Objective To construct the luciferase reporter gene vector of cell division cycle 2 (Cdc2) gene promoter and determine its transcriptional activity. Methods Primers were designed based on human Cdc2 promoter sequence from UCSC software. Then Cdc2 promoter from human genome DNA was replicated. After pGL3-Basic vector and Cdc2 promoter were digested with restriction enzymes SacⅠand XhoⅠseparately, Cdc2 promoter was inserted into pGL3-Basic vector. The recombinant plasmid named pGL3-Cdc2-promoter was transiently co-transfected into U2OS cells with control vector pRL-SV40, and then the activity of dual luciferase was detected. Results pGL3-Cdc2-promoter was constructed successfully. The restriction analysis and sequencing proved the entirely correct sequencing results. The luciferase activity was higher in pGL3-Cdc2-promoter/pRL-SV40 group than that of pGL3-Basic/pRL-SV40 group (1.591 5±0.199 8 vs 0.049 9±0.010 4). Conclusion pGL3-Cdc2-promoter can be transcribed and activated in U2OS cells. This study provided an important basis for screening and evaluation of anticancer drugs.

AIM: To investigate the changes of cardiac function, oxidative stress and apoptosis in myocardium of rat model of heart failure induced by adriamycin (ADR). METHODS: At the end of the study, we observed content of malondialdehyde (MDA), activity of superoxide dismutase (SOD) and apoptosis. Expression of P53 protein and p53 mRNA were measured with immunohistochemical method and RT-PCR, respectively. RESULTS: Our data showed that content of MDA increased, activity of SOD decreased and apoptosis in myocardium happened while cardiac function decreased after ADR treatment. p53 gene expression and P53 protein obviously increased in heart failure group. CONCLUSION: There were oxidative stress and apoptosis occurred significantly in a model of heart failure induced by ADR. p53 gene might play an important role in the apoptosis. Correlation analyses suggested that apoptosis in myocardium is related to oxidative stress in ADR-induced heart failure.

Objective To determine the levels of total antioxidative capacity(TAOC) and malondialdehyde(MDA) in liver exposed to hyperoxia,and to explore whether oxygen inhalation could cause liver injury in newborn rats.Methods Sixty-four newborn rats which were less than 12-hour-old were enrolled in this study.The rats were randomly divided into hyperoxia group(FiO2=0.85,n=32) and control group(air,n=32).Eight rats in each group were randomly sacrificed to obtain liver tissues at 1d,3d,7d and 14d.The TAOC of liver homogenates was detected by chemical colorimetry,and the MDA level of liver homogenates was measured by thiobarbituric acid test.Results In the hyperoxia group,TAOC in liver increased on the 1st day[(3.60±0.28)U/mg prot vs(3.39±0.19)U/mg prot,P<0.05];TAOC began to decreased on the 3rd day,and significantly lower than that of control group on the 14th day [(3.10±0.15)U/mg prot vs (3.56±0.14)U/mg prot,P<0.01].In the hyperoxia group,the MDA level increased on the 3rd day[(3.58±0.11)nmol/mg prot vs(2.82±0.14)nmol/mg prot,P<0.01],and reached a peak on the 7th day[(3.58 ±0.11)nmol/mg prot vs(2.82±0.14)nmol/mg prot,P<0.01],then decreased but still remained higher than control group on the 14th day [(2.92±0.18)nmol/mg prot vs(2.77 ±0.09)nmol/mg prot,P<0.01].Conclusion Too more MDA in liver and TAOC decrease may cause liver injury in newborn rats exposed to hyperoxia.With the oxygen inhalation time prolonging,the liver injury aggravation.

As a kind of additive in feed of monogastric animals, the application of natural phytase is limited due to its disadvantages. In this paper, the strategies of phytse reform was introduced. Furthermore, the research and progress on protein engineering of feed phytase was reviewed, including phytase over-expression, phytase thermostability, catalytic efficiency and optimum pH.

Parkinson's disease(PD) is a common neurodegenerative disorder with no effective protective treatment,characterized by a massive degeneration of dopaminergic neurons in the substantia nigra(SNpc) and the subsequent loss of their projecting nerve fibers in the striatum.The major neurochemical manifestation of this disorder is the loss of the neurotransmitter dopamine(DA) in the striatum as a result of the progressive degeneration of the dopaminergic neurons in the substantia nigra.There have been significant progresses in recent years reporting on the use of mesenchymal stem cells(MSCs)in gene therapy,with specific application towards PD.MSCs,a kind of multipotent adult progenitor cells,are considered as a useful vehicle for cell and gene therapy because of their multiple differentiation potentiality and self-transplantation.The present study was focused on treating rat model of PD using human tyrosine hydroxylase gene(hTH),human aromatic L-amino acid decarboxylase gene(hAADC) and human GT Pcyclohydrolase I gene(hGCH-I) engineered MSCs,in order to provide a better understanding about the application of these cells in the therapeutic benifit of PD.The gene of hTH,hAADC and hGCH-I were introduced via recombinant adeno-associated virus(rAAV) infection into the MSCs in vitro.The genetically modified MSCs expressing hTH,hAADC and hGCH-I were transplanted into the striatum of PD rat models.The behavior,the nigra-striatal level of DA and its metabolite were detected.The results of present study were shown as follows:hTH,hAADC,hGCH-I and LacZ gene were transfected into MSCs with adeno-associated virus vectors.The HEK293 packaging cells(ATCC) were transfected with the plasmids of pAAV-hTH,pAAV-hAADC,pAAV-hGCH-I,pAAV-LacZ,pAAV-RC,pHelper by using calcium phosphate precipitation.Titer was detected using HT1080 cells.Viral particles were collected and used to infect MSCs.The purified modified MSCs expressing the three kinds of genes were selected separately and were grafted in the striatum of the PD model rats in the lesion side.The MSCs genetically modified suvived well 12 weeks after transplantation.The improvements of the behavior were observed every week after transplantation.Compared with the control group,the rounds of asymmetric rotation after apomorphine administration decreased in the groups double or triple genes engineered MSCs grafted(p

reression of M 2 receptor mRNA of rat myocardium intoxicated by soman under high altitude hypoxia. Methods The expressions of M 2 receptor mRNA under hypoxia, soman intoxication and soman intoxication under hypoxia were detected by semi quantitative RT PCR, respectively. Results The expression of M 2 receptor mRNA increased in the high altitude hypoxia group. Both simple soman intoxication and combined soman intoxication and hypoxia decreased the expression of M 2 receptor rapidly. But under hypoxia, the expression increased significantly at 12 h and 24 h. Conclusion M 2 receptor was sensitive to nerve agents. Compared to simple soman intoxication group, the expression of M 2 receptor increased in combined soman intoxication and hypoxia group. This may be one of the major factors leading to aggravation of the injury of heart function by nerve agents in high altitude area.

BACKGROUND:Restoration of tendon integrity and tenacity, and maintenance of ankle joint flexibility, play a crucial role on the rupture of Achiles tendon. OBJECTIVE: To evaluate the muscular force using isokinetic muscle strength test after the ruptured Achiles tendon is repaired, and to assess the repairing effect. METHODS: Muscle power including peak torque, peak torque/body weight, total work, average peak torque, set total work of flexor and extensor of 17 patients with Achiles tendon rupture were assessed after reconstruction using Biodex system 4 at 60 (°)/s and 120(°)/s. RESULTS AND CONCLUSION:The peak torque, peak torque/body weight, total work, average peak torque, set total work of flexor muscle group at the injured side were significantly decreased compared with uninjured side at 60 (°)/s and 120 (°)/s (P < 0.05). Isokinetic muscle strength test can reflect the muscular force after the ruptured Achiles tendon is repaired, and evaluate the repairing effect.

Objective To assess the value of susceptibility weighted imaging (SWI)in the diagnosis of different cerebral diseases complicated with cerebral microbleeds (CVBs)and cerebral vascular malformations (CVMs).Methods The MRI data of 76 cases with CMBs and 25 cases with CVMs were retrospectively analyzed and the data were translated as follows:(1 )To compare the counts and detecting sensitivity of CMBs on routine MRI sequences (T1 WI,T2 WI,T2 FLAIR,DWI)with SWI.(2)To assess the difference of SWI,MIP and Phase in detecting the size,counts,intensity and edge features of CMBs.(3)To assess the advantages of SWI in the diagnosis of different CVMs.Results (1)In all 1 524 CMBs detected on SWI,294,87,361,391 CMBs were detected on T1 WI,T2 WI,T2 FLAIR and DWI respectively.The sensitivities were 19.3%,5.7%,23.7% and 5.7% on T1 WI,T2 WI,T2 FLAIR and DWI respectively.It was of statistical significance (P <0.05)in the counts and detecting sensitivity on different MRI sequences.(2) 1 524 and 1 539 CMBs were detected on SWI and MIP respectively.CMBs showed oval,dotted hypointensity or hypointensity domi-nated mixed intensity lesions which were ≤10 mm in size on SWI and MIP.1 521 CMBs were detected on Phase,which appeared as hyperintensity or hyperintensity dominated mixed intensity lesions.MIP and SWI were similar in detecting the size,counts and edge features of CMBs.And Phase was inferior compared with MIP and SWI .(3)12 cases of cerebral venous malformation were detec-ted,which all appeared as the classic Medusa head sign on SWI.6 cases of intracerebral capillary telangiectasia were detected , which were characterized as multiple small rounded even hypointense lesions ortarget signon SWI .2 cases of arteriovenous malformation (AVM)were detected on SWI,in which one case only showed multiple small vessels and gross drainage vein,and the other showed clear nidus and gross drainage veins into the sigmoid sinus.4 cases of cerebral cavernous angioma were detected.Mulberry sign, which was characterized as dotted mixed intensity lesion with hypointense ring,was noted in all the cases on SWI.One case of Sturge-Weber syndrome was detected,which showed gyriform low signal lesion on MIP imaging.And many broadened,tortuous veins in the cerebral hemispheres and near the tentorium were also noted.Conclusion SWI can be used as an important sequence for detecting CMBs.Different CVMs show their specific signs in SWI sequence.Combined with conventional MRI sequences an accurate diagnosis can be achieved most of the time.

Objective To establish a nerve stem cell model possessing the potentiality of differentiating intoγ-aminobutyric acid neuron-like cells(GABAergic-like cells),and provide eligible investigative vector for study on GABAergic neurons degenerative diseases. Methods Dibutyryl cyclic adenosine monophosphate (dbcAMP)was applied to induce human neuroblastoma SH-SY5Y cells,and the SH-SY5Y cells were divided into control group (0 mmol·L-1 dbcAMP),0.3 mmol·L-1 dbcAMP group,0.6 mmol·L-1 dbcAMP group,1.0 mmol·L-1 dbcAMP group and 2.0 mmol·L-1 dbcAMP group;the morphological changes of SH-SY5Y cells were observed.The Image-Pro Plus 5.0 software was used to measure the length of neurites of the neuron-like cells, and the percentage of the SH-SY5Y cells with neurites longer than 30 μm was calculated. The immunofluorescence cytochemistry technique was utilized to test GAD65 positive cells,and the positive rate of immune response was calculated.Results The results of light microscope observation showed that the cells in control group were polygonal,circular or shuttle type with smooth membrane and clear boundaries. With the increasing of the concentrations of dbcAMP and the prolongation of time,the morphology of SH-SY5Y cells’bodies became smaller with longer processes in dbcAMP groups.The cells interwined each other and showed mature neuron phenotype in 1.0 mmol·L-1 dbcAMP group.Compared with control group(31.4%±4.2%),the percentages of the cell with neurites longer than 30 μm in 0.3, 0.6, 1.0, and 2.0 dbcAMP groups (40.1%± 5.7%, 47.5%± 6.2%, 73.1%±3.2%,and 74.3%± 6.1%)72 h after induction were significantly increased(P<0.05 or P<0.01). Compared with control group (10.2%± 2.1%), the GAD65 positive expression rates in 0.3, 0.6, 1.0,and 2.0 mmol·L-1,dbcAMP groups(22.1%±2.4%,46.9%±3.2%,70.7%±3.4%,and 72.3%±3.7%)72 h after induction were significantly increased(P<0.05 or P<0.01).Conclusion The SH-SY5Y cells have the potentiality of differentiating into GABAergic-like cells, and 1.0 mmol · L-1 is the optimal concentration of dbcAMP.

Objective Comparing the effects of steam sterilization on cyclic fatigue of stainless steel files. Meth-ods Fifty instruments of 25# stainless steel K files were randomly divided into 5 groups, which include 10 in each group. Group 1 was the blank control group, group 2 to 5 were subjected to 1, 3, 4, 5 steam sterilization cycles, then the files were tested by a custom-made device to assess cyclic fatigue and the number of cycles of failure (NCF) was calculated. Microstruc-ture of each file’s fracture surface was analyzed by Scanning Electron microscope (SEM). Results NCF in the 5 groups were 4 345.2 ± 384.2,3 937.9 ± 645.4,3 812.9 ± 532.5,3 626.2 ± 380.0,3 625.9 ± 565.6 respectively, and the differences among 5 groups were significantly different(F=2.598, P<0.05). After 4 or 5 times of steam sterilization, cyclic fatigue de-creased if compared with that in control group (P<0.05). Fracture surface in group 1 and group 2 was tough dimple structure and large numbers of regular, deep dimples were distributed on the surface. You could also see micro cavities clearly formed by fracture;Fracture surface in group 4 was dimple structure in brittle intergranular morphology. It is characterized by the presence of thin brittle precipitates, clean and smooth crystal interface, and a great sense of polyhedral stereo. Conclusion After 4 times of steam sterilization, cyclic fatigue strength of 25# stainless steel K files declined, which possessed the poten-tial risk of fracture.