The Study Of The Impacts Of CXCL10 Gene Therapy Mediated By PCL-PEI Nanoparticles On Killing Effect Of Radiotherapy On Cervical Cancer Cells

Objective: Cervical cancer is one type of malignant tumor arising from cells originating in the cervix of the uterus. It is a very common female malignant tumor and still remains one of the leading malignancies in the worldwide.The purpose of our study was to explore the impacts of CXCL10 gene therapy mediated by PCL-PEI polymer nanoparticles on killing effect of radiotherapy on cervical cancer cells.Method: p o l y mer P C L-P EI was synthesized successfully by Mi chael addition reaction between polycaprolactone(PCL) diacrylate and polyethylenimine(PEI,1.8k Da). the compositions of synthesized PCL-PEI was confirmed through 1H-NMR spectroscopy.The particle size and zeta potential of nanoparticles were measured by laser particle sizer. The morphology of PCL-PEI and PCL-PEI condensed plasmid CXCL10 were comfirmed by Transmission Electron Microscope(TEM). MTT assay was applied to study the cytotoxicity of polymer PCL-PCL.After comfirmed the polymer was synthesized successfully,The nano-composites of PCL-PEI+p DNA was prepared by PCL-PEI nanoparticles and p EGFP,then,agarose gel electrophoresis was applied to investigate the best mass ratio of polymer and p DNA(plasmid EGFP) for transfection.Inverted Fluorescence Microscopewas utilized to observe the expression of green fluorescent protein after transfection of PCL-PEI+ p EGFP nano-composites(according to the best mass ratio).PCR assay was utilized to amplify CXCL10 gene fragments of Hela cells which was transfected by PCL-PEI+p EGFP nano-composites,and agarose gel electrophoresis assay verified the success of transfection. Finally, four types of treatment were applied to Hela cells as followed: Control group(cell+ radiotherapy),PCL-PEI +radiotherapy group, PCL-PEI+p PUC4.1(vector plasmid p PUC4.1) + radiotherapy group, PCL-PEI+p CXCL10+radiotherapy group. After 24 h, each group was received radiotherapy for 6Gy. Then after 48 h, the apotosis of each group were detected by Flow Cytometry. Colony formation assay was utilized to evaluate the colony formation abilities within 4h after the radiotherapy.Results: A biodegradable polymer PCL-PEI was successfully synthesized which could form nano particles in P B S s o l u t t i o n. 1 H- N M R s p e c t r o s c o p y a n d T r a n s m i s s i o n E l e c t r o n Microscope(TEM) comfired the polymer was synthesized successfully. Laser particle sizer showed the Zeta potential and partical size of PCL-PEI was +15mv and 213 nm respectively. Agarose gel electrophoresis showed that the polymer possesses a good ability to condense DNA effectively, where p EGFP is completely retarded when the mass ratio is 1:1. MTT assay shows the polymer is low cytotoxicity and exhibited almost nontoxicity at working concentration. Fluorescence microscope confirrmed p EGFP delivered by PCL-PEI was transfected and expressed in Hela cells successfully. PCR-agarose gel electrophoresis assay confirmed the target gene of CXCL10 deliveried by PCL-PEI was transfected into Hela successfully. At last, Flow cytometry comfirmed that the apoptosis rate of PCL-PEI+p CXCL10+ radiotherapy group was higher than the other groups. And colony formation as say also proved the colony formation ability of PCL-PEI+p CXCL10+ radiotherapy group is lower than the other groups. Conclusion: 1.Polymer PCL-PEI was synthesized successfully. The cytotoxicity and concentration of the polymer were positively correlated. The polymer is low cytotoxicity and exhibited almost nontoxicity at working concentration which indicates that PCL-PEI is a safety and low toxicity gene vector. 2. The polymer could form nanoparticles in PBS. The surface of the particles took positive charges with a uniform distribution of particle size. 3.The PCL-PEI nanoparticles could condense and delivery plasmid DNA, expressing corresponding proteins. 4. It was initially confirmed that comparing to the other groups, the apoptosis rate was higher and colony forming ability was lower in Hela cells which were transfected by PCL-PEI +p CXCL10, which indicated that CXCL10 gene therapy mediated by PCL-PEI polymer nanoparticles could enhance the killing effect of radiotherapy on cervical cancer cells...