The Effect Of Co-culture Conditions Of Fibroblasts And Esophageal Cancer EC9706 On The Sensitivity Of Cisplatin

Objective1.To observe the co-culture model of fibroblasts and human esophageal cancer EC9706 cells was preliminarily established by in vitro experiments,in order to elucid ate the molecular mechanism of fibroblast mediated drug resistance in the tumor micr o-environment of esophageal cancer.2.To detect culture condition downward gasification decoction and represe ntati on of formula Qige Powder combined with cisplatin on regulation of es ophageal canc er EC9706 by proliferation inhibition,Apoptosis cycle and rel ated genes PDCD4 PTE N and Protein Expression based on miR-21 signaling pathway increase the sensitivity of human esophageal cancer EC9706 cells to cisplatin and re verse the resistance of tu mors.3.To provide theoretical basis for the clinical application of gas-phase phleg m-assisted chemotherapy for esophageal cancer.Methods1 Using 24-well Transwell cell culture plates,the upper chamber was inoculated with different proportions of fibroblasts,and the lower chamber was inoculated with esophageal cancer EC9706 cells.The optimal cell ratio of fibroblasts and human esophageal cancer EC9706 co-culture was detected by MTT assay;2 The effect of different cisplatin concentrations on the proliferation inhibition of esophageal cancer EC9706 cells under different culture conditions of single culture of esophageal cancer EC9706 cells under co-cu ltureconditions of fibrocytes and human esophageal cancer EC9706 cells was detected by MTT assay;3 MTT assay was used to detect the effect of different Qige Powder drug-co ntaining serum and Qige Powder ethyl acetate extract combined with cisplatin on proliferation inhibition of fibroblasts and human esophageal cancer EC9706 cellsco-cultured.4 Flow cytometry?FCM?PI staining was used to detect the effect of Qige Po wder ethyl acetate extract combined with cisplatin fibroblasts and human es ophageal cancer EC9706 cells on the cell cycle.5 Flow cytometry?FCM?Annexin V/PI double staining method was used to detect Qige Powder ethyl acetate extract combined with cisplatin fibroblasts and human esophageal cancer EC9706 cells under the conditions of co-cultured cells The effect of apoptosis;6 Quantitative reverse transcription poly merase chain reaction?RT-PCR?was used to detect miR-21 in EC9706 cells under the condition of co-culture of human esophageal cancer EC9706 cells with Qige Powder ethyl acetate extract combined with cisplatin fibroblasts.And its downstream target molecule PDCD4 mRNA,PTEN mRNA expression;7 Western blot method was used to detect the expression of PDCD4 and PTEN protein in EC9706 cells under the conditions of co-culture of human esophageal carcinoma EC9706 cells with ethyl acetate extract and cisplatin fibroblasts.Results1 The results of MTT colorimetric assay showed that the rate of proliferation of esophageal cancer EC9706 cells varied with the proportion of cells inoculated with two cells when transwells were vaccinated with 24-we ll Transwell,among which the ratio of cells?HEF/EC9706?was 1:1.5 and 1:2 The difference between the two groups was statistically significant?P<0.05?.From the results,the proportion of cells?HEF/EC9706?in the 1:2 group was significantly higher than that of esophageal cancer EC9706 cells?P<0.05?.2 The results of MTT assay showed that the IC₅₀0 of cisplatin in the cultured group was 2.63±0.38?g·mL^-1,and the IC₅₀ of cisplatin in the co-culture con ditions between fibroblast HEF and EC9706 was 3.41±0.33?g·mL^-1?P<0.05?.Under the same concentration of cisplatin,the OD value of esophageal cancer EC9706 group was significantly higher than that of EC9706 group cultured alone?P<0.05?.3 The results of MTT colorimetric assay showed that compared with the same concentration of blank control serum group,the effect of Qige Powder dru g-containing serum was not obvious;and the same concentration of blank control serum combined with cisplatin Compared with the group,the effect of Qige Powder drug-containing serum combined with cisplatin was not obvious;the results showed that the extracts of ethyl acetate at different concentrations of Qige Powder were co-cultured under the same conditions.Esophageal cancer EC9706 cells have obvious inhibitory effect,there is a clear do se-effect relationship curve,concentration-dep endent manner.4 The results of MTT colorimetry showed that:compared with cisplatin alone IC₃₀48h group,combined use of drugs for 48h group showed that the effect of combined effects of Qige Powder ethyl acetate and cisplatin was not obvious.There was a significant trend of synergistic effects of combined use of drugs in the sequential admi nistration group?P<0.05?.5 From the apoptotic point of view,compared with the EC9706 alone group,the co-cultured condition group down-regulated the total apoptotic rate?P<0.05?,the dow n-regulation of the late apoptosis rate was obvious;and the c o-culture model Compa red with the control group,the early apoptosis rate of EC9706 was significantly increa sed in the cisplatin group,while the late apoptosis rate of EC9706 was increased in the Qige Powder ethyl acetate group,the combination group and the sequential medication group,and there were differences compared with the co-culture model?P<0.05?,in which the sequential administration of the drug group increased the late apoptosis rate of EC9706,but the early apoptosis rate was not significantly different from the model group.6 The results of cell cycle experiments showed that compared with the EC9 706group alone,the co-cultured model group increased the proportion of S phase?P<0.05?;Compared with the co-cultured model group,Cisplatin group,Qige Powder.The ethyl acetate group and the sequential administration group significantly decreased the proportion of EC9706 cells in the G₀/G₁ phase,and increased the proportion of EC9706 cells in the S phase?P<0.05?.Among them,the sequential drug group was the most effective.the combination group increased the proportion of G₀/G₁ phase obviously?P<0.05?.7 From the results of RT-PCR experiments,compared with the EC9706 alone group,the expression level of miR-21 in the co-cultured model group was signific antly increased,while the expression levels of PDCD4 mRNA and PTEN mRNA were significantly decreased?P<0.05?;Compared with the co-cultured model group,Qige Powder ethyl acetate group and sequential drug group can significantly down-regulate the expression of miR-21,which can inhibit miR-21 to some extent.The expression of PDCD4 mRNA and PTEN mRNA in cisplatin group,Qige Powder ethyl acetate group and sequential medication group were up-regulated?P<0.05?.8 From the experimental results of WB,compared with the EC9706 alone group,the protein expression levels of PDCD4 and PTEN in the co-cultured model group were significantly down-regulated?P<0.05?;the co-cultured model group In compariso n,cisplatin group,Qige Powder ethyl acetate group and sequential drug group could significantly up-regulate the protein expression of PDCD4 and PTEN,and the effect of sequential drug administration was the most obvious?P<0.05?.Conclusions1.The fibroblast HEF can proliferate esophageal cancer EC9706,and it has a resistance to cisplatin under the co-culture conditions of fibroblast HEF and esophag eal cancer EC9706,which reduces the sensitivity to cisplatin.2.In the co-culture conditions of fibroblast HEF and esophageal cancer EC9706,Qige Powder has a certain synergistic effect,which can reverse the resistance of esophageal cancer EC9706 to cisplatin to a certain extent,and increase the sensitivity to cisplatin.3.The combination of fibroblast HEF and esophageal cancer EC9706 co-cul tured Qige Powder can increase the late apoptosis rate of EC9706 in esophageal cancer and up-regulate the S phase and G₀/G₁ phase of EC9706 cells.4.The combination of fibroblast HEF and esophageal cancer EC9706 co-cult ured Qige Powder combined with cisplatin can down-regulate the expression of miR-21 in esophageal cancer EC9706 cells and up-regulate the expression of target gene PDCD4mRNA and PTEN mRNA and protein.Qige Powder can increase the sensitivity of EC9706 cells to cisplatin under co-cultur e conditions and reverse partial resistance to cisplatin,and this mechanism may be achieved through miR-21 and its regulated target gene PDCD4 mRNA and PTEN mRNA.