BACKGROUND: AdiponecUn (apM1) has been a new target for gene therapy of ischemic diseases; apM1 gene cloning is the key of successful apM1 gene therapy. There are two main gene cloning methods: directional cloning and T-A cloning method. Directional cloning method is complicated, while the T-A cloning method is relatively simpler, with higher successful rate. OBJECTIVE: T-A cloning of apM1 gene coding region was applied to verify the sequence in comparison with GenBank. DESIGN, TIME AND SETTING: The gene verification experiment was performed at the laboratories of Fujian Hypertension Research Institute which is in The First Affiliated Hospital of Fujian Medical University and Academy of Integrative Medicine which is in Fujian University of Traditional Chinese Medicine, from June 2006 to December 2008. MATERIALS: Adipose tissue sample of omental fat pad was obtained from a surgical patient in the First Affiliated Hospital of Fujian Medical University. Trizol produced by Invitrogen company; M-,-MLV, Gel Extract Kit produced by PROMEGA company; Taq enzyme produced by TIANGEN company; Restriction Endonucleases BamH Ⅰ and Sal Ⅰ, pMD18-T Vector produced by TAKARAcompany were used in this study.METHODS: Total mRNA was extracted from human greater omentum adipose tissue. The coding region of human apM1 (hapM1) gene was amplified by RT-PCR. The coding region of hapM1 gene was cloned into pMD18-T vector .The recombinant plasmid was identified with restriction enzyme digestion analysis and nucleotide sequencing. MAIN OUTCOME MEASURES: The electrophorasis verification of the recombinant plasmids coding target gene using double enzyme digestion, comparison of the similarity between the sequences of the plasmids and hapM1 in GenBank. RESULTS: Sense and antisense coding region of hapM1 gene were cloned. The sequencing results showed that the sequences of the cloned DNA were completely identical to that of hapM1 in GenBank.CONCLUSION: The coding region of hapM1 gene was successfully cloned.

Aim To investigate the expression of the lentiviral vector carrying a human apM1(hapM1) gene in 293 T cells and its bioactivity.Methods The CDS region of hapM1 gene was subcloned into the lentiviral vector from a cloning vector,and the transfer plasmid(PNL-hapM1-IRES2-EGFP)of the lentiviral vector was constructed.The three plasmids of the lentiviral vector: PNL-hapM1-IRES2-EGFP,HELPER,and VSVG were cotransfected to 293 T cells to produce the recombinant lentivirus;293 T cells were infected by the recombinant lentivirus and were identified with fluorescent microscope,RT-PCR and western blot.Inhibitory effect of the recombinant hapM1 on proliferation of vascular smooth muscle cells(VSMCs) was identified.Results The sequencing results showed that the sequence of the subcloned hapM1 gene was completely identical to that reported in GeneBank.The virus titre of the recombinant hapM1 lentivirus was 2.0?105 TU?L-1.After the recombinant lentivirus infected 293 T cells,RT-PCR and Western blot results showed that the expression of hapM1 in the hapM1-293 T group could be measured.The recombinant hapM1 inhibited the proliferation of VSMCs.Conclusions The recombinant lentivirus carrying a human apM1 gene was constructed successfully.The recombinant hapM1 could be expressed in 293 T cells and had its bioactivity.

Objective To investigate the utility of plasma D-dimer and fibrinogen (FIB) for the severity assessments and predicting the prognosis of patients with community-acquired pneumonia (CAP).Methods The clinical data of patients with CAP admitted to First Affiliated Hospital of Fujian Medical University were retrospectively analyzed.The patients were divided into Ⅰ-Ⅴ level groups according to pneumonia severity index (PSI),and they were divided into non-survivors and survivors according to 30-day prognosis.The data including gender,age,PSI score,platelets count (PLT),white blood cell count (WBC),D-dimer,FIB,and C-reactive protein (CRP) were compared among groups.The correlations between PSI score and D-dimer,CRP as well as FIB were analyzed by Spearman or Pearson correlation analysis.Receiver operating characteristic curve (ROC) was used to assess the prognostic value of these indicators.Results A total of 499 patients with CAP were enrolled with 298 male and 201 female,the average age was (63.4 ± 17.8) years old,and the 30-day mortality was 6.4％ (32/499).There were 77,80,104,162 and 76 patients in PSI Ⅰ-Ⅴ level groups,and there were more male patients in PSI Ⅵ and Ⅴ level groups.There were no significant differences in PLT and FIB among the groups of different PSI levels,but the levels of WBC,D-dimer and CRP were significantly increased as PSI level increased from Ⅰ to Ⅴ (F1 =3.810,x 22 =102.361,F3 =7.070,all P ＜ 0.01).Compared with survivors,the non-survivors were elder (t =-4.773,P ＜ 0.001) with lower PLT (t =3.026,P =0.003)and higher WBC,PSI score,D-dimer and CRP levels (t1 =-2.545,t2 =-8.421,Z3 =-6.947,t4 =-3.770,all P ＜ 0.05).Plasma D-dimer levels in elderly patients (≥ 65 years old) were statistically higher than those in younger patients (＜ 65 years old;Z =-5.338,P ＜ 0.01).It was shown by correlation analysis that PSI score was positively correlated with D-dimer and CRP (r values were 0.475 and 0.260,both P ＜ 0.001),and no correlation was found between PSI score and FIB (r =-0.062,P =0.170).The area under the ROC curve (AUC) for predicting 30-day death of PSI score,D-dimer and CRP was 0.858 [95％ confidence interval (95％CI) =0.802-0.914],0.867 (95％CI =0.812-0.922) and 0.732 (95％CI =0.641-0.823).The combination of D-dimer and PSI score was better than any single indicator for predicting the prognosis with higher AUC up to 0.905 (95％CI =0.867-0.944),all P ＜ 0.001.The sensitivity and specificity for PSI in predicting 30-day death respectively were 78.1％ and 82.4％ with the cut-off of greater than 122,and those for D-dimer were 75.0％ and 82.9％ with the cut-off of greater than 2.10 mg/L,50.0％ and 84.4％ for CRP with the cut-off of greater than 100.50 mg/L.Conclusions D-dimer could well reflect the severity of CAP and be a good indicator for predicting the prognosis.The combination of D-dimer and PSI might improve the accuracy in predicting prognosis.

AIM: To investigate the effect of sodium ferulate on phosphorylation of heat shock protein 27(HSP27) in vascular smooth muscle cells(VSMCs) induced by angiotensin Ⅱ(AngⅡ) and platelet derived growth factor-BB(PDGF-BB).METHODS: Cultured VSMCs derived from rat thoracic aorta were used.The activity of HSP27 was evaluated by Western blotting with specific phospho-HSP27 antibody.RESULTS: The phosphorylation of HSP27 in response to AngⅡ and PDGF-BB was suppressed by sodium ferulate in a dose-dependent manner,with maximal inhibition rates of 39.0%(P

0.05). However, monocrotaline significantly increased WT% and WA% of pulmonary arterioles (WT:39.1%?2.8% vs 50.8%?3.1%, WA:51.2%?3.0% vs 74.5%?2.9%, P

Objective To investigate the effects of angiotensinⅡ(AngⅡ), Benazepril(Bena) and Losartan on proliferation and collagen synthesis of cultured rat cardiac fibroblasts(CFb) derived from SHR (CFbSHR) and WKY (CFbWKY). Methods CFb derived from 12-week-old SHR and WKY was cultured by outgrowth of tissue block. Cell proliferation of CFb was determined by direct cell counting .3H-Proline incorporation of CFb was measured after incubation with AngⅡ,Bena and Losartan. Results In serum free(0.4%FCS) medium, cell number of CFb derived from SHR and WKY were not influenced by AngⅡ(10-10mol～10-6mol)and Bena(10-9mol～10-5mol). Increased 3H-proline incorporation wa s induced by AngⅡ in a concentration dependent manner. Benazepril caused an decrease in 3H-Proline incorporation in CFb derived from SHR and WKY. The increased collagen synthesis induced by AngⅡ(10-7mol) was inhibited by Losartan((10-10mol～10-6mol） i n a concentration dependent manner. Conclusion Proliferation of CFb were not influenced by AngⅡ and Bena. Collagen synthesis of CFb was promoted by AngⅡ and inhibited by Bena. Collagen synthesis of CFb in duced by AngⅡ was inhibited by losartan.

Objective To explore the predictive value of response to neoadjuvant chemotherapy(NAC)in local advanced breast cancer with contrast-enhanced ultrasound(CEUS)of axillary lymph node.Methods CEUS of metastatic axillary lymph nodes in 58 patients stacng Ⅱ-Ⅲ breast cancer was performed before and after NAC treatment. The enhancement patterns and parameters of time-intensity curve were assessed and compared with the pathology.Results The clinic response evaluation were drug-effective in 35 cases and no change in 23 ones.There Were no significant differences in enhancement patterns between no-change and drugeffective groups.Lymph node cortex arriving time was longer in drug-effective cases than that in no-change ones after NAC,whereas it showed no significant differences before NAC.Statistical significant difierence in enhancement duration(ED)was found between the two groups before NAC,which decreased markedly in drug-effective case8 after NAC.Histopatholngic response could be predicted with a sensitivity of 77% and a specificity of 90% by standardized ED below 275 seconds after NAC.No significant difference was found in time to peak(TP),peak intensity(PI)between the two groups.Conclusion The perfusion pattern of axillary lymph node CEUS after NAC Was insufficient to predict curative effect.But the lymph node cortex arriving time and enhancement duration may be of value in the prediction of clinical response to chemotherapy.

Objective To investigate the influence of Angiopoietin-1(Ang1)gene modified mesenchymal stem cells on the expression of adhension factors from endothelial cells,to clarify the feasibility of modulating inflammation reaction by gene modification after stem cells transplantation.Methods Genetic engineering rMSCs were constructed by lentivirally-tranduced Ang1 gene,the expressions of ICAM-1 and VCAM-1 at different time points after 20 ?g/L VEGF stimulating were investigated,the supernatants of gene modified rMSC were collected and then added into HUVEC culture with different concentrations of Ang1,the expressions of adhension factors after VEGF stimulated were observed at the same time.Results Genetic engineering rMSCs were successfully constructed,the expressions of ICAM-1 and VCAM-1 mRNA were significantly increased after VEGF stimulated,the maximal value was at 8 h,the ICAM-1 and VCAM-1 mRNA expressions were decreased after incubation with Ang1 at different concentrations,Ang1 suppressed the VEGF-stimulated protein levels of ICAM-1 and VCAM-1.Conclusion The supernatants of Ang1 gene modified stem cells can suppresse VEGF-stimulated inflammation reaction of HUVEC.

Aim To construct the recombinant lentiviral RNA interference(RNAi) sequence targeting rat heat shock protein 27(HSP27)gene.Methods Three complementary DNA sequences targeting rat HSP27gene were designed and synthesized.After phosphorylation and annealing,these double strands DNA were cloned into vector pSUPER-basic respectively.Next the ds-oligo DNA which expressed short hairpin RNA(shRNA) was subcloned into lentiviral vector.The transfer plasmid(pNL-HSP27-IRES2-EGFP) was constructed and confirmed by electrophoresis and sequencing.Then the three plasmids of the lentiviral vector: pNL-HSP27-IRES2-EGFP,VSVG,and pHeler were cotransfected to 293T cells by Lipofectamine 2000 to package lentiviral particles.Forty eight hours later the supernatant was collected and the titer of virus was measured by detecting expression leve1 of enhanced green fluorescent protein(EGFP).Results The analysis using restriction endonuclease digestion and sequencing confirmed that the ds-oligo DNA was successfully inserted into the lentiviral vector.The recombinant lentivirus was harvested from 293T cells with a viral titer of 3.12 ?109 fu?L-1.Vascular smooth muscle cells(VSMCs) were infected by lentivirus,and the interfering efficiency was detected by RT-PCR and Western blot.The plasmid with the highest interfering efficiency was pNL-HSP27-IRES2-EGFP-1,and the interfering efficiency was 0.771.Conclusions The recombinant lentivirus containing RNAi targeting HSP27 gene has been successfully constructed,which lay a foundation for further study of the function and gene therapy of HSP27.

[ABSTRACT]AIM:ToevaluatetheeffectsofantisenseTGF-β1oligodeoxynucleotide(ASTGF-β1)ontheex-pression of TGF-β1 , deposition of extracellular matrix ( ECM) and the neointima formation in the arteries after balloon inju-ry.METHODS:The unmodified and phosphorothioate-modified AS TGF-β1 which containing 15 bases and surrounding the initiation codon region (ATG) of rat TGF-β1 complementary DNA (cDNA) were designed.At the same time, sense TGF-β1 oligodeoxynucleotide ( S TGF-β1 ) with the base sequence complement to AS TGF-β1 was synthesized as a control . The oligodeoxynucleotides were introduced into in vivo and in vitro experiments , respectively .RESULTS:The AS TGF-β1 significantly inhibited the protein expression of TGF-β1 in a concentration-dependent manner , and S TGF-β1 did not have the same effect.Furthermore, no effect of the AS TGF-β1 on the mRNA expression of TGF-β1 in injured VSMCs was ob-served.Moreover, for the injured VSMCs, AS TGF-β1 significantly and concentration-dependently inhibited the basal DNA synthesis.Both AS TGF-β1 and S TGF-β1 did not exhibit dose-dependent effects on DNA synthesis in uninjured VSMCs . Fibronectin ( FN) mRNA expression in injured VSMCs was significantly decreased by AS TGF-β1 in a concentration (0.01~1 μmol/L)-dependent manner .AS TGF-β1 significantly increased the mRNA expression of contractile marker SM 22α, and decreased the mRNA expression of synthetic markers osteopontin and matrix Gla , especially at the concentration of 0.01μmol/L and 0.1 μmol/L.After treatment with AS TGF-β1 (90 μg· kg-1 · d-1 ) for 28 d, the neointima formation was significantly inhibited , and the area ratio of intima/media was markedly decreased by 68% compared with untreated group , but S TGF-β1 had no effect on neointimal formation .CONCLUSION:The AS TGF-β1 specifically inhibits the pro-tein expression of TGF-β1 in the VSMCs derived from injured arteries .Moreover , it significantly inhibits DNA synthesis and cell proliferation, and decreases the expression of FN .Therefore, AS TGF-β1 dramatically attenuates neointima formation after balloon njury .The effects of AS TGF-β1 on the injured VSMCs may be associated with its reverse effects on the altera-tion of VSMC phenotype after balloon injury .