2003年 · 第116卷第08期

Objective To evaluate the effectiveness of small interfering RNA(siRNA)on inhibiting severe acute respiratory syndrome(SARS)-associated coronavirus replication, and to lay bases for the future clinical application of siRNA for the treatment of viral infectious diseases. Methods Vero-E6 cells was transfected with siRNA before SARS virus infection, and the effectiveness of siRNA interference was evaluated by observing the cytopathic effect(CPE)on Vero-E6 cells. Results Five pairs of siRNA showed ability to reduce CPE dose dependently, and two of them had the best effect. Conclusion siRNA may be effective in inhibiting SARS-associated coronavirus replication. Chin Med J 2003; 116(8): 1262-1264

Objective To investigate the role of nuclear factor kappa B (NF-κB) pathway inhibition in lipopolysaccharide(LPS)-stimulated apoptosis of polymorphonuclear neutrophils(PMNs). Methods Rats with acute lung injury induced by LPS intratracheal instillation and cultured human venous PMNs were studied. Pyrrolidine dithiocarbamate(PDTC)and gliotoxin were used as NF-κB inhibitors. Additionally, to explore the role of extracellularly regulated protein kinase as an upstream signal in NF-κB pathway on regulating LPS-stimulated PMN apoptosis, PD098059, the specific inhibitor of extracellularly regulated protein kinase, was also applied. The lung injury was determined by protein content and PMN numbers in bronchoalveolar lavage fluid. PMN apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate(dUTP)end labeling and DNA fragmentation. IkBα degradation was analyzed by Western blot. NF-κB DNA binding activity was detected by an electrophoretic mobility shift assay. Results (1)The increase of protein content and PMN numbers in bronchoalveolar lavage fluid induced by LPS(100 μg per rat)intratracheal instillation were alleviated by PDTC(50, 100, or 200 mg/kg, i. p.)in a dose-dependent manner.(2)PMNs apoptosis in vivo or in vitro was delayed by LPS, and accelerated by PDTC, gliotoxin or PD098059 pretreatment.(3)IkBα degradation and increased NF-κB DNA binding activity mediated by LPS were inhibited by PDTC, gliotoxin or PD098059 pretreatment. Conclusion Inhibition of either NF-κB itself or the upstream signals in NF-κB pathway such as extracellularly regulated protein kinases has therapeutic effect on LPS-induced acute lung injury, in which the dysregulation of PMN apoptosis plays an important role. Chin Med J 2003; 116(8): 1257-1261

Objective To investigate the changes in respiratory and circulatory functions in chronic obstructive pulmonary disease(COPD)patients during sequential invasive-noninvasive mechanical ventilation therapy, and evaluate the effects of this new technique. Methods Twelve COPD patients with type Ⅱ respiratory failure due to severe pulmonary infection were ventilated through an endotracheal tube. When the pulmonary infection control window(PIC-Window)occurred, the patients were extubated and were ventilated with a facial mask using pressure support ventilation combined with positive end-expiratory pressure. The parameters of hemodynamics, oxygen dynamics, and esophageal pressure were measured at the PIC-Window during invasive mechanical ventilation, one hour after oxygen therapy via a naso-tube, and three hours after non-invasive mechanical ventilation. Results The variation in esophageal pressure was 20. 0 ±6 cmH2O during naso-tube oxygen therapy, and this variation was higher than that during non-invasive mechanical ventilation(10 ±6 cmH2O, P<0. 01). The changes in respiratory and circulatory parameters were not significantly different between invasive mechanical ventilation and noninvasive mechanical ventilation(P>0. 05). Conclusions The respiratory and circulatory functions of COPD patients remained stable during sequential invasive-non invasive mechanical ventilation therapy using PIC-Window as a switch point for early extubation. The COPD patients can tolerated the transition from invasive mechanical ventilation to noninvasive mechanical ventilation. Chin Med J 2003; 116(8): 1253-1256

Objective To observe the ability of triple helix-forming oligonucleotides(TFOs)modified with manganese porphyrin to combine with and cleave HBV DNA fractions. Methods TFO were modified with manganese porphyrin and acridines, and then reacted with the 32 P labeled HBV DNA fragments at 37℃ in vitro(pH 7. 4). Electrophoretic mobility shift assays and DNase I footprinting tests were used to show the affinity and specificity of TFO to bind to target sequences. The ability of TFO to cleave HBV DNA fragments was tested by cleavage experiments. Results TFO modified with manganese porphyrin and acridine could bind to the target sequence in a sequence-dependent manner, with a Kd value of 3. 5 × 10-7 mol/L and a relative affinity of 0. 008. In the presence of potassium monopersulfate(KHSO5), TFO modified with manganese porphyrin and acridine could cleave the target sequence where the triplex DNA was formed. Conclusion In the presence of KHSO5, TFO modified with manganese porphyrin and acridine could bind and cleave the target HBV-DNA in a sequence-dependent manner. Chin Med J 2003; 116(8): 1248-1252

Objective To investigate the status of the drug resistance and the ampC gene expression of Enterobacter cloacae. Methods Disk diffusion tests were made for detecting the susceptibility of antimicrobial agents against Enterobacter cloacae. AmpC gene was amplified by polymerase chain reaction(PCR)and verified by DNA sequencing. AmpC gene expression was analyzed according to antimicrobial agent sensitive phenotype. Results The sensitivity rates of 144 strains to imipenam, cefepime and cefoperazone/sulbactam were 98.61%, 65. 97% and 63.89%, respectively. The sensitivity rates of 144 strains to other antimicrobial agents were lower. Among the 144 strains 120 were found to be positive by PCR for ampC. The PCR product showed high homology to the GenBank ampC sequence. Stably derepressed strains, hyperinducible strains and unexpressing or lower level expressing strains accounted for 30. 0%(36/120), 37. 5%(45/120), and 32. 5%(39/120), respectively. Fifty-six out of 120 strains(46. 67%)also produced extended spectrum β-lactamases(ESBLs). The hyperinducible strains were highly sensitive to all the antimicrobial agents except amoxicillin/clavulanic acid and cefuroxime, while the stably derepressed strains were only sensitive to imipenam and cefepime. However, sensitivity to cefepime decreased if the strains also produced ESBLs. Conclusions The durg resistant status of Enterobacter cloacae is severe. Clearing out the expressive status of ampC gene will be helpful in selection of antimicrobial agents in the treatment of clinical infection. Chin Med J 2003; 116(8): 1244-1247

Objective To identify the egg antigens related to the formation of hepatic granulomas and fibrosis of Schistosomiasis japonica. Methods The egg cDNA library of Schistosoma japonicum(S. japonicum)was constructed and screened by immunological methods with the pooled sera of advanced schistosomiasis patients. The inserted foreign DNA fragments of positive clones were sequenced. The sequence data were analyzed using Wdnasis 2. 5 and compared with Genebank data using blast software. Results Eighty-one clones containing recombinant DNA fragments were obtained from the egg cDNA library of S. japonicum by immunological screening. The DNA sequences of all clones belonged to the miracidial antigen family. The longest cDNA fragment was 1604 bp, which contained an open reading frame of 351 bp, which encoded a protein of 1 2913. 35 daltons. Conclusion The cDNA sequence of the miracidial antigen of S. japonicum(Chinese strain)was obtained for the first time. Chin Med J 2003;116(8): 1239-1243

Objective To investigate the features of regional cerebral metabolic rate of glucose(rCMRglc)in patients with mild cognitive impairment(MCI)by positron emission-tomography and its relationship with neuropsychological test. Methods Positron emission tomography, mini-mental state examination and Wechsler memory scale were applied in 10 patients with MCI and 10 healthy volunteers as the control group. Results Scores of mini-mental state examination and Wechsler memory scale in MCI patients were lower than those in the control group(P<0. 01). rCMRglc of the left orbital gyrus, right middle temporal gyrus and right putamen was lower in the MCI group than in the control group(P<0. 05). Correlation analysis in the MCI group indicated that rCMRglc of many brain regions such as the orbital gyrus, putamen, left hippocampus and parahippocampal gyrus, cingulate gyrus, left amygdaloid body, precentral gyrus, postcentral gyrus, and medial occipitotemporal gyrus in MCI patients, were correlated negatively with age; while the rCMRglc of many parts of the brain such as the left putamen, temporal lobe, anterior cingulate gyrus, left insular lobe, amygdaloid body, precentral gyrus, postcentral gyrus and medial occipitotemporal gyrus were correlated positively with mini-mental state examination; and rCMRglc of the left putamen, temporal lobe, left insular lobe, precentral gyrus and postcentral gyrus were correlated positively with Wechsler memory scale. The right putamen, the right inferior temporal gyrus, precentral gyrus, and left postcentral gyrus were correlated positively with the length of education. However, only rCMRglc of the left amygdaloid body were correlated positively with gender. Conclusion The rCMRglc was lower in the orbital gyrus and putamen of MCI patients. Their rCMRglc were correlated with their cognitive impairment severity, age, length of education and sex. Chin Med J 2003; 116(8): 1235-1238

Objective To seek an appropriate culture condition in which porcine hepatocytes can continuously proliferate and express their normal differentiation phenotypes for a longer period of time. Methods Different types and dosages of reagents, transferrin, epidermal growth factor and nicotinamide were used to culture porcine hepatocytes in serum-free Dulbecco's modified Eagle's medium(DMEM). And the proliferative effects and functions of the cells were detected at different culture times. Results Transferrin at 5 ng/ml, nicotinamide at 10 mmol/L and epidermal growth factor at 10 ng/ml had better effects on the viability of hepatocytes than DMEM, DMEM+ 10% fetal cattle serum FCS and other dosages of these reagents(P<0. 05). OD values of MTS were still high in culture at day 7 and day 10, while nearly 30%-35% cells went into the S phase. Good hepatocyte funcitons were found in these groups, and the secretion of albumin was positively correlated with OD value of MTS. The levels of aspartate transaminase and ammonia in these media were lower than those in DMEM and DMEM + FCS. Conclusion Transferrin at 5 ng/ml, epidermal growth factor at 10 ng/ml and nicotinamide at 10 mmol/L are benefitial to the viability and proliferation of hepatocytes. Chin Med J 2003; 116(8): 1223-1227

Objective To explore the frequency and significance of ApoE gene polymorphisms in patients with sporadic Alzheimer's disease(AD). Methods Single nucleotide polymorphisms of the ApoE gene were analyzed in 32 cases of AD and 26 controls, using PCR and gene sequencing. Results The single nucleotide polymorphism of ApoE gene 462C/G was significantly associated with AD(P<0. 05). Conclusions The 462C/G polymorphism might be a specific genotype in Chinese patients with sporadic AD. Chin Med J 2003; 116(8): 1220-1222

Objective To evaluate the success rates of immediate and delayed placement of implants with respect to the causes of tooth extraction and implant positions. Methods A total of 310 dental implants(immediate implants:delayed implants = 76 :234) were inserted into 80 patients. The types, sizes and positions of the implants and the causes of tooth extraction were recorded. We then investigated the relationship of implant loss with the causes of tooth extraction and placement methods. Results A higher failure rate was found for the implants in the posterior region of the maxilla, and when periodontitis was cited as a reason for tooth extraction. The overall success rates were 93. 4% and 95. 7%in the immediate and delayed implant placement groups, respectively, after a 2-year follow-up. No obvious relationship of success rate was observed with the implant placement method, cause of tooth extaction, and implants' position. Conclusion The immediate placement of implants into fresh extraction sockets could offer advantages over the delayed implant placement. It seems to be a safe and predictable method for patients. Chin Med J 2003; 116(8): 1216-1219

Objective To investigate pre-and postoperative changes of regional cerebral cortical blood flow in patients with cerebral arteriovenous malformation. Method Twenty-two adult patients with arteriovenous malformation(AVM)were recruited into this study at Beijing Tiantan Hospital from September 2001 to May 2002. Eight patients had giant cerebral AVM and the other 14 had a small one. Cortical cerebral blood flow(CBF)was measured by laser Doppler flowmetry(LDF)before and after AVM resections. After surgery, the probe of LDF was implanted adjacent to the area of AVM and monitored for 24 hours. Results CBF increased significantly after the resection in all patients regardless of AVM size. In patients with small AVM, CBF returned to the baseline level within 4 hours, but in patients with giant AVM, CBF remained high even after 24 hours. Conclusions Monitoring CBF is helpful to understand pre-and postoperative changes of regional cortical CBF in patients with cerebral AVM. Chin Med J 2003; 116:(8)1273-1275

Objective To study the gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells. Methods Total RNA extracted from human bone marrow derived mesenchymal stem cells and tendon cells underwent reverse transcription, and the products were labeled with α-32 P dCTP. The cDNA probes of total RNA were hybridized to cDNA microarray with 1176 genes, and then the signals were analyzed by Atlaslmage analysis software Version 1. 01a. Results Fifteen genes associated with cell proliferation and signal transduction were up-regulated, and one gene that takes part in cell-to-cell adhesion was down-regulated in tendon cells. Conclusion The 15 up-regulated and one down-regulated genes may be beneficial to the orientational differentiation of mesenchymal stem cells into tendon cells. Chin Med J 2003; 116(8): 1270-1272

Objective This study was conducted to investigate the function of the first intron of porcine growth hormone(pGH)gene in the gene expression. Methods PCR method was used to amplify the first intron from pig genomic DNA. The intron was then inserted into pGH cDNA to construct pGH cDNA-intron(pGH cDNA-in). The recombinant adenoviruses containing pGH cDNA and pGH cDNA-in genes under control of CMV promoter were generated by homologous recombination method in HEK 293 cells respectively. The effect of the first intron on gene expression was evaluated by comparing the expression levels of pGH cDNA-in and pGH cDNA mediated by adenovirus vectors in CHO cells. Results The expression level of pGH cDNA containing the first intron increased by 117%, which was significantly higher than that of pGH cDNA without the intron(P<0. 001). Conclusion The first intron of pGH gene has the function to improve pGH gene expression. Chin Med J 2003;116(8): 1267-1269

Objective To explore digestive system manifestations in patients with severe acute respiratory syndrome(SARS). Method The clinical data of 96 cases with SARS admitted into our hospital from February 6, 2003 to March 28, 2003 were retrospectively analyzed. Results Among the 96 cases, 26 cases(27%)had diarrhea, 17(18%)had nausea, 6(6%)had vomiting, 16(17%)had bellyache, and 8(8%)had ALT elevation. Conclusions Patients with SARS may have digestive system manifestations; diarrhea is the most common symptom. Chin Med J 2003; 116(8): 1265-1266