A Positively Charged Peptide Derived From Placental Growth Factor Potentiate The Antitumor Of Interferon Gamma In Human Glioblastoma

Human brain glioblastomas multiforme(GBM)is a primary intracranial neoplasm with high morbidity.Since GBM infiltrates surrounding normal tissues and is characterized by the high grade malignancy,its complete cure is impossible.So the prognosis is bad and there's a high mortality.It's reported that the GBM is a median survival of less than 15 months after surgical resection and a multimodal treatment of concomitant chemotherapy and radiotherapy.The present standard treatments for GBM include surgery,radiotherapy and chemotherapy,or combinations of these treatments.These treatments are palliative and prolong patients' life partly,but the tumors couldn't be killed completely.After the therapies,the GBM often is recurrent,that results in the bad prognosis,and hence the need for novel approaches is urgent.Interferon gamma has been focused when it's discovered because it possesses many biological activities including antiproliferative activity and immunomodulation.These activities provide a potential choice for the researchers on oncology.Interferon gamma has been marketed to treat cancers clinically as it could inhibit directly the proliferation of tumor cells and augment the immunological responses against a tumor,thereby indirectly suppressing the tumor progression.It's a useful therapeutic agent to treat cancer.However,the short in vivo half-life of interferon gamma limits its clinical application.It has been reported that the half-life of interferon gamma is 30 minutes and 4.5 hours after either intravenous or intramuscular injection in human,respectively.Gene therapy of Interferon gamma is known to be a viable method to maintain the concentration of Interferon gamma for a long period.But the ubiquitous expression of interferon gamma receptors limits greatly its therapeutic application clinically and results in the low efficiency of interferon gamma.There's a approach to solve the problem by controlling the tissue distribution.It's feasible to design a new interferon gamma fusion protein with lower cytotoxicity to decrease its reverse responses and enhance the antiproliferative activity.We designed a new synthetic interferon gamma(SIF?)to enhance the tumor cells' antiproliferative activity of interferon gamma by fusing the placental growth factor(PGF)derived positively charged peptide(PCP)to interferon gamma DNA's 3' terminal.To test whether SIF? affect the GBM's antiproliferative activity,we use designed vector together with the envelope vector and package vector to transfect transiently 293 T cell,and infect the U87 cell by the lentivirus from the transfected 293 T cell.Finally we get the U87 cell that express the SIF? by screening with the double selection markers builte-in the SIF? vector.We do some tumor relative experiments with infected U87 cells to test whether the PCP potentiate the antitumor activity of IFN? and study the activation of downstream interferon gamma signal pathway.The study includes three parts as bellows.Part I Constructing the SIF? expressing vector and getting the U87 that express SIF? stably.Objective: To construct SIF? expressing vector and study whether it's possible to make U87 cell express SIF? stably.Methods: Acquiring the encoded DNA sequence of IFN? and PCP derived from PGF by RT-PCR and making them bind by PCR to construct the SIF? encoded DNA sequence.The SIF? sequence is connected to the 3' end of the expressed plasmid built-in the double selection markers of GFP and puromycin.Verify it after amplification by cutting with endonuclease.We transfect transiently the 293 T cell with lipofectamine 2000 together with constructed vector,envelope vector and package vector to produce the lentivirus.Concentrate the lentiviral particles from the transfected 293 T cell,and then infect U87 cells by the lentivirus.Finally,we get the U87 cell that express SIF? stably by screening with puromycin and GFP.Results: Construct the SIF? expressing vector successfully and acquire the U87 cells to express SIF? stably.Conclusion: The new constructed SIF? vector could be expressed stably in the U87 cells and those are ready for the next experiments.Part II Study how SIF? affect the biologic behavior of U87 cellsObjective: Study changes of the biologic behavior of U87 cells that express SIFg stably and observe the effects of TMZ to the infected U87 cells.Methods: Detect the proliferative activity of U87 cells by MTT assay,detect the ability of clone forming by clonogenic assay,test tumor invasion ability,detect the cell cycle and apoptosis with flow cytometry.Results: By contrast with the control,cell viability of SIF? decreases,the ability of clone forming reduces,the cell numbers that cross the membrane decrease distinctly.There's no difference on cell cycle,but apoptotic cells increase obviously.SIF? enhance the activity of TMZ,so that lesser dose TMZ could be used to kill the tumor cell to reduce its side effects.That's to say there's a synergistic effect between SIF? and TMZ.Part III Studies of SIF? stimulating interferon gamma downstream signal pathwayObjective: Study activation of interferon gamma downstream JAK/STAT signal pathway by contrast with wild interferon gamma.Methods: Detect activation of IFN? GAS site by double luciferase detection kit,observe the expression of IFN? regulated genes,ie CLDN7?ISG15?SERPINB1 and STAT1 by RT-PCR,analysis the expression of these genes' proteins by western blotting.Results: By contrast with the control,the expression of the chosen IFN? regulated genes(CLDN7?ISG15?SERPINB and STAT1)increase obviously both in the level of genes and proteins,and the signal pathway of JAK/STAT is increased obviously.Conclusion: The new synthetic SIF? fusion protein activated IFN? downstream signal transduction pathway more than wild IFN?.Thus,it is highly possible that the SIF? may use a similar mechanism to activate the downstream signal pathway mediated by IFN?.