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Reversal of Multi-drug Resisitance in Paclitaxel Resistant Cell Line SKOV3/TR30 by RNA Interferencing

Author: OuYangYiQin
Tutor: LiHuaiFang
School: Tongji University
Course: Obstetrics and Gynaecology
Keywords: RNA interference RNA, small interfering Ovarian tumors Genes, MDR Multidrug resistance Reversal
CLC: R737.31
Type: Master's thesis
Year: 2007
Downloads: 58
Quote: 1
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Abstract


Objective: To investigate the inhibitory effect of small interfering RNA (small interference RNA, siRNA) against multi-drug resistant ovarian epithelial cancer SKOV3/TR30 MDR1 gene is highly expressed. By in vitro experiments to understand the use The siRNA ability specificity silence the timeliness of the MDR1 gene and its role. Method: 1, the use of computer software to design PCR primers of MDR1 gene. Corresponding fragments of the MDR1 gene was amplified in the resistant cell subline SKOV3/TR30, the establishment of the amplified fragment T vector plasmid and sequencing. 2, the use of liposomes GAPDH the the siRNA transfection into SKOV3/TR30 cell, to the the GAPDH amount of protein in the Western-blot assay interference, which explore siRNA effective concentration and the effective duration of action. 3 liposome method transfected with MDR1 siRNA design, the use of fluorescence quantitative polymerase chain reaction (fluorescencequantitative PCR), immunofluorescence, Western-blot method detects interference MDR1 gene mRNA at different time points P-gp protein expression. Made by the MTT assay cell growth curve in the use of Taxol to calculate the rate of cell growth inhibition and taxol IC 50 Understanding interfere with cell biological behavior. Results: 1, using the PCR method to determine the paclitaxel resistant subline SKOV3/TR30 has high expression of MDR1 characteristics. Construct the plasmid sequence identification by restriction enzyme digestion and DNA sequencing methods are correct. MDR1 PCR amplified fragment can be used for subsequent experiments. Transfected with GAPDH siRNA grasp the effective concentration of siRNA role and the corresponding role in the onset time. Apply master transfected MDR1 gene siRNA and 48h of total cellular RNA extracted turn MDR1PCR results show that the interference successful. 3, PCR results show interference MDR1 mRNA levels significantly lower its MDR1mRNA accounts not interfere SKOV3/TR30 0.24% (P <0.001), and GAPDH showed no significant change (P> 0.05). Immunofluorescence showed that P-gp on the cell membrane of the interference significantly reduced. The above results demonstrate the effectiveness and specificity of RNA interference effect. Western-blot detection of P-gp lowest interference 48h interference effects, interference 120h when the amount of P-gp restored to nearly the level of interference. 4, MTT assay cell growth inhibition rate plus paclitaxel and paclitaxel IC 50 . The results showed a significant difference in the before and after interference SKOV3/TR30 cell growth inhibition rate, paclitaxel IC 50 difference was statistically significant. 2noml / L, 1250nmol / L concentration of paclitaxel interfere with cell growth inhibition rate were 18.50% and 33.68%. Interference rose to 46.46%, 56.99% (P <0.05). Paclitaxel IC 50 interference before 3467.73nmol / L decreased 344.67nmol / L (P <0.05). Conclusion: MDR1 mRNA sequence specific siRNA introduction efficient, specific reduction of the resistant cell SKOV3/TR30 in the amount of the corresponding target gene mRNA, thereby reducing the expression of the cell P-pg, reversed the resistant cells characteristic. RNA interference (RNA interference, RNAi) technology is expected to become effective adjuvant therapy for ovarian cancer chemotherapy.

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