Objective To compare the three kinds of methods for in vitro primary culturing of rheumatoid arthritis synovial fibroblast-like cells (RASFs), in order to get fast and effective culture methods. Methods Synovial tissue from RA synovial arthroscopic resection were treated by collagenase digestion method, modified tissue culture method, double enzyme digestion method respectively. By using an inverted phase contrast microscope, cell morphology and growth characteristics were observed and identified with vimentin staining. Trypan blue was used to count the number of living cells after culturing for 14d. Results The three primary methods could successfully isolate and culture RASFs, and RASFs met the morphological characteristics of vimentin-positive cells>95%, namely, the proportion of RASFs cell confluence was 70% after 16-20days by the collagenase digestion method,whose cell confluence proportion reached 95%after 4 weeks;and the cell confluence proportion was above 70%after 10-14days by modified tissue culture method,and the cell confluence proportion reached 85%after 4 weeks by the double enzyme digestion method. The comparison of the viable cells number cultured same number of synovial tissue by the three methods show the viable cells number cultured by the modified tissue culture method were (1.60±0.08) ×106, those by the collagenase digestion method were (1.41±0.08) ×106, those by the double enzyme digestion method were (1.19 ±0.05) ×106, which were with significant difference among them (P<0.05) .The comparison of incubation time of RASFs primary cells showed it took (267.50±16.58) mins by the collagenase digestion method, (183.75 ±11.08) mins by the double enzyme digestion method, and 149.10 ±13.71mins by the modified tissue culture method, with significant differences (P<0.05) .Conclusion Modified tissue culture for RASFs is an efficient and fast culture method, the number and purity of RASFs can meet the requirements for biology experiments.

Objective To study the targeting survivin small interfering RNA ( siRNA ) to inhibit proliferation and apoptosis survivin gene expression in rheumatoid arthritis synovial fibroblasts ( RASFs) .Methods RA patients were isolated and cultured in vitro synovial fibroblasts ( RASFs) , designed and synthesized siRNA targeting survivin and negative control, by liposome transfection RASFs cell; real-time quantitative polymerase chain reaction (PCR) and Western blot RASFs detect mRNA expression and protein levels of survivin.Tetrazolium blue (MTT) assay of cell proliferation;TUNEL assay apoptosis.Results The experimental group compared with the negative control siRNA group and control group, 48h after transfection of synovial fibroblasts survivin mRNA and protein expression levels were significantly decreased ( P<0.05 ) .The experimental group compared with the negative control siRNA group and control group, synovial fibroblast proliferation after transfection significantly decreased ( P<0.05 ) . After the experimental group transfected 24h, 48h, 72h growth inhibition rates were (11.5 ±2.6)%, (26.2 ±3.4)%, (47.6 ±4.1)%, at 72 hours after transfection most significant.The rate of apoptosis in experimental group (23.87 ±1.6)%, significantly higher than the negative control group (9.72 ± 1.15)% and the control group (8.70 ±1.09)% (all P<0.05).Conclusion siRNA targeting survivin expression levels through reducing survivin, inhibit synovial fibroblast proliferation and promotes apoptosis.

Objective:To investigate the protective effect of Yiqi Huazhi recipe Quqian granules on rat renal tubular cell apoptosis induced by lead poisoning. Methods:Totally 60 Wistar rats were divided into 2 groups, 12 in the control group and the others in the model group. Chronic lead poisoning model was made by drinking 0. 02% lead acetate water for 60 days. Then the lead poisoning rats were randomly divided into four groups, high-dose Quqian granules group (3. 0 g·kg-1·d-1), low-dose Quqian granules group (0. 6 g·kg-1 ·d-1 ) , positive control group ( calcium disodium edentate plus procaine, im, 50 mg·kg-1 ·d-1 ) and model group. Seven treatment courses were carried out in the first three groups with every 4-d as one course and 4-d withdrawal period between every two courses. After 60 days, the change of lead in blood and kidney was observed by atomic absorption spectrometry,the apoptosis of kidney tissues was studied by TUNEL, the expression of Bcl-2 protein was detected by immunohistochemical methods and the expression of p53 was studied by Western Blotting. Results:Compared with the control group, the body weight, hemoglobin and the expression of Bcl-2 in the model group were decreased significantly(P<0.01)those in, and Pb in blood(0.990 ±0.443)μg·ml-1, Pb in kidney(51.33 ± 5. 16)μg·ml-1 , the apoptosis of tubular epithelial cell(4. 148 ± 0. 414) and the expression of p53 protein (1. 868 ± 0. 139) were significantly higher (P<0. 05). Compared with the those in model group, the body weight, hemoglobin and Bcl-2 in high-dose group were increased significantly(P<0.01), and the blood lead level (0.082 ±0.015)μg·ml-1, the kidney lead level (6.38 ±0.97)μg ·ml-1 , the apoptosis of tubular epithelial cell(1. 412 ± 0. 109) and p53 protein expression(1. 164 ± 0. 172) were significantly lower (P<0. 05). Conclusion:Lead may induce high expression of p53,low expression of Bcl-2 and promote the apoptosis of renal tubular epithelial cells. It is proven that Yiqi Huazhi recipe Quqian granules can inhibit the expression increase of p53 and the expression de-crease of Bcl-2 resulting in the reduction of the renal tubular apoptosis to allivate the renal injury caused by lead.

Objective To investigate the effect of short hairpin RNA(shRNA) eukaryotic expression vector‐mediated silen‐cing of the survivin‐gene on proliferation and apoptosis of human umbilical vein endothelial cells(HUVEC) .Methods The shRNA vector targeting the survivin gene and negative control vector were transfected into human umbilical vein endothelial cells(HUVEC) incubated with 50 ng/mL of recombinant VEGF in vitro by lipofectamine 2000 .Transfection after 48 h ,the expression of survivin mRNA and protein was detected by quantitative real‐time PCR and Western blot ,respectively .HUVEC proliferation was assayed by four methylthiazolyl tetrazolium(MTT) and cell apoptosis was detected by TUNEL .Results (1)Transfection with survivin‐shR‐NA vector significantly down‐regulated the expression of survivin mRNA and protein as compared with the control group ,after transfection of 48 h(P<0 .05) .(2)After survivin‐shRNA vector transfected ,the proliferation of HUVEC decreased significantly . After transfection 24 ,48 ,72 h ,the growth inhibition rate were (13 .53 ± 3 .91)% ,(38 .97 ± 1 .82)% ,(65 .75 ± 1 .83)% respective‐ly ,at 72 hours after transfection was the most significant .(3)The apoptosis rate of experimental group was (28 .07 ± 1 .71)% , which was higher than the negative control group (11 .45 ± 1 .52)% and blank control group (10 .04 ± 1 .46)% (P<0 .05) .Conclu‐sion The shRNA‐mediated mediated silencing of the survivin‐gene could significantly inhibit proliferation and promote the apopto‐sis of rheumatoid arthritis synovial fibroblasts by regulating survivin expression .

Objective To compare the efficacy and safety of gefitinib, erlotinib, and afatinib in the first-line treatment of advanced non-small cell lung cancer (NSCLC). Methods PubMed, EMBASE, and The Cochrane Library were searched to identify the relevant literatures published from December 2008 to December 2018. Bayesian network meta-analysis was carried out to rank the three treatments. Results A total of ten eligible studies involving 2275 patients were enrolled. In terms of efficacy, the surface under the cumulative ranking (SUCRA) indicated that erlotinib performed best in progression-free survival(PFS)(0.88), afatinib performed best in objective response rate(ORR)(0.82) and disease control rate(DCR) (0.86), gefitinib performed worst in PFS (0.45), ORR(0.42), and DCR(0.45). For safety, the differences of grade 3 or 4 adverse events rate (OR=0.29,95%CI:0.08-0.98) and discontinuation rate(OR=0.14,95%CI:0.01-0.8) between erlotinib and the platinum-based doublet chemotherapy were statistically significant. The ranking results also supported that erlotinib was the safest. SUCRA results suggested that gefitinib (0.31) had a lower grade 3 or 4 adverse events rate than afatinib (0.57), and the possibility of discontinuation in gefitinib (0.44) was similar to that of afatinib (0.41). Conclusion Erlotinib might be the preferred first-line treatment for advanced NSCLC after weighing and balancing the benefits and risks.

Objective To explore the expression levels and significance of T cell subgroups in the peripheral blood of patients with primary Sj?gren′s syndrome (PSS).Methods Flow cytometer was applied to detect CD3 +,CD3 +CD4 +CD8 -,CD3 +CD4 -CD8 +,CD3 +CD4 -CD8 - and CD4 +CD25 +high expression levels in 32 PSS patients (observation group)and another 35 healthy people tak-ing physical examination (control group),which were then compared between groups.Results There were no significant differences between two groups in CD3 + and CD3 + CD4 - CD8 + expressions (P >0.05).The ratios of CD4 +/CD8 + and CD3 +CD4 +CD8 - subgroups increased evidently (P <0.01), while those of CD3 + CD4 - CD8 - double-negative T cells and CD4 + CD25 +high subgroup decreased markedly in observation group (P <0.01).Conclusion T cell subgroup expressions are abnormal in PSS patients,and over proliferation and infiltration of CD4 + cells is the risk factor of auto-immunologi-cal diseases,while the decrease of CD3 +CD4 -CD8 - double-negative T cells and CD4 +CD25 +high sub-group can lead to the damage of body immunological tolerance,which may be one of the important fac-tors for the development of PSS.

Objective To explore the expression levels and significance of T cell subgroups in the peripheral blood of patients with primary Sj?gren′s syndrome (PSS).Methods Flow cytometer was applied to detect CD3 +,CD3 +CD4 +CD8 -,CD3 +CD4 -CD8 +,CD3 +CD4 -CD8 - and CD4 +CD25 +high expression levels in 32 PSS patients (observation group)and another 35 healthy people tak-ing physical examination (control group),which were then compared between groups.Results There were no significant differences between two groups in CD3 + and CD3 + CD4 - CD8 + expressions (P >0.05).The ratios of CD4 +/CD8 + and CD3 +CD4 +CD8 - subgroups increased evidently (P <0.01), while those of CD3 + CD4 - CD8 - double-negative T cells and CD4 + CD25 +high subgroup decreased markedly in observation group (P <0.01).Conclusion T cell subgroup expressions are abnormal in PSS patients,and over proliferation and infiltration of CD4 + cells is the risk factor of auto-immunologi-cal diseases,while the decrease of CD3 +CD4 -CD8 - double-negative T cells and CD4 +CD25 +high sub-group can lead to the damage of body immunological tolerance,which may be one of the important fac-tors for the development of PSS.

OBJECTIVETo assess the association of three polymorphisms (14-bpINS/DEL, +3035C/T and +3142C/G) in the 3' untranslated region (3'UTR) of HLA-G gene and systemic lupus erythematosus (SLE) in Yunnan.

METHODSA case-control study has been carried out on 206 SLE patients and 212 healthy controls. Genotypes of 14-bpINS/DEL (rs1704), +3035C/T (rs17179108) and +3142C/G (rs1063320) loci of 3'UTR of the HLA-G gene were determined with DNA sequencing.

RESULTSAllelic and genotypic frequencies of 14-bpINS/DEL and +3142C/G did not differ significantly between the two groups (P > 0.05). The frequencies of +3035T allele was significantly higher in the SLE group compared with the control group (P < 0.01, OR=1.604, 95% CI: 1.186-2.169). With a dominant inheritance model, the odd ratio of dominant genotype (CT+TT) was 2.004 (95% CI: 1.345-2.987, P=0.0006) in the SLE group.

CONCLUSIONThe 14-bpINS/DEL and +3142C/G polymorphisms in the 3'UTR of the HLA-G gene are not associated with susceptibility to SLE in Yunnan, whilst the T allele of +3035C/T may be a risk factor for SLE.

3' Untranslated Regions ; genetics ; Case-Control Studies ; China ; Female ; Genetic Predisposition to Disease ; HLA-G Antigens ; genetics ; Humans ; Lupus Erythematosus, Systemic ; genetics ; Male ; Polymorphism, Genetic

The purpose of this study was to investigate the effects of Celecoxib on the proliferation of the FLT3-ITD positive and negative acute myeloid leukemia cells and its mechanism. The proliferation inhibition effect of Celecoxib with different doses on the FLT3-ITD positive cells MV4-11 and the FLT3-ITD negative K562 cells was detected by CCK-8 method, the cell apoptosis was determined by flow cytometry, and the MEK, Mcl-1, pAKT expression was tested by Western blot. The results showed that Celecoxib inhibited the proliferation of both MV4-11 and K562 cells, but the IC50 for MV4-11 was (29.14 ± 2.4) µmol/L, which was significantly lower than that of K562 cells (39.84 ± 1.0) µmol/L (P < 0.05); The induced apoptosis rate of Celecoxib at 20-80 µmol/L on MV4-11 was not observed, but there was apparent influence on K562 at the same concentration. Western blot showed that Celecoxib down-regulated the expression of MEK and Mcl-1 but did not change the expression of pAKT obviously on MV4-11 cells, while the expression of Mcl-1 was reduced a little, but no obvious change were found in the expression of MEK and pAKT on K562 cells. It is concluded that the Celecoxib can inhibit the proliferation of FLT3-ITD positive AML cells distinctly, and the potential mechanism may be related to the inhibition of the MEK/Mcl-1 signaling pathway.
Apoptosis ; drug effects ; Celecoxib ; Cell Proliferation ; drug effects ; Cyclooxygenase 2 Inhibitors ; pharmacology ; Gene Expression Regulation, Leukemic ; Humans ; K562 Cells ; Leukemia, Myeloid, Acute ; drug therapy ; metabolism ; pathology ; MAP Kinase Kinase 1 ; genetics ; Myeloid Cell Leukemia Sequence 1 Protein ; genetics ; Proto-Oncogene Proteins c-akt ; genetics ; Pyrazoles ; pharmacology ; Signal Transduction ; Sulfonamides ; pharmacology ; fms-Like Tyrosine Kinase 3 ; genetics

OBJECTIVETo map the disease-causing gene in a Chinese family with autosomal dominant retinitis pigmentosa.

METHODSTwenty-seven micro-satellite markers were randomly selected from the region around the known loci of causative genes, and haplotypes were determined by ABI3100 genetic analyzer. Two-point linkage analysis was performed using MLINK.

RESULTSThe Lod score of each marker vs adRP was below 1.

CONCLUSIONThe phenotype of this family may not be caused by mutation of the known disease-causing genes.

Asian Continental Ancestry Group ; genetics ; China ; Female ; Genes, Dominant ; Genetic Linkage ; Genetic Testing ; Humans ; Male ; Microsatellite Repeats ; genetics ; Mutation ; Pedigree ; Phenotype ; Retinitis Pigmentosa ; diagnosis ; genetics ; pathology