Volume 115  ·   Issue 03  ·  2002

Objective To investigate the antagonistic effect and mechnism of the effect of cyproheptadine (Cyp) on endotoxic shock in rats. Methods Endotoxic shock was produced in rats by iv injection of lipopolysaccharides (LPS) (5 mg/kg). Tumor necrosis fator (TNFα) mRNA expression was assessed by Northern blot. Plasma TNFα content was measured by radioimmunoassay. Plasma superoxide dismutase (SOD) activity and malondialdehyde(MDA) content were measured. The intracellular free calcium concentration ([Ca2+]i) in single endothelial cells was determined by laser scanning confocal microscopy (LSCM). Results Cyp 5 mg/kg injected immediately after iv LPS raised the mean arterial blood pressure (MABP)of shocked rats and improved their 24 h survival rate. Meanwhile, Cyp markedly decreased TNFα mRNA levels in rat liver (18 ± 10 vs LPS+saline 38 ± 10, P <0 .01) as well as plasma TNFα content [(7 . 8 ± 2 . 4)ng/L vs LPS + saline (21.5 ± 3.2) μg/L, P <0.01)] . It enhanced plasma SOD activity [(1037.2 ± 112.8) NU/L vs LPS + saline (615.4±92.6) NU/L, P <0.01], reduced the MDA content [(5.2 ±1.1)nmol/L vs LPS + saline (9.8±1 .5) μmol/L, P <0.01], and inhibited TNFα-induced [Ca2+]i elevation. Conclusion Cyp exerts an anti-endotoxic shock effect by inhibiting TNFa gene expression, enhancing SOD activity, reducing lipid peroxidation, and preventing [Ca2+]i overload. Chin Med J 2002; 115(3): 443-445

Objective To establish a method for rapid detection and sub-typing of human papilloma virus (HPV). Methods We utilized the piezoelectric genosensor (PG) technique, which is a combination of the piezoelectric biosensor and gene chips for HPV identification in 22 recurrent biopsy specimens and 22 corresponding original biopsy specimens. The control samples came from normal tissue of healthy persons. A combined reaction took place on the sensor surface between the target genes and probes. The frequency of the piezoelectric sensor will decrease when such reactions occur, and the frequency decrease depends on the concentration of the target gene. Specimens were also analyzed with conventional PCR and dot blot. Results Of the 22 recurrent specimens, 15 contained HPV6 DNA, 2 HPV11 DNA, and 4 HPV16 DNA. Only one specimen was negative. All the 22 original specimens were positive: 17 harbored HPV6 DNA, 3 sequence homologous HPV11 DNA, and 2 HPV16 DNA. No HPV18 DNA was detected in any specimen. When compared with PCR and dot blot analysis, the results were essentially the same except for one specimen, which was shown to contain other sub-types of HPV. Conclusion Our results show that the piezoelectric genosensor technique is a rapid and specific method to analyze HPV. Chin Med J 2002; 115(3): 439-442

Objective To examine the effect of angiotensin Ⅱ (Ang Ⅱ) on nuclear factor-kappa B (NF-κB) activation in human endothelial cell line ECV304 and the molecular mechanism by which Ang Ⅱ activates NF-κB. Methods ECV304 cells were transiently cotransfected with an NF-κB/ luciferase reporter gene and inactive NF-KB-inducing kinase (NIK), IκB kinase a (IKKα), IκB kinase β (IKKβ) mutants or vectors, respectively. The effect on NF-κB was detected by using an electrophoretic mobility shift assay (EMSA) and overexpression of the mutants enabled blocking of reporter gene activation induced by Ang Ⅱ. With immunofluorescence and immuno-electronic microscope techniques, including confocal microscopy and gold particle labeled electronic microscopy, definite cytoplasmic-to-nuclear translocations of NF-κB activation were detected using subunits p50 and p65 induced by Ang Ⅱ . Results The translocation of p50 in nuclei was highly remarkable 2 hours after Ang Ⅱ stimulation, and the activity was somewhat reduced 6 hours after stimulation to the 18th hour. Northern blot also showed PDGFB mRNA increased by stimulation of Ang Ⅱ for 18 hours. Conclusion Ang Ⅱ is effective in stimulating NF-κB activation through a pathway dependent on NIK, IKKα and IKKβ, and induces PDGF-B transcription in the endothelial cell line, ECV304. Chin Med J 2002; 115(3): 433-438

Objective To detect DNA and chromosomes instabilities during the progression of tumors and screen new molecular markers coupled to putative or unknown oncogenes and/or tumor suppressor genes. Methods Five kinds of tumors, in a total of 128 specimens, were analyzed by random amplified polymorphic DNA (RAPD) PCR. Bands representing instabilities were recovered, purified, and cloned,labeled as probes for Southern and Northern blot analysis and DNA sequencing. Results Sample 5 and 3 of the gastric cancer tissues showed the highest genomic changes and the average detectability in five cancers was up to at least 40%(42.2%- 49.4%). There were significant differences in the ability of each primer to detect genomic instability, which ranged from 27%(primer 8) to 68%(primer 2). Band B is a single copy fragment, and was found to be an allelic loss in gastric and colon cancers. It is a novel sequence and was registered in GenBank with Accession Number AF151005 . Further analysis revealed that it might be part of a cis- regulatory element of a new tumor suppressor gene,containing a promoter of cis-action "CACA" box, an enhancer of "GATA" family and a start codon. Conclusions It was impossible or difficult to get great achievements for cancer treatments with the procedure of gene therapy only to one oncogene or one tumor suppressor gene because the extensive DNA variations occurred during the progression of tumor. RAPD assay connected with other techniques was a good tool for the detection of genomic instabilities and direct screening of some new molecular markers related to tumor suppressor genes or oncogenes. Chin Med J 2002; 115(3): 430-432

Objective To investigate the effects of antisense glutamic acid decarboxylase (GAD67) oligodeoxynucleotide (ODN) on behavior, seizure threshold and EEG of hippocampus in the epileptic rats induced by pentylenetetrazol (PTZ). Methods A model of chronic epilepsy in rats was established by PTZ. The inhibition of GAD67 mRNA expression in hippocampus was selectively induced by antisense oligodeoxynucleotide of GAD67. The effect of antisense GAD67 ODN on behavior, seizure threshold and EEG recording of kindled rats was examined. Results Antisense GAD67 ODN could inhibit the expression of GAD67 mRNA and the concentration of GABA. It also could significantly shorten the latencies of seizure and increase the level of seizure and the frequency of epileptiform discharges. Conclusion The gene of GAD67 may be an anti-seizure gene, which might inhibit epileptiform discharge. The treatment of epilepsy by GAD67 gene will have a bright future. Chin Med J 2002; 115(3): 425-429

Objective To investigate the compositions of Th1/Th2/Th3 cells in chronic hepatitis B virus (HBV)-infected individuals by determining the expression of interleukin-4 (IL-4), inetrferon-y (IFN-y), and transform growth factor-β (TGF-β) in single CD4+ T cells isolated from peripheral blood mononuclear cells(PBMCs) and the role of polarized Th cell populations in chronic HBV-infection was discussed. Methods PBMCs from chronically infected HBV individuals were isolated, stimulated by PMA/lonomycin/Monensin, and IL-4, IFN-y and TGF-β production by CD4+ T cells was determined by using fluorescence activated cell sorter (FACS) analysis. Results The percentage of IFN-γ-producing T cells, IL-4-producing T cells and TGF-β-producing T cells ranged from 2.3%- 18.6%, 1.1%- 8.7%and 0.7%-7.1%respectively in CD4+ T cells from non-infected individuals. Most of CD4+ T cells from PBMCs in chronically infected HBV individuals were Th0 cells. The proportion of Th1 cells increased significantly with hepatic inflammatory activity, and in the active period of chronic hepatitis B infection were higher than those in the non-active period (P <0.05) . Th2 cell percentage in CD4+ T cells from HBV-infected individuals did not differ significantly (P> 0.05), but were higher than that from controls (P <0.05). Th3 cell percentage in CD4+ T cells from asymptomatic carrier(AsC) group was higher than that in the chronic hepatitis B (CHB) and control groups (P <0.05). Conclusions Th1 phenotype cytokines were positively correlated with hepatic inflammatory activity in chronic hepatitis B and Th2 cells may be associated with the persistence of HBV infection. Th3 cells cooperating with Th2 cells can negatively regulate immune responses and may be associated with the immune tolerant state of chronic HBV infection. Chin Med J 2002; 115(3): 422-424

Objectives To assess the tendency of manometric asymmetry, and to quantitatively evaluate the function of the anal sphincter. Methods Using the PC polygram HR at the states of relax and squeeze, we compared the values of pressure asymmetry in the anorectum of 27 normal children with that of 12 children with fecal incontinence. Results In the two states, the anal sphincter in normal children showed not only the gradient of pressure,but also a gradient of pressure asymmetry. Children with fecal incontinence did not show any gradients. Conclusion Gradients of pressure and pressure asymmetry can quantitatively evaluate the function of the anal sphincter. Chin Med J 2002; 115(3): 413-417

Objective To test the possibility of modification of human degenerative lumbar disc cells by the exogenous growth factor gene, transforming growth factor β1, (TGF-β1,) cDNA, and the expression of the encoded protein. Methods Nucleus pulposus samples were surgically obtained from 8 patients with degenerative lumbar disc disease. The cells were cultured and directly infected by two adenoviral constructs, Ad/CMV-EGFP containing the enhanced green fluorecence protein (EGFP) gene (marker gene) and Ad/CMV-TGF-β1 containing the potentially therapeutic TGF-β1 gene. Transgene expression was analyzed by fluorescence production and immunohistochemical staining (Ad/CMV-TGF-β1). Results Culture cells transducted by Ad/CMV-EGFP showed specific green fluorescence under the fluoroscope and expression sustained for at least 4 weeks. When infected by Ad/CMV-TGF-β1 approximally 30% of cultured cells were staind brown (+) with TGF-β1, staining. Conclusion This study established the strategy of delivering a potentially therapeutic gene, TGF-β1, by using an adenoviral vector to human degenerative lumbar intervertebral disc cells. Chin Med J 2002; 115(3): 409-412

Objective To determine the perioperative and late outcomes for carotid endarterectomy (CEA) in treatment of patients with high-grade stenotic lesions of the extracranial internal carotid artery. Methods Twenty patients underwent 21 CEAs at the Zhongshan Hospital between May 1993 and June 2000. They were 19 men and 1 woman, with a mean age of 64 ±9 years. Seven patients were performed for stroke, 11 for transient ischaemic attacks (TIAs) and 2 for asymptomatic disease. Duplex scan was the primary tool of evaluation prior to surgery. Perioperative digital subtraction angiography and magnetic resonance angiography were done for 19 and 18 patients, respectively. The percentage of stenosis was calculated using NASCET criteria. Of the 21 lesions operated, 19 had a stenosis of 70%or greater, 2 had ulcerative lesions with a stenosis ranging from 60%to 69%. All CEAs were performed under cervical block anaesthesia with selective intraoperative shunting and patch angioplasty. The patients were followed up regularly with duplex scan surveillance. Results There was no mortality or stroke during 30 days postoperatively. A TIA occurred in one patient and cranial nerve injury in 2 patients perioperatively. All patients were followed up for a mean interval of 31 ±20 months (range: 1 - 63 months). The 2-year survival rate and risk of stroke were 92.3%and 0%,respectively, and the 5-year survival rate and risk of stroke were 79.1 %and 12.5%. respectively. Two asymptomatic recurrent stenoses ranging from 50%to 60%were detected on follow-up duplex scan. Conclusions For the patients in this study, CEA is associated with an acceptable perioperative outcome as well as a satisfactory long-term beneficial effect in stroke prevention. Chin Med J 2002; 115(3): 405-408

Objective To study the effect of dehydroepiandrosterone sulfate (DHEAS) treatment of osteoporosis in men with TBMD ≥2.5SD. Methods Eighty-six patients were randomly divided into two groups: treatment group (n=44) and control group (n=42). DHEAS (100 mg q. d.) was given to the treatment group for 6 months. Bone mineral density, (BMD), biochemical markers of bone absorption and formation and other serum biochemical markers were measured before and after DHEAS treatment. Drug side effects were also evaluated. Results After oral administration of DHEAS (100 mg q. d.) for 6 months, the serum concentrations of DHEAS and IGF-Ⅰ in the treatment group were 93.75%± 16.1% and 17.715± 4.2%higher respectively than those in the control group (P <0.01). The BMD of L2, L3, L4, L2-4 and Neck sections increased in the treatment group by 2.65% ± 0.65%, 2.70% ± 0.48%, 3.10%±0.41%, 2.82% ± 0.37% and 2.32%± 0. 31%, respectively, as compared with that the control group (P<0.05 or 0.01). No significant changes were observed in serum FT, E2 and PSA concentrations in the treatment group as compared with the control group. Conclusion The treatment of osteoporosis in men with DHEAS is safe and effective. Chin Med J 2002; 115(3): 402-404

Objective To establish rat C6 brain-tumor models and find a simple reliable index to judge tumor volume. Methods C6 cell suspension (10 μl) containing 10 g/L agarose and 1 x 106 cells was injected into the right caudate nucleus of the rat brain by a stereotaxic method. After implantation, rats were observed and given MRI scans. Rats were perfused with paraformaldehyde trans-aorta at the 10th, 15th, 20th and 25th day and before natural death. All brains, lungs, spinal cords and tumors were sectioned and inspected. Tumor-containing samples were prepared histologically by hematoxylin and eosin (HE) stains. Results Fifty implanted rats had 100%yield of intracerebral growth, with distant metastasis of 0%to 4%. Conclusion A rat C6 brain-tumor model was successfully established. Rat survival time is correlated with tumor volume and may be useful as an index of tumor size. Chin Med J 2002; 115(3): 455-457

Objective Because maternal epidermal growth factor (EGF) may be an adaptive response to accelerate growth and maturation in premature infants, we compared the EGF content in fresh cow's milk and cow's milk-based infant formulas with full and preterm mother's milk. Methods EGF content of 57 human colostrum from mothers delivering prematurely and at term, 4 different fresh cow's milk and 8 different cow's milk-based infant formulas was determined by radioimmunoassay(RIA). Results Human milk from mothers of premature infants had a higher EGF content compared to that from mothers of term infants (28 .2 ± 10 . 3 nmol/L vs 17.3 ± 9 . 6 nmol/L). EGF content in human milk negatively correlated with gestational age and birth weight of neonates. EGF content in fresh cow's milk (13.8-18.2 nmol/L) was similar to that in human term milk. EGF levels in non-hydrolyzed protein formulas were much lower (5.6-8.6 nmol/L), and were undetactable in hydrolyzed protein formulas. Conclusion The high EGF content in premature milk may represent a maternal compensatory mechanism to accelerate the growth and development of immature infants. Feeding infants with breast milk from their own mother should be advocated since there is lack of EGF in cow's milk-based infant formulas. Chin Med J 2002; 115(3): 451-454

Purpose To review the recent developments in the structure and function of Smad proteins. Data Sources Both Chinese- and English-language literatures were searched using MEDLINE/CD-ROM(1997-2000) and the Index of Chinese-Language Literature (1997-2000). Study Selection Data from published articles about TGF-β signal transduction in recent domestic and foreign literature were selected. Data Extraction Data were mainly extracted from 22 articles which are listed in the reference section of this review. Results Smad proteins mediate signal transduction induced by the TGF-β superfamily. Based on their structural and functional properties, Smad proteins are divided into three groups. The first group, receptor-regulated Smads (R-Smads), are phosphorylated by activated type I receptors and form heteromeric complexes with the second group of Smads, common mediator Smads (Co-Smads) . These Smad complexes translocate into the nucleus to influence gene transcription. Inhibitory Smads (I-Smads) are the third group and these antagonize the activity of R-Smads. In the nucleus, Smads can directly contact Smad-binding elements (SBE) in target gene promoters. Through interaction with different transcription factors, transcriptional co-activators or co-repressors, Smads elicit different effects in various cell types. The aberrance of Smad proteins has been noted in several human disorders such as fibrosis, hypertrophic scarring and cancer. Conclusion The structure of Smads determines their function as transcriptional factors which translocate signals from the cell surface to the nucleus where Smads regulate TGF-β superfamily-dependent gene expression. Chin Med J 2002; 115(3): 446-450