2007年 · 第120卷第09期

Abstract：Background Porcine liver performing efficient physiological functions in the human body is prerequisite for successful liver xenotransplantation. However, the protein differences between pig and human remain largely unexplored. Therefore,we investigated the liver expression profile of a highly inbred minipig line.Methods A cDNA library was constructed from liver tissue of an inbred Banna minipig. Two hundred randomly selected clones were sequenced then analysed by BLAST programme.Results Alignments of the sequences showed 44% encoded previously known porcine genes. Among the 56% unknown genes, sequences of 72 clones had high similarities with known genes of other species and the similarities to human were mostly above 0.80. The other 40 clones showing no similarity to genes in National Centre for Biotechnology Information are newly discovered, expressed sequence tags specific to liver of inbred Banna minipig. Twenty-two of the 200 clones had full length encoding regions, 38 complete 5' terminal sequences and 140 complete 3' terminal sequences.Conclusion These newly discovered expression sequences may be an important resource for research involving physiological characteristics and medical usage of inbred pigs and contribute to matching studies in xenotransplantation。

Abstract：Background Vascular endothelial growth factor plays a key role in human colorectal carcinoma invasion and metastasis. However, the regulation mechanism remains unknown. Recent studies have shown that several cytokines can regulate the expression of vascular endothelial growth factor in tumor cells. In this study, we investigated whether hepatocyte growth factor can regulate the expression of vascular endothelial growth factor in colorectal carcinoma cells.Methods Hepatocyte growth factor and vascular endothelial growth factor in human serum were measured by ELISA.The mRNA level of vascular endothelial growth factor was analyzed by reverse transcription-PCR. Western blot assay was performed to evaluate levels of c-Met and several other proteins involved in the MAPK and PI3K signaling pathways in colorectal carcinoma cells.Results Serum hepatocyte growth factor and vascular endothelial growth factor were significantly increased in colorectal carcinoma subjects. In vitro extraneous hepatocyte growth factor markedly increased protein and mRNA levels of vascular endothelial growth factor in colorectal carcinoma cells. Hepatocyte growth factor induced phosphorylation of c-Met, ERK1/2 and AKT in a dose-dependent manner. Specific inhibitors on MEK and PI3K inhibited the hepatocyte growth factor-induced expression of vascular endothelial growth factor in colorectal carcinoma cells.Conclusion This present study indicates that hepatocyte growth factor upregulates the expression of vascular endothelial growth factor in colorectal carcinoma cells via the MEK/ERK and PI3K/AKT signaling pathways。

Abstract：Background Percutaneous kyphoplasty (PKP) using balloon expander has been proved to be effective in the treatment of painful vertebral compression fractures. Recently, Sky-bone expander, an alternative bone expander for PKP has been developed. The purpose of this study was to show our preliminary clinical outcomes of PKP with Sky-bone expander.Methods PKP with Sky-bone expander was performed in 25 patients (30 vertebrae). The operation time, bleeding volume, cement volume injected were recorded. The pain and functional activities of the patients before and after the operation were compared using Wilcoxon signed-rank test. The cement distribution in the vertebrae, vertebral height restoration, and kyphosis correction after the procedure were evaluated by radiography. The pre- and post-operative absolute values of the vertebral height and kyphotic angle were compared by paired-sample t test. All the patients were followed up by telephone or clinic consulting after being discharged from our hospital.Results The procedure was performed successfully in all the patients. Bipedicular injection was used in 2 of the patients, and unipedicular injection was made in the others. The operation time ranged from 25 to 120 minutes (45 minutes per vertebra on average). The average bleeding volume was about 20 ml. Polymethylmethacrylate1.5 - 5.0 ml (mean, (3.15±0.78) ml) was injected through each pedicle into all the patients except one, who received calcium sulphate 3.5 ml instead. The patients were followed up for 12-15 months (13.5 months on average). The mean visual analogue scale (VAS) score, Oswestry Disability Index, anterior, midline, and posterior vertebral height, and kyphotic angle of the patients were improved significantly at the end of the follow-up compared with those before the operation.(2.5±1.3, 35.1%, (20.94±6.15) mm, (20.26±4.59) mm, (26.72±3.49) mm, and 8.2 degrees vs. 8.5±1.9, 61.2%, (19.11±6.72) mm, (15.88±5.73) mm, (25.78±3.67) mm, and 17.3 degrees; all P＜0.05). The cement distribution with unipedicular injection was mostly limited within the injection site in the vertebral body. Cement extravasation was seen at ten levels (33.3%).Conclusions PKP with Sky-bone expander is an effective and relatively safe alternative to the PKP using balloon expander. It can relieve pain, improve physical function, and restore the height of the collapsed vertebrae, but the cement extravasation is unsolved。

Abstract：Background Bone age development is one of the significant indicators depicting the growth status of children.However, bone age assessment is an heuristic and tedious work for pediatricians. We developed a computerized bone age estimation system based on the analysis of geometric features of carpal bones.Methods The geometric features of carpals were extracted and analyzed to judge the bone age of children by computerized shape and area description. Four classifiers, linear, nearest neighbor, back-propagation neural network,and radial basis function neural network, were adopted to categorize bone age. Principal component and discriminate analyses were employed to improve assorting accuracy.Results The hand X-ray films of 465 boys and 444 girls served as our database. The features were extracted from carpal bone images, including shape, area, and sequence. The proposed normalization area ratio method was effective in bone age classification by simulation. Besides, features statistics showed similar results between the standard of the Greulich and Pyle atlas and our database.Conclusions The bone area has a higher discriminating power to judge bone age. The ossification sequence of trapezium and trapezoid bones between Taiwanese and the atlas of the GP method is quite different. These results also indicate that carpal bone assessment with classification of neural networks can be correct and practical。

Abstract：Bckground Stem cells, which have the ability to differentiate into insulin-producing cells (IPCs), would provide a potentially unlimited source of islet cells for transplantation and alleviate the major limitations of availability and allogeneic rejection. Therefore, the utilization of stem cells is becoming the most promising therapy for diabetes mellitus (DM). Here,we studied the differentiation capacity of the diabetic patient's bone marrow-derived mesenchymal stem cells (MSCs) and tested the feasibility of using MSCs for β-cell replacement.Methods Bone marrow-derived MSCs were obtained from 10 DM patients (5 type 1 DM and 5 type 2 DM) and induced to IPCs under a three-stage protocol. Representative cell surface antigen expression profiles of MSCs were analysed by flow cytometric analysis. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect multiple genes related to pancreatic β-cell development and function. The identity of the IPCs was illustrated by the analysis of morphology, ditizone staining and immunocytochemistry. Release of insulin by these cells was confirmed by immunoradioassay.Results Flow cytometric analysis of MSCs at passage 3 showed that these cells expressed high levels of CD29 (98.28%), CD44 (99.56%) and CD106 (98.34%). Typical islet-like cell clusters were observed at the end of the protocol (18 days). Ditizone staining and immunohistochemistry for insulin were both positive. These differentiated cells at stage 2 (10 days) expressed nestin, pancreatic duodenal homeobox-1 (PDX-1), Neurogenin3, Pax4, insulin, glucagon, but at stage 3 (18 days) we observed the high expression of PDX-1, insulin, glucagon. Insulin was secreted by these cells in response to different concentrations of glucose stimulation in a regulated manner (P＜0.05).Conclusions Bone marrow-derived MSCs from DM patients can differentiate into functional IPCs under certain conditions in vitro. Using diabetic patient's own bone marrow-derived MSCs as a source of autologous IPCs for β-cell replacement would be feasible。

Abstract：Background p38 mitogen-activated protein kinases (MAPK) in ischemic preconditioning (IPG) may be essential to cardioprotection. We assessed whether protective effect of morphine-induced preconditioning (MPC) on myocardial ischemia and reperfusion injury in rat hearts involved p38 MAPK activation.Methods Male Spargue-Dawley rats (weighing 300-350 g) were randomly assigned to 1 of the following 8 groups:control (CON, saline vehicle, n=9), SB 203580 (SB, a p38 MAPK inhibitor, n=-6), MPC (n=-6), IPC (n=-9), SB+MPC,SB+IPC, MPC+SB, and IPC+SB (n=6). Infarct sizes (IS), a percentage of the area at risk (AAR), were determined by triphenyltetrazolium (TTC) staining. Tissue samples were processed from the entire AAR of left ventricle for the determination of p38 MAPK protein expression (5 hearts/group). The bands representing the proteins were visualized using an enhanced chemiluminescence detection system.Results The IS/AAR was significantly reduced by IPC (12.9±1.6)% or MPC (25.3±2.9)% compared to the control (52.7±5.5)%. SB 203580 administered prior to preconditioning abolished the effect of IPC (SB+IPC: (43.8±2.6)%,P＞0.05 vs CON, P＜0.01 vs IPC), but not MPC (SB+MPC: (30.7±0.9)%, P＜0.01 vs CON, P＞0.05 vs MPC). Treatment with SB 203580 prior to sustained ischemia diminished the protective effect of both MPC (MPC+SB: (42.4±2.9)%,P＞0.05 vs CON) and IPC (IPC+SB: (52.0±2.5)%, P＞0.05 vs CON) on IS/AAR. In the IPC group, phospho-p38 MAPK protein increased significantly within 5 minutes into ischemia and remained elevated at 30 minutes into reperfusion,while phospho-p38 MAPK protein in the MPC group only increased significantly at 30 minutes into reperfusion.Conclusion The activation of p38 MAPK just acts as a mediator of MPC,whereas it acts as both a trigger and a mediator in IPC。

Abstract：Background The renin-angiotensin-aldosterone system (RAAS) is important for the development of essential hypertension, and many antihypertensive drugs target it. This study was undertaken to determine whether polymorphisms in the renin-angiotensin-aldosterone system are related to the blood pressure (BP) response to diuretic treatment in a Chinese Han ethnic population.Methods Fifty-four patients with essential hypertension received hydrochlorothiazide (12.5 mg, once daily) as monotherapy for four weeks. Seven polymorphisms in RAAS genes were genotyped by gene chip technology. The relationship between these polymorphisms and the change in blood pressure was observed after the 4-week treatment.Results The patients with angiotensinogen (AGT) -6G allele showed a greater reduction in diastolic BP (P= 0.025) and mean BP (P=0.039) than those carrying AA genotype. Patients carrying aldosterone synthase (CYP11B2) CC genotype exhibited a greater BP reduction than those carrying CT and TT genotypes (systolic BP: P= 0.030; diastolic BP: P= 0.026; mean BP: P=0.003). In addition, patients with a combination of CYP11B2 CC genotype and angiotensin converting enzyme (ACE) D allele might have a more pronounced reduction of systolic BP than those with any other genotypic combinations of the two genes (P= 0.007).Conclusions AGT-6G allele, CYP11B2 -344CC genotype and its combination with ACE D allele are associated with BP response to hydrochlorothiazide treatment. Larger studies are warranted to validate this finding。

Abstract：Background Advanced glycation end products (AGEs) play a critical role in the development of diabetic nephropathy. Reactive oxygen species (ROS) may play a critical role in AGEs induced growth factor expression. In this study, the effects of AGEs on transforming growth factor β1 (TGF-β1), connective tissue growth factor (CTGF) and fibronectin (Fn) mRNA expression and oxidative stress in cultured NRK-49F cells were examined.Methods NRK-49F cells were incubated with medium containing different doses of AGEs (50, 100 or 200 μg/ml) for 24 hours, or with AGEs 100 μg/ml for different times (0, 12, 24 or 48 hours). Cells in the serum-free medium or medium containing 25 mmol/L glucose were controls. Cells were treated with 25 mmol/L glucose and 100 μg/ml AGEs for 24 hours to determine the effects between AGEs and glucose. We clarified the role of antioxidant by pretreating cells with N-acetylcysteine (10 mmol/L), ginkgo biloba extract (50 or 100 mg/L) for 24 hours and with 100 μg/ml AGEs for further 24 hours. Alamarblue dye assay was used to analyze cell growth; intracellular ROS generation was measured by flow cytometry; intracellular glutathione by fluorescence spectrophotometry; expressions of TGF-β1, CTGF and Fn mRNA by semiquantitative RT-PCR.Results AGEs significantly increased the expressions of TGF-β1, CTGF, Fn mRNA and intracellular ROS generation,and decreased the glutathion level in NRK-49F cells in dose- and time-dependent manners. High glucose and AGEs together significantly increased the expression of TGF-β1, CTGF and Fn mRNA, compared with AGEs and high glucose separately. Preincubation with N-acetylcysteine or ginkgo biloba extract increased GSH level, suppressed AGEs-induced oxidative stress and TGF-β1, CTGF and Fn mRNA overexpression.Conclusions AGEs can significantly increase expression of TGF-β1, CTGF, Fn mRNA in NRK-49F cells through enhancement of oxidative stress. The accumulation of AGEs may play a pivotal role in the pathogenesis of tubulointerstitial fibrosis in diabetic nephropathy. Suppression of AGEs induced TGF-β1, CTGF and Fn mRNA overexpression in renal fibroblasts through inhibition of oxidative stress may be a mechanism underlying effect of ginkgo biloba extract in diabetic nephropathy. In addition, antioxidant therapy may help prevent AGEs accumulation and its induced damage。

Abstract：Background The function of peroxisome proliferator-activated receptor γ (PPARγ) in hepatic fibrogenesis remains largely unknown. Curcumin is a natural substance extracted form Curcuma Longa Linn and has a variety of pharmacological effects. In this study, the effects of curcumin on the proliferation, activation and apoptosis of rat hepatic stellate cells (HSCs) through PPARγ signaling were investigated.Methods HSCs were isolated from the normal Sprague Dawley rats through in situ perfusion of the liver with Pronase E and density-gradient centrifugation with Nycodenz. Cells were treated with curcumin, troglitazone, salvianolic acid B or GW9662. The effect on HSCs proliferation was determined by MTT colorimetry. Total RNA was extracted by TRizol reagent and gene levels were determined by semi-quantitative RT-PCR. Total cellular and nuclear protein were isolated and separated by 10% sodium dodecy Isulfate polyacrylamide gel electrophoresis. Protein levels were determined by Western blot. Cell apoptosis was detected by Hoechst 33258 staining. PPARγ subcellular distribution was detected by immunofluorescent staining. The activities of MMP-2 and 9 were measured by Gelatin zymograph assay.Results Curcumin suppressed HSCs proliferation in a dose-dependent manner. As HSCs underwent gradual activation with culture prolongation the PPARγ nuclear expression level decreased. Curcumin up-regulated PPARγ expression and significantly inhibited the production of α-SMA and collagen I. PPARγ is expressed in the cytoplasm and nucleus and is evenly distributed in HSCs, but accumulated in the nucleus of HSCs and disappeared from cytoplasm after curcumin treatment. Hoechst 33258 staining showed that curcumin induced the apoptosis of culture-activated HSCs and significantly increased pro-apoptotic Bax expression and reduced anti-apoptotic Bcl-2 expression. Cyclin D1 gene, activated NFκB p65 protein and TGFβR-I protein expression were down-regulated significantly by curcumin. The activities of MMP-2 and MMP-9 were enhanced significantly by curcumin.Conclusions Curcumin can inhibit the proliferation and activation of HSCs, induce the apoptosis of activated HSCs and enhance the activities of MMP-2 and MMP-9. The effects of curcumin are mediated through activating the PPARγ signal transduction pathway and associated with PPARγ nuclear translocation/redistribution。

Abstract：Background Currently, resistance and relapse are still major problems in acute promyelocytic leukemia (APL) cases.Thus, new agents that override the resistance are crucial to the development of curative therapies for APL. In this study,we investigated the effects of berbamine on the proliferation of APL cell line NB4 and its possible mechanisms.Methods NB4 cells were treated with berbamine at different concentrations (0-64 μg/ml) for 72 hours. MTT assay was used to determine proliferation inhibition of NB4 cells. Cell apoptosis was evaluated by both flow cytometry (FCM) and morphological examination. PML/RAR-α and survivin mRNAs were measured by nested-RT-PCR and RT-PCR,respectively. Activated-caspase 3 was determined by FCM.Results Berbamine greatly inhibited the proliferation of NB4 cells in dose- and time-dependent manners, and its IC50 value was 3.86 μg/ml at 48 hours. Both morphological observations and FCM results showed that berbamine induced apoptosis of NB4 cells with concomitant increase of activated caspase-3 and decrease of survivin mRNA. After treatment with berbamine at 8 μg/ml for 48 hours, the percentage of apoptotic cells increased from 2.83% to 58.44% (P＜0.01), and the percentage of cells with activated-caspase 3 elevated from 2.06% to 70.89% (P＜0.01), whereas, level of survivin mRNA was reduced to 38.24% of control (P＜0.01). However, no significant change was observed in PML/RAR-α mRNA.Conclusions Berbamine induces caspase-3-dependent apoptosis of leukemia NB4 cells via survivin-mediated pathway, suggesting that berbamine may be a novel potential agent against APL with a mechanism distinct from that of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO)。

Abstract：Background In most colorectal carcinomas, the level of phospholipase C (PLC)-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the mechanism is not well known. The aim of this study was to evaluate the role of PLC-gamma 1 in colon carcinogenesis by using recombinant lentivirus that stably suppressed the PLC-gamma 1 expression in human colorectal carcinoma LoVo cells.Methods Recombinant lentivirus producing PLC-gamma 1 siRNA were prepared. After LoVo cells were transduced by each lentivirus, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC-gamma 1 were examined by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the effects of the lentivirus on the cell adhesion, migration and apoptosis were analyzed.Results Stable LoVo cell line deficient in PLC-gamma 1, was established. Notably, PLC-gamma 1 was silenced without affecting the levels of other subtypes of PLC so that the role of PLC-gamma 1 in colon carcinogenesis could be examined.Silencing of endogenous PLC-gamma 1 resulted in efficient inhibition of the adhesion and migration of LoVo cells in vitro and a great increase of 5-fluorouracil induced apoptosis (30%-40%) of LoVo cells.Conclusions PLC-gamma 1 may play an important role in metastasis and anti-apoptosis in human colorectal carcinomas。

Abstract：Background Telomerase activity is found in 85%-90% of all human cancers but not in their adjacent normal cells.Human telomerase reverse transcriptase (hTERT) is an essential component in the telomerase complex that plays an important role in telomerase activity. This study investigated the effect of the telomerase inhibition with an hTERT antisense oligodeoxynucleotide (ODN) in bladder cancer cells (T24) on tumor necrosis factor-o (TNF-α)-induced apoptosis.Methods Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA expression was measured by reverse transcription polymerase chain reaction (RT-PCR) assay and a gel-image system.hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by a morphological method and determined by flow cytometry.Results AS PS-ODN significantly inhibited telomerase activity and decreased the levels of hTERT mRNA which preceded the decline in the telomerase activity. AS PS-ODN significantly reduced the percentage of positive cells expressing hTERT protein following the decline of hTERT mRNA levels. There was no difference seen in the telomerase activity, hTERT mRNA expression or the protein levels between the sense phosphorothioate oligodeoxynucleotide (SPS-ODN) and the control group. AS PS-ODN treatment significantly decreased the cell viability and enhanced the apoptotic rate of T24 cells in response to TNF-α while there was no difference in cell viability and apoptotic rate between the S PS-ODN and the control group.Conclusions AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression. Treatment with AS PS-ODN may be a potential and most promising strategy for bladder cancer with telomerase activity。

There are two types of Human Immunodeficiency Virus (HIV): HIV-1 and HIV-2. HIV-1 dominates epidemics in many different parts of the world, and HIV-2 is principally responsible for the epidemic in western Africa. In China, Zeng et al1 have reported the first individual infected with HIV-1 in 1985. And in the 1990s,there was a severe epidemic involving the HIV-1 B' strain among people who sold blood and plasma in Henan,Hubei and adjacent provinces.2 To further study in HIV/AIDS vaccines and HIV-1 drug resistance for people in these regions, we need to construct an infectious HIV-1 B' molecular clone which is representative of the virus in these areas.3 To this end, we have isolated a HIV-1 B' virus from a child who was infected with HIV-1 from his mother in Hubei province, and have constructed a full-length clone from this genome。

Hereditary spastic paraplegia (HSP) (MIM#182600) is a group of heterogeneous neurodegenerative disorders, with 35 underlying loci recognized by the HGNC (HUGO Gene Nomenclature Committee;http://www.gene.ucl.ac.uk/nomenclature/) and 10 identified genes ( http://www.gene.ucl.ac.uk/cgi-bin/nomenclature/searchgenes.pl plus NIPA1, last search July 2006). The mode of inheritance may be autosomal dominant,autosomal recessive or X-linked. Among these, autosomal dominant spastic paraplegia (AD-HSP) is the most common type, accounting for 70%-80% of all families.1The disease is characterized by lower limb spasticity,hyperreflexia, progressive spastic gait and an extensor plantar response. Pes cavus is one of the commonly reported foot phenotypes.2

Desmoid-type fibromatoses are clonal fibroblastic proliferations that arise in deep soft tissues and are characterized by infiltrative growth and a tendency toward local recurrence。

Post-transplant lymphoproliferative disorder (PTLD), a rare disease, is characterized by an abnormal proliferation of lymphoid cells after solid organ transplantation.1 This complication is usually caused by the immunosuppressive therapy following transplantation.Though the techniques of early detection and diagnosis of the disease are well established, treatment is not so straightforward and poses a real challenge. At this time,options include anti-viral therapy, cytotoxic chemotherapy, cellular immunotherapy, and reduction of immunosuppression. But the effects of these therapies are not satisfying. Rituximab, a chimeric monoclonal anti-CD20 antibody used for the treatment of B cell lymphoma with a good effect, is rarely, especially in combination with chemotherapy, used for PTLD. In this report, we describe a case of PTLD treated with rituximab and chemotherapy resulting in complete remission。

Primitive neuroectodermal tumors (PNETs), derived from the primitive neural crest, are highly malignant and mainly exist in the central nervous system (CNS),chest wall, lower extremities, trunk, kidney, and orbit but rarely in the spine. Though multidisciplinary treatments have been well established as the standard therapy for intracranial PNETs, little is known about the optimal treatment strategy and therapeutic results for intraspinal PNETs. The following report illustrates the operative and non-operative management of an extradural PNET at the level of L5 in a young girl。

Airway-centered interstitial fibrosis (ACIF), a novel form of diffuse interstitial lung disease (ILD) of unknown cause, was recently presented.1 There is no final conclusion on its property and denomination, and it might be a new type of idiopathic interstitial pneumonia (ⅡP)。

Acro-dermato-ungual-lacrimal-tooth syndrome (ADULT syndrome, OMIM 103285) is a rarely seen ectodermal dysplasia disorder first described by Propping and Zerres in 1993.1 ADULT syndrome is known as an autosomal dominant disorder. Only a family constellation1,2 and four independent cases3-6 were reported worldwide up to now. Here, we report a case of ADULT syndrome, which is the first case reported from China。

Tuberous sclerosis complex (TSC) is one of the most commonly identified neurocutaneous disorders with a prevalence of 1/6000 to 1/9000 in general population1,2 In the patients with TSC, 10%-15% have subependymal giant cell astrocytoma (SGCA) .3