Child ; Evidence-Based Medicine ; Humans ; Nephrotic Syndrome ; diagnosis ; etiology ; therapy ; Practice Guidelines as Topic ; Recurrence

Background Research comfirmed that second mitochondrial activator of caspase (Smac) is a promoting tumor cell apoptosis protein.Our previous study showed that the expression level of Smac in LECs is obviously higher in cataract than that in normal eyes.We assumed that silencing Smac gene in LECs can inhibit the apoptosis of LECs.The way to transfect Smac siRNA into LECs is a key step.Objective This study was to construct siRNA lentiviral vector of Smac and identify its silencing efficiency in human lens epithelial B3 cell line (HLE-B3) and establish low-expressed Smac HLE-B3 line.Methods Based on the genebank and our previous study,siRNA sequence of Smac was designed and composed.The synthetic double-stranded DNA was linked to the lentiviral vector GVll8 by T4 DNA ligase and then transformed DH5α competent cells.The plasmids were transformed into the DH5α competent cells.Recombinant colonies were screened by PCR and sequenced.Recombinant plasmids and two other auxiliary plasmids were used to infect 293T cells.Cell culture supernatant was collected for the measurement of viral titer.Recombinant lentiviral vector was used to infect HLE-B3 cells to calculate the viral multiplicity of infection (MOI) under the fluorescence microscope.Transfection efficiency was examined by calculating the GFP-positive cells.HLE-B3 cells were divided into negative control group,siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group.The relative expression levels of Smac mRNA in the cells were detected and compared among the three groups by real-time fluorescent quantitative PCR.Results GV118-Smac-siRNA was successfully constructed with the positive colonies 340 bp and blank vector colonies 299 bp,and viral titer was 3.0× 108 TU/ml.At a MOI of 100,the infecting efficiency of the vector on HLE-B3 cells was about 82％ and the cytotoxicity was low.The relative expression levels of Smac mRNA were (101.290±8.349)％,(92.330±6.320)％ and (32.540±4.221)％ in the negative control group,siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group,respectively,showing a significant difference among the three groups(F =32.871,P＜0.01),and the relative expression level of Smac mRNA was significantly lower in the GV118-Smac-siRNA1 tranfected group than that in the negative control group (P =0.000).However,no significant difference was found in the Smac mRNA expression between the blank plasmid group and the negative control group (P=0.535).Conclusions GV118-Smac-siRNA lentiviral vector is successfully constructed.Smac-siRNA can effectively inhibit the expression of Smac mRNA in human LECs.

ObjectiveTo investigate the expression of heme oxygenase-1 (HO-1) in lung tissue of mice with acute paraquat poisoning, and discuss its pathological mechanism.Methods Fifty-eight healthy male mice were randomly divided into control group (n = 8) and poisoned group (n = 50). The mice in poisoned group were lavaged with 20% paraquat (50 mg/kg), and those in control group with equal amount of normal saline. The mice were sacrificed on the day of experiment in control group, and those in poisoned group at 6 hours and 1, 3, 7, 14 days after poisoning. The lung tissue was harvested to observe the changes in pathology of lung with hematoxylin and eosin (HE) staining. The positive expression of HO-1 was determined with immunohistochemistry, and the protein expression of HO-1 was determined with Western Blot. The contents of superoxide dismutase (SOD) and malonaldehyde (MDA) were determined.Results The mice showed shortness of breath and signs of exhaustion 1 hour after poisoning, getting worse on 3-5 days, but returned to normal 14 days after poisoning. Under the light microscope, it showed that the control group had no significant pathological changes in lung tissue. One day after the ingestion, pulmonary alveolar structure disorder, obvious hemorrhage, edema and infiltration of inflammatory cells were found. At 3 days, the pathological changes in the lung tissue were more pronounced. They were less pronounced on 7 days, and inflammatory changes disappeared on 14th day, but alveolar structure disorder remained. Immunohistochemical test showed that HO-1 was seldom expressed in the lung tissue, and a little amount was expressed in the mucosal epithelial cells of the airway in control group. It was shown that inflammatory cell and endothelial were mainly distributed in the mucosal epithelial cells of airway 1 day after poisoning followed by a gradually decrease tendence, and came to normal level of control group 7 days after poisoning. It was shown by Western Blot that HO-1 (gray value) in lung tissue increased 6 hours after poisoning (2.438±0.467 vs. 0.475±0.167,P< 0.01), peaked at 1 day (9.200±0.940 vs. 0.475±0.167,P< 0.01), continued to increase till 7 days after poisoning, and it lowered to normal level thereafter (0.825±0.260 vs. 0.475±0.167,P> 0.05). The SOD activity (μU/L) in lung tissue was lowered 6 hours after poisoning, and it was significantly lower than that of control group (649.681±13.951 vs. 1 167.051±15.744,P< 0.01), and it continued to decrease up to 14 days after poisoning (859.733±121.079 vs. 1 167.051±14.744,P< 0.01). MDA content (μmol/L) in the lung tissue homogenate was elevated 6 hours after poisoning with significant difference compared with that of the control group (4.542±0.266 vs. 3.705±0.176,P< 0.01). It peaked on day 1 (5.956±0.281 vs. 3.705±0.176,P< 0.01), then it declined and reached normal level 3 days after poisoning (4.134±0.168 vs. 3.705±0.176,P> 0.05).Conclusion HO-1 expression was increased significantly in lung tissue of mice with acute paraquat poisoning, which may be considered as an important protection mechanism against paraquat poisoning.

Objective To investigate the role of CD4+CD25+FoxP3+ in severe Mycoplasma pneumonia among children.Methods One hundred and forty children with M.pneumoniae pneumonia (65 severe and 75 non-severe) who were hospitalized were enrolled along with forty other children as controls.X-ray was assessed.The proportions of peripheral blood CD4+CD25+FoxP3+cells were determined by flow cytometry.Results Both severe and non-severe children had decreased CD4+CD25+FoxP3+cells as compared with control subjects in acute phase (0.87 ± 0.66％ vs.3.88 ± 2.00％,P ＜ 0.01 and 1.17 ± 0.70％ vs.3.88 ±2.00％,P ＜0.01,respectively).The levels of CD4+CD25+FoxP3+cells in severe children were lower than those in non-severe children in acute phase and recovery phase (0.87 ±0.66％ vs.1.17 ±0.70％,P ＜0.05 and 1.66 ±0.85％ vs.3.61 ± 1.45％,P＜0.01,respectively).Both severe children and non-severe children expressed higher CD4+CD25+FoxP3+cells in recovery phase than in acute phase (1.66 ± 0.85 ％ vs.0.87 ± 0.66％,P ＜0.01 and 3.61 ± 1.45％ vs.1.17 ±0.70％,P ＜0.01,respectively).Conclusion The expression of CD4+CD25+FoxP3+Tregs may play a role in the onset of severity of mycoplasma pneumonia and the low express of CD4+CD25+FoxP3+Tregs in children infected with M.pneumonia may increase the susceptibility to severe mycoplasma pneumonia.

Objective To determine the value of the apparent diffusion coefficient (ADC) of magnetic resonance diffusion-weighted imaging (MRDWI) combined with squamous cell carcinoma antigen (SCC) and carcinoembryonic antigen (CEA) in the evaluation of the efficacy and prognosis of concurrent chemoradiotherapy for cervical carcinoma.Methods A total of 80 patients with cervical squamous cell carcinoma confirmed by histology or cytology in our hospital from 2013 to 2016 were included in this study.Of the 80 patients, 39 were FIGO stage ⅡB, 7 were stage ⅢA, 26 were stage ⅢB, and 8 were stage ⅠVA.MRDWI examination and SCC and CEA measurements were first performed for the patients following group assignment, and the patients were then given extrapelvic radiotherapy (45-50 Gy)+platinum-based chemotherapy plus brachytherapy (20-25 Gy) based on their conditions.MRDWI, SCC, and CEA examinations were performed again after treatment to determine the changes in ADC, SCC, and CEA.In addition, ADC, SCC, and CEA were examined in the middle stage of treatment for 40 patients.Data were analyzed using the paired t-test or ANOVA.Results The overall response rate of the 80 patients after concurrent chemoradiotherapy was 100%.No disease progression was identified in any of the patients until the end of treatment, and the overall survival time of the patients was all above 6 months.Serum SCC and CEA were reduced after treatment (P=0.000,0.000), whereas the ADC value was increased after treatment (P=0.000).The increase in ACD following the decreases in SCC and CEA after treatment (P=0.000, 0.000) was indicative of increased efficacy of the concurrent chemotherapy and radiotherapy.Conclusions MRDWI combined with SCC and CEA is highly reliable for the evaluation of efficacy and prognosis of concurrent chemoradiotherapy for cervical cancer.

AIM:To evaluate the efficacy and safety of methazolamide in treating refractory uveitic macular edema.METHODS: Retrospective self-controlled study was designed.A total of 15 patients (20 eyes) with refractory uveitic macular edema which used methazolamide as adjuvant therapy were enrolled in Shanghai First People`s Hospital from January 2015 to June 2016.The changes of central macular thickness (CMT) and best corrected visual acuity (BCVA) were observed at baseline and 2, 4, 8wk after treatment.We also focused on the incidence of complications and relapse.RESULTS: The CMT was 445.95±154.10μm, 338.83±138.34μm, 251.50±40.20μm, 244.90±35.68μm at baseline, 2, 4 and 8wk after treatment, respectively.The differences among them were statistically significant (F=15.467, P<0.05).The BCVA (log MAR) were 0.40±0.17, 0.28±0.21, 0.19±0.20, 0.18±0.21 at baseline, 2, 4 and 8wk respectively, with a significant difference among them (F=5.208, P<0.05).When the cumulative dose reached to 700mg and 1400mg, no one had methazolamide-related complications;and when it came to 2800mg, 5 patients (33%) had methazolamide-related complication.After the withdrawal of methazolamide 1wk, 1 and 3mo, 3 patients (20%), 5 patients (33%) and 8 patients (53%) relapsed, respectively.CONCLUSION: Methazolamide is beneficial in improving macular edema and vision in 4wk.When the cumulative dose is more than 1400mg, we need pay attention to the complications.After discontinuing methazolamide for 1wk, macular edema relapsed in some patients, and more than half of patients recurred after 3mo.So the patients should be followed closely in 3mo after withdrawal of methazolamide.

This research aims to construct a lentiviral expression vector carrying the extracelluar domain (ED) of human hepatocyte growth factor receptor (C-Met), and to express it in transfected 293T cells. The extracellular domain of C-Met was amplified by RT-PCR, ligated with lentiviral expression vector p RRL-CMV-ED, and then expressed in 293T cell line. The expressed protein was purified and identified by RT-PCR and Western blot. The enzyme digestion and sequence analysis showed that the lentiviral expression vector p RRL-CMV-ED was constructed correctly. The size of amplified genes was about 2 700 bp. The purified protein with Ni-affinity column was about 105 kD analyzed by SDS-PAGE. The Western blot and ELISA results showed that the expressed protein which could bind to HGF specifically was the extracelluar domain of human hepatocyte growth factor receptor. This research may lay a foundation for further study of anti-C-MET monoclonal antibody and neutralizing antibody.
Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; Proto-Oncogene Proteins c-met ; genetics ; metabolism ; Transfection

Objective To investigate the effect of platelet-derived growth factor-D (PDGF-D) on the prostate cancer cells migration and its possible mechanism. Methods The expressions of PDGF-D in LNCaP and PC-3 cells were detected with western blot.PDGF-D siRNA was synthesized according to mRNA sequence of PDGF-D gene and was transfected into PC-3 cell.The cells were treated with PDGF-D and PDGF-D siRNA,the cell migration was examined by Boyden chamber migration assay.The expression changes of VEGF and MMP-9 mRNA were detected by RT-PCR. Results The results of western blot indicated that the PDGF-D protein expression level was lower in LNCaP cells (29.47 ± 1.68) than that in PC-3 cells (63.43 ±2.10),(P ＜ 0.05).PDGF-D siRNA could down-regulate the PDGF-D protein expression in the transfected group (35.19 ± 1.51).The exogenous PDGF-D could promote migration of LNCaP and PC-3cells,and up-regulate the expression of VEGF,MMP-9 mRNA in PC-3 cells (P ＜ 0.05,compared with control group).PDGF-D siRNA inhibited PC-3 cells' migration and decreased the level of VEGF,MMP-9mRNA expression (0.72 ± 0.09 vs 0.43 ± 0.18,0.65 ±0.07 vs 0.22 ± 0.08) (P ＜ 0.05). Conclusion PDGF-D is involved in the promotion of prostate cancer invasion and angiogenesis.

Effects of main environmental factors, such as temperature, pH , metal ions and anions, on expression and activity of bacterial extracellular car bonic anhydrase(CA) were studied, exemplified by a bacterium named GLRT102Ca, wh ich was separated from karst ecosystems of Southwest China. The results showed that the tested strain could express different activity of extracellular carboni c anhydrase within the scope of experimental temperature (10℃~50℃) and pH (5. 5~9.0). The activity of extracellular carbonic anhydrase was higher at tempera ture of 20℃~30℃ and at neutral and alkaline trending condition. Moreover, the expression of activity of extracellular carbonic anhydrase could be generally p romoted at the experimental range of concentration of 4 kinds of metal ions such as Ca2+, Mg2+, Zn2+ and Co2+, along with 8 kinds of ani ons such as SO2-_4, H_2PO-_4, NO-_3, NO-_2, Cl-,Br-, I- and HCO-_3. This research provides a cert ain theoretical basis for further study on the role of microbial CA in karst pro cesses.

Objective To investigate the expression of ADAM8 in patients with hepatocellular carcinoma (HCC) and its clinical significance.Methods The protein expression of ADAM8 in HCC tissues was analyzed using immunohistochemical analysis.Serum levels of ADAM8 were measured by ELISA in 126 patients with HCC,50 patients with liver cirrhosis (LC) and 50 healthy individuals.The relationship between patients' pathological features and serum ADAM8 level was analyzed.Results Immunohistochemical analysis showed that ADAM8 expression was associated closely with serum AFP elevation,tumor size,histological differentiation,and tumor stage.The ELISA assay showed that the serum levels of ADAM8 in the HCC were significantly higher than those in LC and healthy groups.Kaplan-Meier survival analysis showed that high expression of serum ADAM8 exhibited a significant correlation with poor prognosis for HCC patients.Multivariate analysis revealed that serum ADAM8 expression is an independent prognostic parameter for the overall survival rate of HCC patients.Conclusion ADAM8 expression was closely associated with tumor size,serum AFP elevation,tumor differentiation,tumor stage and prognosis in hepatocellular carcinoma.Therefore,ADAM8 expression may serve as a biomarker for predicting the prognosis of patients in hepatocellular carcinoma.