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Experimental Study of SKOV3 Cells with UC-MSCS Transduced with an Recombinant Adenoviral Vector Expressing Interleukin12 in Vitro

Author: ShiPengFei
Tutor: ChengJianXin
School: Hebei Medical University
Course: Obstetrics and Gynaecology
Keywords: UC-MSCs IL-12 Gene therapy Transfection SKOV3 Recombinant adenovirus
CLC: R737.31
Type: Master's thesis
Year: 2011
Downloads: 39
Quote: 0
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Abstract


Objective: In this study, the proposed in vitro cultured ovarian cancer cell line SKOV3 (ovarian serous adenocarcinoma cell line) and human umbilical cord mesenchymal stem cells (UC-MSCs), UC-MSCs as gene therapy targeting delivery vehicles, building UC-MSCs load IL-12 adenovirus vector, and in vitro to produce biologically active MIL-12. Observation UC-MSCs load IL-12 recombinant adenovirus (AdIL-12-MSC) the SKOV3 cells growth morphology, proliferation, cell cycle and apoptosis, and to explore UC-MSCs as gene therapy targeting vehicle the efficacy of ovarian cancer as well as its mechanism to provide new targeted gene therapy of ovarian cancer. Method: 1 cultured in vitro ovarian cancer cell line SKOV3 (ovarian serous adenocarcinoma cell line), human umbilical cord mesenchymal stem cells (UC-MSCs) and human embryonic kidney epithelial 293 cells, and daily Morphological changes were observed with an inverted microscope and proliferation. 2 of the recombinant adenovirus repeatedly amplified to the required amount, application adeno-virus-mediated IL-12 gene transfection UC-MSCs (AdIL-12-MSCs) as transfection group, set up an empty viral vector transfection UC-MSCs cells group (Ad-MSCs group) and UC-MSCs group. Using Western Blotting detection with AdIL-12-MSCs group, the IL-12 protein expression in Ad-MSCs group and the group of UC-MSCs. 4 the technical detection with AdIL-12-MSCs group using reverse transcription-polymerase chain reaction (RT-PCR), Ad-MSCs group and UC-MSCs group of IL-12mRNA in expression. SKOV3 cells were seeded in 24h after transfection AdIL-12-MSCs supernatant as transfection Group, SKOV3 cells seeded in UC-MSCs after 24 h of culture supernatant as a control group. 6 inverted microscope contrast observed transfection group and the control group SKOV3 cells 24 ~ 72h growth morphology to clear AdIL-12-MSCs form of ovarian cancer SKOV3 cell growth. 7 detection AdIL-12-MSCs proliferation of SKOV3 cells in vitro by MTT. 8 use of AdIL-12-MSCs of the cell cycle and apoptosis of SKOV3 cells by flow cytometry. 9 using SPSS 13. The 0 statistical software, t inspection group differences significant data \Results: 1 Western Blotting detection after transfection AdIL-12-MSCs in the IL-12 protein expression shows: with AdIL-12-MSCs cells clear bands appear in control Ad-MSCs group and UC-MSCs group cells was not detected in the expression of the IL-12 protein, indicating that AdIL-12-MSC within the IL-12 gene was successfully expressed at the protein level. 2 RT-PCR results show that: with AdIL-12-MSCs in 389bp at the positive bands appear in line with the IL-12 amplified fragment length, indicating that the IL-12 gene within AdIL-12-MSCs successfully expressed at the mRNA level, and in the IL-12 mRNA expression is not detected in the control group Ad-MSCs and UC-MSCs cells. 3 inverted microscope was observed under the SKOV3 cell culture growth after 24 ~ 72h visible: group SKOV3 cell growth were inhibited at different times of the day, transfection, cells adherent rate, lower cell density, cell processes reduce; the control group SKOV3 cell morphology period showed no significant change. 4 MTT assay in vitro proliferation of SKOV3 cells transfected group and the control group 24 ~ 72h as follows: With time, the group of transfected SKOV3 cells suppress the proliferative capacity significantly improved, 72 h after inhibition rate (47.56 ± 2.31)%, statistically significant (P lt; 0.05). 5 AdIL-12-MSCs by flow cytometry on SKOV3 cell cycle affect the results show that: the transfected group compared with the control group, SKOV3 cells in the proliferation of S G2 phase of the cell decline in the proportion of the arrest in the G0/G1 phase of the cell ratio increase, prompted AdIL-12-MSCs can inhibit the proliferation of SKOV3 cells. Flow cytometry: transfection group the SKOV3 cell apoptosis rate (9.72 ± 1.38)%, compared with the control group SKOV3 cell apoptosis rate (2.69 ± 0.45)% AdIL-12-MSCs SKOV3 cell apoptosis results apoptosis rate was significantly higher with statistical significance (P lt; 0.05). Conclusion: In this experiment, the UC-MSCs as gene therapy targeted delivery systems, successfully constructed UC-MSCs load IL-12 recombinant adenovirus vector-with AdIL-12-MSCs cell lines, and observed the secretion of exogenous IL- 12 significantly inhibited proliferation and induced apoptosis of SKOV3 cells. This cell line tumor research can be applied to gene therapy, a new targeted for epithelial ovarian cancer gene therapy and cell immunotherapy.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Female genital tumors > Ovarian tumors
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