Isolation and identification of urine-derived stem cells from female patients with interstitial cystitis/ bladder pain syndrome / 中华泌尿外科杂志

Objective To establish the method for isolation and culture of urine-derived stem cells (USCs) from female patients with interstitial cystitis/bladder pain syndrome (IC/BPS).Methods The USCs were collected from fresh midstream urine samples from 6 female IC/BPS patients admitted to our hospital from June 2018 to December 2018.The 6 patients were 33-55 years old (average 41.5 years old),and their course of illness was 2-18 years (average 8 years).The USCs were isolated from the urine through times of centrifugation and cultured in specific medium.Growth curve and cell cycle of USCs were observed.The expression of surface markers of USCs was analyzed by flow cytometry and immunofluorescence,the smooth muscle and epithelial differentiation potential of USCs were detected by immunofluorescence staining of surface markers of smooth muscle cells and epithelial cells.Results USCs were successfully extracted from 3 of 6 female patients,and the success rate reached 50％ by once extraction.USCs showed a "rice-grain" spindle appearance and showed logarithmic growth.USCs expressed surface markers associated with mesenchymal stem cells (e.g.CD44,CD73,CD105,CD133) and embryonic stem cells [e.g.stage-specific embryonic antigen 4 (SSEA4)] and pericytes[e.g.CD146,platelet derived growth factor beta receptor (PDGFRB) and NG2],but didn't express hematopoietic stem cell surface markers(e.g.CD31,CD34 and CD45).When induced to smooth muscle cells or epithelial cells,the cells expressed the surface markers of smooth muscle cells [e.g.desmin,myosin,alpha-smooth muscle actin(otSMA) and vimentin] and epithelial cells(e.g.uroplakin 1A,uroplakin 3B,AE1/AE3 and cytokeratin 13).Conclusions The method of isolation and culture of USCs from female IC/BPS patients was successfully established,and it provides a preliminary technical method for exploring the application of USCs in the clinical study of IC/BPS patients with autologous treatment.

Cyclophosphamide-induced HCN1 channel upregulation in interstitial Cajal-like cells leads to bladder hyperactivity in mice

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are confirmed to be expressed in bladder interstitial Cajal-like cells (ICC-LCs), but little is known about their possible role in cystitis-associated bladder dysfunction. The present study aimed to determine the functional role of HCN channels in regulating bladder function under inflammatory conditions. Sixty female wild-type C57BL/6J mice and sixty female HCN1-knockout mice were randomly assigned to experimental and control groups, respectively. Cyclophosphamide (CYP)-induced cystitis models were successfully established in these mice. CYP treatment significantly enhanced HCN channel protein expression and I(h) density and significantly altered bladder HCN1 channel regulatory proteins. Carbachol (CCH) and forskolin (FSK) exerted significant effects on bladder ICC-LC [Ca²⁺]i in CYP-treated wild-type (WT) mice, and HCN1 channel ablation significantly decreased the effects of CCH and FSK on bladder ICC-LC [Ca²⁺]i in both naive and CYP-treated mice. CYP treatment significantly potentiated the spontaneous contractions and CCH (0.001-10 µM)-induced phasic contractions of detrusor strips, and HCN1 channel deletion significantly abated such effects. Finally, we demonstrated that the development of CYP-induced bladder overactivity was reversed in HCN1 -/- mice. Taken together, our results suggest that CYP-induced enhancements of HCN1 channel expression and function in bladder ICC-LCs are essential for cystitis-associated bladder hyperactivity development, indicating that the HCN1 channel may be a novel therapeutic target for managing bladder hyperactivity.

Inhibitory effect of interferonα-1b and cyclooxygenase-2 inhibitor on renal cancer cell and xenografts in nude mouse / 局解手术学杂志

Objective To investigate the inhibitory effect on human ACHN cell line and its mice xenograft by using interferon α-1b combined with cyclooxygenase-2 inhibitor and the relevant mechanism in vitro and vivo experiment .Methods ACHN cell and the xenograft mice were devided into 4 groups(IFN-α1b,NS398,IFNα-1b+NS398 and control group).The inhibitory effects were tested by CCK8(Cell Counting Kit 8)assay after AHCN were treated for 24 h and 48 h.The expression of bcl-xl and COX-2 were detected by Western blot .The vol-ume of the xenografts of ACHN cell line and testing the expression of VEGF in xenografts were measured by immunohistochemistry assay .Re-sults Both IFNα-1b and NS398 exerted inhibitory effects on ACHN and this effects showed a rising trend with a increasing concentration of drugs.The combined group was more significant than monotherapy group (P<0.05).Western blot assay showed that IFNα-1b and NS398 downregulated the expression of bcl-xl and COX-2 in ACHN.The combined group was more significant than monotherapy group (P<0.05). The combined group has the greatest inhibitory effects on the xenografts of ACHN cell line compared with monotherapy group and control group(P<0.05).The expression of VEGF in tumor was obiviously inhibited in combined group compared with monotherapy group and con -trol group (P<0.05).Conclusion IFNα-1b combined with NS398 can inhibit the proliferation of ACHN and suppress the tumor growth .