Objective To construct prokaryotic expression vector carrying Japanese encephalitis virus (JEV) NS1 gene, and to express the vector in E. coli, so to lay a foundation for the further development of JEV early diagnosis kit. Methods The NS1 gene was amplified by RT-PCR, the target gene and prokaryotic expression plasmid pET28a(+) were digested by BamH I and Hind Ⅲ respectively. The target gene was then purified by DNA extraction kit. A 1∶3 molar ratio of vector: insert DNA ligated with T4 DNA ligase to construct the recombinant plasmid pET28a(+)-NS1. The ligated products were transformed into E. coli BL21 (DE3) and the colonies were selected on LB medium with karamycin. After cultivation, positive colonies were picked out and the recombinant plasmid were identified by endonuclease digestions, PCR rand sequencing. The target protein was expressed with induction of IPTG. The expressed proteins mentioned above were then identified and analyzed by SDS-PAGE and Western-blotting respectively. Results The sequencing results of amplified products showed that JEV-NS1 RNA fragments were about 1 300bp in length which were similar as respected. Compared with the published sequence of SA14-14-2 with Blast, the homology of the nucleotide sequence was 100%. The molecular weight of expressed protein was about 45kD, the result of Western blotting proved the specific antigenicity of the protein. Conclusion The specific JEV nonstructural protein 1 is expressed in E. coli successfully and shows high specificity to the antibody. The stable expression of the protein and the analysis of its antigenic specificity provide foundation to develop the early stage diagnosis kit.

[Objective]To report the surgical managements and their outcomes in high pressure injection injuries of the hand(HPIIH) in 5 cases.[Method]Open wound technique and series debridement in all 5 cases were performed.The residual skin defects following debridement were repaired with cross-finger island flap in 1 case.[Result]From November 2001 to November 2005,5 cases of HPIIH were treated in our department and the wounds were healed satisfactorily.The flap was survived.Amputation of the finger was executed in 1 case.[Conclusion]The time of the first debridement after injury determines the prognosis.Aggressive wound debridement is a key point for good wound healing in HPIIH.

Objective To explore the effects of earlier oral-motor exercise on swallowing and feeding capability of children with cerebral palsy. Methods 36 children accepted oral-motor exercise performed by speech-language therapist once a day for 3 months. They were assessed for oral motor and feeding before and after treatment, and compared with other 36 controls matched with age and type. Results The oral motor in treatment group improved significantly （t=2.184,P<0.05） after treatment, but merely in control. The difference of scores of oral motor before and after treatment was (9.89±4.33) in treatment group, and (3.38±1.56) in control group (t=7.423, P<0.01). Conclusion Early oral-motor exercises is effective to improve oral motor function and feeding capability.

Objective To investigate the intelligence level and characters in children with cerebral palsy of 4~6 years old, and the factors related with their intelligence. Methods 215 cerebral palsy children of 4~6 years old were assessed with Wechsler Intelligence Scale for children,Gross Motor Function Classification System (GMFCS). Results 210 children finished their intelligence test, and 113 presented mental retardation. The intelligence level was higher in the children with spastic diaplegia, hemiplegia and dyskinetic type than those with quadriplegia and ataxia type. The difference was significant between VIQ and PIQ in the spastic diplegia (P<0.05). The mental retardation was more frequent in the children with GMFCS Ⅳ~Ⅴ, or with epilepsy or microcephaly than those with GMFCS Ⅰ~Ⅲ or without the complications (P<0.05). Conclusion The incidence of mental retardation in children with cerebral palsy was related with the neurotypes, motor function and complications.

Objective To evaluate hepatectomy for liver metastasis in patients of gastric carcinoma.Methods Clinical data of 32 gastric cancer cases undergoing hepatectomy for hepatic metastatic tumor were reviewed retrospectively from 2006 to 2012.16 cases underwent radical gastrectomy and synchronous hepatectomy for liver metastasis,the remaining 16 cases underwent radical resection of gastric cancer and liver resection heterochronously.The relationship between prognosis and clinicopathology was analyzed.Results The overall survival rates were 84％,50％ and 37％ in 1 year,3 years and 5 years.The median survival time was 32 months.Gastric cancer invasion depth,intravascular tumor thrombi,lymphatic metastasis and intraoperative blood transfusion was related to poor prognosis by single factor analysis,while gastric serosal invasion,tumor thrombus and liver metastasis tumor ＞ 5 cm related to poor prognosis by multiple factors analysis.Conclusions Gastric cancer patients with liver metastasis who underwent hepatic resection can achieve good prognosis if hepatic metastatic tumor ＜ 5cm or the primary gastric cancer does not invade the serosa and without tumor thrombus.

Objective To explore related factors affecting bone mineral density (BMD) and osteoporosis (OP) in patients with type 2 diabetes mellitus (T2DM). Methods Dual-energy X-ray absorptiometry was used to measure BMD of the femoral neck and the lumbar vertebrae in 220 patients with T2DM. All the patients were divided into three groups:normal bone mass group, decreased bone mass group and OP group according to the value of BMD. The clinical data and biochemical indicators were compared and analyzed between these groups, and the influencing factors of T2DM and OP were explored. Results Compared with normal bone mass group and decreased bone mass group, there were more older patients, longer course of T2DM, lower BMI and high density lipoprotein cholesterol (HDL-C), and higher glycated hemoglobin (HbA1c) level in OP group. There were significantly higher age, longer course of T2DM, higher level of HbA1c, and lower levels of BMI, HDL-C in decreased bone mass group than those of normal bone mass group (P＜0.05). There were no significant differences in waist-to-hip ratio, systolic blood pressure (SBP), diastolic blood pressure (DBP), postmenopausal women (PMW) ratio, fasting plasma glucose (FPG), 2-h postprandial blood glucose (2hPG), blood phosphorus, calcium, total cholesterol (TC), three acyl glycerin (TG) and low-density lipoprotein cholesterol (LDL-C) between three groups. The risk factors for T2DM combined with OP were older, female, long duration of T2DM, higher levels of FPG, 2hPG and HbA1c. Conclusion Older and female T2DM patients were high-risk group of OP. The treatment plan should be timely adjusted by regularly monitoring indexes of blood glucose and HbA1c.

Objective To varify contrast-enhanced ultrasound (CEUS) quantitative analysis plaque enhancement is able to assess the intraplaque neovascularization.Methods The abdominal aortas were examined with CEUS in 9 atherosclerosis rabbits established by feeding high-fat diet.Sonographic examinations were performed with ultrasound instruments equipped with low mechanical index contrast imaging software.Contrast agent SonoVue was used with a dose of 0.2 ml,The enhanced intensity (EI) with the plaque and the ratio of EI within the plaque to that in the lumen of the carotid artery was calculated with QLab software,Each aorta specimen was stained with F8 (von willebrand factor) for mierovessel.The quantitative parameters EI and ratio were compared with the immunohistochemical study results.Results The mean value of EI and ratio were (3.91 ± 0.87)dB and 0.30 ± 0.06,respectively.Five specimens showed positive F8 staining and 4 showed negative.EI and ratio were significantly greater in positive group than in negative group [(4.47 ± 0.60) dB vs (3.22 ± 0.62) dB,0.34 ± 0.05 vs 0.25 ± 0.05].CEUS quantitative parameters EI ( r =0.779,P =0.013) and ratio ( r =0.693,P =0.019) were correlated to microvessels marked with F8,respectively.Conclusions It is histologically validated that CEUS quantifies the neovascularization in atherosclerosis plaques.CEUS provides a window for plaque risk stratification.

BACKGROUND:Cytological studies show that bone marrow mesenchymal stem cel s play an important role in postmenopausal osteoporosis mechanism.
OBJECTIVE:To study the osteogenic differentiation in vitro of bone marrow mesenchymal stem cel s from ovariectomied osteoporotic rats.
METHODS:The osteoporotic animal model was established by performing ovariectomy in the 6-month-old female Sprague-Dawley rats. There were four groups:bone marrow mesenchymal stem cel s control group, bone marrow mesenchymal stem cel s osteoporosis group, bone marrow mesenchymal stem cel s osteogenic induction group and oseogenesis induction group. Bone marrow mesenchymal stem cel s were isolated from the rats of control group and oseogenesis induction group by means of the whole bone marrow adherence method and cultured to the 3rd generation. Then the bone marrow mesenchymal stem cel s were used in al the experiments. Cel morphology was observed under the inverted phase contrast microscope, cel cycle and proliferation index of bone marrow mesenchymal stem cel s were detected by flow cytometry. After osteogenic induction, the expression level of alkaline phosphatase was detected, and the fornation of calcium nodes of bone marrow mesenchymal stem cel s were marked by alizarin red staining.
RESULTS AND CONCLUSION:The cel s in the osteogenic induction group and oseogenesis induction group had the morphology of osteobalsts, and the change of morphology of the cel s in the oseogenesis induction group was relatively tardiness. The proliferation index in the control group was higher than that in the osteoporosis group (P<0.05);expression level of alkaline phosphatase in the osteogenic induction group was significantly higher than that in the oseogenesis induction group (P<0. 05), and the control group was significantly higher than the oseogenesis group (P<0.05). The alizarin red staining of the cel s in the osteogenic induction group was positive, while negative in the control group and the oseogenesis group;the staining in the osteogenic induction group was stronger than that in the oseogenesis induction group. These findings indicate that both the proliferative potential and the osteogenic potential of bone marrow mesenchymal stem cel s from the ovariectomized osteoporotic rats are decreased, which may be related with the ostoeporosis pathogensis of ovariectomied rats.

Objective To determine whether the supplementation of thyroid hormone(TH)improves β-adrenoceptors(β-AR)density and expression of peripheral blood lymphocyte in elderly patients with chronic heart failure(CHF). Methods A total of 42 elderly patients with advanced CHF(NYHA classⅢorⅣ)due to dilated cardiomyopathy(DCM)or ischemic cardiomyopathy (ICM)were randomly divided into a treatment group and a control group.Low-dose L-thyroxine(LT50)was added to the treatment group,both groups had routine anti-heart-failure treatment.Before and after 1 month of treatment,125I-Pindolol radioligand binding assay was used to measure β-AR in peripheral lymphocytes.Reverse-transcriptase polymerase chain reaction(RT-PCR)was used to measure β1-AR expression in peripheral lymphocytes. Results The density and expression of β-AR of lymphocytes in patients with CHF due to ICM or DCM were lower than those in normal health people.and the serum TH level in patients with CHF was lower than that in normal health people.The L-T4 administration induced an up-regulation of β-AR density and expression in peripheral lymphocytes. Conclusions Thyroxine administration induces an up-regulation of β-AR density and expression in peripharal lymphocytes.

Objective To explore the content change of Alpha Fetal Protein(AFP) and Alpha Fetal Protein Variants(AFP-L3) pre-and post-liver transplantation operation for liver cancer patients.Methods AFP-L3 was de-termined by micro centrifugal column from twelve cases of successive sera(For each case,one original shale pre-oper-ation,and mole shares post-operation successively),and preserved from patients who were diagnosed as fiver cancer and completed fiver transplantation operation.simultaneously the content of AFP and AFP-L3 in the original serum were also determined.and the ratio of AFP-L3 in AFP was calculated in order to compare the content change of AFP and the ratio of AFP-L3 pre-and post-liver transplantation operation.Results Compared with twelve cases of pre-and post-operation,the AFP in only five cases turned tO negative;The AFP in other cases keeped positive though AFP-L3%tumde to negative.and the content of AFP in some specific cases still keeped in a high level.Regarding AFP-L3,besides two casses turning to negative immediately after operation,the content of AFP-L3 in other patients de-creased gradually after operation,and it approached to negative(AFP-L3%<10%).Through statistical analysis,be-tween the half lives of AFP and AFP-L3%,there waa no statistic significance.Therefore,AFP-L3% could be considered as an independent reference item.Conclusion After a radical cure of liver resection or liver transplantation operation,AFP-L3%usually turned to negative in a rather short period (about 1 to 20 days).The ratio of AFP-L3 in AFP became be-low 10%,and it indicated that the operation would be quite complete,remaining liver would have hepatitis or hepatocirrho-sis.Otherwise,the ratio was not decrease during a long period,and it indicated the operation had not eliminated focuses completdy.