Objective To investigate the effects of minocycline on cell viability and neurite outgrowth of pheochromocytoma cells (PC12) after oxygen‐glucose deprivation(OGD) injury .Methods PC12 cells were exposed to OGD insult for 2 ,4 ,6 ,8 h to estab‐lish a cerebral ischemia model in vitro .High‐differentiated PC12 cells were cultivated and randomly divided into three groups :con‐trol group ,OGD group and various doses of minocycline(0 .1 ,1 .0 ,10 .0 μM) treated group .24 h after OGD‐reperfusion ,PC12 cells viability was assessed by CCK‐8 assay ,the neurite was labeled with MAP‐2 by immunofluorescence and neurite length was meas‐ured by the Image‐Pro Plus 7 .0 software ,GAP‐43 protein expression was determined by Western blotting .Results Compared to the OGD groups ,minocycline induced a concentration‐dependent increase in cells viability [(46 .1 ± 2 .9)% vs .(77 .0 ± 2 .5)% ,P<0.01],improvedneuriteoutgrowthandincreasedtheexpressionofGAP‐43proteininPC12cellsafterOGDinjury([(0.34±0.04) vs .(2 .11 ± 0 .10) ,P<0 .01] .Conclusion Minocycline could protect against oxygen glucose deprivation injury and promote neurite outgrowth .This finding suggests minocycline may be a novel therapy for cerebral ischemia .

Objective To investigate the effects of minocycline on regional cerebral blood flow and the expression of endothe-lin-1(ET-1) in ischemic penumbra of rats after focal cerebral ischemia reperfusion injury .Methods Middle cerebral artery occlusion (MCAO) with nylon suture was used to be established as focal cerebral ischemia reperfusion mode (I/R) ,a total of 35 male Spra-geue-Dawley rats were randomly divided into 3 groups :sham-operated group(n= 10) ,model group(n= 15) and minocycline group (n= 15) .After 24 hours of I/R ,The neurobehavioral function of rats was evaluated by Longa′s test ,the regional cerebral blood flow in ischemic penumbra was assessed with laser-Doppler flowmetry .After 6 and 24 hours of I/R ,the expression of ET-1 in peri-in-farct region was measured by both immunohistochemistry and radioimmunoassay .Results Compared with sham-group ,the Longa′s test scales ,ET-1 protein expression increased and the rCBF decreased in ischemic penumbra in model group(P< 0 .05) .the Longa′s test scales ,ET-1 protein expression decreased and the rCBF decreased in ischemic penumbra in minocycline group when compared to the model group(P< 0 .05) .Conclusion Minocycline could promote neurological functional recovery of rats after MCAO ,which might be attributed to increase the cerebral blood flow and regulate the endothelin-1 expression in ischemic penumbra .

To isolate and culture the purified monoclonal neural stem cells from the cerebral cortex of new born mice, new-born mice cerebral cortex was isolated and dissociated to single-cell suspension by mechanical trituration. The dissociated single cells were cultured in serum-free medium. After the formation of neurospheres, single-cell clone culture was performed by limiting dilution and the proliferated single-cell clones were harvested for subculture. Immunocytochemistry was used to detect the specific marker of neuroepithelial stem cells (Nestin) of the primary and monoclonal neurospheres. In the differentiated cells we detected the specific antigen of NF-200 and GFAP. Our results showed that the primary neurospheres expressed Nestin antigen positively. By limiting dilution, we cultured the cell lines from single-cell clone and the monoclonal neurospheres expressed Nestin and had capabilities of self-renewal, proliferation and the potentiality of differentiation into neurons and glial cells. It is concluded that monoclonal neural stem cells which have the ability of proliferation and multi-directional differentiation can be isolated and cultured from the cerebral cortex of new-born mice by limiting dilution.

Objective To investigate the role of poly(ADP-ribose) polymerase (PARP) inhibitor PJ34 in regulating blood-brain barrier (BBB) permeability and matrix metalloproteinases-9 (MMP-9) expression in a mouse model of traumatic brain injury (TBI).Methods A total of 136 adult male BALB/c mice were randomly divided into sham-operated group,injured group and PJ34-treated group according to the random number table.Controlled cortical impact in mice was established.At 6 and 24hours postinjury,neurological deficit was evaluated,including motor,sensory,reflex and beam balance tests ; BBB permeability and brain water content were detected using Evans blue test and gravimetric technique; brain contusion volume was measured using HE staining; levels of MMP-9 in cytosolic fractions were detected using Western blotting.Results At 6 and 24 hours postinjury,neurological severity score in PJ34-treated group (8.00 ± 0.26,7.50 ±0.25) were lower than those in injured group (12.50 ±0.39,11.80 ± 0.32) ; brain contusion volume in PJ34-treated group [(11.25 ± 0.91) mm3,(13.55 ±1.06) mm3] was lower than those in injured group [(25.37 ± 1.75) mm3,(28.24 ± 1.51) mm3] ; BBB permeability in PJ34-treated group [(440.08 ± 3.10) μg/mg,(860.46 ± 3.86) μg/mg] was lower than those in injured group [(936.96 ± 4.71) μg/mg,(1 302.23 ± 5.89) μg/mg] (all P ＜ 0.01).Brain water content lowered significantly in PJ34-treated group than in injured group at 6 hours postinjury [(80.77 ± 0.76) ％ vs (82.55 ± 0.73) ％,P ＜ 0.0l],but between-group difference was not significant at 24 hours postinjury.Lower levels in MMP-9 were also observed in PJ34-treated group compared with injured group at 6 and 24 hours postinjury(P ＜ 0.05 or 0.01).Conclusion PARP inhibitor PJ34 can attenuate MMP-9 up-regulation,inhibit BBB injury and hence protect the brain against TBI in mice.

Objective To study the effect of siRNA targeting survivin and tumor necrosis factor related apoptosis-inducing ligand on the proliferation and apoptosis of hepatocellular carcinoma ( HCC) cells. Methods siRNA eukaryotic expression vector was constructed and the effects of soluble tumor necrosis factor related apoptosis-inducing ligand ( sTRAIL) on HCC cells were observed. Results The recombinant plasmid Psilence ( + )-survivin was successfully constructed. Survivin mRNA expression inhibition ratio reached 73% by RT-PCR. Observed by MTT method sTRAIL failed to inhibit the survival rate of HepG2、HepG2/Silence( - ) cells at 12 h、24 h、48 h when compared to control groups. With the survivin gene being inhibited, the survival rate of HepG2/Silence( + ) cells(0. 518?0. 017) decreased in 12 h compared to control groups (0. 741?0. 005 ) and reached the lowest level in 48 h ( P

Objective To investigate the distribution characteristics of pathogens isolated fromcerebrospinal fluid of neurosurgical patients with intracranial infection following open craniotomy and thetherapeutic effect influenced by these pathogens,in order to give a reference to the clinical treatmentmeasures.Methods A retrospective analysis was made on the pathogen distribution and therapeuticeffect of 43 patients with intracranial infection and positive cerebrospinal fluid cultures after open cranioto-my from May 2007 to May 2013.Cerebrospinal fluid was cleared using the intraventricular catheter orlumbar catheter combined with intraventricular (ventricular irrigation) or intraspinal (intrathecalirrigation) injection of antibacterial agents.Results To test bacteria in cerebrospinal fluid pathogencultures,34 cases were infected with single strain (26 Gram-positive bacteria and 8 Gram-negativebacteria) and 9 cases had mixed infection with multiple strains.Fifty-two pathogen strains were isolated,including 32 (62％) Gram-positive bacteria,18 (35％) Gram-negative bacteria,2 (4％) fungi.A totalof 29 cases were cured (67％),7 improved (16％),and 7 ineffective (16％).Conclusions Cere-brospinal fluid pathogen infection is primarily Gram-positive bacterial infection,usually staphylococcusepidermidis and staphylococcus aureus.Gram-negative pathogens are acinetobacter,klebsiella,andpseudomonas aeruginosa.Ventriculoperitoneal shunting surgery and craniocerebral surgery are often asso-ciated with mixed infection of pathogens.Ventricular irrigation allows better results than intrathecal irriga-tion.Indications of intrathecal irrigation treatment used to control intracranial infection after ventriculoper-itoneal shunting surgery and craniocerebral surgery should be strictly performed.

Objective To explore the value of fluid-attenuated inversion recovery (FLAIR)sequence and gradient echo T2 ?weighted image (GRE-T2 ? WI)in diagnosis of acute traumatic extra-axial hemorrhage.Methods 50 patients who were diagnosed as acute traumatic extra-axial hemorrhage by plain CT underwent FLAIR and GRE-T2 ? WI in acute stage.The diagnosis consistency (Kappa ),detection rate of subarachnoid hemorrhage(SAH),epidural hemorrhage(EDH)and subdural hemorrhage(SDH)were compared.The detection rates of SAH in 8 locations among FLAIR,GRE-T2 ? WI and combination of two sequences were analyzed by Chi-square test.Results 48 patients were enrolled in the study.The diagnosis consistency of SAH between FLAIR and GRE-T2 ? WI was high (k =1.0).The detection rate of SAH was 100% for both FLAIR and GRE-T2 ? WI.Comparing with GRE-T2 ? WI and combi-nation of two sequences,FLAIR tended to misdiagnose SAH in perimesencephalic cistern (P <0.05).The diagnosis consistency of EDH between FLAIR and GRE-T2 ? WI was high (k =1.0).3 patients with EDH were all detected by FLAIR and GRE-T2 ? WI. The diagnosis consistency of SDH between FLAIR and GRE-T2 ? WI was high (k =0.943).The detection rate of 12 patients with SDH was 100% in FLAIR,and 1 patient with SDH was missed by GRE-T2 ? WI.Conclusion The detection rate of acute traumatic extra-axial hemorrhage is high for both FLAIR and GRE-T2 ? WI.Combination of two sequences can improve the accuracy of acute traumatic extra-axial hemorrhage in clinic.

BACKGROUND: Deletion of glial cell-derived neurotrophic factor and endothelin receptor type B gene will induce abnormal development of enteric nervous system. Neural stem cell transplantation can repair nervous system from anatomy and function,and be considered as a vector of gene transfection.OBJECTIVE: To transfect recombinant adenovirus carrying glial cell-derived neurotrophic factor and endothelin receptor type B gene into mouse neural stem cells, and to observe expression of target gene.DESIGN: A cell-gene study.MATERIALS: New-born Kunming mice were provided by the Animal Center of Tongji Medical College, Huazhong University of Science and Technology, China. jetPEI reagent was purchased from PolyPlus Co, France. The pAdTrack-CMV-GE with green fluorescent protein (GFP) was gifted by Doctor Sun Nianfeng and Zhang Jinghui in our laboratory.METHODS: Neonatal mouse brain tissues were sterilely obtained to prepare monoplast suspension. Adenovirus expressing glial cell-derived neurotrophic factor and endothelin receptor type B gene with GFP was dissolved in NaCI to prepare JetPEI/DNA complex. Subcultured neural stem cells in DMEM/F12 were regulated to 5×10~8/L, and 400 μL cell suspension and 100 μL JetPEI/DNA complex were seeded on a 24-well plate at 37 ℃ in 5% CO_2 incubator. Neural stem cells were harvested at 24, 48 and 72 hours following transfection.MAIN OUTCOME MEASURES: The efficiency of transfection was detected using fluorescence microscope and flow cytometry.Target gene expression in neural stem cells was determined using RT-PCR.RESULTS: Bright green fluorescence of the transfected cells could be observed under fluorescence microscope after 24 hours of transfection. The positive rate of GFP was 15.36%, 24.67%, 25.73% at 24, 48 and 72 hours following transfection respectively.Neural stem cells expressed glial cell-derived neurotrophic factor and endothelin receptor type B gene at various time points.Strap brightness was low at 24 hours, and exogenous gene expression was great at 72 hours.CONCLUSION: The target genes were successfully transfected into neural stem cells by using jetPEI reagent. Moreover, glial cell-derived neurotrophic factor and endothelin receptor type B gene effectively transcribed and expressed in target cells.

Objective To evaluate the antitumor effect of SEB-scFv fusion Drotein on gastric cancer. Methods Typical changes of morphologic features and super-microstructure were observed when SEB-scFv fusion protein was used in SGC7901 cens;and the effects of SEB-scFv and its concentration on the cell growth were examined by methyltetrazolium(MTT)assay;DNA ladder and flow cytometry were employed respectively to detect the inhibition phenomenon or apoptosis.We produced a subcutaneous gastric tumor model in baby SD rats by implanting SGC7901 cells.The SEB and SEB-scFv were injeeted to the vena caudalis in the trial groups,and normal saline to the control group.The weight of the tumor and the survival were recorded after treatment. Results Cell inhibitory rate was increased along with increased concentration of SEB-scFv fusion protein.Electron microscopy revealed that the cell presented typical changes of apoptosis.FCM indicated that the apoptotic rate of SGC7901 cell lines significantly increased with increasing dose of SEB-scFv fusion protein,and agarose gel electrophoresis appeared marked DNA ladder.The average weight of tumor in the SEB-scFv group was lower than that in control groups(P＜0.05).The tumor inhibition rate was 61.3%,and the mean survival period of rats in SEB-scFv group was longer than that of other group(P＜0.05)with a survival prolongation rate of 54.6%. Conclusion The resuhs indicate that SEB-scFv fusion protein has an obvious antitumor effect on gastric cancer.

Objective To evaluate the feasibility,safety and short-term outcome of laparoscopic-assisted surgery for colorectal cancer.Methods From August 2001 to November 2004,laparoscopic resection of colorectal carcinoma were performed in 112 cases,including right hemicolectomy(n=23),left himicolectomy(n=7),radical resection of sigmoid cancer(n=15),Dixon procedure(n=49),and Miles procedure(n=18).Results One hundred and five patients underwent laparoscopic resection successfully,7 cases were converted to open surgery because of hemorrhage,obesity or adhesion with adjacent organ,6 of which were left colon or rectal cancer.The mean operating time was(161.2?48.6)min,and the mean operative blood loss was 78.5 mL.There were 8 cases occurred postoperative complications,and no mortality during perioperative period.The length of upper and lower segment of resection for colonic cancer was (14.5?3.2)cm and(11.0?2.6)cm respectively.The length of upper and lower segment of resection for rectal cancer was(15.3?2.7)cm and(2.8?1.6)cm,respectively.The mean number of lymph nodes dissected was(8.2?4.6),and lymph node metastases were found in 49 cases.One hundred and seven cases(95.5%) were followed up for 8-44 months,of which,7 cases had local recurrence and 6 cases had distant metastases.No case of trocar port tumor implantation was observed.Conclusions Laparoscopic surgery for colorectal cancer is feasible and safe,can result in the same outcome as open radical surgery,and has the advantages of mini-invasive procedure.