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ObjectiveTo investigate the intervention mechanism of Dendrobium officinale leaf fermentation fluid in mice with alcoholic hepatitis. MethodsA total of 70 healthy male C57BL/6J mice， aged 6-8 weeks， were randomly divided into normal group， model group， liquid feed control group， silybin group， and low-， middle-， and high-dose Dendrobium officinale leaf fermentation fluid groups， with 10 mice in each group. The mice in the normal group were given normal diet， and those in the other groups were given Lieber-DeCarli classic liquid diet for 8 weeks to induce alcoholic hepatitis. During modeling， the mice in the low-， middle-， and high-dose Dendrobium officinale leaf fermentation fluid groups were given Dendrobium liquid manufactured by Warmen Pharmaceutical， and the mice in all the other groups were given pure water； the mice in the normal group， the model group， and the liquid feed control group were given normal saline by gavage， those in the silybin group were given silybin 0.25 mL/10 g by gavage， and those in the low-， middle-， and high-dose Dendrobium officinale leaf fermentation fluid groups were given Dendrobium officinale leaf fermentation fluid at a dose of 0.125 mL/10 g， 0.250 mL/10 g， and 0.375 mL/10 g， respectively， by gavage， once a day. At week 8， chloral hydrate was injected intraperitoneally for anesthesia， and blood samples were collected from the eyeball. After serum was separated， the biochemical method was used to measure the levels of aspartate aminotransferase （AST） and alanine aminotransferase （ALT）； HE staining and oil red staining were used to observe liver histopathology and lipid accumulation in mice； multiplex Luminex assay was used to measure the serum levels of interleukin-6 （IL-6）， interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， and CCL2； quantitative real-time PCR， Western blot， and immunofluorescence assay were used to measure the protein expression levels of NLRP3， caspase-1， caspase-11， gasdermin D （GSDMD）， N-terminal gasdermin D （GSDMD-N） in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the normal group， the model group had significant increases in the serum levels of AST， ALT， IL-6， IL-1β， TNF-α， and CCL2 （all P<0.05）， and compared with the model group， the high-dose Dendrobium officinale leaf fermentation fluid group had significant reductions in the serum levels of AST， ALT， IL-6， IL-1β， TNF-α， and CCL2 （all P<0.05）. HE staining showed that the model group had disordered structure of hepatic lobules， with a large number of steatosis vacuoles and massive cell necrosis， and compared with the model group， the high-dose Dendrobium officinale leaf fermentation fluid group had alleviation of liver histopathological injury， intact structure of most hepatic lobules， and a small amount of inflammatory cell infiltration. Oil red staining showed that the model group had accumulation of large and small lipid droplets in the liver and a significant increase in liver fat content， and compared with the model group， the high-dose Dendrobium officinale leaf fermentation fluid group had significant alleviation of hepatic steatosis， with the presence of sporadic small lipid droplets. Immunofluorescence assay of liver tissue showed that compared with the normal group， the model group had a significant increase in the ratio of GSDMD-positive staining area in hepatocyte cytoplasm （P<0.001）， and compared with the model group， the high-dose Dendrobium officinale leaf fermentation fluid group had a significant reduction in such ratio in hepatocyte cytoplasm （P<0.001）. Quantitative real-time PCR showed that compared with the normal group， the model group had significant increases in the protein expression levels of NLRP3， caspase-1， caspase-11， GSDMD， GSDMD-N， interleukin-18 （IL-18）， and IL-1β in liver tissue （all P<0.05）， and compared with the model group， the high-dose Dendrobium officinale leaf fermentation fluid group had significant reductions in the protein expression levels of NLRP3， caspase-1， caspase-11， GSDMD， GSDMD-N， IL-18， and IL-1 （all P<0.05）. Compared with the model group， the high-dose Dendrobium officinale leaf fermentation fluid group had significant reductions in the protein expression levels of caspase-1 and caspase-11 （both P<0.05）， with a relative expression level of caspase-1 of 1.757 （reduced by 26.6% compared with the model group） and a relative expression level of caspase-11 of 0.455 （reduced by 70.3% compared with the model group）， suggesting that caspase-11 showed a greater reduction than caspase-1. ConclusionDendrobium officinale leaf fermentation fluid can alleviate alcoholic hepatitis in mice， possibly by inhibiting the non-classical cell pyroptosis pathway mediated by caspase-11.
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【Objective】 To conduct a case-control study on precocious puberty as an example to introduce the establishment and design of the electronic Data capture and management platform using Research Electronic data Capture (REDCap) system and support the development of clinical research. 【Methods】 Based on the clinical REDCap system, the case-control research project of precocious puberty was created, the case report forms were designed, the user rights were set, and the data quality control rules were formulated. 【Results】 We established the electronic data capture and management platform for our research, which had 15 case report forms, to collect the data of the participants, including sociodemographic information, time for rest and activities, diet, exposure to environmental internal-secretion interfering-substances, physical examination and biochemical indicators. We conducted project management by setting up features such as user permissions and workgroups, and added data quality verification rules to control data quality. The data could be exported in various file formats for analysis and sharing. 【Conclusion】 The application of REDCap to establish the data capture and management platform of precocious puberty case-control study has promoted the efficient implementation of clinical research, which can be further popularized and applied to clinical researches in other fields.
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Objective:To evaluate the expression of Vimentin and its relationship with clinical pathological data and prognosis in patients with metastatic renal cell carcinoma (mRCC).Methods:A total of 269 mRCC patients, from June 2007 to June 2017, treated with tyrosine kinase inhibitors (TKI) in Zhongshan Hospital, Fudan University were included in the study. Vimentin was assessed by immunohistochemistry on continuous paraffin embedded slides. To analyzed the relationship between Vimentin expression and clinicopathological data and prognosis. Measurement data were expressed as mean±standard deviation ( Mean± SD). The relationship between Vimentin expression and clinicopathological data of mRCC patients was determined by Chi-square test. Survival analysis used Kaplan-Meier survival curve, Log-rank test to judge the relationship between Vimentin expression and patient′s overall survival, and COX regression analysis was used for univariate and multivariate analysis. Results:Vimentin was expressed in cytoplasm of tumor cells. Vimentin expression in mRCC patients′ tissues was positively correlated with tumor Fuhrman grade ( P=0.002), and negatively correlated with prognostic overall survival ( P=0.001). Multivariate regression analysis showed that Vimentin expression was an independent risk factor for overall survival in patients with mRCC ( P=0.006). Conclusions:Vimentin expression can be used as a prognostic indicator of mRCC. It is expected that Vimentin can provide prediction for the prognosis of mRCC patients treated with TKI.
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Objective To evaluate the effect and mechanism of tribulus terrestris in kidney calcium oxalate stone in rats.Methods Sisty healthy male Wistar rats were divided into control group,model group,low-dose prevention group,high-dose prevention group,low-dose treatment group,high-dose treatment group by random number table method,each group of 10 rats.The control group was had free access to water,the model group was given 1％ ethylene glycol had free access to water,and the low-dose prevention group was given 1％ ethylene glycol had free access to water while was added in Tribulus terrestris extract 250 mg/(kg · d) treated by gavage,the high-dose prevention group was given 1％ ethylene glycol had free access to water while was added in Tribulus terrestris extract 500 mg/(kg · d) treated by gavage.The low-dose treatment group received 1％ ethylene glycol had free access to water while was added in tribulus terrestris extract 250 mg/(kg · d) treated by gavage on the 15th day.The high-dose treatment group received 1％ ethylene glycol had free access to water while was added in tribulus terrestris extract 500 mg/(kg · d) treated by gavage on the 15th day.At the end of the experiment on the 28th day,the body weight,blood urea nitrogen,creatinine,P,Ca2+,Mg2+ content,24 h urine volume,24 h urine Ca2+,Mg2 +,P,oxalic acid secretion,renal tissue homogenate detection of superoxide dismutase (SOD),glutathione peroxidase (GPX),malondialdehyde content of each group were detected.The crystal deposition and pathological changes of calcium oxalate were observed under the microscope.Measurement data were expressed as mean ± standard deviation (Mean ± SD),multiple comparisons between groups were performed using LSD-t test,and comparison between groups was performed by one-way anova analysis of variance.Results Compared with the control group,the rats in the model group had blood urea nitrogen (28.93 ±3.37) mmol/L,creatinine (162.31 ± 34.08) μmol/L,24 h uric acid (132.67 ± 27.96) μmol,Ca2+ (18.24 ± 1.94) μmol/24 h,the content of malondialdehyde (191.04 ± 20.56) nmol/mgprot in kidney homogenate was significantly increased,blood Ca2+ (2.01 ±0.28) mmol/L,renal GPX (479.82 ± 136.97) μmol/gprot,SOD(1.54 ±0.21) U/mgprot decreased significantly,these differences were statistically significant (P ＜ 0.05).Renal histopathology showed low-dose and high-dose prevention and treatment groups kidney calcium oxalate crystals and kidney damage were less than the model group.Conclusion The extract of Tribulus terrestris has a beneficial effect against calcium oxalate urinary stone formation induced by ethylene glycol.
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Objectives: To evaluate the tumor-infiltrating PD-1 positive lymphocytes and regulatory T cells (Treg) as prognosis predic-tors of metastatic renal cell carcinoma (mRCC), and investigate the expression of PD-1 and Treg cells in mRCC and elucidate their corre-lation with clinicopathological parameters and prognosis. Methods: A total of 269 mRCC patients from June 2007 to June 2017 in Zhongshan Hospital, Fudan University, were included in the study. The expression of PD-1 and Tregs in mRCC samples were detected by immunohistochemistry. The relationship between the expression of PD-1 and Tregs was analyzed. Results: PD-1 positive expression in mRCC was 31.60% (85/269) and it was positively correlated with the tumor Fuhrman grade and negatively correlated with progno-sis. Tumor infiltration of Tregs in mRCC was 36.80% (99/269), and it was also positively correlated with the tumor Fuhrman grade and negatively correlated with prognosis. Univariate analysis showed that PD-1 positive lymphocytes and high Treg infiltration numbers were negatively correlated with overall survival (OS) and progression free survival (PFS) rates. Thus, PD-1 positive lymphocytes and high Treg infiltration numbers are independent prognostic indicators of OS and PFS and when combined, they can render a better pre-diction for prognosis. Conclusions: Intra-tumoral infiltration of PD-1 positive lymphocytes and Tregs can be used as significant prognos-tic indicators of mRCC, and the combined predictive effect is better than the individual predictive effect. Therefore, evaluating the number of PD-1 positive lymphocytes or infiltrating Tregs in mRCC is helpful in clinically estimating mRCC prognosis in patients.
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Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB 1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB 1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648-2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL2 1. The expression of His tagged PB 1 protein was induced by IPTG, and His-PB 1 proteins were purified by Ni-NTA resin. For preparation of PB 1 protein antiserum, rabbits were immunized with His-PB 1 fusion protein 3 times. Then the titer of PB 1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB 1 protein from WSN virus infected or pCMV FLAG-PB 1 transfected cells. Meanwhile, PB 1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB 1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1.
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Animais , Coelhos , Anticorpos Antivirais , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Metabolismo , Vetores Genéticos , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza A Subtipo H9N2 , Plasmídeos , Proteínas Virais , Alergia e ImunologiaRESUMO
BACKGROUND:There is lack of effective diagnostic methods for early knee osteoarthritis. Proteomics refers to the large-scale experimental analysis of proteomes from the overal level of intracel ular protein compositioon, expression and modification, based on which, we can understand protein-protein interaction and relationship, thereby revealing protein functions and cel activity patterns. OBJECTIVE:To apply the isobaric tags for relative and absolute quantitation (iTRAQ) to identify proteins differential y in serums of osteoarthritis Kel gren-Lawrence classification at each stage, and to find out potential molecular markers at each stage of osteoarthritis. METHODS:Sixty patients with knee osteoarthritis were enrol ed, and according to Kel gren-Lawrence (K-L) grading, they were subdivided into K-L 0, II and IV groups. There were 10 males and 10 females randomly selected in each group. Proteins differential y regulated in serums were identified by the stable isotopes 116, 117, 118 of the iTRAQ labeled, reversed-phase column separation, mass spectrometry and Swissport database search, after the serums were subjected to high abundance proteins depletion. Final y, we analyzed the proteins identified by using bioinformatics software. RESULTS AND CONCLUSION:Total y 169 proteins were identified through iTRAQ peptides experiments of different sample tags, Q-star mass spectrum identification and MASCOT library search. 153 proteins were identified as K-L 0 to K-L IV, 153 proteins were identified as K-L 0 to K-L II, and 145 proteins were identified as K-L II to K-L IV. iTRAQ technology may help to identify novel serum markers for early diagnosis of knee osteoarthritis, indicating iTRAQ technology for proteomics serum biomarkers of osteoarthritis study has good application prospect.