Objective To observe the effect of DNA methyltransferase 1 (DNMT1 ) gene silencing by RNA interfering technology on the proliferation and apoptosis of HeLa cells. Methods Recombinant plasmid pshRNA-DNMT1-A, B and C were respectively transfected into HeLa cells by lipofectamine 2000, while cells transfected plasmid vector pSilencer3.1-HI and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and western blotting was used to detected the mRNA and protein expression of DNMT1 in HeLa cells transfected for 24, 48 and 72 hours. Cell counting kit-8 (CCK-8 ) assay was used to investigate the proliferation of the HeLa cells after transfection, while apoptosis was detected by flowcytometry(FCM ) method. Results Three DNMT1-targeted short hairpin RNA (shRNA) A,B and C were successfully inserted into the plasmid vector PShRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragments. The results indicated that both recombinant plasmid pshRNA-DNMT1-A and B could effectively knock down the expression of DNMT1 gene in human cervical cancer cells, of which pshRNA-DNMTI-B was the better choice. While no effect of pshRNA-DNMTI-C was seen. BT-PCR results showed that the relative mRNA expression of DNMT1 gene in Helm cells transfected with pshRNA-DNMT1 for 24, 48 and 72 hours were 0.406±0.057,0.191±0.036 and 0.104±0.015, which were significantly lower than that in Helm cells transfected by empty vector and non-transfected cells (0.520±0.020, 0.537±0.041, respectively, P ＜ 0.05 ). The western blotting analysis manifested that the relative expression of DNMT1 protein of Helm cells transfected by pshRNA-DNMT1 for 24, 48 and 72 hours were 0.197±0.024, 0.075±0.015, 0.040± 0.013, which were significantly lower than that in transfected cells by empty vector and non-transfected cells (0.273±0.010, 0.283±0.016, respectively, P ＜0.05). The CCK-8 results showed that the cell survival rates of HeLa cells transfected by pshRNA-DNMT1 for 24, 48, 72, 96 and 120 hours were 70.8%, 64.8%, 51.6%, 45.3% and 38.0%, there were statistically different compared with cells transfected by empty vector and non-transfected cells at different time-points (P ＜ 0.01 ). The results of FCM indicated that the apoptesis rate of HeLa cells trandected with pshRNA-DNMTI for 24, 48 and 72 hours were (17.7± 1.3 ) %, (35.3±1.3 ) %, (47.6±1.6 ) %, which were significantly higher than empty vector transfected cells and non-transfected cells [(4.9±0.5 ) %, (5.1±0.7 ) %, respectively, P ＜ 0.05]. Conclusions DNMT1 can be successfully silenced by RNA interfering in cervical Helm cells. Downregulation of DNMT1 can inhibit cervical cancer cells proliferation and induce cell apoptosis.

OBJECTIVE:To systematically review the efficacy and safety of Danshu capsule in the treatment of chronic chole-cystitis,and provide evidence-based reference for clinical treatment. METHODS:Retrieved from PubMed,EMBase,Cochrane Li-brary,Web of science,CJFD,CBM,VIP Database and Wanfang Database,randomized controlled trials(RCT)about Danshu capsule versus other medicines in the treatment of chronic cholecystitis were collected. Meta-analysis was performed by using Rev Man 5.2 software after data extracting and quality evaluating by Cochrane 5.1.0. RESULTS:Totally 12 RCTs were enrolled,involving 1140 patients. Results of Meta-analysis showed the cure rate of Danshu capsule in the treatment of chronic cholecystitis was higher than control group,there was statistically significant difference between 2 groups [RR=1.62,95%CI(1.33,1.96),P<0.001];total effec-tive rate in Danshu capsule group was higher than Ursodeoxycholic acid tablet group[RR=1.37,95%CI(1.14,1.64),P<0.001], Xiaoyan lidan tablet group [RR=1.40,95%CI(1.24,1.58),P<0.001],Jindan tablet group[RR=1.13,95%CI(1.04,1.23),P=0.005] and Danning tablet group[RR=1.16,95%CI(1.05,1.28),P=0.004],there were statistically significant differences among groups. Incidence of adverse reactions of Danshu capsule was lower than control group,there was statistically significant difference between 2 groups [RR=0.20,95%CI(0.12,0.34),P<0.001]. CONCLUSIONS:Both efficacy and safety of Danshu capsule are good in the treatment of chronic cholecystitis.

ObjectiveTo investigate the clinical value of serum complement C3 and C4 levels for predicting the severity of hepatic fibrosis in patients with chronic hepatitis B.MethodsHistopathological diagnosis was confirmed in 442 patients with chronic hepatitis B.Serum complement C3 and C4 levels were determined by Beckman-Coulter Immage 800 immunochemistry system.ROC curve was used to analyze the value of serum complement C3 and C4 levels in predicting the severity of hepatic fibrosis.ResultsThe areas under ROC curve of complement C3 and C4 for predicting significant fibrosis ( ≥ S2),severe fibrosis ( ≥ S3) and cirrhosis (S4) were all significantly larger than the area under diagonal reference line ( P =0.009,0.000,0.000 and P =0.005,0.000,0.000,respectively).According to ROC curves,the optimal cut-offs of serum complement G3 for predicting severe fibrosis and cirrhosis were ≤0.74 g/L and ≤0.64 g/L,and the corresponding sensitivity,specificity,positive predictive value,negative predictive value,accuracy were 0.585,0.681,0.617,0.650,0.636 and 0.509,0.775,0.423,0.830,0.710,respectively.The optimal cut-offs of serum complement C4 for predicting severe fibrosis and cirrhosis were ≤0.14 g/L and ≤0.12 g/L,and the corresponding sensitivity,specificity,positive predictive value,negative predictive value,accuracy were 0.565,0.634,0.576,0.623,0.602 and 0.463,0.781,0.407,0.818,0.704,respectively.ConclusionSerum complement C3 and C4 may be used for predicting severe fibrosis and cirrhosis in patients with chronic hepatitis B,but its stability and reliability need to be improved.

Objective To investigate the role of a transcription factor p 53 in dengue virus infec-tion.Methods A plasmid expressing siRNA specific for p 53 gene was constructed and then used to prepare HepG2 cell line with a suppressed expression of p 53 protein.The expression of p53 protein was detected by Western blot assay .A wild type control group and a siRNA group were set up by infecting wildtype HepG 2 cells and p53 low expressing HepG2 cells with type 2 dengue viruses,respectively.The virus titers in two dif-ferent cells were determined by plaque forming assay using Vero cells .Indirect immunofluorescence assay was performed to detect virus multiplication .The apoptosis of virus infected cells were analyzed by flow cytome-try.ELISA was performed to analyze the levels of IFN-βsecreted by infected cells from two groups .Results Compared with wildtype control group ,the cells in siRNA group showed a suppressed expression of p 53 pro-tein,suggesting that the HepG2 cell line with low p53 protein expression was successfully established .The vi-rus titer in supernatants of the cells from siRNA group was about 100-fold higher than that of wildtype control group at 24 hours after viral infection .Fluorescence activated cell sorting analysis showed that the numbers of green fluorescence labeled cells were remarkably increased in siRNA group .We speculated that p53 protein might play a role in the inhibition of dengue virus infection as indicated by the observed results .The numbers of apoptotic cells showed no significant difference between two groups .However,the level of IFN-βsecreted by wildtype HepG2 cells was six times higher than that of the cells in siRNA group .Conclusion p53 pro-tein might inhibit dengue virus infection through the activation of type Ⅰ interferon signaling pathway rather than enhance cell apoptosis .