3.Establishment and application of a high-throughput drug screening model based on COL1A1 promoter for anti-liver fibrosis.
Shuang-Shuang ZHAO ; Ju-Xian WANG ; Yu-Cheng WANG ; Rong-Guang SHAO ; Hong-Wei HE
Acta Pharmaceutica Sinica 2015;50(2):169-173
For screening the potential drugs as anti-liver fibrosis candidates, we established a high- throughput drug screening cell model based on COL1A1 promoter. The activity of COL1A1 promoter and luciferase reporter gene can be elevated by TGF-β1, and inhibited by candidate drugs. We constructed a recombined plasmid with COL1A1 promoter and luciferase reporter gene pGL4.17, the activity of COL1A1 promoter was reflected by fluorescence intensity. COL1A1 promoter activity was detected by Dual-Luciferase Reporter Assay System, it came that the relative luciferase activity of COL1A1 promoter was 15.98 times higher than that of control group induced by TGF-β1, showing the recombined plasmid could be used in cell model. The recombined plasmid was transfected into human hepatic stellate cells LX2, detected the effect of potential drugs, and obtained a stable expression system through stable transfection and monoclonal cell culture. A sample which could reduce COL1A1 promoter activity signally by our cell model, decreased collagen I mRNA and protein expression detected by real-time RT-PCR and Western blotting. It indicates this novel cell model can be used in high-throughput drug screening of potential anti-liver fibrosis drugs.
Collagen Type I
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genetics
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Drug Evaluation, Preclinical
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methods
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Genes, Reporter
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Hepatic Stellate Cells
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High-Throughput Screening Assays
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Humans
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Liver Cirrhosis
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drug therapy
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Luciferases
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Plasmids
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Promoter Regions, Genetic
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RNA, Messenger
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Transfection
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Transforming Growth Factor beta1
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pharmacology
4.Decoration reconstruction for finger defect in emergency
Ji-Hui JU ; Rui-Xing HOU ; Yue-Fei LIU ; Qiang ZHAO ; Lei LI ; Guang-Zhe JIN ;
Chinese Journal of Microsurgery 2006;0(06):-
Objective To discuss methods of decoration reconstruction for finger defect in emergency and to observe the elinical effects.Methods Of the 41 cases of finger injuries of different degrees,15 were repaired with part of the skin flaps of the big toenails or skin flaps of the second toenalis,8 were repaired with part of the skin flaps of the big toenails,7 were reeonstructed with the second tiptoes,11 were repaired with the abdominal skin flaps of the big toes or lateral flaps of the second toes.Results All the 41 fingers sur- vived.One skin flap of the big toe was somewhat swelling and a decorating operation was performed.The 4~18 months of follow-up visitation of the rest cases revealed good function and shapes.No obvious functional ab- norality was found in the donating feet.Conclusion Various kinds of decoration reeonstruetion for finger defects are available to recover the hand shape and function as much as possible.
5.Single-center report of 5-year follow-up on 94 patients underwent transmyocardial laser revascularization.
Zheng QU ; Ju-bing ZHENG ; Zhao-guang ZHANG
Chinese Medical Journal 2007;120(22):1982-1985
BACKGROUNDTransmyocardial laser revascularization (TMLR) has been used in the treatment of patients with end-stage coronary artery disease (CAD) since 1990. The aim of this study was to evaluate the long-term effectiveness of TMLR in patients with diffuse CAD.
METHODSNinety-four consecutive patients underwent TMLR in one center from July 1997 to December 2000. The follow-up data of these patients were obtained through face-to-face, mail questionnaires, or telephone interviews in July 2004 and December 2004. Four cases failed to respond. Mean follow-up time was (5.5 +/- 1.0) years.
RESULTSMean Canadian Cardiovascular Society (CCS) angina scores of TMLR patients were 3.1 +/- 0.8 at baseline, 1.7 +/- 0.9 at 1 year (P < 0.05), 1.7 +/- 0.9 at 3 years (P < 0.05), and 1.9 +/- 0.9 at 5 years (P < 0.05). At an average of 5 years, 69% of the patients had > or = 1 angina class reduction, mean NYHA class level (1.9 +/- 0.9) ameliorated compared to the baseline (2.5 +/- 0.7, P < 0.001), the rate of re-hospitalization was 2.7 times/person. Kaplan-Meier survival rate was 87% at 1 year, 69% at 3 years and 64% at 5 years. The causes of death were attributed more to heart failure (58.9%) and myocardial infraction (14.7%) after TMLR. The patients with no angina relief, or who died after TMLR, had a higher percentage of preoperative unstable anginas or prior myocardial infraction compared to the survivors. The assorted shapes of myocardial laser channels were detected in some patients by the color Doppler velocity technique.
CONCLUSIONSTMLR provided a long-term improvement in the quality of life, including CCS angina class or NYHA heart functional class for about 70% of Chinese patients with severely disabling angina pectoris. The various myocardial laser channels would always be visible after TMLR. 5-years after TMLR as a sole therapy, the survival rate of the patients was 64%.
Aged ; Coronary Artery Disease ; mortality ; psychology ; surgery ; Female ; Follow-Up Studies ; Humans ; Laser Therapy ; methods ; Male ; Middle Aged ; Myocardial Revascularization ; methods ; Quality of Life ; Tomography, Emission-Computed, Single-Photon ; Ventricular Function, Left
6.Expression of MICA/B protein in esophageal cancer and its clinical significance.
Jia-zhuan MEI ; Ji-zhi ZHAO ; Guang-ying YANG ; Fang-fang GAO ; Gui-ju LIU
Chinese Journal of Oncology 2012;34(10):745-747
OBJECTIVETo explore the expression of MICA/B in human esophageal cancer, and to analyze its correlation with clinicopathological features.
METHODSThe expression of MICA/B in 40 cases of esophagus carcinoma and corresponding normal esophageal mucosa tissues were examined by immunohistochemistry.
RESULTSThe positive rate of expression of MICA/B protein in the esophageal carcinoma was 75.0% (30/40), and that in the corresponding normal esophageal mucosa was 0 (0/40). Up-regulation of MICA/B expression was found in the esophageal carcinomas. The expression of MICA/B was related with histological grade of the esophageal carcinoma (P = 0.012).
CONCLUSIONMICA/B protein plays an important role in the esophageal carcinogenesis, and my become a useful molecular marker for the diagnosis of esophageal carcinoma.
Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; metabolism ; Esophageal Neoplasms ; diagnosis ; metabolism ; pathology ; Female ; Histocompatibility Antigens Class I ; metabolism ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Neoplasm Grading ; Up-Regulation
7.Effect of thalidomide in a mouse model of paraquat-induced acute lung injury and the underlying mechanisms.
Dong LI ; Li-yan XU ; Zi-juan CHANG ; Guang-ju ZHAO ; Chao NAN ; Zhong-qiu LU
Chinese Journal of Industrial Hygiene and Occupational Diseases 2013;31(3):178-183
OBJECTIVETo investigate the intervention effect of thalidomide on paraquat-induced acute lung injury in mice and its mechanism.
METHODSMale ICR mice were randomly allocated to negative control group (n = 30), thalidomide control group (n = 30), paraquat poisoning group (n = 30), 50 mg/kg thalidomide treatment group (n = 30), 100 mg/kg thalidomide treatment group (n = 30), and 150 mg/kg thalidomide treatment group (n = 30). The negative control group was intraperitoneally injected with the same volume of saline; the thalidomide control group was intraperitoneally injected with thalidomide (150 mg/kg); the paraquat poisoning group was intraperitoneally injected with diluted paraquat solution (22 mg/kg); each thalidomide treatment group was intraperitoneally injected with the same volume of paraquat solution (22 mg/kg) and was injected with thalidomide (50, 100, or 150 mg/kg) 1 h later. All mice were anesthetized and sacrificed at 1, 3, or 7 d after paraquat poisoning, and their lung tissue was collected. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in lung tissue were measured by double-antibody sandwich ELISA; the mRNA expression of nuclear factor-kappa B (NF-κB) was measured by RT-PCR; the protein expression of nuclear NF-kgr;B p65 was measured by Western blot. The pathological changes of lung tissue were observed under light microscope; the wet/dry ratio of the lung was calculated.
RESULTSCompared with the negative control group, the paraquat poisoning group had significantly increased levels of TNF-α, IL-1β, IL-6, NF-κB mRNA, and nuclear NF-κB p65 and wet/dry ratio of the lung (P < 0.05). Compared with the paraquat poisoning group, the thalidomide treatment groups had significantly decreased levels of TNF-α, IL-1β, IL-6, NF-κB mRNA, and nuclear NF-κB p65 and wet/dry ratios of the lung (P < 0.05), and the 150 mg/kg thalidomide treatment group showed the most significant decrease in the levels of TNF-α, IL-1β, IL-6, NF-κB mRNA, and nuclear NF-κB p65. The observation of pathological changes showed that the paraquat poisoning group had the most marked lung tissue damage at 3 d after poisoning, and the lung tissue damage was lessened in the thalidomide treatment groups.
CONCLUSIONThalidomide can reduce paraquat-induced acute lung injury and lung edema. The mechanism may include inhibition of NF-κB activation and expression and downregulation of TNF-α, IL-1β, and IL-6.
Acute Lung Injury ; chemically induced ; drug therapy ; Animals ; Cytokines ; metabolism ; Disease Models, Animal ; Male ; Mice ; Mice, Inbred ICR ; NF-kappa B p50 Subunit ; metabolism ; Paraquat ; poisoning ; Thalidomide ; pharmacology ; Transcription Factor RelA ; metabolism
8.Expression and diagnostic value of plasma miR-145 and miR-183 in children with lupus nephritis.
Lie-Ju LIN ; Lang-Jun MAI ; Guang CHEN ; Er-Nong ZHAO ; Ming XUE ; Xian-Du SU
Chinese Journal of Contemporary Pediatrics 2020;22(6):632-637
OBJECTIVE:
To study the expression and diagnostic value of plasma miR-145 and miR-183 in children with lupus nephritis (LN).
METHODS:
A total of 92 children with LN who were admitted from January 2016 to May 2019 were enrolled as the LN group, among whom 17 had type II LN, 15 had type III LN, 36 had type IV LN, 18 had type V LN, and 6 had type VI LN. Forty healthy children who underwent physical examination were enrolled as the healthy control group. According to Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), the 92 children with LN were further divided into a stable LN group with 34 children (SLEDAI score <10) and an active LN group with 58 children (SLEDAI score ≥10). RT-PCR was used to measure the expression of miR-145 and miR-183 in plasma. The receiver operating characteristic (ROC) curve was used to analyze the value of plasma miR-145, miR-183, and anti-dsDNA antibody in the diagnosis of LN. Pearson correlation analysis was used to investigate the correlation of the expression levels of miR-145 and miR-183 in plasma with laboratory markers.
RESULTS:
The LN, active LN, and stable LN groups had significantly higher levels of anti-dsDNA antibody, C-reactive protein, serum creatinine (Scr), and blood urea nitrogen (BUN) than the control group (P<0.05). The active LN group had significantly higher SLEDAI score, anti-dsDNA antibody, Scr, and BUN than the stable LN group (P<0.05). The LN, active LN, and stable LN groups had significantly lower levels of complement C3, complement C4, and serum albumin (Alb) than the control group (P<0.05). The active LN group had a significantly lower level of Alb than the stable LN group (P<0.05). The LN, active LN, and stable LN groups had significantly lower plasma levels of miR-145 and miR-183 than the control group (P<0.01). The active LN group had significantly lower plasma levels of miR-145 and miR-183 than the stable LN group (P<0.01). The children with difference types of LN had significantly lower plasma levels of miR-145 and miR-183 than the control group (P<0.01), and the type V-VI group and the type IV group had significantly lower plasma levels of miR-145 and miR-183 than the type II-III group (P<0.01). The ROC curve analysis showed that the optimal cut-off values of plasma miR-145, miR-183, and anti-dsDNA antibody were 1.05, 0.62, and 186.30 IU/mL respectively, in the diagnosis of LN, and the combination of these three indices had the largest area under the ROC curve of 0.896 (95%CI: 0.835-0.955), with a sensitivity of 90.5% and a specificity of 84.2%. In the children with LN, the plasma levels of miR-145 and miR-183 were negatively correlated with SLEDAI score, anti-dsDNA antibody, Scr, and BUN (P<0.05) and were positively correlated with complement C3, complement C4, and Alb (P<0.05).
CONCLUSIONS
There are significant reductions in the expression levels of miR-145 and miR-183 in plasma in children with LN, which are correlated with the activity level and pathological typing of LN. Combined measurement of miR-145, miR-183, and anti-dsDNA antibody has a high value in the diagnosis of LN.
Biomarkers
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Child
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Complement C4
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Humans
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Lupus Nephritis
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genetics
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MicroRNAs
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genetics
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ROC Curve
9.Construction and Expression of Plasmid Coexpressing Human Papillomavirus Type 11 E7 and Human IFN?-2b
Wen-Zhong XIANG ; Fei WANG ; Guang-Ju LI ; Xin-Jun WANG ; Qun WANG ; Feng LIU ; Zhao-Song ZHANG ; Zhi-gang Bi
Chinese Journal of Dermatology 2003;0(10):-
Objective To construct an expression plasmid of human papillomavirus type 11 E7 (HPV11-E7)/hurnan IFN?-2b fusion gene, to express the fusion gene in E.coli BL21, and pave way for further immunological study. Methods The recombinant plasmid was introduced into E.coli BL21, then the expression product was analyzed by SDS-PAGE and Western blotting after induction with isopropy-?-D-thiogalactoside (IPTG). Results The fusion gene of HPV11-E7 and human IFN?-2b was successfully cloned into pET-32a by a linker with the same sequence as we expected. The expressed fusion protein was confirmed by SDS-PAGE and Western blotting. Conclusions The successful construction of prokaryotic expression plasmid and expression of HPV11-E7/human IFN?-2b fusion gene enable further immunological study.
10.Clinical and Genetic Analysis for 10 Patients with 17? Hydroxylase/17, 20 Lyase Deficiency
jun, YANG ; xiao-ying, LI ; shou-yue, SUN ; jie, QIAO ; yong-ju, ZHAO ; jian-min, LIU ; guang, NING ; man-yin, XU ; jia-lun, CHEN
Journal of Shanghai Jiaotong University(Medical Science) 2006;0(01):-
Objective To analyze the clinical and genetic characteristics of 10 Chinese patients with 17? hydroxylase/17,20 lyase deficiency (17OHD). Methods Clinical features and laboratory data were collected from 7 kindreds with 17OHD. PCR products and subclone sequencing were performed to screen the mutation of CYP17A1 gene. Results All patients had typical clinical presentation of sexual infantilism, hypertension and hypokalemia. The laboratory examinations indicated decreased plasma cortisol, 17-hydroxy progesterone, estradiol and testosterone, and elevated blood adrenocorticotrophic hormone(ACTH), follcie-stimulating hormone(FSH) and luteinizing hormone(LH). CT scan showed bilateral adrenal hyperplasia. 5 CYP17A1 mutations were identified, 4 of which are novel types D487_F489del, the most frequent mutation, was identified in 4 families and 45% alleles. Conclusion Our study indicates that 17OHD should be considered in the diagnosis of patients with sexual infantilism. D487_F489del is the most frequent mutation in Chinese 17OHD patients.