Construction Of Proliferation And Tumor Cell Specific Operating System For The Gene Therapy Of Gliomas

Objectives:Glial tumors were the most common tumors of primary central nervous system.The infiltration of glioma cells is a pathological characteristic of gliomas.Surgery,radiotherapy and/or chemotherapy,as generally available,had been of limited value.The current studies showed that gene therapy could become an effective means for the future treatment of gliomas.But the key problem of gene therapy is its specificity and safety,and the specificity depends largely on its specific promotor.The promotors,such as desmin,M6 and PSMA,etc,could only solve the problem of tumor cell specificity,whilst,hTERT promotor could only solve the problem of proliferative specificity.The combination of proliferative and tumor cell specificity,may further improve the specificity and safety of gene therapy,leading to the transition of theroy to clinic and ideal to application.The main obiective of our studies was to construct a proliferative and tumor cell specific operating system for malignant glioma gene therapy on the basis of recombinant proliferating cell nuclear antigen promoter PCNATAI and glial fibrillary acid protein promoter GFAPp.Methods:PCNATAI-LoxP-stop-LoxP operating system under the help of GFAP promoter controlled Cre recombinase could initiate the expression of the downstream gene in malignant glioma cells in a proliferation and tumor cell specific manner.The fragment PCNATAI was inserted into pBluescript SK vector,and then transcriptional termination sequence 'stop' equipped with Cre recombinase recognition sites(LoxP)on both ends was inserted into the downstream of PCNATAI promoter to construct the PCNATAI-LoxP-stop-LoxP operating system.Qualitative analysis plasmid and quantitative analysis plasmid using EGFP and luciferase as the reporter genes were respectively constructed with this operating system.The full length coding region of Cre recombinase and GFAP promotor spanning-1660 to +16 were PCR ampified utilizing pBluescriptKS-Cre and human genome as templates;The plasmid vector pEGFP-1 was used as a cloning backbone,GFAP promotor was inserted into the multiple clone site and the region EGFP was replaced by Cre recombinase;Then an accessory plasmid(pGFAP-Cre)mediating the expression of Cre recombinase under the control of GFAPp promoter was also established.The transcriptional activity and specificity of this operating system was analyzed in TJ905 glioblastoma cell line(TJ905),HEK293 immortal cell line(HEK293),NGF induced PC12 cell line(PC12)and in vitro cultivated rat astrocytes.The mRNA level of this operating system was tested in these four cell lines by RT-PCR.Results:? PCNATAI-LoxP-stop-LoxP operating system was identified by restrictive endonuclease digestion,agarose gel electrophoresis demonstrated that the inserted fragments were at right position,the magnitude and orientation were both correct,its validity was further verified by DNA sequencing.And based on it the reconstruction plasmid pPCNATAI-stop-EGFP,p-B-Pstop and p-E-Pstop were also successfully constructed.? The reconstructed plasmid pGFAP-Cre was identified by restrictive endonuclease digestion,and the fidelity of the inserted fragment were verified by sequencing.? Qualitative analysis showed that the single transfection of pPCNATAI-stop-EGFP can't gave out green fluorescent,only in TJ905,co-transfe ction of the qualitative analysis plasmid and pGFAP-Cre can gave out green fluorescent,in the other three cell lines can't express green fluorescent either.The single transfection of p-B-Pstop or p-E-Pstop had low transcriptional efficacy in all the cell lines and had no significant difference(P>0.05).High transcriptional activity was found only in TJ905 co-tranfected with the quantitative analysis plasmid and pGFAP-Cre,and the transcriptional efficacy could be further improved by SV40 enhancer.? RT-PCR of total RNAs demonstrated that Cre recombinase and PCNATAI-EGFP only expressed in the co-transfected TJ905,not in the co-transfected HEK293 and PC 12,and Cre recombinase only expressed in the co-transfected vitro cultivated rat astrocytes.The reverse product of PCNATAI-EGFP was about 600bps in size,it showed that the stop seguence had been successfully deleted during the process of Cre-mediated recombination.Conclusions:? When single transfection of pPCNATAI-stop-EGFP?p-B-Pstop or p-E-Pstop(regulated by PCNATAI-LoxP-stop-LoxP),the 'stop' seguence could efficiently repress the transcription of the downstream gene.? When co-transfection of pGFAP-Cre and the three analysis plasmids,the 'stop' seguence could be deleted successfully and induced the transcription of the downstream gene in a proliferation and tumor cell specific manner.? Only in TJ905,SV40 could further increase the transcriptional activity without influencing the proliferative and tumor cell specificity.? The result of RT-PCR demonstrated that the transcriptional progress of this operating system was completely consistent with anticipation.