Preliminary Study On The Application Of Apoptin In Detecting CTCs From Human Peripheral Blood

BackgroundCirculating tumor cells(CTCs)are tumor cells that are released from the primary tumor into the peripheral circulation system and play a key role in cancer metastasis.Studies have shown that patients with CTCs are poorly diagnosed compared to patients without CTCs.Compared with patients with elevated CTCs after treatment,the prognosis of patients with reduced number of CTCs after treatment was significantly improved.Therefore,CTCs detectionhas a certain application value in diagnosis of cancer patients,survival prediction,treatment monitoring and so on.However,due to the scarcity of CTCs,CTCs detection is difficult.At present,based on the nature of CTCs(such as Ep CAM),a series of separation and detection techniques have been developed,but there still exists some defects such as lack of sensitivity.It is necessary to develop a technique that can accurately and sensitively detect CTCs from peripheral blood cells.Apoptin is a small molecule protein derived from chicken anemia virus(CAV)and couldspeciallyinduce apoptosis of tumor cells.Studies have found that the location of Apoptinin tumor cells and normal cells is different,and Apoptin is mainly located in the nucleus of tumor cells,whereas in normal cells it is mainly located in the cytoplasm.This characterization of Apoptin provides a theoretical basis for the identification of tumor cells and normal cells,which can detect CTCs from peripheral blood cells.Fluorescein is an organic synthetic dye capable of producing fluorescence.It can release fluorescence under excitation light.It can bind to protein molecule.After labeling,it does not affect the activity of protein.Its fluorescence efficiency is high and the requirement of protein binding is small.It is stable and easy to preserve.Fluorescein can be used in conjunction with protein to achieve protein visibility in scientific experiments.Green fluorescent protein(GFP)is a 27 k Da size protein,this protein will emit green fluorescence under excitation light.GFP is commonly used as a biomarker in biology research.GFP can be combined by molecular biology on the substance to be labeled.The labeled substance can be observed under a fluorescence microscope.ObjectiveThe purpose of this study is to uitilyze the special location of Apoptin in tumor cells and normal cells and the visibility of fluorescence / green fluorescent protein to identify normal cells and tumor cells,after the Apoptin protein waslabeled with fluorescent substances,or fused with green fluorescent protein directly.Methods1.Construction of prokaryotic expression vector p ET-28b(+)-TAT-Apoptin and expression of protein.2.Construction of eukaryotic expression vectors p EGFP-N1-Apoptin,pc DNA3.1(+)-Apoptin.3.Apoptin-1T gene(Thr-108 mutates to Gly)and Apoptin-3T gene(Thr-106,107,108 mutate to Gly,Pro,Gly)were amplified by overlapping extension technique to construct the eukaryotic expression vectors p EGFP-N1-Apoptin-1T and PEGFP-N1-Apoptin-3T.4.The eukaryotic expression vectors were transfected into tumor cells saos-2,H460 by lipo2000.Cells viability were detected by CCK8 after 48 h.And the eukaryotic expression vector with the least apoptosis of tumor cells was screened.5.The eukaryotic expression vectors were transfected into tumor cells and normal cells by electroporation.The cells were stained with Hoechst 33342 at 24 h,48h,72 h.The fluorescence was used to observe the distribution of green fluorescence in the cells.The percentage of cells with geen,blue fluorescence at the same time gathered in the nucleus / cellscontain both green and blue fluorescence could reflect the specificity of Apoptin recognizesnormal cells and the sensitivity of Apoptin recognizes tumor cells.Comparison ofthe sensitivity at different time points,the most sensitive time to identify tumor cells was screened.6.PBMCs were transfected with p EGFP-N1-Apoptin-3T.After 48 h,the cells were stained with Hoechst 33342.The distribution of green and blue fluorescence in the cells was observed by fluorescence microscopy.According to the overlap of green,blue fluorescence,CTCs in PBMCs were determined.Results1.The p ET-28b(+)-TAT-Apoptin plasmid was successfully constructed.The target protein was purified by Ni-NTA affinity chromatography,but the protein was poor in stability,easy to precipitate,not suitable for in vitro labeling.2.PEGFP-N1-Apoptin-1T,p EGFP-N1-Apoptin-3T,p EGFP-N1-Apoptin-1T and p EGFP-N1-Apoptin-3T were successfully constructed.3.Compared with wild-type Apoptin-EGFP,Apoptin-EGFP-3T had the least effect on apoptosis of tumor cells,suggesting that Apoptin-EGFP-3T decreased the apoptosis of CTCs compared with wild-type Apoptin-EGFP.4.Apoptin-EGFP is present in the cytoplasm of normal cells,and most of the tumor cells Saos-2 and H460 accumulate in the nucleus.Apoptin-EGFP,Apoptin-EGFP-1T and Apoptin-EGFP-3T were the most sensitive to the recognition of tumor cells at 48 h,and the sensitivities of three types of Apoptin-EGFP Apoptin-EGFP to tumor cells were not statistically different.5.There was a statistically significant difference in the ratio of CTCs between lung cancer patients and normal subjects recognized by Apoptin-EGFP-3T(P <0.05).The ratio of CTCs in tumor patients was significantly higher than that in normal subjects.Conclusions1.The prokaryotic expression system based on p ET-28b(+)plasmid was able to express TAT-Apoptin,but the stability of protein was not sufficient to support subsequent experiments.2.The specificity of Apoptin-EGFP specificity can reach 100% when identifying normal cells.The sensitivity of Apoptin-EGFP was 49.53% ± 3.55% and 40.82% ± 4.26% in the identification of tumor cells Saos-2 and H460.The optimal detection time was 48 h after transfection3.The CTCs in peripheral blood of lung cancer patients treated with Apoptin-EGFP-3T were significantly higher than that in normal controls(P <0.05),which indicating that this method has certain potential application value in CTCs detection.