| Object:Urine-derived stem cell(USC),also known as Urine-derived Mesenchymal Stem Cells(UDMSC),is a kind of mesenchymal stem cell that can be isolated from urine and have great application prospects.As one of the most common tumors of urinary system,bladder cancer will inevitably interact with USCs in urine during its occurrence and development.Although many kinds of mesenchymal stem cells have been identified to have different effects on the genesis and progression of tumors,the specific mechanism of action between USCs and bladder cancer has not yet been clarified.This paper aims to cultivation USCs from people urine source samples,and co-culture USCs with bladder cancer cells to detect the interreactions between two kinds of cells in the aspects of growth,invasion,migration influence each other.Clear the role of USCs in bladder urothelial carcinoma’s process of disease development,and explore the possible regulatory mechanism.This study may provide an important theoretical basis for the application of USCs in the related fields of urinary tumors.Methods:1.Urine-derived mesenchymal stem cells(USCs)were isolated and cultured from normal human urine tissues.Cell surface antigen molecules were identified by flow cytometry,and the viability of USCs in urine was determined by fluorescence staining.2.By collecting 5637 and T24 bladder tumor cells in the conditioned medium for USCS culture,the proliferation ability of USCS was measured by MTT assay.Transwell co-culture was used to detect the migration ability of USCs and bladder cancer cell lines5637 and T24,respectively,and q PCR method was used to detect the classical homing inducing factor SDF1-α of T24 and 5637 mesychmal stem cells.To evaluate the effect of bladder tumor cells on USCS.3.USCS and bladder cancer cell lines 5637 and T24 were co-cultured in Transwell chamber or USCS conditioned medium was collected for 5637 and T24 culture,cell proliferation and cell invasion were measured.The protein expression levels of ERK,Akt pathway and E-cadherin and other EMT-related factors were detected by q PCR and Western blot to evaluate the effect of USCS on malignant phenotypes of bladder tumors.Results:1.USCs were successfully isolated from urine,and a culture system suitable for their growth was established to obtain cell lines that could be stably expressed and passed down.The USCS cell lines were identified by flow cytometry,and CD29,CD73,and CD90 were positive,while CD31,CD34,and HLA-DR were negative,indicating that the cells extracted from urine in this study were indeed USCs.Cell active fluorescence staining showed that USCs could survive for more than 6 hours in artificial urine environment and maintain a high survival rate.2.After cultured in the conditional media of 5637 and T24,the cell activity analysis showed that both types of bladder cancer cell lines could promote the proliferation of USCs.Transwell chamber was used to co-culture the bladder cancer cell lines 5637 and T24 with USCs,respectively.It was found that the migration ability of USCs was enhanced after co-culture with bladder cancer cell lines.The q PCR results showed that the m RNA expression of stromal derived factor 1-α(SDF1-α)was significantly enhanced in T24 and 5637 cells after co-culture,which induced the homing of USCs to tumor tissue.3.Cell activity analysis after cultured with USCS conditional medium in 5637 and T24 cell lines showed that the proliferation activity of both types of bladder cancer cell lines was up-regulated.USCs were co-cultured with bladder cancer cell lines 5637 and T24 in Transwell chamber,and it was found that USCs significantly enhanced the invasion ability of T24 and 5637 cells.Western blot and q PCR were used to detect invasion related indexes of T24 and 5637 cells.It was found that USCs could promote epithelial mesenchymal transformation(EMT)in T24 cells,and down-regulate the expression of E-cadherin in T24 and 5637 cell lines at the same time.This may be an important reason for the enhancement of T24 cell invasion ability.Western blot method was used to detect cell proliferation-related pathways,and it was found that the phosphorylation level of ERK protein in T24 and 5637 cells was up-regulated after co-culture with USCS.We speculated that the up-regulation of ERK pathway may be one of the reasons for the enhanced proliferation activity of bladder cancer cells.Conclusion:In this study,USCs subcultured were successfully isolated from human urine tissue andcould be stably expressed,and their source was identified and confirmed to be correct.In addition,USCs could survive stably for more than 6 hours in simulated urine environment.At the same time,in vitro co-culture showed that USCS could promote the growth of tumor cells by upregulating the level of ERK phosphorylation in T24 and 5637 cell lines,and could enhance the invasion ability of bladder cancer cell lines by inducing EMT transformation.Meanwhile,two bladder cancer cell lines,T24 and 5637,can also induce homing of USCs by secreting SDF1-α to enhance the migration ability of USCs.In this study,the basic characteristics of USCS and its mechanism of action in the growth and invasion of urinary urothelial cancer cells are explored,which can provide a new research platform and clinical strategy for cancer treatment and recurrence prevention,and also has important significance for the application of USCS in the field of urinary system. |
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