2004年 · 第117卷第08期

Background Chemokine-like factor 1 (CKLF1) was recently identified as a novel cytokine. The full-length CKLF1 cDNA contains 530 bp encoding 99 amino acid residues with a CC motif similar to that of other CC family chemokines. Recombinant CKLF1 exhibits chemotactic activity on leucocytes and stimulates proliferation of murine skeletal muscle cells. We questioned whether CKLF1 could be involved in the pathogenesis of inflammation and proliferation in the lung. Therefore we used efficient in vivo gene delivery method to investigate the biological effect of CKLF1 in the murine lung. Methods CKLF1-expressing plasmid, pCDI-CKLF1, was constructed and injected into the skeletal muscles followed by electroporation. Lung tissues were obtained at the end of week 1, 2, 3 and 4 respectively after injection. The pathological changes in the lungs were observed by light microscope. Results A single intramuscular injection of CKLF1 plasmid DNA into BALB/c mice caused dramatic pathological changes in the lungs of treated mice. These changes included peribronchial leukocyte infiltration, epithelial shedding, collagen deposition, proliferation of bronchial smooth muscle cells and fibrosis of the lung. Conclusions The sustained morphological abnormalities of the bronchial and bronchiolar wall, the acute pneumonitis and interstitial pulmonary fibrosis induced by CKLF1 were similar to phenomena observed in chronic persistent asthma, acute respiratory distress syndrome and severe acute respiratory syndrome. These data suggest that CKLF1 may play an important role in the pathogenesis of these important diseases and the study also implies that gene electrotransfer in vivo could serve as a valuable approach for evaluating the function of a novel gene in animals. Chin Med J 2004; 117(8):1123-1129

Background Previous studies showed that blood B-type natriuretic peptide (BNP) level could predict the prognosis of acute coronary syndromes (ACS). This study investigated the evaluation value of circulating BNP for early percutaneous coronary intervention (PCI) in patients with ACS. Methods Nine hundred and sixty consecutive patients with ACS were enrolled. Circulating BNP level was measured when each patient arrived at the emergency room. All patients underwent PCI in 90 minutes in spite of contraindication. Cardiac events (death from any cause, heart failure, and recurrence of acute myocardial infarction or ACS) were recorded during follow-up. Results In patients with BNP ≥80 pg/ml, mortality from all causes within 1 month and 6 months in those underwent delayed PCI (≥6 hours) was significantly higher than those received early PCI (<6 hours) (9. 53% vs 3. 49%, P = 0. 027; 13. 61% vs 5. 24%, P = 0. 010, respectively). Similarly, the incidence rate of heart failure in delayed PCI patients was significantly higher than those received early PCI within 1 month and 6 months (12. 93% vs 4. 66%, P = 0. 008; 14. 97% vs 6. 98%, P = 0. 021, respectively). The recurrence rate of acute myocardial infarction or ACS, however, was not significantly different between early PCI and delayed PCI patients in group BNP ≥ 80 pg/ml. In patients with BNP <80 pg/ml, no significant difference was observed between early PCI and delayed PCI patients with any of the above cardiac events within 1 month or 6 months. Conclusion While early level of circulating BNP ≥80 pg/ml, the incidence of mortality and heart failure, but not recurrence of acute myocardial infarction, is significantly reduced in patients with ACS provided by early PCI. Chin Med J 2004; 117(8):1130-1134

Background The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway and the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway are the two major independent signal transduction pathways. However, it has recently been found that STAT3 may be negatively regulated by ERK1/2 in gp130-dependent signaling. Cardiotrophin-1 (CT-1), a potent novel hypertrophic cytokine, depends on gp130 to induce signaling and depends on STAT3 to exert hypertrophic effect. In this study, we examined whether STAT3 activity was negatively regulated by ERK1/2 during CT-1-induced signaling in rat cardiomyocytes and, if so, whether such crosstalk interfered with the hypertrophic effect of CT-1 and, furthermore, whether the mechanism underlying the crosstalk involved phosphorylation of serine 727 (S727) in STAT3. Methods The activities of ERK1/2 and STAT3 were assessed by in-gel kinase assay and Western blot analysis, respectively. The role of S727 phosphorylation in the crosstalk between ERK1/2 and STAT3 was determined by a transient transfection study using a STAT3S727A mutant. Cardiomyocyte hypertrophy was evaluated by the cellular protein-to-DNA ratio and [3H]-leucine incorporation. Results CT-1 simultaneously activated both ERK1/2 and STAT3 in rat cardiomyocytes. Inhibition of ERK1/2 by U0126 resulted in an increase of CT-1-induced tyrosine phosphorylation of STAT3 and, consequently, the protein-to-DNA ratio and [3H]-leucine incorporation. Transient transfection of the cells with STAT3S727A had no significant effect on CT-1-induced tyrosine phosphorylation of STAT3. Conclusions STAT3 is activated by CT-1 in rat cardiomyocytes, but full activation is mitigated by the simultaneous activation of ERK1/2. The inhibition of ERK1/2 increases the activity of STAT3, which, in turn, enhances the hypertrophic effect of CT-1. The crosstalk between ERK1/2 and STAT3 is independent of the phosphorylation of the S727 in STAT3. Such crosstalk may contribute to the development of adequate cardiac hypertrophy. Chin Med J 2004; 117(8):1135-1142

Background The release of Weibel-Palade Bodies (WPB) is a form of endothelial cell activation. But the signal transduction pathway leading to WPB release is not yet defined. We hypothesized that small G-protein rac1 and reactive oxygen species (ROS) mediate the ligand induced release of Weibel-Palade Bodies. Methods We tested this hypothesis by using wild-type and mutant adenoviral rac1 expression vectors, and by manipulating the production and destruction of superoxide and hydrogen peroxide in human aortic endothelial cells (HAEC). Results Thrombin (1.0 Unit, 30 min) induced the increase of WPB release by 3. 7-fold in HAEC, and that H202 (0. 1 mmol/L, 30 min) induced by 4. 5-fold. These results correlated with thrombin-stimulated activation of rac-GTP binding activity by 3. 5-fold, and increase of ROS production by 3. 4-fold. The dominant negative adenoviral rac-N17 gene transfer dramatically inhibited the release of WPB by 64. 2% (control) and 77. 3% (thrombin-stimulation), and decreased ROS production by 65. 5% (control) and 83. 6% (thrombin-stimulation) compared with non-infected cells, respectively. Anti-oxidants, catalase and N-acetyl-cysteine significantly decreased the release of WPB by 34% and 79% in control cells, and further decreased by 63. 6% and 46. 7% in rac-N17 transferred cells compared with non-infected cells. We also confirmed that rac1 was located upstream of ROS in the WPB release pathway. Conclusions Small G-protein rac1 medicates ligand-induced release of Weibel-Palade Bodies in human aortic endothelial cells, and the signal pathway of WPB release is a rac1-dependent ROS regulating mechanism. Chin Med J 2004; 117(8):1143-1150

Background Cyclo-oxgenase 2 (COX-2) is involved in prostaglandin synthesis in central nervous system, and it also plays a role in human carcinogenesis. Our purpose of this study is to investigate the COX-2 expression in different development stages of colorectal cancer, and to discuss the relationship between the gene expression and clinicopathological features of the cancer. Methods COX-2 expression was examined by immunohistochemical staining in 76 surgical specimens of colorectal cancer (44 of advanced stage and 32 of early stage), thirty-three adenomas and 18 normal colonic mucosal tissues taken by endoscopic biopsy. Kaplan-Meier survival curves and Cox proportional hazards regression were used to evaluate the relation of COX-2 to prognosis. Results COX-2 expression, divided into 4 grades from "-" to " + + + ", is respectively 83. 3%, 16. 7%, 0% and 0% in normal colonic mucosal tissues; 12. 1%, 42. 4%, 36. 4% and 9. 1 % in adenomas; 6. 3%, 28. 1 %, 46. 9% and 18. 7% in early colorectal cancers (ECCs), and 6. 8%, 20. 5%, 18. 2% and 54. 5% in advanced colorectal cancers (CRCs). The differences in COX-2 expression between advanced CRCs and early colorectal cancers (ECCs) as well as between the advanced CRCs and adenomas were statistically significant (P <0. 01); but there was no significant difference between ECCs and adenomas. Kaplan-Meier survival analysis showed a significant difference in the survival curves between low high COX-2 groups (P < 0. 05). Cox proportional hazards regression showed that COX-2 expression was related to poorer long-term outcome with a hazard ratio of 2. 665 unadjusted for other variables (P < 0. 05), and COX-2 expression was an independent risk factor of poor prognosis. Conclusions COX-2 expression is gradually up-regulated in the development from normal epithelium to adenomas and from ECCs to advanced CRCs. Alhough the COX-2 protein can not be regarded as a tumor marker to diagnose CRCs early, COX-2 expression can be regarded as an independent risk factor of poor prognosis for postoperative patients with advanced CRCs. Chin Med J 2004; 117(8):1151-1154

Background People have more and more concerned about allitridum as studies have shown that taking more raw garlic associated with a lower risk for cancers of the alimentary system. In the present study, we tried to examine whether a large dose of allitridum and a microdose of selenium prevent gastric cancer. Methods A double-blind intervention study was performed on the participants aged (35-74) years, who had matched at least one of the following criteria: (1) a medical history of stomach disorder, (2) a family history of tumour, or (3) smoking and/or alcohol consumption. A total of 2526 and 2507 persons were randomly enrolled into intervention group and control group respectively from 288 natural villages of seven communities in Qixia County, Shandong Province, China. Each person of the intervention group orally took 200 mg synthetic allitridum every day and 100 μg selenium every other day for one month of each year during November 1989 to December 1991. At the same time, people in control group were given 2 placebo capsules containing corn oid with the identical appearance to that in the intervention group. Results For all subjects the large dose of allitridum was accepted and no harmful side effects were found during the study. In the first follow-up five years (1992-1997) after stopping the intervention, the morbidity rates of malignant tumours in the intervention group declined by 22%, in contrast to the control group, declined by 47. 3%. After adjusting for age, gender, and other potential confounders, relative risks (RRs) for all tumours and gastric cancer of the whole population were 0. 67 (95% CL: 0. 43 -1. 03) and 0. 48 (95% CL: 0. 21 - 1. 06), respectively, and for male group they were 0. 51 (95%CL: 0. 30 -0. 85) and 0. 36 (95%CL: 0. 14 -0. 92), respectively. No signigicantly protective effect was found for the female subgroup. Conclusion The present study proves that large doses of allitridum and microdorse of selenium may effectively prevent gastric cancer, especially in men. Chin Med J 2004; 117(8):1155-1160

Background An important aspect of Alzheimer's disease (AD) is loss or impairment of cholinergic neurons. It is controversial whether there is a similar cholinergic impairment and cerebral deficit of acetylcholine (ACh) in the case of vascular dementia (VD). The purpose of this study was to explore the levels of ACh and choline (Ch) in the cerebrospinal fluid (CSF) of patients with AD and VD, and their possible relationship with cognitive impairment. Methods Twenty-two AD patients, twenty-two VD patients, and twenty normal controls were recruited and scored with a Mini-Mental State Examination (MMSE). CSF concentrations of ACh and Ch were measured using high-performance liquid chromatography with an electrochemical detector (HPLC-ECD) and the results were then compared to cognitive status. Results ACh concentrations in CSF of AD patients [ (10. 7 ±5. 1) nmol/L] and VD patients [(16. 8 ±7. 4) nmol/L] were both significantly lower than in controls [ (34. 5 ±9. 0) nmol/L, t = 10. 67, P < 0. 001; t = 6. 91, P<0. 001]. Both results correlated positively with MMSE scores (rs =0. 88 and rs = 0. 85, respectively, P<0. 01). The CSF concentration of Ch was significantly higher in VD patients [(887. 4 ±187. 4) nmol/L] compared to AD patients [(627. 6 ±145. 1) nmol/L, t = 6. 4, P<0. 001] and controls [(716. 0 ±159. 4) nmol/L, t = 4. 2, P = 0. 002]. CSF Ch concentration showed no difference between AD patients and normal controls, nor did it correlate with MMSE score in any of the three groups. Conclusions The positive correlation between ACh deficit and cognitive impairment suggests that ACh is an important neurotransmitter for memory. The similar decrease in ACh concentration in AD and VD patients may imply a similar pathogenesis for the process of cognitive impairment involved in these two disorders. The elevated CSF levels of Ch in VD patients compared to AD patients may be useful diagnostically. Cholinesterase inhibitors may be helpful not only for AD patients, but also for VD patients. Chin Med J 2004; 117(8):1161-1164

Background The most intimidatory pathological changes in patients with DM are cardiovascular illnesses, which are the major causes of death in diabetic patients and are far more prevalent than in nondiabetics because of accelerated atherosclerosis. In this study, we tried to clarify the changes in macrovascular endothelial ultrastructure and in the gene expression of endothelial nitric oxide synthase (eNOS) mRNA in diabetic rats. Methods The study was conducted on 52 of 10-week old Sprague Dawley (SD) rats with body weight of (320 ± 42) g. SD rats were divided into: experimental group treated with a single intraperitoneal injection of streptozotocin (STZ, 60 mg/kg), (male, n = 20, diabetes mellitus (DMM)); female, n = 12, diabetes mellitus female (DMF)) and control group (male, n = 10, diabetes mellitus male control (DMMC); female, n =10, diabetes mellitus female control (DMFC)). Four weeks after treatment, half of the rats were sacrificed; the remainders were sacrificed ten weeks after treatment. One part of the abdominal aortic sample was stored under glutaraldehyde (volume fraction ΨB = 2. 5 %). After the process of chemical fixation, chemical dehydration, drying and conductivity enhancement, all samples were observed and photographed using scanning electron microscopy (Leica-Stereoscan 260, England). The other part of the abdominal aortic sample was treated with liquid nitrogen and the expression of eNOSmRNA was assessed by semi-quantitative RT-PCR. Results The aortic lumen of both experimental groups adsorbed much more debris than that of either control group. The endothelial surfaces of diabetic rats were coarse, wrinkled and protuberant like fingers or villi. The vascular endothelial lesions of diabetic male rats were very distinct after 4 weeks, and as obvious as those at 10 weeks. The vascular endothelial lesions of diabetic female rats were not severe at 4 weeks and only became marked after 10 weeks. In both males and females, the abdominal aortic eNOSmRNA content of 4 weeks and 10 weeks diabetic rats was very significantly lower (P<0. 01) than that of controls. Conclusions Aortic endothelial ultrastructure in DM rats is injured compared with controls. Abnormal changes of aortic endothelia in male DM rats are more obvious than those in females. Expression of abdominal aortic eNOSmRNA content of DM rats is significantly lower than that of controls. Chin Med J 2004; 117(8):1165-1169

Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs. Smad4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors. The aim of this study was to assess the effects of the antisense Smad4 gene on Ito cell line, LI90. Methods The recombinant retroviral vector pLXSN-Smad4 was constructed by cloning the rat antisense Smad4 cDNA into the retroviral vector pLXSN. Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad4 and pLXSN vectors into PA317 cells. Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418. The expression of Smad4 was detected by Northern and Western blots. Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed. Results mRNA and protein expressions of Smad4 in LI90 cells transfected with retrovirus containing the antisense Smad4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells. Cells hypoexpressing the Smad4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline (P < 0. 01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus. Conclusions The antisense Smad4 gene can suppress the expression of the Smad4 gene, reduce endogenous production of Smad4 mRNA and protein, block TGF-β1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM). Our results may provide a basis for the development of antifibrotic gene therapy. Chin Med J 2004; 117(8):1170-1177

Background This study was to evaluate whether anergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways can act as potent immunoregulatory cells in vitro and prolong cardiac allograft survival after adoptive transfer. Methods Anergic cells were induced in vitro by the addition of anti-CD154 and anti-CD80 monoclonal antibodies (mAbs) to primary MLR (mixed lymphocyte reaction) consisting of BALB/c as responder and C3H as stimulator. Anergic cells were added to a newly formed MLR in assessing the regulatory capacity and antigen specificity of anergic cells. The ability of anergic cells to respond to antigen and/or exogenous recombinant mouse interleukin-2 (rmlL-2) was tested. For in vivo studies, anergic cells were intravenously injected into 3. O-Gy y-irradiated BALB/c mice immediately after heterotopic abdominal cardiac transplantation. To prolong allograft survival, recipient mice injected with anergic cells received rapamycin therapy (1 mg • day-1 • kg-1). Results Anergic cells strongly suppressed the proliferation of naive BALB/c splenocytes against the original (C3H) stimulator in a dose-dependent manner, but they failed to suppress the proliferation of naive BALB/c splenocytes against the third-party (C57BL/6J) stimulator. The anergic state was reversed by both original (C3H) stimulator and additional exogenous IL-2. In in vivo studies, untreated irradiated BALB/c mice rejected C3H cardiac allografts with a mean survival time of (8. 6 ± 1. 1) days, whereas those injected with the anergic cells rejected the allografts with a mean survival time of (11. 8 ± 1. 9) days, which was slightly longer than that of the untreated mice. The protocol based on anergic cells injection plus rapamycin therapy could prolong allograft survival significantly [(29.6±4.4) days]. Conclusions Anergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways can act as potent immunoregulatory cells in vitro, and prolong cardiac allograft survival after adoptive transfer in the presence of rapamycin therapy. This procedure might be clinically useful for prolonging allograft survival if optimal protocols are developed. Chin Med J 2004; 117(8):1178-1183