Objective To investigate the effect of Fas and FasL abnormal expression on immune escape mechanism of renal cell carcinoma(RCC). Methods The expression of Fas and FasL was detected in 44 cases of RCC and 10 cases of normal kidney tissue by immunohistochemical method. Tumor infiltrating T lymphocyte's apoptosis in 44 cases of RCC was examined by TUNEL assay. FCM was used to detect the expression of Fas and FasL as well as apoptosis in RCC cell line 786-0. In addition, the apoptosis of Jurkat T cell with Fas exprssion was also analysed with FCM. Results Fas expression rate (22.8%) in RCC was significantly lower than that (53.8%) in normal kidney tissue(P

BACKGROUND:In vitro culture of sufficient vaginal epithelial cels and smooth muscle cels is the key for vaginal tissue engineering. However, the culture, purification and passage of vaginal epithelial celsin vitro are difficult. Primary culture and passage of vaginal epithelial cels from large animals such as canines has not been reported. OBJECTIVE:To establish a stable method of culturing canine vaginal epithelial cels and smooth muscle cels. METHODS: Vaginal epithelial cels were isolated from the vaginal specimens by enzymatic digestion with Dispase and trypsin separately, and cultured in keratinocyte serum-free medium. Vaginal smooth muscle tissue were minced and digested with colagenase type II; the colected smooth muscle cels were cultured in DMEM culture medium containing 10% fetal bovine serum. The cultured cels were passaged regularly. Cel morphology and proliferation characteristics were observed and cel phenotypes were confirmed by morphology and immunohistochemistry staining. RESULTS AND CONCLUSION: Primary vaginal epithelial cels began to adhere after 24-36 hours, grew logarithmicaly after 4-5 days, and reached 70% confluence after 7-8 days; the epithelial cels showed a typical cobblestone, with no fibroblasts. Cultured epithelial cels passaged every 4-5 days and subcultured to 6-7 generations continuously. Immunohistochemical staining confirmed a positive staining for anti-pancytokeratin (AEl/AE3). Primary cultured smooth muscle cels adhered and grew after 24 hours. The smooth muscle cels were spindle-shaped and proliferated logarithmicaly. After 4 days, primary cultured smooth muscle cels were confluent and showed a typical shape of “peaks and valeys”, and then the cels could be passaged every 3-4 days and passaged 7-8 generations. Immunohistochemistry staining showed α-actin staining was positive. These findings indicate that canine vaginal epithelial cels and smooth muscle cels could have a long-term stable culture and proliferation, to provide adequate seed cels for vaginal tissue engineering.

Objective To evaluate the efficacy and safety of Sun's tip-flexible ureterorenoscopy (tf-URS) combined with holmium laser lithotripsy in treatment of renal and upper ureteral calculi.MethodsClinical data of 48 patients (29 males and 19 females) with renal and upper ureteral calculi treated by tf-URS with holmium laser lithotripsy in our hospital from November 2015 to May 2016 were retrospectively analyzed.Among 48 patients there were 29 males and 19 females with a mean age of (37.8±12.5) years (21-67), including 31 cases of upper ureteral calculi and 17 cases of renal calculi with a mean stone size of (1.4±0.6) cm (0.8-2.1).The tf-URS was advanced through the ureter under visual direction with rigid mode.When the endoscope reached the ueteropelvic junction, the inner flexible part was extended out to explore the pelvicaliceal stones and then the holmium laser lithotripsy was performed.The therapeutic results were evaluated by KUB 2 days after operation and color ultrasonography or CT 4 weeks after operation.Results The success rate of insertion of tf-URS was 94% (45/48).The calculi were detected in 45 cases, and laser lithotripsy succeeded in 43 cases (96%).The rate of total stone clearance was 87% (39/45).The procedure was transformed to laparoscopic or percutaneous nephrolithotomy (PCNL) in 3 patients with ureterostenosis or ureteral distortion.No serious complications occurred.Conclusion The tf-URS with holmium laser in the treatment of renal and upper ureteral calculi is safe,reliable and effective.

Objective To evaluate the feasibility of bladder extracellular matrix (BACM) seeding with autologous cultured vaginal smooth muscle cells (VSMC) repaired rabbit vaginal defect.Methods This study included 24 female rabbits.BACM and vaginal acellular matrix (VACM) were obtained from 8 rabbits by decellularization process.The other 16 rabbits were randomly divided into experimental and control groups.Vaginal tissue biopsies ( ～ 1 cm2) were harvested from female New Zealand rabbits.VSMC were cultured and stained with a-smooth muscle actin antibodies. Cultured VSMC cells were seeded on BACM ( experimental group) or VACM ( control group) at a cell density of 1 × 107 cells/cm2.The cell-seeded matrixes were cultured for 5days.In experimental group of 8 rabbits,a 2 cm segment of vagina was resected and replaced with BACM seeding with VSMC.Then the regenerative segment was studied with histological technique by hematoxylin-eosin staining after 3,6 and 12 weeks postoperative aud vaginography was performed at 12 weeks postoperative.The 8 rabbits in control group underwent the exact same procedure as above but the vaginal defect was repaired with VACM seeding with VSMC,Results The prepared BACM and VACM were transparent,HE staining and scanning electron microscopy showed the two acellular matrix was both consisted of abundant network of fibers with the regular arrangement,without cellular debris.Primary culture of VSMC was successfully established and passaged,and was uniformly spindle-shaped in the confluent state and showed a characteristic hill and valley'formation.Immunohistochemical staining showed VSMC in culture stained positively with a-smooth muscle actin antibodies.VSMC began to adhere to the BACM 5 hour after implanting and the number of the adhered cells increased with time.Cells gradually expanded and showed the typical morphology of smooth muscle cells.Three weeks after implantation in vivo,the luminal surface of matrix was completely covered by vaginal epithelial tissue,a layer of smooth muscle cells was formed in the outer surface of the matrix.Multilayered vaginal epithelial and improved development of organized muscle bundles was observed after 6 weeks.The regenerative tissue was equivalent to the normal vaginal tissue at 12 weeks postoperatively.Vaginography demonstrated the maintenance of full patency and a wide vaginal caliber without fibrosis and graft rejection.There was no significant difference in all evaluated items between experimental and control groups.Conclusions BACM has the same regenerative process as VACM in the replacement of vaginal defect.Moreover,BACM has wider source and appears to be an suitable material for vaginal replacement.

OBJECTIVETo investigate the mechanism of immune escape in renal cell carcinoma(RCC).

METHODSFas and FasL expressions were examined by immunohistochemical technique in 44 RCC patients, with the Ki67 expression and apoptosis of tumor infiltrating lymphocytes(TIL) monitored simultaneously. Cytokines including IL2 and IFN alpha were used to induce the expression of the renal carcinoma cell lines 786-0 cells. Combination treatment of 786-0 with cytokines and Anti-Fas monoclonal antibody (FasAb) was used to induce apoptosis. FasL function was assessed by in vitro co-culture assays using renal cancer cells 786-0 and Fas-sensitive Jurkat T-cells.

RESULTS(1) Fas expression rate in RCC(22.8%) was lower than that in the controlled normal kidney tissues(53.8%, P < 0.01). FasL expression rate in RCC (46.5%) was higher than that in the controlled normal kidney tissues (23.2%, P < 0.01). That of Ki67 was 32.8%, with the expressions of Fas and Ki67 showing a negative correlation (r = -0.62, P < 0.05). In contrast, the expressions of FasL and Ki67 showed a positive correlation. (r = 0.93, P < 0.01). The Fas expression of stage I was significantly higher than that of stages III and IV. The expression rate of FasL in RCC was significantly increased with RCC stage (P < 0.01). (2) The apoptotic rate of TIL in RCC (33.9%) was significantly higher than that of the normal kidney tissues (3.5%, P < 0.01). The expression of FasL and the apoptotic rate of TIL in RCC gave a positive correlation (r = 0.96, P < 0.01). (3) Fas expression rate in 786-0 cells was 13.7%. The apoptotic rate mediated by FAsAb was 9.6%. IFN alpha was able to up-regulate the Fas expression and subsequently augment the FasAb-mediated apoptosis in 786-0 cells. But IL2 did not show similar effects. (4) The FasL expression rate of 786-0 was 18.6%. FasL expressed by 786-0 cells was able to induce apoptosis of Jurkat T-cells in co-culture assays and the apoptosis of Jurkat T-cells was significantly lowered after blocking the effect of FasL with Fas-neutralizing antibody NOK-2, giving the apoptotic rates of 14.9% and 2.0%, respectively, the difference therein is statistically significant (P < 0.01).

CONCLUSIONDown-regulation of Fas expression and up-regulation of FasL-expression are the mechanisms through which the RCC cells escape from immune attack.

Adult ; Aged ; Carcinoma, Renal Cell ; immunology ; Fas Ligand Protein ; Female ; Humans ; Ki-67 Antigen ; immunology ; Kidney Neoplasms ; immunology ; Male ; Membrane Glycoproteins ; immunology ; Middle Aged ; fas Receptor ; immunology

Objective:To investigate the effect of tumor-associated macrophage(TAM) on proliferation of renal carcinoma cells and its related mechanism.Methods:The model of TAM was established by stimulating human monocytic leukemia cell line THP-1 with phorbol myristate acetate (PMA), bacterial endotoxin (LPS) and interferon-γ (IFN- γ). Then the TAM model was co-cultured with carcinoma cell lines ACHN and 786-O in vitro .The cytokines IL-6, TNF-α and IL-1β in TAM supernatant were detected by enzyme-linked immunosorbent assay (ELISA). MTT method was used to detect the proliferation of ACHN and 786-O cells treated with supernatant of TAM or TAM/Tocilizumab. Western blot was used to detect lactate dehydrogenase A (LDHA) expression of both renal cancer cells co-cultured with TAM or TAM/Tocilizumab. The ACHN and 786-O cells with LDHA-overexpression and LDHA-knockdown were cultured in TAM supernatant in vitro. The cell proliferation was detected by MTT and the relative proliferation rate was calculated.Results:THP-1 cells was differentiated into TAM through the treatment of 80 ng/ml PMA combined with 20 ng/ml LPS and 20 ng/ml IFN- γ.The expression rate of CD68, a cell surface marker on TAM, was (36.2 ±4.5)%. When TAM was co-cultured with ACHN cells, the results of ELISA showed that the secretion of IL-6 in the supernatant was significantly elevated compared with that in the supernatant when ACHN cells cultured alone [(138.0 ±12.4) pg/ml and (19.7±4.9) pg/ml], and the secretion of TNF- α [(122.5 ±14.2) pg/ml and (12.6 ±2.3) pg/ml] and IL-1 β [(89.2 ±6.4) pg/ml and (69.2 ±3.5) pg/ml] were also significantly increased. The secretion of IL-6 [(119.2 ±14.8) pg/ml and (17.1 ±3.3) pg/ml], TNF- α [(122.6 ±14.4) pg/ml and (45.7 ±7.2) pg/ml] and IL-1 β [(95.1 ±11.8) pg/ml and (88.2 ±12.7) pg/ml] in the supernatant were also significantly elevated when 786-O cells co-cultured with TAM compared with 786-O cells cultured alone. After treated with the supernatant of TAM for 72 hours, the relative proliferation rates of ACHN and 786-O cells [(128.6 ±21.4)% and (124.2 ±19.7)%] were significantly higher than that of the control group (100.0%). At the same time, the expression of LDHA in ACHN and 786-O cells increased significantly. After 72 hours of treatment with the supernatant of TAM combined with tocilizumab, the relative proliferation rates of ACHN and 786-O cells [(76.5±13.7)% and (74.8±12.5)%] were significantly lower than that of the control group(100.0%), and the expression of LDHA was also significantly decreased at the same time. The relative proliferation rates of ACHN and 786-O cells in LDHA overexpression group [(121.5 ±17.2)% and (122.7±21.6)%]were significantly higher than that in blank-vector-transfection group[(93.3±10.7)% and (89.8±11.2)%], while the relative proliferation rates in LDHA-knockdown group [(61.4±11.2)% and (58.0 ±10.6)% ]were significantly lower than that in blank-vector-transfection group.Conclusions:By secreting IL-6, TAM can up-regulate the expression of LDHA and promote the proliferation of renal cancer cells.

Objective To explore the role of B7-H3 in the Tie2 expressing monocytes (TEMs) mediated angiogenesis of clear cell renal cell carcinoma (ccRCC).Methods Level of B7-H3 expression on TEMs surface was detected by flow cytometry in ccRCC tissues and normal renal tissues,which were obtained from April 2016 to August 2016 from 20 patients.Microvessel density (MVD) labeled by CD34 in high B7-H3 + TEMs group and low B7-H3 + TEMs group was detected by immunohistochemical examination in ccRCC specimens.B7-H3 + TEMs and B7-H3-TEMs were co-cultured with the 786-O cell lines,and B7-H3 + TEMs and B7-H3-TEMs culture supernatants were collected as conditioned medium,then the effect of B7-H3 + TEMs on angiogenesis was tested by tubule formation assay and mouse aortic ring assay.Results Flow cytometry showed that the frequency of B7-H3 expression on TEMs in ccRCC was (45.10 ± 17.78)％,and the frequency of B7-H3 expression in normal kidney tissues was (10.28 ± 4.28) ％.The frequency of B7-H3 expression on TEMs was significantly higher than that in normal renal tissues (P ＜ 0.001).The MVD of high B7-H3 + TEMs group (103.81 ± 29.28) was higher than that of low B7-H3 + TEMs group (76.55 ± 20.80) (P =0.027).The results of tube formation assay showed that the number of tubule formation of HUVEC in B7-H3 +TEMs group(55.25 ± 11.48) was significantly greater than that of B7-H3-TEMs group (31.34 ± 8.45) and blank control group (25.00 ± 6.74) (P ＜ 0.001).The results of mouse aortic ring assay showed that the number of neovascularization in B7-H3 + TEMs group(77.35 ± 18.47) was significantly greater than that of B7-H3-TEMs group (39.42 ± 8.29) and blank control group (28.79 ± 7.63) (P ＜0.001).Conclusions B7-H3 + TEMs can promote angiogenesis in ccRCC,and might act as an effective target in anti-angiogenic therapy for ccRCC.