Objective To explore the inhibition effect of recombinant mutant type(mt) p27 gene adenovirus (Ad-mt p27) transfection on the proliferation of colorectal cancer cell line SW480. Methods The recombinant mt p27 adenovirus vector was transfected into SW480 cells, western blotting analysis was applied to determine the p27 expression, and cell cycle distribution was analyzed using flow cytometry, and growth curve was drawn to observe the cell growth. Results There was high expression of p27 protein in the transfected SW480 cells. 77 9% cells were blocked in G 1/G 0 phase in the transfected group, which the percentage of the G 1/G 0 phase of Ad-LacZ transfected group and normal control group were 27 5% and 25 29% respectively. Ad-mt p27 transfection markedly inhibited SW480 cell growth in 24h and the inhibition effects was maintained for 7 days. Conclusion Recombinant adenovirus could efficaciously trasfect mt p27 gene into SW480 cells, and obtain high level mt p27 exprerssion. mt p27 markedly bolcked SW480 cell cycle progression and prominently inhibited SW480 cell growth. The mutant p27 gene transfection may be a new way of gene therapy for colorectal cancer.

0.05),and the levels of Fib and D-dimer were obviously higher but AT-Ⅲ was obviously lower in 2 NS groups than those in normal control group(P

BACKGROUND: The incidence rate of colon cancer is on an obvious increase. Previous treatments are primarily surgical therapy; radiotherapy and chemotherapy. Gene therapy has been applied in the clinical practice for treating colon cancer.OBJECTIVE: To construct replication deficient hp27mt recombinant adenovirus and detect its expression in SW480 cell in order to investigate the viability of gene therapy based on adenovirus and study the anti-tumor characteristic of mutant p27.DESIGN: A non-randomized controlled trial.SETTING: Department of Gastroenterology, Affiliated People' s Hospital of Yunyang Medical College, Institute of Clinical Medical Science of Yunyang Medical College; Department of Gastroenterology, Zhongnan Hospital of Wuhan University.MATERIALS: The experiment was conducted in the Institute of Clinical Medicine, Yunyang Medical College from January 2002 to September 2003. Restriction endo-nucleases Age Ⅰ, Nhe Ⅰ, Kpn Ⅰ, Pac Ⅰ and Pme Ⅰ;Taq polymerase; T4 DNA ligase; Western blot kit; mouse anti-human p27kipl polyclonal antibody, horseradish-peroxidase conjugated goat anti mouse second IgG monoclonal antibody; pORF9-hp27mt plasmid, adenovirus framework plasmid pAdeasy-1, shuttle plasmid pShuttle-CMV, LacZ recombinant adenovirus Ad-LacZ, liposome, colon cancer cell line SW480.METHODS: hp27mt was excised from vector pORF9-hp27mt by restriction endonucleases, and inserted into shuttle plasmid pShuttle-CMV after two cycles of subclone, forming plasmid pShuttle-CMV-hp27mt. Then it was transformed by linear terminus(cut by PmeI) plasmid pShuttle-CMV-hp27mt into competent E. coli BJ5183 which contained adenovirus framework plasmid pAdeasy-1 to ensure that homologous recombination occurred. After correct identification of the transformant, it was digested by PacⅠ and transfected Ad293 cells, and packed into recombinant adenovirus Ad-hp27mt. Recombinant adenovirus was identified by polymerase chain reColon cancer cell line SW480 was infected with recombinant adenovirus Ad-p27mt, and expression of p27 protein was detected by Western blot.of expressed p27 protein after Ad-p27mt transfected colon cancer cell.novirus framework plasmid pAdeasy-1 with pShuttle-CMV-hp27mt, 30%combinant adenovirus DNA contained the target gene. Virus titer of the recancer cells, p27 was highly expressed in SW480 cells, as compared to low expression in non-transfected cells and Ad-LacZ transfected cells.CONCLUSION: Adenovirus, a type of gene transferring vector, can efficiently mediate p27 expression in tumor cells, which provides effective gene transfecting carrier for treating colorectal cancer with mutation p27 gene.

Objective To investigate the effect of cyclic tensile stress (CTS) on the metabolism inand apoptosis of rat chondrocytes.Methods Primary rat chondrocytes were cultured on a Bioflex plate for one day and then stretched cyclically for 24 hours at a frequency of 0.5 Hz using a Flexcell-5000T apparatus.The cells were divided into 5 groups according to their stretching ratio:0％ (the control group),2％,6％,10％ and 14％.After the stretching,Col Ⅱ,Aggrecan,MMP-13 and ADAMTS-5 mRNA were measured using qPCRs,and the NO and PGE2 levels were measured using ELISA kits.Moreover,TUNEL staining and Annexin V-FITC/PI were used to analyze the apoptosis of chondrocytes.Results Compared with the control group,the average levels of Col Ⅱ and Aggrecan mRNAdecreasedin 10％ and 14％ groups [(0.738±0.11) and (0.58±0.13),(0.75±0.11) and (0.55±0.09)].In those groups,the MMP-13 [(2.47±0.47) and (2.88±0.36)] and ADAMTS-5 mRNA level [(2.39±0.33) and (2.75±0.49)],the NO [(6.96±0.96) and (8.28±0.82)] and PGE2 level [(6.83±0.66) and (7.15±0.71)] had increased significantly.In the 6％ group the average levels of Col Ⅱ(1.76±0.30) and Aggrecan mRNA (1.93±0.14)of 6％ group were significantly higher than the control group,but the NO level of the former (3.07±0.20) was significantly lower than the control group (3.89 ± 0.33).The apoptosis rate of chondrocytesin 2％ and 6％ groups were (0.065±0.013) and (0.063 ± 0.147),without significant differences to that of the control group (0.045 ± 0.008).However,compared with the control group,apoptosis in the 10％ and 14％ groups [(0.135 ±0.026) and (0.184±0.020)] increased significantly.Conclusion The effect of cyclic tensile stress on chondrocyte metabolism and apoptosis was magnitude-dependent.Ten percent and 14％ CTS can increase the catabolism and apoptosis of chondrocytes.Ten percent and 14％ strain can increase the catabolism and apoptosis of chondrocytes.Cyclic 6％ strain can increase the anabolism of chondrocytes,but 2％ strain has no impact on metabolism or apoptosis.

Objective To explore the difference of the clinical manifestations between the elderly and non-elderly patients with non-massive pulmonary thromboembolism (PTE) and the significance of D-dimer in the diagnosis of PTE and its dynamic change after anticoagulant therapy.Methods The clinical manifestations of 83 cases with PTE were retrospectively analysed and divided into two groups:39 elderly and 44 non-elderly.The dynamic changing of D-dimer content was determined by immunoturbidimetry(ITM) method before and 3 d after anticoagulant therapy in the two groups.Results There were no significant statistical differences in the incidence of the main symptoms:dyspnea,cough,emptysis,syncope,palpitations between the elderly and the non-elderly (x2 =2.74,0.06,0.10,0.49,0.01,P＞0.05) except for the incidence of chest pain [14 cases (35.9 ％) vs.30 cases (68.2 ％),x2 =4.95,P ＜ 0.05].No differences were found in the the main signs:shortness of breath,tachycardia,accentuation or split of second pulmonary valve sound,cyanosis,and engorgement of neck veins between the two groups (x2 =2.60,0.03,0.61,0.06,0.33,0.11,P＞0.05).D-dimer content was lower in the elderly than in the non-elderly [(1.89±1.21) mg/L vs.(4.93±3.88) mg/L,Z=-2.55,P=0.01] before anticoagulant therapy.But there was no difference in D -dimer content between the two groups 3 d after anticoagulant therapy [( 1.28 ±1.11) mg/L vs.(2.09±2.22) mg/L,Z=-7.07,P=0.50].The decreasing level of D-dimer was less prominent in the elderly than in the non-elderly [(0.61±1.01) mg/Lvs.(2.84±2.95) mg/L,Z=-3.54,P=0.001].Conclusions The main clinical manifestations are similar between the elderly and non-elderly with non-massive PTE,but the incidence of chest pain is less in the elderly than in the non-elderly.The content of D-dimer is lower in the elderly than non-elderly after PTE and its decrements are less prominent in the elderly than the non-elderly after anticoagulant therapy.

Objective To investigate the impact flat deformity of the cerebral cortex induced by congenital hydrocephalus on motor functioning and cognition.Methods Tomography was used to confirm the presence of flat cortex deformity in three congenital hydrocephalus patients ranging in age from 20 to 33.Their motor control,balance,cognition and ability in the activities of daily living (ADL) were evaluated using the Fugl-Meyer assessment (FMA),the Berg balance scale (BBS),the mini-mental state examination (MMSE) and the modified Barthel index (MBI).Speech dysfunction was judged on the basis of clinical communication.The patients were scanned using a 64-slice spiral CT and size-of-ventricle indices were calculated.Results All 3 patients underwent ventrideperitoneal shunt.Their FMA scores were 75.5,83 and 100,with BBS scores of 4,24 and 56,MMSE scores of 14,23 and 26,and the MBIs of 40,90 and 100.CT images showed obvious ventricle enlargement and a thinner cortex layer in all three,with the thinnest part 0.18 cm,0.22 cm and 0.57 cm.Their ventricle indexes were 303％,288％ and 192％,respectively.Conclusion Although there is no systematic rehabilitation therapy for such patients,their motor,cognition and speech functioning were good,indicating the great potential for plasticity of the human brain.