2000年 · 第113卷第02期

Objective To test the potential role of an insertion/deletion polymorphism of angiotensin converting enzyme (ACE) gene on the renoprotective responsiveness to benazepril in type 2 diabetes mellitus with nephropathy. Methods The insertion/deletion polymorphisms of ACE gene were determined by polymerase chain reaction(PCR). Ninety cases with diabetes nephropathy were classified according to the genotype of the ACE gene into 30 cases withⅡ genotypes, 29 with ID and 31 with DD. Mean arterial blood pressure (MABP), urinary albumin excretion rate (UAER), serum creatinine (Scr) and ACE levels were measured before and after the six-month treatment of benazepril (no differences in drug dose between groups). Results At baseline the three groups with different genotypes were similar with regard to sex, age, duration of diabetes, frequency of retinopathy, body mass index, HbA1c, MABP, UAER, serum creatinine, total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein, insulin dose. The dose of benazepril and the number of patients receiving nifedipine after starting ACEI were also similar in the patients with II, ID, DD genotypes, respectively. The serum levels of ACE in patients with DD genotype were the highest, the intermediate with ID and the lowest with II genotype ( P < 0.05). Initiation of 6-month treatment with benazepril induced a significant drop in MABP, UAER and ACE levels in all three groups (P < 0.05), but the reduction was significantly greater in patients with Ⅱ genotype (UAER57.9%, MABP21.4 mm Hg, ACE74.8%), compared to patients with either ID or DD genotype (P < 0.01, respectivly). A mild increase in the serum creatinine was seen in all groups after the initiation of benazepril, but the rate of increase in DD group was the highest (8.9%) and in II group lowest (5.4%) (P < 0.05). A multiple linear regression analysis revealed that the ACE gene polymorphism influenced the decline in albuminuria after initiation of ACE inhibition (R2 = 0.69, P < 0.001). Conclusion The patients with II genotype are particularly susceptible to commonly advocated renoprotective treatment.

Objectives To examine the expression of p16 gene and the clinicopathological characteristics of 31 pituitary adenomas. To investigate the relationship between p16 expression and cell proliferative activity by analyzing the expression of proliferating cell nuclear antigens (PCNA). Methods Immunohistochemistry and in situ hybridization were performed on freshly-prepared paraffin-embedded specimens of 31 pituitary adenomas. Results Varying degrees of overexpression of the p16 gene was found in the tumors specimens. A higher proportion of p16 positive tumor cells was associated with bigger size, more frequent invasion and higher recurrence of the tumors. The expression of p16 was found to be significantly associated with the degree of PCNA expression. Conclusion Though inactivation of the p16 gene is known in wide range of tumors, our study suggested that p16 overexpression might play a role in the development and progression of pituitary adenomas. Chin Med J 2000; 113(2): 162-166

Objective To screen for mutation hotspot of Wilson disease (WD) gene in Chinese population. Methods Cycle sequencing was used to detect mutation in exon 8 of WD gene in 30 patients with Wilson disease. Results The same missense mutation, Arg779Leu, was identified in 14 WD patients, four of whom were homozygous and the other heterozygous for this mutation. The frequency of this mutation in Chinese patients was 30%. Conclusion The codon 779 (CCG→CTG) of exon 8 of WD gene was one of mutation hotspots in Chinese. Chin Med J 2000; 113(2): 172-174

Objective To investigate the manifestations that heat shock proteins(HSPs) possess in the pathogenesis of asthma and chronic bronchitis. Methods Using reverse transcription-DNA polymerase chain reaction (RT-PCR), we investigated the expression levels of HSP70, HSP90α and HSP90β genes in peripheral blood mononuclear cells(PBMC) at natural state and after heat shock in 14 healthy volunteers, 21 patients with asthma and 18 patients with chronic bronchitis. Results No HSP70 gene but HSP90α and HSP90β expressions were found in non-heat-shocked PBMC of normal control; HSP90α and HSP90β genes may be expressed in PBMC of patients no matter whether they were in acute episode or not. Expression of HSP70 was found in PBMC of patients in acute episodes and of three symptoms-free patients with Aas 3, step 2. No expression of HSP70 gene was found in PBMC of patients in convalescent period but in PBMC of patients in acute episode. HSP90α and HSP90β genes were expressed in PBMC of the two patients groups; After heat shock, expressions of the three genes increased in amount significantly in PBMC of all normal controls and patients. Conclusion Expression of HSP70 gene in PBMC of asthmatic patients and chronic bronchitis was different, indicating that HSPs, especially HSP70 might be involved in the pathogenesis of asthma. Chin Med J 2000; 113(2): 175-177

Objective To compare the curative effects of three different antifungal regimens in the treatment of cryptococcal meningitis. Methods Twenty-two patients were divided into 3 groups: Group I was given intravenous amphotericin B alone or combination with flucytosine therapy. GroupⅡ received intravenous fluconazole alone or combination with flucytosine. The treatment of GroupⅢ was divided into two steps, where the patients received intrathecal amphotericin B plus intravenous amphotericin B with or without intravenous fluconazole until the mycological culture of cerebrospinal fluid (CSF) turned negative, followed by oral fluconazole or itraconazole as maintenance therapy until direct microscopic examination of CSF showed negative once a week for three consecutive weeks. Results Of the twenty-two patients, 17 (77.3%) were cured, 2 (9.1%) improved, 3 (13.6%) died, and one (4.5%) relapsed. Of the 8 patients in GroupⅠ, 5 were cured, 2 improved, one died and one relapsed; Of the 4 patients in Group Ⅱ, 2 were cured, and 2 died; All the 10 patients in Group Ⅲ were cured without any recurrence. Conclusion The two-step therapeutic regimen may be suited to the treatment of cryptococcal meningitis. Chin Med J 2000; 113(2): 178-180

Objective To assess the prevalence of Chlamydia pneumomia DNA in patients with otolaryngic disease. Methods PCR assay was used to detect Chlamydia pneumonia specific Pst I 474 fragment DNA in swabs from patients with acute or subacute pharyngolaryngitis or rhinitis and sinusitis. C. pneumonia specific antibodies in sera were also assayed with microimmuno-fluoresence (MIF). Results About 28% (49/175) of the patients were PCR positive and 25.7% (45/175 ) were MIF antibodies positive. The accordance rate of the two methods was 91.8%. Conclusion It is suggested that the C. pneumonia infection was common in this group of patients and the C. pneumonia Pst I 474 specific PCR was sensitive and specific for detecting C. pneumonia in pharyngolaryngitis or rhinitis and sinusitis. Chin Med J 2000; 113(2): 181-183

Objective To investigate the effects of arotinoid acid (Rol3-7410) on the morphological and functional alterations of leukemia HL-60 cell line and compared with those of RA. Methods Differentiation of HL-60 cells was assessed by morphology and by NBT reduction. Trypan blue exclusion was used to determine viability. Apoptosis was assessed by changes in cell morphology and by measurement of fragmented DNA using the PCD-assay-kit. Telomerase PCR ELISA-kit tested telomerase activity. The cell cycle was analyzed by flow cytometry. Results Incubation of the HL-60 cells with 10-6-10-8 mol/L Rol3-7410 resulted in suppression of cell growth. Apoptotic cells were detected following exposure to 10-6 mol/LRo13-7410 for 3 hours by measurement of the "in situ" enzymatic labeling of DNA breaks with biotinylated dUTP. Ultrastructural examination of Ro13-7410-treated samples showed cells with chromatin compaction and cytoplasm condensation and the presence of "apoptotic bodies". Cells induced into apoptosis were accompanied by increase of intracellular free Ca2+. Percentage of HL-60 cells reduced NBT following incubation with Rol3-7410 was lower than with all-trans retinoic acid (RA) (27% vas 85%). Telomerase PCR-ELISA assay showed that HL-60 cells cultured in the absence of inducing agents had significant telomerase activity. Telomerase activity declined gradually after 10-6 mol/L Rol3-7410 treatment, and changes becoming evident at 1 day. The inhibition of telomerase activity at day 5 of treatment with Rol3-7410 was less effective than with RA. DNA flow cytofluorimetric analysis revealed that Rol3-7410 caused partial cell arrest in the G2/M phase after a 2-day treatment and the percentage of cells arrested in the G2/M phase increased after 4-days treatment. With RA-treated cells, a reduction in the percentage of cells in the G2/M phase was observed after 2-day of treatment. Conclusion Our study shows that Rol3-7410 suppresses HL-60 cells growth mainly via the induction of apoptosis and is less effective than RA in induction differentiation. Rol3-7410 dramatically inhibits telomerase activity during the course of induction and results in G2/M arrest within 2 days. These findings suggest that Rol3-7410 is worthy of further study for its effects on leukemic cells.

Objective To establish bio-11-photosoralen (BP) labeling primer reverse transcription in situ polymerase chain reaction (PCR), and to detect the location and distribution of hepatitis C virus in 30 cases liver tissues embedded with paraffin. Methods BPs were labeled in tymidine (T) position under ultraviolet lamp. The method was compared with indirect RT-in situ PCR and in situ hybridization for detecting hepatitis C virus (HCV) RNA. Results Serum HCV PCR and southern blot showed that BP labeling psimer PCR was possible, and had a good specificity. The HCV positive rate was 53% (16/30) by indirect in situ PCR, 50% (15/30) positive specimens were found by BP labeling primer in situ PCR. Statistical analysis revealed P ≥ 0.05 and the two methods had no dominant differences. Meanwhile, only 23% (7/30) positive signals were seen by in situ hybridization, which was lower than two in situ PCR( P≤0.05). HCV was mainly located in hepatic-plasmas, and positive signals were found in monocytes and cholangiolar epithelia. Conclusions Both indirect in situ PCR and BP labeling in situ PCR have good sensitivity and specificity for detecting HCV RNA of liver tissues. HCV RNA is located in hepatocytes, monocytes and cholangiolar epithelia. Chin Med J 2000; 113(2): 103-106

Objective To investigate all-trans retinoic acid (ATRA) and As2O3 which were found to be able to selectively induce differentiation and apoptosis in acute promyelocytic leukemia (APL) and recently became standard treatment for de novo or relapsed APL. The results of long-term follow up in 72 APL patients were presented and prognostic factors discussed. Methods Seventy-two newly-diagnosed patients with APL entering CR with ATRA were consolidated with chemotherapy alone (31 patients), ATRA + chemotherapy (30 patients) and ATRA alone (11 patients). Univariate analysis was done to identify the potential prognostic factors. A total of 40 cases of patients relapsed after their first complete remission, including 3 groups of patients: group A, patients treated with ATRA and chemotherapy after relapse (8 patients); group B patients treated with As203 alone for 2nd CR and consolidation (21 patients); group C patients treated with As2O3 for 2nd CR and both As2O3 and chemotherapy for consolidation (11 patients). Univariate analysis was also done to identify the potential prognostic factors. Results With a median follow-up of 45 months (5-75 months), the median event-free survival was 21 months and median overall survival was not achieved. The estimated 3- and 5-year event-free survival (EFS) and over-all survival (OS) were 32.5 ± 10.5％, 18.4 ± 7.5% and 73.8 ± 17.5%, 58.5 ± 15.2%. In denovo patients, the combination of ATRA and chemotherapy in both induction and post-remission treatment was found to be statistically significant for EFS (P = 0.023), and initial peripheral leukocyte count was significantly related to OS. In relapsed patients, only the treatment of AS2O3 with or without chemotherapy in consolidation after relapse was statistically significant for CR and both EFS (P = 0.0061) and OS (P = 0.0013). Conclusion ATRA is an effective induction therapy and can be considered as first choice of treatment in denovo APL. Addition of chemotherapy in both induction and post-remission therapy can delay or decrease the possibility of relapse compared to ATRA alone. AS2O3 is an effective agent for relapsed APL and remains an important prognostic factor for relapsed APL. Chin Med J 2000; 113(2): 107-110

Objective To explore the relationship between type 2 diabetes mellitus and the mutation(s) in mitochondrial DNA. Methods According to the previous literature, the fragment of mitochondrial DNA from nucleotide 3153 to 3551, which had shown high frequency of point mutation, was scanned with the technique of polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) in Chinese normal control, type 2 diabetic population, and 12 families suffered from maternally inherited type 2 diabetes mellitus. Direct sequencing was applied to detect the fragments with abnormal conformation. Results No special band was found in SSCP electrophoreses in Chinese normal control, and only one subject (No. 81) of diabetic population indicated the abnormality in SSCP study, which was affirmed to be a silent point mutation of T to C at nucleotide 3336 inducing no change in amino acid (ATT→ATC, Ile). Pedigree 25 001 was the only family that exhibited strongly different SSCP characteristic from the other 11 ones, which was confirmed to be caused by a single point mutation mt3285T→C/T in the coding region of tRNALeu(UUR) gene by the technique of direct sequencing. Conclusions The variation within mt DNA 3153 -551 is not the major cause of type 2 diabetes in Chinese population suffered from this disease in this study. The point mutation T→C/T at the mitochondrial nucleotide 3285, which was found in pedigree 25 001, is located in the highly conservative region of tRNALeu(UUR) gene. It is strongly suggested that this mutation cause the conversion in the 3-dimentional structure of tRNALeu(UUR), which might disturb the normal translation and lead to the impairment in mitochondrial oxidative phosphorylation characterized by the defects of the polypeptides involved in the respiratory chain. Thus, insulin secretion deficiency and insulin resistance might occur. Chin Med J 2000; 113(2): 111-116

Objective To determine the location of a putative tumor suppressor gene (TSG), and evaluate the frequency loss of heterozygosity (LOH) of the long arm of chromosome 9 (9q) in bladder cancer. Methods We analyzed 25 patients with bladder cancer for LOH of 9q using 25 high-density microsatellite markers. Results Twenty-three samples (92%) showed LOH at least at one locus on 9q. We identified that the commonly deleted region were at 9q12-q21, 9q22, and 9q34. The rate of LOH was 44.0%, 22.7%, 22.7%, 16.0%, 12.0% on DBH, D9S15, D9S1815, D9S1831, D9S176 locus, respectively, and was not significantly related with grades and stages of tumor. Conclusion These data suggest that alteration of a TSG at DBH of 9q may play an important role in the development of bladder cancer. Chin Med J 2000; 113(2): 117-119

Objective To investigate expressions of gp-91phox and IL-8 gene and the function of neutrophils in hemodialysis (HD) patients, and to understand the relationship between uremia and immune. Methods Gene expression was studied by means of reversal transcription polymerase chain reaction (RT/PCR) from the untreated and treated with lipopolysaccharide (LPS) neutrophils in controls and HD patients. Phagocytosis and bactericidal assays were measured as a decrease in the number of viable intracellular and extracellular bacteria by colony counts. Results Freshly isolated neutrophils express gp-91phox mRNA in controls while no gp-91phox mRNA was detected in HD patients. When challenged with LPS, gp-91 phox mRNA exhibited decreased expression. IL-8 mRNA in HD patients could be spontaneously expressed. Phagocytosis and intracellular killing of neutrophils, when exposed to Staphylococcus aureus, were decreased obviously compared with controls. Conclusions The impaired gp-91phox gene expression of neutrophils indicates the impaired NADPH-oxidase system and the untreated neutrophils spontaneously express IL-8 mRNA in the HD patients, which is consistent with suppressed phagocytosis and intracellular killing capacity. Chin Med J 2000; 113(2): 120-123

Objective To study the effect of 1, 25-dihydroxyvitamin D3(1, 25(OH)2D3) on the growth and apoptosis of breast cancer cell line MCF-7. Methods Cell number was determined using the MTT method. Flow cytometric analysis was performed on cell cycles, and the percentage of apoptosis was counted. Apoptotic cells were quantified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and bc1-2 protein expression was estimated with Western blotting. Results After incubation with 1, 25(OH)2D3 10-7 mol/L for 48 hours, MCF-7 cells exhibited significant growth in a dose- and time-dependent manner. Flow cytometric analysis indicated that cell numbers in G0/G1 increased along with increasing apoptotic peak and percentage. With microscope and electron microscope observation, characteristics of apoptosis such as typical apoptotic bodies were commonly found. TUNEL also showed that 1, 25(OH)2D3 10-8mol/L and 10-7mol/L groups had significantly high apoptosis percentage than control group with dose-dependence on induction apoptosis. And Western blot showed that 1, 25(OH)2D3 10-8mol/L could down-regulate bcl-2 protein and 10-7mol/L could almost block bcl-2 protein expression. Conclusions 1, 25(OH)2D3 can inhibit cell growth with G0/G1 arrest, enhance the proliferation inhibition action of adriamycin, and induce apoptosis which may result from the down-regulation of the anti-apoptotic bcl-2 protein. Chin Med J 2000; 113(2): 124-128

Objective To elucidate the diagnosis and surgical treatment of the posterior fossa solid hemangioblastomas (PFSHs). Methods The data from 22 patients with PFSHs verified by surgery and pathology were analyzed retrospectively and a review of relevant literature was conducted. Results The preoperative definitive diagnosis rate was 13/22. Total tumor removal was achieved in 20 patients. Two of the 22 patients died after surgery, one of brain-stem injury and the other from an intracranial hemorrhage. Twenty patients were followed up for 2 months to 8 yeas (average, 2 years). Karnofsky scale was ≥ 80 in 15 patients, 60 - 70 in 1 patient and not measured in 4 patients who died during follow-up. The causes of death were pneumonia (2 patients) and purulent meningitis (2). Conclusion MRI and DSA (digital subtraction angiography) are major preoperative diagnostic modalities for PFSH, but PFSH remains a challenging benign neoplasm. Special microsurgical techniques and improved operative manipulation can improve surgical efficacy. Chin Med J 2000; 113 (2): 129-132

Objective To determine the pattern of malalignment and the extent of chondral damage, the patellofemoral joint was evaluated by CT scanning and the findings were proved by arthroscopic examination. Methods In 20 cases, 40 knees were examined by CT scanning with and without quadriceps contraction in order to differentiate between static and dynamic abnormalities. The CT images were analyzed. The arthroscopic examination was carried out for 27 knees in 20 cases. Results Ten knees had static patellar lateral subluxation and 16 knees dynamic lateral subluxation. Eleven knees had static patellar lateral tilt, and 7 knees dynamic tilt. All of the 27 knees showed arthroscopic patellar maltracking and chondral damage. Conclusions The patellofemoral alignment, affection of quadriceps, and the extent of tight retinaculum may be precisely evaluated by CT measurement and proved by arthroscopic examination. Chin Med J 2000 ; 113(2): 133-135

Objective To investigate the mechanism of Chinese medicine ligustrazini against thrombosis, and the effects of ligustrazini on plasminogen activator inhibitor (PAI-1) expression in normal endothelial cells and lipopolysaccharide (LPS) stimulated endothelial cells. Methods Human umbilical vein endothelial cells (HUVECs) were cultured by trypsin digestion method. PAI-1 protein in HUVEC conditioned medium was measured by Sandwich enzyme-linked immunosorbent assay (ELISA), and PAI-1 mRNA expression was determined by Northern blot analysis. Using electrophoretic mobility shift assay (EMSA), we observed HUVEC nuclear factor-kappa B (NF-kB) nuclear translocation. Results LPS treatment of cultured HUVECs resulted in a significant increase in PAI-1 protein and mRNA expression by these cells. However, when HUVECs were incubated with LPS plus ligustrazini, the upregulation of PAI-1 by LPS was abated. Moreover, ligustrazini could decrease the basal level of PAI-1 protein and mRNA in HUVECs as compared with control. Nuclear extracts prepared from HUVECs stimulated by LPS demonstrated that binding to the NF-kB oligo nucleotide increased as compared with the unstimulated cells, but ligustrazini did not change those binding in the absence or presence of LPS. Conclusions Ligustrazini inhibited both basal and LPS-induced PAI-1 protein and mRNA expression in endothelial cells, and the modulation of PAI-1 in HUVECs by ligustrazini might have other mechanisms rather than NF-kB pathway. Chin Med J 2000; 113(2): 136-139

Methods Two effective ribozymes (HCRZNL and HCRZC), targeted to HCV 5' NC region and C region, were designed and synthesized. Eukaryotic expression vectors, pSV2-gpt, CD-SR α, containing either HCRZNC or HCRZC were constructed and transfected into MT-2 cells, Objective To determine the nucleotide sites of hepatitis C virus (HCV), which can be cleaved by ribozymes? and to obtain a highly effective, nontoxic and inexpensive antisense ribozyme specific for HCV. which had been infected by HCV. Quantitative polymerase chain reaction (PCR) and hybridization were used to determine the effect of inhibition of HCV by ribozymes. Results HCRZNC and HCRZC suppressed the replication of HCV by 54.7% and 62.1%, respectively. Furthermore, when both ribozymes were cotransfected into cells, they suppressed replication by 78.8%. Conclusion The specific antisense ribozymes, have strong inhibitory effects on the replication of HCV in cultured cells, and have better effect when used together.