| Objective: Exploring the role of Corilagin in affecting the malignant progression of glioma stem cells through inhibition of Wnt7B/β-catenin.Methods: 1.The human glioma U251 cell line was cultured using conventional methods,isolated,cultured into stem cells and the cultured glioma stem cells were identified.The experimental group model was constructed by treating cultured U251 glioma stem cells with a transcriptional lentivirus that knocked down Wnt7 B,and the blank group model was constructed by treating cultured U251 glioma stem cells with a blank transcriptional lentivirus.A control group model was constructed using untreated U251 glioma stem cells.Lentiviral transfection efficiency was assessed using q PCR and immunofluorescence microscopy.Corilagin at a concentration of 100 μg/ml was chosen to intervene in the three models based on the results of previous experiments.The expression of Wnt7B/β-catenin in the 3 groups of U251 glioma stem cells after the intervention was obtained using protein blotting technique.Cell proliferation in the 3groups was detected using CCK-8.Apoptosis and cell cycle changes were detected in the 3 groups of models using flow cytometry.2.The experimental model with knockdown of U251 glioma stem cells and the control model without knockdown of U251 glioma stem cells were obtained according to the above method,3 rats per intervention concentration,12 rats per group,24 male nude mice at 4 weeks of age were used to construct the nude mouse glioma stem cell subcutaneous implantation tumor model.Corilagin was administered at different concentrations(0μg/ml,100μg/ml,200μg/ml,400μg/ml)to the rats by gavage on days 6,9,15 and 18.Tumour growth was measured every 3 days and tumour growth curves were plotted.At 21 days after implantation,the rats were killed and the subcutaneous tumours were collected.The tumours were stained with HE and immunohistochemically stained for GFAP,P53,ATRX,IDH-1,Ki-67,Wnt7 B and β-catenin expression.Results: 1.Immunofluorescence expression was evident in both the experimental and blank groups.The expression of Wnt7 B and β-catenin in the experimental group was significantly lower than that in the blank group,demonstrating the completion of lentiviral transfection.2.After intervention with Corilagin in the experimental and control groups.The survival rate was significantly lower in the Corilagin intervention than in the non-intervention group(P<0.05).3.After intervention with Corilagin all 3 groups of stem cells showed arrest in the G1 phase,the blank group was comparable to the control group and the number of arrests was lower in the experimental group compared to the other 2 groups(P<0.05).4.After intervention with Corilagin all 3 groups of stem cells showed arrest in the G1 phase,the blank group was comparable to the control group and the number of arrests was lower in the experimental group compared to the other 2 groups(P<0.05).After intervention with Corilagin,all 3groups of stem cells showed a decrease in proliferative capacity,with the blank group showing a comparable decrease to the control group and the experimental group showing a lower decrease than the other 2 groups(P<0.05).5.A hard mass could be palpated in the axilla 3-4 days after the axillary implantation of the stem cell spheres in nude rats.Thereafter,the masses increased in size,the rats ate less than before,their mental state was worse than before,no significant changes in body weight,no significant changes in gait,no significant changes in group living or fighting behaviour were observed.6.Before gavage,the tumors in the axilla of the experimental mice were significantly smaller than those in the control group.As the concentration of Corilagin increased,the tumor size of both groups decreased gradually,and the tumor size of the experimental group was significantly smaller than that of the control group(P<0.05).7.HE staining of subcutaneous implantation tumour tissue from nude mice showed variable cell morphology with marked anisotropy and large,darkly stained nuclei.This is consistent with the microscopic appearance of glioma.Immunohistochemistry showed positive results for GFAP,P53,ATRX,IDH-1 and Ki-67,which were consistent with the immunohistochemical manifestation of glioma.The expression of Wnt7 B and β-catenin in the control group and the experimental group decreased gradually with the increase of the intervention concentration,and when0μg/ml Corilagin was used,the expression of both in the experimental group was significantly lower than that in the control group.Conclusion: Corilagin significantly inhibits the proliferation and cell cycle progression of glioma stem cells by inhibiting the Wnt7B/β-catenin signaling pathway and promotes apoptosis of glioma stem cells. |
PDF Full Text Request