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Study on Reversal of Multidrug Resistance on B-MD-C1(ADR +/+) by RNA Interference on the Suppression of CtBP1 Gene

Author: YangHongXia
Tutor: AiJun；DanBaoEn
School: Hebei Medical University
Course: Oncology
Keywords: Multidrug resistance MDR1 P-gp CtBP1 shRNA Transfection
CLC: R737.33
Type: Master's thesis
Year: 2010
Downloads: 74
Quote: 0
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Abstract


Objective: CtBP1 target genes, design and construct the recombinant plasmid vector capable of generating short hairpin RNA (shRNA), in vitro transfection of human endometrial cancer resistant strain B-MD-C1 (ADR / ) cells using RNA interference silencing the CtBP1 gene, to study the impact on human endometrial cancer cell gene expression of MDR1 and P-gp protein, as well as human endometrial cancer cell sensitivity to ADM changes to explore the significance of the the CtBP1 gene in the reversal of multidrug resistance. Method: 1 tetrazolium blue method (MTT) to detect endometrial cancer resistant strain B-MD-C1 (ADR / ) resistance multiples. 2 by reverse transcription - polymerase chain reaction (revers transcription PCR, RT-PCR) detection of endometrial cancer resistant strain B-MD-C1 (ADR / ) and sensitive cell lines B- MD-C1 (wt), the intracellular expression of the MDR1 gene. 3 immunoblotting (Western blot) detection of endometrial cancer resistant cell lines B-MD-C1 (ADR / ) and sensitive cell lines B-MD-C1 (wt) cells P -gp protein expression. CtBP1 target genes, designed and constructed able to generate short hairpin RNA (shRNA) RNA interference eukaryotic expression recombinant plasmid vector, the recombinant plasmid was transiently transfected into human endometrial cancer resistant strain B-liposome method MD-C1 (ADR / ). 5 by reverse transcription - polymerase chain reaction (revers transcription PCR, RT-PCR) detection of human endometrial cancer resistant strain B-MD-C1 (ADR / ) restructuring were transiently transfected with 48 hours after intracellular expression of MDR1 gene for detecting RNA interference MDR1 gene silencing. 6 immunoblotting (Western blot) detection of human uterine endometrial cancer resistant strain B-MD-C1 (ADR / ) recombinant plasmid was transiently transfected cells after 72 hours of P-gp protein expression, detection of RNA interference closed P-gp protein expression. The 7-methyl thiazolyl tetrazolium (MTT) detection of recombinant plasmid transient transfection 0,24,48 and 72 hours of human endometrial cancer cell sensitivity to ADM, for the detection of RNA interference can reverse the endometrial cancer resistant cell multidrug resistance. Results: 1 ADM B-MD-C1 (ADR / ) and half of the B-MD-C1 (wt) cells inhibitory concentration IC50 of the 21.57 ± 0.77μg/ml and 2.32 ± 0.34μg / ml, B-MD-C1 (ADR / ) resistant cells in multiples of approximately B-MD-C1 (wt) cells up to 9.3 times, has a very significant difference (p lt; 0.01). 2 B-MD-C1 (ADR / ) cells have a strong expression of the MDR1 gene, while the B-MD-C1 (wt) cells were not detected to the expression of the gene, has a very significant difference between the two (p lt; 0.01). 3 B-MD-C1 (ADR / ) P-gp in the cells showed high expression, and the significantly reduced P-gp expression in B-MD-C1 (wt) cells than the former, both having very significant difference (p lt; 0.01). 4 to successfully design and build two for the the CtBP1 gene can produce the shRNA eukaryotic expression recombinant plasmid vector, both by RNA interference effectively inhibit the expression of the gene (inhibition rates were 47.18% and 41.75%). 5 recombinant plasmid was transiently transfected 48 hours after intracellular expression of MDR1 gene with the resistant cell line B-MD-C1 (ADR / ) significantly weakened, they have significant differences. 6 recombinant plasmid was transiently transfected 72 hours after the P-gp protein expression in the cells with the drug-resistant cell lines B-MD-C1 (ADR / ) significantly weakened, they have significant differences. 7 recombinant plasmid was transiently transfected with 24, 48 and 72 hours after the cell proliferation is inhibited, wherein the 48,72 hrs restrained obvious highly significant difference (p lt; 0.01), compared with control group. Conclusion: 1 endometrial cancer resistant strain B-MD-C1 (ADR / ) has obvious resistance, one of the resistance mechanisms of multidrug resistance gene MDR1 and its expression product of P-gp protein expression. 2 shRNA expression vector designed and constructed by RNA interference effectively closed the CtBP1 Gene Expression. 3 for CtBP1 the shRNA cut human endometrial cancer resistant strain B-MD-C1 (ADR , / ) in the MDR1 gene and P-gp protein expression. 4 for CtBP1 the shRNA can improve human endometrial cancer resistant strain B-MD-C1 (ADR / ) AMD sensitivity.

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