2005年 · 第118卷第20期

Abstract：Methods hMSCs were isolated from human bone marrow by density gradient fractionation and adherence to plastic flasks. Individual colonies were selected and cultured in tissue dishes. Packaging cells PT67 were transfected by PLEGFP-N1 retroviral vector , and hMSCs were transduced by viral supernatant infection. Meanwhile, hMSCs-EGFP were identified by immune phenotypes and whether it could differentiate into osteoblasts or adipocytes under conditioned media was investigated. Results The rate of stably transduced hMSCs-EGFP was up to 96% after being screened by G418. hMSCs-EGFP exhibited fibroblast-like morphological features. Flow cytometric analyses showed that hMSCs-EGFP were positive for CD73, CD105, CD166, CD90 and CD44, but negative for CD34 and CD45. In addition, it could functionally be induced into osteocytes or adipocytes under conditioned media. These biological features of hMSCs-EGFP were consistent with those of hMSCs.Conclusions hMSCs transduced by PLEGFP-N1 retroviral vector can be used in vivo securely because they can maintain their biological characteristics and differentiation. It is a simple and reliable way to trace the changes of hMSCs in vivo by EGFP during cell transplantation and gene therapy.

Abstract：Background This study aimed at assessing the effect of noninvasive limb preconditioning on myocardial infarct size, and determining whether nitric oxide and neurogenic pathway play an important role in the mechanism of acute remote ischemic preconditioning (IPC).Methods Forty Wistar rats were randomly divided into four experimental groups. In Group Ⅰ, the rats underwent 30-minute occlusion of the left anterior descending coronary artery, and 120-minute reperfusion. In Group PL, the rats underwent four cycles of 5-minute occlusion and reperfusion of both hind limbs using a tourniquet before the experiment was continued as in Group Ⅰ. In Group PL-N and Group PL-H, we administered L-nitro-arginine methyl ester (L-NAME) 10 mg/kg or hexamethonium chloride 20 mg/kg intravenously, 10 minutes before IPC. Infarct size as a percentage of the area at risk was determined by triphenyltetrazolium chloride staining. Results There were no statistically significant differences in mean arterial pressure and heart rate among these groups at any time point during the experiment (P>0.05). The myocardial infarct size (IS) was decreased significantly in Group PL and Group PL-H compared with Group Ⅰ, and the IS/AAR was 34.5%±7.6%, 35.9%±8.6% and 58.5%±8.5%, respectively (P<0.05). The IS/AAR was 49.1%±6.5% in Group PL-N, and there was no significant difference compared with Group Ⅰ (P>0.05).Conclusions Noninvasive limb IPC is effective in protecting the myocardium from ischemia reperfusion injury. Nitric oxide plays an important role in the mechanism of acute remote IPC, in which the neurogenic pathway is not involved。

Abstract：Background We investigated the pathogenesis of atherosclerosis in diabetes, and detected the expression of scavenger receptor CD36 in monocytes in patients with type 2 diabetes.Methods According to the criteria by WHO, diabetic patients were classified into two groups: well controlled diabetic patients (WCP) and poorly controlled diabetic patients (PCP). The expression of CD36 protein and mRNA were evaluated by flow cytometry and reversal transcription polymerase chain reaction (RT-PCR). Plasma levels of accumulution of oxidized LDL (oxLDL) were directly measured by sandwich enzyme-linked immunosorbent assay (ELISA) method.Results Flow cytometry and RT-PCR showed that the mean fluorescence intensity (MFI) of CD36 in monocyte and CD36 mRNA were significantly higher in the PCP and WCP in comparison with healthy controls (P<0.01). CD36 MFI and mRNA in the PCP were increased by 78% and 36% compared to the WCP. In both groups, CD36 MFI and mRNA were significantly higher in patients with diabetic atherosclerosis in comoparison with those without diabetic atherosclerosis (P<0.05). No significant difference was found in CD14 expression between the groups (P>0.05). The concentrations of plasma oxLDL were higher in the PCP group compared to WCP and control group (P<0.05), whereas oxLDL average values did not differ significantly between WCP and control groups (P>0.05). In the WCP and PCP groups, oxLDL levels were higher in patients with diabetic atherosclerosis than those without diabetic atherosclerosis (P<0.05).Conclusions The increased expression of scavenger receptor CD36 may be one of the mechanism of accelerated atherosclerosis in diabetic. The poorly controlled diabetes patients are at higher risk for the vascular complications than the well controlled diabetic patients。

Abstract：Background The palate is differently regulated and developed along the anterior-posterior axis. The Bmp signal pathway plays a crucial role in palatogenesis. Conditioned-inactivation of Bmp type I receptor Alk2 or Alk3 in the neural crest or craniofacial region leads to palatal cleft in mice. However, how different Bmp members are involved in palatogenesis remains to be elucidated. In the present study, mRNA expression patterns of Bmp2, Bmp3 and Bmp4 in the developing anterior and posterior palates were examined and compared, focusing on the fusion stage. Methods To detect the expression of Bmp mRNA, antisense riboprobes were synthesized by in vitro transcription. Radioactive in situ hybridization was performed on sagital and coronal sections of mice head from E13 to E18. Results The expression of these Bmps were developmentally regulated in the anterior and posterior palates prior to, during and after palatal fusion. During palatal fusion, Bmp4 expression shifted from the anterior to the posterior palate, Bmp2 was highly expressed in both the anterior and posterior palates in this process, whereas Bmp3 was only localized in the posterior palate. They showed generally non-overlapping pattern in their expression domains. Thereafter, their expression was detected in both the anterior and posterior palates regulating osteogenesis and myogenesis respectively. Conclusions Bmp signalling is involved in palatogenesis in multiple stages and has multiple roles in regulating anterior and posterior palatal development. Disturbances of Bmp signalling during palatogenesis might be a possible mechanism of cleft palate。

Abstract：Background Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta superfamily, are powerful regulators of cartilage and bone formation. This study investigated the biological changes of NIH3T3 cells incubated with secretive BMP2 that was induced by gene transfection through transwell. Methods Eukaryonic expression vector (pcDNA3.1-B2) was transfered into NIH3T3 cells with SofastTM,a positive compound transfection agent. The positive cell clones were selected with G418. The cytoplasmic and extracellular expressions of BMP2 were determined by immunohistochemical stain and enzyme-linked immunosorbent assay. NIH3T3 cells were co-cultured with hBMP2 gene transfecting cells through transwell, and the ultrastructure, alkaline phosphatase activity and the expression of osteocalcin (the marker of osteogenetic differentiation) changes were observed. Results There were cytoplasmic and extracellular expressions of BMP2 in transfecting NIH3T3 cells. The ultrastructural changes, the high activity of alkaline phosphatase and the positive stain of osteocalcin suggested the osteogenetic differentiation tendency of NIH3T3 cells co-cultured with transfecting NIH3T3 cells. Conclusion Secretive BMP2 that is induced by gene transfection could promote the osteogenetic differentiation of fibroblast cells。

Abstract：Background The severity of hypoalbuminemia has been shown to be related to morbidity and mortality in some critical illnesses, illustrating the need for better understanding of molecular mechanism of hypoalbuminemia. Lipopolysaccharide (LPS) is a key mediator inducing hypoalbuminemia in sepsis and septic shock. The present study was designed to identify if the reduction of albumin expression is directly induced by LPS and modulated by activated extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in rat hepatocytes.Methods Primary rat hepatocytes were divided into five groups. In two of them, hepatocytes were treated with normal saline or 1 μg/ml LPS, then albumin mRNA expression was observed at 0, 2, 8, 12 and 24 hours after treatment. In another group, hepatocytes were pretreated with 100, 40 or 20 μmol/L of cycloheximide (CHX, an inhibitor of protein synthesis) for 30 minutes followed by 1 μg/ml LPS for 24 hours. Then the RNA was extracted from the cells for RT-PCR to detect the expression of albumin. The other two groups were administered 1 μmol/L, 10 μmol/L and 50 μmol/L of SB203580 (p38 MAPK inhibitor) or PD98059 (ERK inhibitor) 30 minutes prior to 1 μg/ml LPS treatment. After 24 hours of LPS treatment, the supernatant was collected and assayed for albumin concentrations. Data were analyzed by one-way analysis of variance, followed by the Newman-Keul test; a P<0.05 was considered significant.Results There was no marked change in albumin mRNA expression in the control group during 24-hours treatment with normal saline. The reduction did not occur until 24 hours after LPS treatment, and albumin mRNA decreased by 30% approximately compared to the control group at 24 hours (0.587 vs 0.832, P=0.007). CHX could inhibit the decline of albumin mRNA induced by LPS and the effect was correlated with the dose of CHX. The ERK inhibitor PD98059 caused a significant increase in LPS-induced albumin production at the three concentrations (119.7, 111.4 and 80.0 ng/ml vs 44.4 ng/ml, P=0.0013, 0.0025 and 0.009, respectively), whereas SB203580 obviously blocked albumin reduction in LPS-treated cells at the concentrations of 10 and 50 μmol/L (87.5 and 93.6 ng/ml vs 44.4 ng/ml, P=0.0076 and 0.0049, respectively).Conclusions LPS can induce the reduction of albumin expression by new synthesized proteins indirectly, and the process may be related to the signal proteins of ERK and p38 kinase. The ERK and p38 kinase are critical signaling pathways in LPS-induced hypoalbuminemia which is worthwhile to understand in studying the molecular mechanism of hypoalbuminemia in sepsis and septic shock。

Abstract：Background Thalidomide is reviving for its antiangiogenic effect on corneal neovascularization models. Recently, it has been employed in tumor research in several types of solid carcinomas. However, its effect on hepatocellular carcinoma (HCC) has not yet been clarified. Methods A total of 48 nude mice bearing human HCC with a high metastatic potential were randomly divided into 4 groups. Thalidomide (200 mg/kg), paclitaxel (13 mg/kg), or their combination, which was dissolved in 0.5% sodium carboxyl methyl cellulose (CMC) suspension, was intraperitoneally injected in each group since the second day of the establishment of animal model. The group simply administered with 0.5% CMC was set as placebo-control. The mice were sacrificed on the 30th day, for the measurement of tumor size, weight and metastasis in the lungs. The levels of CD34 and endothelial growth factor (VEGF) mRNA in tumor tissues were detected by immunohistochemistry and semiquantitative RT-PCR, respectively, and microvessel density (MVD) was evaluated. Results No statistical difference was found in tumor weight and volume between the thalidomide group and control (P>0.05). Paclitaxel showed a growth-inhibiting effect on tumors (P<0.05). The value of MVD and VEGF mRNA and metastases to the lungs in each group were lower than those in the placebo-control group (P<0.05); such difference in the combination group was statistically significant (P<0.05). Conclusions Paclitaxel, but not thalidomide, has significant growth inhibitory effect on tumors, but both significantly inhibit angiogenesis and metastasis of human HCC in nude mice, such effects of paclitaxel can be amplified by thalidomide。

Abstract：Background Introperitoneal hemorrhage is one of the most common complications of radiofrequency (RF) ablation of hepatic tumors. This study was designed to investigate the reason and management of intraperitoneal hemorrhage occurred during or after percutaneous RF ablation of hepatic tumors.Methods Three hundred and fifty-six patients with hepatic tumors have been treated at 592 procedures of ultrasound guided RF ablation. Intraperitoneal hemorrhage occurred in 5 patients (0.8%). The reasons and management of intraperitoneal hemorrhage in these 5 cases were retrospectively analyzed. Results Two patients with liver metastasis and one hepatocellular carcinoma (HCC) patient suffered from hemorrhage during the RF treatment. Two patients with recurrent HCC after surgery developed hemorrhage 20 minutes or 4 hours after RF treatment. One case of hemorrhage was due to the inappropriate electrode positioning induced liver laceration while treating a 1 cm liver metastasis near the liver capsule. One was due to the injury of a small vessel by the RF needle in another liver metastasis patient. Three cases were due to tumor rupture with two cases induced by cough or position change after treating large protruding HCC lesions. Four (80%) of the 5 cases of hemorrhage were rapidly identified by ultrasound. The causes and sites of bleeding during the RF treatment in three cases were confirmed through ultrasound, which were successfully treated using RF coagulation to achieve hemostasis of the bleeding site. Two patients with post-ablation hemorrhage recovered in one hour and 24 hours, respectively after given blood transfusion and other conservative measures. No surgical intervention was required. Two patients died of wide spread metastasis 23-36 months afterwards and the other three patients have lived for 18-25 months to date.Conclusions It is important to perform close monitoring during and after RF ablation in order to identify intraperitoneal hemorrhage in time. RF ablation of the bleeding sites was a simple and effective management when the bleeding site could be confirmed by ultrasound. The hemorrhage due to the rupture of large and protruding liver tumors could be serious and should be considered as contraindication for RF treatment.

Abstract：Background The true risk of choronic villus sampling (CVS) is poorly defined. The objective of this study was to review the clinical outcome of transabdominal CVS performed in a university teaching unit, with an emphasis on the complication rate.Methods A comprehensive audit database was maintained for 1351 pregnant women, including 17 sets of twin pregnancies, who had a CVS. Details and outcome of all CVSs made in the unit between May 1996 and May 2004 were reviewed. All CVSs were performed by one of 5 operators using the identical techniques.Results All procedures were performed transabdominally. A total of 1355 CVSs were performed because there were 4 dichorionic twin pregnancies which required 2 punctures. The mean gestation at CVS was (11.8±0.7) weeks, and 97.3% of the procedures were performed between 11 and 13 completed weeks. The majority (96.2%) required only 1 puncture to achieve correct needle placement. The procedure failed to obtain an adequate sample in 4 subjects (0.30%). A total of 1351 chromosomal studies were requested and there was 1 case (0.07%) of culture failure. The results of chromosomal studies were available within 14 days in 36.7% of the cases and within 21 days in 94.0%. Overall, 77 chromosomal abnormalities (5.7%) and 5 cases of thalassemia major were detected. Pregnancy outcome was unknown in only 13 singleton subjects (0.96%). In the remaining 1355 fetuses, there were 76 pregnancy terminations (5.56%), 10 fetal losses with obvious obstetric causes (0.73%), and 21 potentially procedure-related fetal losses (1.54%). In the last group, the majority had one or more co-existing obstetric complications. The background fetal loss rate for pregnancies at similar gestational age in the unit was about 0.8%. Therefore, the procedure-related fetal loss rate was estimated to be at the maximum of 0.74%.Conclusions In experienced hands, first trimester transabdominal CVS is an accurate and safe invasive prenatal diagnostic procedure. It should be one of the treatment options available to pregnant women who require prenatal genetic diagnosis。

The trabecular meshwork is located at the anterior chamber angle, and is the main route for the outflow of aqueous humor. It is composed of perforated sheets of collagen and elastic tissue covered by trabecular meshwork (TM) cells, forming a filter with decreasing pore size as the canal of Schlemm is approached. TM cells have some endothelial properties, such as the presence of intercellular junctional complexes, particularly tight junctions (TJs). TJs form paracellular seals between adjacent cells and act as fences that segregate protein (and partially lipid) components of the apical and basolateral plasma membrane domains. Under the electron microscope, TJs appear as a series of discrete contacts between the lateral membranes of adjacent cells。

The situation of tuberculosis (TB) and Mycobacterium tuberculosis (M. tuberculosis) drug resistance is still very serious in China. Improper treatment for TB may lead to a prolonged course of antimicrobial therapy and spreading of drug resistant strains. Thus, rapid detection of drug resistant strains will allow prompt initiation and adjustment of effective chemotherapeutic treatment of TB. Ethambutol (EMB) is a first line anti-TB drug. embB Met306 is located in a cytoplasmic loop that forms an EMB resistance determining region,1 Lety et al2 have demonstrated that a missense mutation in the M. smegmatis embB gene confers EMB resistance. The identification of mutations in embB gene may offer a means to rapidly screen M. tuberculosis isolates for EMB resistance. Thus, in this study, we analysed 197 isolates of M. tuberculosis from Chinese people and characterized mutations in the 258 bp region of the embB gene.

Atrial fibrillation (AF) is the most common cardiac arrhythmia with debilitating compli-cations of stroke. Multiple-wavelet re-entry and focal activation from pulmonary vein foci are two dominant electrophysiological theories of AF. Atrial electrical remodeling plays a role in the maintenance of AF. However, molecular mechanisms of the arrhythmia are still poorly understood.

Fulminant hepatitis (FH) refers to liver diseases that have severe state and complicated manifestations. It will endanger patients'lives and health largely. The incidence of fulminant hepatitis in pregnancy (FHP) is 66 times of patients not in pregnancy,1 which is more dangerous and is one of the major causes of maternal and perinatal death in China. In order to predict the prognoses of FHP and decrease maternal and perinatal death rate, we summarized clinical information of 25 cases of FHP admitted to our hospital and investigated into prognostic factors that influenced FHP。

Congenital aortic arch anomalies occur most commonly in children. The disease can be classified into three types: ① obstructive congenital abnormalities, including coarctation of aorta (CoA) and interruption of aortic arch (IAA); ② non-obstructive congenital abnormalities, including double aortic arch and others; ③ congenital shunt abnormalities, including different types of patent ductus arteriosus (PDA). Management of patients with congenital aortic arch anomalies relies on imaging. Routine imaging modalities, such as conventional X-ray plain film and transthoracic echocardiography (TTE), have been recently complemented by magnetic resonance imaging (MRI)。

Bifurcation lesions are still technically challenging even in the era of modern stents.1 High incidence of restenosis both in main vessel and side branch limits the long-term prognosis although several kinds of techniques have been identified to be successful for coronary bifurcations.2-5 Reports have demonstrated the main reason for higher incidence of ostial side branch even though drug-eluting stent used in side vessel lies in that there were gaps in metal coverage and drug application.6-9 Therefore, new technique ensuring complete vessel scaffolding without gaps in drug delivery at the bifurcation is crush technique which is similar to other techniques including T- and Y- stenting still needing postdilatation of kissing balloon angioplasty to expand the stent fully in the ostial side branch and to prevent stent distortion in main vessel.10 As a result, kissing balloon angioplasty is a key step to improve the final result and to reduce the restenosis after stenting bifurcation lesions. However, kissing angioplasty is difficult to be underwent or impossible because operators usually fail to rewire two layers of metal strut, which would result in suboptimal stent deployment, a main reason of high incidence of restenosis, and acute- or-late-thrombosus. The present study reports modified DK crush technique improving success rate of kissing balloon angioplasty under the guidance of intravascular ultrasound (IVUS)。

Intranodal palisaded myofibroblastoma (IPM), a benign, rare primary mesenchymal neoplasm, located in the inguinal lymph nodes, was described as "palisaded myofibroblastoma" by Weiss et al, and "intranodal hemorrhagic spindle-cell tumor with amianthoid fibers" by Suster and Rosai.1,2 Similar neoplasms have been previously reported as intranodal schwannoma.1 The age of the patients ranged between 19-70 years, and more men are affected than women.2 Even though most of the cases occurred in inguinal lymph nodes, the submandibular, cervical and mediastinal lymph nodes location also have been reported.2,3 IPM is generally characterized by proliferation of spindle cells, interstitial hemorrhage and amianthoid fibers. Metaplastic bone formation and calcification have been also reported.3,4 IPM has also been reported as multicentric1 and recurrent.2 The neoplasm appears to be benign. Metastases have not been reported. Local excision is adequate and curative.5