AIM:The homologous recombination takes place in E.coli BJ5183 between shuttle vector and adenoviral backbone vector.Recombinants are selected for kanamycin resistance,and the linearized recombinant plasmid is transfected into 293 cells.In this study,AdEasy system was used to generate recombinant adenovirus vector carrying rat interleukin-10(IL-10) gene.METHODS:The experiment was conducted in Department of Microbiology,Qingdao University Medical College from August 2006 to May 2007.①The materials included five clean-grade SD rat,a shuttle vector pAdTrack-CMV,an adenoviral backbone plasmid pAdEasy-1,E.coli.BJ5183 and DH10B,and human embryo kidney 293 cells,which were given as a present by professor Luo.All animal treatment was accorded with the ethical standards.②Total RNA was extracted from spleen cells of rat stimulating by lipopolysaccharide.IL-10 cDNA was amplified by using RT-PCR.The gene of interest was firstly cloned into a shuttle vector pAdTrack-CMV.The resultant plasmid was linearized by digesting with restriction endonuclease Pme Ⅰ,and subsequently transformed into E.coli.BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1.Finally,the linearized recombinant plasmid was transfected into adenovirus packaging cell lines 293 cells.Recombinant adenovirus vAd-IL-10 was obtained.The expression of green fluorescent protein(GFP) was observed under fluorescent microscope.293 cells were cultured in 96-well culture plate with 1 ?104 cells per well.Condensed virus stock solution was added into the plate at different ratios.Recombinant adenoviruses titer was determined.Three days later,total RNA was extracted from 293 cells by TRIzol one-step method,and contaminant DNA was digested by DNaseⅠ.Electrophoresis detected the expression of IL-10 mRNA.RESULTS:①GFP expression was observed 16 hours after packing of the linearized recombinant adenoviruses in 293 cells.The amplification product of replicationdeficient Ad-IL-10 virus was transfected into adenovirus packaging cell lines 293 cells.When the fourth and fifth generations were seeded in virus for 24-48 hours,fluorescence was found in almost cells,and 5.5?108 pfu/mL titer of Ad-IL-10 was obtained.Titer of vAd-GFP was 9.0?108 pfu/mL.②Total RNA was extracted from transfected 293 cells.Electrophoresis showed that 580bp amplification product was expressed obviously and recombinant adenovirus was duplicated in 293 cells.CONCLUSION:The recombinant adenoviral vector carrying IL-10 gene is successfully constructed using AdEasy system.Moreover,vAd-IL-10 recombinant adenovirus with high titer is prepared and transcribed in 293 cells.

Objective To study the expressions and clinical significance of E cadherin(E CD) and CD44v6 in bladder transitional cell cancinoma. Methods The expressions of E CD and CD44v6 of 96 cases of bladder transitional cell cancer were detected by streptavidin peroxidase immunohistochemical method. Results The positive expression rates of E CD and CD44v6 were as follows:Grade Ⅰ,86.7%(26/30),80.0%(24/30);Grade Ⅱ,52.8%(19/36),69.4%(25/36);Grade Ⅲ,26.7%(8/30),33.3%(10/30), P

Yin-Yang1 (YY1),as a ubiquitous nuclear transcription factor with double transcriptional activity in eukaryotic cells,regulates many genes transcription and plays an important role in the cellule biological processes.Overexpression of YY1 is found in several tumor types recently,and may play a role in tumor development and progression by regulating oncogenes,tumor suppressor genes,angiogenesis related factors,as well as apoptosis.YY1 may become a new kind of tumor markers,is conducive to estimate the prognosis of patients with tumor and gain important breakthrough in cancer targeted therapy.

Objective To explore surgical strategy for patients with hilar cholangiocarcinoma (HCC) and study prognostic factors after curative treatment. Methods We retrospectively reviewed medical records of 41 patients with HCC surgically treated in our department during the 9-year period from January 1999 to February 2007. Clinicopathological factors were evaluated for their association with post-operational survival by univariate and multivariate analysis using Cox proportional hazard model. Results All the 41 patients underwent laparotomy following preoperative assessment of extent of disease and 21 patients (resectability rate 51.2%) ultimately underwent resection with curative in-tent. In the resection group, R0 radical resection was possible in 11 patients, while R1 resection in 6and R2 in 4. Different types of hepatectomy were combined to accomplish resection. Meanwhile, por-tal vein wedge resection or reconstruction was needed in two patients. The 1-, 3-and 5-year survival rates were 41.5%, 14.6% and 4.9% in the overall group and 71.3%, 28.6%, 9.5% in the resection group, respectively. In R0-resection, Rl-resection and R2 resection group, the 1-,3-and 5-year sur-vival rates were 81.8% ,45.5% ,18.2% ;66.7% ,16.7% ,0 and 50% ,0,0, respectively. Survival rates after resection were significantly higher than those after palliative drainage and exploratory laparotomy (P<0. 001). Higher survival rates were seen in R0-resected patients when compared with Rl-or R2-resected patients (P<0.001). Multivariate analysis revealed that tumor-free margins, pTNM stage and combined hepatectomy were independent prognostic factors affecting survival. Conclusion Only surgery can provide chance to achieve the possibility of cure and long-term survival. Tumor-free margins, pTNM stage and combined hepatectomy are the most important prognostic factors affecting the survival.

ATM: To prepare Shenfuqing Lyophilized Powder (Radix Ginsenp Rubra, Pericarpium Citri Reticulatae Viride, ect.) METHODS:Shenfuqing Lyophilized Powder was made by freeze drying. The concentration of Shenfuqing solution and the varieties and quantities of filling material were selected as well as the stability of Shenfuqing solution and powder for injection were examined primarily. RESULTS: The better prescription is 10% mannitol and 6% drug components. CONCLUSION: The quality of Shenfuqing Lyophilized Powder is more stable than that of solution for injection.

AIM To study the anti inflammatory and immunomodulatory actions of total glucosides of Mudan cortex(TGM)on adjuvant arthritis(AA)rats and its mechanisms. METHODS To observe the change of secondary inflammation and immune function on AA rats treated with TGM and to detect the effect of TGM on immune function in normal and AA rats in vitro. RESULTS TGM (25,50,100 mg?kg -1 ig) could significantly inhibit secondary inflammatory reaction(secondary inflammatory swelling, multiple arthritis, pathologic change of AA rats ankle arthrosis)of AA rats. Among them, TGM 50 mg?kg -1 ig had the best therapeutic effect, and could make the weight of atrophied thymus of AA rats restore to normal. Treated with TGM ig, not only the lower response of splenocytes to Con A and decreased IL 2 synthesis of AA rats were restored to normal but also the elevated IL 1 synthesis and PGE 2 release of PM? were reduced. The production of IL 1 by LPS induced PM? from normal rats were enhanced by in vitro treatment with lower concentrations (0 4～10 mg?L -1 )of TGM, but diminished with higher concentrations(50～250 mg?L -1 )of it. The production of PGE 2 were increased with dose independent manner(TGM 0 4～250 mg?L -1 ). In the range of 10～250 mg?L -1 TGM in vitro could significantly decreased the production of IL 1 and PGE 2 from AA rats and enhanced Con A induced proliferative response of splenocytes and IL 2 synthesis from splenocytes of AA rats. CONCLUSION TGM has anti inflammatory and immunomodulatory actions on AA rats mainly through inhibiting cell mediated immunity.

OBJECTIVE:To study the general pattern and characteristics of adverse drug reactions (ADR) induced by cimetidine. METHODS: A total of 306 ADR cases induced by cimetidine reported by domestic journals of 1983～2007 were analyzed statistically. RESULTS: The clinical manifestations of ADR induced by cimetidine were complicated and various and characterized by lesions of central nervous system (27.78%), cardiovascular system (21.24%), endocrine disturbance (8.82%) and hematological system (8.82%) etc. CONCLUSION: Great importance should be attached to the ADR induced by cimetidine to guard against the serious ADR such as aplastic anemia, cardiac arrest and allergic shock.

Objective To establish an inductively coupled plasma‐mass spectrometry (ICP‐MS) method for analysis of elements in peripheral blood of children .Methods A total of 474 healthy children in Hunan area were enrolled in this study ,and six ele‐ments ,including Ca ,Mg ,Fe ,Cu ,Zn and Pb ,in peripheral blood specimens were detected by using ICP‐MS method .Results The levels of elements ,including Ca ,Mg ,Fe ,Cu ,Zn and Pb ,in peripheral blood of healthy children showed skewed distributions ,and no significant differences were found in levels of these elements between male and female children(P>0 .05) .The reference intervals of Ca ,Mg ,Fe ,Cu ,Zn and Pb in peripheral blood of healthy children in this area were 57 .30 -81 .40 mg/L ,30 .40 -44 .80 mg/L , 361 .20-531 .40 mg/L ,848 .10-1 469 .20 μg/L ,2 .68 -6 .54 mg/L and 0 .00 -100 .00 μg/L respectively .Conclusion The ICP‐MS method for simultaneously detecting Ca ,Mg ,Fe ,Cu ,Zn and Pb in peripheral blood of children and reference interval of each ele‐ment are successfully established .

Objective To optimize the technique of isolating and extracting oligosaccharide from Radix Rehmanniae.Methods The optimum conditions for the water extracting technique of Radix Rehmanniae were studied by orthogonal test design.Then the water extract was isolated by macroporous resins and active charcoal successively,meanwhile the pattern of macroporous resins,the volume of the eluents,and the decolorizing methods of active charcoal were studied.And then the oligosaccharide was obtained after being purified by Sephadex gel column chromatography.The content of stachyose was determined by HPLC.Results The optimal preparation process was as follows: 12 fold water,three times,0.5 h for each time.Then the water extract was isolated and purified by D-101 macroporous resins,0.1% active charcoal and Sephadex G15 column chromatography were successively used to obtain the oligosaccharide.The analysis of oligosaccharide indicated its main constituents are stachyose,rafinose,and manninotriose,and the content of stachyose is more than 60%.Conclusion This preparation technique of extracting oligosaccharide from Radix Rehmanniae is reasonable and feasible.And the yield of the oligosaccharide from Radix Rehmanniae achieves to 26%.

OBJECTIVE:To establish the method for determining the contents of neferine and liensinine in plumula nelumbini.METHODS:Neferine and liensinine in plumula nelumbini were extracted by ultrasound method and determined by TLCs.RESULTS:The average recovery and relative standard deviation were(97.91?2.49)% and (99.28?3.22)%,respectively.The correlation coefficient were 0.996 and 0.999,respectively.CONCLUSION:The method,operated simply and attained the reliable results with good reproducibility,can be used to determine the contents of bioactive alkaloid in plumula nelumbini or other compound preparations.