With the global development of the research of medicine,more and more new drug safety evaluation institutions have been certified by Good Laboratory Practices (GLP) and Association for Assessment and Accreditation of Laboratory Animal Care International (AAALACi).Laboratory animal,which is the carrier of drug safety evaluation,its survival condition and animal welfare will directly effect the experimental results.Dealing with laboratory animal husbandry by scientific methods in two systems is the requirement to make sure the accuracy of experimental data of animal experiments.With the certification processes and practical experiences in two certification systems of Tianjin Institute of Pharmaceutical Research New Drug Evaluation Co.,Ltd.,also accompanied by experience exchangement with peers,the relationship of practices in two certification systems will be preliminarily discussed;Through strengthening the management of laboratory animal,it can help the institutions get the certificates.

Objective To discuss repairing effects of articular cartilage defects by nitric oxide synthase inhibitor (S-methylisothiourea, SMT), and explore the role of nitric oxide in cartilage repair. Methods Full-thickness defects of cartilage were created in the intercondylar trochlear groove of femur of thirty-six adult New Zealand white rabbits, and were divided into three groups. Twenty-four defects were untreated as the control, twenty-four were filled with fibrin glue and impregnated with rhBMP as rhBMP group, the rest twenty-four were filled with fibrin glue and impregnated with rhBMP, and hypodermic injection with SMT as SMT group. The animals were sacrified at sixteen weeks postoperatively, and the gross appearance of the defect was estimated. The repair tissue was examined histologically and was evaluated according to the grading scale of histology. The amount of released NO and the activities of nitric oxide synthase (NOS) were examined by chemical colorimetry. The distribution of type-Ⅰ,Ⅱcollagen were examined by Sirius-Red. The proteoglycan synthesis was assessed by incorporation of radio-labelled sodium sulphate Na35SO42-. RT-PCR examined the expression of iNOSmRNA and MMP9mRNA. Results The filled extent of the defect in SMT group and rhBMP group had no significant difference from the control group, and the marginal integration, cellular morphology, architecture within the defect and subchondral plate repair were better than the untreated defects (P

Pointing to the contradiction features existent in teaching of clinical practice in the beginning stage of establishing affiliated hospital of university,it primarily discusses the management method in teaching of clinical practice,which gets first achievements after 3-year trial.

Objective To study the effect of nitric oxide synthase inhibitors S methylisothiorurea (SMT) on cartilage metabolism after intraarticular injection of interleukin 1? (IL 1?) and lipopolysaccharides (LPS) in rabbits.Methods The experiments were performed in three groups:normal group,control group and SMT group.The effects of SMT on IL 1? and LPS induced iNOS mRNA expression ,NO production and NOS activity in synovium,synovial fluid and cartilage were detected after 8 h.Proteoglycan synthesis was measured by ex vivo incorporation of Na 2 35 SO 4 into cartilage after 48 h.Results IL 1? and LPS induced iNOS mRNA expression and increased NO release;SMT could reduce NO release and inhibit iNOS mRNA expression in synovium,synovial fluid and cartilage,and partly restore proteoglycan synthesis of cartilage.Conclusion iNOS inhibitor SMT can protect IL 1? and LPS induced cartilage damage in high concentration.

Objective To obtain recombinant human granulysin using prokaryotic expression system. Methods Total RNA was extracted from cultured PBMC. Granulysin gene segments were obtained with granulysin-specific primers by RT-PCR and then inserted into pET32a(+) plasmid. After identification by DNA sequence, pET-GN-LY9K and pET-GNLY15K were transferred to E. Coli Rosetta (DE3). The fusion protein was identified by SDS-PAGE and Western blot. The bioactivity of granulysin fusion protein was measured by MTT assay. Results The prokaryotic expression vectors pET-GNLY9K and pET-GNLY15K were successfully constructed. The corresponding protein was highly expressed in E. Coli. Recombinant protein was specifically bound by anti-granulysin antibody. GNLY9K fusion protein significantly inhibited the growth of A549 cells in a dose-dependent manner, while GN-LY15K had little effect on the growth of A549. Conclusion Granulysins with different mw were successfully expressed using prokaryotic expression system, which might be helpful for the further study of granulysin.

An inner-bioreactor rapid filtration sampling probe was designed to resolve the problems of continuous sampling the fermentation broth in on-line measurement the fermentation process. The probe consists of a support, a plug valve, an O-ring, a filter membrane and a pipe cover. It can be inserted directly into the bio-reactor withstanding high-temperature sterilization before fermentation, or preventing the bacteria's invasion during sampling. Fermentation broth in tank is cross-flow filtrated by a cylindrical ceramic membrane which is used as filtration membrane, and then the sterile permeated liquid is transported to the analyzer by a peristaltic pump. The sampling experiments using this device was described for sampling glucose solution indicating it is suitable for fermentation online sampling. The sampling rate is up to 3. 0 mL/ min, glucose permeability maintains 100% , and the delay time caused by the probe is about 2 min.

Objective To investigate the effects of nitric-oxide synthase inhibitor on matrix metalloproteinases (MMPs) expression in osteoarthritis (OA) cartilage by Luminex analysis,and to explore the mechanism of nitric-oxide synthase inhibitor on modification of the metabolism of OA cartilage.Methods Fifteen specimens of articular cartilage taken from the patients with OA were cultured and divided in two groups.The control group was those with no intervention.L-NIL group was co-cultured with NOS inhibitor L-NIL.After 72 h cultivation,the release of NO and the activity of NOS on OA cartilage were measured by Griess reaction and spectrophotometric methods.MMPs (MMP-1,MMP-2,MMP-3,MMP-9,MMP-13) expression was measured by Luminex analysis.Comparisons between groups were performed with paired sampies t test.Results After cultured for 72 h,spectrophotometric analysis showed high concentration of NO release[(216±47) μmol/L ] and high level of active NOS [(5.7±1.3)U/ml]in supernatants of the control,1 mmol/L concentration L-NIL could evidently reduce NO release [(55±20)μmol/L,P<0.01] and NOS activity [(1.7±0.7)U/ml,P<0.01 ].Luminex analysis demonstrated high MMP-1,MMP-2,MMP-3,MMP-9,MMP-13 expression in cartilages of the control group [respectively for (10.8±5.4)ng/ml,(9.2±3.3) ng/ml,[11.6±4.2 )ng/ml,(1.27±1.07)ng/ml,(3.6±1.3)ng/ml] and 1 mmol/L concentration L-NIL could evidently inhibit MMP-1,MMP-2,MMP-3,MMP-9,MMP-13 expression [respectively for (3.6±1.8)ng/ml,(2.3±1.2)ng/ml,(3.6±1.4)ng/ml,(0.65±0.21)ng/ml,(1.8+0.5)ng/ml,P<0.05 ].Conclusion Luminex analvsis has shown that NOS inhibitor can reduce NO release and NOS activity and modify metabolism of articular cartilage by inhibiting the over-expression of MMPs.

Objective To obtain recombinant human granulysin using prokaryotic expression system.MethodsTotal RNA was extracted from cultured PBMC. Granulysin gene segments were obtained with granulysin-specific primers by RT-PCR and then inserted into pET32a(+) plasmid. After identification by DNA sequence,pET-GNLY9K and pET-GNLY15K were transferred to E. coli Rosetta (DE3). The fusion protein was identified by SDS-PAGE and Western blot.The bioactivity of granulysin fusion protein was measured by MTT assay.Results The prokaryotic expression vectors pET-GNLY9K and pET-GNLY15K were successfully constructed.The corresponding protein was highly expressed in E.coli. Recombinant protein was specifically bound by anti-granulysin antibody. GNLY9K fusion protein significantly inhibited the growth of A549 cells in a dose-dependent manner,while GNLY15K had little effect on the growth of A549.Conclusion Granulysins with different mw were successfully expressed using prokaryotic expression system,which might be helpful for the further study of granulysin.

Objective To analyze the correlation of vertebral osteophyte and lumbar disc degeneration in the elderly people and explore the possible mechanism of osteophyte formation. Methods X-ray and MRI data of 120 elderly people with backache or leg pain were retrospectively analyzed. Osteophyte was classified into four grades by X-ray according to the method proposed by Nathan. Discs with osteophyte were defined as occurring when osteophyte of grade II or greater were present. Lumbar disc degeneration was classified into five grades by MRI according to the method proposed by Pfirrmann. The obtained parameters were statistically treated and analyzed. Results Osteophyte and age were positively correlated at every lumbar disc level (P < 0.05). The proportion of osteophyte in L3/4 (76.7%) and L4/5 (70.08%) were more severe than that in L1/2 (31.7%) and L2/3 (46.7%). Osteophyte and lumbar disc degeneration were positively correlated at every lumbar disc level (P < 0.01). Conclusion Osteophyte becomes more severe with the increasing of lumbar disc degeneration. The vertebral stress after lumbar disc degeneration may be the main cause of osteophyte.

Objective To outline the classification of spinal tuberculosis based on MRI findings (Southern Medical University classification,SMU classification) and explore its use in the diagnosis,surgical protocols.Methods The MRI data from 230 cases with spinal tuberculosis were analyzed retrospectively.Our classification system was based on clinical and radiological criteria (abscess formation,disc degeneration,vertebral collapse,kyphosis,sagittal index,instability and neurological problems).The surgical strategies were made according with this classification.Results Two hundred and thirty cases with spinal tuberculosis were classified into the 5 types.There were signal change type (type Ⅰ) in 28 cases,abscess formation type (type Ⅱ) in 39,vertebral collapse type (type Ⅲ) in 78,canal compression type (type Ⅳ) in 46 and kyphosis type (type Ⅴ) in 39 respectively.In type I lesion,25 patients had been followed up.Twenty patients were treated medically.Recurrence of tuberculosis was found in 2 cases.Surgical meticulous debridements were done in 5 cases without recurrence.In type Ⅱa lesion,6 patients were treated medically.The other 6 patients underwent surgical meticulous debridement with recurrence occurred in one patient.There was no difference between medical and surgical treatment regarding outcomes in patients with type Ⅰ and Ⅱa lesion.In type Ⅱb-Ⅴ,surgical treatments were carried out according to the pathological changes.There was no difference between medical and surgical treatment regarding outcomes in the patients with type Ⅱb-Ⅴ.Conclusion The SMU classification helps in differentiating the various manifestations of spinal tuberculosis and appears to correlate with the surgical treatment of spinal tuberculosis.We believe that this new classification system can be used as a practical guide in the treatment of spinal tuberculosis.