| Tumor is one of the most significant disease threat to human life and health, inrecent years, with the development of stem cell and cancer biology research, tumor stemcells (tumor stem cell, TSC) theory has been proposed. TSC is a special type of stemcell which has self-renewal capacity. TSC come from stem cells and play a key role intumor development. TSC also has closely related with tumor metastasis, drug resistance,and relapse after treatment. The number of TSC is seldom and difficult to detect, so theresearch of tumor’s marker molecules can deepen the understanding of the developmentand clinical diagnosis of the tumor.In recent years, Leucine rich repeat containing G protein coupled receptor5(Lgr5)was focused as a new marker molecule of stem cell. Lgr5, also known as GPR49, HG38,FEX, is a member of G-protein-coupled receptor superfamily. It has18leucine-richrepeat units and7transmembrane regions. And it was expressed in the brain, spinal cord,mammary glands, hair follicles, eyes, reproductive organs and the gastrointestinal tract.In2007, Lgr5was first reported as an cancer stem cell surface markers and the targetgene of Wnt signaling pathway, and thus, it was suggested to have a great correlationwith tumor formation and development. So develop specifical anti-human Lgr5monoclonal antibody is not only provide a new perspective for a thoroughunderstanding of the delicate regulation of Lgr5in the immune system, but also havepotential application value in clinical application.This study aims to clone human Lgr5gene, establish transgenic cell lineCHO/Lgr5, and then use this cell line as an immunogen to prepare mouse anti-humanLgr5monoclonal antibody. Establish Lgr5transient transfected SW480cells, andinvestigate the effect of over-expression Lgr5on the biological characteristic of coloncancer cells. The research has provided solid experiment foundation for further studiesin the biological characteristic of Lgr5and its role in the development of cancer. Part1: Establishment of transgenic cell line and investigatethe effect of over-expression Lgr5on the biological characteristicof colon cancer cellsObjective: To clone human Lgr5gene from the human colon cancer tissure,establish stable transfection CHO cell line and transient transfection SW480cell linewith Lgr5over-expression. Investigate the effect of over-expression Lgr5on thebiological characteristic of colon cancer cells.Methods: The human Lgr5gene was cloned from the colon cancer tissue byRT-PCR and then inserted into the eukaryotic expression vector PIRES2-EGFP toconstruct the recombinant pIRES2-EGFP-Lgr5after double digestion with Xho I andBamHI.The recombinant plasmid was transfected to CHO cells after induction withlipfectamineTM2000and the cells were further selected with G418. RT-PCR,Western-blot and Immunohistochemistry were used to identify the expression oftransfected CHO/Lgr5. Establish transient transfection SW480cell line, then analyzedby Western blot and Immunocytochemistry. Using CCK-8and hanging-drop experimentto analyze the affect of over-expression Lgr5in colon cancer cell’s proliferation rate.Scratche experimental was used to analysis the affect of Lgr5in colon cancer cell’smigration ability.Results:(1) The recombinant pIRES2-EGFP/Lgr5expression vector wasconstructed, stable transfection CHO cell line and transient transfection SW480cellline with Lgr5over-expression was established;(2) Colon cancer cells with Lgr5over-expression grew faster, packed into more compact spheroids resistant tomechanical disruption and had a weaker ability to migrate compared to the cellsinfected control vector, thus, Lgr5gene transfection have a certain impact on hebiological behavior of colorectal cancer cells. Part2: Preparation of mouse anti-human Lgr5monoclonalantibodies and discussion the role of Lgr5in human colorectalcarcinomaObjective: To prepare mouse anti-human Lgr5monoclonal antibodies, analysis theexpression of Lgr5in colon cancer tissue and its clinical significance.Methods: BALB/c mice were immunized with Lgr5transfected cell CHO/Lgr5which stably expressed human Lgr5molecule. The spleen cells of immunized mousewere fused with myeloma cells SP2/0, and then were cultured with HAT selectivemedium. Hybridoma cells were screened with CHO/Lgr5cells by Flow Cytometry.Meanwhile, CHO/mock cell which were respectively transfected with wild vectorpIRES2-EGFP were used as negative control. The positive hybridoma cells wererepeatedly subcloned, until one hybridoma cell line which was sustainably and stablysecreting specific anti-Lgr5mAb was obtained. mAb Ig subclass was detected withRapid Qualitative Test Strip, Indirect Immunofluorescence assay, Immunocytochemistryand Western blot was used to identify the specificity of the obtained mAb. Theexpression of Lgr5in colon carcinoma, adjacent tissue and distal normal tissue wasdetected by using mAb9B3and the clinical significance was analyzed.Results:(1) After multiple screening and subcloning, one monoclonal antibodynamed9B3was obtained. The hybridoma cells grew well after long-term storage inliquid nitrogen and culture in vitro. Chromosome analysis showed that the chromosomeof hybridoma is more than100, so the cell is hybridoma;(2) The mAb9B3was provedto be IgG1with κ light chain. Indirect Immunofluorescence assay,Immunocytochemistry and Western blot showed that9B3mAb could recognize Lgr5protein specially;(3) Using the method which establish by our laboratory to inducedascites and preparated monoclonal antibody. After the ascites was purified by protein Gaffinity chromatography, the concentration of the protein was about2.5milligram(mg)/ml and the ascitic titer was over1:1000dilution by Indirect Immunofluorescenceanalysis;(4) Immunohistochemistry results show that the the mAb9B3can recognizethe Lgr5molecules expressed in colon cancer specifically, Lgr5expression in coloncancer tissue has a significant higher level than that in distal normal tissues (P <0.0001), and has no significant difference with that in adjacent tissues (P=0.5237).Conclusion: In summary, this study has successfully cloned human Lgr5gene,established stable transfection CHO cell line and transient transfection SW480cell linewith Lgr5over-expression. The effect of over-expression Lgr5on the biologicalcharacteristic of colon cancer cells was investigate. One hybridoma cell line sustainablyand stably secreting specific mouse anti-human Lgr5mAb was obtained successfully.The obtained anti-human Lgr5mAb can be used for Flow Cytometry, Western blot andImmunohistochemistry. Finally the expression of Lgr5in colon carcinoma, adjacenttissue and distal normal tissue was detected by using mAb9B3. The research hasprovided solid experiment foundation for further studies in the biological characteristicof Lgr5, Lgr5-mediated signal pathway and its role in the development of cancer. |
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