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Effect of siRNA Targeting VEGF on Cell Apoptosis and the Expression of Survivin of K562 Cells

Author: LiFangFang
Tutor: XuYouHua
School: Chongqing Medical University
Course: Pediatrics
Keywords: Adenovirus Vascular endothelial growth factor (VEGF) Transfection Survivin Apoptosis Cells Vascular endothelial growth factor
CLC: R733.7
Type: Master's thesis
Year: 2009
Downloads: 41
Quote: 0
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Abstract


The first part of the targeting VEGF siRNA Construction and identification of recombinant adenovirus carrying Objective To construct targeting vascular endothelial growth factor small interfering RNA (VEGF siRNA) of recombinant plasmids, harvest high titer of recombinant adenovirus vectors provide tools for further study. Methods directional cloning method will insert targeting VEGF siRNA shuttle plasmid pSES-HUS, restriction enzyme digestion and DNA sequencing, named pSES-VEGFsi; using homologous recombination methods pSES-VEGFsi with adenoviral backbone plasmid pAdEasy-l connection, Construction of recombinant plasmid pAd5-VEGFsi. Restriction endonuclease pAd5-VEGFsi, liposome-mediated method to import packaging cells HEK293, fluorescence microscopy pAd5-VEGFsi packaging and amplified in the cell by infection with ping-pong cesium chloride density gradient centrifugation to obtain high titer adenovirus Ad5-VEGFsi. Results (1) restriction enzyme digestion and gene sequencing confirmed pAd5-VEGFsi recombinant plasmid was constructed successfully. (2) fluorescence microscopy confirmed that recombinant adenovirus Ad5-VEGFsi successfully imported HEK293 packaging cells, is well expressed in the cells. (3) to obtain a high titer recombinant adenovirus Ad5-VEGFsi, the virus titer of approximately 4.6x l011 pfu / ml. Conclusion We successfully constructed a recombinant adenovirus carrying plasmid targeting VEGF siRNA and harvest high titer recombinant adenovirus Ad5-VEGFsi, for further research has laid a good foundation. The second part of the adenovirus-mediated targeting VEGF siRNA on K562 cell apoptosis and expression of survivin gene Objective adenovirus-mediated VEGF siRNA (ie, recombinant adenovirus carrying VEGF siRNA) silencing K562 cell expression of VEGF, testing K562 cell proliferation and apoptosis, and further detecting intracellular expression of survivin gene explore VEGF in the pathogenesis of leukemia and VEGF and survivin in the pathogenesis of leukemia relationship. Methods successfully constructed the first part of a portable targeting VEGF siRNA recombinant adenovirus (Ad5-VEGFsi) infection in K562 cells. Were divided into three groups: the experimental group (K562/Ad5-VEGFsi), empty vector (K562/Ad5), blank control group (K562). By RT-PCR in K562 cells were detected within the VEGF mRNA and expression of survivin mRNA; ELISA method for detection of K562 cell culture supernatant VEGF protein secretion; Western blot assay K562 cells survivin protein expression; MTT assay and flow of K562 cells cytometry to detect apoptosis. Results (1) three groups of cells in both VEGF mRNA expression and protein secretion; Meanwhile, in the three groups of cells was also detected in survivin mRNA and protein expression. (2) the experimental group VEGF mRNA expression and protein concentrations were (0.25 ± 0.06), (1120.74 ± 14.55) pg / ml, with the empty vector group and blank control group were significantly lower, the difference was statistically significant ( p lt; 0.01). (3) the experimental group survivin mRNA and protein expression levels were (0.42 ± 0.03), (0.26 ± 0.01), with the empty vector group and blank control group also decreased (p lt; 0.01). (4) the experimental group apoptosis rate (16.45 ± 0.14)% compared with the empty vector group and blank control group was significantly increased (p lt; 0.01); experimental group decreased cell viability, proliferation was inhibited, and the empty vector group and compared to the control group were significantly different (p lt; 0.01). Conclusions (1) human leukemia K562 cells expressing VEGF mRNA and secreted VEGF protein. (2) carry targeting VEGF siRNA recombinant adenovirus Ad5-VEGFsi K562 cells can inhibit expression of VEGF mRNA and VEGF protein secretion. (3) K562 cells were infected with Ad5-VEGFsi, the apoptosis was increased, decreased viability, proliferation was inhibited. VEGF overexpression in cells, the tumor cells are resistant to apoptosis and malignant proliferation ability. (4) at the same time as the expression of VEGF decreased intracellular survivin mRNA and protein expression decreases, suggesting that VEGF and survivin expression are closely related. In summary, VEGF overexpression of the leukemia cells resistant to apoptosis and malignant proliferation ability, and this ability of VEGF may be through increased intracellular survivin gene expression to achieve.

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CLC: > Medicine, health > Oncology > Hematopoietic and lymphoid neoplasms > Leukemia
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