本文選題：膽管癌細胞　切入點：LOXL2　出處：《昆明理工大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:(1)構(gòu)建出穩(wěn)定過表達LOXL2基因的膽管癌細胞系QBC939,以期進一步深入探索LOXL2蛋白的功能及作用機制。(2)在穩(wěn)定細胞系QBC939-EX-LOXL2中轉(zhuǎn)入SUMO-2/3干擾基因,觀察沉默SUMO-2/3對QBC939細胞Snail蛋白表達量的影響。(3)在穩(wěn)定細胞系QBC939-EX-LOXL2中轉(zhuǎn)入過表達SENP1基因,觀察過表達SENP1對QBC939細胞Snail蛋白表達量的影響。方法:(1)設(shè)計以G418為研究目的的劑量——反應分析實驗,摸索G418對于QBC939細胞系的最低致死濃度。(2)通過脂質(zhì)體轉(zhuǎn)染的方法構(gòu)建穩(wěn)定過表達LOXL2基因的QBC939細胞系,利用實驗得到的G418最低致死濃度篩選QBC939細胞以獲取陽性克隆,擴增培養(yǎng)陽性細胞,并以普通QBC939細胞和QBC939-vector-LOXL2細胞作為對照組,運用熒光定量PCR實驗和蛋白免疫印跡Western Blot技術(shù)檢測LOXL2的mRNA的表達以及LOXL2的蛋白翻譯情況,從而確保過表達LOXL2基因轉(zhuǎn)染的QBC939細胞模型最終構(gòu)建成功。(3)以穩(wěn)定過表達LOXL2的QBC939細胞作為對照組,QBC939-EX-LOXL2+Control si RNA 細胞作為空載組,QBC939-EX-LOXL2+SUMO-2/3 siRNA作為實驗組,運用蛋白免疫印跡Western Blot技術(shù)檢測在干擾SUMO-2/3蛋白的影響下,Snail蛋白的表達變化量。(4)以穩(wěn)定過表達LOXL2的QBC939細胞作為對照組,QBC939-EX-LOXL2+Vector-SENP1 1 胞作為空載組,QBC939-EX-LOXL2+EX-SENP1作為實驗組,運用蛋白免疫印跡Western Blot技術(shù)檢測在SENP1蛋白過表達的影響下,Snail蛋白的表達變化量。結(jié)果:(1)G418劑量-反應分析實驗結(jié)果表明,濃度為600-1000ug/ml的G418將培養(yǎng)的QBC939細胞全部殺死,而600ug.ml以下均有細胞存活,故600ug/ml為G418篩選QBC939細胞的最低致死濃度。(2)600ug/ml的G418篩選轉(zhuǎn)染過表達LOXL2基因的QBC939細胞7-11天后,可見陽性細胞的克隆,以普通QBC939細胞和Vector-LOXL2細胞作為對照組,運用熒光定量PCR實驗檢測LOXL2的mRNA的表達,蛋白免疫印跡Western Blot技術(shù)檢測有LOXL2蛋白的翻譯。(3)以穩(wěn)定過表達LOXL2的QBC939細胞作為對照組,QBC939-EX-LOXL2+Control si RNA 細胞作為空載組,QBC939-EX-LOXL2+SUMO-2/3 siRNA作為實驗組,運用蛋白免疫印跡Western Blot技術(shù)檢測在干擾SUMO-2/3蛋白的影響下,Snail蛋白的表達量在實驗組下降。(4)以穩(wěn)定過表達LOXL2的QBC939細胞作為對照組,QBC939-EX-LOXL2+Vector-SENP1 1 胞作為空載組,QBC939-EX-LOXL2+EX-SENP1作為實驗組,運用蛋白免疫印跡Western Blot技術(shù)檢測在SENP1蛋白過表達的影響下,Snail蛋白的表達量下降。結(jié)論:(1)穩(wěn)定過表達LOXL2基因的QBC939細胞系構(gòu)建成功,為進一步探索LOXL2蛋白的功能及作用機制及其與膽管癌細胞侵襲轉(zhuǎn)移的關(guān)系奠定了深厚的基礎(chǔ)。(2)進一步證明LOXL2蛋白通過介導SUMOylation提高膽管癌細胞Snail蛋白的穩(wěn)定性,為預防和治療膽管癌細胞的侵襲和轉(zhuǎn)移提供了新的靶點。
[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.
【學位授予單位】：昆明理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.8

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