Objective To explore bladder carcinoma eystectomy and orthotopic ileal neobladder postoperative,the impact of new bladder on upper urinary function.Methods Twenty-eight patients with muscle invasive bladder urothelial carcinoma undergoing cystectomy W-shaped orthotopic ileal neobladder in Department of Urology of the Nuclear Industry 215 Hospital of Shaanxi Province(Mar.2006-Jun.2010)were investigated.All patients were followed for over 2 years.Assessment items included creatinine determination,urinary B-Ultrasound determination of the amount of residual urine volume and hydronephrotic cystography.Results Four weeks after the operations,all patients were removed bladder catheter angiography and found no obvious contrast extravasation.After 3 months mild hydronephrosis was found in 8 cases (28.6％),including 2 cases (7.1％) before surgery associated with hydronephrosis,the difference being not statistically significant(x2=0.49,P ＞ 0.05).The mild hydronephrosis was found in 5 cases (17.9％) 2 years postoperation,whithout statistically significant difference compared with preoperation (x2 =0.22,P ＞ 0.05).Preoperative serum creatinine was (72.92 ± 14.58) mmol/L,while 3 months after surgery serum creatinine was (83.42 ± 15.18) mmol/L (t =-6.43,P ＜ 0.05).Preoperative and postoperative serum creatinine was within the normal range.Two years after surgery serum creatinine was (82.50 ± 14.39) mmol/L,with significant difference compared with that of preoperation (t =-4.67,P ＜ 0.05),but were in the normal range,no clinical significance,the postoperative 3 months bladder capacity (160 ± 23) mL,while 2 years later bladder capacity residual urine volume of (58.7 ± 9.7) mL and (430 ± 21) mL,residual urine volume (61.3 ± 37.1) mL(t =-0.37,P＞ 0.05).Conclusion Orthotopie ileal bladder ideal substitute for postoperative cystectomy with less impact on the upper urinary tract function.

[Abstract ] Objective Difficulties with the preparation of gastric cancer stem cells (CSCs) have been a main obstacle to the studies of gastric cancer .This article addresses the technology of the serum-free medium suspension cultivation of the MKN-45 gastric cancer cell line and screening of stem cells from the cell line based on the biomarkers of gastric CSCs . Methods MKN-45 cells were cultured in serum-free medium for 8 weeks and those in the logarithmic phase cultivated with hoechst 33342 followed by detection of the side cells by flow cytometry .When the side population cells reached 25%, all the cell microspheres were collected , hatched with CD133 and CD44, and subjected to fluorescence-activated cell sorting.The CDl33 +and CD44 +cells were selected as gastric CSCs . Results About 40%of the MKN-45 gastric CSCs were alive , prolif-erated, and formed floating cell balls .Side population cells constitu-ted 3.4% of the MKN-45 cells and 26.9% of the cell balls.The CDl33 +and CD44 +cells made up 11.2% of the MKN-45 cells and 90.3%of the cell balls. Conclusion Cell balls rich in CSCs can be successfully obtained by serum-free medium suspension culti-vation and CSCs can be screened out with hoechst 33342 and surface markers , which may serve as an experimental ground for the stud-ies of gastric CSCs .

BACKGROUND: Integrin α5β1 has been shown to be related to acetabular cartilage degeneration due to developmental dysplasia of the hip.OBJECTIVE: To investigate the expression of integrin α5β1 in differently degenerated cartilage tissues of the knee after knee osteoarthritis.METHODS: Seventy-five degenerated articular cartilage specimens at the weight-bearing surface of femoral lateral and medial condyles were removed from patients with knee osteoarthritis who underwent total knee replacement, and were then allotted to four groups based on Mankin's grading system: normal (n=6), mild degeneration (n=24), moderate degeneration (n=26) and severe degeneration (n=19) groups. The expression of integrin α5β1 in each group was detected using real-time PCR, immunohistochemistry and western blot assay.RESULTS AND CONCLUSION: The detected results were consistent in the three kinds of assays, and the expression level of integrin α5β1 was ranked as follows: severe degeneration group > moderate degeneration group > mild degeneration group > normal group (P < 0.05). To conclude, differential expression levels of integrin α5β1 are found in different degenerative degrees of articular cartilage in knee osteoarthritis. Furthermore, the expression level of integrin α5β1 is increased with the degenerative severity.

Objective To explore the immune-microenvironment of the retinas at different stages of retinal degeneration in Royal College of Surgeon (RCS) rats. Methods RCS-rdy--P+(RCS) rats at early stage (P20), middle stage (P40) and late stage (P60) were involved,12 rats at each post-natal day,RCS-rdy+-P+rats severed as control. Relative concentrations of rat cytokines in rat retina homogenate were detected by using Bio-Plex Suspension Array System. Relative expressions of interleukin-2 (IL-2),C-C motif ligand 2 (CCL2),chemokine (C-X-C motif) ligand 9 (CXCL9),CXCL10,CXCL11 and interferon-γ(IFN-γ) mRNA in rat retina were analyzed by real-time PCR. Expressions of IFN-γ and immune cells surface marker CD4,CD8 and CD161 in the retinas were detected by immunohistochemical staining. Percentage of IFN-γ positive T lymphocytes and natural killer(NK) cells in rat retina were analyzed by flow cytometry. The concentrations of IFN-γ in rat retina homogenate were evaluated by enzyme-linked immunosorbent assay ( ELISA ). The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results Lymphocytes related cytokines and chemokines mRNA expression levels in the RCS rat retinas showed increase trends with the extension of time. The expression levels of IL-2,CCL2,CXCL9,CXCL10,CXCL11 and IFN-γ mRNA in P60 RCS rat retinas were significantly increased than those in the P20 RCS rat retinas and the control rat retinas (all at P<0.05).The positive rates of CD4,CD8 and CD161 cells in the retinas of P60 RCS rats was (9.09±0.89)%, (18.77±0.38)% and (9.41±0.38)% ,respectively. The proportion of IFN-γ positive cells in the retinas of P60 RCS rats was (8.29±0.27)%,which was significantly higher than that of the control rats ([0.28±0.02]%),with a significant difference between them (t=29.03,P=0.00). CD4+,CD8+and CD161+lymphocytes were mainly distributed in the retinas of P60 RCS rats, and the expressions of IFN-γ were co-located with lymphocyte surface markers. There were significant differences in the concentrations of IFN-γ in the retinas of RCS rats and control rats at different day ages (Fgroup=16.49,P<0.01; Ftime=21.05,P<0.01),the concentration of IFN-γ in retinas of P60 RCS rats was significantly higher than that of P20 RCS rats, P40 RCS rats and control rats, and the differences were statistically significant ( all at P<0.05). Conclusions Along with the process of retinal degeneration,immune privilege balance in the retinas is disrupted, the expressions of lymphocytes related chemokines and cytokines are elevated. Lymphocytes infiltration and activation are appeared in the retina highly activated at the late stage of RP, leading to the significant up-regulation of inflammatory cytokine IFN-γ in microenvironment, which indicates that lymphocytes mediated immune response may take part in retinal degeneration.

Objective To explore the relationship between phenotypic changes of retinal microglia and retinal ganglion cells(RGCs)death after optic nerve injury.Methods Male C57BL/6J mice(6 to 8 weeks old)were randomly divided into 1-,3-,7-,and 14-day injury groups and sham operation group,with 4 mice in each group.The eyes in the injured groups were inflicted with optic nerve crush(ONC),while the eyes of the sham operation group were treated with the same operation procedure but without optic nerve clamp.Flash visual evoked potential(fVEP)and immunofluorescence staining were employed to evaluate the impact of optic nerve injury on visual function and number of RGCs.RT-qPCR and immunofluorescence staining were applied to detect the effecy of optic nerve injury on phenotypic changes in retinal microglia.Results fVEP results showed that the visual conduction of the injured eye was gradually decreased over time when compared with that of the sham group(P＜0.01).Immunofluorescence staining revealed that the number of RGCs was lost mainly within 7 d after injury(P＜0.01).At the same time,the number of retinal microglia reached its peak at 7 d after injury(P＜0.01).RT-qPCR indicated that the expression of disease-associated microglia(DAM)and interferon-responsive microglia(IRM)specific genes were significantly increased when compared with the sham group at 7 d after ONC(P＜0.01).Immunofluorescence staining displayed that the number of DAM peaked at 3 d after ONC(P＜0.01),but the proportion was decreased gradually with the progress of time(P＜0.05).The number and proportion of IRM peaked 7 d after ONC(P＜0.01).Correlation analysis suggested that the number of IRM was strongly correlated with the loss of ganglion cells(P＜0.01).Conclusion The conversion of retinal microglia from DAM type to IRM type after optic nerve injury may be an important cause of ganglion cell loss.

Objective: To explore the method of extracting chaperone antigen peptide complexes from gastric cancer stem cells and its immune function. Methods: Gastric cancer stem cells and gastric cancer cells were screened by low temperature ultrasonic lysis. After salting out and dialysis, the lysate supernatant was processed with SDS-PAGE to analyze the expression of chaperone antigen peptide complexes, and then was separated and purified with CNBr-activated SepharoseTM 4B. Reverse high pressure liquid chromatography (HPLC), SDS-PAGE and Western blotting were used to analyze the purity and nature of the acquired albumen. Lymphocyte proliferation assay and lymphocytotoxicity assay were used to ditermine the immunological activity of the chaperone-antigen peptide complexes. Results: The chaperone antigen peptide complexes of gastric cancer stem cells were prepared and identified successfully, of which the main components were the antigen peptides of HSP60, HSP70, HSP90 and HSP110. 0.75 μg and 1.00 μg HSP70-antigen peptide and 1.00 μg HSP90-antigen peptide activated lymphocytes significantly. Their A₄₉₀ values were 0.26±0.03, 0.45±0.05 and 0.32±0.04, respectively, while the corresponding doses of HSP60-antigen peptide and HSP110-antigen peptide did not activate lymphocytes. The killing rates of 1.00 μg HSP70-antigen peptide and 1.00 μg HSP70 were (45.0±2.0)% and (16.0±2.0)%, respectively, showing a significant difference (P=0.012). Similarly, the killing rates of 1.00 μg HSP90-antigen peptide and 1.00 μg HSP90 were (36.0±5.0)% and (13.0±4.0)%, respectively, also showing a significant difference (P=0.048). Conclusions The amount of chaperone antigen peptide complexes in gastric cancer cells is extremely low, but it is obviously increased in gastric cancer stem cells. After purification, the chaperone antigen peptide complexes with high purity can be prepared. The extracted chaperone antigen peptide complexes have stronger immunogenicity, and can be used to make tumor vaccine in vitro, which may have a good application value in the targeted therapy of gastric cancer.