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The Study of Inhibition of bFGF siRNA to STAT3in Glioma

Author: YangJiao
Tutor: LiMu
School: Tianjin Medical University
Course: Surgery
Keywords: bFGF siRNA glioma STAT3 CyclinD1 Bcl-xl
CLC: R739.41
Type: Master's thesis
Year: 2008
Downloads: 1
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Abstract


Objective:To Study the inhibition of basic fibroblast growth factor (bFGF) siRNA on STAT3and its downstream molecule cyclinD1and bcl-xl of JAK-STAT singal pathway in glioma cell line U251. This method provides a new gene therapy for glioma.Methods:A bFGF-targeting small interfering RNA(siRNA)expression vector named pGPU6/GFP/Neo-shFGF2was constructed and transfected into U251glioma cell by lipid (lipofectamine2000) mediated method. We elect the optimum dosage of bFGF siRNA with MTT when the dosage of bFGF siRNA was34ug、4ug and5ug, the dosage of liposomes waslOul in the experiment. and then detect MTT absorption value after transfection24hours、48hours、72hours and96hours with the optimum dosage of bFGF siRNA to reflec the proliferation activity of U251. Immunohistochemistry was used qualitatively (after transfection24h) and western blot (after transfection24h,48h,72h) relative quantitatively to detect STAT3, cyclinD1and bcl-xl protein expression changes after transfection.Results:1.MTT showed that lOul liposomes and bFGF siRNA4ug was the best transfection conditions.after transfection24hours, normal control group, the negative control group, the experimental group MTT absorbance was no significant difference; the experimental group and the normal control group and negative control group were significantly different after transfection48hours and72hours, showed that the inhibition of cells proliferation was significant; after transfection96hours, the experimental group and the negative control group had statistical significance, with normal control group had no statistical significance.2. Immunohistochemistry showed that:the positive rate expression of STAT3, cyclinDl and bcl-xl of experiment group in glioma cell line U251of were lower than the normal control group after transfection24hours, after transfection24hours, and the normal control group of STAT3expression (+++), positive cells was46.27%, the expression of the experimental group (+), the positive cells was14.23%, the negative control group (++), expression of positive cells was28.39%; the normal control group of cyclinDl expression (+++), positive cells was51.3%, the expression of the experimental group (+), the positive cells was10.56%, the negative control group (++), expression of positive cells was27.82%; the normal control group of bcl-xl expression (+++), positive cells was48.79%, the expression of the experimental group (+), the positive cells was8.89%, the negative control group (++), expression of positive cells was31.36%.3. Western blot results:after transfection24h、48h、72h showed the experimental group of STAT3cyclinDl and bcl-xl protein band width and brightness than the normal control group and the negative control group, with FlourchmeFC2analysis software can be noted that the inhibition of plasmid was strongest after transfection48h, followed by72hours in the last24hours.Conclusion:The bFGF-targeting small interfering RNA (siRNA)expression plasmid can successfully down-regulate the expression of STAT3, CyclinDl and Bcl-xl, inhibits cell proliferation and induces apoptosis in glioma cell line U251. This study was the basic for further multi-gene therapy for glioma.

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CLC: > Medicine, health > Oncology > Nervous system tumors > Intracranial tumors and brain tumors
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