2006年 · 第119卷第22期

Abstract：Background Since October 1997, an international standard for hepatitis C virus (HCV) nucleic acid amplification technology assay, 96/790, has been available. We compared a series of lyophilized standards with known HCV RNA concentrations against the international standard in fluorescence quantitative PCR detection.Methods A series of lyophilized sera were calibrated by ROCHE COBAS AMPLICOR HCV Monitor test against the international standard and sent to various manufacturers to analyse the samples using their own kits.Then calibration curves from the series were compared with that obtained from the external standard calibration curve with the manufacture's series.Results The standard calibration curve with the series of lyophilized serum showed an excellent correlation(R2＞0.98), slope and intercept that were similar to those from the manufacture's series. When the standard calibration curve from the series of lyophilized standards were used to define the values of the given sample,lower coefficients of variation between kits from different manufactures were obtained.Conclusion The results showed that the lyophilized standards could be used to setup the standard calibration curve for clinical HCV RNA quantitative PCR detection。

Abstract：Background Salivary nitrate is positively correlated with plasma nitrate and its level is 9 times the plasma level after nitrate loading. Nitrate in saliva is known to be reduced to nitrite by oral bacteria. Nitrate and nitrite levels in saliva are 3-5 times those in serum in physiological conditions respectively in our previous study. The biological functions of high salivary nitrate and nitrite are still not well understood. The aim of this in vitro study was to investigate the antimicrobial effects of nitrate and nitrite on main oral pathogens under acidic conditions.Methods Six common oral pathogens including Streptococcus mutans NCTC 10449, Lactobacillus acidophilus ATCC 4646, Porphyromonas gingivalis ATCC 33277, Capnocytophaga gingivalis ATCC 33624, Fusobacterium nucleatum ATCC 10953, and Candida albicans ATCC 10231 were cultured in liquid medium. Sodium nitrate or sodium nitrite was added to the medium to final concentrations of 0, 0.5, 1, 2, and 10 mmol/L. All of the microorganisms were incubated for 24 to 48 hours. The optical densities (OD) of cell suspensions were determined and the cultures were transferred to solid nutrient broth medium to observe the minimum inhibitory concentration and minimum bactericidal/fungicidal concentration for the six tested pathogens.Results Nitrite at concentrations of 0.5 to 10 mmol/L had an inhibitory effect on all tested organisms at low pH values. The antimicrobial effect of nitrite increased with the acidity of the medium. Streptococcus mutans NCTC 10449 was highly sensitive to nitrite at low pH values. Lactobacillus acidophilus ATCC 4646 and Candida albicans ATCC 10231 were relatively resistant to acidified nitrite. Nitrate at the given concentrations and under acidic conditions had no inhibitory effect on the growth of any of the tested pathogens.Conclusion Nitrite, at a concentration equal to that in human saliva, is both cytocidal and cytostatic to six principal oral pathogens in vitro, whereas nitrate at a similar concentration has no antimicrobial effect on these organisms。

Abstract：Background Type 1 deiodinase (D1) plays an important role in the metabolism of thyroid hormone and has close relationship with thyroid function. In this study we explore the effects of iodine intake on D1 activity and its mRNA expression and its possible mechanism.Methods Forty-eight Wistar rats were randomly divided into six groups with 8 in each: low iodine (LI), normal iodine (NI), five-fold iodine (HI5), ten-fold iodine (HI10), fifty-fold iodine (HI50), one hundred-fold iodine (HI100)group. Three months, six months and twelve months after admistration of potassium iodate, they were sacrificed and thyroids were excised. The expression of D1 mRNA in the thyroid tissue was determined by RT-PCR and D1 activity was analyzed by 125I-rT3 as substrate. The thyroid hormone was measured with radioimmunoassay method.Results Compared with NI group, D1 mRNA expression in LI groups slightly decreased, and D1 activity greatly increased. Both T3 and T4 in thyroid tissue significantly decreased, but the T3/T4 ratio increased. D1 mRNA expression decreased in all HI groups, and D1 activity was significantly lower in HI groups. There was a tendency of decrease in D 1 activity with increased doses of iodine intakes. There was no significant difference in T4 in thyroid tissue between HI groups and NI group, but a tendency of decrease in T3 level was found in all HI groups.Conclusions In the case of iodine deficiency, D1 activity increased greatly in order to convert more T4 to T3.Excess iodine can inhibit both D1 mRNA expression and its activity to protect organism from being injured by excessive T3。

Abstract：Background The mechanism of mucosal damage induced by ischemia-reperfusion (IR) after hemorrhagic shock is complex; mast cells (MC) degranulation is associated with the mucosal damage. Astragalus membranaceus can protect intestinal mucosa against intestinal oxidative damage after hemorrhagic shock, and some antioxidant agents could prevent MC against degranulation. This study aimed to observe the effects of astragalus membranaceus injection on the activity of intestinal mucosal mast cells (IMMC) after hemorrhage shock-reperfusion in ratsMethods Thirty-two Wistar rats were randomly divided into the normal group, model group, low dosage group,(treated with Astragalus membranacaus injection, 10 g crude medication/kg) and high dosage group (treated with Astragalus membranacaus injection, 20 g crude medication/kg). The rat model of hemorrhagic shock-reperfusion was induced by hemorrhage for 60 minutes followed by 90 minutes of reperfusion. The animals were administrated with 3 ml of the test drug solution before reperfusion. At the end of study, intestinal pathology,ultrastructure of IMMC, and expression of tryptase were assayed. The levels of malondisldehyde (MDA), TNF-α,histamine, and superoxide dismutase (SOD) activity in intestine were detected, and the number of IMMC was counted.Results The Chiu's score of the rats in the model group was higher than in other groups (P＜0.01). The Chiu's score in the high dosage group was higher than that in the low dosage group (P＜0.05). Hemorrhage-reperfusion induced IMMC degranulation: Astragalus membranaceus injection attenuated this degranulation. Expression of tryptase and the number of IMMC in the model group increased compared with the other groups (P＜0.01) and was significantly reduced by the treatments of Astragalus membranaceus injection at both doses. There was no significant difference between the two treatment groups (P＞0.05). MDA content and concentration of TNF-α in the model group were higher than that in the other three groups (P＜0.05), and the concentration of TNF-α in the low dosage group was higher than that in the high dosage group (P＜0.05). SOD activity and the concentration of histamine in the model group were lower than the other three groups (P＜0.05). There was a negative correlation between the Chiu's score and the concentration of histamine and a positive correlation between the Chiu's score and the concentration of TNF-α and between the SOD activity and the concentration of histamine in the four groups (P＜0.05).Conclusion Astragalus membranaceus injection may reduce the damage to small intestine mucosa by inhibiting the activated IMMC after hemorrhagic shock。

Abstract：Background People with metabolic syndrome are at higher risk for developing coronary artery disease (CAD).The effect of the metabolic syndrome on outcomes in patients with preexisting CAD has not been well studied.This study was conducted to assess the prevalence, characteristics, in hospital and long term prognosis of CAD with metabolic syndrome and to determine the factors influencing the prognosis of the disease.Methods The DESIRE registry contains data of 3696 patients with CAD between 2001 and 2004. Mean long term followup was (829±373) days. Diagnosis of metabolic syndrome was based on modified International Diabetes Federation (IDF) Worldwide Definition of the Metabolic Syndrome, using body mass index (BMI)instead of waist circumference.Results Of 2596 patients with complete records of height, weight, and so on, 1280 (49.3%) were identified with metabolic syndrome. The patients with metabolic syndrome had higher level of body mass index, systolic blood pressure, diastolic blood pressure, fasting glucose and disordered blood lipid (all P＜0.0001), with higher creatinine [(10.5±4.3) mg/L vs (9.9±2.9) mg/L, P＜0.0001] and the number of white blood cells [(7.49±2.86)× 109/L vs (7.19 ± 2.62) × 109/L, P=0.008) compared with those without metabolic syndrome. The patients with metabolic syndrome showed severer coronary angiographic alterations (left main artery and/or ≥2-vessel)(73.6% vs 69.6%, P=0.031). There were no significant differences of major adverse cardiac and cerebral events(MACCE) or mortality in hospital between the two groups. During followup, the ratio of MACCE in CAD with metabolic syndrome patients increased significantly (11.8% vs 10.0%, P=0.044). Fasting blood glucose (≥ 1000mg/L) and triglyceride (TG, ≥ 1500 mg/L) were responsible for most of the increased risk associated with the metabolic syndrome (adjusted OR 1.465, 95% CI 1.037-1.874, P=0.032; OR 1.378, 95% CI 1.014-1.768,P=0.044).Conclusions The prevalence of metabolic syndrome was very high in CAD patients. The metabolic syndrome confers a higher risk of long term MACCE in patients with CAD, and dysglycaemia and hypertriglycaemia appear to be responsible for most of the associated risk。

Abstract：Background Pulse wave velocity and flow-mediated vasodilation (FMD) are widely used as noninvasive modalities for evaluating atherosclerosis. However, it is not known whether pulse wave velocity is related to FMD in patients with coronary artery disease (CAD). Therefore, the present study was designed to investigate the alteration in brachial-ankle pulse wave velocity (baPWV) and endothelial function in CAD patients.Methods Thirty-three patients with CAD and thirty control subjects were recruited for this study. baPWV was measured non-invasively using a VP 1000 automated PWV/ABI analyzer (PWV/ABI, Colin Co. Ltd., Komaki,Japan). Endothelial function as reflected by FMD in the brachial artery was assessed with a high-resolution ultrasound device.Results baPWV was increased in CAD patients compared with control subjects [(1756.1±253.1) cm/s vs(1495.3 ± 202.3) cm/s, P＜0.01]. FMD was significantly reduced in CAD patients compared with control subjects[(5.2±2.1) % vs (11.1 ±4.4) %, P＜0.01]. baPWV correlated with FMD (r =-0.68, P＜0.001). The endothelium-independent vasodilation induced by sublingual nitroglycerin in the brachial artery was similar in the CAD group compared with the control group.Conclusions CAD is associated with increased baPWV and endothelial dysfunction. Increased baPWV parallels diminished endothelial function. Our data therefore suggest that baPWV can be used as a noninvasive surrogate index in clinical evaluation of endothelial function。

Abstract：Background The concentration of serum fructosamine is correlated with plasma glucose level. The aim of this study was to determine whether the level of serum fructosamine can be diagnostic for abnormal glucose tolerance in pregnant women.Methods Serum samples were collected from 161 pregnant women between November 2004 and April 2005.The women were divided into three groups according to the gestational age (16-20 weeks group, 56 patients; 28-34 weeks group, 72; and 37-41 weeks group, 33). Each group was subdivided into normal and abnormal glucose tolerance subgroups. The levels of serum fructosamine were measured. Differences among the groups were assessed by ANOVA and Student-Newman-Keuls test. Correlations between the level of fructosamine and other variables including the results of glucose challenge test (GCT), oral glucose tolerance test (OGTT), and glycosylated hemoglobin (HbA1c) test, and infant's birth weight were analyzed by Pearson correlation.Results The level of serum fructosamine decreased with gestational age [(223.25 ±48.90) μmol/L, (98.44±29.57)μmol/L, and (53.99±29.94) μmol/L, respectively. P＜0.05]. It was higher in women with abnormal glucose tolerance than that in women with normal glucose tolerance, however, the difference reached statistical significance only in the 28-34 weeks group (P＜0.05). In this group, the level of serum fructosamine correlated positively with the GCT result (r=0.28, P＜0.05). No correlation was found between fructosamine level and OGTT result, HbA1c level, or neonatal weight.Conclusions Fructosamine can be used to monitor the glucose level of pregnant women with abnormal glucose tolerance, and to identify the patients at high risk of abnormal glucose tolerance, but can not be used to predict gestational diabetes mellitus (GDM) in early stage of pregnancy。

Abstract：Background Early enteral feeding (EF) may result in fever, elevated white blood cell count, increased serum levels of liver enzymes, and diarrhea. We name the complications "enteral refeeding syndrome", as a subtype of refeeding syndrome, because they are likely to result from long-term lack of lumen nutrition. The aim of this study was to investigate the characteristics of enteral refeeding syndrome after long-term total parenteral nutrition(TPN), and the solution for the disease.Methods We collected the clinical data of 100 patients with gastrointestinal fistula, who were cured from Apirl2001 to July 2002. Their fasting time, daily stool frequency, body temperature, heart rate, respiratory rate, levels of transaminases, alkaline phosphatase (AKP), and γ-glutamylcyclotransferase (γ-GT), white blood cell count,and systemic inflammatory reaction syndrome (SIRS) score were recorded before and 1, 3, 5, 10, and 15 days after EF. Student's t test and analysis of variance were used to analyze the data.Results Of the 100 patients, 56 were cured after selective resection of intestinal fistula, 15 were cured by emergency operation, and 29 recovered spontaneously. The levels of AKP and γ-GT increased significantly on the 3rd day after EF [On the 3rd day after EF, (243.0± 121.6) U/L and (177.2± 109.9) U/L vs. before EF (181.5± 127.5) U/L and (118.4±94.2) U/L, P＜0.05], and decreased gradually afterwards. The SIRS scores on the 1st day (1.05±1.08) and 3rd day (0.96± 1.11) after EF were significantly higher than that before EF (0.72±0.84),then decreased to 0.83±0.91, 0.49±0.73 and 0.32±0.60 on the 5th, 10th and 15th days after EF. The number of patients with diarrhea at 1, 3, 5, 10 and 15 days post-EF were 31, 26, 12, 13, and 7, respectively.Conclusions The longer the TPN lasts, the more severe the enteral refeeding syndrome becomes. Continuous EF is effective for the syndrome. Early enteral nutrition is useful in preventing it。

Abstract：Background Multiple sclerosis (MS) is a continuously disabling disease and it is unresponsive to high dose steroid and immunomodulation with disease progression. The autologous haematopoietic stem cell transplantation (ASCT) has been introduced in the treatment of refractory forms of multiple sclerosis. In this study, the clinical outcomes followed by ASCT were evaluated for patients with progressive MS.Methods Twenty-two patients with secondary progressive MS were treated with ASCT. Peripheral blood stem cells were obtained by leukapheresis after mobilization with granulocyte colony stimulating factor. Etoposide,melphalan, carmustin and cytosine arabinoside were administered as conditioning regimen. Outcomes were evaluated by the expanded disability status scale and progression free survival. No maintenance treatment was administered during a median follow-up of 39 months (range, 6 to 59 months).Results No death occurred following the treatment. The overall confirmed progression free survival rate was77% up to 59 months after transplantation which was significantly higher compared with pre-transplantation (P=0.000). Thirteen patients (59%) had remarkable improvement in neurological manifestations, four (18%)stabilized their disability status and five (23%) showed clinical recurrence of active symptoms.Conclusions ASCT as a therapy is safe and available. It can improve or stabilize neurological manifestations in most patients with progressive MS following failure of conventional therapy。

Abstract：Background The hepatitis B virus (HBV) genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 (human gene 5 transactivated by pre-S1 protein of HBV) is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory (GenBank accession: AY427953). In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS 1TP5 protein.Methods The reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then "bait" plasmid pGBKT7-PS 1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization.After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH 109 with Y 187 containing a leukocyte cDNA library plasmid.The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis.Results Forty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes with known functions were obtained, including Homo sapien leukocyte adhesion protein p150, 95, interleukin 2receptor gamma chain, PALM2-AKAP2 protein (PALM2-AKAP2), eukaryotic translation initiation factor 4A,beta-2-microglobin, solute carrier family 9 (sodium/hydrogen exchanger), calreticulin, asialoglycoprotein receptor 1 (ASGR1), MHC class Ⅱ lymphocyte antigen, cytochrome c oxidase subunit 1, lymphocyte antigen 86(LY86) and lymphocyte cytosolic protein 1. One novel gene with unknown function was found and named as PS1TP5BP1. After being electronically spliced, it was deposited in GenBank (accession number: DQ471327).Conclusions Genes of proteins interacting with PS1TP5 were successfully screened from leukocyte cDNA library. These results suggested that PS1TP5 was closely correlated with immunoregulation,carbohydrate metabolism, signal transduction, the formation of hepatic fibrosis and initiation and development of tumors and also brought some new clues for further studying the biological functions of the pre-S 1 protein。

Abstract：Background In vitro fertilization (IVF) researches have suggested that cystathionine β synthase (CBS) is involved in oocyte development. However, little is known about the regional and cellular expression patterns of CBS in the ovary. The purpose of this study was to analyze the localization of CBS in mice ovaries and to investigate the expression profile during follicular development.Methods We used in situ hybridization and immunohistochemical analysis to determine CBS expression in the ovaries of female Balb/c mice. Then the follicles were collected from F1 (C57BL × Balb/c) mice and cultured in vitro. With the method of semi-quantitative RT-PCR, we also investigated the expression profile of CBS during follicular development.Results CBS was absent in the oocytes, although it was ubiquitously expressed in the ovary with the strongest expression in follicular cells at all stages. In late antral follicles, CBS expression was markedly higher in granulosa cells located close to the antrum and in cumulus cells around the oocyte. The semi-quantitative RT-PCR showed that CBS mRNA was detected in follicles at all stages in vitro. In cumulus-oocyte complexes superovulated, CBS expression also increased rapidly.Conclusions CBS was located mainly in the follicular cells in the ovaries. The level of CBS expression is high in follicles during folliculogenesis in mice. Differences in the CBS expression profile between oocyte and follicular cells suggest a role for CBS as a mediator in interactions between oocyte and granulosa cells。

Abstract：Objective To review the history and recent development of research on autosomal dominant polycystic kidney disease (ADPKD) in China.Data sources Both Chinese and English literatures were searched in MEDLINE/CD ROM (1979 - 2006) and the Chinese Biomedical Literature Disk (1979 - 2006).Study selection Published articles about ADPKD from mainland of China were selected. Data were mainly extracted from 58 articles which are listed in the reference section of this review.Results Some preliminary reports on cyst decompression surgeries and mutation analysis represent the contribution to the ADPKD research from China in the history. A serial of basic research and clinical studies on ADPKD in recent years also have been summarized. A technique platform for ADPKD research was firstly established. The genomics/proteomics/bioinformatics approach was introduced, which provide a lot of valuable information for understanding the pathogenesis. By denature high performance liquid chromatography (DHPLC)technique the entire PKD1 and PKD2 gene sequence screening system for Chinese Han population has been successfully established. Based on the characteristic data of Chinese patients, an integrated therapy protocol was put forward and won an advantage over the traditional therapy. Some novel experimental studies on therapy also were encouraging. Conclusions Remarkable progress of ADPKD research in China have been made recently. Still many works, including the government support, international collaboration and active participation of more Chinese nephrologists, should be enhanced to advance this process in the near future。

The HLA genes, located on the short arm of human chromosome 6, encode peptides involved in host immune response, are important in tissue transplantation and are associated with a variety of infectious, autoimmune, and inflammatory diseases. Moreover, the HLA loci display an unprecedented degree of diversity and the distribution of HLA alleles and haplotypes among different populations is considerably variable.1 The expression of particular HLA alleles may be associated with the susceptibility or resistance to some diseases.2 In this study, the genetic polymorphism of HLA-DQA1 and -DQB1 in Jiangsu Han population was analyzed by polymerase chain reaction-sequence-based typing (PCR-SBT)。

Ketogenic diet (KD) is a high fat, low protein, low carbohydrate diet. Its antiepileptic effect is certain but the underlying mechanism is unknown.1Mossy fiber sprouting in the inner molecular layer of the dentate gyrus causes the synaptic reorganization in the hippocampus, which is an important cause of temporal lobe epilepsy in animals and humans.1,2 It is also essential to the genesis and development of epilepsy. As the predominant excitatory neurotransmitter in the central nervous system, glutamate plays a role in synaptic reorganization and development of epilepsy. In recent years, the role of glutamate receptor 5 (GluR5) in the genesis of seizures has attracted more and more attention of researchers and this receptor has become a candidate target of new antiepileptic drugs.3,4 In this study, we investigated the possible antiepileptic mechanism of KD in terms of synaptic reorganization and GluR5 expression, attempting to provide a theoretical basis for the development of new antiepileptic drugs。

The incidence of the inferior vena cava (IVC)tumor thrombus is reported to be 4%-10% in patients with renal cell carcinoma (RCC). Tumor thrombus may extend through to the right atrium.Management of patients with level Ⅲ/Ⅳ tumor thrombus is usually difficult. We report two cases of level Ⅳ thrombus in our hospital in 2002 and 2004。