Objective To investigate the effect of matrix metalloproteinase(MMP-2) in the attack of exprimental autoimmune myositis(EAM) rats, and the effects of methylprednisolone on expressions of gene mRNA and protein of MMP-2. Methods The expression levels of MMP-2 in the peripheral blood, spleen lymphocytes and muscles were detected by using RT-PCR and immunohistochemistry techniques and was compared among EAM group,EAM with methylprednisolone treatment group (EAMM) and control group. Results ⑴ The degree of episode in EAMM group was lower than that of EAM group , and the infiltration of the inflammatory cells and the necrosis of the muscles were also mild in EAMM group. ⑵There was significant upregulation of the expression of the MMP-2 gene mRNA in the lymphocytes of peripheral bood and spleen of EAM rats as compared with that of the control,Elevation of the expression of MMP-2 protein in rats muscles tissues of EAMM group was observed obviously. However, the expression of the mRNA and the protein of MMP-2 was suppressed significantly in EAM group compared with control group. Conclusions The upregulation of the expression of MMP-2 may be correlated with the onset of EAM. Methylprednisolone may relieve the degree of the pathology of EAM by the downregulation of MMP-2 .

AIM　Diclofenac potassium sustained-release tablets were developed as a gel matrix for using twice daily and evaluated in vitro release characteristics. METHODS　The formulations were screened according to the designed in vitro release rate by the use of HPMC as the gel matrix and the hydrophobic retarding agent to modify the release. RESULTS　The release was properly characterized by the diffusion mechanism, influenced by pH of the media, slightly affected by the basket rotating speed, without the influences from the pressure exerted during the tabletting procedure. CONCLUSION　The selected formulation of diclofenac potassium sustained-release tablets could ensure the desired in vitro release rate. In addition, it proved a good manufacturing reproducibility.

Audiometry, Speech ; Humans ; Language

Objective Liver metastasis model of human primary gastric lymphoma in nude mice was reproduced for an experiment to evaluate the inhibitory effect of cytokine-induced killer (CIK) cells on the growth and liver metastasis of primary gastric lymphoma. Methods Surgical orthotopic implantation of a histologically intact liver metastasis fragment derived from a surgical specimen of a patient with metastatic gastric lymphoma was initally implanted, in order to reprodueing a liver metastasis model of human primary gastric lymphoma. Peripheral blood mononuclear cells (PBMC), isolated by blood cell separator from healthy donors and patients with primary gastric lymphoma, were incubated in vitro. rhIFN-?, rhIL-2 and anti-CD3 McAb were added to PBMC in order to prepare CIK cells as well as lymphokine-activated killer (LAK) cells. CIK and LAK cells from different donors were used in treating gastric malignant lymphoma, so as to investigate the inhibitory effect of 2 kinds of effector cells on the growth of the tumor and liver metastasis. Results The liver metastasis model of human primary gastric malignant lymphoma in nude mice was successfully reproduced. After administration of different agents continuously for 20 days (0.3ml/d), the inhibitory rates of the following 4 groups, healthy donors LAK group (2?1010/ml), healthy donors CIK group (2?1010/ml), patients CIK group (1?1010/ml) and patient CIK group (2?1010/ml), were 39.28%, 53.57%, 40.38% and 56.42%, respectively. The liver metastasis rates in control group, healthy donors LAK group, healthy donors CIK group, patients CIK group (1?1010/ml) and patient CIK group (2?1010/ml), were 100.0%, 62.5%, 50.0%, 62.5% and 37.5%, respectively. Tumor weights of all treatment groups were lighter than that of saline group (P

AIM:To express recombinant hCD154-GST fusion protein, to prepare anti-hCD154 monoclonal antibody, and to investigate the effect of anti-hCD154 monoclonal antibody on graft rejection. METHODS AND RESULTS: Total RNA was prepared from human peripheral blood mononuclear cell (PBMC) activated with 10ng/mL PMA and 1 ?g/mL PHA for 8h, the total RNA was reversetranscribed to cDNA. The entire coding region and a part of the 3'non-coding regions were amplified by PCR using a pair of primers designed and synthesized according to the sequence of human CD154 gene from gene bank. The amplified product, a 820bp DNA fragment was cloned into pGEX-4T-1 plasmid expressing glutathione S-transferase(GST). The cloned insert was identified by double digestion of the cloned pGEX-4T-1 plasmid with retriction enzymes BamHⅠand EcoRⅠ.The fusion protein expression plasmid of PGEX-4T-1/hCD154 was constructed, then transformed to E coli BL21. The human CD154-GST fusion protein expression was induced by IPTG in BL21. The expression of recombinant 26kD GST and 55kD human CD154-GST fusion protein were confirmed by SDS-PAGE. CONCLUSION: We have express the recombinant human CD154-GST fusion protein. The expressed hCD154-GST fusion protein will be used to prepare anti-hCD154 monoclonal antibody, to investigate the role of anti-CD154 monoclonal antibody on graft rejection.

AIM:To obtain the light chain region(VL) and heavy chain region(VH) genes from the hybridoma cell line and analyse their sequence for construction of the engineering antibody against hCD154. METHODS: In this research ,total RNA was extracted from the hybridoma cell line 7E8, which secretes McAb against hCD154, and subjected to reverse transcription. The VL gene and VH gene were amplified by PCR, cloned into puc18 vector and sequenced by Sanger's dideoxymediated chain-termination method. RESULTS: The VL cDNA of 7E8 McAb consists of 341 bp encoding 113 amino acid residues. Compared with mouse Ig database, the VL region is in accord with the characterization of DNA sequence present in the mouse Ig Vk region , it belong to mouse V?2 light chain. The VH cDNA of 7E8 McAb consists of 354 bp encoding 118 amino acid residues. Compared with mouse Ig database, the VH region is in accord with the characterization of DNA sequence present in the mouse Ig VH region. CONCLUSION: The DNA squence analysis showed that the cloned genes code the light and heavy chain variable region of mouse respectively.

Objective　To obtain mammalian cell expression vector of human CD154 gene. Methods　A 820 bp cDNA fragment was amplified by RT-PCR method from total RNA of human peripheral blood mononuclear cell（PBMC） activated with 10 ng／mL PMA and 1 μg／mL PHA for 8 hours. The fragment was cloned into pcDNA3.1（＋） plasmids.The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes BamH Ⅰ and EcoR Ⅰ and sequenced by Sangers-dideory-mediated chain termination. Results　This cDNA fragment included 820 bp entire coding region and a part of the 3 non-coding region. The recombinant mammalian cell expression vector of pcDNA3.1（＋）／hCD154 was constructed， the sequence of the insert was identical to the published sequence encoding human CD154 antigen. Conclusion　The recombinant mammalian cell expression vector of pcDNA3.1（＋）／hCD154 was successfully constructed.

Objective To analyze the potential etiologies and risk factors of childhood stroke. Methods This study retrospectively reviewed the clinical data of 159 children who were admitted from Jan.2006 to Jan.2014. Results The 159 children were composed of 100 boys and 59 girls , with median onset age of 1.8 years (ranged from 1 day to 12 years old) and median peak age of 0.9 years (ranged from 3 months to 2.8 years old). Their initial symptoms included limb hemiplegia,language dififculties and convulsion. The common causes included infections found in 46 cases (central nervous system infection in 32 cases, respiratory and gastrointestinal tract infection in 14 case), head injury in 42 cases, vitamin K deifciency in 29 cases, Moyamoya disease in 8 cases, heart diseases in 11 cases, spontaneous hemorrhage in 11 cases and 12 cases of unknown reason. Infectious diseases were the most common cause of children acute ischemic stroke in toddler period;and vitamin K1 deifciency were the most common cause of children hemorrhage stroke in infancy. The most common region of infarction is basal ganglia and middle cerebral artery in neuronal imaging. The median age at the time of diagnosis was 1.4 days. The median time of inhospital was 28 days. The median apex time was 4.3 days. Conclusions Among 159 cases, acute ischemic stroke is much more common than hemorrhagic stroke in children stroke, and the major risk factors are infections and head injury;Vitamin K1 deifciency is a major risk factor in infants with hemorrhagic stroke.

Objective The study was conducted to investigate the acute phase of convulsion related problem on the clinical manifestations,imaging and electroencephalograph (EEG) examination of purulent meningitis.Methods Cluster sampling method was employed to select children in our hospital,a total of 301 cases with purulent meningitis was analyzed retrospectively.Among them,62 cases had convulsion.The incidence of convulsion in the acute phase of the purulent meningitis,risk factors,and prognosis were analyzed.Results The convulsion incidence rate of acute purulent meningitis was 20.60％.The partial seizure was eight cases (12.90％).The secondarily generalized seizure following partial seizure was 15 cases (24.19％).The generalized seizure was 32 cases (51.61％).The convulsive status was 7 cases (11.29％).The EEG abnormality was significantly different between the convulsion group and the no convulsion group (P ＜ 0.05).The incidence of brain organic damage was significantly different between two groups (P ＜0.05).The multivariate unconditional logistic regression analysis showed,cause of disease,first symptom,disturbance of consciousness,obvious signs,and cerebrospinal fluid culture with convulsion were the relevant factors (P ＜ 0.01).Conclusions The most common seizure of purulent meningitis was the generalized seizure.Brain organic damage easily resulted in convulsion of purulent meningitis.The days of hospitalization,cause of disease,first symptom,disturbance of consciousness,obvious signs,and cerebrospinal fluid culture with convulsion were the positively relevant factors.Those positively relevant factors in combination of the clinical manifestations,imaging,and EEG examination in children would play an important role in diagnosis,treatment,and prognosis evaluation of convulsion derived from purulent meningitis.Moreover,convulsion affects the disease recovery in children with purulent meningitis.

Objective To evaluate the endoscopic managements of acute cholangitis of severe type (ACST) accompanied with multiple organ dysfunction syndrome (MODS). Methods A total of 122 ACST patients accompanied with MODS from January 2000 to October 2008 underwent endoscopic treatment in two time periods. In critical phase, emergent endoscopic retrograde cholangiopancreatography (ERCP) plus en-doscopic naso-biliary drainage (ENBD) were performed to correct critical situation of the patients. After sta-bilization, endoscopic sphincterotomy (EST) plus stone removal, EST plus stent placement, or laparoscopy was performed according to the causes of ACST. Results Emergent endoscopic managements succeeded in all patients of critical phase. At third day post-operation, a reduction in white blood cell count, serum total bilirubin, body temperature, and rate of patients with shock, mental symptoms and purulent bile juice was a-chieved. Recovery rate of dysfunction organs was 60.2% at one week after emergent procedure, and 82. 6% at 2 weeks post-operation. Selective EST plus stone removal was performed in 36 patients with a success rate n one session at 91.7%. Laparoscopic cholecystectomy was performed in 85 patients with a success rate of 95.3%. Stent was placed in 16 patients with an effective rate of 81.3% at 3 months post the procedure. No severe complication or death occurred during the whole therapeutic course. The 6-month survival rate of 10 cancer cases was 70%. Conclusion Therapeutic ERCP plus ENBD is the first choice for acute severe cholangitis accompanied with MODS, while EST plus biliary lithotomy, or EST plus stent placement, or com-bined laparoscopy are ideal methods for subsequent treatment.