| PART I: Effect of human umbilical cord mesenchymal stem cell culture supernatant on angiogenesis of human umbilical vein endothelial cell induced by thyroid papillary carcinoma cellsObjective The purpose of this study is to explore the effect of human umbilical cord mesenchymal stem cell culture supernatant(hUCMSC-CS)on the angiogenesis of human umbilical vein endothelial cell(HUVEC)induced by papillary thyroid carcinoma cell line TPC-1.Methods 1.Characterization of hUCMSCs was identified by adipogenic differentiation assay,osteogenic differentiation assay and flow cytometry assay.2.TPC-1 cells were treated with hUCMSC-CS in different concentrations.3.The effect of hUCMSC-CS-treated TPC-1 cells on the angiogenesis of human umbilical vein endothelial cells was detected by the endothelial tube formation assay.4.The proliferation of TPC-1 cells was detected by CCK-8 assay.5.RT-PCR and Western blotting were used to detect the expression of vascular endothelial growth factor(VEGF)m RNA and protein in hUCMSC-CS-treated TPC-1 cells;and the concentration of VEGF in the culture supernatant of hUCMSC-CS-treated TPC-1 cells was measured by ELISA.Results 1.hUCMSCs have the ability to differentiate into osteocytes and adipocytes,and the positive rate of CD73 and CD90 on the cell surface was more than 95%,while the positive rate of CD14 and CD19 was less than 2%.These results were consistent with the definition of MSC by ISCT,suggesting the successful extraction of hUCMSCs.2.Compared with the control group,hUCMSC-CS significantly inhibited the proliferation of TPC-1 cells(P < 0.05).hUCMSC-CS-treated TPC-1 cells were significantly down-regulated angiogenesis of HUVEC(P < 0.001).At the same time,the expressions of VEGF m RNA and protein in hUCMSC-CS-treated TPC-1 cells were significantly reduced(P < 0.001).The concentration of VEGF in the culture supernatant of hUCMSC-CS-treated TPC-1 cells was also decreased(P < 0.05).Conclusion Our data suggested that hUCMSC-CS down-regulated the expression and secretion of VEGF in TPC-1 cells and suppressed the angiogenesis of HUVEC induced by TPC-1.PART II: Effect of human umbilical cord mesenchymal stem cellderived extracellular vesicles on thyroid papillary carcinoma cellsObjective The purpose of this study is to explore the effect of human umbilical cord mesenchymal stem cell-derived extracellular vesicles(hUCMSC-EVs)on thyroid papillary carcinoma cells.Methods 1.hUCMSC-EVs was isolated and purified by ultracentrifugation from the culture supernatant of hUCMSCs.The morphology and diameter of hUCMSC-EVs were observed under transmission electron microscope and nanoparticle tracking analysis system.The surface markers of hUCMSC-EVs were detected by Western blotting.2.PTC cells were treated with hUCMSC-EVs in gradient concentrations.3.The effect of hUCMSC-EVs on the proliferation ability of PTC cells was detected by CCK-8 assay and Ed U proliferation assay,and the effect of hUCMSC-EVs on the apoptosis of PTC cells was detected by flow cytometry.4.The effect of hUCMS-EVs on migration and invasion of PTC cells was detected by Transwell assay.5.To evaluate the effects of hUCMSC-EVs on PTC cells growth,we established a xenograft model in which W3 cells were injected into nude mice.Then the mice were treated with intratumor injection of hUCMSC-EVs or a control solution and the therapeutic effect was observed.6.The expression of related proteins in hUCMSC-EVs-treated PTC cells and tumor tissues of nude mice was detected by Western blotting and immunohistochemical staining.Results 1.hUCMSC-EVs were successfully isolated and purified.The hUCMSC-EVs were typical saucer-shaped membrane particles with diameters ranging from 55 to 450 nm,averaging 142 nm.The representative marker for EVs,CD63,was expressed in the hUCMSC-EVs,whereas the marker of endoplasmic reticulum,calreticulin,was not.Moreover,hUCMSC-EVs were internalized by PTC cells.2.Compared with the control group,hUCMSC-EVS significantly inhibited the proliferation(P < 0.05)of PTC cells and promoted their apoptosis(P < 0.001).Moreover,the activation of Erk signaling pathway in PTC cells was inhibited,the expression of anti-apoptotic protein Bcl-2 in PTC cells was down-regulated,and the expression of pro-apoptotic protein Bax was up-regulated.3.Compared with the control group,hUCMSC-EVs significantly inhibited the migration and invasion of PTC cells(P < 0.001).Moreover,the expression of Ecadherin in PTC cells was up-regulated,while the expression of Vimentin was downregulated.4.Compared with the control group,hUCMSC-EVs significantly inhibited tumor growth in nude mice(P < 0.001).There were lower Ki-67 positive cells cells in tumor sections,and the expression of E-cadherin protein in tumor tissues was up-regulated,while the expression of Vimentin protein was down-regulated.Conclusion Our data suggested that hUCMSC-EVs significantly inhibited the proliferation,migration and invasion of PTC cells,and promoted their apoptosis.Moreover,hUCMCEVs inhibited tumor growth in nude mice.These results suggested that hUCMC-EVs could inhibit PTC progression. |
PDF Full Text Request