人Lgr5基因转染细胞株的构建及其单克隆抗体的研制

发布时间：2018-06-27 09:46

  本文选题：Lgr5 + 基因转染　； 参考：《苏州大学》2013年硕士论文

【摘要】：肿瘤是威胁人类生命健康的最重大疾病之一，近年来，随着对干细胞及肿瘤生物学研究的深入，提出肿瘤干细胞（tumor stem cell，TSC）理论。TSC是一种特殊类型的干细胞，具有自我更新能力。TSC在肿瘤的发生发展中起关键性作用，它与肿瘤的转移、抗药性的产生以及治疗后的复发有密切关系。TSC数量少且难以检测，因此对其标记物的研究必将对肿瘤发生发展的了解及肿瘤的临床诊断、治疗都带来深远的影响。 近年来，G蛋白偶联受体Lgr5（leucine rich repeat containing G protein coupledreceptor5）作为新兴的标记分子成为干细胞的研究热点。Lgr5，又名GPR49，HG38，FEX，是G-蛋白偶联受体超家族成员，是由具有18个富含亮氨酸的重复单位和7个跨膜区域组成的大分子蛋白，在人体内分布广泛，大脑、脊髓、乳腺、毛囊、眼睛、生殖器官及胃肠道等都有表达。2007年首次提出Lgr5是结肠肿瘤干细胞表面标志物，是最新发现的Wnt信号传导的目标基因，与肿瘤的形成和发展有很大相关性，可以作为新的治疗靶点。鉴此，研制特异性抗人Lgr5单克隆抗体不仅在理论研究中具有重要意义，而且在临床应用中也具有重要潜在价值。 本课题研究旨在通过克隆人Lgr5全长基因，建立CHO/Lgr5稳定基因转染细胞株，以CHO/Lgr5稳定转染细胞株为免疫原，常规免疫BALB/c小鼠，研制获得鼠抗人Lgr5单克隆抗体，同时构建SW480/Lgr5瞬时基因转染细胞株，以研究Lgr5基因对结肠癌细胞株体外生物学特性的影响，为进一步研究人Lgr5分子的生物学特性及其在肿瘤发生发展中的作用奠定基础。 本论文分为两个部分： 一、人Lgr5基因转染细胞株的构建及Lgr5基因对结肠癌细胞株生物学特性影响的研究 【目的】构建pIRES2-EGFP-Lgr5重组表达载体，，获得能稳定表达人Lgr5分子的CHO/Lgr5基因转染细胞株，为研制鼠抗人Lgr5单克隆抗体提供免疫原。构建SW480/Lgr5瞬时基因转染细胞株，探讨转染Lgr5基因对结肠癌细胞生物学特性的影响。 【方法】运用逆转录聚合酶链式反应（RT-PCR）技术从结直肠癌组织中获得Lgr5全长基因，经双酶切（XhoI和BamHI）连接入真核表达载体pIRES2-EGFP中，利用脂质体法转染CHO细胞，经G418长期加压筛选获得稳定表达Lgr5分子的CHO基因转染细胞。经RT-PCR、Western blot、免疫细胞化学法检测Lgr5分子的表达。利用脂质体法将pIRES2-EGFP-Lgr5重组表达载体转染SW480细胞，经Western blot、免疫细胞化学法检测Lgr5分子的表达。CCK-8法和悬滴实验分析Lgr5对结肠癌细胞增殖率和成球能力的影响，划痕实验分析Lgr5对结肠癌细胞迁移能力的影响。 【结果】（1）成功克隆人Lgr5全长基因，构建真核表达载体pIRES2-EGFP/Lgr5，并构建稳定表达人Lgr5的基因转染细胞株CHO/Lgr5；（2）获得SW480/Lgr5瞬时基因转染细胞株，转染Lgr5基因的结肠癌细胞体外增殖速度加快，形成的细胞球大而紧密，迁移能力下降，由此提示Lgr5基因转染对结肠癌细胞的生物学行为有一定的影响。 二、鼠抗人Lgr5单克隆抗体的研制及Lgr5在结直肠癌组织中的表达及其临床意义 【目的】研制鼠抗人Lgr5单克隆抗体，分析Lgr5在结直肠癌组织中的表达及其临床意义，为进一步探讨人Lgr5的生物学特性奠定物质基础。 【方法】以稳定表达人Lgr5的基因转染细胞株CHO/Lgr5为免疫原，常规免疫BALB/c小鼠，采用B淋巴细胞杂交瘤技术，将免疫后小鼠的脾脏细胞与小鼠骨髓瘤细胞SP2/0进行细胞融合，经HAT选择培养，以CHO/Lgr5细胞株作为阳性筛选细胞株，以转染pIRES2-EGFP空载体的CHO/Mock细胞作为阴性对照细胞株，经间接免疫荧光标记和流式细胞术分析、反复筛选和多次克隆化培养，获得1株持续、稳定分泌鼠抗人Lgr5单克隆抗体的杂交瘤细胞株；利用细胞核染色体计数、Ig亚型快速定性试纸法、Western-blot、免疫细胞化学法等试验，对获得的杂交瘤细胞株及单克隆抗体进行生物学特性的鉴定；利用单克隆抗体9B3检测Lgr5在结直肠癌组织、癌旁组织及远端正常组织的表达差异。 【结果】（1）成功获得一株稳定特异性分泌鼠抗人Lgr5单克隆抗体的杂交瘤细胞株，命名为9B3。经体外连续培养半年以上，液氮冻存后复苏，细胞的生长状态良好，分泌抗体的性能稳定。染色体数目分析显示，杂交瘤细胞株的染色体在100条以上，大于小鼠B细胞和SP2/0细胞的染色体数目，表明为融合体；（2）经Ig亚型快速定性试纸条分析显示，单抗轻链为κ链，重链为IgG1亚型。流式细胞术、免疫细胞化学法证明单抗9B3能特异性结合CHO/Lgr5基因转染细胞株表面Lgr5分子。Western-blot分析显示，单抗9B3与基因转染细胞株CHO/Lgr5蛋白裂解液特异性结合，形成大小约为90Kd的特异性条带，与Lgr5蛋白大小一致；（3）采用本室建立的腹水诱生方法制备单克隆抗体，腹水形成阳性率约为90%，腹水的产量平均为3.5ml/只小鼠，经Protein G亲和层析柱分离纯化抗体，单克隆抗体纯化后蛋白含量为2.5mg/ml，间接免疫荧光法分析表明，其效价在1：1000以上；（4）免疫组织化学分析结果显示，单抗9B3能够特异性识别结直肠癌组织中的Lgr5分子，Lgr5在结直肠癌组织中的表达水平显著高于远端正常组织（P0.0001），与癌旁组织中的表达无显著差异（P=0.5237）。 【全文结论】本课题成功构建稳定表达人Lgr5的基因转染细胞CHO/Lgr5株和瞬时表达人Lgr5的基因转染细胞SW480/Lgr5，初步探讨Lgr5基因对结肠癌细胞生物学特性的影响。在此基础上研制出能稳定分泌特异性鼠抗人Lgr5单克隆抗体的杂交瘤9B3，所得单克隆抗体可用于流式细胞术、Western blot、免疫组织化学等多种检测。利用单克隆抗体9B3检测Lgr5在结直肠癌组织、癌旁组织及远端正常组织的表达差异。为进一步研究人Lgr5分子的生物学特性及其在肿瘤中的作用机制奠定基础。
[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 .
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

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