Aim Vascular endothelial cell growth inhibitor(VEGI) is a recently discovered novel member of the TNF superfamily,which is expressed predominantly in endothelial cells.As an endothelial cell-specific negative regulator of angiogenesis,the relationship between structure and function of VEGI is not understood at present.Methods In order to explore the functional key amino acids of VEGI,four mutants of VEGI(E45→R,G47→A,Y111→F,Y111→T) were construced by site-directed mutagenesis,and recombinant proteins were generated from E.coli.Four mutant proteins behaved similar to the wild type VEGI in various physico-chemical assays.The proliferation of HUVEC and chick choriallantic membrane assay were performed to study the activity of four mutants.Results The mutant E45→R significantly decreased the biological activity,and the mutant G47→A caused a slight drop on activity,but the mutants Y111→F,Y111→T almost completely abolished biological activity.Conclusion It suggests that Y111 is an important residue in biological activity,which may play a direct role in receptor recognition.Moreover,the tyrosine ring and hydroxy group of the amino acid are important determinant of biological activity.Additionally,E45 also plays an important role in biological activity of VEGI.

Objective To explore the management therapy of pediatric rhabdomyosarcoma.Methods Retrospective analysis of treatment of 29 cases with rhabdomyoscroma from Oct.1998 to Jun.2006.Primary sites included 5 cases of neck,1 chest wall,2 abdominal wall,12 retroperitoneal pelvic cavity and 9 extremity.Pathologic types included embryonal type 19(65.5%),alveolar 8(27.6%),pleomorphic 2(6.9%).According to IRS staging system they were Ⅰstage(n=2),Ⅱstage(n=6),Ⅲstage(n=9)and Ⅳstage(n=12).Most tumors were large than 5 cm(n=26).Results Twenty-seven cases were followed up but 2 of the cases were lost,19 cases got complete remission(4 cases relapsed or died after stopping treatment for 3 months to 3 years and 1 case relapsed after giving up treatment for 3 months),4 cases got partial remission(3 cases relapsed after 6 months diagnosed),4 cases got no response(3 of which deteriorated or relapsed and 1 case surved with tumor).Fourteen cases had constant remission between 5-77 months,averaging 22.9 months with average guitting time of treatment 16.9 months.Conclusion Total tumor resection,chemotherapy and radiotherapy play an important role in management of pediatric rhabdomyosarcoma.

Objective To study the diagnosis and treatment of the model of multidisciplinary team treatment on hepatoblastoma in children.Methods Retrospective analysis of treatment and long term follow-up of 16 patients with hepatoblastoma(8 cases were boys,8 cases were girls;aged 3 months to 11 years old,average age was 2 years old) during Aug.1998 to Jan.2006 in Shanghai Children's Medical Center.Special in multidisciplinary team treatment,preoperative evaluate and operation.Results The morbility locum of 8 cases were in right liver lobe,8 cases were in left liver lobe.Fifteen cases of the clinical presentation were abdominal mass,1 case was peritonitis.According to International Society of Pediatric Oncology(SIOP) Pretext staging system,43.7%(7/16)cases were stage Ⅱ,43.7%(7/16)cases were stage Ⅲ,12.5%(2/16)cases were stage Ⅳ.In 7 cases of stage Ⅱ,6 cases were accepted tumor total resection,1 case was accepted emergency surgery because tumor was fracture.Patients were given ICE protocal.In 7 cases of stage Ⅲ,6 cases were able to undergo delayed tumor resection following open biopsy and subsequent treatment with chemotherapy 2-3 courses.One case gave up treatment because its had poor response to chemotherapy.In 2 cases of stage Ⅳ,1 case was received liver transplantation due to poor respond to chemotherapy.One case was not received any treatment.In 7 cases of stage Ⅱ,6 cases were alive,1 case died,3-year free survival rate was 100%(4/4).In 7 cases of stage Ⅲ,5 cased were alive,3-year free survival rate was 75%(3/4).Two cases of stage Ⅳ all died.Conclusions Surgery play a very important role in the treatment of hepatoblastoma in children.Chemotherapy has improved the resectability of the tumor.Multidisciplinary team treatment is an effective model for diagnosis and treatment on childhood hepatoblastoma.

Objective To explore the role of fibrogenic cytokines and inflammatory cytokines in the pathogenesis of frozen shoulder. Methods From September, 2014 to April, 2016, 20 patients with frozen shoulder accepted arthroscopic surgery were included, ten of them were diagnosed as primary frozen shoulder (group A), the other ten were secondary frozen shoulder (group B). Other ten patients undergo-ing shoulder arthroscopy for instability (4 cases), rotator cuff injury (3 cases) and subacromial impingement (3 cases) were as the controls (group C). The mRNA levels of matrix metalloproteinase (MMP) 1, MMP3, tumour necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, IL-8, granulocyte-macrophage colony stimulating factor (GM-CSF) and M-CSF in synovium were analyzed with quantitative polymerase chain reaction. Results The expression of mRNA of MMP1, MMP3, TNF-α, IL-1, IL-6, IL-8, GM-CSF and M-CSF were more in group A and group B than in group C (P<0.05). There was no significant difference between group A and group B (P>0.05). Conclusion The fibro-genic cytokines and inflammatory cytokines may play a role in the pathogenesis of frozen shoulder.

Objective To provide the reference basis for reducing the occurrence of nutritional risk by analyzing possible risk factors for nutritional risk status and investigating the relationship between preoperative nutritional risk status and prognosis.Methods We retrospectively analyzed the clinical data of 894 patients(nutritional risk group of 491 cases,non-nutritional risk group of 403 cases) with esophageal cancer resection.The univariate analysis was used to analyze the relationships between nutritional risk status with postoperative complications and length of hospitalization.And the multiple Logistic regression model analysis was applied to analyze the risk factors of nutritional risk status.Results The nutritional risk group had a higher rate of postoperative complications (33.60 ％ vs.19.60 ％,U =-3.429,P =0.001),higher incidence of serious complications (23.01％ vs.8.68％,U =-3.611,P =0.000) and longer hospital stays [(37.20 ± 13.89) days vs.(31.69 ± 13.49) days,t =4.124,P =0.000] than that of non-nutritional risk group.The results of the multiple logistic regression analysis showed that the preoperative nutritional risk factors were associated with the patients' age (OR =1.58,95％ CI:1.101-2.268),number of symptoms entries(OR =7.97,95 ％ CI:6.071-10.463),symptom severity (OR =0.26,95％ CI:0.186 -0.385),and dietary intake (OR =0.62,95 ％ CI:0.482-0.813),P ＜ 0.05 for all.Conclusion The older patients with more severe symptoms and poor diet are more likely to suffer from nutritional risk.Prolonged hospital stay and postoperative complications easily happen in patients with nutritional risk.So patients with preoperative nutritional risk should be given timely and effective nutrition intervention measures,in order to reduce postoperative complications and length of hospitalization.

Objective To explore the effect of surgical treatment on primary gastrointestinal non-Hodgking lymphoma(NHL) in children.Methods Nine cases of clinical and follow-up data of primary gastrointestinal NHL were studied retrospectively to evaluate the effect of surgical treatment on primary gastrointestinal NHL in children.Results Seven cases were male and 2 cases were female.The mean age was(5.59?3.27)years old.The clinical manifestation included abdominal mass (7 cases),abdominal pain (5 cases),fever (2 cases),haematemesis and melena (2 cases),constipation (1 case) and paroxysmal abdominal pain with vomiting (1 case).Nine cases were diagnosed as primary gastrointestinal NHL,including 1 case of intussusception,1 case of acute appendicitis,2 cases of gastrointestinal obstruction,2 cases of gastrointestinal bleeding and 3 cases of abdominal mass.One case received the operation of intussusception reduction,tumor resection and intestinal anastomosis.One case received appendectomy.One case received the operation of tumor biopsy and transverse colon colostomy.Six cases received laparotomy.Six cases were diagnosed as Burkitt lymphoma.One case was anaplastic large cell lymphoma and 2 cases were diffuse large B-cell lymphoma.One case was at stage Ⅰ,1 case was at stage ⅠE,2 cases were at stage Ⅱ,3 cases were at stage ⅡE and 2 cases were at stage Ⅲ.Nine patients had received operation.One case died after operation and 8 cases had received combined chemotherapy.The 1 and 3 years survival rates were 75.0% and 37.5%,respectively.Conclusions Acute abdomen is often the first symptom of primary gastrointestinal NHL in children and comprehensive surgical treatment is an effective procedure for it.

Objective To build the experimental basement for the clinical use of cytokines induced killer(CIK)cells from the cord blood mononuclear cells(CBMNC)in tumor adoptive cellular immunotherapy, an effective protocol for their proliferation in vitro and cytotoxicity of CIK cells was established.Methods The lymphocytes from umbilical cord blood were isolated by density gradient centrifugation and suspended in medium with CD_3 mAb,rIL-2,rIL-1 and IFN-? as inducing agents to prepare CIK cells.At the same time, the lymphokine activated killer(LAK)and CBMNC were set as controls,which were only added IL-2 and not any cytokines during the whole culture.The changes of CIK cells before and after induction were observed with microscope and the phenotypes of the cells were analyzed by using flow cytometry.The proliferation of CIK cells were determined by trypan blue exclusion assay and the cytotoxic activity to lung cancer cell were tested with MTF method.Results According to the experiment,combining use of four types of cytokines could generate a great deal of CIK cells possessing highly cytotoxicity.From day 5 CIK cells became to prolif- erate and reached the peak at day 14.During the whole period,the relative percentage of CD_3~+ CD_(56)~+ cells in- creased significantly.Compared with LAK cells,which reached the proliferation peak at day 7 and then showed no evident proliferation.The control cells(CBMNC)showed no evident change of phenotypes and proliferation.CIK cells showed a higher antitumor activity on the tumor cells than LAK cells and CBMNC in vitro.Conclusion Umbilical cord blood can generate a great deal of CIK cells combining used with cy- tokines.Compared with classic LAK cells,umbilical cord blood CIK cells have the advantages of rapid prolif- eration speed and powerful cytotoxicity.CIK cells will be promising as a new strategy for the adoptive cellular immunotherapy of tumor.

One hundred Pseudomonas aeruginosa quorum-sensing-related genes were selected and their primers were synthesized. The fragments of specific sequences which are related QS genes were amplified by PCR. These verified sequences were inserted into the vector pMD-18T for sequencing. These DNA fragments were dotted onto glass slides to make cDNA microarray. Hybridization was performed with cy3/cy5-dCTP labeled probes. The scanning data of early stationary phase and mid-logarithmic phase indicated that 9 genes were up-regulated and 6 genes were down-regulated. Undergoing the different medicines,we took tobramycin as an example to compare the expression diversity. The results confirm that the QS cDNA chip is useful,and may contribute to better understand the mechanism of quorum-sensing,and can help us find the new targets for restraining the growth of Pseudomonas aeruginosa.

Objective To explore the efficacy of arthroscopic treatment for acute avulsion fracture of posterior cruciate ligament (PCL) using double titanium plate TightRope. Methods From December 2014 to January 2015, two cases with acute avulsion fracture of PCL were treated with double titanium plate TightRope under arthroscopy, after assessment of three dimensional reconstructive CT and MRI. Rehabili-tation was carried out after operation. Results One case succeeded with double titanium plate TightRope, and rehabilitation could be per-formed early postoperatively. Another case failed in TightRope fixation, and accepted two internal hollow screws fixation instead. Rehabili-tation was delayed until two weeks postoperatively. The function of the knee was satisfactory in the former patient after three months, who could extend the knee in a normal range, without pain or locking. The same effect was gained in the latter patient after four months. Conclu-sion For acute tibial avulsion fracture of PCL with larger and intact fragment without osteoporosis, arthroscopic restoration and fixation with double titanium plate TightRope offers the opportunity to achieve anatomic reconstruction and rigid fixation for early rehabilitation in adult patients.

Objective To evaluate the influential factors of iron acquisition during Francisella tularensis LVS infection of mouse macrophages.Methods F.tularensis LVS expressing green fluorescent protein was used to infect murine macrophage J774A.1 cells.Transferrin receptor 1(Tfr1)was detected with mono-antibody and visualized with a goat-anti mouse IgG conjugated to Alexa 594.The expression profile of 5 iron metabolism related genes of J774A.1 murine macrophages uninfected or infected with F.tularensis LVS was determined with real-time PCR.Immunoblot analysis was used to compare the Tfr1 expression of live Francisella infected macrophage with dead bacteria.Tfr1 knock-off in J774A.1 cells was performed with siRNA.The transfected cells were infected with Francisella for immunoblotting and microscopy and infection assay.Results It was revealed that the live vaccine strain of F.tularensis induced the expression of Tfr1 in host macrophages.Gene expression analysis indicated that F.tularensis LVS drove an active iron acquisition program with induction of Tfr1 and iron regulatory proteins(Irp1 and Irp2).It was shown by Western-blotting that the siRNA-Tfrc-1 could knock off about 75% of Tfr1 in J774A.1 cells.It was determined by infection assay that,Tfr1 was knocked off,the bacteria number at 1h infection with Francisella was not different from that of control(F=1.06,P=0.326 5),while it was decreased significantly after 24h of infection(F=24.12,P=0.000 6).Conclusions It is demonstrated that upregulation of the Tfr1 may be mediated by post-transcriptional regulation during early infection,but sustained later through increased expression of Irp 1 and Irp 2.Increased expression of Tfr1 expands the intracellular iron pool through transferrin-mediated delivery and may thus be readily available for uptaking by Francisella.Knocking off the expression of Tfr1 does not affect bacterial invasion.Francisella,however,may fail to proliferate in macrophages in which the expression of transferrin receptor has been suppressed.