Objective To assess the efficacy of pirfenidone combined with PD-L1 inhibitor for treatment of bladder cancer in a mouse model and its effect on tumor immune microenvironment modulation.Methods Forty C57BL/6 mouse models bearing ectopic human bladder cancer xenografts were randomized into control group,PD-L1 inhibitor group,pirfenidone group and combined treatment group(n=10).After successful modeling,PD-L1 inhibitor treatment was administered via intraperitoneal injection at 12.5 mg/kg every 3 days,and oral pirfenidone(500 mg/kg)was given on a daily basis.The survival rate of the mice and tumor growth rate were compared among the 4 groups.The expressions of CD3,CD8,CD45,E-cadherin and N-cadherin in the tumor tissues were detected with immunohistochemistry after the 21-day treatment,and bone marrow-derived suppressor cells(MDSCs)were observed with immunofluorescence staining;serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),urea nitrogen(BUN),creatinine(CRE)and lactate dehydrogenase(LDH-L)were analyzed using an automated biochemical analyzer.Results Treatment with PD-L1 inhibitor and pirfenidone alone both significantly decreased tumor growth rate and tumor volume at 21 days(P<0.05),but the combined treatment produced an obviously stronger inhibitory effect(P<0.05).PD-L1 inhibitor and pirfenidone alone significantly increased E-cadherin expression and decreased N-cadherin expression in the tumor tissue(P<0.05).The two treatments both significantly increased the percentage of CD3+,CD8 and CD45+ T cells and decreased the percentage of Ly-6G+CD11b+MDSCs in the tumor tissue,and these changes were more obvious in the combined treatment group(P<0.05).No significant differences were found in serum ALT,AST,BUN,CRE or LDH-L levels among the 4 groups(P>0.05).Conclusion Combined treatment with pirfenidone and PD-L1 inhibitor significantly inhibits the progression of bladder cancer in mice possibly by regulating tumor immune microenvironment and inhibiting epithelial-mesenchymal transition of the tumor cells.

Objective To assess the efficacy of pirfenidone combined with PD-L1 inhibitor for treatment of bladder cancer in a mouse model and its effect on tumor immune microenvironment modulation.Methods Forty C57BL/6 mouse models bearing ectopic human bladder cancer xenografts were randomized into control group,PD-L1 inhibitor group,pirfenidone group and combined treatment group(n=10).After successful modeling,PD-L1 inhibitor treatment was administered via intraperitoneal injection at 12.5 mg/kg every 3 days,and oral pirfenidone(500 mg/kg)was given on a daily basis.The survival rate of the mice and tumor growth rate were compared among the 4 groups.The expressions of CD3,CD8,CD45,E-cadherin and N-cadherin in the tumor tissues were detected with immunohistochemistry after the 21-day treatment,and bone marrow-derived suppressor cells(MDSCs)were observed with immunofluorescence staining;serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),urea nitrogen(BUN),creatinine(CRE)and lactate dehydrogenase(LDH-L)were analyzed using an automated biochemical analyzer.Results Treatment with PD-L1 inhibitor and pirfenidone alone both significantly decreased tumor growth rate and tumor volume at 21 days(P<0.05),but the combined treatment produced an obviously stronger inhibitory effect(P<0.05).PD-L1 inhibitor and pirfenidone alone significantly increased E-cadherin expression and decreased N-cadherin expression in the tumor tissue(P<0.05).The two treatments both significantly increased the percentage of CD3+,CD8 and CD45+ T cells and decreased the percentage of Ly-6G+CD11b+MDSCs in the tumor tissue,and these changes were more obvious in the combined treatment group(P<0.05).No significant differences were found in serum ALT,AST,BUN,CRE or LDH-L levels among the 4 groups(P>0.05).Conclusion Combined treatment with pirfenidone and PD-L1 inhibitor significantly inhibits the progression of bladder cancer in mice possibly by regulating tumor immune microenvironment and inhibiting epithelial-mesenchymal transition of the tumor cells.

African swine fever(ASF)is an acute and highly pathogenic hemorrhagic disease of pigs,causing huge economic losses to pig industry.In order to quantitatively detect clinical samples of ASF and inactivated ASFV antigens,the IgG of ASF positive serum was used as capture anti-body and the HRP-labeled p72 monoclonal antibody was used as detecting antibody.The standard curve was drawn with the cell-cultured ASFV,and a sandwich ELISA detection of antigen was es-tablished.The specificity,sensitivity and stability of the method were evaluated.The effects of dif-ferent inactivation methods and adjuvant addition on antigen detection were further evaluated.The results showed that the minimum detection limits of the recombinant protein and the ASFV were 0.1 mg/L and 103.7 TCID50/mL,respectively.There was no cross-reaction with five common porcine pathogenic viruses,and the coefficient variations between batches was less than 10％.The total co-incidence rate with real-time fluorescence quantitative PCR was 92％(23/25).The sensitivity of antigen detection was significantly reduced when antigen was treated by BEI inactivation,and the detection results were severely interfered by aluminum adjuvant and nano-adjuvant.In summary,the sandwich ELISA antigen detection method established is specific,sensitive,and repeatable,with a good consistency to the qPCR method,which provides an effective clinical diagnostic meth-od for ASFV antigen.

In order to obtain a cell line which high expressed of rabies virus glycoprotein the produc-tion of rabies subunit vaccine,the RABV glycoprotein gene sequence was amplified by RT-PCR and cloned into the lentiviral expression vector.The recombinant plasmid pLV-RVG,envelope plasmid pMD2.0G and packaging plasmid psPAX2 were co-transfected into HEK-293T cells to harvest lentivirus and infect new HEK-293T cells.The 293T-RVG cell line was obtained by puro-mycin pressure screening.Indirect immunofluorescence assay(IFA)and Western blot showed that the 67 kD RABV-G protein was highly expressed in the cells.The neutralizing antibody titer reached 2.19 IU/mL within 14 days after immunization,which was higher than the internationally recognized protection threshold(0.5 IU/mL).This study showed that the cell line 293T-RVG with stable and high expression of rabies virus glycoprotein was successfully constructed.The cell line could protect mice against rabies virus challenge and could be used as a subunit vaccine with poten-tial for large-scale production and application.

Objective:To observe any effect of an enriched environment (EET) on cognitive functioning and sonic hedgehog (SHH) signaling in rats modeling cerebral ischemia.Methods:Sixty adult male Sprague-Dawley rats were randomly divided into a blank control group, a model group and a training group. A model of cerebral ischemia was established in the model and training groups by thread thrombus. The training group was given an EET. After 7 and 14 days, the rats′ cognition was tested using the Morris water maze and the platform jumping test. Apoptosis of brain cells in the hippocampus was detected by using TUNEL staining, and the expression of SHH, Gli2 and PTCH1 proteins in the hippocampus were measured using qRT-PCRs, western blotting and immunohistochemistry.Results:After 14 days the average escape latency in the Morris water maze test had shortened more in the training group than in the model group, while the average swimming speed, the number of platform crossings and the time spent in the quadrant had increased significantly more. They also received fewer electric shocks and spent significantly less time on the platform in the platform jumping test on average. Apoptosis in the hippocampus after 14 days was significantly less in the training group with significantly greater expression of SHH and Gli2 protein and significantly less PTCH1 protein expression.Conclusion:An EET can significantly improve cognition after cerebral ischemia, at least in rats. Its mechanism may be related to enhanced activation of the SHH signaling pathway.

Objective To assess the value of four different techniques of detecting the Mycobacterium tuberculosis (MTB) in bronchoalveolar lavage fluid (BALF) in the diagnosis of tracheobronchial tuberculosis. Methods A total of 98 patients diagnosed as tracheobronchial tuberculosis were selected from May 1,2013 to June 30,2016. The clinical data was analyzed retrospectively,and the positive rates of MTB of the 960 cultrue, the direct smears , the modified Ziehl?Neelsen stain method and Xpert MTB/RIF assay were compared. Results The positive rates of the 960 cultrue,the direct smears,the modified Ziehl?Neelsen stain method and Xpert MTB/RIF assay were 20.4%(20/98),15.3%(15/98),70.4%(69/98) and 74.5%(73/98),respectively. Among the four techniques ,the positive rates of the modified Ziehl?Neelsen stain method and Xpert MTB/RIF assay were significantly higher than those of the 960 cultrue and the direct smears(P<0.05,respectively). However,no significant difference was found between the modified Ziehl?Neelsen stain method and the Xpert MTB/RIF assay (P > 0.05). Conclusions The modified Ziehl?Neelsen stain method and Xpert MTB/RIF assay for detecting the MTB in BALF have high clinical value in the diagnosis of tracheobronchial tuberculosis.

Objective To test the immune therapeutic effects of PICKCa adjuvant rabies vaccine for the post-exposure mice and beagle dogs and immune memory effect for human.Methods For the mice and beagle dogs first to infect with rabies wild virus and then immunize with PICKCa rabies vaccine.For volunteers, first to immunize with PICKCa rabies vaccine after 2.5 years to take peripheral blood mononuclear cell and then to check cells secreted interferon-gwith flow cytometry. Results In 3 independent assays of post-explosure immunization of mice and beagle dogs PICKCa rabies vaccine were much better then commercial adjuvant-free rabies vaccines, the protective rate from 20%-30%to 70%-100%Also, in the assay of cells secreted IFN-γby peripheral blood mononuclear cells from volunteers immunized by PICKCa rabies vaccine the IFN-γcell levels were much higher than control.Conclusions Compared with commercial adjuvant-free rabies vaccines the PICKCa rabies vaccine had significant immune therapeutic effects in the mice and dogs.Also PICKCa rabies vaccine had good immune memory effect in human.

OBJECTIVETo inhibit the proliferation and metastasis of colon cancer cells by increasing the expression level of B-cell translocation gene-2 (BTG2).

METHODSWestern blot assay was used to detect the expression level of BTG2 protein in the normal intestinal epithelial HIEC cells and three colon cancer cell lines SW620, HT-29 and LS174T. The expression of BTG2 protein in normal colonic epithelial tissue, colon adenoma and colon cancer tissue was detected by immunohistochemistry. The plasmid with BTG2 gene full-length sequence was transfected into colon cancer SW620 cells, and the expression of BTG2 protein was detected by Western blot. The cell growth curve was drawn by MTT test. The Ki-67-positive rate was calculated using immunofluorescence staining. The cell migration of colon cancer cells was detected by scratch test and Transwell double chamber culture system, and the pseudopodia growth of tumor cells was detected by Matrigel 3D culture system.

RESULTSWestern blot results showed that BTG2 relative expression levels were 0.83 ± 0.12, 0.18 ± 0.04, 0.20 ± 0.05 and 0.36 ± 0.07 in normal human intestinal epithelial cells HIEC, and human colon cancer cell line SW620, HT-29 and LS174T, respectively. The results of immunohistochemistry showed that the positive expression of BTG2 protein in normal colorectal tissue, colorectal adenoma and colorectal carcinoma tissues were 82.5% (33/40), 77.5%(31/40) and 17.5% (7/40), respectively, with a significant difference between two groups (P < 0.05). Immunofluorescence results showed that the positive rate of Ki-67 in the control group, empty vector group and BTG2 transfection group was (76.2 ± 8.0)%, (81.4 ± 9.7)% and (50.1 ± 7.1)%, respectively, showing a significant difference between two groups (P < 0.05). The scratch test results showed that in the control group, empty vector group and BTG2 transfection group, the distance of SW620 cells between two sides was (79.27 ± 11.24) µm, (80.65 ± 12.17) µm and (124.77 ± 19.63) µm, respectively, with a significant difference between two groups (P < 0.05). Transwell results showed that in the control group, empty plasmid group and BTG2 transfection group, the SW620 cell migration rate was (78.5 ± 13.1)%, (73.2 ± 12.9)% and (47.4 ± 9.1)%, respectively, showing a significant difference between two groups (P < 0.05). The number of neurospheres of BTG2 transfection group was decreased SW620, which had poor ductility.

CONCLUSIONSBTG2 gene is involved in colon cancer cell proliferation and metastasis, and effectively restores the function of BTG2 protein. Therefore, it may be expected to become a new option in gene therapy for colon cancer.

B-Lymphocytes ; physiology ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; genetics ; Colonic Neoplasms ; Genetic Vectors ; Humans ; Immediate-Early Proteins ; genetics ; Immunohistochemistry ; Plasmids ; Transfection ; Tumor Suppressor Proteins ; genetics

BACKGROUND:Previously deep burn wound or skin defects are generaly repaired with skin grafting or flap of skin grafting. Obvious scar hyperplasia usualy appears after operation, which requires multiple surgeries. Meanwhile, patients have to suffer from great pain and bear high cost. OBJECTIVE: To observe the clinical effects on deep wounds by continuous traction of self-designed skin-stretching device (patent No. ZL 2012 2 0022443.7). METHODS: Thirty patients with deep burn wound, skin defect or funicular scar were enroled, including 22 males and 8 females, aged 18-49 years, and randomly divided into two groups. Skin-stretching device was adopted for skin traction treatment. Twenty cases underwent skin traction from 1 kg puling force to 5 kg, with an increase of 1 kg per 2 days, 6 hours a day for 10 days. Blood flow at the beginning, 1, 5, 10, 15, 20, 30, 60 minutes of the skin traction, and the changes of wound edge skin as wel as histological changes of the skin were observed. Of the remaining 10 cases, 2, 6, and 2 cases underwent skin traction of 2, 4, 7 kg, respectively. Blood flow and skin changes were also observed to find out the most suitable and safe force. RESULTS AND CONLUSION:Al the 30 cases achieved primary healing without necrosis of skin, infection or peripheral circulatory disorders, and the appearance and function recovered wel. The healing time was 8-24 days. The skin-stretching device was most safe under 4 kg puling force, by which, there was neither blood circulation obstacle nor tear of skin. After traction, the skin blood flow and the number of cels increased, especialy the epithelial basal cels. The colagen fibers became thicker and denser, and the elastic fibers regenerated significantly; the fibroblasts and capillary density increased. It has been proved that we can better close the wound and reduce scar formation effectively with the self-designed skin-stretching device for skin traction.

Objective Clinical effects of dot from body skin irradiated xenogeneic skin improved composite graft.Methods Selected 80 patients with severe burn patients randomized after admission on the basis of conventional treatment,3-5 days line Crust treatment group transplanted autologous point-like skin irradiation pigskin coverage.Results Treatment of patients in group one week dressing see irradiated pigskin Tiefu full two weeks the pigskin was dry scab-like.Wound healing treatment group (29 ± 5) days (P ＜ 0.01) was significantly shorter than the control group(39 ±4) days.Wound healing rate of treatment is significantly higher than the control group (P ＜0.01).Wound infection rate reduced greatly reduce labor intensity and dressing,and reduce pain.1 year after scar formation in patients with good flexibility and functionality.Conclusions Punctate since improved composite body skin irradiated xenogeneic skin transplantation can improve skin graft survival rate,promote wound healing,treatment of a large area of the burn wound repair is feasible and effective.