2007年 · 第120卷第15期

Abstract：Background Enhanced apoptosis of cytotrophoblasts in early pregnancy is associated with high risk of intrauterine growth retardation and preeclampsia, which are two common pregnant complications. Its etiological factors remain unclear. Cytotrophoblasts share some traits with innate immune cells and may show response to lipopolysaccharide. This study was conducted to demonstrate whether lipopolysaccharide has apoptosis-inducing effects on cytotrophoblast and the role of innate immune reaction in this process.Methods Cytotrophoblasts were isolated from early pregnant villous tissues and cultured with serum-free medium.Subsequently, cytotrophoblasts were treated with lipopolysaccharide at the concentrations of 0 (control), 25, 50, 100 and 200 ng/ml for 24 hours. Apoptosis of cytotrophoblasts was determined by light microscopy, Hoechst 33258 DNA staining with a fluorescent microscope, transmission electron microscope and annexin V-fluorescein isothiocyanate-conjugated /propidium iodide (PI) staining with flow cytometry. Then expression of caspase-3 was detected by Western blot. Confocal immunofluorescence technique was used to detect tumor necrosis factor α expression in cytotrophoblasts. The levels of tumor necrosis factor α in the culture medium were detected by enzyme-linked immunosorbent assay.Results Under light, fluorescence microscope and transmission electron microscope, characteristic alternations of apoptosis in cytotrophoblasts were observed after lipopolysaccharide treatment. Flow cytometry results showed that lipopolysaccharide significantly increased apoptosis indexes of cytotrophoblasts. Significant statistical differences were found in the above groups (P≤0.01). The mean relative densities of bands corresponding to caspase-3 were significantly increased in groups treated with lipopolysaccharide, as compared with the normal control (P＜0.001). Tumor necrosis factor α expression was found to increase in cytotrophoblasts by confocal immunofluorescence technique and in culture medium by enzyme-linked immunosorbent assay after lipopolysaccharide treatment. A positive correlation was found between tumor necrosis factor α expression and apoptosis indexes of cytotrophoblasts (r=0.747, P＜0.001).Conclusion Apoptosis of cytotrophoblasts could be induced by lipopolysaccharide, in which innate immune reaction is the important mechanism。

Abstract：Background Pancreatic cancer is one of the most common tumors and has a 5-year survival for all stages of less than 5%. Most patients with pancreatic cancer are diagnosed at an advanced stage and therefore are not candidates for surgical resection. In recent years, investigation into alternative treatment strategies for this aggressive disease has led to advances in the field of gene therapy for pancreatic cancer. E. coli purine nucleoside phosphorylase/6-methylpurine deoxyribose (ePNP/MePdR) is a suicide gene/prodrug system where PNP enzyme cleaves nontoxic MePdR into cytotoxic membrane-permeable compounds 6-methylpurine (MeP) with high bystander activity, hTERT is expressed in cell lines and tissues for telomerase activity. In this study we examined the efficacy of ePNP under the control of hTERT promoter sequences and assessed the selective killing effects of the ePNP/prodrug MePdR system on pancreatic tumors.Methods Recombinant pET-PNP was established. The protein of E. coli PNPase was expressed and an antibody to E.coli PNPase was prepared. Transcriptional activities of hTERT promoter sequences were analyzed using a luciferase reporter gene. A recombinant phTERT-ePNP vector was constructed. The ePNP/MePdR system affects SW1990 human pancreatic cancer cell lines in vitro.Results The hTERT promoter had high transcriptional activity and conferred specificity on cancer cell lines. The antibody to E. coli PNPase was demonstrated to be specific for the ePNP protein. The MePdR treatment induced a high in vitro cytotoxicity on the sole hTERT-ePNP-producing cell lines and affected SW1990 cells in a dose-dependent manner.Conclusions The hTERT promoter control of the ePNP/MePdR system can provide a beneficial anti-tumor treatment in pancreatic cancer cell lines including a good bystander killing effect。

Abstract：Background It has been reported that hydrogen sulfide (H2S) could relax vascular smooth muscle by direct activation of KATP channels and hyperpolarization of the membrane potential. Recently, our study has shown that H2S facilitated carotid baroreflex. This study was conducted to investigate the effect of H2S on carotid baroreceptor activity (CBA).Methods The functional curve of carotid baroreceptor (FCCB) was constructed and the functional parameters of carotid baroreceptor were measured by recording sinus nerve afferent discharge in anesthetized male rats with perfused isolated carotid sinus.Results H2S (derived from NarHS) 25, 50 and 100 μmol/L facilitated CBA, which shifted FCCB to the left and upward.There was a marked increase in peak slope (PS) and peak integral value of carotid sinus nerve charge (PIV) in a concentration-dependent manner. Pretreatment with glibenclamide (20 μmol/L), a KATP channel blocker, the above effects of H2S on CBA were abolished. Pretreatment with Bay K8644 (an agonist of calcium channels, 500 nmol/L) eliminated the role of H2S on CBA. An inhibitor of cystathionine Y-lyase (CSE), DL-propargylglycine (PPG, 200 μmol/L) inhibited CBA in male rats and shifted FCCB to the right and downward.Conclusions Our results suggest that exogenous H2S exerts a facilitatory role on isolated CBA through opening KATP channels and further closing the calcium channels in vascular smooth muscle. Endogenous H2S may activate CBA in vivo。

Abstract：Background Hepatocellular carcinoma (HCC) ranked the second among the causes of cancer mortality in China since the 1990s. Up to now, medication still plays an important role in the treatment of HCC. The therapies based on the allicin as a potential chemopreventive analog although is in its infancy at the present time, may have a significant role in the future management of HCC. Diallyl trisulfide (DATS) is a natural compound derived from garlic. In this study, we investigated the inhibitory effects of hepatic targeted polybutylcyanoacrylate nanoparticles of diallyl trisulfide(DATS-PBCA-NP) on orthotopic transplanted HepG2 hepatocellular carcinoma in nude mice.Methods DATS-PBCA-NP were detected by transmission electron microscope (TEM) and high-performance liquid chromatography (HPLC). The orthotopic transplantation HCC models were established by implanting HCC HepG2 xenograft bits under the envelope of the mice liver. Successful models (n=29) were divided into 4 groups: normal saline(NS), empty nanoparticles (EN), DATS and DATS-PBCA-NP were intravenously administered to the mice respectively for 2 weeks. In vivo antitumor efficacy was evaluated by the measurement of tumor volume. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and protein levels of apoptosis and cell proliferation proteins by immunoblotting in tumor tissues were performed to elucidate the possible mechanism.Results DATS-PBCA-NP possessed smooth and round appearance, dispersed well, and released in vitro in accord with double phase kinetics model. DATS-PBCA-NP changed the tissue/organ distribution of DATS in vivo. The successful rate of tumor implantation was 100%. Intravenous administration of DATS-PBCA-NP significantly retarded the growth of orthotopically transplanted hepatoma in BALB/c nude mice (compared with the other three groups, all P＜0.05) without causing weight loss (P＞0.05). TUNEL staining showed that the tumors from DATS-PBCA-NP treated mice exhibited a markedly higher apoptotic index compared with control tumors. Western blot analysis of tumor tissue revealed that the down-regulated expression of proliferation cell nuclear antigen (PCNA) and Bcl-2 proteins in DATS-PBCA-NP group, and there were no significant differences in the expression of Fas, FasL and Bax proteins among the four groups (P＞0.05).Conclusions DATS-PBCA-NP has good prolonged release effect in vivo and hepatic-targeted activity, and significant anti-tumor effect on the orthotopic transplantation HCC model in mice in association with the suppression of proliferation and the induction of apoptosis of tumor cells. These advantages are probably due to their liver targeting characteristics and consequently bring a higher anti-tumor activity。

Abstract：Background Current prosthetic, small diameter vascular grafts showing poor long term patency rates have led to the pursuit of other biological materials. Biomaterials that successfully integrate into surrounding tissue should match not only the mechanical properties of tissues, but also topography. Polyglycolic acid (70/30) has been used as synthetic grafts to determine whether human vascular smooth muscle cells and endothelial cells attach, survive and secrete endothelin and 6-keto-prostaglandin F1α (6-keto-PGF1α).Methods Endothelial cells and smooth muscle cells were isolated from adult human great saphenous vein. They were seeded on polyglycolic acid scaffold in vitro separately to grow vascular patch (Groups A and B respectively) and cocultured in vitro to grow into vascular patch (Group C). Smooth muscle cells and endothelial cells were identified by immunohistochemical analysis and growth of cells on polyglycolic acid was investigated using scanning electron microscopy. The levels of endothelin and 6-keto-PGF1α in the culturing solutions were examined by radioimmunology to measure endothelial function.Results Seed smooth muscle cells adhered to polyglycolic acid scaffold and over 28 days grew in the interstices to form a uniform cell distribution throughout the scaffold. Then seed endothelial cells formed a complete endothelial layer on the smooth muscle cells. The levels of endothelin and 6-keto-prostaglandin F1 alpha in the culturing solution were (234±29) pg/ml and (428+98) pg/ml respectively in Group C and (196+30) pg/ml and (346±120) pg/ml in Group B; both significantly higher than in Groups A and D (blank control group, all P＜0.05 ).Conclusions Cells could be grown successfully on polyglycolic acid and retain functions of secretion. Our next step is to use human saphenous vein smooth muscle cells and endothelial cells to grow tubular vascular grafts in vitro。

Abstract：Background CD4+ T cells play a crucial role in the pathogenesis of aplastic anaemia. However, the mechanisms of over-proliferation, activation, infiltration of bone marrow and damage to haematopoietic cells of CD4+ T cells in aplastic anaemia are unclear. Therefore, we screened differentially expressed genes of bone marrow CD4+ T cells of aplastic anaemia patients and normal donors by suppressive subtractive hybridization to investigate the pathogenesis of aplastic anaemia.Methods The bone marrow mononuclear cells of a first visit aplastic anaemia patient and a healthy donor of the same age and sex were isolated using lymphocyte separating medium by density gradient centrifugation. With the patients as "tester" and donor as "driver", their CD4+T cells were separated with magnetic bead sorting and a cDNA library established by suppressive subtractive hybridization. Then 15 of the resulting subtracted cDNA clones were randomly selected for DNA sequencing and homological analysis. With semiquantitative RT-PCR, bone marrow samples from 20 patients with aplastic anaemia and 20 healthy donors assessed the expression levels of differentially expressed genes from SSH library.Results PCR detected 89 clones in the library containing an inserted fragment of 100 bp to 700 bp. Among 15 sequenced clones, 12 were known genes including 3 repeated genes. Compared with normal donors, there were 9/12 genes over-expressed in bone marrow CD4+T cells of patients with aplastic anaemia. The effects of these genes included protein synthesis, biology oxidation, signal transduction, proliferative regulation and cell migration. Not all these genes had been reported in the mechanisms of haematopoietic damage mediated by CD4+ T cells in aplastic anaemia.Conclusions Screening and cloning genes, which regulate functions of CD4+ T cells, are helpful in elucidating the mechanisms of over proliferation, activation, infiltrating bone marrow and damaging haematopoietic cells of CD4+ T cells in aplastic anaemia。

Abstract：Background T cell immune abnormalities in patients with dilated cardiomyopathy (DCM) has been intensively studied over the past 10 years. Our previous study has suggested that immunization of mice with the peptides derived from human adenine nucleotide translocator (ANT) result in the production of autoantibodies against the ANT and histopathological changes similar to those in human DCM. The ANT peptides can induce autoimmune cardiomyopathy like DCM in Balb/c mice. In this study we aimed to focus on the molecular mechanism of T cells in the autoimmune cardiomyopathy mouse model by detecting the expression of the two T cell signaling molecules.Methods The ANT peptides were used to cause autoimmune cardiomyopathy in Balb/c mice. Anti-L3T4 or rat anti-mouse IgG was administered to the mice (n=6 in each group) simultaneously immunized with ANT. ELISA analysis was used to detect autoantibodies against the ANT peptides and the percentages of interferon-Y and interleukin-4 producing cells among splenic CD4+ lymphocytes was determined by using flow cytometry analysis. The expression of CD45 in spleen T cells was determined by immunohistochemistry and the mRNAs of T cell signaling molecules were detected by real-time PCR.Results Treatment of ANT immunized Balb/c mice with anti-CD4 mAb caused a reduction in the gene expression of P56lck and Zap-70 and a lower level of CD45 expression by spleen T cells. Aiso, a reverse of the Th1/Th2 ratio that results in the reduced production of antibodies against ANT was found in the anti-CD4 monoclonal antibodies (mAb)group. Whereas irrelevant antibody (rat anti-mouse IgG) did not suppress T cell signaling molecules nor inhibit CD45 expression, and control-antibody mice did not show any significant differences compared with the DCM group.Conclusion The results show that anti-CD4 mAb is a powerful inhibitor of the early initiating events of T cell receptor(TCR) signal transduction in mouse autoimmune dilated cardiomyopathy。

Abstract：Background Human rickettsioses are worldwide zoonoses and it is not easy to differentiate them from other infectious diseases because of their atypical manifestation. In recent years the number of patients with fever of unknown causes from Hongta District CDC, Yuxi city of Yunnan Province has been increasing significantly in the summer. Diagnosis of scrub typhus was made by local clinicians. In order to ascertain the disease, we undertook a laboratory investigation for such patients from August 18 to 26, 2005.Methods Active surveillance was conducted by Hongta District CDC Yuxi city of Yunnan Province from 2002 to 2004 and basic data were obtained from cases confirmed according to clinical definitions. Average incidences and town-level incidences were calculated during the study periods. Blood samples were analyzed by PCR and serological test. Based on the groEL gene sequences a paired general outer primers (Gro-1 and Gro-2) targeting typhus, spotted fever as well as scrub typhus and two paired inner primers (SF1, SR2 and TF1, TR2) for typhus together with spotted fever and scrub typhus, respectively, were designed to perform a multiplex-nested PCR. Serological assay was carried out by indirect immunofluorescence assay with 7 different rickettsial antigens, i.e., R. mossori, R. sibirica, R. conorii, O. tsutsugamushi,B. quintana, B. henselae and Coxilella burnetii phase Ⅱ Ag.Results Epidemiological surveillance showed that from 2002 to 2004, the average incidences of the scrub typhus or scrub typhus with murine typhus were 222.1/105, 204.3/105 and 109.6/105, respectively. Of 13 blood samples taken during acute stage of illness, 6 showed the amplified products for scrub typhus and the sequenced products showed 100%, 99%, 99%, 99%, 99%, 99% similarity to O. tsutsugamushi Karp but they shared the same deduced amino acid sequences, which indicated 100% identity with the heat shock protein of the O. tsutsugamushi Karp strain. Five yielded PCR products for murine typhus and their corresponding nucleotide sequences exhibited 100%, 100%, 99%, 99% and 99% similarity to R. mossori Wilmington and the analyses of predicted amino acid sequences indicated 100%, 100%,98%, 98% and 98% identity with the heat shock protein of R. mossoriWilmington strain. Of the 8 PCR positive patients, 3 showed a co-infection of scrub typhus with murine typhus. All the 13 serum samples from febrile patients were positive against O. tsutsugamushi and 8 of them were positive against R. mossori. All of the 8 paired specimens had four-fold elevation of antibody against O. tsutsugamushi, and seroconversion for typhus was demonstrated in 3 paired serum samples. Another finding in the study was that a high seropositive prevalence (76.9%) of Q fever was detected.Conclusion It's confirmed that co-prevalence of scrub typhus with murine typhus are occurring in Yuxi city of Yunnan province, China. Other rickettsial diseases also need to be investigated in these areas。

Abstract：Background There are few studies on the arterial compliance of noise exposure. The purpose of this study was to understand the relationship between hearing loss, blood pressure and arterial compliance of female workers who exposed to occupational noise in a textile mill.Methods The noise levels in the workplace were measured with a HS6288 sound level meter. Cumulated noise exposure (CNE) was calculated according to the noise intensity and the exposure period. Hearing ability and arterial compliance were measured in 618 noise exposed workers. The database was set up with EpiData and the statistical analysis was performed with SAS software 9.1.3.Results The noise levels were 80.1dB(A) to 113.5dB(A), of which the levels at 92.5% of the noise monitoring sites were over the national standard. The incidence of high frequency hearing loss (HFHL) was 24.43% and language frequency hearing impairment (LFHI) was 0.81%. The incidence of hypertension was 7.93%. Both systolic blood pressure (SBP)and diastolic blood pressure (DBP) in the high frequency hearing loss group were significantly higher than those in the normal hearing group (P＜0.05), while C1 (large artery compliance) and C2 (small artery compliance) were significantly lower (P＜0.05). The high frequency hearing threshold (HFHT) of the hypertension group was significantly higher than in the normal blood pressure group (P＜0.05), while C1 and C2 were significantly lower (P＜0.05). C1 and C2 had a negative correlation with HFHT, SBP, DBP, mean of arterial pressure (MAP), pulse pressure (PP) and pulse rate (PR) (P＜0.05).The multiple regression analyses showed that blood pressure and PR were the main influencing factors on C1 and C2.LFHT was an influence on C2 and HFHT on C1.Conclusions Textile mill noise pollution is very serious and has an obvious influence on worker's auditory function. The female workers with Iow artery compliance or with high blood pressure might be suffering from hearing loss; those with noise induced hearing loss might be suffering from hypertension if she is constantly exposed to loud industrial noise。

Abstract：Background Despite low morbidity, acute or subacute spinal epidural hematoma may develop quickly with a high tendency to paralysis. The delay of diagnosis and therapy often leads to serious consequences. In this study we evaluated the effects of a series of methods for the diagnosis and treatment of the hematoma in 11 patients seen in our hospital.Methods Of the 11 patients (8 males and 3 females), 2 had the hematoma involving cervical segments, 2 cervico-thoracic, 4 thoracic, 1 thoraco-lumbar, and 2 lumbar. Three patients had quadriplegia, including one with central cord syndrome; another had Brown-Sequard's syndrome; and the other seven had paraplegia. Five patients were diagnosed at our hospitals within 3-48 hours afterappearance of symptoms, and 6 patients were transferred from community hospitals within 21-106 hours after development of symptoms. Key dermal points, key muscles and the rectal sphincter were determined according to the American Spinal Injury Society Impairment Scales as scale A in two patients,B in 5 and C in 4. Emergency MRI in each patient confirmed that the dura mater was compressed in the spinal canal, with equal intensity or hyperintensity on T1 weighted image and mixed hyperintensity on T2 weighted image. Preventive and curative measures were taken preoperatively and emergency operation was performed in all patients. Open laminoplasty was done at the cervical and cervico-thoracic segments, laminectomy at the thoracic segments, laminectomy with pedicle screw fixation at the thoraco-lumbar and lumbar segments involving multiple levels, and double-sided laminectomy with the integrity of articular processes at the lumbar segments involving only a single level. During the operation, special attention was given to hematoma evacuation, hemostasis and drainage tube placement.Results Neither uncontrollable hemorrhage nor postoperative complications occurred. All patients were followed up for 1-6 years. A marked difference was noted between postoperative and preoperative scales (u=3.66, P＜0.01). Most patients recovered after therapy, but the recovery of patients treated at our hospitals was superior to that of those transferred from community hospitals (t =2.95, P＜0.05). Of the patients treated at our hospitals, 4 were cured and 1 was upgraded with scale from A to D, whereas none of those transferred from community hospitals recovered completely,even one remained scale C.Conclusions Physical examination plus MRI is essential to early diagnosis of acute or subacute spinal epidural hematoma. Preventive and curative measures including emergency operation are helpful to the recovery of patients'nerve function。

Abstract：Objective To review the transmission models of tuberculosis in heterogeneous population.Data sources The data used in this review were adopted mainly from the studies of models of tuberculosis reported from 1995 to 2006.Study selection Relevant literature on transmission models of tuberculosis in heterogeneous populations are referenced.Results Casual/random factors and genetic factors are the main reasons for epidemics of tuberculosis in recent years.Mass public transport is playing the primary role in casually close contact which can facilitate the transmission of tuberculosis. Genetic susceptibility not only varies endemic prevalence levels, but also drastically alters the effects of treatment for tuberculosis patients. Detailed studies further exhibit that casual contact and genetic factor are responsible for over 30%-40% of the total new cases in recent years. The prevalence of tuberculosis could double (from 33% to 60%)if a genetically susceptible phenotype is present in only 30% of the population. And some challenges have emerged along with these exciting results.Conclusions Casual/random contact, public transport and genetic susceptibility are responsible for most new tuberculosis cases and a wide variation in endemic tuberculosis levels between regions. Hence, the transmission model of tuberculosis in a heterogeneous population can provide more clues to underlying mechanism of tuberculosis transmission than in a homogeneous population. However, many challenges remain for us in understanding transmission of disease。

Since Parodi et al1 reported the first successful case of endovascular treatment for abdominal aortic aneurysm (AAA) in 1991, the endovascular repair has become an important option for the surgical treatment of aortic aneurysms.2-4 The occluder is a newly-developed device introduced intraluminally to block the blood flow in certain arteries。

Neurilemmoma, also named Schwannoma, is usually a benign tumour composed of Schwann cells.1,2 It is commonly located in the retroperitoneum, mediastinum,head and neck, and is rarely seen in the genitourinary organs。

Endometriosis is a common benign disease affecting about 10% to 20% of women. The frequency of intestinal endometriosis varies between 3% and 37% in women with proven endometriosis。

Tuberculosis (TB) is a systemic infection caused by Mycobacterium tuberculosis. Extrapulmonary TB accounts for 10% to 27% of all the patients with TB.1 Here we report an unusual case of extrapulmonary TB in a pregnant woman。

Situs inversus (SI) is a congenital anomaly characterized by the mirror-image orientation of the inner organs. The estimated incidence of SI is 1/15 000 to 1/10 000 live births.1 The disease is often complicated with biliary atresia, polysplenia, intestinal nonrotation,preduodenal portal vein, aberrant hepatic artery, or absence of the suprarenal inferior vena cava.2,3 In 2004,we performed a liver transplantation on a patient with SI complicated with hepatolithiasis, end-stage liver cirrhosis and liver failure, because of the unavailability of donor organs, the operation became more complicated and difficult。

Areverse headgear is thought to be an effective treatment approach for skeletal class Ⅲ malocclusion with retruded maxilla. Clinical studies have shown that the changes of occlusion are a combination of skeletal and dental changes: forward movement of the maxilla, proclination of the maxillary incisors, clockwise rotation of the mandible, and retroclination of the mandibular incisors.1,2 However, the undesirable dental effects, such as excessive mesial movement and extrusion of maxillary molars and labial tipping of maxillary incisors, have been reported。

Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults. Its insidious onset and progression often hinder timely renal biopsy and early diagnosis delaying treatment while worsening prognosis. The complication of malignant hypertension(MHT) is rarely seen in idiopathic MN. To provide a better understanding of the disease we report a case of idiopathic MN diagnosed by biopsy six years after onset。