Objective To investigate the combined effects of fluoride (NaF) and arsenate (NaAsO2) exposure on proliferation,differentiation and bata-catenin expression in SD rat osteoblasts.Methods Osteoblasts were isolated from calvarias of twelve SD rats born in 1-3 days and cultured.The method was divided into 9 groups [F0.0As0.0 (control group),F0.5As0.0,F4.0As0.0,F0.0As0.1,F0.0As10.0,F0.5As0.1,F0.5As10.0,F4.0As0.1,F4.0As10.0] by factorial experiment design (3 factors and 2 levels).Osteoblasts were exposed to NaF (F-:0.0,0.5,4.0 mmol/L,F0.0,F0.5,F4.0),NaAsO2 (As3+:0.0,0.1,10.0 μmol/L,As0.0,As0.1,Asi10.0) and cultured for 72 hours.The proliferation and alkaline phosphatase (ALP) was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl (MTT) and Enzyme-linked immunosorbent assay (ELISA).The expression of beta-catenin was analyzed by quantitative real-time PCR (qPCR) and Western blotting after 72 hours of experiment.Results ①There was significant difference in cell proliferation and the activity of ALP among groups after 72 hours (F =14.022,14.425,all P ＜ 0.05).Compared with control group,the proliferation and the activity of ALP were significantly induced in F0.5 treated osteoblasts groups (0.313 ±0.023 vs0.455 ± 0.152,4.46 ± 0.72 vs 6.09 ± 0.68,all P ＜ 0.05),and the proliferation was significantly suppressed in F4.0As0.0 group (0.029 ± 0.014,P ＜ 0.05),the activity of ALP was significantly induced in F0.0As10.0 group (0.156 ± 0.010,6.29 ± 0.67) and the proliferation was significantly suppressed in F0.0As0.1 group (0.370 ± 0.029,3.68 ± 0.45,all P ＜ 0.05).②There was statistical difference in beta-catenin mRNA and protein expressions among groups with F-(F =7.782,559.455,all P ＜ 0.05) at As0.0 condition.There was significant difference in the expression of betacatenin mRNA and beta-catenin protein in F0.5As0.0 group compared with control group (1.00 ± 0.32 vs 1.99 ± 0.14,3.56 ± 0.15 vs 5.11 ± 0.26,all P ＜ 0.05),the beta-catenin protein was significantly suppressed in F4.0As0.0 group (1.10 ± 0.02,P ＜ 0.05).There was significant difference in the expression of beta-catenin protein of all groups with As3+ (F =154.736,P ＜ 0.05) at F0.0 condition.③Factorial analysis showed that fluoride or arsenic alone could affect the proliferation and the expression level of beta-catenin mRNA and protein (F =82.081,11.991,514.741;19.302,8.753,523.698,all P ＜ 0.05),the effect of arsenic on ALP activity of osteoblasts was also the main effect (F =17.444,P ＜ 0.05);and there was an interaction between fluoride or arsenic to cell proliferation and the activity of ALP and the expression of beta-catenin mRNA and protein (F =13.085,18.157,4.936,426.036,all P ＜ 0.05).Conclusions A biphasic pattern of fluoride or arsenic on proliferation and differentiation has been induced in SD rat osteoblasts.Fluoride or arsenic can affect bone metabolic by beta-catenin.

Objective To observe the inhibitory effect of caspase\|3 mRNA antisense oligodeoxynucleotides (ASODN) on the apoptosis of HL\|60 cells, and to screen the effective ASOND. Methods By means of the lipofectimine\|mediating transfection, caspase\|3 mRNA ASODNs of four different sequences were introduced into HL\|60 cells followed by ? irradiation.The gel electrophoresis was performed for the DNA ladder, Hoechst 33258\|propidium iodide fluorecent staining for the percentage of apoptotic cells, and FCM for quantitative analysis of apoptosis. Results When HL\|60 cells were transfected with caspase\|3 mRNA ASODNs targeting 5'noncoding region (sites -62 to -46) and initiative coding region (sites -1 to 16) at the final concentration of ≥3?? mol/L, the DNA ladder was not detected with the gel electrophoresis, nor was the apoptotic peak found with FCM. Fluorescent staining analysis showed that the percentage of apoptotic cells was significantly lower than that in the control groups including non\|transfection group and mismatched oligodeoxynuleotide group ( P

BACKGROUND:Pulmonary aspergilosis is a disease caused by pulmonary fungal infection. Its diagnosis and treatment is usualy delayed because of nonspecific clinical symptoms, physicial sign and imaging changes as wel as uncertainties of histological and bacterial findings. Therefore, it is necessary to establish mouse models of invasive pulmonary aspergilosis to investigate the underlying pathological mechanism and novel therapeutic methods. OBJECTIVE: To establish mouse models of invasive pulmonary aspergilosis, detect the expression ofγ-interferon, Tol-like receptor 2 and Tol-like receptor 4, and discuss the mechanism of action underlying invasive pulmonary aspergilosis. METHODS:Seventy-five female BALB/c mice of clean grade, aged 6-8 weeks, were randomly and evenly divided into five groups: blank control group (group A), immunosuppressive model group treated with high concentrations of Aspergilus fumigatus spore suspension (group B), normal infection group treated with high concentration of Aspergilus fumigatus spore suspension (group C), immunosuppressive model group treated with low concentration of Aspergilus fumigatus spore suspension (group D), normal infection group treated with low concentration of Aspergilus fumigatus spore suspension (group E). First, mice in the groups B and D were intraperitonealy injected with cyclophosphamide to establish immunosuppressive models. The mice in the groups D, E (108 cfu/mL) and groups B, C (109 cfu/mL) were treated with 12 mL Aspergilus fumigatus spore suspension through the use of nebulizer. Mice in the group A were treated identicaly with sterile PBS. At 1, 3, 5 days of infection, the pathological change of lung tissue was observed, the mass concentration of γ-interferon in bronchoalveolar lavage fluid and the expression levels of γ-interferon mRNA and Tol-like receptor 2 and Tol-like receptor 4 mRNA and protein in the lung tissue were determined. RESULTS AND CONCLUSION:Abscess, spores and very severe bleeding and congestion, widenened alveolar septum and tracheal epithelial cel shedding and necrosis were observed in the mouse lung tissue in the group B. At 5 days of infection, the mass concentration of γ-interferon in bronchoalveolar lavage fluid and the expression ofγ-interferon mRNA in the lung tissue in the group B were significantly decreased compared with the group A (P < 0.05). Tol-like receptor 2 expression was strongly positive in the group B. Tol-like receptor 2 expression in the group C was significantly lower than that in the group B (P< 0.05). Tol-like receptor 4 expression was positive in the groups B and C, and its expression in the group C was significantly greater than in the group B (P < 0.05). The expression of Tol-like receptor 2, 4 mRNA in the mouse lung tissue of group B was significantly increased at 1, 3, 5 days of infection (P < 0.05). These results suggest that atomizing high concentration of aspergilus fumigatus spore suspension to immunosuppressive mice can establish stable invasive pulmonary aspergilosis models with typical pathological features. The infection of aspergilus fumigatus can activate tol-like receptor 2, 4 at the same time, and the pathological mechanism is closely related to organism’s immune defense function.

Objective:To investigate the effects of interleukin-17 on tumor,we transfected interleukin-17 gene into mouse colon cancer cells(C26)and set up an animal model in tumor.Methods:By plasmid vector,IL-17 gene was transfected into C26.Meanwhile, empty plasmid vector(pcDNA3.1)and C26 cells were transfected as control groups.C26/pcDNA3.1-IL-17,C26/pcDNA3.1,and C26 cells were subcutaneously inoculated into mice respectively and the tumor volume and the survival time were observed.Proliferation of splenocyte and NK activity were detected.Detect the characteristic cytokines and transcriptional factors of Th1,Th2,Th17 and Treg cells in splenic lymphocyte.Proliferation of TIL was detected.The characteristic cytokines IL-10 of M1 and the characteristic cytokines IL-12 of M2 in tumor infiltrating macrophages were detected.Results: The growth of tumor in mice inoculated with C26/pcDNA3.1-IL-17 cells was significantly retarded ( P<0.05 ) , and the growth of tumor in male mice inoculated with C26/pcDNA3.1-IL-17 cells was significantly retarded than female mice ( P<0.05 ).The mice survival time of C26/pcDNA3.1-IL-17 group was similar with C26/pcDNA3.1 and C26 groups(P>0.05).The proliferation of the splenocytes from C26/pcDNA3.1-IL-17 inoculated mice was higher than those of C26/pcDNA3.1,C26 groups(P<0.05),but was similar with the normal group(P>0.05),the proliferation of the splenocytes from C26/pcDNA3.1 and C26 inoculated mice was slow than those of normal groups(P<0.05).The NK(separate from spleen) activity of C26/pcDNA3.1-IL-17,C26/pcDNA3.1 and C26 inoculated mice was lower than those of normal groups when the ratios of effector cells and target cells were 40∶1,20∶1(P<0.05),the NK(separate from spleen) activity of C26/pcDNA3.1-IL-17 inoculated mice was higher than those of C26/pcDNA3.1 and C26 groups when the ratios of effector cells and target cells were 40∶1(P<0.05),there′s no difference among every groups when the ratio of effector cells and target cells were 10∶1 ( P>0.05 ).The splenocytes from the mice inoculated with C26/pcDNA3.1-IL-17 cells secreted more IFN-γ( the characteristic cytokines of Th1 ) , IL-4 ( the characteristic cytokines of Th2),GATA-3,ROR-γt,IL-10(the characteristic cytokines of Treg)mRNA(P<0.05).The proliferation of TIL from C26/pcDNA3.1-IL-17 inoculated mice was higher than those of C26/pcDNA3.1,C26 groups(P<0.05),the proliferation of TIL from C26/pcDNA3.1-IL-17,C26/pcDNA3.1 and C26 inoculated mice was lower than those of normal groups( P<0.05).And there′s no differences among every groups of the express of cytokines IL-10 and IL-12 mRNA in tumor infiltrating macrophages(P<0.05).Conclusion: The transfection of IL-17 gene inhibited tumor growth in the mice,inoculated with enhancing the immune function.

Objective To evaluate the safety and clinical efficacy of retroperitoneal laparoscopic partial nephrectomy (LPN) in patients with incidental renal cell carcinoma.Methods Twenty-six patients with T1 incidental renal cell carcinoma treated with retroperitoneal LPN from Dec.2011 to Oct.2013 were analyzed retrospectively.The operation time,warm ischemia time,intraoperative blood loss,complications and prognosis of perioperative periods were collected.Results All 26 cases were operated successfully without conversion to open or other surgery.The operation period was 90-190 minutes.Blockage of renal artery was applied in all 26 cases,and the warm ischemia time was 15-30 minutes.The intraoperative blood loss was 100-360 ml.The drainage was removed 3-7 days postoperatively,and the hospitalization period was 7-12 days.All cases were clear cell renal cell carcinoma by pathological examinations,and showed negative surgical margins.No complications such as postoperative bleeding,inflammation and leakage of urine were occurred.All cases had normal renal function during the follow-up of 3-25 months without local recurrence or distant metastasis.Conclusion Retroperitoneal LPN for the treatment of T1 incidental renal cell carcinoma is worthy of application with the advantages of safety,minimal invasiveness,fewer complications,quick recovery and good nephron-sparing functional recovery.

Objective To investigate the effects of 14-3-3 phosphorylation (p-14-3-3) induced by C-Jun N-termi-nal kinase (JNK) on ischemic brain injury in rats. Methods Twenty rats were divided into 4 groups:sham operation group, ischemia-reperfusion group, SP600125 group and solvent control group. The rat model of cerebral ischemia was established. The p-14-3-3, the binding of 14-3-3 and Bax and the protein expression of Bax in cytoplasm and mitochondria in hippo-campal CA1 region were detected by immunoprecipitation (IP) and immunoblotting 12-hour after ischemia-reperfusion in four groups. Results Compared with the sham operation group, protein expression levels of p-14-3-3 in cytoplasm and Bax in mitochondria were significantly increased, the binding of 14-3-3 and Bax was significantly decreased in ischemia-re-perfusion group, solvent control group and SP600125 group. The protein expressions of p-14-3-3 and Bax were significantly lower in SP600125 group than those of ischemia-reperfusion group and solvent control group. The binding of 14-3-3 and Bax was significantly higher in SP600125 group than that of ischemia-reperfusion group and solvent control group (P <0.05). Conclusion 14-3-3 phosphorylation induced by JNK plays important effects on ischemic brain injury in rats.

Objective:To observe the outcome of posterior staphyloma (PS) marginal retinal photocoagulation in pars plana vitrectomy (PPV) for high myopia macular hole retinal detachment eyes accompanied with PS.Methods:From January 2017 to June 2019, 49 patients (49 eyes) with high myopia macular hole retinal detachment accompanied with PS who were undergone PPV operation from Tianjin Eye Hospital were included in this study. There were 13 males (13 eyes) and 36 females (36 eyes). All patients underwent best corrected visual acuities (BCVA) and optical coherence tomography examinations. The standard logarithmic visual acuity chart was used for BCVA examination, and the visual acuity was converted to minimum resolution angle in logarithmic (logMAR) when recorded. The patients were randomly divided into two groups according to surgical options: conventional PPV with internal limiting membrane (ILM) peeling (group A, 24 eyes), PS marginal retinal photocoagulation in PPV with ILM peeling (group A, 25 eyes). The mean preoperative logMAR BCVA of group A and B were 1.87±0.28 and 1.80±0.37, the difference was not statistically significant ( t=0.604, P=0.551). The patients in the group A received 23G PPV, triamcinolone acetonide staining during the operation, the epiretinal membrane was peeled off, indocyanine green assisted staining, the posterior macular ILM was peeled off, and the peripheral retina was examined in detail during the operation. Areas with retinal degeneration were reinforced by laser photocoagulation, and the subretinal fluid was drained through the macular hole and filled with silicone oil. The eyes of the group B were subjected to retinal photocoagulation for 2 to 3 rows at the edge of the PS in addition to the usual surgical procedures. The average follow-up time was 8.34±3.21 months. Surgical outcome were estimated by the average number of operation, retinal reattachment rate, macular hole closure rate and BCVA. The χ2 test or Fisher exact probability was used to compare the count data. Independent sample t test was used to compare the measurement data. Results:Retinal reattachment was obtained in 17 eyes (70.8%, 17/24) and 24 eyes (96.0%, 24/25) in group A and B after first surgery respectively, the difference was statistically significant ( χ2=3.984, P=0.046). Final retinal reattachment was obtained in all 49 eyes. Final macular hole closure was in 15 eyes (62.5%, 15/24) and 19 eyes (76.0%, 19/25) in group A and B, respectively, the difference was not statistically significant ( χ2=1.051, P=0.305). The mean postoperative logMAR BCVA of group A (1.20±0.47) and B (1.08±0.39) were all improved than preoperative BCVA, the differences were all statistically significant ( t=2.899, 5.327; P=0.001, 0.000), the differences of mean postoperative logMAR BCVA between two groups was not statistically significant ( t=0.675, P=0.506). The mean number of operation of group A (2.63±0.88) was more than group B (2.08±0.28), the difference was statistically significant ( t=3.003, P=0.006). Conclusion:In comparison with conventional PPV, combined PS marginal retinal photocoagulation can improve retinal reattachment rate after first surgery, and reduce the number of reoperations.

Objective:To observe the changes of macular microvessels in patients with retinal vein occlusion (RVO) and macular edema (ME) after intravitreal injection of aflibercept (IVA), and analyze its correlation with best corrected visual acuity (BCVA).Methods:A retrospective case study. Thirty patients (30 eyes) with monocular RVO with ME (RVO-ME) who were diagnosed in the clinical examination of Tianjin Eye Hospital from April 2019 to February 2020 were included in the study. Among them, there were 12 males (12 eyes) and 18 females(18 eyes); the average age was 54.30±13.17 years. The average course of disease was3.43±1.97 months. Both eyes were examined by BCVA and optical coherence tomography (OCTA). The on-demand injection was adopted after the first injection in IVA treatment regimen. The macular area 6 mm×6 mm in both eyes was scanned with an OCTA instrument, and the area of the foveal avascular area (FAZ), FAZ circumference (PERIM), and out-of-roundness were measured at baseline and 1, 3, and 6 months after treatment. Index (AI), blood flow density within 300 μm width of FAZ (FD-300), foveal retinal thickness (CMT), superficial retinal capillary plexus (SCP), deep retinal capillary plexus (DCP) blood flow density. The paired t test was used to compare the quantitative parameters of the affected eye and the contralateral healthy eye at baseline; the changes of the quantitative parameters at baseline and 1, 3, and 6 months after treatment were analyzed by repeated measures analysis of variance. Pearson correlation analysis was used to analyze the correlation between BCVA, retinal perfusion, and macular blood supply parameters at 6 months after IVA treatment. Results:At baseline, compared with the contralateral healthy eye, the FAZ area ( t=-4.091), PERIM ( t=-5.098) and AI ( t=-9.093) of the RVO-ME eye were enlarged, and FD-300 ( t=7.237) and overall SCP and DCP blood flow density ( t=8.735, 9.897) decreased, the difference was statistically significant ( P<0.001). Six months after treatment, the BCVA of RVO-ME eyes was significantly increased, CMT decreased, FAZ area expanded, and AI decreased ( t=8.566, 16.739, -6.469, 9.719; P<0.001), the difference was statistically significant. There was no significant change in the blood flow density of FD-300 and overall SCP and DCP, and the difference was not statistically significant ( t=1.017, 1.197, 0.987; P>0.05). Compared with baseline, the FAZ area of RVO-ME eyes gradually expanded at 3 and 6 months after treatment, and the difference was statistically significant ( F=21.979, P<0.001). Correlation analysis results showed that BCVA at 6 months after treatment was positively correlated with the overall SCP and DCP blood flow density at baseline and 6 months after treatment ( r=-0.538, -0.484, -0.879, -0.854; P<0.05). There was a negative correlation with the area of FAZ 6 months after treatment ( r=0.544, P=0.001). The number of ME recurrences was negatively correlated with BCVA and overall SCP and DCP blood flow density 6 months after treatment ( r=0.604, -0.462, -0.528; P<0.05), it was positively correlated with FAZ area ( r=0.379, P=0.043). Conclusion:Within 6 months of IVA treatment in RVO-ME eyes, ME is significantly reduced and visual acuity is improved; SCP blood flow density decreases, and FAZ area expands.

Objective To detect the immune effect of FbaAmAb2 against the surface protein of group A Straptococcus (GAS),and explore the pathogenesis and therapy of GAS infections.Methods By subclonal and bacterial ELISA,the positive hybridoma cells were screened that can produce better titers of FbaAmAb2 against GAS-surface FbaA protein,and were injected into the peritoneal cavities of BALB/c mice to produce ascites.The collected ascites were performed to dilute,as follows,original ascite,1:2,1:4,1:8,and 1:16 to test tube agglutination.Based on the results,we selected appropriate dilution to passively immunize mice,and then challenged the mice with GAS,evaluating FbaAmAb2 neutralizing ability with GAS in mice by the survival rate of the immunized mice.Whether FbaAmAb2 could inhibit the binding of factor H to GAS was confirmed by the invasive inhibition assay.Results The IgG titer of bacteria solution ELISA is 1:160 and the titer of tube agglutination is 1∶8.The protect rates of FbaAmAb2 on preventing mice with GAS infections are as follows:66.67％ in original ascite and 1:2 diluted groups,and 50％ in 1:4 diluted group.Mice in each experimental group were evoked significantly protective immune responses compared with the PBS control by SPSS analysis.FbaAmAb2 can competitively inhibit factor H binding to the surface proteins FbaA of GAS,which decreased the entry of GAS into the cytoplasm of human epithelial cells through the binding of factor H.Conclusion FbaAmAb2 is promising to be used in emergent prevention or the clinical therapy for GAS infection and it is promising starting points for pharmacologic targeting and further development of new therapeutic agents for GAS.

Objective To explore the effect of QizhiJiangtang Capsule on the insulin resistance (IR)in the diabetic rats,and to clarify the action mechanism.Methods The diabetes rat models were induced by high fat diet combined with STZ injection.The successful models of the rats were randomly divided into diabetes group (DM), ShenqiJiangtang Granule group (SQ)and high (QJH),middle-(QJM),low (QJL)doses of QizhiJiangtang Capsule groups;at the same time control group (NC)was established. The drug concentrations in high, middle and low-doses of QizhiJiangtang Capsules groups were 1.35, 0.68, and 0.34 g · kg-1 respectively;and the concentration of ShenqiJiangtang Granule was 0.27 g·kg-1.After the diabetic model was established successfully, the rats were treated for 8 weeks on the basis of drug dose.Then the levels of fasting blood glucose (FBG),fasting insulin (FINS),insulin resistance index (IRI)and biochemical indexes related to lipid metabolism of the rats were measured using blood glucose detector and automatic biochemistry analyser.The gene expression of insulin receptor substrate-1 (IRS-1),phosphatidyl inositol 3-kinase (PI3K),and glucose transporter 4 (GLUT4)in liver tissue were examined by Real Time PCR.The levels of tumor necrosis factorα(TNF-α)and adiponectin (ADPN)in serum were detected using ELISA.Results Compared with control group,the levels of FBG,FINS and IRI of the rats in diabetes group were significantly increased (P<0.05 or P<0.01 );the serum total cholesterol (TC), triglyceride (TG)and low density lipoprotein (LDL)levels were significantly increased (P<0.05 ), while the serum high-density lipoprotein (HDL)level was significantly decreased (P<0.05);the mRNA expression levels of IRS-1,PI3K and GLUT4 in liver tissue were decreased (P<0.05);the level of serum TNF-αwas increased (P<0.05),but the ADPN level was decreased (P<0.05).Compared with diabetes group,the FBG level and IRI of the rats in QizhiJiangtang Capsule and ShenqiJiangtang Granule groups were significantly decreased (P<0.01);the levels of FINS of the rats middle and high doses of in QizhiJiangtang Capsule groups and ShenqiJiangtang Granule group were significantly decreased (P<0.05);the levels of serum TC,TG and LDL of the rats in middle dose of QizhiJiangtang Capsule group and ShenqiJiangtang Granule group were significantly decreased (P<0.05 or P<0.01),but the HDL level was increased (P<0.05);the mRBA expression lvels of IRS-1,PI3K and GLUT4 inliver tissue were increased (P<0.05);the levels of serum TNF-αof the rats in middle dose of QizhiJiangtang Capsule group and Shenqijiangtang Granule group were significantly decreased (P<0.05),but the serum ADPN levels were increased (P<0.05 ). Conclusion QizhiJiangtang Capsule can significantly improve the IR in the diabetic rats,and the pharmacological mechanisms are related to adapting the blood lipid component and insulin signal transduction pathways.