Objective To investigate the function of bone-marrow mesenehymal stem eells(BMSCs) differ-entiating into neuron-like cells in vitro after transfected by human brain-derived neurotrophie factor (BDNF) gene. Methods Human BDNF genes were cloned and recombinant pcDNA3. 1(-)-BDNF plasmids were construc-ted. BMSCs from five guinea pigs were isolated and cultured while their morphologies were observed by microscope. The surface antigen was detected by flowcytometry. BDNF genes were transfected into BMSCs with electroporation, and the transfected BMSCs were induced by ratinoie acid(RA)after bolted by Geneticin-418 (G418), then the dif-ferentiated BMSCs were identified by immunocytochemistry. Results The culture ceils demonstroted the typical mor-phology and surface antigen of BMSCs. The transfected cells expressed neuron- specific enolase(NSE), Nestin and glial fi-brillary acid protein(GFAP) also secreted BDNF. Conclusion BMSCs transfected by BDNF genes can differentiate into neu-ron- like cells in vitro, electroporation can enhame the transfection efficiency, RA can promote cell induction.

0.05),while the levels of IL-8 and IL-6 declined,but were still significantly higher than that at immediately after induction of anesthesia(P

Objective To investigate the function of bone-marrow mesenchymal stem cells(BMSCs) differentiating into neuron-like cells in vitro after transfected by human brain-derived neurotrophic factor (BDNF) gene.Methods Human BDNF genes were cloned and recombinant pcDNA3.1(-)-BDNF plasmids were constructed. BMSCs from five guinea pigs were isolated and cultured while their morphologies were observed by microscope. The surface antigen was detected by flowcytometry. BDNF genes were transfected into BMSCs with electroporation,and the transfected BMSCs were induced by ratinoic acid(RA)after bolted by Geneticin-418(G418),then the differentiated BMSCs were identified by immunocytochemistry. Results The culture cells demonstroted the typical morphology and surface antigen of BMSCs. The transfected cells expressed neuron-specific enolase(NSE),Nestin and glial fibrillary acid protein(GFAP) also secreted BDNF.Conclusion BMSCs transfected by BDNF genes can differentiate into neuron-like cells in vitro,electroporation can enhame the transfection efficiency,RA can promote cell induction.

Objective To explore the pediatric emergency medical mode in critical ill children by pediatrics specialty and nurses,using equipments of ICU for adults in second class general hospital. Methods We retrospectively analyzed the effect of establishing the pediatric observation unit in the adult observationdistrict and the prognosis and disease spectrum of pediatric critical patients in our emergency ICU in the past five years. Results 5 076 pediatric patients had been admitted to the emergency observation unit accounting for 3.40% of outpatient yearly. There were 464 critically ill children,accounting for 9. 14% of children into the observation unit,251 cases (54. 09%) were transported to other hospitals,35 cases (7.54%) were admitted to emergency ICU due to transport high-risk, 14 cases required ventilator support. The disease spectrum based mainly on childhood accident,including trauma,poisoning and drowning,etc. The other major diseases were severe infection and fulminant myocarditis. After the treatment such as stopping bleeding, respiratory supporting ,correcting shock, and maintaining the function of important organs,77. 14 % were improved or recovered. The survival rate of children with mechanical ventilation was > 85%. Conclusion In our country,the pediatric emergency medical service system is inadequate. The critical illness treatment model of using the advantages of equipments and nurse' s team of adult ICU in second class general hospital ,combining with pediatrician trained in PICU is feasible and consistents with our national conditions.

BACKGROUND:Gene transfection of cells includes virus and non-virus vector.As virus vector has some issues,such as safety and immunological rejection,the present study explored lipofectamine and electroporation transfection methods.OBJECTIVE:To establish genetic engineering cells using human brain-derived neurotrophic factor(hBDNF)gene transfected bone marrow mesenchymal stem cells(BMMSCs)by lipofectamine or electroporation,and explore its characteristics and expression in vitro.METHODS:Lipofectamine method:The BMMSCs were obtained from the tibias and femurs of the guinea pigs.The third passage BMMSCs were cultured with plasmid-lipofectamine mixture for 6 hours,followed by fetal bovine medium for 48 hours.Immunohistochemistry was performed for transient expression.G418 was added after 48 hours.Electroporation method:BMMSCs were trypsinized and resuspended with serum-free medium.Cell suspension was added into electrotransformation pool,and plasmid was added.The electrotransformation pool was moved between electrodes.After transfection for 48 hours,gene transient expression was detected.G418 was added after 48 hours.Brain-derived neurotrophic factor expression was detected by immunohistochemistry and RT-PCR.RESULTS AND CONCLUSION:Immunohistochemistry showed that BDNF transient expression was 5.80% by lipofectamine and 24.29% by electroporation.Cells almost died at 14 days following lipofectamine transfection.Stable expression cell lines of BDNF engineered BMMSC were successfully established by electroporation,with 90% expressive rate by immunohistochemistry and expression of BDNF mRNA by RT-PCR.Genetic engineering cells using BDNF transected BMMSC were established by electroporation whereas failed by lipofectamine,and the expressed BDNF was confirmed by immunohistochemistry and RT-PCR in vitro.

Objective To investigate the appropriate setting up of normal reference ranges of lymphocyte subsets in some flow cytometry laboratories and to study the effects of different flow cytometers and various reagents by different manufacturers on the analysis of peripheral blood lymphocyte subsets. Methods Three FCM labs (named A, B and C) in Beijing region were selected representing 3 commonly used flow cytometers (Beckman Coulter Epics XL, Beckman Coulter Cytomics FC500, BD FACS Calibur). 50 samples from healthy donors were distributed to 3 labs and tested according to individual lab's standard operating procedure to verify whether the normal reference ranges of peripheral blood lymphocyte subsets established were appropriate. The application of internal quality control was also investigated. Commercial blood quality control reagents were given to the 3 FCM labs and tested within 20 working days paralleled with routine samples. In addition, 20 patients' samples were prepared using 4 different combinations of reagents ( a , b , c and d). The results from combination a, which used the Beckman Coulter reagents and instrument, were compared to the results from combination b, c and d, which used reagents from different manufacturers. Then the prepared samples were tested on Beckman Coulter Epics XL to evaluate the effects of different combinations of reagents on the results of peripheral blood lymphocyte subsets analyzed by the same instrument. Furthermore, 24 patients' samples prepared by same reagents from Beckman Coulter company were tested on both Beckman Coulter Epics XL and BD FACS Calibur respectively to assess the effects of different instruments on peripheral blood lymphocyte subsets. 20 patients' samples prepared by same reagents and instruments were analyzed by Beckman Coulter Epics XL analytic system and BD FACS Calibur analytic system respectively to assess the effects of the two analytic systems on the lymphocyte subsets. Results Over 10% of the results for NK and T4/T8 in lab A as well as T4 in labs B and C fell outside of their normal reference ranges. The probabilities exceeding corresponding normal reference ranges were 16% ( 9/50 ), 24% ( 12/50 ), 22% (11/50) and 12% ( 6/50 ), respectively. The results using internal blood quality control in 3 FCM labs within 20 working days were all within the reference ranges of the quality control provided by the kit. The biases from b and c reagent combinations were substantial compared with that of reagent a combination. Among the biases from b and c reagent combinations, the lowest probability of bias exceeding 10% was T8 of combination b, which had probability of 70% (14/20). The highest probabilities of hias exceeding 10% were T3 and T4 of b and c reagent combinations, which reached 100% (20/20) . Furthermore, the biases of T3, T8 and B of d reagent combination compared with that of reagent a combination were also substantial. The probabilities of bias exceeding 10% were 35% (7/20) ,85% (17/20) and 75% (15/20), respectively. Comparing the results of samples prepared and analyzed by reagents and instruments from different manufacturers to that of samples prepared and analyzed by the same company's reagents and instruments showed that there were great discrepancies in T3, T4 , T8 , B and NK. The probabilities of bias exceeding 10% were 71% ( 17/24), 80% (19/24) ,38% (9/24), 33% (8/24) and 92% (22/24), respectively. The biases of T8, NK and B were substantial when compared the results from Beckman Coulter Epics XL analytic systems and BD FACS Calibur analytic systems. The probabilities of bias exceeding 10% were 55% (11/20 ), 70% ( 14/20 ) and 55% (11/20), respectively. Conclusions FCM labs should set up their own normal reference range for peripheral blood lymphocyte subsets. The normal reference range should be verified periodically. It is important to apply internal blood quality control regularly and accumulate the quality control results. The reagents and instrument for preparing peripheral blood samples should be from the same manufacturers.

Object To investigate and compare the scavenging effect of Cordyceps growing in different environment on hydroxyl radical. Methods 10-Phennanthroline-Fe 2+ oxidative assay was used to observe the effect of scavenging hydroxyl radical from H_2O_2/Fe 2+ system by the aquenous extracts of Cordyceps. Results Cordyceps from Naqu of Xizang,northwest Yunnan,and northwest Sichuan all showed us the significant effect on scavenging of hydroxyl radical produced from Fenton Reaction. The biggest values of MDR among Cordyceps specimens from various microecological environment surpassed that among specimens of Naqu of Xizang,northwest Yunnan,and northwest of Sichuan. Conclusion The aquenous extracts of Cordyceps could scavenge the hydroxyl radical from Fenton Reaction. The microecological environment for Cordyceps growing has an significant effect on its scavenging action.

Objective To study the postoperative delirium in predicting dementia in elderly patients with femoral neck frac‐ture and provide prevention advises for postoperative delirium .Methods 120 elderly patients with femoral neck fracture were in‐cluded .All patients were tested normal by Clinical Dementia Rating (CDR) preoperative .Basic imformation and postoperative deliri‐um in predicting dementia were recored in detail .After one year of follow up ,all patients were tested by CDR again and divided into dementia group and without dementia group .Results There were 40 patients (33 .3% ) with delirium postoperative with dementia 1 week after operation;there were 16 patients got 0 .5 -3 .0 CDR score after one year follow up (40 .0% ) .There were 80 patients (66 .7% ) did not experience delirium postoperative 1 week after operation ,and 4 patients (5 .0% ) with dementia got CDR score higher than zero after one year folloew up;the difference was statistically significant (P<0 .05) .Single factor analyse showed that there were close correlation between age ,introverted ,level of education < 6 years ,diabetes mellitus ,delirium ,LDL‐C level and de‐mentia (P<0 .05) .Multiple factors showed that age ,diabetes history and delirium were the independent risk factors of dementia in elderly patients with femoral neck fracture (P<0 .05) .ROC curve showed that the AUC area of postoperative delirium in predic‐ting dementia in elderly patients with femoral neck fracture was 0 .878 .Conclusion In elderly patients without the history of de‐mentia ,age ,diabetes history and delirium after hip fracture surgery are the major predictor of dementia within half years .

Objective To observe the effects of hydrotalcite on histological ulcer healing quality in patients with Helicobacter pylori (Hp) positive active gastric ulcer .Methods A total of 145 patients with H p positive active gastric ulcer were divided into two groups .The control group was treated with esomeprazole ,amoxicillin and furazolidone triple therapy .The treatment group was treated with above triple therapy and hydrotalcite .After four-week treatment ,gastroendoscopy was repeated .The sections of gastric biopsy specimens were stained with hematoxylin-eosin (HE) staining ,Van Gieson staining , Alcian blue-periodic acid stiff (AB-PAS ) staining , streptavidin-perosidase (SP ) immunohistochemistry staining and computer imaging analysis technology were applied to observe maturity type of mucosal structure at the margin of ulcer ,the content of collagen and neutral mucus ,and the changes of expression of epithelial growth factor receptor (EGFR) and basic fibrobast growth factor (bFGF) before and after treatment .Paired samples t test was performed for comparison before and after treatment .F test and Chi-square test were used for comparison between two groups .Results The percentage of maturity type of regenerated mucosal structure of treatment group was 62 .9% (39/62) ,however that of control group was 40 .6% (26/64) ,and the difference was statistically significant (χ2 =13 .09 , P=0 .03) .Compared with those before treatment , the content of collagen in granulation tissue and neutral mucus in regenerated mucosa increased in both groups after treatment , and the increase was more significant in treatment group ((55 .1 ± 10 .4)% and (45 .8 ± 7 .1)% ,respectively);and the differences were statistically significant (F= 12 .85 and 18 .17 ,both P<0 .01) .Compared with those before treatment ,the percentage of EGFR and bFGF positive cells of both two groups increased , and the increase was more significant in treatment group ((49 .5 ± 8 .4)% and (48 .8 ± 9 .4)% ,respectively) ,and the differences were statistically significant (F=12 .17 and 18 .73 ,both P<0 .01) .Conclusion Hydrotalcite combined with anti-H p triple therapy can improve the maturity degree of structure and function of regenerated mucosa at the margin of ulcer in patients with H p positive active gastric ulcer and then improve the healing quality of ulcer .

Objective:To investigate the expression of TAZ and KLF5 and their clinical significance in hepatocellular carcinoma ( HCC).Methods:We freshly collected 76 samples of surgically resected HCC and matched normal tumor-adjacent tissues and detected TAZ and KLF5 expression in these samples using immunohistochemical staining.The clinical significance of TAZ and KLF5 protein expression were analysed.Results:The protein expression of TAZ and KLF5 in HCC tissues was significantly higher than those in matched normal tumor-adjacent tissues ( P=0.001;P=0.035 ).Clinicopathological analysis suggested that TAZ and KLF5 protein expression were associated with histopathological differentiation ( P=0.007;P=0.047 ) and TNM stage ( P=0.009;P=0.040).TAZ was positively correlated with KLF5 protein in HCC tissues (r=0.651,P=0.003).Conclusion:The high-expression of TAZ and KLF5 are correlated with poor clinicopathological characteristics,and TAZ is positively associated with KLF5 in HCC tissues, suggesting that TAZ may promote tumor progression through inhibition of KLF5 protein degradation in HCC.