Objective:To investigate the role of autophagy mediated by mTOR signaling pathway in the inhibition of osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) induced by cadmium.Methods:HBMSCs were divided into 0, 2.5 or 5.0 μmol/L groups according to the exposure dose of cadmium chloride (CdCl 2), and each group was treated for 1 day, 4 days and (or) 7 days. The ALP activity and mRNA and protein expression levels of osteogenesis markers (ALP, RUNX2 and OSTERIX), autophagy-related proteins (LC3 and Beclin-1) and mTOR signaling pathway related proteins (mTOR, p-mTOR and p-p70S6K) expression, alkaline phosphatase staining and alizarin red staining were detected. MHY 1485 was selected as the signaling pathway activator. The control group, CdCl 2 group (5.0 μmol/L), MHY 1485 group and CdCl 2+MHY 1485 combined treatment group were set. After 7 days of treatment, the expression levels of autophagy related proteins and mTOR signaling pathway related proteins of hBMSCs in each group were detected. Results:There was no significant difference in ALP activity between 0, 2.5 and 5.0 μmol/L groups on day 1 and 4 ( P>0.05); On day 7, compared with the 0 μmol/L group, the ALP activity, expression of osteogenic markers (ALP, RUNX2, OSTERIX) and mTOR signaling pathway related proteins (mTOR, p-mTOR, p-p70S6K) expression decreased in the 2.5 and 5.0 μmol/L group ( P<0.05). Compared with the 0 μmol/L group, the staining of the 2.5 and 5.0 μmol/L groups became lighter, and the formation of ALP and mineralized nodules was reduced. Compared with the CdCl 2 group, the autophagy related protein expression in the CdCl 2+MHY 1485 combined treatment group decreased, and the mTOR signaling pathway related protein expression increased. The difference was statistically significant ( P<0.05). Conclusion:The inhibition of osteogenic differentiation of hBMSCs by cadmium may be related to autophagy mediated by mTOR signaling pathway.

Objective:To investigate the role of autophagy mediated by mTOR signaling pathway in the inhibition of osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) induced by cadmium.Methods:HBMSCs were divided into 0, 2.5 or 5.0 μmol/L groups according to the exposure dose of cadmium chloride (CdCl 2), and each group was treated for 1 day, 4 days and (or) 7 days. The ALP activity and mRNA and protein expression levels of osteogenesis markers (ALP, RUNX2 and OSTERIX), autophagy-related proteins (LC3 and Beclin-1) and mTOR signaling pathway related proteins (mTOR, p-mTOR and p-p70S6K) expression, alkaline phosphatase staining and alizarin red staining were detected. MHY 1485 was selected as the signaling pathway activator. The control group, CdCl 2 group (5.0 μmol/L), MHY 1485 group and CdCl 2+MHY 1485 combined treatment group were set. After 7 days of treatment, the expression levels of autophagy related proteins and mTOR signaling pathway related proteins of hBMSCs in each group were detected. Results:There was no significant difference in ALP activity between 0, 2.5 and 5.0 μmol/L groups on day 1 and 4 ( P>0.05); On day 7, compared with the 0 μmol/L group, the ALP activity, expression of osteogenic markers (ALP, RUNX2, OSTERIX) and mTOR signaling pathway related proteins (mTOR, p-mTOR, p-p70S6K) expression decreased in the 2.5 and 5.0 μmol/L group ( P<0.05). Compared with the 0 μmol/L group, the staining of the 2.5 and 5.0 μmol/L groups became lighter, and the formation of ALP and mineralized nodules was reduced. Compared with the CdCl 2 group, the autophagy related protein expression in the CdCl 2+MHY 1485 combined treatment group decreased, and the mTOR signaling pathway related protein expression increased. The difference was statistically significant ( P<0.05). Conclusion:The inhibition of osteogenic differentiation of hBMSCs by cadmium may be related to autophagy mediated by mTOR signaling pathway.

Objective To evaluate the efficacy and safety of autologous peripheral blood stem cell transplantation (APBSCT) in treatment of patients with decompensated hepatitis B cirrhosis.Methods Sixty two patients with decompensated hepatitis B cirrhosis admitted in Ningbo Second Hospital during January 2010 and December 2013 were enrolled in the study.Patients were randomly assigned in two groups: 50 patients in control group received comprehensive medical treatment only, and 12 patients in combination group received APBSCT on the basis of medical treatment.The levels of serum total bilirubin (TBil), albumin (Alb), alanine aminotransferase (ALT) and prothrombin time (PT) in two groups were mearsured at the 4th,12th,24th week.Overall survival (OS),progression-free survival (PFS) and complications were compared between two groups after 3 years follow-up.SPSS17.0 software was used to analyze the data.Results After APBSCT treatment, the level of Alb and PT at week 4,12 and 24 in combination group improved significantly(tAlb=-4.437,-5.210 and-6.915,tPT=12.083,11.251 and 10.640,all P<0.01),there were also significant differences between combination group and control group (tAlb=4.985, 5.565 and 6.260,tPT =-3.013、-3.727 and-3.983,all P<0.01).The 3-year OS and 3-year PFS of patients in combination group were higher than those of control group [(90.9±8.7)%vs.(60.7±7.4)%, (75.8±12.5)% vs.(47.9±7.3)%](χ2=6.887 and 5.565,P<0.05).Besides,APBSCT had more advantages than control group in reducing ascitic fluid and hepatic encephalopathy(χ2=7.992 and 4.681,P<0.05 or <0.01).Conclusion APBSCT combined with medical treatment can improve liver function and 3-year survival rate with mild adverse reaction in patients with decompensated hepatitis B cirrhosis.

Objective To study the facial cosmetic zonation and its application of adipose injection for facial juvenescence.Methods According to standard of aesthetics,we divided total face into ten cosmetic units and 49 cosmetic sub-units.23 patients,injected adipose for facial juvenescence,were evaluated.Results 149 cosmetic units from 23 patients were injected with adipose granules,and involved 300 sub-units.Each unit was injected with adipose from 1 to 35 ml.After injection,the faces showed swelling in varying degrees,which resolved from ten to fourteen days.Local ecchymosis occurred in 11 cases,and resolved from five to twenty days.No any complication appeared,such as infection,scleroma and vascular embolism and so on.The follow-up results showed that one month later post-operation,swelling on injective sites resolved,and appearances were stable.3-6 months later post-operation,the injective sites were fuller.One year later,the good j uvenescence effect achieved,and the patients felt satisfactory.Conclusions On the basis of facial cosmetic zonation,the whole cosmetic evaluation combined with local cosmetic evaluation might be helpful to carry out adipose injection for facial juvenescence,and reach a satisfactory result.

Objective To prepare the virus-like particles (VLPs) of Chikungunya virus (CHIKV) and evaluate the immunogenicity.Method CHIKV structural protein C-E3-E2-6K-E1 encoding gene were amplified by fusion PCR,and cloned into an insect cell expression vector.The recombinant Baculovirus were recovered by co-transfection of the expression plasmid with baculovirus linear DNA into SF9 insect cells,and CHIKV VLPs were prepared from suspension culture SF9 cells.Structural proteins expression were analyzed using IFA,SDS-PAGE and Western-Blot,and morphological analysis via electron microscopy.Result Which showed that CHIKV structural proteins were secreted into the cell culture supernatant and assembled into virus-like spherical particles.BALB /c mice were immunized with the VLPs,high levels of CHIKV specific antibodies were detected in the sera,and CHIKV infection of Vero cells could be effectively neutralized.Couclusion The results showed that CHIKV VLPs can be efficiently produced by the baculovirus expression system in insect cells,and specific IgG and neutralization antibodies could be induced in mice after VLPs immunization.This research laid the foundation for the development of CHIKV VLPs based on immunological detection reagents and even vaccines.

Objective To investigate the application value of recombinant human growth hormone ( rhGH) in the treatment of severe burns .Methods 36 patients with severe burns were selected and randomly divided into the observation group and control group ,18 cases in each group .Both two groups used the same anti-infection therapy , nutritional support and surgical methods .The observation group received rhGH subcutaneous injection therapy , the control group used the same amount of daily injections of 0.9%sodium chloride.The levels of tumor necrosis factor-α( TNF-α) and interleukin 6 ( IL-6 ) , wound healing time , hospitalization time and other indicators were compared between the two groups.Results 7d,14d after treatment,the TNF-αand IL-6 levels of the two groups were (18.4 ± 3.3)ng/L vs (24.9 ±7.3)ng/L,(133.2 ±66.9)ng/L vs (271.4 ±88.9)ng/L,(15.4 ±3.2)ng/L vs (25.4 ± 4.9)ng/L,(90.9 ±20.8) ng/L vs (200.7 ±62.2) ng/L,the differences were significant between the two groups (t=3.74,4.75,4.02,4.69,all P<0.05).The healing time and hospital stay in the observation group were signifi-cantly shorter than those of the control group (t=3.75,3.86,4.75,4.64,all P<0.05).Conclusion rhGH therapy can significantly improve the wound healing of severe burn patients ,reduce hospitalization time,it has good clinical results.

ObjectiveTo discuss the clinical characteristic and perioperative management of colonic obstruction caused by colonic carcinoma in elderly.MethodsThe clinical characteristic and perioperative management of 30 patients with colonic obstruction caused by colonic carcinoma from January 2000 to November 2010 were collected and analyzed retrospectively.Among 30 cases,11 cases were accepted emergency operation,19 cases were accepted limited operation.Resection and anastomosis at stage Ⅰ was in 26 cases (86.7％),shortcut surgery and simple fistulation was in 4 cases ( 13.5％ ).ResultsThe length of stay was 22-56 days.Postoperative complication including wound infection in 5 cases,wound liquescence in 3 cases,wound dehisce in 2 cases,anastomotic fistula in 2 cases,pulmonary infection in 3 cases.Two cases died during perioperative period.Conclusions Besides diagnosis as early as possible,appropriate perioperative management should be taken to the elderly patients,including reasonable operative time and surgical approaches and these are the keys to improve clinical curative effect.

Objective To investigate the feasibility of fast-track surgery in colorectal surgery.Methods Eighty consecutive patients with colorectal cancer admitted for colorectal surgery were divided into two groups by random digits table with 40 cases each. Group A was treated with the new concept of fasttrack surgery and group B was treated with the traditional methods of operation. The time of postoperative bowel venting and defecation,hospital stay time, the rate of complication, the rate of readmission and the total cost during hospitalization were compared. Results The time of postoperative bowel venting and defecation,hospital stay time were shorter in group A [(2.7 ± 0.9), (2.9 ± 0.1 ), (5.8 ± 1.0) d,respectively]than those in group B [( 3.9 ± 0.5 ), (4.2 ± 0.3 ), ( 8.3 ± 1.2) d, respectively] and the rate of complication and the total cost during hospitalization in group A [7.5%(3/40), ( 1.83 ± 0.22) ten thousand yuan] were lower than those in group B[27.5%(11/40), (2.35 ± 0.36) ten thousand yuan](P＜ 0.05). Conclusion The new concept of fast-track surgery can accelerate recovery after colorectal resection,reduce the rate of overall complications and total cost during hospitalization.

Objective To investigate the effect of Tip60 on the cellular radiosensitivity,and to explore the related mechanism.Methods siRNA and anacardic acid (AA,an inhibitor of Tip60 acetyltransferase) were used to inhibit Tip60 expression and its acetyltransferase activity,respectively.Radiosensitivity was analyzed by colony-forming ability assay.γ-H2AX foci were detected to analyze the DNA double-strand break (DSB).Immunoprecipitation was used to determine the interaction of proteins.Results siRNA-mediated silencing of Tip60 led to enhanced sensitivity of U2OS cells at 1,2 Gy after γ-ray irradiation,but had no significant effect at 4 Gy post-irradiation ( t =3.364,3.979,P ＜ 0.05 ).γ-H2AX foci detection indicated that Tip60 silencing resulted in a decreased capability of DNA doublestrand break repair at 1,4 and 8 h after irradiation( t =3.875,3.183 and 3.175,respectively,P ＜ 0.05 ).The interaction of Tip60 and DNA-PKcs was prompted by ionizing radiation.Anacardic acid largely abrogated the phosphorylation of DNA-PKcs at T2609 site induced by irradiation.Conclusions Tip60plays a role in the cellular response to ionizing radiation-induced DNA damage through,at least in part,interacting with DNA-PKcs and regulating its phosphorylation.

Objective To investigate the dose-response pattern on the inducible expression of PIG3 mRNA in normal human lymphoblastoid AHHI cells by 60Co γ-rays,and its possibility for developing novel radiation biodosimeter.Methods Laser confocal fluorescent microscopy was used to detect the γ-H2AX foci,a biomarker of DNA double-strand break.After irradiation with 0,1,2,4,6,8 and 10 Gy of 60Coγ- rays,AHH-1 cells were harvested at 4,10 and 24 h post-irradiation,and subjected to total RNA extraction and detection of PIG3 mRNA expression by quantitative real-time PCR.Results PIG3 protein foci were formed in the nuclei at 30 min after irradiation,and a part of these PIG3 foci were colocalized with γH2AX foci.After irradiation,PIG3 mRNA level was enhanced with the increased time of postirradiation,and remained an increased level at least till 24 h.The radiation-inducible expression of PIG3 mRNA was demonstrated in a dose-dependent manner.The dose-dependent range at 4 h post-irradiation was 0 - 6 Gy,and at 10 h and 24 h was 0 - 10 Gy.Conclusions PIG3 involves in the cellular response to DNA double-strand break.The dose-dependent inducible response of PIG3 mRNA expression might provide a valuable candidate for developing a novel radiation biodosimeter.