Investigate The Relationship Between Interleukin 6 And Thyroid Tumors Based On Cancer Stem Cells Research

ObjectiveTo observe the changes of interleukin 6 levels in patients with thyroid tumors with different TCM clinical types.Method1.Records the name of patients,and number of hospital or outpatient,,gender,age,course of the disease,and general information.2.Detection of FT3、TF4、TSH、TPOAb、TgAb.3.IL-6 levels was detected by chemiluminescence method.4.Thyroid nodule was evaluated by ultrasonography,and patients with malignant signs were treated with a fineneedle aspiration and cytological examination.Results1.Compared with ROUYING disease,IL-6 levels of SHIYING disease significantly higher,there was statistical difference.2.Compared with GYTN Group,IL-6 level increased in patients with QZTN Group and TYHJ group,there was statistical difference.3.Compared with GYTN Group,Ratio of thyroid cancer increased in patients with QZTN Group and TYHJ group,there was statistical difference.4.Compared with benign nodules,the level of IL-6 patients with thyroid cancer is significantly increased,there was statistical difference.5.Compared With the benign nodules<1.0 cm,IL-6 levels increased significantly in patients with benign nodules≥1.0 cm,and IL-6 levels were significantly increased in patients with thyroid cancer,there was statistical difference.Conclusion1.Elevated IL-6 levels has a certain guiding significance in the clinical diagnosis and clinical syndromes of TCM in thyroid tumor.2.Elevated IL-6 is a closely associated with benign and malignant thyroid nodule and size,and elevated IL-6 is one of the risk factors of thyroid cancer.ObjectivesTo observe the effect of IL-6 on the proliferation and differentiation of human thyroid stem cells,and to determine the role of IL-6 in the pathogenesis of thyroid tumors.Methods1.Isolation and primary culture of thyroid cells and thyroid stem cells.2.The proliferation of thyroid stem cells was detected by BrdU ELISA.3.Effect of IL-6 on the formation of thyroid sphere cells was detected by the sphere formation experiment.4.The expression of thyroid stem cell related genes OCT4,NIS,Tpo,Tg,and differentiation-related genes NIS,TG,TPO,TSHR,PAX8 was detected by real-time quantitative(PCR).5.The effect of IL6 on the expression of OCT4 in thyroid stem cells was detected by immunofluorescence staining.Results1.IL-6 promotes formation of thyroid sphere cell.Human thyroid cells were isolated and cultured as suspension for 24 hours.Different doses of IL-6 were added to the cells to stimulate the formation of the sphere cell.After 7 days stimulation,the formation rate of sphere cells was increased in a dose-dependent manner.2.IL-6 promotes the proliferation of thyroid stem cells.The BrdU ELISA result suggested that after 48 hours of IL-6 stimulation,the proliferation of thyroid stem cells accelerated in a dose-dependent manner.3.IL-6 monoclonal antibody(Anti-IL-6)inhibits the proliferation of thyroid stem cells.After 48 hours of blocking with Anti-IL-6,the proliferation of thyroid stem cells was significantly inhibited in dose-dependent manner of Anti-IL-6.4.IL-6 promotes the expression of genes related to thyroid stem cells.Real-time quantitative PCR showed that 20 ng/ml IL-6 significantly promoted the expression of OCT4 mRNA and ABCG2 mRNA in thyroid stem cells,but had no significant effect on GATA4.5.Fluorescent immunoassay of OCT4 in thyroid stem cells.The fluorescence of OCT4 enrichment in IL-6 cultured thyroid stem cells was significantly higher than that in the blank control group,but the fluorescence was significantly reduced after blocking with IL-6 monoclonal antibody.6.The effect of IL-6 on the morphology of thyroid stem cell differentiation cells.Compared to TSH cell culture,thyroid cells by IL-6 treated were small and compact from primary thyroid cells.7.IL-6 inhibits the expression of thyroid stem cells differentiation marker genes.20 ng/ml IL-6 significantly inhibited the expression of NIS mRNA,TG mRNA and TPO mRNA,but had no significant effect on TSHR mRNA and PAX8 mRNA.Conclusions1.IL-6 can promote the thyroid sphere cells formation and the proliferation of thyroid stem cells.2.IL-6 can promote the expression of genes related to thyroid stem cells,inhibit the differentiation of thyroid stem cells into thyroid cells,which may lead to cell variability and thyroid tumor.ObjectivesTo observe the effect of IL-6 on the differentiation of thyroid cancer stem cells and epithelial-mesenchymal transition,in order to explore the mechanism of IL-6 on the proliferation and invasion of thyroid tumors.Methods1.The thyroid undifferentiated cancer cell line HTh74 and drug resistant strain HTh74R stem cells were cultured by thyroid cancer stem cell culture technology.2.The formation rate of IL-6 on thyroid tumors were detected using tumor cell formation technique.3.The proliferation of HTh74 and HTh74R cells in thyroid undifferentiated carcinoma were detected by MTT assay.4.The effect of IL-6 on the formation of clonal undifferentiated cancer cells was observed by colony formation assay.5.The expressions of stem cell-associated genes OCT4,ABCG2 and CD133 and the expression of epithelial-mesenchymal transition-related genes E-cadherin,Vimentin and Snail were detected by Real-time quantitative PCR.6.The expression of IL-6/JAK1/STAT3/OCT4 protein in the cellular signaling pathway by Western blot.7.The effect of STAT3-siRNA blocking IL-6 on the proliferation of thyroid cancer stem cells by siRNA interference technology.Results1.IL-6 can promote the formation of HTh74 and HTh74R sphere cells in thyroid undifferentiated carcinoma.After adding s-IL-6R100ng/ml and different concentrations of IL-6(0-20ng/ml)for 7 days in culture medium,the formation rate,volume and number of sphere cells were all increased with the increase of IL-6 concentration.2.IL-6 can promote the proliferation of HTh74 and HTh74 cancer stem cells.With the increase of IL-6 concentration,the proliferation activity of HTh74 and HTh74R cancer stem cells also increased significantly in a dose-and time-dependent manner.3.IL-6 monoclonal antibody can significantly inhibit the proliferation activity of HTh74 and HTh74R cancer stem cells.As the concentration of IL-6 monoclonal antibody gradually increased,the proliferation activity of HTh74 and HTh74R cancer stem cells decreased significantly.4.IL-6 can promote the clonal formation of HTh74 and HTh74R cells.Through tumor cell cloning experiments,it was found that the cloning rate of HTh74 and HTh74R cells gradually increased with the increase of IL-6 concentration.5.IL-6 can promote the expression of stem cells related gene levels of HTh74 and HTh74R cancer stem cells.IL-6 can significantly promote the expression of OCT4 and ABCG2 genes in HTh74 and HTh74R cancer stem cells,and also promote the expression of CD 133 gene in HTh74R cells.6.IL-6 can promote the expression of E-cadherin,an epithelial-mesenchymal transition-related gene,meanwhile the expression levels of Vimentin and Snail were increased.7.IL-6 significantly promoted the level of phosphorylated JAK1/STAT3,and the expression level of OCT4 protein also increased significantly.The IL-6 monoclonal antibody can block JAK1/STAT3 phosphorylation and OCT4 protein levels.8.STAT3-siRNA can reduce the expression of STAT3 protein,and siRNA transfection can reduce STAT3 phosphorylation.STAT3-siRNA transfection significantly inhibited the proliferation of thyroid cancer stem cells.Conclusions1.IL-6 can promote and maintain the formation of HTh74 and HTh74R sphere cells,and the formation of cell clones,It is suggested that IL-6 can promote the proliferation of thyroid cancer stem cells.2.IL-6 can increase the expression of stem cells related genes of cancer stem cell and increase the characteristics of cancer stem cells.It is suggested that IL-6 can increase the self-replication and unlimited proliferation of cancer stem cells.3.IL-6 can promote the epithelial-mesenchymal transition process of thyroid cancer stem cells.It is suggested that IL-6 can increase the invasion and metastasis of thyroid cancer stem cells.4.IL-6 regulated the proliferation of thyroid cancer stem cells through the JAK1/STAT3/OCT4 cell signaling pathway.It promotes the occurrence and development of thyroid cancer.