Objective To obtain eukaryotic expression vectors containing coding region of nephroblastoma overexpression gene (NOV) and detect its expression in COS-7 cells. Methods A 1 165-bp cDNA fragment was amplified from the total RNA of normal rat brain tissue by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1/Myc-His(+)/lacZ. The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes HindⅢ and BamHⅠ. The recombinant plasmid was transfected into COS-7 cells with liposome. The expression of NOV gene was detected by Western blotting and immunocytochemistry. Results Eukaryotic expression vectors containing 1 165 -bp coding region of NOV gene was constructed. COS-7 cells transfected with the recombinant plasmid expressed high level of NOV protein in cytoplasm. Conclusion That eukaryotic expression vectors containing coding region of NOV gene was constructed can provide a strong molecular tool for the studies of effect of NOV gene.

BACKGROUND:Previous studies have demonstrated that hepatocyte growth factor (HGF) gene transfection can improve the effectiveness of bone marrow mesenchymal stem cel transplantation, but the mechanism is stil unclear. OBJECTIVE:To observe the effects of HGF gene transfection on c-MET, Bax, Bcl-2, Caspase-3 of bone marrow mesenchymal stem cel s cultured under hypoxia and serum-free conditions. METHODS:(1) Bone marrow mesenchymal stem cel s were isolated and amplified in vitro by differential adhesion method. The infection efficiency of recombinant adenovirus Ad-HGF in bone marrow mesechymal stem cel s was tested by x-gal staining. (2) Bone marrow mesenchymal stem cel s were cultured under hypoxia and serum-free conditions for 0, 3, 6, 9, 12 hours. RT-PCR and western blot assays were used to evaluate the expression of Bax, Bcl-2, Caspase-3. (3) Bone marrow mesenchymal stem cel s were cultured under hypoxia and serum-free conditions for 6 hours, and RT-PCR and western blot assays were adopted to detect HGF, c-Met, Bax, Bcl-2 and Caspase-3. (4) Cel scratch test was used to detect the effect of HGF transfection on the migration of bone marrow mesenchymal stem cel s cultured under hypoxia and serum-free conditions for 6 hours. RESULTS AND CONCLUSION:(1) Transfection efficiency of bone marrow mesenchymal stem cel s was increased with multiplicity of infection in a dose-dependent manner. When the multiplicity of infection was 150, the transfection efficiency was 96.4%. (2) Expressions of Bax and Bcl-2 were gradual y increased with hypoxia time (P<0.05). The Bax/Bcl-2 ratio and Caspase-3 expression reach the minimum at 6 hours of hypoxia (P<0.05). (3) Compared with the control and Ad-LacZ groups, the expressions of HGF, c-Met, Bcl-2 increased, and the expressions of Bax and Caspase-3 decreased in the Ad-HGF group after 6 hours of culture under hypoxia and serum-free conditions (P<0.05). There was no significant difference between the control and Ad-LacZ groups. (4) The mobility of bone marrow mesenchymal stem cel s was higher in the Ad-HGF group than the control group and Ad-LacZ groups after 6 hours of culture under hypoxia (P<0.05). These findings indicate that transfection of HGF in bone marrow mesenchymal stem cel s can increase the expression of c-Met, Bcl-2 and decrease the expression of Bax, Caspase-3 under hypoxia and serum-free conditions, which also enhance the mobility of bone marrow mesenchymal stem cel s under hypoxia and serum-free conditions.

BACKGROUND:Adrenomedulin gene transfection can strength the anti-apoptotic ability of bone marrow mesenchymal stem cels under ischemia and hypoxia, but its mechanism is not yet clear.
OBJECTIVE:To investigate the effect of adrenomedulin on the expression of apoptosis-related proteins, Bax, Bcl-2 and Caspase-3, in bone marrow mesenchymal stem cels under hypoxia and ischemia.
METHODS:Bone marrow mesenchymal stem cels of Sprague-Dawley rats were isolated, cultured and purified, and then cultured in serum-free medium under hypoxic condition for 0, 3, 6, 9, 12 hours. Then, western blot assay was employed to detect the expression of Bax, Bcl-2 and Caspase-3 so as to determine the optimal hypoxia time that was determined at 6 hours of hypoxia. Depending on whether adrenomedulin pretreatment was done, the cels were divided into control group (with no adrenomedulin pretreatment before hypoxia and ischemia) and adrenomedulin groups with different concentrations (1, 10, 100 μg/L). Afterwards, the expression of Bax, Bcl-2 and Caspase-3 was detected by using western blot assay.
RESULTS AND CONCLUSION:(1) After cultured in serum-free medium under hypoxia for 0, 3, 6, 9, 12 hours, the expression of Bax, Bcl-2 and Caspase-3 in bone marrow mesenchymal stem cels were increased (P < 0.05);at 6 hours of hypoxia, the Bax/Bcl-2 ratio and Caspase-3 expression reached the minimum value (P < 0.05). (2) At 6 hours of hypoxia, the expression of Bax and Caspase-3 protein as wel as Bax/Bcl-2 ratio became the lowest in the 100 μg/L group compared with the 1 and 10 μg/L groups, but the expression of Bcl-2 protein reached the peak (P < 0.05). These findings indicate that adrenomedulin can reduce the expression of Bax/Bcl-2 ratio and Caspase-3 protein in bone marrow mesenchymal stem cels cultured in serum-free medium under hypoxic conditions, which is in a dose-dependent manner.

OJECTIVE To investigate the preventive effect of Fengliao extract on ulcerative colitis of mice. METHODS Using the intestinal propulsion rate experiment and senna induced diarrhea model , the intestinal propulsion rate, diarrhea rate and index of diarrhea were observed. Mice were randomly divided into normal group,model group,mesalazine hydrochloride group and Fengliao extract 11.7, 23.4 and 46.8 g · kg-1 group. The mouse colitis model was induced by 4% dextran sulfate sodium. The mice were administraed once daily for 7 d while the disease activity index(DAI)score was calculated and the activity of tumor necrosis factor α(TNF-α),interleukin-1β(IL-1β), myeloperoxidase(MPO) and content of malondialdehyde(MDA) and nitric monoxide (NO) in colon tissue were determined. RESULTS Fengliao extract 46.8 g · kg-1 inhibited the intestinal propulsion rate(P<0.05),reduced the frequency of diarrhea and the diarrhea index(P<0.05). Results of colitis showed that the body mass of mice in the model group was significantly decreased but the DAI score increased compared with normal group(P<0.05). The activity of MPO and the contents of IL-1β,TNF-α,MDA and NO in colon mucosa were increased(P<0.01). Compared with the model group,Fengliao extract 46.8 g·kg-1 decreased the DAI score(P<0.05)while Fengliao extract 46.8 and 23.4 g · kg-1 reduced MPO activity in colonic mucosa and content of IL-1β,TNF-α,MDA and NO in colonic homogenate(P<0.05). CONCLUSION Fengliao extract can significantly improve the DSS induced colitis in mice,which is probably associated with its antispasmodic and anti-diarrheal effect as well as the reduced release of inflammatory mediators and antioxidants.

OBJECTIVE:To study the percutaneous absorption of 0.03% Tacrolimus ointment,and to compare the difference of domestic test preparation and imported reference preparation. METHODS:Modified Franz diffusion cells were adopted in trans-dermal test in vitro;HPLC-MS method was used to determine permeation amount and rate in vitro,delay time of domestic test preparation and imported reference preparation 0.03%Tacrolimus ointment. RESULTS:24 h in vitro permeation amount of test and reference preparations were(3 907±1 191)and(3 896±1 064)ng/cm2;permeation rates were 186.7 and 182.9 ng/(cm2·h);de-lay time were 1.95 and 2.00 h,respectively(P>0.05). CONCLUSIONS:Test preparation shows good percutaneous property,and is similar to reference preparation in penetration absorbency through nude mice skin.

BACKGROUND: NOV protein encoded by nephroblastoma overexpression gene(NOV) is IGF(insulin-like growth factor) -binding protein. What is its impact on human neural stem cell(hNSC) proliferation and differentiation?OBJECTIVE: To investigate the impacts of NOV protein on hNSCs proliferation and differentiation.DESIGN: A single factor analysis of variance experimental study using cells as subjectsSETTING: Department of histology and embryology, and department of neurobiology in a military medical university.MATERIALS: Study was conducted in the Department of Histology and Embryology of the Third Military Medical University of Chinese PLA. Subjects were hNSCs cultured from 10 to 14 weeks human embryo cerebral cortex.INTERVENTIONS: COS-7 cells were transfected by NOV gene recombined plasmid. COS-7 cell and COS-7 cell modified by NOV gene conditioned culture media(COS-CM and NOV-CM) were collected and reacted with the cultured HNSCs.MAIN OUTCOME MEASURES: hNSCs proliferation was detected by 3H-TdR scintillation analysis, and hNSCs differentiation was detected by immunocytochemistry and flow cytometer(FCM).RESULTS: Both COS-CM and NOV-CM could significant promote the intake of 3H-TdR by HNSCs, of which the 1/minute of NOV-CM group was significantly higher than that of COS-CM group(P ＜ 0.05), which indicated that NOV-CM contained component that could facilitate hNSCs proliferation, and moreover, there was certain dose-effect relationship in NOV-CM' s facilitation of cellular proliferation. The results of immunocytochemistry and FCM revealed that there were more NF-200 positive cells in NOV-CM group, while many glial fibrillary acidic protein positive cells could be seen in COS-CM group.CONCLUSION: NOV protein might have facilitative effects on hNSCs proliferation and differentiation into neurons.

Objective To observe the effects of different membrane proteins and dimethylsulfoxide on neurite outgrowth of cerebellum granule cells(CGC).Methods Membrane proteins were extracted from the liver,sciatic nerve and brain white matter of adult rats and coated on the cover slips.CGC were dissociated from newborn rats and inoculated on the coated cover slips,while dimethylsulfoxide(DMSO) was added into the CGC suspension.Results The neurite outgrowth was inhibited by membrane protein of brain white mater and the effect was concentration-dependent.Low concentration(

Objective To optimize purification technology of total flavonoids in Callicarpa nudiflora Hook. et Arm. by macroporous adsorption resin. Methods Macroporous resin models including AB-8, D-101, HP-20, HP2MG, were optimized by static adsorption and desorption experiments regarding to adsorption rate and desorption rate of total flavonoids. Purification technology parameters of total flavonoids were optimized by single factor test. Results HP-20 macroporous resin presented the best purification efficiency,the optimum purification conditions were that taking 4. 46 mg·mL-1 of total flavonoidsat pH 3. 0, loading at 3 BV·h-1, washed with 3BV of water at 3 BV·h-1,then eluted with 4 BV 75% ethanol at 2 BV·h-1, finally obtaining the total flavonoids from the dry extract of Callicarpa nudiflora Hook. et Arn. with the purity of 47. 4%. Conclusion HP-20 macroporous resin is suitable for preliminary purification of total flavonoids in Callicarpa nudiflora Hook. et Arn.

OBJECTIVE:To study the protective effects of Polygonum hydropiper extract on acute gastric mucosal lesion (AGML)in rats. METHODS:48 rats were randomly divided into normal group(normal saline),model group(normal saline), positive group(ranitidine hydrochloride,0.05 g/kg),P. hydropiper extract low-dose,medium-dose and high-dose groups(2.7, 8.1,24.3 g/kg by crude drug),i.g. for consecutive 7 d,once a day. Except for normal group,other groups were given absolute ethyl alcohol to induce AGMI model after 1 h of last administration. 1.5 h after modeling,gastric mucosal lesion index of rats was calculated;the pathological changes of gastric tissue in rats were observed;nuclear factor E2 related factor 2(Nrf2)content and SOD activity in gastric tissue of rats were determined by ELISA. RESULTS:Compared with normal group,the gastric mucosa of model group was damaged obviously,there was blood capillary rupture in submucosa,gastric mucosal lesion index was increased significantly(P<0.01);Nrf2 content and SOD activity were significantly decreased in gastric tissue of rats(P<0.05 or P<0.01). Compared with model group,gastric mucosal lesion of rats was relieved to different extent;in positive group,P. hydropiper extract medium-dose and high-dose groups,gastric mucosal lesion index was decreased significantly(P<0.05),and Nrf2 content and SOD activity were increased significantly(P<0.05 or P<0.01). CONCLUSIONS:P. hydropiper extract has good protective effect on absolute ethyl alcohol-induce AGMI,the mechanism of which may be associated with raising Nrf2 content and enhancing SOD activity in gastric mucosal tissue.