2003年 · 第116卷第11期

Objective To detect the expression of cytokines by acute promyelocytic leukemia (APL) cells before and after exposure to arsenic trioxide. Methods Diagnoses were performed according to the FAB cytological classification criteria and cytogenetic criteria. Bone marrow or blood samples from APL patients were collected in heparinized tubes, then primary APL cells were separated by traditional Ficoll-Hypaque density centrifugation and purified after adherence to plastic surfaces. IL-1β, IL-6, IL-8, TNFα and G-CSF levels in the leukemia cell culture supernatants were detected by ELISA. At the same time, nitro blue tetrazolium (NBT)reduction test was used to detect the differentiation of APL cells. Results After 96 hours exposure to arsenic trioxide, 10-6 mol/L in vitro or 10 mg/d in vivo, APL cells showed a significant increase of IL-1 β (P <0. 05) and G-CSF (P <0. 05) production, and a significant decrease of IL-6 (P<0. 05) and IL-8 (P <0. 05). However, there was no obvious variation of TNFα when compared with APL cells without exposure to arsenic trioxide. On the other hand, the proliferation ratio of APL cells in vitro was statistically correlated to the IL-1β secretion ratio or G-CSF secretion ratio. The cell number ratio in patients with detectable IL-1β or G-CSF was higher than that without detectable IL-1β or G-CSF. Conclusion IL-1β and G-CSF secretion may play an important role in the proliferation of APL cells after exposure to arsenic trioxide. Chin Med J 2003; 116(11): 1639-1643

Objective To investigate the effects of arsenic trioxide (As2O3) on the apoptosis and p-glycoprotein(P-gp) expression of multidrug-resistant human leukemia cells. Methods Human multidrug-resistant leukemia cell line K562/ADM overexpressing the MDR1 gene, was used as the target cells. The cell proliferating activity was assessed using the MTT colorimetric assay. Cytomorphology was investigated under light, confocal and electron microscopes. DNA fragmentation was examined using agarose gel electrophoresis, while p-gp expression, cell cycle status and sub-G1 cells were determined using flow cytometry. Results Zero point five to 20 μmol/L As2O3 inhibited the proliferation of K562/ADM cells, and K562/ADM cells were more sensitive to As2O3 than the parental K562 cells. As2O3-induced apoptosis of K562/ADM cells was determined by the observance of typical morphological changes and the appearance of DNA ladder and sub-G1 cell populations. As2O3 significantly inhibited the P-gp expression of K562/ADM cells, and synergistically enhanced the sensitivity of the drug-resistant cells to adriamycin. Conclusions As2O3 induces growth-inhibition and apoptosis, down-regulates P-gp expression and exerts a synergistic effect in combination with adriamycin in multidrug-resistant leukemia cells. Chin Med J 2003; 116(11): 1644-1648

Objective To prevent Graft-versus-host disease (GVHD) in rat model, we evaluated the feasibility of mesenchymal stem cells (MSCs) as a gene transfer target and studied the efficiency of recombinant adenovirus mediated gene therapy. Methods We constructed the recombinant adenovirus containing CTLA4lg gene. Rat MSCs of passages 3 -5 were infected by the adenovirus, and the transfection efficiency was monitored by GFP markers. We performed flow cytometric analysis, immunohistochemical and Western blotting analysis to identify the CTLA4lg expression. The gene transferred MSCs were tested for their ability to inhibit the allogeneic lymphocyte response in vitro and to prevent GVHD in a rat model. Results Recombinant adenovirus pAd-CTLA4lg was correctly constructed and confirmed. After MSCs were infected by the adenovirus, the CTLA4lg protein was detected not only in transgenic MSCs, but also in the culture medium. In a mixed lymphocytes response (MLR) test, the transgenic MSCs could significantly inhibit the allogeneic lymphocyte response compared with the control groups(P <0. 05). A model of GVHD was developed by transplanting bone marrow cells and spleen lymphocytes of F344 rats to lethally irradiated SD rats. The onset of GVHD could be ameliorated or prevented by co-administration of transgenic MSCs. All the rats in the control groups suffered severe acute GVHD. CTLA4lg expression was observed in the liver, intestine, kidney and spleen 30 days post- transplantation. Conclusions Our results indicate that adenoviral vectors could efficiently transfer CTLA4lg gene into MSCs and sustain long-term stable expression in vitro and in vivo. Chin Med J 2003; 116(11): 1649-1654

Objectives To assess the feasibility and efficacy of eliciting leukemia-specific T-cell responses in syngeneic mice in vitro and in vivo using dendritic cells (DCs) pulsed with total RNA from leukemia cells. Methods DCs generated from bone marrow culture in vitro in the presence of combined cytokines were pulsed with cellular total RNA isolated from cultured L615 cells by cationic lipid 1,2-dioleoyloxy-3-(trimethylammonium) propane (DOTAP). T-cell responses were evaluated by in vitro proliferation, and cytotoxicity assay. And in vivo immune protection and proghosis of mice with leukemia were studied. Results DCs pulsed with total RNA isolated from cultured L615 cells (DCs/RNA) were remarkably effective in stimulating L615-specific T-cell response in vitro, but did not cross-react with other leukemia cells from syngeneic mice. Vaccination of naive mice with viable DCs/RNA vaccine was able to partly protect from challenge with a lethal dose of live L615 cells, leading to low leukemia incidence and overall survival prolongation. Statistically significant survival was also observed in a low lethal dose of L615-bearing mice that received treatment using viable DCs / RNA vaccine alone, suggesting that systemic administration of IL-2 could enhance the anti-tumor efficacy of leukemia RNA/DCs vaccine. Conclusions These data support the use of DCs/RNA vaccine as a feasible and effective route to elicit leukemia immunity against unidentified leukemia-associated antigens for treatment of leukemia-bearing animals. Chin Med J 2003; 116(11): 1655-1661

Objective To investigate the effect of Ca2+ on lipopolysaccharide (LPS)-induced NF-κB activation in pancreatic acinar cells and the role of NF-κB in LPS-induced acinar cell injury. Methods Male rat pancreatic acinar cells were isolated by collagenase digestion, then exposed to varying concentrations of LPS (from 1 to 20 mg/L) in the presence or absence of EGTA. At various time points (30 minutes, 1 hour, 2 hours, 4 hours and 10 hours) after treatment with the agents, cell viability was determined by MTT. Nuclear translocation of NF-κB's subunit p65 was visualized by immunofluorescence staining and nuclei protein was extracted to perform EMSA which was used to assay the activity of NF-κB binding to the DNA sequence containing the recognition site of NF-κB. Results LPS induced cell damage in a time- and concentration-dependent manner while EGTA attenuated LPS-induced cell damage (P <0. 05). NF-κB p65 immunofluorescence staining had increased intensity in the cytoplasm and indicated that nuclear translocation occurred within 30 minutes and its zenith was reached at 1 hour after LPS (10 mg/L) treatment. Testing of NF-κB DNA binding activity showed the same alteration phase as p65 immunofluorescence staining. NF-κB activation preceded the pathological alteration of pancreatic acinar cells. The Ca2+ chelator EGTA inhibited LPS-induced NF-κB activation. Conclusions NF-κB activation is an important early event in LPS-induced injury to pancreatic acinar cells. Ca2+ is an important mediator in the process of LPS-induced NF-κB activation. Chin Med J 2003; 116(11): 1662-1667

Objective To investigate the relationship between NF-κB activity and IFN-y gene expression, as well as the histopathological changes following liver transplants, both with and without cyclosporin A (CsA)treatment. Methods Sixty male Wistar and Thirty male SD rats were subjected to orthotopic liver transplants. Fourty-five of the Wistar rats were used as recipients, and were divided into 3 groups: group I,syngeneic control (Wistar-to-Wistar); group II, acute rejection (SD-to-Wistar); and group III: acute rejection treated with cyclosporin A by intramuscular injection (SD-to-Wistar + CSA). After the liver transplants, electrophoretic gel mobility shift assay was used to analyze NF-κB activity in the splenocytes of recipient rats, and RT-PCR was used to measure IFN-γ gene expression in grafted liver specimens. In addition, histopathological examinations were performed to assess the severity of acute liver rejection. Results In group I, low levels of NF-κB activity were only detectable on day 5 and 7 post-transplant, and only weak IFN-γ mRNA expression was observed at all time points. By contrast, both high NF-κB activity and high expression levels of IFN-γ mRNA were detected at all time points in group II . In group III, NF-κB activity and IFN-γ mRNA expression were significantly inhibited, as compared to group II (P <0. 05). A good correlation was found between NF-κB activity and IFN-γ mRNA expression (r=0. 815) . In addition, NF-κB activity and IFN-γ mRNA expression mirrored histopathological changes in all three experimental groups. Conclusions Changes in IFN-γ mRNA expression levels after liver transplantation are at least partially due to changes in levels of NF-κB activity. CsA appears to downregulate NF-κB activity, thus inhibiting IFN-γ gene transcription. Blocking the NF-κB mediated transcription pathway may be of benefit in preventing liver transplant rejection. Chin Med J 2003; 116(11): 1668-1672

Objective To investigate whether pretreatment with α1-antitrypsin (AAT) can attenuate acute lung injury (ALI) in rabbits induced with endotoxin. Methods Thirty-two healthy adult New Zealand rabbits were anaesthetized, tracheotomized and mechanically ventilated. They were then randomly divided into four groups (n=8): ① Infusion of Escherichia coli endotoxin [Lipopolysaccharide (LPS) 500 ng/kg] without AAT (Group LPS) .②Infusion of AAT 120 mg/kg at 15 minutes after LPS (Group LAV). ③ Infusion of AAT 120 mg/kg without endotoxin (Group AAT).④ Infusion of saline 4 ml/kg as control (Group NS). Arterial blood gases, peripheral leukocyte counts and airway pressure were recorded every hour for eight hours. Physiologic intrapulmonary shunting (Qs/Qt) was measured every four hours. After eight hours,blood samples were collected for measurement of plasma concentration and activity of AAT. Then, the animals were sacrificed, and bronchoalveolar lavage fluid (BALF) was collected for measurement of concentrations of total protein (TP), interleukin-8 (IL-8), tumor necrosis factor (TNFα, the activities of NE and AAT, total phospholipids (TPL) and disaturated phosphatidylcholine (DSPC). In addition,the wet-to-dry lung weight ratio (W/D) was measured. Results The infusion of endotoxin induced decreases in arterial oxygen pressure (PaO2), peripheral leukocyte counts, total respiratory compliance (TLC) and the increases in peak pressure ( Ppeak), Qs/Qt compared with the baseline values (P <0. 05) . The increased plasma concentration but reduced activity of AAT was also found in contrast to that in Group NS (P <0. 05). In the BALF, the activity of AAT, TPL, DSPC/TPL were lower than those in Group NS (P <0. 05), but the concentrations of albumin, IL-8, TNFα, the activity of NE and the ratio of W/D were higher than those in Group NS (P<0. 05). The pretreatment of AAT attenuated the deterioration of oxygenation, the reduction of compliance and the deterioration of other physiological and biochemical parameters mentioned above. Conclusion Pretreatment with AAT could attenuate endotoxin-induced lung injury in rabbits. Those beneficial effects of AAT might be due, in part, to reduction in the levels of mediators that could activate neutrophils, in addition to the direct inhibitory effect on neutrophil elastase. Chin Med J 2003; 116(11): 1678-1682

Objective To observe triclabendazole effect on Paragonimus skrjabini in experimentally infected rats,and to develop a new drug for treating paragonimiasis. Methods Metacercariae of Paragonimus skrjabini were isolated from crabs (Sinopotamon) collected from endemic area. Wistar rats were infected intraperitoneally. One and two months after infection, they were treated with triclabendazole at the dosage of 300 mg • kg-1•2d-1, 450 mg • kg-1 • 3d-1and 600 mg • kg-1 • 3d-1 respectively. Five patients with Paragonimus skrjabini were treated, with Triclabendazole dosage of 10 mg/kg bid x3 days. Results The worm reduction rates were 50. 3%, 80. 8%and 86. 7%respectively one month after completion of treatment. Dead worms of sesame size recovered from muscles, liver, abdominal cavity, chest cavity and lung were greatly diminished in size and weight in comparison with that of the control group. Many large (about 1 cm) black-colored distended worm cysts were found in the lungs of the control rats. Usually there were two adult worms pairs with numerous eggs in each worm cyst. Most worm cysts in the treated groups of rats were changed into hemorrhagic-necrotic patches. All five patients were cured. Conclusion Triclabendazole was highly active against Paragonimus skrjabini in rats experimentally infected and patients. Chin Med J 2003; 116(11): 1683-1686

Objective To study the duration of prourokinase gene expression in vein grafts and the role of the prourokinase gene in protecting vein grafts from neointimal hyperplasia. Methods Fifty-four Wistar rats were used in this study. In each rat, the jugular vein was excised and distended for 30 minutes using a solution containing either Adv5-CMV (control group) or Adv5-CMV/Pro-UK (treatment group). Next, the jugular vein was reversed and interposed into the divided carotid artery of the same rat. On the 14th day after transfection, vein grafts of the control group were collected in order to perform a fibrinolysis test for prourokinase (Pro-UK) activity. On the 2nd, 7th,14th, 28th, and 60th day, the vein grafts of the treatment group were likewise collected in order to detect prourokinase activity. On the 28th day, the vein grafts of both groups were explanted to evaluate the 3H-TDR incorporation so that pathologic analysis could be performed. Results Pro-UK activity could not be detected in the control group, while in the treatment group, the Pro-UK activity could be detected from the 2nd day onwards, peaking on the 7th day and declining from the 14th day, but yet persisting at a low level for a further month. The amount of 3H-TDR incorporated in the control group was higher than that in the treatment group. Pathologic analysis demonstrated that vein grafts of both groups exhibited wall thickening, but that the degree of graft neointimal hyperplasia and reduction of the graft lumen was greater in the control group than that in the treatment group. The occlusion rate of grafts in the control group was 20%. All grafts in the treatment group were patent. Conclusions Pro-UK gene transfer before vein grafting in vitro results in a high level of gene expression in the vein graft from the 7th day to 14th day. And its gene expression in the vein graft could reduce neointimal hyperlasia in the vein graft. Chin Med J 2003; 116(11): 1687-1690

Objective To investigate the effects of BmkTXKβ, a newly purified 'long chain' peptide inhibitor of K+ channels from the Chinese scorpion Buthus martensi Karsch (BmK), on the electrophysiological properties of isolated rabbit atrial myocytes. Methods The standard whole-cell patch-clamp technique was used to study the effects of multiple concentrations of BmkTXKβ on potassium currents and action potentials. Results BmkTXKβ produced concentration-dependent prolongation of action potential duration at 20%, 50%, and 90%repolarization (APD20,50,90) without any use-dependence. Meanwhile, it had no significant effect on RMP, APA, or Vmax(n=9). At a dose of 1 μmol/L, BmkTXKβ decreased lto by 41.4%(n=10, P <0. 01) at a membrane potential of+50 mV [from (13. 63 ± 0. 87) pA/pF to(7. 98 ±0. 78) pA/pF]. Ito was reduced significantly with an IC50 value of 1. 82 μmol/L (95%confidence interval: 1. 47 - 2. 17 μmol/L), in a clear concentration-dependent manner. BmkTXKβblocked lKs and lKS,tall with an IC50 of 20. 15 μmol/L and a 95%confidence interval of 16. 93 -23. 37nmol/L. At a concentration of 10 μmol/L, BmkTXKβ blocked both lKs(mean reduction 37. 3%±4. 2%,P<0. 01, n=7) and lKs,tail(mean reduction 35. 8%±4. 1%, P<0. 01, n=7). At 0 mV, 10 μmol/L BmkTXKβ inhibited both lKr (mean reduction 40. 5%±2. 6%, P <0. 01, n=6) and lKr,tail (mean reduction 42. 3%±2. 9%, P <0. 01, n=6). Blocking of lKr by BmkTXKβ occurred in a concentration-dependent manner, with an IC50 of 17. 21 µmol/L (95%confidence interval: 14. 76 -19. 66 µmol/L). An absence of effects on IK1 was observed for BmkTXKβ, with no change in reversal-potential (n=6,P>0. 05). Conclusions BmkTXKβ exerts direct blocking effects on several potassium channels involved in cardiac repolarization, and has a strong effect on prolonging the repolarization of rabbit cardiomyocytes without reverse frequency dependence. This finding suggests that BmkTXKβ could be a promising class III drug for anti-arrhythmic therapy without the risk of proarrhythmia. Chin Med J 2003; 116(11): 1691-1696

Objective To investigate the cardiac effect of QT interval prolongation in the treatment of acute promyelocytic leukemia (APL) with arsenic trioxide (As2O3), and the relationship between QT and serum arsenic concentration. Methods Blood serum arsenic concentrations of thirty APL patients were determined at 2 hours, 4hours, 8 hours, and 24 hours after As2O3 injection using atomic fluorophotometry. Cardiac functions were measured simultaneously using a 12-lead body-surface electrocardiogram (ECG). Q-T intervals were manually measured, and then corrected using Bazett's formula (QTc). QT dispersion (QTd)was also calculated. In order to assess the effects of arsenic on the symptoms of anemia, twenty-four anemia patients were divided into two groups on the basis hemoglobin concentration: Groupl (Hb≥90g/L), and Group 2 (60 g/L≤Hb <90 g/L). QTc and QTd of these patients were also manually measured. Results All QT intervals of APL patients treated with As2O3 injection were prolonged [32. 2 ms (27,41 ms); P <0. 05], but the changes of QTd were not prominent [3 ms (-8, 7 ms), P> 0. 05]. There was a delay of 2 hours in maximum QTc following peaks in serum arsenic concentration. Changes in QTc and QTd of the two anemic groups were not prominent. Conclusions As2O3 can prolong QTc intervals in APL patients, but the effects are delayed compared to peak serum arsenic concentrations. As2O3 has no prolongation effect on QTd. Mild and moderate anemia do not effect QTc and QTd. Chin Med J 2003; 116(11): 1764-1766

Objective To investigate the effect of micronized fenofibrate on vascular endothelial function in patients with hypertriglyceridemia. Methods Using high-resolution ultrasound, we measured flow- and nitroglycerin-induced dilatation of the brachial artery in 30 patients with hypertriglyceridemia before and after treatment with micronized fenofibrate at a dose of 200 mg once daily for 4 weeks. Simultaneously, both serum lipid and plasma endothelin (ET) levels were determined. Results After micronized fenofibrate therapy, serum triglyceride (TG) levels decreased significantly(P <0. 05) . Plasma ET levels also decreased markedly [(82. 66 ± 15. 46) μg/L vs. (106. 22 ±19. 16) μg/L, P <0. 001]. Flow-induced vasodilatation was much improved (11. 0%± 9. 0% vs 2. 7%±2. 0%, P<0. 01). However, no significant changes in vasodilatation occurred in response to nitroglycerin (16. 2%±6. 0%vs 15. 0%±5. 0%, P>0. 05) in patients with hypertriglyceridemia. Conclusions Micronized fenofibrate can improve impaired endothelium-dependent vasodilatation in patients with hypertriglyceridemia. Improving endothelial function may also be the mechanism responsible for the beneficial effects of micronized fenofibrate. Chin Med J 2003; 116(11): 1767-1769

Objective To determine whether the extent of prolongation of the transisthmus interval after ablation predicts complete bidirectional block. Methods Since 1996 to 2002, 30 consecutive patients underwent ablation procedures for isthmus-dependent atrial flutter. There were 23 males and 7 females [mean age (47. 85 ±9. 35) years]. With the use of fluoroscopic view of anatomy, radiofrequency ablation was performed during coronary sinus pacing at a cycle length of 600 ms. Results Bidirectional block was achieved with ablation in 29 (97%) of 30 patients. The transisthmus intervals before ablation and after complete transisthmus block were (73. 82 ± 13.01) ms and(140. 47 ±20. 48) ms, respectively, in the clockwise direction (P <0. 0001), and (77. 63 ±8. 36) ms and (138. 17 ±15. 55) ms, respectively, in the counterclockwise direction (P <0. 0001). A period of incomplete isthmus block was observed during 17 (58%) of the 29 ablation procedures. The clockwise transisthmus intervals during incomplete block [(107. 65 ± 21.33) ms] were (45. 5 ±8. 7)% longer than the baseline transisthmus intervals. An increase in the transisthmus interval by ≥50%in both directions after ablation predicted complete bidirectional block with 100. 0%sensitivity and 83. 3%specificity. The positive and negative predictive values were 90. 6%and 100.0%, repectively. The diagnostic accuracy of a ≥50%prolongation in the transisthmus interval was 83. 3%. Conclusion The analysis of transisthmus interval is a valuable method for determining complete bidirectional isthmus block. Chin Med J 2003; 116(11): 1770-1772

Objective To establish a new diagnostic method for Down's syndrome using polymerase chain reaction (PGR). Methods DNA extracted from five healthy individuals and five Down's syndrome patients was amplified in six specific tetranucleotide repeat loci on chromosome 21 using PGR. An accurate diagnosis was made by analyzing allelic distribution at each locus. Results All Down's syndrome patients were identified as having at least two loci with three alleles,while none of the healthy individuals had three alleles. In addition, when two alleles were identified for a particular locus in the Down's syndrome samples, it was more likely that the intensity ratio between the two alleles was close to 2: 1. Conclusion The molecular method can provide a fast, accurate, and economical alternation for the traditional cytogenetic diagnostic method for Down's syndrome. Chin Med J 2003; 116(11): 1773-1775

Objective To investigate the protective effects of vitamin B12, ginseng saponin, and folic acid on mouse embryos subjected to high heat. Methods Mice were used for the experiment. Results After exposure of pregnant mice to high heat, the rates of teratism, stillbirth, and fetal absorption were markedly lower in mice treated with ginseng saponin and folic acid following heat exposure than in untreated mice. There were no significant differences in these rates when comparing mice treated with vitamin B12 with the untreated mice. Conclusions Ginseng saponin and folic acid can lessen injuries to murine embryos caused by high heat, while vitamin B12 has little protective effect against high temperature except for promoting overall embryonic growth. Chin Med J 2003; 116(11): 1776-1778

Objective To investigate the relationship between the presence of the TNF2 allele and plasma concentrations of tumor necrosis factor-α (TNFα) and soluble TNF receptor (sTNF-R) with the development of acute severe pancreatitis (ASP) and severe sepsis. Methods Genomic DNA was prepared from peripheral blood leukocytes. The TNF1 and TNF2 biallelic polymorphisms were identified by analyzing Ncol-digested DNA fragments obtained from PCR products. Plasma levels of TNFa and sTNF-R were measured by EASIA. Results The overall TNF2 allele frequency in ASP patients was comparable to that found in healthy volunteers (29.2%vs. 29. 3%, P>0.05). Severe sepsis occurred in 26 of 72 patients. Patients with severe sepsis showed a significantly higher prevalence of TNF2 than those without (46.2% vs. 19. 6%, P<0.05). Plasma TNFa, sTNF-R I, and sTNF-R II levels were (36±31) pg/ml, (5.4 ±3.5) ng/ml, and (11.2 ±7.8) ng/ml, respectively, in patients with severe sepsis, and (31 ±25)pg/ml, (4.6 ±3. 8) ng/ml, and (8. 8 ±6. 6) ng/ml in non-severe sepsis subjects. Differences in TNF levels were not statistically significant between patients with ASP and control group (P>0. 05). Moreover, there was no correlation between TNF2 allele frequency and TNFa levels [(37 ±31) pg/ml vs. (31 ±25) pg/ml in TNF2 group and TNF1 group, respectively, P>0. 05]. Conclusions Our results suggest that there is no relationship between ASP and the TNF2 allele, but that the TNF2 allele is associated with a susceptibility to severe sepsis as a result of ASP. Chin Med J 2003; 116(11): 1779-1781

Objective A retrospective study was undertaken to examine therapeutic strategies used in 6 patients with left renal venal nutcracker phenomenon. Methods Three patients underwent surgical transposition of the superior mesenteric artery. Three patients underwent endograft stent implantations with intervention. Results All 6 cases were completely relieved of left renal vein compression after the operations, leading to improved clinical symptoms and normal urine tests. Conclusions Transposition of the superior mesenteric artery is a feasible method, but leads to complications and great trauma. Treating left renal venal nutcracker phenomenon with endografting techniques is minimally invasive in certain cases, and seems to have a prospective future. Chin Med J 2003; 116(11): 1782-1784