| At present, therapeutic regimen including surgery, radiotherapy, chemotherapy and hormonal therapy greatly improved the survival rate of cancer patients, but the rate of recurrence and metastasis remains high. Pre-clinical study of biologic therapy for ovarian cancer had made great progress,and it has brought new hope to the advanced ovarian cancer patients.IL-24 is the cytokine being thought of an available tumor suppressor. When transfected into melanoma cells, IL-24 can selectively inhibit the differentiation of melanoma cell and induce the apoptosis of the cancer cell. If the IL-24 do not work, the melanoma tumor will growth and invasion soon. Foreign research show that adenovirus-IL-24 transfected into 20 cancer cell lines and found that 19 cancer cell lines were apoptotic.IL-24 gene can induce apoptosis in a variety of tumor cells, but did not affect normal cells, and it can play the anti-tumor activity through stimulating the immune response . Therefore, IL-24 and its receptor was considered to have brighter prospects in clinical application. The research of IL-24 gene expression was both important in the theory and clinical aspect.This study is divided into four parts. In order to understand the function of IL-24,We constructed and identified the eukaryotic expression vector pcDNA3.1-myc-his (-) B-IL-24 in Part one .We observed whether IL-24 inhibit the growth of epithelial ovarian cancer cells after the recombinant plasmid pcDNA3.1-myc-his (-) B-IL-24 was transfected into epithelial ovarian cancer SKOV3 cells with electric shock,and was stable expressed in SKOV3 cells in Part two. The cell cycle, the apoptosis rates and the changes of apoptogene of the epithelial ovarian cancer cells transfected with exogenous IL-24 gene be detected through various methods in Part two,too. In Part three, In order to observe that whether it can improve the sensitivity of tumor cells to radiotherapy, after pcDNA3.1-myc-his (-) B-IL-24 eukaryotic expression vector transfected into epithelial ovarian cancer. Epithelial ovarian cancer xenograft model in nude mice were established. In order to observe whether it can inhibit the growth of ovarian cancer-bearing nude mice when the nude mice treated by pcDNA3.1-myc-his (-) B-IL-24 eukaryotic expression vector In Part four. Objective: To construct and identify the eukaryotic expression vector pcDNA3.1-myc-his (-) B-IL-24 in this part.Methods: Lympholeukocyte of peripheral blood was isolated. Primer was design by Primer Premier 5.0, then the total RNA was extracted from lympholeukocyte of peripheral blood.IL-24 gene was acquired by RT-PCR from the total RNA of lympholeukocyte of peripheral blood, and was confirmed by sequencing, then was cloned into plasmid pcDNA3.1-myc-his( - )B by DNA recombination technique to generate the eukaryotic expression vector named pcDNA3.1-myc-his(-)B-IL-24. The eukaryotic expression vector was amplificated when the sequence was proved correct by PCR and enzyme digest.Results:1,Lympholeukocyte of peripheral blood was isolated successfully. The total RNA was extracted and was analyzed by electrophoresis. Three bands can be detected in Agarose gel .They are 28s,18s,5s,and the optical density value of 28s are more than doubled of 18s. The RNA sample was integrity and less degradation.2,Reverse transcription PCR (RT-PCR) products of IL-24 was identified by agarose gel electrophoresis. About 600~+bp was amplified by RT-PCR. It was the expected size of IL-24 cDNA length.3,When the eukaryotic expression vector pcDNA3.1-myc-his (-) B-IL-24 was used as a template, PCR products is about 600~+ bp with 1% agarose gel electrophoresis identification. The size of gene sequence was consistent with the size of IL-24 gene.4,The eukaryotic expression vector was identificated by the digest of enzyme BamH I and Hind III , two bands can be detected by 1% agarose gel electrophoresis: about 5.4Kb, 600~+bp. The size of gene sequence was consistent with the size of pcDNA3.1-myc-his (-) B and IL-24 gene. The eukaryotic expression vector was identificated by the digest of enzyme XbaI , two bands can be detected by 1% agarose gel electrophoresis: about 5.4Kb, 600~+bp. The size of gene sequence was consistent with the size of pcDNA3.1-myc-his (-) B and IL-24 gene.5,The eukaryotic expression vector was sequenced by Invitrogen Company after identificated by the digest of enzyme. As analysised and comparied by the NCBI BLAST program, the IL-24 gene sequence was completely consistent with the genebank.Conclusions:1,There is IL-24 mRNA in human peripheral blood lymphocytes.2,IL-24 gene was successfully cloned into plasmid pcDNA3.1-myc-his(-)B and its eukaryotic expression vector pcDNA3.1-myc-his (-) B-IL-24 was constructed successfully. Objective: We will further observe whether IL-24 inhibit the growth of epithelial ovarian cancer cells ,and investigate the effects of IL-24 gene on the growth of SK0V3 cell,after the recombinant plasmid pcDNA3.1-myc-his (-)B-IL-24 was transfected into epithelial ovarian cancer SK0V3 cells with electric shock,and was stable expressed in SK0V3 cells. The cell cycle, apoptosis rates and the changes of apoptogene in the epithelial ovarian cancer cell transfected with exogenous IL-24 gene will be detected through various methods in this part,too.Methods: The concentration of G418 was optimized after SKOV3 cells was cultured successfully. The recombination plasmid pcDNA3.1-myc-his (-) B-IL-24 and empty vector pcDNA3.1-myc-his (-) B were transfected into SK0V3 cells with electric shock. Cell clones with stable expression of IL-24 were selected by G418, and were detected the expression of IL-24 mRNA by RT-PCR, and were detected the expression of IL-24 protein by Western Blot. Inhibition of proliferation was measured by cell counting,MTT assay and plate colony formation assay. We observed the morphology of apoptotic cells by the inverted microscope. cell apoptosis be detected by Hoechest 33258 staining,and measured the apoptotic rates by flow cytometry. PI fluorescence staining can detect the cell cycle,and AnnexinV-FITC and PI fluorescence staining can detect the changes of cell apoptosis rate. p53 mRNA and caspase-3 mRNA expression changes can be detected by RT-PCR.Results:1,The cells were selected by G418 after they were transfected by pcDNA3.1-myc-his (-) B-IL-24.all the negative group died, but some small cell cloning in the IL-24 transfected group and empty-vector transfected group were formed after one week,and more big or small cell cloning in the IL-24 transfected group and empty-vector transfected group were formed after two weeks. 2,The expression of IL-24 mRNA and GADPH mRNA were both confirmed by RT-PCR at about 600~+bp and 400~+bp. The IL-24 transfected SKOV3 cells can observed two bands,but the other SKOV3 cells can be only observed the GADPH specific band.3,The expression of IL-24 protein were confirmed by Western Blot at 23.8KD. The IL-24 transfected SKOV3 cells can observed one band at 23.8KD,but the other SKOV3 cells can not be observed the specific band.4,The growth curve of cells was measured by cell counting. The transfection of IL-24 gene into human ovarian carcinoma cell line can inhibited vitality of the cell (P=0.000). the number of the SKOV3-IL-24 cell compared with the other two groups, the difference was significant (P=0.000). The non-transfected SKOV3 cells compared to the transfected with empty-vector SKOV3 cells, the difference was not statistically significant (P=0.145).5,The value of absorbance can be measured by MTT assay. The transfection of IL-24 gene into human ovarian carcinoma SKOV3 cell line can inhibited the growth of the cell (P = 0.000). the proliferation of the SKOV3-IL-24 cell compared with the other two groups, the difference was significant (P = 0.000).The non-transfected SKOV3 cells compared to the transfected with empty vector SKOV3 cells, the difference was not statistically significant (P = 0. 326).6,The rate of colony formation can be measured by plate colony formation assay . the rates of colony formation of the SKOV3- IL-24 cell,the SKOV3-empty-vector cell and the SKOV3 cell were (51.67±0.04) %,(60.33±0.07) % and (62.00±0.05) %, respectively, the difference was not statistically significant (P=0.12). the cells per colony of the SKOV3- IL-24 cell,the SKOV3-empty-vector cell and the SKOV3 cell were (303±15.67),(295±13.54) and (99±11.97) , respectively. the cells per colony of the SKOV3-IL-24 cell compared with the other two groups, the difference was significant (P=0.000).The non-transfected SKOV3 cells compared to the transfected with empty vector SKOV3 cells, the difference was not statistically significant (P=0.326). The transfection of IL-24 gene into human ovarian carcinoma SK0V3 cell line can inhibited the ability of colony formation of the cell.7,the cells which was apoptic increased significantly when we observed by the inverted microscope. The morphology changes of IL-24 transfected cells nucleus detected by Hoechest 33258 fluorescence staining. normal cells and the cells transfected with empty vector can be observed subdued blue fluorescence. The size of cell nucleus was same. The nucleus were round or oval,and numerous too. They stained with blue fluorescence,and no obvious morphological changes such as apoptotic bodies can be observed. the cells transfected with IL-24 can be observed bright blue fluorescence. we can see that more apoptotic cells. The density of cytoplasm increased. The colors of nucleus change deeper, the size smaller, the shape irregular. formation of apoptotic bodies and nuclear fragmentation can be observed usually.8,The cell cycle can be detected by flow cytometry, the rates of G2 / M phase of the SKOV3-IL-24 cell, the SKOV3-empty-vector cell and the SKOV3 cell were 12.88%,4.79% and 4.29%, respectively, the rate of G2 / M phase of the three groups, the difference was significant (F=309.805, P=0.000). The non-transfected SKOV3 cells and the transfected with empty vector compared to the SKOV3- IL-24 cells, the difference was statistically significant (P=0.000).The non-transfected SKOV3 cells compared to the transfected with empty vector SKOV3 cells, the difference was not statistically significant (P=0. 233).9,The apoptosis rate can be detected by flow cytometry. the rates of apoptosis of the SKOV3-IL-24 cells,the SKOV3-empty-vector cells and the SKOV3 cells were 14.51%,3.60% and 3.33%, respectively, the rate of apoptosis of the three groups, the difference was significant (F=1205.073, P=0.000). The non- transfected SKOV3 cells and the transfected with empty vector SKOV3 cells compared to SKOV3-IL-24 cells, the difference was statistically significant (P=0.000).The non-transfected SKOV3 cells compared to the transfected with empty vector SKOV3 cells, the difference was not statistically significant (P=0. 318).10,The change of p53 mRNA expression can be detected by RT-PCR. The expression of p53 mRNA and 6-actin mRNA were both confirmed by RT-PCR at about 600~+bp and 500 ~+bp. the value of p53 mRNA relative expression of the SKOV3-IL-24 cells,the SKOV3-empty-vector cells and the SK0V3 cells were 0.44±0.005,0.44±0.004 and 0.43±0.005,respectively.the p53 mRNA expression of the cells transfected with IL-24 compared with the other two groups, the difference was not significant (F = 3.000, P = 0.125).The change of caspase-3 mRNA expression can be detected by RT-PCR. The expression of caspase-3 mRNA and 6-actin mRNA were both confirmed by RT-PCR at about 260bp and 500~+bp. the value of caspase-3 mRNA relative expression of the SKOV3-IL-24 cells,the SKOV3-empty-vector cells and the SKOV3 cells were 0.64±0.003,0.20±0.004 and 0.26±0.005,respectively.the caspase-3 mRNA expression of the cells transfected with IL-24 compared with the other two groups, the difference was significant (F=1741.000,P=0.000).Conclusions:1,IL-24 gene can be transfected successfully into SKOV3 cells with electric shock.2,the SKOV3-IL-24 cells can stable express IL-24 mRNA and protein after selected by G418,namely, epithelial ovarian cancer cell model of stable expression of IL-24 were established .3,The proliferation of SKOV3 cells can be inhibited by IL-24 gene.4,IL-24 gene can lead to G2 / M arrest of cancer cell.5,IL-24 gene can induce apoptosis of epithelial ovarian cancer SKOV3 cells.6,The effect of IL-24 on the apoptosis in human epithelial ovarian cancer has nothing to do with p53,but with the caspase-3. Objective: In order to observe that whether it can improve the sensitivity of tumor cells to radiotherapy, after pcDNA3.1-myc-his (-) B-IL-24 eukaryotic expression vector transfected into epithelial ovarian cancer.Methods: Epithelial ovarian cancer cells were exposed to the radiation radiated by imported Siemens linear accelerator after epithelial ovarian cancer cells were transfected and selected . Inhibition of proliferation was measured by MTT assay. AnnexinV-FITC and PI fluorescence staining (flow cytometry) can detect the changes of cell apoptosis rate.Results:1,The value of absorbance can be measured by MTT assay. The transfection of IL-24 gene into human ovarian carcinoma SK0V3 cell line after exposed to the radiation can inhibited the growth of the cell (P =0.000). The value of absorbance of the SKOV3-IL-24 cell compared with the other two groups, the difference was significant (P<0.05).The non-transfected SKOV3 cells compared to the transfected with empty vector SKOV3 cells, the difference was not statistically significant (P =0.184).2,The apoptosis rate at the 24th hours after treatment by 6 Gy radiation can be detected by flow cytometry. the rates of apoptosis of the SKOV3- IL-24 cell,the SKOV3-empty-vector cell and the SKOV3 cell were 6.90%, 8.13% and 20.12%, respectively. the rate of apoptosis of the SKOV3- IL-24 cell compared with the other two groups, the difference was significant (P=0.000).The non-transfected SKOV3 cells compared to the transfected with empty vector SKOV3 cells, the difference was not statistically significant (P=0.053).Conclusions:1. Exogenous expression of IL-24 gene can improve the radiation sensitivity of epithelial ovarian cancer SKOV3 cells. Objective: Epithelial ovarian cancer xenograft model in nude mice were established. In order to observe that whether it can inhibit the growth of epithelial ovarian cancer-bearing nude mice when the nude mice treated by pcDNA3.1-myc-his (-) B-IL-24 eukaryotic expression vector.Methods: Epithelial ovarian cancer xenograft model in nude mice were established. tumor size, weight, and the inhibition rate were observed after the nude mice treated by pcDNA3.1-myc-his(-)B-IL-24 eukaryotic expression vector.Results:1,The tumor volume of Balb / c nu / nu nude mice treatment by pcDNA3.1-myc-(-) B-IL-24 plasmid grow slower, compared with the non-transfected group and the empty vector group, the difference is significant (P =0.000).2,The rate of tumor inhibition is 63.21% after IL-24 treatment. The tumor weight of Balb / c nu / nu nude mice treatment by pcDNA3.1-myc-(-)B-IL-24 plasmid reduced,, compared with the non-transfected group and the empty vector group, the difference is significant (P=0.000).Conclusions:1,IL-24 can inhibit the tumor growth of epithelial ovarian cancer SKOV3 cells in nude mice.2,IL-24 gene has great value for the treatment of epithelial ovarian cancer.
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