2005年 · 第118卷第06期

Severe acute respiratory syndrome (SARS) was first identified in November 2002 in China and spread internationally causing more than 910 deaths before being contained in 2003. A novel coronavirus, named SARS coronavirus (SARS CoV) has been identified as the etiologic agent of SARS.1

Abstract：Background Homoharringtonine (HHT) is a cephalotaxine ester derived from an evergreen tree found wildely throughout southern China, which has antileukemic activities against a variety of acute myeloid leukemic cells. For the sake of illustrating the mechanisms of HHT in the treatment of leukemia, we assessed the effect of HHT on the apoptosis of human chronic myeloid leukemic cell line K562.Methods The apoptosis of K562 cells induced by HHT was analyzed by transmission electron microscopy, agarose gel electrophoresis of DNA, flow cytometry and terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick labeling.Results Characteristic apoptosis-related features emerged in K562 cells after exposed to HHT at a concentration 0.05-100 μg/ml. Transmission electron microscopy of HHT treated K562 cells displayed chromatin condensation and aggregation under the nuclear membrane, nuclear fragmentation and apoptosis body formation. Typical DNA ladder in agarose gel electrophoresis was observed in the cells exposed to HHT. The cell cycle analysis measured by flow cytometry showed G1 phase cells decreased with the increase of S phase cells while apoptosis was induced by HHT in K562 cells. The percentage of apoptotic cells in K562 cells treated with 50 μg/ml of HHT decreased significantly when pretreated with 1 μg/ml of cycloheximide, 0.05 μg/ml of Actinomycin D respectively.Conclusions HHT has apoptotic effects on K562 cells. The HHT induced apoptosis mainly of the cells in G1 phase and this process required RNA transcription and protein synthesis。

Abstract：Background Nonmyeloablative allogeneic bone marrow transplantation has been used since the 1990s as a new hematological stem cell transplantation strategy for treating hematological diseases. The purpose of this study was to explore the graft-versus-leukemia (GVL) effects of donor lymphocyte infusions (DLIs) after nonmyeloablative allogeneic bone marrow transplantations, while assessing the declines in treatment-associated morbidity, mortality, and graft-versus-host disease (GVHD).Methods A total of 615 (H-2k) mice were injected with L615 tumor cells and received 500 cGy (60Coγ-ray) irradiation three days later, followed by an allogeneic bone marrow transplantation (allo-BMT). The allo-grafts consisted of 3×107 bone marrow cells and 1×107 spleen cells from BALB/C (H-2d) donor mice. Two days after the allo-BMT, the recipient mice were given 200 mg/kg of cyclophosphamide. Subsequently, recipient mice were infused with either donor spleen cells (2×107) on day 14 or 21, or donor spleen cells (5×107) pretreated with hydrocortisone and cyclosporin A (CsA) in vitro on day 14 post-BMT.Results The median survival time of mice that received DLI on day 21 and pretreated DLI on day 14 post-BMT was longer than that of controls and the day 14 DLI group (P<0.01). No evidence of severe GVHD was observed in the day 21 DLI group nor in the day 14 treated DLI group. Mixed chimerism was confirmed in the day 14 DLI group, the day 14 treated DLI group, and the day 21 DLI group on the thirteenth day post-transplantation; full donor chimerism was observed two weeks after DLI.Conclusion Donor lymphocyte infusion after nonmyeloablative bone marrow transplantation may reduce transplantation-associated morbidity and mortality while strengthening graft-versus-leukemia effects。

Abstract：Background Most of the studies on traditional Chinese medicine (TCM)‘spleen’deficiency syndrome in the recent 30 years were conducted only on the basis of single functional index, neglecting the study on the pathophysiologic internal relationship between spleen deficiency syndrome and gastric diseases in modern medicine. But it was at the subcellular molecular biological level that we explored the pathophysiologic basis of classification of spleen deficiency in chronic gastritis by detecting the bioactive substances in gastric mucosa nuclei and mitochondria. Methods By means of optical microscope, scanning electron microscope (SEM), transmission electron microscopy (TEM) and histochemical staining, we conducted histopathological, subcellular ultrastructural analysis and nuclei and mitochondrial ultrastructural analysis of gastric mucosa of 188 spleen deficiency patients and of 42 voluntary blood donors. At the same time, bioactive substances were measured by means of X-ray energy dispersive analysis system (EDAX) image analysis system, radioimmunoassay method and chemiluminescence method. Results The content of cAMP, superoxide dismutase (SOD), Zn and Cu in gastric mucosa, and the content of Zn and Cu in mitochondria decreased progressively in order of groups: healthy control (HC), spleen Qi deficiency without organic lesion (F-SQD), spleen Yang deficiency without organic lesion (F-SyangD), disease without symptoms group, spleen Qi deficiency with organic lesion (G-SQD), spleen Yang deficiency with organic lesion (G-SyangD), spleen Yin deficiency (SyinD) and spleen deficiency with Qi stagnation (SDQS), chronic spleen deficiency gastritis (CSG) and chronic atrophic gastritis (CAG); decreased in order of HC, intestinal metaplasia (IM)Ⅰa, IMⅠb, IMⅡa and IMⅡb, P<0.05. The content of DNA, Zn and Cu in nuclei progressively increased in order mentioned above, P<0.05.Conclusions The quantitative changes of gastric mucosal cAMP, SOD, Zn, Cu, of mitochondrial Zn, Cu and of nuclear DNA, Zn and Cu are not only the substance base on which the lesion of gastric mucosa tissue structure occurs, but also the substance base on which spleen deficiency is classified. G-SQD and G-SyangD were more likely to be found in low-grade or middle-grade CSG and CAG, while SyinD and SDQS in middle-grade or high-grade CSG, CAG and IMⅡb。

Abstract：Background Almost half of the world's population suffer from the Helicobacter pylori (H. Pylori) infection, but only some individuals develop gastric diseases with clinical symptoms. One reason for the phenomenon may be the different pathogenicity of infected H. Pylori strains. The presence of cytotoxin-associated gene A (cagA) and expression of vacuolating cytotoxin activity encoded by vacuolating cytotoxin gene A (vacA) are considered the two major virulent markers of H. Pylori. The aim of this study was to detect dominant cagA/vacA genotypes and coinfection frequency of H. Pylori in patients with peptic ulceration (PU) or chronic gastritis (CG), and to determine correlations among different cagA/vacA genotypes, coinfection and severity of the diseases. Methods For each of 139 patients in Zhejiang Province who had been diagnosed as PU or CG based on clinical symptoms and gastroscopy, two gastric biopsy specimens (one from antrum and the other from corpus) for H. Pylori isolation were taken by two different disinfected biopsy forceps. One hundred and fifty-six H. Pylori strains were isolated from both the antrum and corpus biopsy specimens of 78 patients (36 PU and 42 CG). PCRs were performed to detect cagA genes, and signal (s) and middle (m) regions of vacA genes in the H. Pylori isolates. The amplified fragments of dominant vacA gene s and m subtypes from representative H. Pylori isolates were sequenced after TA cloning. Dominant cagA/vacA genotypes of the H. Pylori isolates, coinfection frequency and correlations among the different genotypes, coinfection and severity of the diseases were determined.Results Of the H. Pylori strains isolated from the antrum specimens, 96.2% were cagA gene positive, as were 97.4% of the H. Pylori strains isolated from the corpus specimens. Only one s region subtype (s1a) and four m region subtypes m1, m2, m1b and m1b-m2 of vacA gene were found. The proportions of vacA gene subtypes s1a/m1, s1a/m2, s1a/m1b and s1a/m1b-m2 in the 83 strains isolated from the antrum specimens were 7.2%, 61.5%, 30.1% and 1.2%, respectively, while those in the other 84 strains isolated from the corpus specimens were 9.5%, 58.3%, 28.6% and 3.6%, respectively. S1a/m2 (58.3% vs 30.1%, χ2=13.47, P<0.01) and then s1a/m1b (28.6% vs 9.5 %, χ2=9.88, P<0.01) were the dominant vacA gene subtypes in the H. Pylori isolates. The dominant H. Pylori genotype was cagA+s1a/m2 (59.0% from antrum specimens and 57.1% from corpus specimens), and followed by cagA+s1a/m1b (28.9% from antrum specimens and 27.4% from corpus specimens). Sixteen of 78 patients (20.5%) were infected with two or three H. Pylori strains with different genotypes. However, no statistically significant differences among cagA occurrence, the different vacA subtypes and PU or CG could be found (each P>0.05). Similarities of the nucleotide sequences from vacA gene s region PCR products of six isolates and from vacA gene m region PCR products of four isolates were 93.2% to 98.3% and 93.8% to 97.6%, respectively, compared to the reported corresponding sequences.Conclusions The dominant genotypes of H. Pylori in PU or CG patients in Zhejiang area may be cagA+ s1a/m2 and cagA+ s1a/m1b. Numerous coinfections with different H. Pylori strains in PU or CG patients indicate diversity of the infected H. Pylori origins. S and m regions of vacA gene from different H. Pylori isolates show high nucleotide sequence similarities. cagA gene positive rate, different vacA gene subtypes and coinfection with different H. Pylori strains are not closely associated with severity of the diseases。

Abstract：Background Several coronaviruses establish persistent infections in vitro and in vivo, however it is unknown whether persistence is a feature of the severe acute respiratory syndorme coronavirus (SARS-CoV) life cycle. This study was conducted to investigate viral persistence.Methods We inoculated confluent monolayers of Vero cells with SARS-CoV at a multiplicity of infection of 0.1 TCID50 and passaged the remaining cells every 4 to 8 days for a total of 11 passages. Virus was titrated at each passage by limited dilution assay and nucleocapsid antigen was detected by Western blot and immunofluoresence assays. The presence of viral particles in passage 11 cells was assessed by electron microscopy. Changes in viral genomic sequences during persistent infection were examined by DNA sequencing. Results Cytopathic effect was extensive after initial inoculation but diminished with serial passages. Infectious virus was detected after each passage and viral growth curves were identical for parental virus stock and virus obtained from passage 11 cells. Nucleocapsid antigen was detected in the majority of cells after initial inoculation but in only 10%-40% of cells at passages 2-11. Electron microscopy confirmed the presence of viral particles in passage 11 cells. Sequence analysis at passage 11 revealed fixed mutations in the spike (S) gene and ORFs 7a-8b but not in the nucleocapsid (N) gene. Conclusions SARS-CoV can establish a persistent infection in vitro. The mechanism for viral persistence is consistent with the formation of a carrier culture whereby a limited number of cells are infected with each round of virus replication and release. Persistence is associated with selected mutations in the SARS-CoV genome. This model may provide insight into SARS-related lung pathology and mechanisms by which humans and animals can serve as reservoirs for infection。

Abstract：Background Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emerging virus that gives rise to SARS patients with high rates of infectivity and fatality. To study the humoral immune responses to SARS-CoV, the authors evaluated IgG and IgM specific antibodies in patients' sera.Methods Two methods, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent assay (IFA), were used to detect specific serum IgG and IgM against SARS-CoV in 98 SARS patients and 250 controls consisting of patients with pneumonia, health-care professionals and healthy subjects. The serum antibody profiles were investigated at different times over one and a half years in 18 of the SARS patients. Results The sensitivity and specificity of ELISA for detecting IgG against SARS-CoV were 100.0% and 97.2% and for IgM 89.8% and 97.6% respectively; the figures using IFA for IgG were 100.0% and 100.0% and for IgM 81.8% and 100.0% respectively. During the first seven days of the antibodies trace test, no IgG and IgM were detected, but on day 15, IgG response increased dramatically, reaching a peak on day 60, remaining high up to day 180 and decreasing gradually until day 540. On day 15, IgM was detected, rapidly reached a peak, then declined gradually until day 180 when IgM was undetectable. Conclusion The detection of antibodies against SARS virus is helpful in the clinical diagnosis of SARS.

Abstract：Background The B7/CD28 pathway provides critical costimulatory signals for complete T cell activation, and members of this pathway have served as useful targets for immunotherapeutic strategies. In this study, we investigated the RNA interference (RNAi) effect induced by small interfering RNA (siRNA) targeting CD28 mRNA on human lymphocytes and its specificity.Methods According to CD28 gene sequence, we designed and synthysized three different siRNAs (siRNA-1, siRNA-2, siRNA-3) containing 21 bases using SilencerTM siRNA construction kit. These siRNAs were transfected into freshly isolated human lymphocytes with Lipofectamine 2000 reagent. At 24-hour, 48-hour and 72-hour post transfection, these cells were collected and analyzed. The changes of surface expression of CD28 gene were detected by flow cytometry, and the changes of CD28 mRNA levels were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The cell viability of transfected lymphocytes was determined by methyl thiazolyl tetrazolium (MTT) assay and trypan blue dye exclusion assay. Results Three siRNAs (siRNA-1, siRNA-2, siRNA-3) specifically targeting CD28 mRNA were successfully designed and constructed. Flow cytometry analysis showed that a decrease in CD28 expression was detectable at 24-hour post transfection. Different siRNA showed different inhibition effects on CD28 expression. At 48-hour post transfection, the degrees of reduction with siRNA-1, siRNA-2 and siRNA-3 were 22.10%±1.63%, 73.50%±1.02% and 42.90%±0.89% respectively compared with the control (P<0.001).Neither of the groups transfected only with siRNA or lipo showed marked reduction in CD28 expression (3.15%±0.75% and 4.55%±0.80%) (P>0.05). Moreover, lymphocytes treated with siRNA-co showed no marked reduction in CD28 expression (5.07%±0.96%) (P>0.05). The results of semi-quantitative RT-PCR assay indicated CD28 mRNA level was inhibited after transfection of specific siRNAs. At least 4-fold of reduction in siRNA-2 group occurred at 48-hour post transfection compared with the control (P<0.001). MTT assay and trypan blue dye exclusion assay demonstrated that the viable cell rations of transfected lymphocytes were significantly reduced in siRNA-1, siRNA-2 and siRNA-3 groups at 48-hour post transfection (P<0.01). The control groups showed no marked reduction in cell viability (P>0.05). Conclusions Three different siRNAs were synthesized and transfected into lymphocytes. They could reduce the expression of CD28 and the CD28 mRNA level. siRNA-2 was the most efficient. The cell viability reduced correspondingly. Therefore, the silencing effect on CD28 mRNA induced by siRNA may contribute to costimulatory blockade. This result show that siRNA may be useful for further study on graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT)。

It is believed that the degeneration of cholinergic cells in the nucleus basalis of Meynert (NBM) and the loss of cortical cholinergic innervation cause dementia of Alzheimer's disease (AD).1 Currently available therapeutic interventions are mainly aimed at alleviating the cholinergic deficits. Unfortunately, these strategies do not prevent the disease, but instead offer limited symptomatic improvement.2 A recent study demonstrated that transplantation of in vitro expanded neural stem cells (NSCs) in an animal model of Parkinson's disease (PD) resulted in functional recovery of the animals to some extent,2 suggesting that such neural precursors might offer a useful future therapy for AD. In this study, we tried to find whether mouse embryonic stem (ES) cell derived cholinergic NSCs grafted in the prefrontal and parietal cortex have effects on the disruption of spatial memory following development of lesion in NBM。

The main active components of Shenfu injection (SFI), an extract of traditional Chinese herbs, are ginsenoside and higenamine. Ginsenoside can improve ischemic myocardium metabolism, scavenge free radicals, protect myocardial ultrastructure and reduce Ca2+ overload. Higenamine can enhance heart contractility, improve coronary circulation and decrease the effect of acute myocardial ischemia. SFI was found to have had some cardiac protective effect during cardiopulmonary bypass.1,2 SFI was added into St. Thomas crystal cardioplegic solution in this study to investigate the protective effect of SFI on ischemic-reperfused rabbit heart and to observe the influence on mitochondrial oxygen free radical (OFR), Ca2+ and mitochondrial ultrastructure.

Worldwide estimates establish gastric carcinoma as the second most frequent cause of cancer deaths. Tumour invasion and metastasis is the biggest impediment to gastric carcinoma cure. Active migration of tumour cells is now considered as the pivotal step in cancer invasion and metastasis. RhoC is a member of the Ras-superfamily of small guanosine triphosphatases that can regulate many cellular functions, especially cytoskeletal organization and cell locomotion. Overexpressing RhoC in vitro in the poorly metastatic cell line from human melanoma may induce a highly metastatic phenotype.1 The recent development of laser capture microdissection (LCM) affords the opportunity to further evaluate the role RhoC plays in the invasion and metastasis of gastric carcinoma cells in their native tissue environment.

CT observations and the cellular factors of molecular biology each enable one to recognize macro/micro changes in lung cancer. Growing pattern, characteristic shapes, degree of malignancy, relapse and metastasis of lung cancer are mainly determined by their molecular biology. Change of tumour shape, determined by the tumour's biological behaviour, is the basis of CT observations. That is, the pathological change acts as a bridge which links the CT observation to molecular biology and makes the investigation of internal relationship between CT sign and molecular biology behaviour possible. As a tumour suppressor gene, nm23 gene is located in chromosome 17q21.3, encoding a nucleoside diphosphate kinase.1,2 We studied the expression of nm23 in peripheral nonsmall cell lung cancer (NSCLC) using the streptavidin peroxidase (SP) immunohistochemical method and investigated retrospectively the relationship between nm23 gene, CT observation, biological behaviour and prognosis of NSCLC。

Severe acute respiratory syndrome (SARS) is the first severe viral epidemic we encountered this century, which once spread in more than thirty countries in 2003.1 The etiological agent of SARS has been confirmed to be a novel coronavirus, namely SARS coronavirus (SARS-CoV),2,3 and the first outbreak of SARS has been successfully controlled worldwide, but the identification of SARS-CoV isolated from wild animals, the emergence of some sporadic SARS cases later after that outbreak, all suggest that the recurrence of such an epidemic is not unlikely in the future. In this case, development of SARS vaccines and specific drugs is undoubtedly essential to the control and prevention from the possible outbreak.4,5

Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease caused by a novel coronavirus, firstly broke out in November 2002 in Guangdong and prevailed quickly in Beijing, Hong Kong, Taiwan and other regions of China. It was one of the most potential pandemic diseases and had affected more than 20 other countries.1,2 There have been a lot of resear-ches2-7 in terms of its etiology, epidemiology, pathogenesis, clinical characteristics, diagnostics, treatment and prevention, vaccines and so on。

The femoral neck in children is much stronger than that in adults and can only be fractured by a severe force. It is therefore rare and often associated with severe injury once it takes place. Besides, as the blood supply to the femoral head is precarious, the fracture can lead to a high incidence of post-traumatic avascular necrosis of the femoral head.1,2 So, much attention should be paid to the treatment of femoral fractures in children. The authors have successfully cured an old femoral neck fracture of a girl by open reduction and internal fixation with 3 Kirschner wires supplemented by an external fixator across the hip joint and cancellous allograft at the fracture site. The patient has gotten a satisfactory functional recovery of the hip。

Tentorial dural arteriovenous fistula (DAVF) is a rare disease, accounting for some 4% of all cases of intracranial DAVF.1 Because of a high risk of intracranial hemorrhage, patients with DAVF need aggressive treatment. Despite recent advances in endovascular technology, many researchers2-4 advocate open surgery for the treatment of tentorial DAVF. In this report, we present a case of tentorial DAVF with pure leptomeningeal venous drainage successfully treated by interruption of the draining vein。

Von Hippel-Lindau (VHL) disease is an autosomal-dominant hereditary familial neoplasm syndrome characterized by development of a variety of benign and malignant tumors in multiple organ systems, such as the brain, kidney, pancreas, adrenal gland, and epididymis, with a prevalence of one in 39000-53000.1-4 Hallmarks of the condition include retinal angiomas, hemangioblastomas of the cerebellum and the spinal cord, renal cell carcinoma and cysts, and pheochromocytomas. In this article, we report imaging findings in three cases of VHL disease。

The WHO has classified malignant mixed tumours of salivary glands into noninvasive carcinoma in pleomorphic adenoma, invasive carcinoma in pleomorphic adenoma, carcino-sarcoma and metastasizing mixed tumour.1 Carcinosarcoma, or true malignant mixed tumour, is a tumour composed of both carcinomatous and sarcomatous elements. It is an exceedingly rare tumour of the salivary glands and only about 60 cases have been reported.2 In this report we describe a case of carcinosarcoma of a parotid gland that contained an unusual mesenchymal component (follicular dendritic cell sarcoma, FDCS) in a 55-year-old man with cytological, histological and immunohistochemical findings. To our knowledge, this histological pattern has not been reported previously in the English literature。