Objective To study exogenous expression and subcellular localization of wild type (WT ) and mu-tant PAX3 proteins in vitro by generating their expression plasmids for further study of pathogenesis of Waarden-burg syndrome (WS) .Methods The plasmids pECE-PAX3 and pcDNA3 .0-HA were ligased after they were cut by double enzyme digestion using molecular cloning technique to generate recombinant eukaryotic expression plasmid pcDNA3 .0-PAX3-HA ,which was as a template to generate expression plasmids pcDNA 3 .0 -H80D -HA and pcDNA3 .0-H186fs-HA of novel mutations H80D and H186fs of PAX3 gene .All constructs were verified by di-rect nucleotide sequencing .NIH3T3 cells were transfected transiently with the expression plasmids of PAX3 ,H80D and H186fs respectively .The exogenous expression of WT PAX3 protein and mutant H80D ,H186fs proteins were analysed using Western blot assay ,while their subcellular distribution were observed using immunofluorescence as-say .Results The DNA sequences of expression plasmids of PAX3 and its mutant H80D ,H186fs were correct . Both WT and mutant PAX3 proteins were detected at the expected size .WT PAX3 and H80D proteins were only lo-calized in the nucleus ,whereas H186fs protein showed aberrant localization in both cytoplasm and nucleus .Conclu-sion We successfully generated the recombinant eukaryotic expression plasmids of PAX 3 gene and its mutants and drew preliminary conlusion of gene mutation having effect on subcellular distribution of WT PAX 3 proteins in vitro , which lays experimental basis for further study of the moceluar mechanism of WS caused by PAX3 gene mutations in China .

Platelet-activation factor (PAF), one of the potent proinflammatory mediators, is produced from a large range of cells, including polymorphonuclear neutrophils, monocytes, and natural killer cells. To study the role of PAF in the pathogenesis of silicosis, we determined the PAF in silicotic patients and in healthy persons. The results showed that the concentration of PAF in the plasma of silicotic patients was significantly higher than that of healthy persons. Our in vitro experimental results showed that the total numbers of fibroblasts were markedly raised with added PAF from 0 to 1 μ g/ml. Adding 1 μ g/ml PAF significantly increased the total numbers of fibroblasts after culture for 48, 72, 96 hrs. Therefore, we suggest that PAF be possibly involved in the pathogenesis of silicosis. However, the mechanism remains to be further elucidated.
Platelet Activating Factor ; g <3> ; Pathogenesis ; /mL ; Effective

Aim To investigate the role of TGF-β3 in the anti-proliferation effect of ursolic acid(UA)in co-lon cancer cells and the possible molecular mechanism underlying this effect.Methods We introduced crys-tal violet staining,flow cytometry and Western blot as-say to determine the effect of UA on proliferation and apoptosis in HCT1 1 6 cells.The levels of TGF-β3, Smad2 /3 and β-catenin in HCT1 1 6 cell were evaluated by RT-PCR and Western blot.Finally,TGF-β3 inhibi-tor and recombinant adenovirus,and luciferase reporter assay were used to analyze the possible mechanism through which TGF-β3 mediated the anti-cancer effect of UA in HCT1 1 6 cells.Results UA inhibited the proliferation and induced apoptosis apparently in HCT1 1 6 cells.UA down-regulated TGF-β3 both in mRNA and in protein level.Meanwhile,UA decreased the phosphorylation of Smad2 /3 concentration depend-ently,although no significant effect was found on the total protein level of Smad2 /3 in HCT1 1 6 cells.Over-expression of TGF-β3 attenuated the inhibitory effect of UA on the proliferation of HCT1 1 6 cells,while the TGF-β3 inhibitor potentiated this effect. UA sup-pressed the transconduction of Wnt/β-catenin signaling in HCT1 1 6 cells through decreasing the level of β-catenin.Exogenous expression of TGF-β3 increased the level of β-catenin and partly reversed the UA-in-duced decrease of β-catenin.However,TGF-β3 inhib-itor potentiated the inhibitory effect of UA on β-catenin in HCT1 1 6 cells.Conclusion The anti-proliferation activity of UA in colon cancer may be partly mediated through down-regulating TGF-β3 to suppress Wnt/β-catenin signaling at least.

Aim To study the anti-proliferation effect of resveratrol (Res)and the role of Res-induced bone morphorgenetic protein 9 (BMP9 )in this process in colon cancer cells.Methods Crystal violet staining and flow cytomtry were introduced to assay the anti-proliferation effect of Res in LoVo cells.The effect of Res on apoptosis in LoVo cells was also detected with flow cytometry.Then,RT-PCR and Western blot assay were employed to unveil the effect of Res on the ex-pression of BMP9 .The effect of BMP9 on the anti-pro-liferation of Res in LoVo cells was analyzed with crystal violet staining and flow cytometry too.Finally,the effect of Res on the expression of ALK2 and ALK3 was assayed with RT-PCR,and the inhibitor of ALK2 and ALK3 was used to figure out the possible mechanism of BMP9 on Res-induced proliferation inhibition in LoVo cells.Results Res apparently inhibited the prolifera-tion,arrested the cell cycle at S phase in LoVo and in-creased the percentage of apopotic cells in LoVo cells. Res increased the expression of mRNA and protein of BMP9 concentration dependently. Exogenous ex-pressed-BMP9 enhanced the anti-proliferation and ap-optosis inducing effects of Res in LoVo cells, but BMP9 knockdown decreased these effects of Res.Al-though Res had no apparent effect on increasing the phosphorylation of Smad1/5/8,it increased the ex-pression of ALK2 and ALK3 .Inhibition of ALK2 and ALK3 decreased the anti-proliferation effect of Res partly in LoVo cells.Conclusion Res is potent to in-hibit the proliferation of LoVo cells,Which may be mediated by up-regulating the expression of BMP9 and its receptor at least.

Aim To investigate the relationship be-tween the anti-proliferation effect of resveratrol ( Res ) and p38 MAPK in colon cancer cells .Methods Crys-tal violet staining , Western blot and flow cytometry were employed to analyze the effect of Res on the pro-liferation in LoVo cells.Western blot assay was used to detect the effect of Res on the apoptosis of LoVo cells and the phosphorylation of p 38 MAPK.Crystal violet staining and Western blot assay were used to analyze whether p38 MAPK was involved in the Res-induced proliferation inhibition and apoptosis in LoVo cells .Re-sults Res inhibited the proliferation , arrested cell cy-cle at S phase , and increased the protein level of PC-NA in LoVo cells apparently .Res increased the level of Bad in LoVo cells, but decreased the level of Bcl-2. Although Res exerted no substantial effects on total lev-el of p38 MAPK, it markedly increased the phospho-rylation level of p38 MAPK in LoVo cells.p38 MAPK inhibitor promoted the proliferation , and decreased the anti-proliferation effect of Res on LoVo cells .Moreo-ver , the effects of Res on the level of Bcl-2 and Bad were both reduced by the p 38 MAPK inhibitor .Con-clusions Res can inhibit the proliferation of LoVo cells, which may be partly mediated by promoting the phosphorylation of p38 MAPK.

Objective To evaluate the immunogenicity and safety of a booster dose of live attenua-ted measles-mumps-rubella (MMR) vaccine for 4-year-old children and to provide references for reasonable arrangement of MMR immunization schedule. Methods Children aged 4 years (54-60 months) old were recruited and divided into three groups as follows: Group 8 months MR [receiving live attenuated measles and rubella(MR) vaccination at 8 months and MMR vaccination at 18 months],Group 8 months MMR(re-ceiving MMR vaccination at both 8 and 18 months) and Group 12 months MMR(receiving MMR vaccination at both 12 and 22 months). Active follow-up was conducted for safety evaluation after immunization of all subjects with the booster dose of MMR vaccine. Blood samples were collected before and 35 days after vacci-nation and analyzed by ELISA to detect serum antibodies to measles,mumps and rubella. Results A total of 514 subjects were enrolled in this study of safety evaluation and 469 of them received serologic detection of antibodies twice. The rate of adverse reactions following vaccination was 17.12% (general reactions accoun-ted for 94.21%) and no severe adverse reactions were reported. No significant difference in the rates of ad-verse reactions was found among the three groups (χ2=4.82, P=0.090). Subjects who were seropositive for measles, mumps and rubella increased after immunization with MMR vaccine, accounting for 100%, 99.79% and 99.79%,respectively. Geometric mean concentrations (GMC) against measles, mumps and rubella in all subjects were 1.35,3.05 and 2.13 times higher than what they were before the immunization. Levels of antibodies to measles,mumps and rubella were all increased significantly in the three groups after immunization with the booster dose of MMR vaccine (Fisher Exact Test, P=0.000). Conclusion The booster dose of MMR vaccine increases the levels of serum antibodies in children aged 4 years old with high safety. It suggests that two doses of MMR vaccine should be encouraged in the immunization program in China.

Objective To investigate the clinical efficacy of three-dimensional visualization technique (3DVT) combined with enhanced recovery after surgery (ERAS) in the treatment of hepatolithiasis.Methods The retrospective cohort study was conducted.The clinicopathological data of 64 patients with hepatolithiasis who were admitted to Zhujiang Hospital of Southern Medical University from November 2015 to August 2018 were collected.There were 17 males and 47 females,aged from 30 to 82 years,with a median age of 55 years.Of the 64 patients,23 who completed preoperative assessment and planning using 3DVT,and furthermore received ERAS for perioperative management were divided into 3DVT + ERAS group,and 41 who received preoperative assessment merely under the guidance of 3DVT,combined with conventional perioperative management were divided into 3DVT + conventional group.Observation indicators:(1) preoperative CT and 3DVT assessment;(2) perioperative conditions;(3) follow-up.The follow-up was conducted by outpatient service,e-mail or telephone interview to detect the postoperative recurrence of hepatolithiasis up to March 2019.The measurement data with normal distribution were expressed as Mean±SD,and the t test was used for comparison between groups.The measurement data with skewed distribution were expressed as M (P25,P75),and the Mann-Whitney U test was used for comparison between groups.The count data were expressed as absolute numbers or percentages,and the comparison between groups was pedormed using the chi-square test or Fisher exact probability.Results (1) Preoperative CT and 3DVT assessment:23 patients in the 3DVT + ERAS group underwent preoperative CT examination and 3DVT assessment,the consistency between CT results and intraoperative findings was 91.3％ (21/23),and the consistency between 3DVT results and intraoperative findings was 95.7％(22/23).Fourty-one patients in the 3DVT + conventional group underwent preoperative CT examination and 3DVT assessment,the consistency between CT results and intraoperative findings was 90.2％ (37/41),and the consistency between 3DVT results and intraoperative findings was 95.1％ (39/41).(2) Perioperative conditions:the volume of intraoperative blood loss,duration of postoperative hospital stay,postoperative total bilirubin,postoperative direct bilirubin,postoperative albumin,postoperative alanine aminotransferase,postoperative aspartate aminotransferase and postoperative hemoglobin were 50 mL (10 mL,100 mL),8 days (7 days,9 days),12 μmol/L (9 μmol/L,16 μmoL/L),6 μmol/L (4 μmoL/L,8 μmol/L),(37±4)g/L,44 U/L (18 U/L,85 U/L),32 U/L (20 U/L,65 U/L),(117±18)g/L in the 3DVT + ERAS group,and 100 mL (50 mL,300 mL),13 days (10 days,16 days),17 μmol/L (12 μmoL/L,33 μmoL/L),11 μmoL/L (7 μmoL/L,21 μmol/L),(29±6)g/L,78 U/L (43 U/L,122 U/L),121 U/L (72 U/L,176 U/L),(106±13)g/L in the 3DVT + conventional group,respectively;there were significant differences between two groups (Z =-3.084,-4.827,-2.953,-3.632,t =5.261,Z=-2.960,-4.625,t =2.773,P＜0.05).Two patients had pulmonary infection and 2 had pleural effusion in the 3DVT + ERAS group,and all the 4 patients were cured after treatment.One case of biliary fistula,4 cases of pulmonary infection and 5 cases of pleural effusion occurred in the 3DVT + conventional group,and these patients were cured by adequate abdominal drainage,antibiotic therapy and thoracocentesis,respectively.There was no perioperative death in either group.(3) Follow-up:64 patients were followed up for 6-36 months,with a median time of 23 months.During the follow-up,no recurrent hepatolithiasis in the 3DVT + ERAS group,and 1 case of recurrent hepatolithiasis was confirmed by ultrasound in the 3DVT + conventional group.No cholangiocarcinoma occurred in either group.Conclusion The combination of 3DVT and ERAS is effective,safe and feasible in the management of hepatolithiasis,which can accelerate the postoperative recovery of liver function,thus enhancing perioperative recovery and improving the prognosis of patients simultaneously.