| As protein gene of anti- apoptosis, Bcl-2 can protect cells which itswere avoided apoptosis by virus,oxidant stimulated and induced .Thehigh expression of Bcl-2 is one of important reasons that it lead tooccurrence of tumor and development of tolerance .Therefore, theobjective that utilization of RNA technique and expression of anti-Bcl-2gene achieves and accelerates apoptosis of tumor cells is a kind of newpathway investigated actively at present. RNA interference is a kind of conservative response of defensefrom low organism to mammal existed. Double-stranded RNA was cutabout 21~23nt siRNA by endonuclease RNaseâ…¢ in cell, and formattedRNA-induced silencing complex, RISC identified and degraded mRNAof homological rank, it lead to ultimately specific silence of geneexpression. To construct carrier that it can transcript functional siRNA incell, and it was advantaged to confuse expression of Bcl-2, to establishbackground for further investigation of gene function and therapeutictool. PART â… CONSTRUCTION OF RECOMBINANT PLASMID CARRIER Objective: RNA interference (RNAi) has recently been used to silencegene expression in various species, it can inhibit evidently expression ofobjective gene. The connection between siRNA of Bcl-2 gene and plasmidpGenesil-1 of fluorescent protein expression , construct siRNArecombinant plasmid to aim directly at Bcl-2 (pshRNA-Bcl-2)andrecombinant plasmid of negative contrast (pshRNA-Yx) . Method :After designing and checking siRNA rank of aim directly atBcl-2 of objective gene and negative contrast, to design and synthesiscorresponding shRNA (short hairpin RNA, shRNA). The shRNA andplasmid pGenesil-1 of fluorescent protein expression connect to constructpshRNA-Bcl-2 and pshRNA-Yx. According to behavior that pGenesil-1plasmid produced green Fluorescence after expression, to observe andcount green Fluorescent cells and count of all cells with fluorescencemicroscope after transfecting cells, so, the incidence of green Fluorescentcells is also transfection efficiency. Results: By rank analyzed and electrophoresis, to confirm successionthat construct pshRNA-Bcl-2 and pshRNA-Yx to observe and count greenFluorescent cells and count of all cells by using fluorescence microscopeafter transfecting cells , transfection efficiency of green Fluorescent cellsreach 86%. Conclusion: pshRNA-Bcl-2 and pshRNA-Yx constructed canefficiently effect at human ovary carcinoma cell. This experiment that it advantaged to produce a marked effect thatDNA carrier transcript mRNA in cell , overcome shortcoming thatexpensive cost and low transfection efficiency of Synthesized RNA ,advantage application and extensibility of RNAi technique . PARTâ…¡ The effect on inhibition of RNAi-mediated Bcl-2 tocellproliferation of skov3 human ovary carcinoma Objective: Through using transfective cells shRNA of targetBcl-2 gene constructed, to observe its inhibition to endogeneticexpression of Bcl-2 gene in SKOV3 human ovary carcinoma cell andeffect of biological character on SKOV3 human ovary carcinoma cell . Method : Through utilizing MTT , electron microscope ,RT-PCR,FCM etc , to investigate change of transcriptive level of Bcl-2mRNA and Bcl-2 expression after transfecting to aim directly at shRNAof Bcl-2 in SKOV3 human ovary carcinoma cell , meanwhile observeinfluence on SKOV3 apoptosis after action of shRNA . To detect protein expression of cells , include Bcl-2,Bcl-xl,Baxand Caspase-3 , after RNA interference , through immunohistochemicalmethod, detect effect after interference on protein expression of Bcl-2,through MTT detect effect of vital force of SKOV3 cell after RNAi ;RT-PCR detects mRNA level of Bcl-2 gene; electron microscopeobserves Alteration of microstructure of SKOV3 cell . Results : RNAi-mediated Bcl-2 gene lead to protein expressiondecrease evident ,apoptosis and inhibit evidently growth of Tumorcells . Conclusion:Because Biological function of Bcl-2 protein isn`tdemand absolute in normal cells , inhibition of expression of Bcl-2protein possibly produce little effect to body . Th... |
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