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Study on the Activity and Mechanism of Mouse Beta-defensin 2 in Anti-uterine Cervical Cancer

Author: WeiXiaoLi
Tutor: LiMingYuan
School: Sichuan University
Course: Immunology
Keywords: Murine beta defensins Uterine cervix cancer Stably transfection Cell apoptosis Flow cytometry
CLC: R737.33
Type: PhD thesis
Year: 2007
Downloads: 258
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Abstract

ObjectivesAnti-bacterial peptides are important small peptides of the innateimmune system, which have many anti-bacterial activities. Defensins are oneof the most important anti-bacterial peptides, and comprise four families, thatare alpha-defensins, beta-defensins, plant defensins and insect peptides. Theexpressions of beta-defensins (BD) are specificity in different tissues, whichplay an important role in mucosal immune. Beta-defensins 2 (BD2) is animportant family of defensins. It provides a critical link between the innateimmune system and the adaptive immune response. At present, manyscientific researchers pay more attention to anti-tumor activity of defensins,such as multiple myeloma, gastric cancer, nasopharyngeal carcinoma, etc. Inthis study, we hope to provide a new idea for biotherapy of uterine cervixcancer by that constructed the eukaryote expression vector of mBD2, andobserved the inhibitory effects of mBD2 on uterine cervix cancer in vivo andin vitro.Methods(1) Mice total RNA was extracted from the lungs of BALB/c mice whichwere injected LPS by intraperitoneal before. The gene of mBD2 wasamplified by reverse transcription-polymerase chain reaction (RT-PCR).The fragment amplified by RT-PCR was inserted into the eukaryotic expression plasmid pcDNA3.1 (+) to construct pcDNA3.1 (+)/mBD2.(2) The pcDNA3.1 (+)/mBD2, pcDNA3.1 (+)/rmBD2 and pcDNA3.1 (+) weretransformed into the SiHa, an uterine cervix cancer cell line. Theexpression of target genes and proteins were detected with RT-PCR,Western Blot and immuno-flourescence respectively. Cell ViabilityAnalyzer, TUNEL and Annexin V were applied to detect effects of mBD2on SiHa growth and viability.(3) The models of naive mice transplanting tumor were established withstably transfected cell lines, SiHa/M, SiHa/R and SiHa/K. The diametersof tumor were measured and tumor growth curves were draw. mBD2protein expression and pathological changes in tumor tissues wereobserved with HE and immuno-histochemical staining. Apoptosis in thetransplanted tumor was detected by TUNEL method.(4) For evaluation of the induction of protective immunity by mBD2 protein,U14 cells, a cell line from mouse uterine cervix cancer, were injectedhypodermic into BALB/c mice. Tumor-bearing mice were therapied withplasmids purified using a plasmid purification kit. Efficiency of therapywas evaluated by survival rate of tumor-bearing mice and checked byflow cytometry on CD4 and CD8 of peripheral blood.Results(1) Total RNA was extracted successfully from murie lungs. A fragment of250 bp was amplified by RT-PCR and its eukaryotic expression plasmidpcDNA3.1(+)/mBD2 were constructed. The recombinant plasmid wasverified by restriction endonuclease digestion and sequencing. Thesuccessful constructions of eukaryotic expressing plasmids and multigeneexpressing plasmids were confirmed.(2) The recombinant eukaryotic expression plasmid were transfected intoSiHa cells. Through selection of anti-G418, the stably expressing cells, SiHa/M, SiHa/R, SiHa/K were obtained. The target gene of mBD2 and protein was confirmed correctly by immunofluorescence assay, RT-PCR and western Blot. The viabilitys of SiHa/M, SiHa/R showed significant difference with SiHa/K, SiHa (P＜0.05). The apoptotic indexs of SiHa/M, SiHa/R were 40.7％, 30.2％, while SiHa was 7.3％(P＜0.05). The Annexin V~+/PI~+ rate of SiHaJM, SiHa/R and SiHa were 2.4％, 2.3％, 2.3％, and Annexin V~+/PI~- were 0.9％, 1.2％, 1.5％. But there were not statistical difference in three group (P＞0.05). (3) The animal experiment of navie mice showed SiHa/K and SiHa tumor grew faster than SiHa/M and SiHaJR. The diameters difference of the tumor had statistically significant (P＜0.05) at the same two time points. The same result was showed in HE staining and TUNEL assay. (4) The BALB/c U14 model was established successful. By therapy with purific plasmids, the growth of tumor was inhibited and survival time was prolonged. Because of latter phase of tumor progression, immunological function was hurt hardly, so the total count of T cells decreased sharply. But the ratios of CD4/CD8 were more than two after therapy.Conclusions Methods(1) mBD2 gene was cloned successfully and the eukaryotic expression vector pcDNA3.1 (+)/mBD2 was constructed correctly. The mBD2 gene could be expressed in SiHa cells.(2) The transplanting tumor models with SiHa and U14, uterine cervical cancer cell lines, were established successfully.(3) The mBD2 could inhibit SiHa cell growth, and induce its apoptosis. The survival time of model mice-bearing tumor was prolonged and apoptosis was induced by treated with mBD2.

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