本文選題：膽管癌細(xì)胞　切入點(diǎn)：LOXL2　出處：《昆明理工大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:(1)構(gòu)建出穩(wěn)定過(guò)表達(dá)LOXL2基因的膽管癌細(xì)胞系QBC939,以期進(jìn)一步深入探索LOXL2蛋白的功能及作用機(jī)制。(2)在穩(wěn)定細(xì)胞系QBC939-EX-LOXL2中轉(zhuǎn)入SUMO-2/3干擾基因,觀察沉默SUMO-2/3對(duì)QBC939細(xì)胞Snail蛋白表達(dá)量的影響。(3)在穩(wěn)定細(xì)胞系QBC939-EX-LOXL2中轉(zhuǎn)入過(guò)表達(dá)SENP1基因,觀察過(guò)表達(dá)SENP1對(duì)QBC939細(xì)胞Snail蛋白表達(dá)量的影響。方法:(1)設(shè)計(jì)以G418為研究目的的劑量——反應(yīng)分析實(shí)驗(yàn),摸索G418對(duì)于QBC939細(xì)胞系的最低致死濃度。(2)通過(guò)脂質(zhì)體轉(zhuǎn)染的方法構(gòu)建穩(wěn)定過(guò)表達(dá)LOXL2基因的QBC939細(xì)胞系,利用實(shí)驗(yàn)得到的G418最低致死濃度篩選QBC939細(xì)胞以獲取陽(yáng)性克隆,擴(kuò)增培養(yǎng)陽(yáng)性細(xì)胞,并以普通QBC939細(xì)胞和QBC939-vector-LOXL2細(xì)胞作為對(duì)照組,運(yùn)用熒光定量PCR實(shí)驗(yàn)和蛋白免疫印跡Western Blot技術(shù)檢測(cè)LOXL2的mRNA的表達(dá)以及LOXL2的蛋白翻譯情況,從而確保過(guò)表達(dá)LOXL2基因轉(zhuǎn)染的QBC939細(xì)胞模型最終構(gòu)建成功。(3)以穩(wěn)定過(guò)表達(dá)LOXL2的QBC939細(xì)胞作為對(duì)照組,QBC939-EX-LOXL2+Control si RNA 細(xì)胞作為空載組,QBC939-EX-LOXL2+SUMO-2/3 siRNA作為實(shí)驗(yàn)組,運(yùn)用蛋白免疫印跡Western Blot技術(shù)檢測(cè)在干擾SUMO-2/3蛋白的影響下,Snail蛋白的表達(dá)變化量。(4)以穩(wěn)定過(guò)表達(dá)LOXL2的QBC939細(xì)胞作為對(duì)照組,QBC939-EX-LOXL2+Vector-SENP1 1 胞作為空載組,QBC939-EX-LOXL2+EX-SENP1作為實(shí)驗(yàn)組,運(yùn)用蛋白免疫印跡Western Blot技術(shù)檢測(cè)在SENP1蛋白過(guò)表達(dá)的影響下,Snail蛋白的表達(dá)變化量。結(jié)果:(1)G418劑量-反應(yīng)分析實(shí)驗(yàn)結(jié)果表明,濃度為600-1000ug/ml的G418將培養(yǎng)的QBC939細(xì)胞全部殺死,而600ug.ml以下均有細(xì)胞存活,故600ug/ml為G418篩選QBC939細(xì)胞的最低致死濃度。(2)600ug/ml的G418篩選轉(zhuǎn)染過(guò)表達(dá)LOXL2基因的QBC939細(xì)胞7-11天后,可見陽(yáng)性細(xì)胞的克隆,以普通QBC939細(xì)胞和Vector-LOXL2細(xì)胞作為對(duì)照組,運(yùn)用熒光定量PCR實(shí)驗(yàn)檢測(cè)LOXL2的mRNA的表達(dá),蛋白免疫印跡Western Blot技術(shù)檢測(cè)有LOXL2蛋白的翻譯。(3)以穩(wěn)定過(guò)表達(dá)LOXL2的QBC939細(xì)胞作為對(duì)照組,QBC939-EX-LOXL2+Control si RNA 細(xì)胞作為空載組,QBC939-EX-LOXL2+SUMO-2/3 siRNA作為實(shí)驗(yàn)組,運(yùn)用蛋白免疫印跡Western Blot技術(shù)檢測(cè)在干擾SUMO-2/3蛋白的影響下,Snail蛋白的表達(dá)量在實(shí)驗(yàn)組下降。(4)以穩(wěn)定過(guò)表達(dá)LOXL2的QBC939細(xì)胞作為對(duì)照組,QBC939-EX-LOXL2+Vector-SENP1 1 胞作為空載組,QBC939-EX-LOXL2+EX-SENP1作為實(shí)驗(yàn)組,運(yùn)用蛋白免疫印跡Western Blot技術(shù)檢測(cè)在SENP1蛋白過(guò)表達(dá)的影響下,Snail蛋白的表達(dá)量下降。結(jié)論:(1)穩(wěn)定過(guò)表達(dá)LOXL2基因的QBC939細(xì)胞系構(gòu)建成功,為進(jìn)一步探索LOXL2蛋白的功能及作用機(jī)制及其與膽管癌細(xì)胞侵襲轉(zhuǎn)移的關(guān)系奠定了深厚的基礎(chǔ)。(2)進(jìn)一步證明LOXL2蛋白通過(guò)介導(dǎo)SUMOylation提高膽管癌細(xì)胞Snail蛋白的穩(wěn)定性,為預(yù)防和治療膽管癌細(xì)胞的侵襲和轉(zhuǎn)移提供了新的靶點(diǎn)。
[Abstract]:Objective to construct a stable overexpression of LOXL2 gene in cholangiocarcinoma cell line QBC939, in order to further explore the function and mechanism of LOXL2 protein. To observe the effect of silencing SUMO-2/3 on the expression of Snail protein in QBC939 cells. To observe the effect of overexpression of SENP1 on the expression of Snail protein in QBC939 cells. To explore the lowest lethal concentration (LLC) of G418 on QBC939 cell line, we constructed a stable QBC939 cell line with overexpression of LOXL2 gene by liposome transfection, and screened QBC939 cell line by G418 lethality concentration to obtain positive clones. The expression of mRNA in LOXL2 and the protein translation of LOXL2 were detected by fluorescence quantitative PCR assay and Western blot Western Blot technique, and the normal QBC939 cells and QBC939-vector-LOXL2 cells were used as control group. Thus, the model of QBC939 cells transfected with overexpression of LOXL2 gene was successfully constructed.) QBC939 cells stably expressing LOXL2 were used as control group QBC939-EX-LOXL2 Control si RNA cells as control group, QBC939-EX-XL2 SUMO-2/3 siRNA was used as experimental group, QBC939-EX-XL2 SUMO-2/3 siRNA was used as experimental group, and QBC939-EX-XL2 SUMO-2/3 siRNA was used as experimental group. Western blotting Western Blot technique was used to detect the expression of Snail protein under the influence of interfering SUMO-2/3 protein. The QBC939-EX-LOXL2 Vector-SENP1 1 cell was used as the control group, QBC939-EX-LOXL2 EX-SENP1 was used as the experimental group, and QBC939 cells stably expressing LOXL2 were used as the control group, QBC939-EX-LOXL2 EX-SENP1 was used as the experimental group, QBC939-EX-LOXL2 EX-SENP1 was used as the experimental group. Western blot Western Blot technique was used to detect the expression of SENP1 protein under the influence of overexpression of SENP1 protein. Results the results of dose-response analysis of G418 showed that G418 at the concentration of 600-1000ug/ml killed all of the cultured QBC939 cells. However, all the cells below 600ug.ml survived, so 600ug/ml was the lowest lethal concentration of G418 to screen QBC939 cells. G418 was used to screen QBC939 cells transfected with LOXL2 gene for 7-11 days. The positive cells were cloned into normal QBC939 cells and Vector-LOXL2 cells as control group. Fluorescence quantitative PCR assay was used to detect the expression of LOXL2 mRNA, and Western blot Western Blot technique was used to detect the translation of LOXL2 protein.) QBC939-EX-LOXL2 Control si RNA cells were used as the control group, QBC939-EX-LOXL2 Control si RNA cells were used as the experimental group, QBC939-EX-LOXL2 SUMO-2/3 siRNA was used as the experimental group, and the QBC939-EX-LOXL2 SUMO-2/3 siRNA was used as the experimental group. Western blot Western Blot technique was used to detect the expression of Snail protein in the experimental group under the influence of interfering SUMO-2/3 protein. QBC939 cells with stable LOXL2 expression were used as the control group, QBC939-EX-LOXL2 Vector-SENP1 1 cell as the empty load group, QBC939-EX-LOXL2 EX-SENP1 as the experimental group, and QBC939-EX-LOXL2 EX-SENP1 as the experimental group. Western blot Western Blot technique was used to detect the decrease of SENP1 protein expression under the influence of overexpression of SENP1 protein. Conclusion the QBC939 cell line with stable overexpression of LOXL2 gene is successfully constructed. In order to further explore the function and mechanism of LOXL2 protein and its relationship with invasion and metastasis of cholangiocarcinoma cells, it is further proved that LOXL2 protein enhances the stability of Snail protein in cholangiocarcinoma cells by mediating SUMOylation. It provides a new target for the prevention and treatment of invasion and metastasis of cholangiocarcinoma cells.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.8

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本文編號(hào)：1649545