Objective To investigate the effect of nimodipine (ND) injecting into cisterna magna on the mitochondrial pathway in hippocampus in rabbit model of subarachnoid hemorrhage (SAH). Methods Eighteen New Zealand white rabbits were randomly allocated to Sham group, SAH group and ND group, six in each group. All the animals underwent operation under anaesthesia. One mL/kg autologous non-heparinized arterial blood was injected into cisterna magna in SAH group and ND group, and the same dosage of saline was injected into cisterna magna in Sham group. Thirty minutes after injection, 1 mg/kg nimodipine was injected into cisterna magna in ND group, and equal-volume of saline was injected into cisterna magna in Sham group and SAH group. All the animals were assessed for the grade of food intake and neurological impairment, and rats were killed 72 hours after SAH. Their hippocampus were processed for detecting the expressions of Bcl-2, Bax, Caspase-3 and Cyt-C mRNA by qRT-PCR. The protein expressions of Caspase-3 and Cyt-C were detected by Western blot assay. Results Compared with the Sham group, there were lower grade of food intake, varying degrees of neurological impairment and lower ratio of Bcl-2/Bax, while the mRNA levels of Bcl-2, Bax, Caspase-3 and Cyt-C and protein levels of Caspase-3 and Cyt-C showed elevated expressions in SAH group and ND group (P<0.05). Compared with SAH group, there were no significant differences in the score of food intake and neurological impairment in the ND group ( P>0.05). There were higher ratio of Bcl-2/Bax and expression levels of Bcl-2 mRNA and lower expression levels of Bax mRNA, Caspase-3 and Cyt-C mRNA and proteins in ND group than those in SAH group (P<0.05). Conclusion Nimodipine plays a protective role in inhibiting the activity of mitochondrial pathway in hippocampus after subarachnoid hemorrhage.

Objective To compare the effects of two methods of nimodipine administration on mitochondrial pathway in the hippocampus of rabbit models of subarachnoid hemorrhage.Methods Twenty-four New Zealand white rabbits were randomly allocated to sham-operated group,subarachnoid hemorrhage (SAH) group and nimodipine introvenous injection (ND1) group,and nimodipine intracistemal administration (ND2) group (n=6).All animals underwent operation under anaesthesia;one mL/kg autologous nonheparinized arterial blood was injected into cisterna magna in SAH group,ND1 and ND2 group,and one mL/kg saline into cisterna magna in sham-operated group.Thirty minutes after SAH,0.5 mL/kg 0.2％ nimodipine was injected into cisterna magna in ND2 group,and equal-volume saline into cisterna magna in sham-operated group,SAH group and ND1 group.While 0.5 mL/kg 0.2％ nimodipine via introvenously injection was performed in the ND1 group,and equal-volume saline via introvenously injection into the sham-operated group,SAH group and ND2 group.All animals were assessed for the grading of food intake and neurological impairment 72 h after SAH,and then,they were scarified;their hippocampi were processed for detecting the mRNA and protein expressions of Caspase-3 and Cyt-c by using real time-PCR and Western blotting.Results The differences of food intake and neurological impairment between the four groups were statistically significant 72 h after SAH (H=16.664,P=0.001;H=15.411,P=0.001);according mean rank,the food intake and neurological impairment in the ND2 group were decreased as compared with those in the ND2 group.The mRNA and protein expression levels of Caspase-3 and Cyt-c among the four groups were statistically different (P＜0.05).As compared with those in the sham-operated group and SAH group,the mRNA and protein expression levels of Caspase-3 and Cyt-c were significantly higher in the ND1 and ND2 groups (P＜0.05);As compared with ND1 group,ND2 group had significantly lower mRNA and protein expression levels of Caspase-3 and Cyt-c (P＜0.05).Conclusion Intracistemal administration ofnimodipine could decrease mRNA and protein expressions of Caspase-3 and Cyt-c and inhibit activation of mitochondrial pathway in hippocampus in rabbit models of SAH.

Objective To study the feature of neonatal infections and characteristics of antibiotic treatment in a tertiary children ' s hospital. Methods Clinical data including incidence of infection, primary disease,species of bacteria, complication and antibiotic utilization in hospitalized patients from Jan. 2010 to Dec. 2012 were retrospectively reviewed using their medical records. Results Among 1826 patients admitted to neonetal surgery ward, 542 infants ( 29. 7％) were with infection. The incidence of antibiotic resistance was 23. 51％. The top five infectious diseases were:perianal abscess, necrotizing enterocolitis, colicitis, omphalitis and subcutaneous gangrene. 12 cases of multi-resistant infection were cured by non-restricted antibiotics. 109 were cured by restricted antibiotics. And other 7 were cured by special antibiotics. No death nor multi-resistant nosocomial infection were found. Risk factors including multi-site infection, premature or low birth weight infants, liver, kidney or heart dysfunction,fever lasting more than 3 days after antibiotic therapy, septic shock, sepsis, digestive tract perforation and peritonitis,were vital in choosing specific antibiotics. Conclusions Infection is one of the most common diseases in neonatal surgery ward, with major pathogens sensitive to antibiotics. The clinical characteristics and drug sensitive test are conductive to the reasonable use of antibiotics. Special antibiotics can be used directly in patients with risk factors Clinical doses of antibiotics in neonates depend on the monitoring of drug concentration.

Objective To analyze the pattern of antibiotic use and antibiotic resistance tendency of gastrointestinal surgery in a tertiary children′s hospital .Methods 2 625 patients(which account for 27 .52% of all the hospilitalized patients ,the resistant rate was 15 .70% ) detailed morbidity ,entity ,bacteria ,complication ,antibiotic utilization was retrospectively reviewed using the hospital medical records from 2010 to 2012 .Results 2 625 patients the percentages of the top five disease category were :appendicitis ac-counting for 40 .72% ,perianal abscess accounting for 21 .53% ,periappendiceal abscess accounting for 9 .30% ,necrotizing enterocol-itis accounting for 3 .73% ,omphalitis accounting for 2 .93% .The top three pathogen were :escherichia coli ,klebsiella pneumoniae subsp ,staphylococcus aureus respectively .255 multi-resistant bacteria of the superficial infection patients and 157 of the invasive in-fection patients .49 multi-resistant infections were cured by first or second generation of cephalosporins and penicillinase-fast peni-cillin ,and 346 were cured by third or forth generation of cephalosporins and penicillinase-fast penicillin ,and 17 were cured by car-bapenem or vancomycin .No dead or multi-resistant hospital infectious case was reviewed .Conclusion The sensitive rates of surgi-cal infected patient were 84 .3% ,and opportunistic pathogen infection was the main characteristics .To aware the clinical character-istics and drug sensitive test is conductive to the reasonable use of antibiotics of severe infections .The cases of superficial resistant infection or invasive non-resistant infection tend to use restricted antibiotics .The cases of invasive resistant infection tend to use special antibiotics .

Objective:To design and construct the replication-deficient recombinant adenovirus Ad-siCTGF which can silence the expression of connective tissue growth factor (CTGF) by RNA interference and verified its function. Methods:A specific sequence, which was verified to be able to silence CTGF gene with high efficiency, was cloned into pSES-HUS vector to produce the shuttle plasmid pSES-siCTGF. The plasmid after Pme Ⅰ linearization was cotransduced with pAdEasy into BJ5183 E.coli strains to construct recombinant vector Ad-siCTGF. After linearization treatment with Pac Ⅰ enzyme digestion Ad-siCTGF was transfected into HEK293 cells via liposome mediation. The recombinant adenovirus was packaged. The titer of the Ad-siCTGF was increased after three times of cross-infection. 4T1 cells were infected with the adenovirus. The silencing efficiency was tested by real-time fluorescence quantitative (RFQ)-PCR and Western blotting.Results:Pac Ⅰ enzyme digestion electrophoresis indentified that recombinant adenovirus was successfully constructed. The titer of the recombinant adenovirus Ad-siCTGF was 2.6×10~(10) pfu/mL after amplification and purification. The CTGF mRNA and protein expression in 4T1 cells were decreased by 36.27% and 31.56%, respectively, compared with the control groups.Conclusion:The recombinant adenovirus which can silence the expression of CTGF was successfully constructed. It laid a good foundation for further investigation of the action mechanism of CTGF in tumor cells.

Objective To explore the mechanism and safety of miltidrug-resistance gene 1(MDR1)transfection into the bone marrow mononuclear cells of rabbit in vitro with the adenovirus vector promoted by ultrasonic microbubble.MethodsBone marrow mononuclear cells of rabbits were collected and divided into 5 groups after cultured in the 6-well plate according to the different experimental conditions(MDR1 gene was transferred into the cells with or without ultrasound irradiation and microbubbles)：conventional culture group (A)，Ad5-MDR1 group(B)，Ad5-MDR1+ultrasound irradiation group(C)，Ad5-MDR1+microbubbles group(D)，Ad5-MDR1+ultrasound irradiation+microbubbles group(E).The positive transfection rate of MDR1 gene in mononuclear cells of different groups were tested by flow cytometry,and the survival rate of cells in different periods were tested by trypan blue exclusion method.Moreover,the appearance and ultramicrostructure of cells were observed by electronmicroscope.Results①The transfection rate of MDR1 gene in different groups were 0.39%±0.11%，5.03%±0.35%，4.93%±0.38%，5.25%±0.80%and 19.93%±1.51%respectively.The transfection rate of MDR1 gene in group E was higher than those in other groups(P＜0.05).②Compared with those in control groups(group A，B，C and D)，the transfection rate in group E was significant raised by ultrasound irradiation and microbubbles.However,there were no significant difference in survival rate of cells between the five groups(P＞0.05).③After ultrasonic irradiation，there were transient holes on the cell membrane，which could disappear after irradiation by ultrasound for 24 hours.And the temporary swelling of organelles was reversible.Conclusions Microbubbles irradiated by ultrasound can cause small transient holes on eell membrane and increase permeability of it，and enhance the transfection of MDR1 gene in bone marrow mononuclear cells with the adenovirus vector，which is safe and available.

Objective: To investigate the enhancive effect of TNF-α on transfection efficiency of adenovi-rus in mononuclear cells of mouse and the enhancive capability of protection of bone marrow by MDR1. Meth-ods: Before the mononuclear cells of mouse were infected by recombinant adenovirus encoding human MDR1 gene, they had been pretreated by TNF-α. Transfection efficiency of adenovirus was monitored by fluo-rescence microscopy, immunohistochemistry and flow cytometry (FCM). mRNA and protein levels of MDR1 in the mononuclear cells of mouse were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot before and after treatment of TNF-α. Results: After treatment of TNF-α, tansfection rates of adenovirus were obviously increased in the treated group (26.26%) compared with the untreated group (11.96%). mRNA levels and protein levels of MDR1 were obviously increased in the treated group compared with the untreated group. Conclusion: TNF-α could enhance transfection efficiency of adenovirus in mononu-clear cells of mouse and enhance capability of protection of bone marrow by MDR1.

BACKGROUND: Heme oxygenase-1 (HO-1) plays an important role on preventing tissues and organs from oxidant stress injury, which remains currently one of the most active areas of investigation. OBJECTIVE: To study HO-1 over-expression on the survival and fiver function of rats after reduced-size liver transplantation by constructing recombinant adenovims Ad5-HO-1.DESIGN, TIME AND SETTING: Randomized controlled animal study was performed in the Institute of Pediatrics, Children Hospital Affiliated to Chongqing Medical University from September 2003 to March 2005. MATERIALS: Seventy-four SD rats were used to establish in situ reduced-sized liver transplantation models. METHODS: Recombinant adenoviral vector encoding Ha-1 gene (AdS-HO-1) was generatred by molecular biology method, wluch was administered to donors via voln at 48 hours before transplantation. All rats were randomly divided into control group(n=12),saline group(n=12), cobalt protoporphyrin (CoPP)group(n=13),Ad5-HO-1 group(n=13),Ad5-green fluorescent protein(GFP)group(n=12),and Ad5-HO-1+zinc protoporphyrin (ZnPP)group (n=12).Livers of donor wefe harvested and stored for 24 hours at 4℃ in HTK solution. Before it is implantedinto recipient. MA IN OUTCOME MEASURES: Survival rate and liver function; portal vein blood flow monitored 2 hours after transplantation by Color Doppler How Imaging; pathological changes of transplanted liver tissue observed by HE staining;HO-1 activity, tumor necrosis factor-alpha(TNF-α),Bcl-2 and Bax expression detected by immunohistoehemical staining; changes of HO-1,TNF-α,bcl-2 and bax mRNA detected by molecular viewpoint. RESULTS: Survival rate in the Ad5-HO-1 group was significantly higher than that in the saline group at al,7,and 21 days after liver transplantation(P＜0.05).Glutamic pyruvic transaminase decreased in the Ad5-HO-1 group as compared to that in the saline,Ad5-GFP,and Ad5-HO-1+ZnPP groups(P＜0.05);portal venous blood flow significantly increased 2 hours after transplantation(P＜0.05);HO-1 activity also significantly increased(P＜0.05).RT-PCR and immunohistochemical staining showed that HO-1 and bcl-2 expressions increased(P＜0.05),but bax and TNF-αexpressions decreased(P＜0.05).CONCLUSION:Ad5-HO-1 significantly induces high expression of HO-1 increases portal venous blood flow within 2 hours alter liver transplantation, and promotes liver functional recovery so as to prolong survival time of rats after liver transplantion.

BACKGROUND: Compared to those original viruses systems, adeasy adenovirus, a recombinant adenoviral system widely used in recent years, based on viruses with a deletion of both El and E3, reported by T.C. He in 1998, is an improved one. It simplifies the generation and production of such viruses and expedite the process of generating and testing recombinant adenoviruses using homologous recombination in bacteria rather than in eukaryotic cells. Moreover, it can be conveniently followed with the aid of green fluorescent protein encoded by EGFP gene incorporated into the viral backbone.OBJECTIVE: To construct the recombinant adenovirus and to evaluate them by transfect them to mesenchymal stem cells (MSCs)and detect the expression of target gene hlGF-I at gene and protein levels.DESIGN: Repetitive measurement wail.SETTING: The Institute of Pediatric Research, Chongqing University of Medical Science.METHODS: The study was performed at the Institute of Pediatric Research, Chongqing University of Medical Science from November 2004 to March 2005. After the amplification of truncated hlGF-1 gene from pcDNA3.l-hlGF-I by polymerase chain reaction (PCR), the gene fragment was inserted into the shuttle plasmid pAdtrack-CMV for homologous recombination with backbone plasmid pAdeasy-I in bacteria BJ5183 to get adenovirus.Ad-hlGF-1. The high titer adenovirus supernatant was obtained by repeated transducing of HEK 293 cells by adenovirus harvested after confirmation of the adenovirus structure. As target cells,MSCs were infected with adenovirus earned target gene, hIGF-1, to determine the expression of hlGF-1 gene.MAIN OUTCOME MEASURES: ① The construction of recombinant adenovirus vector;② the expression of target gene hIGF-1 in HEK 293 cells and the proper multiplicity of infection (MOI); ③ hIGF-1 gene expression in MSCs.RESULTS: The adenovirus vector based on adeasy system was constructed successfully and the Ad-hlGF transducing was successfully or efficiently expressed in MSCs cells. The ideal expression of harvested recombinant adenovirus in MSCs was detected by fluorescence microscope, RT-PCR, immunocytochemistry, and Western Blot.CONCLUSION: Adenovirus vector is an effective vector tools for gene expression and wansfection of MSCs. MSCs transduced with Ad-hIGF-1 maybe another option to gene-modified seed cells for articular cartilage tissue engineering.

BACKGROUND: Mesenchymal stem cells from bone marrow stroma are able to differentiate into multiple mesoderm-type cell lineages. Because of the ease of their isolation and their extensive differentiated potentiality, mesenchymal stem cells become potential resource of cell and gene therapy. OBJECTIVE: To summarize biological characteristics and application of bone marrow mesenchymal stem cells. RETRIEVAL STRATEGY:An online search of Pubmed was undertaken by using the keywords of "Bone marrow mesenchymal stem cells, gene therapy, transplant"to identify the relevant articles published in English from January 1999 to December 2006. At the same time,Wanfang database was undertaken to identify the relevant articles published between January 1999 and December 2006 with the key words of "Bone marrow mesenchymal stem cells, gene therapy, transplant" in Chinese.The data were selected primarily, and then quotations of each article were checked. Inclusive criterion:The articles related to the biological characteristics and application of bone marrow mesenchymal stem cells were included. Exclusive criteria:the articles with repetitive research or Meta analysis were excluded.Totally 95 relevant articles were selected and 78 of them met the inclusive criterion. The 45 excluded articles were of old or repetitive content. LITERATURE EVALUATION: Selected articles were on basic experiment or clinical study of the biological characteristics, cell transplantation and gene therapy of bone marrow mesenchymal stem cells. Totally 33 articles of the 78 articles met the inclusive criterion were included. Of them, 4 articles were on the isolation of bone marrow mesenchymal stem cells, 3 on surface marker of bone marrow mesenchymal stem cells, 6 on biological characteristics of bone marrow mesenchymal stem cells, 3 on immune response between bone marrow mesenchymal stem cell transplantation and host and 17 on the application of bone marrow mesenchymal stem cells on disease therapy. DATA SYNTHESIS: In the suitable condition, bone marrow mesenchymal stem cells can differentiate into multiple cells, such as bone, chondrocytes, fat, muscle, tendon, neuron-like cells, myocardial cells and stroma-cells that support hematopoietic stem cells. Bone marrow mesenchymal stem cells seem to be the cells of immunosuppression and low-down immunogenicity. It makes bone marrow mesenchymal stem cells used in systemic transplantation, local implantation, as a vehicle for genes in gene therapy protocols or to generate transplantable tissues and organs in tissue engineering protocols. The results of these initial trials are very encouraging in animal test and several clinical trials are successful in some patients. But the enough clinical trails and long-term safety of therapeutics based on bone mesenchymal stem cells are needed. CONCLUSION: Biological characteristics of bone marrow mesenchymal stem cells have been gradually recognized. It is hoped that using bone marrow mesenchymal stem cells in clinic will bring major advances in the therapy of some diseases.