粘蛋白型O-聚糖對人結(jié)直腸癌細(xì)胞的生物學(xué)行為的影響
發(fā)布時(shí)間：2019-06-01 21:26
【摘要】：研究背景和研究目的結(jié)直腸癌是常見的消化道惡性腫瘤之一。在中國,隨著人們生活水平的提高,飲食結(jié)構(gòu)的改變,結(jié)直腸癌發(fā)病率呈逐年上升的趨勢。雖然目前治療方法不斷增多,治療手段不斷提高,但因?yàn)榻Y(jié)直腸癌根治困難且易復(fù)發(fā)轉(zhuǎn)移,仍是嚴(yán)重威脅患者生命的臨床棘手難題。因此,深入研究結(jié)直腸癌的發(fā)生、進(jìn)展和轉(zhuǎn)移等機(jī)制,尋找有效診治結(jié)直腸癌患者的方法,以期對其進(jìn)行“個(gè)體化”治療等均具有非常深遠(yuǎn)的意義。粘蛋白型O-聚糖指由N-乙酰氨基半乳糖轉(zhuǎn)移酶等催化合成的一類修飾糖蛋白的糖類復(fù)合物,它們參與生物體內(nèi)很多重要的生物學(xué)過程,如維持蛋白質(zhì)構(gòu)象,參與細(xì)胞粘附、聚集以及細(xì)胞表面受體激活、信號轉(zhuǎn)導(dǎo)等。大量研究表明,在細(xì)胞癌變及發(fā)展過程中常有粘蛋白型O-聚糖的結(jié)構(gòu)或數(shù)量發(fā)生異常改變,并且可能與腫瘤細(xì)胞的侵襲、粘附、轉(zhuǎn)移、增殖和凋亡等有關(guān)。如在胰腺癌已經(jīng)發(fā)現(xiàn)異常的O-聚糖可促進(jìn)細(xì)胞的增殖、遷移能力。已知粘蛋白型O-聚糖是消化道上皮細(xì)胞粘膜粘液層的主要成分,很多結(jié)直腸腫瘤病人能檢測到O-聚糖結(jié)構(gòu)異常,表現(xiàn)為腫瘤相關(guān)糖類抗原Tn陽性,但尚不清楚粘蛋白型O-聚糖對結(jié)直腸癌的發(fā)生及發(fā)展起何種作用。研究發(fā)現(xiàn),T合酶是粘蛋白型O-聚糖合成的關(guān)鍵糖基轉(zhuǎn)移酶,在人體和其他脊椎動(dòng)物,T-合酶的形成需要特異性分子伴侶Cosmc的參與。一旦Cosmc功能異可導(dǎo)致T-合酶失活,以至于不能形成正常的O-聚糖結(jié)構(gòu),導(dǎo)致Tn抗原暴露。本研究擬從T合酶特異性分子伴侶Cosmc異常為視角探討O-聚糖異常對結(jié)直腸癌細(xì)胞生物學(xué)行為的影響作用。研究方法1.制備穩(wěn)定無Tn抗原暴露的人結(jié)直腸癌細(xì)胞株(1)構(gòu)建慢病毒表達(dá)載體GV367-EGFP-Cosmc;(2)向經(jīng)磁珠分選的異常O-型糖基化的LS174T(Tn抗原陽性)結(jié)直腸腫瘤細(xì)胞株感染GV367-EGFP-Cosmc,并同時(shí)感染GV367-EGFP-blank vector作為對照control組,用嘌呤霉素抗性篩選穩(wěn)定表達(dá)Cosmc及control組的結(jié)直腸癌細(xì)胞株;(3)熒光顯微鏡鏡下觀察細(xì)胞感染效率;(4)Real-time PCR檢測感染細(xì)胞的Cosmc轉(zhuǎn)錄水平;(5)Western blot檢測感染細(xì)胞的Cosmc蛋白水平,以檢測細(xì)胞感染效率;(6)流式細(xì)胞技術(shù)檢測感染細(xì)胞Tn抗原的變化。2.粘蛋白型O-聚糖對結(jié)直腸癌細(xì)胞生物學(xué)特性的影響(1)利用CCK8細(xì)胞增殖實(shí)驗(yàn)觀察Cosmc感染后正常O-型糖基化反應(yīng)對細(xì)胞增殖能力的影響;(2)利用PE Annexin V/7-AAD細(xì)胞凋亡實(shí)驗(yàn)觀察Cosmc感染后正常O-型糖基化反應(yīng)對腫瘤細(xì)胞凋亡能力的影響;(3)利用細(xì)胞劃痕實(shí)驗(yàn)和Transwell細(xì)胞遷移實(shí)驗(yàn)觀察Cosmc感染后正常O-型糖基化反應(yīng)對對細(xì)胞遷移能力的影響;(4)利用Transwell細(xì)胞侵襲實(shí)驗(yàn)觀察Cosmc感染后正常O-型糖基化反應(yīng)對細(xì)胞侵襲能力的影響。研究結(jié)果1.制備穩(wěn)定無Tn抗原表達(dá)的人結(jié)直腸癌細(xì)胞株熒光顯微鏡下觀察幾乎所有感染細(xì)胞可激發(fā)出綠色熒光;Real time-PCR檢測穩(wěn)定感染細(xì)胞株中Cosmc的表達(dá)情況,以2(-△△Ct)統(tǒng)計(jì)學(xué)分析后發(fā)現(xiàn)Cosmc在LS174T(Tn+)+Cosmc組較LS174T(Tn+)+control組表達(dá)有顯著性差異(P=0.0018)。Western blot結(jié)果與Real-time PCR結(jié)果一致。流式檢測細(xì)胞表面抗原Tn表達(dá),結(jié)果提示LS174T(Tn+)+Cosmc細(xì)胞Tn抗原明顯轉(zhuǎn)陰。2.粘蛋白型O-聚糖對結(jié)直腸癌細(xì)胞生物學(xué)特性的影響a.CCK8細(xì)胞增殖實(shí)驗(yàn)相同數(shù)量(10000個(gè)/孔)LS174T(Tn+)+Cosmc與LS174T(Tn+)+control、LS174T(Tn+)接種于96孔板中,隨著時(shí)間的延長,各組細(xì)胞OD值均逐漸增加,三組細(xì)胞隨生長時(shí)間變化差異具有統(tǒng)計(jì)學(xué)意義(P0.001)。根據(jù)三組細(xì)胞同一天測得的數(shù)據(jù)分析,除第1天(P=0.7328)外,LS174T(Tn+)+Cosmc組與LS174T(Tn+)+control組間均存在明顯差異(P0.05),LS174T(Tn+)+Cosmc組增殖明顯緩慢,而LS174T(Tn+)+control與LS174T(Tn+)組組間無明顯差異(P0.05)。b.流式細(xì)胞技術(shù)檢測細(xì)胞凋亡通過流式細(xì)胞技術(shù)檢測發(fā)現(xiàn),以相同劑量及時(shí)間紫外線照射誘導(dǎo)細(xì)胞凋亡后,LS174T(Tn+)+Cosmc和LS174T(Tn+)+control組細(xì)胞早期凋亡率平均值分別是51.24%和34.38%,晚期凋亡率平均值分別是18.48%和14.74%,總凋亡率平均值分別是69.72%和49.12%,可見感染Cosmc對早期凋亡影響更大,感染Cosmc后促進(jìn)細(xì)胞凋亡,統(tǒng)計(jì)其總凋亡率兩組有明顯統(tǒng)計(jì)學(xué)差異(P=0.0010)。c.細(xì)胞遷移能力的檢測細(xì)胞劃痕實(shí)驗(yàn)檢測細(xì)胞遷移能力:對LS174T(Tn+)+Cosmc、LS174T(Tn+)+control、LS174T(Tn+)三組細(xì)胞進(jìn)行相同劃痕處理后,感染Cosmc組較對照組遷移能力減弱(24h:P=0.0215,48h:P=0.0028),control組和blank組無明顯差異(P0.05)。Transwell細(xì)胞遷移實(shí)驗(yàn):感染Cosmc組細(xì)胞較對照組遷移能力明顯減弱(P=0.0008),control組和blank組無明顯差異(P0.05)。d.細(xì)胞侵襲能力的檢測感染Cosmc組細(xì)胞較對照組侵襲能力無明顯差異(P=0.9192)。結(jié)論T合酶特異性分子伴侶Cosmc可修復(fù)LS174T(Tn+)的正常O-聚糖形成過程,使Tn抗原轉(zhuǎn)陰,正常的O-聚糖結(jié)構(gòu)可以抑制細(xì)胞的增殖、遷移能力并促進(jìn)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.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：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.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.34
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