討sCD155產(chǎn)生機(jī)制和功能研究打下實(shí)驗(yàn)基礎(chǔ)。
[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
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392
本文編號：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
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392
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相關(guān)碩士學(xué)位論文 前1條
1 鄧虎平;人可溶型CD155分子產(chǎn)生機(jī)制的研究[D];第四軍醫(yī)大學(xué);2007年
，本文編號：2507761
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