粘蛋白型O-聚糖对人结直肠癌细胞的生物学行为的影响
发布时间：2019-06-01 21:26
【摘要】：研究背景和研究目的结直肠癌是常见的消化道恶性肿瘤之一。在中国,随着人们生活水平的提高,饮食结构的改变,结直肠癌发病率呈逐年上升的趋势。虽然目前治疗方法不断增多,治疗手段不断提高,但因为结直肠癌根治困难且易复发转移,仍是严重威胁患者生命的临床棘手难题。因此,深入研究结直肠癌的发生、进展和转移等机制,寻找有效诊治结直肠癌患者的方法,以期对其进行“个体化”治疗等均具有非常深远的意义。粘蛋白型O-聚糖指由N-乙酰氨基半乳糖转移酶等催化合成的一类修饰糖蛋白的糖类复合物,它们参与生物体内很多重要的生物学过程,如维持蛋白质构象,参与细胞粘附、聚集以及细胞表面受体激活、信号转导等。大量研究表明,在细胞癌变及发展过程中常有粘蛋白型O-聚糖的结构或数量发生异常改变,并且可能与肿瘤细胞的侵袭、粘附、转移、增殖和凋亡等有关。如在胰腺癌已经发现异常的O-聚糖可促进细胞的增殖、迁移能力。已知粘蛋白型O-聚糖是消化道上皮细胞粘膜粘液层的主要成分,很多结直肠肿瘤病人能检测到O-聚糖结构异常,表现为肿瘤相关糖类抗原Tn阳性,但尚不清楚粘蛋白型O-聚糖对结直肠癌的发生及发展起何种作用。研究发现,T合酶是粘蛋白型O-聚糖合成的关键糖基转移酶,在人体和其他脊椎动物,T-合酶的形成需要特异性分子伴侣Cosmc的参与。一旦Cosmc功能异可导致T-合酶失活,以至于不能形成正常的O-聚糖结构,导致Tn抗原暴露。本研究拟从T合酶特异性分子伴侣Cosmc异常为视角探讨O-聚糖异常对结直肠癌细胞生物学行为的影响作用。研究方法1.制备稳定无Tn抗原暴露的人结直肠癌细胞株(1)构建慢病毒表达载体GV367-EGFP-Cosmc;(2)向经磁珠分选的异常O-型糖基化的LS174T(Tn抗原阳性)结直肠肿瘤细胞株感染GV367-EGFP-Cosmc,并同时感染GV367-EGFP-blank vector作为对照control组,用嘌呤霉素抗性筛选稳定表达Cosmc及control组的结直肠癌细胞株;(3)荧光显微镜镜下观察细胞感染效率;(4)Real-time PCR检测感染细胞的Cosmc转录水平;(5)Western blot检测感染细胞的Cosmc蛋白水平,以检测细胞感染效率;(6)流式细胞技术检测感染细胞Tn抗原的变化。2.粘蛋白型O-聚糖对结直肠癌细胞生物学特性的影响(1)利用CCK8细胞增殖实验观察Cosmc感染后正常O-型糖基化反应对细胞增殖能力的影响;(2)利用PE Annexin V/7-AAD细胞凋亡实验观察Cosmc感染后正常O-型糖基化反应对肿瘤细胞凋亡能力的影响;(3)利用细胞划痕实验和Transwell细胞迁移实验观察Cosmc感染后正常O-型糖基化反应对对细胞迁移能力的影响;(4)利用Transwell细胞侵袭实验观察Cosmc感染后正常O-型糖基化反应对细胞侵袭能力的影响。研究结果1.制备稳定无Tn抗原表达的人结直肠癌细胞株荧光显微镜下观察几乎所有感染细胞可激发出绿色荧光;Real time-PCR检测稳定感染细胞株中Cosmc的表达情况,以2(-△△Ct)统计学分析后发现Cosmc在LS174T(Tn+)+Cosmc组较LS174T(Tn+)+control组表达有显著性差异(P=0.0018)。Western blot结果与Real-time PCR结果一致。流式检测细胞表面抗原Tn表达,结果提示LS174T(Tn+)+Cosmc细胞Tn抗原明显转阴。2.粘蛋白型O-聚糖对结直肠癌细胞生物学特性的影响a.CCK8细胞增殖实验相同数量(10000个/孔)LS174T(Tn+)+Cosmc与LS174T(Tn+)+control、LS174T(Tn+)接种于96孔板中,随着时间的延长,各组细胞OD值均逐渐增加,三组细胞随生长时间变化差异具有统计学意义(P0.001)。根据三组细胞同一天测得的数据分析,除第1天(P=0.7328)外,LS174T(Tn+)+Cosmc组与LS174T(Tn+)+control组间均存在明显差异(P0.05),LS174T(Tn+)+Cosmc组增殖明显缓慢,而LS174T(Tn+)+control与LS174T(Tn+)组组间无明显差异(P0.05)。b.流式细胞技术检测细胞凋亡通过流式细胞技术检测发现,以相同剂量及时间紫外线照射诱导细胞凋亡后,LS174T(Tn+)+Cosmc和LS174T(Tn+)+control组细胞早期凋亡率平均值分别是51.24%和34.38%,晚期凋亡率平均值分别是18.48%和14.74%,总凋亡率平均值分别是69.72%和49.12%,可见感染Cosmc对早期凋亡影响更大,感染Cosmc后促进细胞凋亡,统计其总凋亡率两组有明显统计学差异(P=0.0010)。c.细胞迁移能力的检测细胞划痕实验检测细胞迁移能力:对LS174T(Tn+)+Cosmc、LS174T(Tn+)+control、LS174T(Tn+)三组细胞进行相同划痕处理后,感染Cosmc组较对照组迁移能力减弱(24h:P=0.0215,48h:P=0.0028),control组和blank组无明显差异(P0.05)。Transwell细胞迁移实验:感染Cosmc组细胞较对照组迁移能力明显减弱(P=0.0008),control组和blank组无明显差异(P0.05)。d.细胞侵袭能力的检测感染Cosmc组细胞较对照组侵袭能力无明显差异(P=0.9192)。结论T合酶特异性分子伴侣Cosmc可修复LS174T(Tn+)的正常O-聚糖形成过程,使Tn抗原转阴,正常的O-聚糖结构可以抑制细胞的增殖、迁移能力并促进其凋亡。
[Abstract]:Colorectal cancer is one of the most common malignant tumors of the digestive tract. In China, with the improvement of people's living standard and the change of the diet structure, the incidence of colorectal cancer is increasing year by year. Although the current treatment method is increasing, the treatment means is continuously improved, but because of the difficult and easy recurrence of the colorectal cancer, it is still a difficult and difficult problem to seriously threaten the life of the patient. Therefore, it is of great significance to study the mechanism of the occurrence, progression and metastasis of colorectal cancer in order to find effective diagnosis and treatment of colorectal cancer. mucin-type O-glycans are carbohydrate complexes of a class of modified glycoproteins that are catalyzed by N-B-galactosyltransferase and the like, which are involved in many important biological processes in the organism, such as maintaining a protein conformation, participating in cell adhesion, aggregation, and cell surface receptor activation, Signal transduction, and the like. A large number of studies have shown that there is an abnormal change in the structure or quantity of mucin-type O-glycans in the process of cell canceration and development, and may be related to the invasion, adhesion, metastasis, proliferation and apoptosis of the tumor cells. As in the case of pancreatic cancer, an abnormal O-glycan can be used to promote cell proliferation and migration. The known mucin type O-glycans are the main components of the mucosa mucus layer of the digestive tract epithelial cells, and many colorectal tumor patients can detect the abnormal structure of the O-glycan, which is expressed as the tumor-related carbohydrate antigen Tn-positive, However, it is not clear what role of mucin-type O-glycans in the occurrence and development of colorectal cancer. It has been found that T-synthase is a key glycosyltransferase for the synthesis of mucin-type O-glycans, and in humans and other vertebrates, the formation of T-synthase requires the participation of specific molecular chaperones Cosmmc. Once the Cosmmc function results in the inactivation of the T-synthase, it is not possible to form a normal O-glycan structure, resulting in the exposure of the Tn antigen. In this study, the effect of O-glycan on the biological behavior of colorectal cancer cells is discussed from the perspective of the T-synthase-specific molecular chaperone Cosmmc. Study Method 1. a lentiviral expression vector GV367-EGFP-Cosmmc was constructed by the preparation of a human colorectal cancer cell strain (1) with stable non-Tn antigen exposure; (2) an abnormal O-type glycosylated LS174T (Tn antigen-positive) colorectal tumor cell line sorted by magnetic beads was infected with GV367-EGFP-Cosmmc, and the GV367-EGFP-blank vector was simultaneously infected as a control control group, screening and stably expressing the colorectal cancer cell strain of the Comc and the control group by using the resistance of the spectinomycin; (3) observing the cell infection efficiency under the fluorescence microscope; (4) detecting the Cosmmc transcription level of the infected cell by the Real-time PCR; and (5) detecting the level of the Cosmmc protein of the infected cell by Western blot to detect the cell infection efficiency; (6) Flow cytometry was used to detect the change of the antigen of the infected cell Tn. Effects of mucin-type O-glycans on the biological characteristics of colorectal cancer cells (1) The effect of normal O-type glycosylation on the cell proliferation ability after Comc infection was observed by using the CCK8 cell proliferation assay. (2) Using PE Annexin V/7-AAD cell apoptosis experiment, the effect of the normal O-type glycosylation reaction on the apoptosis ability of the tumor cells after Comc infection was observed; (3) the effect of the normal O-type glycosylation reaction on the cell migration ability after the Comc infection was observed by the cell scratch test and the Transwell cell migration experiment; (4) Transwell cell invasion experiment was used to observe the effect of normal O-type glycosylation on cell invasion ability after Comc infection. Study Results 1. the method for preparing the human colorectal cancer cell strain with the stable non-Tn antigen expression can be used for observing that almost all the infected cells can stimulate the green fluorescence; and the real time-PCR is used for detecting the expression of the Cosmmc in the stable infection cell line, The expression of Comc in LS174T (Tn +) + Cosmmc group and LS174T (Tn +) + control group was found to be significant (P = 0.0018), and the results of Western blot were consistent with the results of Real-time PCR. The results suggested that the Tn antigen of LS174T (Tn +) + Cosmmc cell was significantly negative. The effect of mucin-type O-glycans on the biological characteristics of colorectal cancer cells was a. The same number (10,000/ well) of LS174T (Tn +) + Cosmmc and LS174T (Tn +) + control, LS174T (Tn +) were inoculated into a 96-well plate, and the OD values of each group were gradually increased as the time was prolonged. The difference of the three groups was statistically significant with the time of growth (P 0.001). There was a significant difference between the group of LS174T (Tn +) + Cosmmc and LS174T (Tn +) + control group (P0.05), but there was no significant difference between the group of LS174T (Tn +) + control and the group of LS174T (Tn +) (P0.05). Flow cytometry was used to detect the apoptosis of the cells by flow cytometry. The mean values of early apoptosis in the cells of LS174T (Tn +) + Cosmmc and LS174T (Tn +) + control group were 51.24% and 34.38%, respectively, and the average of the late apoptotic rates was 18.48% and 14.74%, respectively. The average apoptotic rate was 69.72% and 49.12%, respectively. The cell migration ability was detected by the cell scratch test: after the same scratch treatment for the three groups of LS174T (Tn +) + Cosmmc, LS174T (Tn +) + control and LS174T (Tn +), the migration ability of the infected Cosmmc group was decreased (24 h: P = 0.0215,48 h: P = 0.0028), and there was no significant difference between the control group and the blank group (P0.05). There was no significant difference between control group and control group (P = 0.0008), control group and blank group (P0.05). There was no significant difference between the invasion ability of Comc group and the control group (P = 0.9192). Conclusion The T-synthase-specific molecular chaperone Cosmmc can repair the normal O-glycan formation of the LS174T (Tn +). The normal O-glycan structure can inhibit the proliferation, migration and apoptosis of the cell.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.34
[Abstract]:Colorectal cancer is one of the most common malignant tumors of the digestive tract. In China, with the improvement of people's living standard and the change of the diet structure, the incidence of colorectal cancer is increasing year by year. Although the current treatment method is increasing, the treatment means is continuously improved, but because of the difficult and easy recurrence of the colorectal cancer, it is still a difficult and difficult problem to seriously threaten the life of the patient. Therefore, it is of great significance to study the mechanism of the occurrence, progression and metastasis of colorectal cancer in order to find effective diagnosis and treatment of colorectal cancer. mucin-type O-glycans are carbohydrate complexes of a class of modified glycoproteins that are catalyzed by N-B-galactosyltransferase and the like, which are involved in many important biological processes in the organism, such as maintaining a protein conformation, participating in cell adhesion, aggregation, and cell surface receptor activation, Signal transduction, and the like. A large number of studies have shown that there is an abnormal change in the structure or quantity of mucin-type O-glycans in the process of cell canceration and development, and may be related to the invasion, adhesion, metastasis, proliferation and apoptosis of the tumor cells. As in the case of pancreatic cancer, an abnormal O-glycan can be used to promote cell proliferation and migration. The known mucin type O-glycans are the main components of the mucosa mucus layer of the digestive tract epithelial cells, and many colorectal tumor patients can detect the abnormal structure of the O-glycan, which is expressed as the tumor-related carbohydrate antigen Tn-positive, However, it is not clear what role of mucin-type O-glycans in the occurrence and development of colorectal cancer. It has been found that T-synthase is a key glycosyltransferase for the synthesis of mucin-type O-glycans, and in humans and other vertebrates, the formation of T-synthase requires the participation of specific molecular chaperones Cosmmc. Once the Cosmmc function results in the inactivation of the T-synthase, it is not possible to form a normal O-glycan structure, resulting in the exposure of the Tn antigen. In this study, the effect of O-glycan on the biological behavior of colorectal cancer cells is discussed from the perspective of the T-synthase-specific molecular chaperone Cosmmc. Study Method 1. a lentiviral expression vector GV367-EGFP-Cosmmc was constructed by the preparation of a human colorectal cancer cell strain (1) with stable non-Tn antigen exposure; (2) an abnormal O-type glycosylated LS174T (Tn antigen-positive) colorectal tumor cell line sorted by magnetic beads was infected with GV367-EGFP-Cosmmc, and the GV367-EGFP-blank vector was simultaneously infected as a control control group, screening and stably expressing the colorectal cancer cell strain of the Comc and the control group by using the resistance of the spectinomycin; (3) observing the cell infection efficiency under the fluorescence microscope; (4) detecting the Cosmmc transcription level of the infected cell by the Real-time PCR; and (5) detecting the level of the Cosmmc protein of the infected cell by Western blot to detect the cell infection efficiency; (6) Flow cytometry was used to detect the change of the antigen of the infected cell Tn. Effects of mucin-type O-glycans on the biological characteristics of colorectal cancer cells (1) The effect of normal O-type glycosylation on the cell proliferation ability after Comc infection was observed by using the CCK8 cell proliferation assay. (2) Using PE Annexin V/7-AAD cell apoptosis experiment, the effect of the normal O-type glycosylation reaction on the apoptosis ability of the tumor cells after Comc infection was observed; (3) the effect of the normal O-type glycosylation reaction on the cell migration ability after the Comc infection was observed by the cell scratch test and the Transwell cell migration experiment; (4) Transwell cell invasion experiment was used to observe the effect of normal O-type glycosylation on cell invasion ability after Comc infection. Study Results 1. the method for preparing the human colorectal cancer cell strain with the stable non-Tn antigen expression can be used for observing that almost all the infected cells can stimulate the green fluorescence; and the real time-PCR is used for detecting the expression of the Cosmmc in the stable infection cell line, The expression of Comc in LS174T (Tn +) + Cosmmc group and LS174T (Tn +) + control group was found to be significant (P = 0.0018), and the results of Western blot were consistent with the results of Real-time PCR. The results suggested that the Tn antigen of LS174T (Tn +) + Cosmmc cell was significantly negative. The effect of mucin-type O-glycans on the biological characteristics of colorectal cancer cells was a. The same number (10,000/ well) of LS174T (Tn +) + Cosmmc and LS174T (Tn +) + control, LS174T (Tn +) were inoculated into a 96-well plate, and the OD values of each group were gradually increased as the time was prolonged. The difference of the three groups was statistically significant with the time of growth (P 0.001). There was a significant difference between the group of LS174T (Tn +) + Cosmmc and LS174T (Tn +) + control group (P0.05), but there was no significant difference between the group of LS174T (Tn +) + control and the group of LS174T (Tn +) (P0.05). Flow cytometry was used to detect the apoptosis of the cells by flow cytometry. The mean values of early apoptosis in the cells of LS174T (Tn +) + Cosmmc and LS174T (Tn +) + control group were 51.24% and 34.38%, respectively, and the average of the late apoptotic rates was 18.48% and 14.74%, respectively. The average apoptotic rate was 69.72% and 49.12%, respectively. The cell migration ability was detected by the cell scratch test: after the same scratch treatment for the three groups of LS174T (Tn +) + Cosmmc, LS174T (Tn +) + control and LS174T (Tn +), the migration ability of the infected Cosmmc group was decreased (24 h: P = 0.0215,48 h: P = 0.0028), and there was no significant difference between the control group and the blank group (P0.05). There was no significant difference between control group and control group (P = 0.0008), control group and blank group (P0.05). There was no significant difference between the invasion ability of Comc group and the control group (P = 0.9192). Conclusion The T-synthase-specific molecular chaperone Cosmmc can repair the normal O-glycan formation of the LS174T (Tn +). The normal O-glycan structure can inhibit the proliferation, migration and apoptosis of the cell.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.34
【相似文献】
相关期刊论文 前10条
1 董超;刘迪;冯业童;刘朋飞;吴璇;吴昊昱;周余来;;结直肠癌细胞外泌体的制备及其促单核细胞增殖作用[J];吉林大学学报(医学版);2013年03期
2 张义平;郑美蓉;殷嫦嫦;吴萍;周许峰;吴建芳;周小鸥;;热休克诱导结直肠癌细胞外泌体的免疫效应[J];肿瘤防治研究;2011年07期
3 崔滨滨,刘明,赵鹏,赵家宏,董新舒;重组腺病毒介导的野生型p53基因对结直肠癌细胞的放射治疗增敏作用[J];中华外科杂志;2005年15期
4 耿焱;姜泊;王伟;马强;;四分子交联体5表达上调对结直肠癌细胞转移能力的促进作用观察[J];解放军医学杂志;2010年06期
5 杨晓东;邢春根;吴永友;赵奎;龚巍;陈博;何腾飞;;结直肠癌细胞辐射敏感性的定量比较蛋白组学研究[J];原子能科学技术;2010年09期
6 张继民,刘明姬,n澗托，
本文编号：2490597
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2490597.html
