1.Effect of CTGF siRNA on Apoptosis of Fibroblast-like Synoviocytes of Rheumatoid Arthritis
Shuang DING ; Fang FANG ; Hongmei DUAN ; Weiguo XIAO
Journal of China Medical University 2016;45(5):430-433
Objective To silence the expression of CTGF by small interfering RNA technology,to observe the influence on fibroblast?like synovial cell apoptosis and several apoptosis?related genes,and to explore the mechanism of action of CTGF in rheumatoid arthritis synovial lesions. Methods Effective CTGF siRNA was screened through real?time PCR. The influence of CTGF siRNA on FLS apoptosis was detected with FITC?PI double staining by flow cytometry. bax,bcl?xl and survivin were detected using real?time PCR when CTGF mRNA has been silenced. Results Compared with other 2 groups of oligo and NC oligo,H1 oligo exhibited the strongest interfering action to CTGF(inhibition ratio>70%),so that it is selected as the effective target gene sequence for the following experiment. Apoptosis of FLS induced by serum deprivation was significantly decreased in the presence of exogenous CTGF. When expression of the CTGFgene was knocked down in FLS,FLS apoptosis was significantly increased,and expres?sion levels of survivin mRNA were decreased significantly(P<0.01). Conclusion FLS survival is positively regulated by CTGF,which may through the sustaining the expression of survivin.
2.Changes of expression of FADD and Daxx following focal cerebral ischemia in rats
Yue-Qiang HU ; Bo XIAO ; Fang-Fang BI ; Ling DING ;
Chinese Journal of Neurology 2005;0(11):-
Objective To investigate the changes of expression of Fas-associated proteins named Fas-associated death domain protein(FADD)and death-associated protein(Daxx)in the ischemic penumbra following transient focal cerebra ischemia in rats.Methods ①Adult male Sprague-Dawley rats were randomly divided into the sham-operated group and the cerebral ischemia model group.Rats underwent right middle cerebral artery occlusion(MCAO)for 2 h and reperfusion for 1,3,6,12 and 24 h using an intraluminal suture technique.The expression of FADD and Daxx mRNA and protein were measured with methods of immunohistochemistry.Western blot and reverse transcription-polymerase chain reaction(RT- PCR)respectively were used in the ischemic penumbra of rats.②Double-label fluorescence confocal laser scanning microscopy(CLSM)was performed to monitor FADD and Daxx intracellular location before and after ischemia.Results RT-PCR,Immunohistochemistry,Western blot experiments indicated that a very low level of FADD mRNA and protein were detected in the cerebral cortex of sham rats.The expression level both of FADD mRNA and protein increased significantly at 3 h after reperfusion,peaked at 12 h,then declined markedly at 24 h in the ischemic penumbra of model rats.RT-PCR,Immunohistochemistry indicated that a relatively high level of Daxx mRNA was detected in the cerebral cotex of sham rats.The expression level of Daxx mRNA increased significantly at 3 h after reperfusion and persisted to 24 h at a high level,whose protein had a same change of expression level in the ischemic penumbra of model rats. Immunofluorescence double-staining laser scanning by CLSM showed that the immunoreactivity of FADD was located in cytoplasm,and the intracellular translocation of the immunoreactivity of Daxx from nucleus to cytoplasm was monitored by measuring the green fluorescence after ischemia.Conclusion The transient upregulation of FADD and the persistant high level of expression of Daxx may contribute to neuronal apoptosis following cerebral ischemia/reperfusion.
4.Evaluation of glucocorticoid-induced osteoporosis in childhood kidney diseases by quantitative ultrasound
Xiaojie PENG ; Fang WANG ; Huijie XIAO ; Jie DING
Chinese Journal of Applied Clinical Pediatrics 2015;30(17):1325-1327
Objective To observe the glucocorticoid-induced osteoporosis (GIOP) in children with kidney diseases by quantitative ultrasound (QUS), and to analyze its influencing factors.Methods The tibia/radius bone mineral density(BMD) was checked obtained in 67 cases with childhood kidney diseases treated with glucocorticoid by QUS,BMD was measured in children over the age of 12 by dual-energy X-ray absorptiometry(DXEA) ,and BMD was measured with QUS consistency and different stages of osteoporsis were compared.The clinical data of gender, age,body mass index(BMI) ,administration duration and daily dosage of glucocorticoid were analyzed.The association between the duration of glucocorticoid use,and daily dosage of glucocorticoid and the different degrees of BMD was analyzed by Logistic regression analysis.Results Sixty-seven patients(male 45 ,female 22) were divided into 4 groups according to the reference standard of Asian children BMD data provided by Sunlight Company:the normal BMD group(41 cases), the mild osteoporosis group (18 cases), moderate osteoporosis group (5 cases), and severe osteoporosis group (3 cases).Both QUS and DEXA were highly correlated with BMD in patients measured (P > 0.05).The duration of glucocorticoid treatment and daily dosage of glucocorticoid in 3 abnormal BMD groups were all significantly longer and larger than those of the normal BMD group (all P < 0.05).Correlation analysis showed that BMI was positively correlated with the bone mass of the tibia(r =0.395 ,P < 0.01).The duration of glucocorticoid treatment and daily dosage of glucocorticoid were negatively correlated with those of radius BMD(r =-0.474,-0.381 ,all P < 0.01).Analysis showed that both the duration of glucocorticoid,and the daily dosage of glucocorticoid were the risk factors for GIOP.Conclusions QUS is a better method for evaluating of BMD and diagnosing of GIOP compared with DEXA in children.The daily dosage of glucocorticoid and the duration of glucocorticoid treatment are both the risk factors for GIOP.
5.Clinical study of the influence on marginal bone levers when open healing of implants with platform ;switching
Shulan XU ; Shuo YANG ; Xijuan XIAO ; Xianglong DING ; Fang JIA
The Journal of Practical Medicine 2016;32(12):2020-2022
Objective The study examined the influence on marginal bone levers at implants which implants were assigned to open healing and implant-abutment connection restored according to a platform-switching concept. Methods One implant were placed in the first molar of mandible of 48 patients , randomly assigned to open (OH) or submerge (SH) healing. Standardized radiographs were obtained after implant surgery, when implant transfer and crown mounting, and 6 ,12 and 24 months after implants restoration, then evaluated for implant-bone-lever alterations(ΔIBL). Results After 6 months after implants restoration,ΔIBL were (-0.25 ± 0.08)mm mm(OH)and (-0.26 ± 0.12)mm(SH). After 12 months follow-up, ΔIBL were (-0.29 ± 0.13)mm (OH)and (-0.31 ± 0.14) mm(SH), and after 24 months later, ΔIBLwere (-0.34 ± 0.15)mm(OH)and (-0.33 ± 0.11)mm (SH). Conclusions Platform-switched implants showed very limited peri-implant bone-lever alterations. The healing-mode neither affected the total amount nor the temporal patterns of ΔIBL.
6.Relationship between hyperhomocysteinemia and chronic kidney disease in children
Jiapeng SUN ; Huijie XIAO ; Jie DING ; Fang WANG ; Jing LIU
Chinese Journal of Applied Clinical Pediatrics 2017;32(11):852-855
Objective To investigate the detection rate and possible factors of hyperhomocysteinemia(HHcy) in children with chronic kidney disease(CKD).Methods The clinical data of children with CKD between July 2012 and September 2016 in the Department of Pediatrics,Peking University First Hospital were retrospectively collected.The homocysteine(Hcy) level of patients were measured.The other data included the general information,diagnosis and laboratory test results.Results Seventy-six pediatric patients with CKD were enrolled including 49 boys and 27 girls.The average age of the patients was (9.9±3.4) years old.The main cause of the patients in the study was primary glomerulopathy(48.7%,37/76 cases),and the rest were congenital and inherited glomerular diseases(36.8%,28/76 cases),secondary glomerular diseases(9.2%,7/76 cases)and renal tubular diseases(5.3%,4/76 cases).Fifty patients (65.8%,50/76 cases) had normal level of Hcy which was 10.40(7.30,11.62) μmol/L.Twenty-six patients(34.2%,26/76) were detected with HHcy whose Hcy level was 17.93(16.76,24.11) μmol/L.The detection rate of HHcy in CKD stage 1,stage 2,stage 3,stage 4 and stage 5 was 13.9%(5/36 cases),22.2%(2/9 cases),50.0%(4/8 cases),57.1%(4/7 cases) and 68.8%(11/16 cases) respectively,and the detection rate increased with CKD stages and the difference was statistically significant (χ2=17.574,P<0.001).The level of Hcy was 10.05(7.04,12.47) μmol/L,11.75(10.78,16.44) μmol/L,13.73(10.09,18.23) μmol/L,15.81(11.12,20.71) μmol/L and 17.39(11.86,24.76) μmol/L in CKD stage 1,stage 2,stage 3,stage 4 and stage 5,respectively.The Kruskal-Wallis test revealed that the distribution of homocysteine in CKD stages had statistically significant difference(P=0.001).Multiple linear regression model showed that creatinine clearance was an independent predicator of HHcy.Conclusions In this study of the CKD patients,the detection rate of HHcy was high and increased with the progression of CKD.HHcy is mainly influenced by creatinine clearance in CKD.The level of Hcy should be monitored regularly in children with CKD and HHcy should be treated with proper measures.
7.Simultaneous determination of amlodipine, benazepril and benazeprilat in human plasma by LC-HESI/MS/MS method.
Hua-Ling PAN ; Li-Shan LIN ; Jue-Fang DING ; Xiao-Yan CHEN ; Da-Fang ZHONG
Acta Pharmaceutica Sinica 2014;49(1):95-100
The study aims to develop a rapid, sensitive and specified method of liquid chromatography with heated electrospray ionization tandem mass spectrometry (LC-HESI/MS/MS) for simultaneous determination of amlodipine, benazepril and benazeprilat in human plasma using amlodipine-d4 and ubenimex as internal standards (ISs). Selected reaction monitoring (SRM) with heated electrospray ionization (HESI) was used in the positive mode for mass spectrometric detection. Analytes and ISs were extracted from plasma by simple protein precipitation. The reconstituted samples were chromatographed on a C18 (100 mm x 4.6 mm, 5 microm) column with mixture of methanol-acetonitrile-5 mmol.L- ammonium acetate-formic acid (30 : 30 : 40 : 0.1) as mobile phase at a flow rate of 0.6 mL.min-1. The standard curves were demonstrated to be linear in the range of 0.02 to 6.00 ng.mL-1 for amlodipine, 0.2 to 1,500 ng.mL-1 for benazepril and benazeprilat with r2>0.99 for each analyte. The lower limit of quantitation was identifiable and reproducible at 0.02, 0.2 and 0.2 ng mL-1 for amlodipine, benazepril and benazeprilat, respectively. The intra-day and inter-day precision and accuracy results were within the acceptable limit across all concentrations. The plasma samples were stable after four freeze-thaw cycles and being stored for 93 days at -20 degrees C. The method was applied to a pharmacokinetic study of a fixed-dose combination of amlodipine and benazepril on Chinese healthy volunteers.
Administration, Oral
;
Amlodipine
;
administration & dosage
;
blood
;
Benzazepines
;
administration & dosage
;
blood
;
Chromatography, Liquid
;
Humans
;
Sensitivity and Specificity
;
Spectrometry, Mass, Electrospray Ionization
;
Tandem Mass Spectrometry
8.The efficacy and impact of recombinant human cytotoxic T lymphocyte-associated antigen-4 fusion protein on human tumor necrosis factor-α and CX3CL1 in active rheumatoid arthritis patients
Rong ZHANG ; Chunling WU ; Liping XIA ; Fang FANG ; Shuang DING ; Hongmei DUAN ; Weiguo XIAO
Chinese Journal of Rheumatology 2012;16(7):458-462
Objective To evaluate the efficacy,safety and impact of recombinant human cytotoxic T lymphocyte-associated antigen (CTLA)-4 fusion proteins (rhCTLA-4Ig) on serum human tumor necrosis factor (TNF)-α and CX3CL1 in active rheumatoid arthritis (RA) patients.Methods Forty-four RA patients were treated with rhCTLA-4Ig and placebo.Clinical response was assessed by American College of Rheumatology (ACR) criteria and disease activity score in 28 joints (DAS28).The levels of serum TNF-α and CX3CL1 were determined in 44 RA patients and 20 healthy controls by enzyme-linked immunosorbent assay (ELISA).Comparisons between groups were performed by t-test or x2 test.Results At week 12,ACR20,ACR50and ACR70 responses in RA patients with rhCTLA-4Ig were achieved by 95%(20/21 ),76%( 16/21 )and 19%(4/21) respectively,but no patient with placebo achieved ACR20,ACRS0 and ACR70 responses.There were significantly statistical differences in ACR20 and ACR50 responses (x2=39.17,26.69,P<0.01 ).At week 12,the mean DAS28 in the rhCTLA4Ig group was 3.1±1.3 versus 6.2±1.1 at baseline (P<0.01).Similarly,health assessment questionnaire (HAQ) improved significantly,declining from 1.4±0.5 at baseline to 0.4±0.5 at week 12 (P<0.01).However,the mean DAS28 in the placebo group was 5.8±1.2 versus 6.0±0.7 at baseline (P>0.05),HAQ declined from 1.6±0.4 to 1.6±0.6 (P>0.05).In addition,there were higher levels of TNF-α and CX3CL1 in the active RA patients than those of the healthy controls (P<0.01).After 12 weeks therapy,Serum TNF-α and CX3CL1 levels in the rhCTLA-4Ig group decreased significantly (P<0.01).There weren't decline in the placebo group (P>0.05).Conclusion This study has shown that rhCTLA-4Ig is very effective in reducing disease activity,improving function during the 12 weeks treatment.rhCTLA-4Ig therapy for 12 weeks can lead to significant decrease of serum TNF-α and CX3CL1.
10.Changes in skeletal metabolism of glucocorticoids-treated children with kidney diseases
Dahai WANG ; Fang WANG ; Yong YAO ; Huijie XIAO ; Xuhui ZHONG ; Xiaoyu LIU ; Jie DING
Chinese Journal of Applied Clinical Pediatrics 2017;32(5):346-349
Objective To understand the skeletal metabolism in glucocorticoids-treated children with kidney diseases and to evaluate the role of Alfacalcidol plus calcium supplements.Methods A retrospective self-controlled study was performed in children with kidney diseases,admitted to Department of Pediatrics,Peking University First Hospital from July 2012 to November 2014,and they conformed to the inclusion and exclusion criteria.Inclusion criteria:children with renal diseases were included when they were administered with glucocorticoids,Alfacalcidol and calcium at the enrollment and follow-up,whose proteinuria was in remission or improved on follow-up,and renal function defined by glomerular filtration rate was more than 80 mL/(min · 1.73 m2),and they were followed up for at least 3 months.Exclusion criteria:whereas on follow-up,children with renal diseases were excluded if glucocorticoids was discontinued,or the follow-up period was less than 3 months,and related parameters were not available at the initial and finial visit.Serum levels of calcium,phosphate,alkaline phosphatase,25-hydroxy-vitamin D (25-OH-VitD) were detected,and bone mineral density at the left radius and mid tibia was measured by using quantitative ultrasound at the initial and finial visit.The levels of calcium,phosphate,alkaline phosphatase,25-OH-VitD and bone mineral density were compared between the initial and finial visit.Results A total of 29 cases (23 male,6 female) were recruited.Their ages at study entry were 2.4 to 14.9 years.Underlying disorders were primary nephrotic syndrome(17 cases),IgA nephropathy (4 cases),lupus nephritis (3 cases),purpura nephritis (2 cases),acute glomerulonephritis (1 case),Sjogren's disease(1 case),and autoimmune disease(1 case).At the time of enrollment,the course of disease was 1 to 69 months (median course of disease was 8 months),the duration of using corticosteroid was 0-60 months (median duration was 5 months),and the follow-up time was 92-409 days.The serum calcium,alkaline phosphatase and 25-OH-VitD levels at finial visit[(2.29 ±0.11) mmol/L,(153.21 ±71.00) U/L,and (40.96 ± 11.40) nmol/L,respectively] were significantly higher as compared with those at initial visit [(2.17 ± 0.16) mmol/L,(114.10 ±64.48) U/L,and (31.64 ± 10.99) nmol/L,respectively],and the differences were significant (all P < 0.01),whereas there was no significant difference in serum level of phosphorate between initial and finial visit[(1.45 ±0.25) mmol/L vs.(1.48 ± 0.25) mmol/L] (t =-0.452,P > 0.05).Meanwhile,there was significant difference in serum level of albumin level between initial and finial visit [(31.41 ± 9.31) g/L vs.(42.09 ± 4.00) g/L,t =-3.670,P < 0.001].Compared with baseline assessment,bone mineral density at the left radius and tibia improved or maintained in 24/29 patients,whereas that of 5/29 patients declined.However,compared with initial and finial visit of bone mineral density in all patients(0.09 ± 1.58 vs.0.22 ± 1.07 at the left radius;0.93 ± 1.13 vs.1.00 ± 1.35 at the mid tibia),there was no significant difference (all P > 0.05).Conclusions Bone loss can be prevented by Alfacalcidol plus calcium supplement in glucocorticoids-treated children with kidney diseases.