Study On The Construction Of IL-8 Gene Eukaryotic Expression Vector And The Cytobiology Mechanism Of OVCAR-3 Cells

Objective: Being one of department of gynecology tumor, ovarian cancer which is seriously attacking women's health has driven professor's attention which is hard to search out in early stage. There are varieties of tumor of the ovary whose organism construction are complicated and owe different bionomics. The malignant degree of epithelium tissue cancer is higher than interstitial substance cancer. While its therapeutic efficacy can not reach the germinocarcinoma. And this tumor which grows very fast, can't be diagnosed easily in the early stage and its death rate is very high is eager to be researched in the region of woman tumor. So a new therapy should be found. Recently, the research that specialists try to find out the cause and developing relationship of some uncommon genes and ovarian cancer is hitting at the district of boss molecular biology research. Ovarian cancer's cause and developing relationship is a complex and mixed-step process which involves quantities genes and proteins'expression.IL-8 which is found by Yoshimur at 1987 is the first chematropism cell factor belonging CXC family that plays an important role in the contribution of inflammation and immunologic process. Recently, it has drawn more and more attention of researchers as malignant tumor correlative molecule. Nowadays, it is known that many tumor cell can secrete IL-8, IL-8 overexpression relates to tumorous growth and metastasis but it is not clear that the mechanism of encouraging tumor appearance and development.Recent years, the construction and development of recombi- nation technology can provide new strategy for ovarian cancer in theory and practice——gene therapy can be expected to become new channel of ovarian cancer therapy. Ideal therapeutic gene can inhibit tumor cell's growth effectively without inhibitory action to normal tissue and cell. In this research, we clone the human IL-8 gene into eukaryotic expression vector pcDNA3.1(+), with the act of liposome, transient transfection OVCAR-3 which is epithelial ovarian cancer cells expressing little IL-8. The functional change and biology mechanism are investigated induced by IL-8, and provide a feasibility to IL-8 gene therapy ovarian cancer.Method: 1 Construct the eukaryotic expression vector of IL-8 gene Total RNA was isolated from PBMC of healthy volunteers, IL-8 gene encoding fragment was amplified by RT-PCR and cloned into the vector PMD-18T. BamHI/XhoI double digested product of PMD-18T/IL-8 was connected with the vector pcDNA3.1(+) which was digested by BamHI/XhoI .2 Transient transfect into OVCAR-3 cells The recombinant plasmid pcDNA3.1(+)/IL-8 was transfected into OVCAR-3 cells which express IL-8 a little with Lipofectamine2000. The expression of IL-8 was tested in OVCAR-3 cells by RT-PCR,ELISA and Western blot assay.3 Detect cell proliferation Detect these cells'reproductive activities by MTT and draw growth curve.4 Detect the cell cycle The cell cycle was detected by Using flow cytometry (FCM) on three group cells: pcDNA3.1(+)/IL-8 transfection group,pcDNA3.1(+) group and OVCAR-3 none transfection group .5 Measure Bax,Bcl-2,VEGF and MMP-9 expression analysis The expression of Bax,Bcl-2,VEGF and MMP-9 were detected by using FCM on three group cells the same asmethod 4. RESULTS: 1 After PCR amplification, BamHI/XhoI digestion and DNA sequencing confirmation, the eukaryotic expression vector pcDNA3.1(+)/IL-8 was constructed successfully.2 The IL-8's expressions in quantity were founded in the cells of transient transfection when detected by the way of RT-PCR. The gray scale value of pcDNA3.1(+)/ IL-8 transfection group which was 226.88±5.47 correspondingly againstβ-actin was 1.55, while OVCAR-3 none transfection group which was 120.38±6.72 correspondingly againstβ-actin was 0.82. The growth rate was 189.02%;The IL-8's expressions in cell sap and the cells after transfecting pcDNA3.1(+)/ IL-8 6h and 48h were respectively 3.69±0.37,19.67±0.24,11.10± 1.14 by using ELISA, while the others were a little, P<0.05; the specific protein strap was detected only in the former by the way of Western-blot.3 The results by MTT indicated that the light absorption value of OVCAR-3 cells transfected IL-8/pcDNA3.1 were respectively 0.56±0.08,0.63±0.06,0.93±0.09,1.20±0.06,1.32±0.12,1.39±0.14, while OVCAR-3 none transfection group were respectively 0.62±0.07,0.70±0.10,0.84±0.12,0.92±0.10,0.96±0.14,1.01±0.10. There was no statistical significance compared pcDNA3.1(+)/ IL-8 transfection group with the ones not transfected within 3days(P>0.05), while pcDNA3.1(+)/ IL-8 transfection group was obviously more active after 3 days(P<0.05), and the former's increasing rate presents an evident tendency of going up along with the time's extension.4 The cellular S stage in pcDNA3.1(+)/IL-8,pcDNA3.1(+) and OVCAR-3 cells were respectively 55.18±2.98,39.72±4.16,31.36±1.46 by FCM. The former was much higher than the others, P<0.05. These proliferation index were 58.83±2.85,44.32±4.20,35.31±1.56.The former PI was much higher and the comparison between them has statistical significance(P<0.05).5 The expression of VEGF and MMP-9 in pcDNA3.1(+)/IL-8,pcDNA3.1(+) and OVCAR-3 cells were respectively 430.44±21.70,358.70±17.77,335.52±19.68 and 356.15±22.70,279.37±27.48,283.56±26.67; while Bcl-2 and Bax in these cells were respectively 380.86±15.27,319.15±17.89,305.04±28.90 and 343.12±26.10,416.27±25.45,420.13±23.65. There was statistical significance against pcDNA3.1(+) transfection group and OVCAR-3 none transfection group (p<0.05), while the comparison between them has no statistical significance.Conclusions: IL-8 could transfect OVCAR-3 cells effectively by liposome. Moreover, IL-8 can also promote cell'proliferation function by increasing the number of cell S stage, that the mechanism was connected with angiogenesis factor and correlation factor about apoptosis, which provides a foundation for researching the mechanism of IL-8 on ovary cancer.