摘要
目的
用单淋巴细胞分析方法(ISAAC)方法,从TransChromo (TC)老鼠,提取出9种特异性结合人类TRAIL-R1的人免疫球蛋白G的单克隆抗体,并分类这9种TRAIL-R1特异性单克隆抗体,筛查抗体在TRAIL-R1相对应的表位。进一步,筛选对TRAIL治疗敏感性较高的肿瘤细胞类型和TRAIL治疗肿瘤较有效的细胞TRAIL-R1表位位点。探讨TRAIL和TRAIL-R1的表位构象和敏感性关系,帮助阐明TRAIL诱导凋亡机制,为应用TRAIL和TRAIL-R1信号靶向治疗恶性肿瘤提供有用资料。
方法
ELISA竞争法分类TRAIL-R1特异性单克隆抗体以及该TRAIL-R1特异性抗体的可能表位。用流式细胞术,在Colo205、K562、Daudi、KMP4、MCF7等14种肿瘤细胞上继续分类和筛查TRAIL-R1特异性单克隆抗体在TRAIL-R1上的可能抗体表位。进一步用细胞生存能力分析,筛选TRAIL-R1抗体表位(TR1-272-、438-和419-表位),并选择对于细胞凋亡作用最强的TRAIL-R1抗体表位。接着,激光共聚焦显微镜分析和验证,所筛选的TRAIL-R1抗体表位在TRAIL结合之后可能发生的变化。进一步分析,各个细胞系TRAIL-R1 cDNA序列表达情况,检测各个细胞系TRAIL-R1的表达差异是否是因为各个细胞系TRAIL-R1碱基序列发生了变化。用衣霉素抑制各个肿瘤细胞TRAIL-R1的N-糖链或benzyl-a-GalNAc抑制肿瘤细胞合成O-糖链,继续用流式细胞术分析各个细胞系抑制TRAIL-R1糖链后的细胞染色结果。我们用Mann-Whitney'sU-检验估计P值。
结果
(1)ELISA竞争法推测9种TRAIL-R1特异性单克隆抗体在TRAIL-R1结合表位,从TR1-272抗体,TR1-419抗体,TR1-404和TR1-416抗体,TR1-407、417-和422-抗体,TR1-412和438-抗体之间,完全阻断、部分阻断或几乎不阻断等特点分析,推测出在重组体TRAIL-R1至少存在5种抗体表位。我们暂时将这些TRAIL-R1表位指定为TR1-272-、419-、416-、438-或417-表位。TR1-272-表位几乎被其他抗体竞争阻断,除了TR1-438-抗体。而TR1-438-表位不能被TR1-272-抗体竞争阻断,而被TR1-416和417-抗体部分竞争阻断。TR1-416-表位被TR1-272-和419-抗体阻断,而几乎不被TR1-417-和438-抗体阻断。TR1-417表位几乎不被TR1-272-、416-、419-和438-抗体阻断。TR1-419表位几乎被TR1-272抗体阻断,被TR1-417-和416-抗体部分阻断,而不被TR1-438抗体阻断。(2)下一步ELISA竞争法分析TRAIL-R1特异性单克隆抗体结合位点和在TRAIL-R1的TRAIL结合位点间的关系。结果显示TRAIL阻断TR1-417-和438-抗体和TRAIL-R1表位的结合,而几乎不阻断TR1-416-、419-和272-抗体的与TRAIL-R1表位的结合。这些结果显示,TR1-417-和438-表位位于TRAIL-结合位点或近处,TR1-272-、416-和419-表位可能位于TRAIL结合位点外侧。从竞争ELISA结果分析显示,TR1-272和438-表位是相互远离的。而抗体结合TR1-416-、417-和438-表位,较少相互干扰,TR1-417-和272-表位和TR1-416-、419-和272-表位是相互靠近的。(3)我们用流式细胞仪分析各个抗体在不同细胞系的结合情况。有趣的是,部分细胞系被TR1-404-、407-、416-、417-或422-抗体染色,而不被TR1-272-、419-、412-或438-抗体染色。这些结果证明,根据竞争ELISA结果分类抗体,符合细胞结合模式分类,在各个细胞系细胞表面TRAIL-R1抗体表位表达是不同的。TR1-416和417-表位在所有细胞系表达,表达TR1-416和417-位的细胞系,不一定表达TR1-22、419-和438-表位。而有趣的是,Colo205、K562、Daudi和Du145细胞不表达TR1-272表位,而强烈表达其他抗体表位。(4)进一步,我们检测不同TRAIL-R1表位(TR1-272-、438-和419-表位)表达是否与TRAIL诱导肿瘤细胞系凋亡的敏感度相关。其中TR1-272-表位缺失的Colo205、K562、Daudi和Du145肿瘤细胞比TR1-22-表位阳性的肿瘤细胞更容易被TRAIL诱导凋亡(p=0.0014)。与此对比,TR1-438-表位构象和TRAIL诱导的凋亡不相关(p=0.41)。TR1-419表位构象和TRAIL诱导的凋亡也显示不相关。我们观察到一些抗体包括TR1-416抗体和TR1-419抗体,不和TRAIL竞争结合TRAIL-R1。在这里,我们又检测了TRAIL和TR1-416抗体联合诱导肿瘤细胞凋亡。分析结果显示,TR1-416抗体明显增强了TRAIL对于诱导Colo205、K562和Daudi等肿瘤细胞的凋亡作用。总之,这些结果显示TR1-272抗体能够预测TRAIL在临床,用TRAIL治疗肿瘤时的敏感性。TR1-416抗体能够增强TRAIL诱导部分肿瘤细胞的凋亡作用。(5)为了解TRAIL诱导细胞死亡的不同敏感性调节机制,在TR1-272-表位阴性或阳性细胞系用TRAIL处理后,用激光共聚焦显微镜分析了细胞表面TRAIL-R1的情况。激光共聚焦显微镜检测结果显示,TRAIL作用后的TR1-272-表位阴性细胞,TRAIL-R1形成了大聚束,而TR1-272阳性细胞不能形成聚束。显示,TRAIL-R1寡聚化作用以及TR1-272-表位构象,可能影响肿瘤细胞对TRAIL诱导肿瘤细胞凋亡的敏感性。(6)进一步分析各个细胞系TRAIL-R1 cDNA序列表达发现,各个细胞系的TRAIL-R1氨基酸序列基本相同,因此说明各个细胞系表位表达差异不是发生在基因水平。(7)下一步继续分析TRAIL-R1的翻译后糖链修饰。在各个细胞,用衣霉素抑制TRAIL-R1的N-糖链或Benzyl-a-GalNAc抑制合成O-糖链,继续用流式细胞术分析,是否各个细胞系TR1-272、419-或438-表位是否被蛋白质翻译后糖链修饰后出现了阴性或阳性染色表达。结果显示,O-糖链或N-糖链的抑制未能改变阴性表达率。观察到糖链对于TRAIL-R1表位的构象无影响,提示糖链可能对表位构象不起关键作用。
结论
TR1-272-抗体可能识别TRAIL-R1构象,而这些构象能释放强大的细胞死亡信号。与此对比,TR1-438-、TR1-419-、TR1-416和TR1-417-表位构象与TRAIL诱导的凋亡不相关。TR1-416抗体能够明显增强TRAIL对于诱导Colo205、K562和Daudi等肿瘤细胞的凋亡作用。各个细胞系TRAIL-R1表位表达差异不是发生在基因水平,而TRAIL-R1翻译后糖链修饰可能对表位构象不起关键作用。进一步分析TRAIL表位构象和敏感性,可帮助阐明TRAIL诱导凋亡机制,并将为TRAIL和TRAIL-R信号治疗肿瘤提供有用资料。
Background
Immunospot array assay on a chip (ISAAC) method, from TransChromo(TC) mice, 9 several to extract specific binding human TRAIL-R1 in human immunoglobulin G monoclonal antibody, and classification of these nine kinds of TRAIL-R1 monoclonal cloned antibodies, antibody screening in TRAIL-R1 corresponding to the epitope. Further, screening of the high sensitivity of TRAIL treatment of the tumor cell type and TRAIL treatment of tumor cells more effectively TRAIL-R1 epitope sites. TRAIL and TRAIL-R1 of the epitope conformation and sensitivity of the relationship, to help clarify the mechanism of TRAIL induced apoptosis, the application of TRAIL and TRAIL-R1 signal provides useful information targeted treatment of malignant tumors.
Method
ELISA competition Category TRAIL-R1 monoclonal antibody and the TRAIL-R1 may be epitope specific antibody. Using flow cytometry, in Colo205, K562, Daudi, KMP4, MCF7 and other 14 kinds of tumor cells to TRAIL-R1 classification and screening of monoclonal antibodies in the TRAIL-R1 on the possibility of antibody epitopes. Further analysis by cell viability, screening TRAIL-R1 antibody epitope (TR1-272-,438-and 419-epitope), and select the most for apoptosis TRAIL-R1 antibody epitope. Then, confocal microscopy analysis and verification, the selection of TRAIL-R1 TRAIL antibody epitope binding may occur after the changes. Further analysis, each cell line the expression of TRAIL-R1 cDNA sequences, detect various cell lines TRAIL-R1 differences expression if in various cell lines is due to TRAIL-R1 base sequence changes. With tunicamycin inhibition of TRAIL-R1 of N-glycosylation in all tumor cellsor benzyl-a-GalNAc inhibition of tumor cell synthesis of O-glycosylation, continue to use the various cell lines by flow cytometry analysis of inhibition of TRAIL-R1 after the glycosylation cells staining. We used Mann-Whitney's U-test P values estimated.
Results
ELISA competition speculated 9 type TRAIL-R1 monoclonal antibody binding epitope of TRAIL-R1, from the TR1-272 antibody, TR1-419 antibody, TR1-404 and TR1-416 antibody, TR1-407,417-and 422-antibodies, TR1-412 and 438-among antibodies, completely blocked, partially blocked or hardly the characteristics of blocking analysis, suggesting that the recombinant TRAIL-R1 at least five kinds of antibody epitopes. We do these TRAIL-R1 epitope designated as TR1-272-,419-,416-, 438-or 417-epitope. TR1-272-epitope was almost blocked by the other antibody competition, in addition to TR1-438-antibodies. The TR1-438-epitope can not be TR1-272-blocking antibody competition, but can be TR1-416 and 417-part antibody blocking competition. TR1-416-epitope-based TR1-272-and 419-blocking antibody, but little has been TR1-417-and 438-antibody inhibition. TR1-417 epitope hardly was TR1-272-,416-,419-and 438-antibody inhibition. TR1-419 epitope was almost TR1-272 antibody blocked by TR1-417-and 416-antibody partially blocked, not blocked by TR1-438 antibody. (2) The Next Competitive ELISA analysis TRAIL-R1 monoclonal antibody binding sites and TRAIL of TRAIL-R1 in the relationship between binding sites. The results show that TRAIL blockade TR1-417-and 438-antibody and TRAIL-R1 epitope binding, but almost no blocking TR1-416-,419-and 272-antibody and TRAIL-R1 binding epitope. These results suggest that, TR1-417-and 438-epitope of TRAIL-binding sites located in or near, TR1-272-,416-and 419-epitope may be located outside of TRAIL binding sites. The results from the competition ELISA analysis showed that, TR1-272 and 438-epitope away from each other's. The antibody TR1-416-,417-and 438-epitope, less interference, TR1-417-and 272-epitope and TR1-416-,419-and 272-epitope is close to each other. (3) we used flow cytometry analysis of each antibody in different cell lines combination. Interestingly, some cells were TR1-404-,407-,416-,417-or 422-antibody staining, without being TR1-272-,419-,412-or 438-antibody staining. These results show that classification according to results of competitive ELISA antibody, consistent with cell-binding pattern classification, in each cell line cell surface TRAIL-R1 antibody epitope expression is different. TR1-416 and 417-epitope in all cell lines expression, the expression of TR1-416 and 417-epitope of the cell lines, not necessarily the expression of TR1-272,419-and 438-epitope. Interesting to note that, Colo205, K562, Daudi and Dul45 cells not express TR1-272 epitopes, and strong expression of other antibody epitopes. (4) Further, we detected different of TRAIL-R1 epitope (TR1-272-, 438-and 419-epitope) expression whether the TRAIL induced apoptosis in tumor cell lines of sensitive related. Among TR1-272-epitope absence Colo205, K562, Daudi and Dul45 tumor cells than TR1-272-epitope positive tumor cells more vulnerable to TRAIL induced apoptosis (p=0.0014). With this contrast, TR1-438-epitope conformation and TRAIL-induced apoptosis to not related (p=0.41). TR1-419 epitope conformation and TRAIL-induced apoptosis also showed no correlation. We observed a number of antibodies, including TR1-416 antibody and TR1-419 antibody, and TRAIL are not competitive binding TRAIL-R1. Here, we again examined TRAIL and TR1-416 antibodies conjunction induced tumor cell apoptosis. The results show that, TR1-416 antibody significantly enhanced the TRAIL to induce Colo205, K562 and Daudi such as tumor cells of apoptosis. Overall, these results show that TR1-272 antibodies can predict the TRAIL in clinical, treatment of tumors with TRAIL sensitivity. TR1-416 antibody can enhance TRAIL induced apoptosis in some tumor cells. (5) In order to understand the different sensitivity adjustment mechanism by TRAIL induced cell death, after treated with TRAIL of TR1-272-epitope-negative or-positive cell lines, using laser scanning confocal microscopy analysis of cell surface TRAIL-R1 situation. Confocal laser microscopy showed that, TRAIL after the role of TR1-272-epitope-negative cells, TRAIL-R1 form a large spotlight, while TR1-272-positive cells can not form a bunch. Show, TRAIL-R1 oligomerization affect and TR1-272-epitope conformation, may affect the tumor cells to TRAIL induced apoptosis of sensitivity. (6) Further analysis of the various cell lines express TRAIL-R1 cDNA sequences found,in various cell lines of TRAIL-R1 is basically the same amino acid sequence, it shows the expression of various cell epitope differences did not occur at the gene level. (7) Next to continue analysis the translation of TRAIL-R1 after the glycosylation is modified. In each cell, with tunicamycin inhibited TRAIL-R1 in the N-glycosylation or Benzyl-a-GalNAc inhibition of synthesis of O-glycosylation, continue to flow cytometry analysis, whether the various cell lines TR1-272,419-or 438-epitope are to be protein post-translational after modification of glycosylations occurrence a negative or positive staining of expression. The results showed that, O-glycosylation or N-glycosylation of inhibition failed to change the negative expression rate. Glycosylation was observed for TRAIL-R1 epitope on the conformation had no effect, indicating glycosylation of conformational epitopes may not play key roles.
Conclusion
TR1-272-antibody may recognize TRAIL-R1 conformation, which there conformational can release a powerful cell death signal.With this contrast, TR1-438-, TR1-419-, TR1-416 and TR1-417-epitope conformation and not related to TRAIL-induced apoptosis. TR1-416 antibody can significantly enhance the TRAIL to induce Colo205, K562 and Daudi such as tumor cells apoptosis. Various cell lines TRAIL-R1 epitope of express differences did not occur at the gene level, while TRAIL-R1 post-translational modification of glycosylations may be the conformational epitope not afford a key role. Various cell lines TRAIL-R1 epitope of express differences did not occur at the gene level, while TRAIL-R1 post-translational modification of glycosylations may be the conformational epitope not the key role. Further analysis of TRAIL epitopes conformational and sensitivity, can help clarify the mechanism of TRAIL induced apoptosis, and TRAIL and TRAIL-R for the signal to provide useful information to treat cancer.
引文
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