本文選題：Lgr5 + 基因轉(zhuǎn)染��； 參考：《蘇州大學(xué)》2013年碩士論文

【摘要】：腫瘤是威脅人類生命健康的最重大疾病之一，近年來，隨著對干細胞及腫瘤生物學(xué)研究的深入，提出腫瘤干細胞（tumor stem cell，TSC）理論。TSC是一種特殊類型的干細胞，具有自我更新能力。TSC在腫瘤的發(fā)生發(fā)展中起關(guān)鍵性作用，它與腫瘤的轉(zhuǎn)移、抗藥性的產(chǎn)生以及治療后的復(fù)發(fā)有密切關(guān)系。TSC數(shù)量少且難以檢測，因此對其標記物的研究必將對腫瘤發(fā)生發(fā)展的了解及腫瘤的臨床診斷、治療都帶來深遠的影響。 近年來，G蛋白偶聯(lián)受體Lgr5（leucine rich repeat containing G protein coupledreceptor5）作為新興的標記分子成為干細胞的研究熱點。Lgr5，又名GPR49，HG38，F(xiàn)EX，是G-蛋白偶聯(lián)受體超家族成員，是由具有18個富含亮氨酸的重復(fù)單位和7個跨膜區(qū)域組成的大分子蛋白，在人體內(nèi)分布廣泛，大腦、脊髓、乳腺、毛囊、眼睛、生殖器官及胃腸道等都有表達。2007年首次提出Lgr5是結(jié)腸腫瘤干細胞表面標志物，是最新發(fā)現(xiàn)的Wnt信號傳導(dǎo)的目標基因，與腫瘤的形成和發(fā)展有很大相關(guān)性，可以作為新的治療靶點。鑒此，研制特異性抗人Lgr5單克隆抗體不僅在理論研究中具有重要意義，而且在臨床應(yīng)用中也具有重要潛在價值。 本課題研究旨在通過克隆人Lgr5全長基因，建立CHO/Lgr5穩(wěn)定基因轉(zhuǎn)染細胞株，以CHO/Lgr5穩(wěn)定轉(zhuǎn)染細胞株為免疫原，常規(guī)免疫BALB/c小鼠，研制獲得鼠抗人Lgr5單克隆抗體，同時構(gòu)建SW480/Lgr5瞬時基因轉(zhuǎn)染細胞株，以研究Lgr5基因?qū)Y(jié)腸癌細胞株體外生物學(xué)特性的影響，為進一步研究人Lgr5分子的生物學(xué)特性及其在腫瘤發(fā)生發(fā)展中的作用奠定基礎(chǔ)。 本論文分為兩個部分： 一、人Lgr5基因轉(zhuǎn)染細胞株的構(gòu)建及Lgr5基因?qū)Y(jié)腸癌細胞株生物學(xué)特性影響的研究 【目的】構(gòu)建pIRES2-EGFP-Lgr5重組表達載體，，獲得能穩(wěn)定表達人Lgr5分子的CHO/Lgr5基因轉(zhuǎn)染細胞株，為研制鼠抗人Lgr5單克隆抗體提供免疫原。構(gòu)建SW480/Lgr5瞬時基因轉(zhuǎn)染細胞株，探討轉(zhuǎn)染Lgr5基因?qū)Y(jié)腸癌細胞生物學(xué)特性的影響。 【方法】運用逆轉(zhuǎn)錄聚合酶鏈式反應(yīng)（RT-PCR）技術(shù)從結(jié)直腸癌組織中獲得Lgr5全長基因，經(jīng)雙酶切（XhoI和BamHI）連接入真核表達載體pIRES2-EGFP中，利用脂質(zhì)體法轉(zhuǎn)染CHO細胞，經(jīng)G418長期加壓篩選獲得穩(wěn)定表達Lgr5分子的CHO基因轉(zhuǎn)染細胞。經(jīng)RT-PCR、Western blot、免疫細胞化學(xué)法檢測Lgr5分子的表達。利用脂質(zhì)體法將pIRES2-EGFP-Lgr5重組表達載體轉(zhuǎn)染SW480細胞，經(jīng)Western blot、免疫細胞化學(xué)法檢測Lgr5分子的表達。CCK-8法和懸滴實驗分析Lgr5對結(jié)腸癌細胞增殖率和成球能力的影響，劃痕實驗分析Lgr5對結(jié)腸癌細胞遷移能力的影響。 【結(jié)果】（1）成功克隆人Lgr5全長基因，構(gòu)建真核表達載體pIRES2-EGFP/Lgr5，并構(gòu)建穩(wěn)定表達人Lgr5的基因轉(zhuǎn)染細胞株CHO/Lgr5；（2）獲得SW480/Lgr5瞬時基因轉(zhuǎn)染細胞株，轉(zhuǎn)染Lgr5基因的結(jié)腸癌細胞體外增殖速度加快，形成的細胞球大而緊密，遷移能力下降，由此提示Lgr5基因轉(zhuǎn)染對結(jié)腸癌細胞的生物學(xué)行為有一定的影響。 二、鼠抗人Lgr5單克隆抗體的研制及Lgr5在結(jié)直腸癌組織中的表達及其臨床意義 【目的】研制鼠抗人Lgr5單克隆抗體，分析Lgr5在結(jié)直腸癌組織中的表達及其臨床意義，為進一步探討人Lgr5的生物學(xué)特性奠定物質(zhì)基礎(chǔ)。 【方法】以穩(wěn)定表達人Lgr5的基因轉(zhuǎn)染細胞株CHO/Lgr5為免疫原，常規(guī)免疫BALB/c小鼠，采用B淋巴細胞雜交瘤技術(shù)，將免疫后小鼠的脾臟細胞與小鼠骨髓瘤細胞SP2/0進行細胞融合，經(jīng)HAT選擇培養(yǎng)，以CHO/Lgr5細胞株作為陽性篩選細胞株，以轉(zhuǎn)染pIRES2-EGFP空載體的CHO/Mock細胞作為陰性對照細胞株，經(jīng)間接免疫熒光標記和流式細胞術(shù)分析、反復(fù)篩選和多次克隆化培養(yǎng)，獲得1株持續(xù)、穩(wěn)定分泌鼠抗人Lgr5單克隆抗體的雜交瘤細胞株；利用細胞核染色體計數(shù)、Ig亞型快速定性試紙法、Western-blot、免疫細胞化學(xué)法等試驗，對獲得的雜交瘤細胞株及單克隆抗體進行生物學(xué)特性的鑒定；利用單克隆抗體9B3檢測Lgr5在結(jié)直腸癌組織、癌旁組織及遠端正常組織的表達差異。 【結(jié)果】（1）成功獲得一株穩(wěn)定特異性分泌鼠抗人Lgr5單克隆抗體的雜交瘤細胞株，命名為9B3。經(jīng)體外連續(xù)培養(yǎng)半年以上，液氮凍存后復(fù)蘇，細胞的生長狀態(tài)良好，分泌抗體的性能穩(wěn)定。染色體數(shù)目分析顯示，雜交瘤細胞株的染色體在100條以上，大于小鼠B細胞和SP2/0細胞的染色體數(shù)目，表明為融合體；（2）經(jīng)Ig亞型快速定性試紙條分析顯示，單抗輕鏈為κ鏈，重鏈為IgG1亞型。流式細胞術(shù)、免疫細胞化學(xué)法證明單抗9B3能特異性結(jié)合CHO/Lgr5基因轉(zhuǎn)染細胞株表面Lgr5分子。Western-blot分析顯示，單抗9B3與基因轉(zhuǎn)染細胞株CHO/Lgr5蛋白裂解液特異性結(jié)合，形成大小約為90Kd的特異性條帶，與Lgr5蛋白大小一致；（3）采用本室建立的腹水誘生方法制備單克隆抗體，腹水形成陽性率約為90%，腹水的產(chǎn)量平均為3.5ml/只小鼠，經(jīng)Protein G親和層析柱分離純化抗體，單克隆抗體純化后蛋白含量為2.5mg/ml，間接免疫熒光法分析表明，其效價在1：1000以上；（4）免疫組織化學(xué)分析結(jié)果顯示，單抗9B3能夠特異性識別結(jié)直腸癌組織中的Lgr5分子，Lgr5在結(jié)直腸癌組織中的表達水平顯著高于遠端正常組織（P0.0001），與癌旁組織中的表達無顯著差異（P=0.5237）。 【全文結(jié)論】本課題成功構(gòu)建穩(wěn)定表達人Lgr5的基因轉(zhuǎn)染細胞CHO/Lgr5株和瞬時表達人Lgr5的基因轉(zhuǎn)染細胞SW480/Lgr5，初步探討Lgr5基因?qū)Y(jié)腸癌細胞生物學(xué)特性的影響。在此基礎(chǔ)上研制出能穩(wěn)定分泌特異性鼠抗人Lgr5單克隆抗體的雜交瘤9B3，所得單克隆抗體可用于流式細胞術(shù)、Western blot、免疫組織化學(xué)等多種檢測。利用單克隆抗體9B3檢測Lgr5在結(jié)直腸癌組織、癌旁組織及遠端正常組織的表達差異。為進一步研究人Lgr5分子的生物學(xué)特性及其在腫瘤中的作用機制奠定基礎(chǔ)。
[Abstract]:Tumor is one of the most important diseases threatening the health of human life . In recent years , with the in - depth study of stem cells and tumor biology , this paper presents the theory of tumor stem cell ( TSC ) . TSC is a special type of stem cell with self - renewal ability .

Lgr5 is a member of G - protein coupled receptor superfamily . Lgr5 is a marker of G - protein coupled receptor superfamily . It is the target gene with 18 leucine - rich repeat units and 7 transmembrane domains . It is the most recently discovered target gene of Wnt signaling . It is a new therapeutic target . In this case , the development of specific anti - human Lgr5 monoclonal antibody has important significance not only in theory study , but also in clinical application .

The aim of this study was to establish a CHO / Lgr5 stable gene - transfected cell line by cloning Lgr5 full - length gene .

This thesis is divided into two parts :

Construction of human Lgr5 gene transfected cell line and study on the effect of Lgr5 gene on the biological characteristics of colon cancer cell line

Objective To construct pIRES2 - EGFP - Lgr5 recombinant expression vector , to obtain CHO / Lgr5 gene transfected cell line stably expressing human Lgr5 molecule , and to provide immunogen for the development of mouse anti - human Lgr5 monoclonal antibody .

The Lgr5 full - length gene was obtained from colorectal cancer by reverse transcription polymerase chain reaction ( RT - PCR ) . The expression of Lgr5 was detected by liposome method . Western blot and immunohistochemical method were used to detect the expression of Lgr5 molecule . Western blot and immunohistochemical method were used to detect the expression of Lgr5 molecules .

The eukaryotic expression vector pIRES2 - EGFP / Lgr5 was constructed and transfected into CHO / Lgr5 stably expressing human Lgr5 gene .
( 2 ) To obtain SW480 / Lgr5 transient gene transfection cell line , the proliferation rate of colon cancer cells transfected with Lgr5 gene was accelerated , and the cell ball formed was large and close , and the migration ability decreased , thus suggesting that Lgr5 gene transfection had a certain influence on the biological behavior of colon cancer cells .

Development of murine anti - human Lgr5 monoclonal antibody and expression of Lgr5 in colorectal cancer tissue and its clinical significance

Objective To study the expression and clinical significance of Lgr5 monoclonal antibody against human Lgr5 in colorectal cancer and to lay a material foundation for further study on the biological characteristics of human Lgr5 .

BALB / c mice were immunized with CHO / Lgr5 cell line as negative control cell line . CHO / Lgr5 cell line was used as negative control cell line , and the hybridoma cell line with sustained and stable secretion of anti - human Lgr5 monoclonal antibody against human Lgr5 was obtained .
The biological characteristics of hybridoma cell lines and monoclonal antibodies were identified by means of cell nuclear chromosome counting , Ig subtype rapid qualitative test paper , Western - blot and immunohistochemical method .
Monoclonal antibody 9B3 was used to detect the expression of Lgr5 in colorectal cancer , adjacent tissues and distal normal tissues .

The chromosome number analysis showed that the chromosome number of hybridoma cell lines was more than 100 , which was larger than that of mouse B cells and SP2 / 0 cells .
( 2 ) The monoclonal antibody 9B3 was specifically combined with the CHO / Lgr5 gene transfected cell line surface Lgr5 . Western - blot analysis showed that the monoclonal antibody 9B3 was specifically bound to the CHO / Lgr5 gene transfected cell line CHO / Lgr5 . Western - blot analysis showed that the monoclonal antibody 9B3 was specifically bound to the CHO / Lgr5 protein lysate of the gene - transfected cell line , and the specific band of about 90Kd was formed , which was consistent with the size of Lgr5 protein ;
( 3 ) The monoclonal antibody was prepared by the method of ascites induced by ascites , the positive rate of ascites was about 90 % , the yield of ascites was 3.5 ml / mouse , purified antibody was purified by protein G affinity chromatography column , the protein content after purification of monoclonal antibody was 2.5 mg / ml , and indirect immunofluorescence analysis indicated that its titer was above 1 : 1000 ;
( 4 ) The results of chemical analysis showed that monoclonal antibody 9B3 can specifically identify Lgr5 molecule in colorectal cancer tissue , and Lgr5 expression level in colorectal cancer tissue is significantly higher than that in the distal normal tissue ( P0.05 ) , but there is no significant difference in expression of Lgr5 in colorectal cancer tissue ( P = 0.5237 ) .

The expression of Lgr5 gene in colorectal cancer tissues , adjacent tissues and distal normal tissues was determined by using monoclonal antibody 9B3 , which lays a foundation for further study on the biological characteristics of Lgr5 molecule and its mechanism in tumor .
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R392

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