Effect Of Qige Powder On Cisplatin Sensitivity Of EC9706 Cells Based On The Exosomes Of Human Umbilical Vein Endothelial Cells

ObjectiveTo study the effect of exosomes secreted by human umbilical vein endothelial cells?HUVEC?on the effect of cisplatin resistance of esophageal cancer cells?EC9706?,and the effect of Qige powder on the miR-21 of exosomes secreted by HUVEC,trying to elucidate the mechanism of cisplatin resistance of coculturation HUVEC with EC9706 and part mechanism of Qige powder on the cisplatin resistance of esophageal cancer based on exosomes.Methods1.Extraction and identification of exosomes secreted by HUVEC:Culture the HUVEC cells,the cells in good growth were used to do the experiment.Collect the supernatant of HUVEC cells.To extract exosomes secreted by HUVEC with the exosome extraction kit ExoQuick-TC Exosome Isolation Reagent,to observe the size and shape of exosomes extracted with transmission electron microscopy,to identify the biomarker protein Alix and CD-9 of exosomes using Western Blot.2.The uptake of HUVEC exosomes by EC9706 cells:Extract exosomes of HUVEC and dye the exosomes with PKH26 Staining Kit.Culture the EC9706 with the dyed exosomes for 24h.To observe whether EC9706 cells had ingestion of exosomes secreted by HUVEC with Confocal laser scanning microscopy.3.Cytotoxicity of cisplatin on EC9706 under coculture of HUVEC exosomes and EC9706 cells was detected by MTT:The cells are divided into two groups:control group?EC9706?,coculturegroup?EC9706+HUVECexosomes?.Set5cisplatin concentrations:0?g/mL,1?g/mL,2?g/mL,4?g/mL,8?g/mL.Every concentration set 3complex holes.The OD values of two groups of cells were detected by MTT method,and the IC₅₀0 values of cisplatin in each group were calculated.Compare the toxicity difference between two groups of cisplatin in EC9706 cells.Repeat the experiment three times.4.Detection of miR-21 expression in HUVEC exosomes by qRT-PCR:HUVEC cells were cultured in 5%Qige powder containing serum group and 5%control serum group respectively.Extract the RNA of exosomes secreted by HUVEC in two groups.To compare the difference of miR-21 expression in the exosomes secreted by HUVEC cells with RT-PCR.Repeat the experiment three times.Results1.The size and morphology of exosomes secreted by HUVEC were observed by transmission electron microscopy,and the biomarker protein Alix and CD-9 of exosomes detected by Western Blot were positive.2.The intake of EC9706 cells to the exosomes secreted by HUVEC was observed by the confocal technique.3.when exosomes of HUVEC and EC9706 were cocultured,the IC50 of EC9706to cisplatin was 3.84±0.45 g/mL.Under the condition of EC9706 alone,the IC50 of EC9706 to cisplatin was 2.69±0.23g/mL,there was a statistically significant difference between them?P<0.05?.4.The miR-21 expression of exosomes of HUVEC in the the 5%control serum group was 1.00±0.00,the miR-21 expression of exosomes of HUVEC 5%Qige powder containing serum group was 7.58±2.51,there was a statistically significant difference between them?P<0.05?.Conclusion1.EC9706 cells have an uptake of exosomes of HUVEC.2.The exosomes of HUVEC increase the resistance of EC9706 to cisplatin3.The 5%concentration of Qige powder serum did not reverse the resistance of EC9706 to cisplatin under coculture conditions and increased the miR-21 of EC9706under coculture conditions,which may be related to the highly expression of miR-21in the HUVEC exosomes at this concentration,and the uptake of EC9706 cells.