2002年 · 第115卷第07期

Objective To establish an experimental model for exploring the role of hepatitis C virus (HCV) in the development of cholangiocarcinoma. Methods Recombinant plasmids of HCV-C gene were constructed by molecular cloning techniques and identified by PCR and restriction enzyme mapping. The plasmids were then transfected into QBC939 cells(a cholangiocarcinoma cell line) by Lipofection. After selection with G418, resistant colonies were obtained and analyzed by immunocytochemistry and Western blotting. The morphology was observed by transmission electron microscopy (TEM). The expression of NF-κB was detected by immunocytochemistry. Results Recombinant plasmid was shown by PCR and restriction enzyme mapping to carry the target gene. Moreover, it could efficiently express HCV-C protein in QBC939 cells. HCV-like particles were found in the cytoplasm by TEM, which were spherical with a diameter of 50 - 80 nm and possessed an outer membrane. Moreover, NF-κB activation could be shown in HCV core-transfected cells. Conclusion Expression of the HCV-C gene in cholangiocarcinoma cells was achieved. Transfected tumor cells (QBC939-HCVc) could be used as a model to study the effect of HCV on the development of cholangiocarcinoma. Chin Med J 2002; 115(7): 998-1001

Objective To search for A3243G point mutations in mitochondrial DNA (mtDNA) from 10 cases of mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes (MELAS). Methods Using PCR-restriction analysis, we investigated A3243G point mutations in mtDNA of muscle and/or blood cells from 10 patients and their 8 maternal relatives. We also quantitated the A3243G mtDNA in samples harboring the mutation. Results A3243G point mutations were identified in all muscle and/or blood samples from 10 MELAS patients. The proportion of mutant mtDNA was 10.8%-47.8%in blood (7 cases), and 39.4%-67.7%in muscle (5 cases). This ratio was invariably higher in muscle than in blood from two patients whose blood and muscle samples were both available. Younger patients usually carried higher proportions of A3243G mutant mtDNA in blood. Eight maternal relatives from 6 families were also examined. Maternal transmission of the disease could be identified in one family. No A3243G point mutations were found in mothers' blood from 3 families and siblings' blood from 2 families. Conclusions All 10 MELAS patients were found to have the mtDNA A3243G mutation in their muscle and/or blood. The A3243G mutation seems to be sporadic in 5 of the families examined, suggesting the mechanism of de novo mutation for the pathogenesis of their MELAS syndrome. Chin, Med J 2002; 115(7): 995-997

Objective To establish an effective laboratory examination system for carrier detection and prenatal diagnosis of haemophilia A (HA) with a variety of molecular biological methods which are simple, rapid and easy to use. Methods Detection of inversion involving intron 22 in the FⅧ gene was completed by long distance polymerase chain reaction (PCR) and linkage analysis was performed by using several genetic polymorphisms including an intragenic Bel I RFLP, 2 STRs and an extragenic St14 VNTR. Results Intron 22 inversion was observed in 10 out of the 21 (47.6%) pedigrees examined. Prenatal diagnosis was completed in 3 pedigrees. A further combination of the four intragenic and extragenic polymophic loci gave an informative rate of 94.7%. Conclusions Female relatives in HA families with inversion can be detected with direct diagnostic procedure. The application of long distance PCR makes the detection much more simple and rapid. For families without inversions, it is easier and more cost-effective to undertake linkage analysis of genetic polymorphism based on PCR. Chin Med J 2002; 115 (7): 991-994

Objective To detect the methylation pattern of the p15INK4B gene and to explore its significance in the pathogenesis of acute leukemia (AL) and leukemic transformation of myelodysplastic syndromes (MDS). Methods A total of 49 AL cases and 22 MDS cases were analyzed by methylation specific polymerase chain reaction (MSP) for methylation patterns in CpG islands of the p15INK4B gene. Results Hypermethylation of the p15INK4B gene was found in 90%(26/29) of newly diagnosed AL,including 46%with complete methylation and 54%with partial methylation. All 3 evolved AL from MDS and 9 relapsed AL showed a methylated p15INK4B gene and the proportion of complete methylation was 67%and 56%respectively. Only 5 of 11 (45%) AL in remission, including 2 in complete remission (CR) and 3 in partial remission (PR), were partially methylated. The frequency of p15INK4B gene methylation in newly diagnosed or relapsed AL was significantly higher than that in AL in the remission stage (P=0.002). P15INK4B gene methylation was found in 5 of 13 (38%) low-risk MDS (RA/RAS) patients and 80%of them showed only partial methylation. However, p15INK4B gene methylation was found in all 9 cases in the highrisk group (RAEB/RAEB-T), including complete methylation in 56%, significantly different from the low-risk MDS group (P=0.002). Conclusions Hypermethylation of the p15INK4B gene occurs frequently in leukemia and high-risk MDS. It is possible that hypermethylation of this gene is related to the pathogenesis and development of AL and MDS. It may be used as a gene marker to detect minimal residual disease, relapse of AL and leukemic transformation in MDS. Chin Med J 2002; 115(7): 987-990

Objective To evaluate the role of thrombopoietin (TPO) in the pathology of chronic thrombocytopenic disease. Methods We measured the endogenous plasma concentration of TPO in 40 patients with acquired aplastic anaemia (AA) and in 32 patients with idiopathic thrombocytopenic purpura (ITP) by a sensitive Sandwich enzyme-linked immunosorbent assay (ELISA) and compared the results. Results Plasma TPO concentrations were significantly higher in AA patients (774 ± 393 pg/ml) in comparison with healthy control subjects (55 ± 34 pg/ml, P <0 . 001), and there was a significant negative correlation between their plasma TPO concentrations and platelet counts. In patients with ITP, however,the TPO levels were normal or only slightly elevated (73 ± 36 pg/ml), and there was no significant difference compared with healthy controls (P>0.05). There was also no relationship between their plasma TPO levels and platelet counts. Conclusions TPO levels may be regulated not only by platelets but also by megakaryocytes in AA and ITP, and measurement of TPO levels is useful for diagnosing thrombocytopenia and understanding the pathophysiology of thrombocytopenia. Chin Med J 2002; 115(7): 983-986

Objective To report the clinical characteristics, biochemical profiles, diagnosis and treatment of one Chinese pedigree with glucocorticoid-remediable aldosteronism (GRA) and to study its molecular mechanism. Methods Plasma and urinary aldosterone, Cortisol and plasma renin activities were dynamically tested and diagnostic therapy with dexamethasone was undergone in 3 affected subjects. Long-distance PGR as well as DNA sequencing were applied to detect the fusion gene in this pedigree. Results In this GRA pedigree, there were 4 affected subjects who had hypertension, hypokalemia and low basic and provoked renin activity. Three patients were given dexamethasone treatment, and had a significant decrease in plasma aldosterone concentrations (PACs) (from 192 ± 9 ng/L to 87 ± 7 ng/L, P < 0.05) after 5 days. Among them, one patient (Ⅱ-3) responded quite satisfactorily to the therapy, with serum K+ rising from baseline value of 2.5 to 2.9, 3.8 and 4.15 mEq/L on the 10th, 28th and 35th days after treatment respectively. Three weeks later, his blood pressure decreased from its original level of 146.3± 10.7/94.6 ± 5.3 mm Hg to 138.3 ± 3.1/87.3 ± 6.1 mm Hg (P <0.05). The other 2 members(Ⅲ-2 and Ⅲ-4) showed modest improvement although their PACs decreased significantly. Using longdistance PCR, we found a 3.9 kb band in all 4 affected individuals, which was absent in 5 unaffected members from this pedigree or 8 patients with aldosterone-producing adenoma (APA) or idiopathic hyperaldosteronism (IHA). By DNA sequence analysis, we found that the breakpoint of "unequal crossing-over" is both within intron 2 of the 11 β-hydroxylase gene (CYP11B1) and the aldosterone synthase gene(CYP11B2). Conclusions The excess of mineralocorticoid in patients with GRA can be inhibited by exogenous glucocorticoids. The fusion gene resulting from unequal crossing-over between the 11β-hydroxylase gene and the aldosterone synthase gene is the pathogenesis of this Chinese GRA pedigree. Chin Med J 2002; 115(7): 979-982

Objective To investigate the effects of the mixed endothelin receptor antagonist, bosentan, combined with the long-acting calcium channel blocker, amlodipine, compared to the angiotensin-converting enzyme inhibitor, cilazapril, on the progressive renal injury in spontaneous hypertensive rats (SHR) with diabetes. Methods Diabetic hypertensive rats (SHR-DM) were induced by streptozotozin injected in male SHR(7-week-old), and divided into an untreated and three treated groups: ① cilazapril treated group; ②bosentan + amlodipine treated group; and ③ amlodipine treated group. Wistar Kyoto rats (WKY) and SHR rats served as normotensive and hypertensive control, respectively. The mean arterial blood pressure,renal function, endothelin and angiotensin Ⅱ levels as well as the protein expression of renal extracellular matrix components and transforming growth factor (TGF)-β1 were determined at the end of the 4th week. Results Mean arterial blood pressure significantly increased in SHR and SHR-DM rats compared to WKY rats. All the therapies reduced the blood pressure to normal levels. However, the enhanced urinary protein excretion, the decreased creatinine clearance as well as the increased plasma and intrarenal endothelin and angiotensin Ⅱ levels were found in the untreated SHR-DM and prevented by treatment with bosentan + amlodipine and cilazapril. Similarly, these two kinds of therapies in SHR-DM abolished the overexpression of renal TGF-β1 by Western blot analysis and reduced the accumulation of collagen type Ⅳ, laminin and fibronectin proteins by an immunochemical approach. Amlodipine monotherapy had no detectable effects on the above parameters . Conclusion Bosentan combined with amlodipine can offer similar renoprotective effects on that of cilazapril and may be a potent therapy to attenuate renal injury by reducing renal protein levels of TGF-β1 in diabetes with a hypertensive state. Chin Med J 2002; 115(7): 972-978

Objective To evaluate the effect of endogenous nitric oxide (NO) on the ability of 5-fluourouracil (5-FU)to induce apoptosis in the liver carcinoma Bel7402 cell line, and to observe the anti-tumor mechanism and effective adjuvant of 5-FU. Methods Cells were cultured under routine conditions with Dulbecco's modified Eagle's medium (DMEM)without L-arginine (L-Arg) . We observed the expression of inducible nitric oxide synthase (iNOS) and apoptosis of cells induced by 5-FU with L-Arg added to the medium. The production of nitric oxide was determined by the cell expression of iNOS detected by immunohistochemical staining, and by the concentrations of nitrite and nitrate in the supernatant. Results 5-fluourouracil significantly increased the iNOS expression to 0.1687 ± 0.01968 (P<0.05, vs control group), and the concentration of nitric oxide to 213 ± 30.2 μmol/L (P <0. 05, vs control group)The apoptotic cell rate increased significantly to 17.85 ± 0.78%, while the necrotic cell rate decreased to 32.99 ± 0.83%(P <0. 05, compared with the 5-FU group). Nω-nitro-L-Arginine methyl ester (L-NAME), the antagonist of L-Arg, can block the apoptotic effects of endogenous nitric oxide. Conclusions 5-FU had a synergistic effects with L-Arg by increasing the production of endogenous nitric oxide. Endogenous nitric oxide plays an important role in the process where 5-FU induces apoptosis in liver carcinoma cells. L-Arg may be a good adjuvant for chemotherapy with 5-FU. Chin Med J 2002; 115(7): 968-971

Objective To determine if aquaporinl (AQP1) and aquaporin5 (AQP5) are expressed in the alveolar capillary membrane in rats. Moreover, to investigate the alteration of AQP1 and AQP5 in acute injured lungs. Methods The distribution of AQP1 and AQP5 in alveolar capillary membrane were investigated by immunohistochemistry and immunoelectron microscopy with affinity-purified antibodies to human AQP1 and AQP5. To study the possibility that alveolar capillary membrane AQP1 and AQP5 undergo altered regulation, we established a rat model using alveolar instillation of lipopolysaccharide (LPS). Results Immunolabelling showed AQP1 was stained primarily in the microvascular endothelia of normal lungs, while AQP5 was expressed in type I pneumocytes. Immunohistochemical analysis showed a significant decrease in the expression of AQP1 and AQP5 in injured lungs at 4h -48h after LPS instillation. AQP1 protein was resumed partly at 24h after LPS instillation and steroid administration, whereas AQP5 was unchanged. Conclusion The decreased expressions of AQP1 and AQP5 in injured lungs suggest that both of them may play a role in abnormal fluid transportation. Chin Med J 2002; 115(7): 963-967

Objective To investigate the effects of sulindac metabolites on proliferation and apoptosis in the human umbilical vein endothelial cell line ECV304 in vitro. Methods The proliferation profile of ECV304 was determined by methyl thiazolyl tetrazolium (MTT)method. Cell cycle distribution, apoptosis and the ultrastructure of ECV304 were detected by flow cytometry(FCM) and electron microscopy, respectively. Results MTT assay showed that the sulfide inhibited the proliferation of ECV304 and its effect was dose-dependent; the IC50 was 200 μmol/L. FCM showed that the sulfide changed cell cycle distribution. The cell cycle distribution was as follows: G1 phase (control group 77.74% ± 1.58%; sulfone group 75.63%± 2.12%; sulfide group 46.12% ± 1.60%); S phase (control group 13.64% ±1.22%; sulfone group 16.40 ± 2.30%; sulfide group 27.26% ±2.08%); G2-M phase (control group 8.61% ± 0.67%; sulfone group 7.98% ± 0.49%; sulfide group 26.62% ± 3.54%). The apoptosis rates in the control group,sulfone group and sulfide group were 6.08% ± 3.39%, 4.81 % ± 2.14% and 51.90% ± 5.67%,respectively. Sulfide reduced the proportion of G1 phase, increased the proportion of S phase, G2-M phase and the apoptosis rate significantly (P <0.01, vs control). In the sulfide-treated cells, there were nuclear fragmentation and chromosomal condensation, shrinkage of the cell and loss of contact with neighboring cells. Apoptotic bodies were observed. Sulfone showed no effect on cell proliferation, cell cycle distribution or cell morphology. Conclusions Sulfide can significantly reduce the proliferation of ECV304, change the cell cycle distribution and arrest cells in G2-M phase where apoptosis may be induced. Sulfone has no such effects on this cell line. Chin Med J 2002; 115(7): 1074-1077

Objective To evaluate the measurement of Samarium-153 ethylenediaminetetramethylene phosphonic acid(153Sm-EDTMP) bone uptake rate using whole-body scintigraphy and analyze the relationship between bone uptake rate and therapeutic effect. Methods Sixty-six patients with painful bony metastases from prostate (n=15), lung (n=20), breast (n =18), nasopharyngeal carcinoma (NPC) (n=5), colon (n=2), kidney (n=2) and unknown cause (n= 4) carcinoma were examined with whole-body scintigraphy 10 min and 5 h post administration of 153Sm-EDTMP. Bone uptake rate was then calculated. (1) Complete response (CR): disappearance of > 2 metastases, Karnofsky Performance Score (KPS) increase >20, moderate or complete remission of bone pain 7 d post injection of 153Sm-EDTMP. (2) Partial response (PR): disappearance of 1 -2 metastases,KPS increase 10 - 20, moderate remission of bone pain in 3 wk. (3) Non-response (NR): no disappearance or shrinkage of metastases, KPS increase <10, no or slight remission of bone pain. Results The range of bone uptake rate in 66 patients was 31.9%- 86.6%(mean=56.0%). The bone uptake rate in the CR group (17 cases, 25.7%), PR group (24 cases, 36.4%). and NR group (25 cases, 37.9%) was 52.4% - 86.6%(mean=68.7%), 43.7%- 70.4%(mean=58.3%), and 31.9%-51.5%(mean=41.0%) respectively. Statistical analysis showed that there was a significant difference between the CR and PR groups (t=4.258, P=0.001) as well as between PR and NR groups (t=8.48,P=0.001). Conclusions Using a simple and reliable whole-body scintigraphic technique to calculate prospectively the bone uptake rate, we have, for the first time in China, reported the relationship between bone uptake rate and therapeutic effect. This allows nuclear medicine physicians to calculate a safe and effective dose of 153Sm-EDTMP in individual patients to palliate bone cancer pain without myelotoxicity. Chin Med J 2002; 115(7): 1096-1098

Objective To study the factors that affect the prognosis of the patients with benign prostatic hypertrophy(BPH) after prostatectomy. Methods Using urodynamic methods, we studied 50 BPH patients with embarrassing outcomes after prostatectomy. Results Detrusor instability (DI) appeared in 20 (40%) patients. Twelve (24%) patients suffered from weak contractility of the detrusor and 18 (36%) patients had bladder outlet obstruction. Conclusions DI, weak contractility of the detrusor and bladder outlet obstruction are the most frequent causes affecting the prognosis of BPH patients after prostatectomy. Urodynamics may play a significant role in the identification of the causes of unsatisfied prognosis of BPH patients. Chin Med J 2002; 115(7): 1093-1095

Objective To study the prevalence of skin positivity to purified protein derivative (PPD) in human immunodeficiency virus (HlV)-infected patients in Hong Kong. Methods Consecutive clients of an HIV clinic were administered the PPD test and 2 units of PPD-RT23 were used. The area of induration was then measured in 48 to 72 h. Results were related to patient characteristics and HIV-related parameters. Results Eight (17.0%) out of 47 clients tested positive to the administration of 2 units of PPD-RT23. If the cutoff were raised to 10 mm according to current practice, only two (4.3%) would test positive. Conclusion The prevalence of PPD positivity in HIV-infected patients in Hong Kong is 17%, when a cutoff of 5 mm is used. This figure may form the basis for further studies on the utility of isoniazid preventive therapy in this group of patients. Chin Med J 2002; 115(7): 1091-1092

Objective To determine whether the transfusion-transmitted virus (TTV) is hepatotropic. Methods Total DNA was extracted from various tissues of 5 experimentally infected Rhesus monkeys during the viremic period. A dot hybridization was done with viral double stranded DNA probes or single antisense probes. Results The double-stranded probe was hybridized with DNA from the liver, bone marrow, spleen,stomach, small intestine and colon. The single-stranded antisense probe was hybridized with DNA from the liver, small intestine and bone marrow of all 5 monkeys, but not with that from other tissues. Conclusions As the viral genome was of negative polarity, the plus-stranded fragment identified in our study might be a replicative intermediate, and was only demonstrated in the liver, small intestine, and bone marrow by dot blot hybridization with single-stranded antisense probes. It is suggested that the TTV replicates in the liver, bone marrow and small intestine, and TTV might be hepatotropic. Chin Med J 2002; 115(7): 1088-1090

Objective To explore the relationship between genetic polymorphisms of the ethanol metabolizing enzymes and the occurrence of alcoholic liver disease (ALD). Methods Sixty-five healthy male controls and 165 alcoholisms (including 122 ALD patients and 43 male alcohol abusers without liver complications defined as alcohol-dependent) were analyzed by polymerase chain reaction and hybridized with oligonucleotide microarray to detect the polymorphisms of the ethanol metabolizing enzymes genes. Results The frequencies of alcohol dehydrogenase gene 2 * 1 (ADH2 * 1) allele were shown as 37.69%, 46.51 %and 59.02%in control, alcohol-dependent and ALD groups respectively; while those of ADH2 * 2 allele were shown as 62.31%, 53.49% and 40.98% respectively. No ADH2 * 3 was detected in any of the subjects. The frequency of ADH2 * 1 was significantly higher in alcoholisms (ALD group and alcohol-dependent group) than in healthy controls (P <0.01), and significantly higher in ALD group than in alcohol-dependent group (P <0.05) . The frequency of ADH3 * 2 was significantly higher in alcohol-dependents than in healthy controls (P <0.05) . The frequencies of ALDH2 * 2 allele mutation were significantly lower in alcoholisms than that in the healthy controls, and the deference between ALD group and alcohol-dependent group was significant. No homozygotes for the mutant ALDH2 * 2 allele were found in either alcoholic groups. Conclusions Polymorphic ADH2, ADH3 and ALDH2 genes can affect the propensity for alcohol drinking in Chinese. The alleles of ADH2 * 2, ADH3 * 1 and ALDH2 * 2 are most likely to play a protective role against excessive consumption. ADH2 * 2 and ALDH2 * 2 may contribute to susceptibility for ALD. Chin Med J 2002; 115(7): 1085-1087

Objective To investigate the prevalence of functional dyspepsia (FD) and the psychological disorders in Chinese population and their relation. Methods A total of 1016 apparently healthy people (study population) randomly selected from population were interviewed. A general questionnaire, Zung self-rating depression scales (SDS), and anxiety scales(SAS) were given to each subject. Seventy-two inpatients with confirmed diagnosis of FD, 84 with organic dyspepsia, and 197 with other organic diseases were also studied. Results Among the study population, 23.5% had FD and 9.1% had disturbances of depression and/or anxiety. The prevalence of psychological disturbances in FD group (15.5%) differed significantly from that in non-FD group (7.1%, P <0.01). In the survey of inpatients, the rate of depression/anxiety in FD group (54.2%) was greater than that in organic dyspepsia group (19.0%) and other organic diseases group (28.9%), P <0.05, and was also significantly higher than that of the FD cases in the study population (15.5%), P <0.01. Further analysis showed that some psychosocial disturbances were risk factors of FD. Conclusion Both FD and depression/anxiety disturbances are common in China, and there is a link between them. Chin Med J 2002; 115(7): 1082-1084

Objectives To review the identified deafness genes related to nonsyndromic hearing loss (NSHL) and summarize their expressions and functions in the cochlea and to introduce the current studies of molecular genetics on NSHL in China. Methods The presented data are based on a review of the literature as well as the author's experience with NSHL and communications with other researchers in China over the past 3 years. Results Currently, 23 deafness genes related to NSHL have been cloned and identified. Some genes are associated with both NSHL and syndromic hearing loss (SHL), in both dominant and recessive deafness. Deafness genes have a highly specific expression pattern in the inner ear. Some functional categories are starting to emerge from a characterization of deafness genes. There are interacting genes in the genetic background that influence the extent of hearing impairment. The GJB3 gene, which is associated with high-frequency hearing impairment, was cloned in a Chinese laboratory. Mutations in some genes, such as GJB2 and mitochondrial 12S rRNA, have been screened in Chinese patients with NSHL. Mapping new deafness gene loci as well as identifying new genes and their functions is an active area of study in China. Conclusions It is challenging for us to continue identifying new deafness genes and analyze gene functions. By identifying genes responsible for monogenic hearing impairment, more insight may be gained into the molecular process of hearing and the pathology of hearing loss. Chin Med J 2002; 115(7): 1078-1081