Volume 121  ·   Issue 05  ·  2008

Abstract：Background Multidrug resistance to chemotherapeutic agents is an important clinical problem during the treatment of leukemia.The resistance process is multifactorial.To realize the totaI factors involved in multidrug resistance,we analyzed the differentially expressed proteins of K562 and K562/ADM cells and we investigated one of the up-regulated proteins(CRKL)using siRNA to determine its role in K562/ADM cells.Methods Altered protein expressions between K562/S(K562 ADM-sensitive cell line)and K562/ADM(K562 multidrug resistant cell line induced by adriamycin)were identified by 2D-DIGE coupled with mass spectrometry. Meanwhile,we confirmed the differential expression of CRKL and Stathmin in both K562 and K562/ADM cells by Western blot analysis.Furthermore,we used RNA interference to silence the CRKL gene expression.Results Among the 9 differentially expressed proteins,3 were up-regulated in K562/ADM cells,while 6 were down-regulated in the K562/ADM cells compared with its parent cell line.The expression of CRKL was up-regulated significantly in K562/ADM cells,and it can be decreased by recombinant lentivirus.Moreover,the multidrug resistance of K562/ADM cells was efficiently reversed by silence of CRKL gene expression.Conclusions The data provided the differentially expressed proteins jn K562 and jts resistant cell line and highlights the power of 2D-DIGE for the discovery of resistance markers in cancer.We found CRKL may be a new protein involved in the multidrug resistanse of leukaemia cells。

Abstract：Background Animal models are needed for the study of rapid tooth movement into the extraction socket through distraction osteogenesis of the periodontal ligament.Methods Modified distraction devices were placed on eight dogs between the first and third mandibular premolars on the left sides;similar placement of traditional straight wise appliances on the right sides served as the control.The experimental distractors were activated(0.25 mm/d)twice a day and the control devices were activated(100 g)for two weeks with consolidation periods at weeks two,three,six,and ten.Two dogs were sacrificed at each consolidation time point;rates and patterns of tooth movement,loss of anchorage,and periapical films were evaluated,and the aftected premolars and surrounding periodontal tissues were decalcified and examined histologically.General observations,X-ray periapical filming and histology examination were performed.Results Distal movement((3.66±0.1 4)mm)measured two weeks after modified distraction exceeded that achieved using the traditional device((1.15±0.21)mm;P＜0.05).Loss of anchorage was minimally averaged(0.34±0.06)mm and (0.32±0.07)mm in the experimental and control sides,respectively.By radiography,apical and lateral surface root resorptions on both sides were minimal.Alveolar bone Iesions were never evident.Fibroblasts were endched in periodontal ligaments and bone spicules formed actively along directions of distraction.Conclusions The canine model is suitable for the study of rapid tooth movement through distraction osteogenesis of the periodontal ligament.The technique accelerates tooth movement,periodontal remodeling,alveolar bone absorption,and may induce fibroblast formation,as compared to the traditional orthodontic method,without adversely affecting root absorption,bone loss,tooth mobility and anchorage loss。

Abstract：Background Cornea epithelial cells play early and crucial roles in the initiation of ocular surface responses to pathogens.Participation of toll-like receptor(TLR)2 and TLR4,which are major forms of fungi receptors,may be involved in Aspergillus fumigatus induced immune responses.The obiective of the present study was to examine whether inactive Aspergillus fumigatus conidia induce NF-κB activation and production of proinflammaory cytokines,and whether the expression of TLR2 and TLR4 were amplified by conidia in cultured immortalized human corneal epithelial cells (THCEs).This may contribute to our knowledge of the mechanism by which the host cornea can successfully defend against invasive fungi.Methods Aspergillus fumigatus conidia were used to challenge THCE cells.THCE cells were harvested after 0.5,1,2or 4 hours incubation.Real-time quantitative PCR was performed to determine the expression of TLR2,TLR4,TNF-α and IL-8.Western blotting was performed to determine the expression of NF-κB.Enzyme-linked immunosorbent assay (EUSA)was performed to determine the expression of TNF-α and IL-8.And the release of TNF-α and IL-8 in the cell supematant were also assessed by ELISA with or without pretreatment with TLR2 and TLR4 neutralizing antibodies.Results Aspergillus fumigatus conidia elicited the expression of TLR2,TLR4,TNF-α and IL-8 mRNA in THCEs.Exposure of THCE cells to Aspergillus fumigatus conidia resulted in NF-κB activation,which increased at 30 minutes (increased from 11.35±2.74 in the controls to 19.12±3.48,P＜0.05)and thereafter increased steadily up to 4 hours after challenge(P＜0.01).Concomitant with NF-κB acfivation,secretion of TNF-α and IL-8 in conidia-challenged cells was increased in a time-dependent manner.Incubation of THCE cells with TLR2 antibody or TLR4 antibody before conidia challenge resulted in jnhibifion of conidia-induced TNF-α and IL-8 secretion(P＜0.05),TLR2 antibody and TLR4 antibody together significantly increased inhibition of the conidia-induced secretion of TNF-α and IL-8 from THCE cells (P＜O.01).Conclusion Aspergillus fumigatus conidia stimulates THCEs inflammatory response through a pathway dependent on TLR2 and TLR4 siqnaling。

Abstract：Background Transcription factor Pokemon,a central regulation gene of the important tumor suppressor ARF gene,exerted its activity by acting upstream of many tumor-suppressing genes and proto-oncogenes.Its expression in non-small cell lung cancer (NSCLC)and its clinical significance remains unclear.The aim of this study was to investigate the expression of Pokemon in NSCLC and to explore its correlation with the clinical pathological characteristics and its influence on patients'prognosis.Methods Fifty-five cases of NSCLC were involved in this study.The expression of Pokemon in the tumor tissue,the corresponding tumor adjacent tissue and the surrounding tissue was detected via reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting,with the aim of investigating the correlation between the expression of Pokemon in tumor tissue of NSCLC and its clinicaI pathological characteristics.Moreover,a prognostic analysis was carried out based upon the immunohistochemical(IHC)detection of the expression of Pokemon gene in archival tumor specimens (5 years ago) of 62 cases of NSCLC.Results Statistical significance of the expression of Pokemon mRNA and protein was determined in the tumor tissue,the tumor adjacent tissue and the surrounding tissue (P＜0.05).The expression of Pokemon was determined not to be associated with the patients'sex,age,smoking condition,tumor differentiation degree,histology and lymph node metastasis condition.However,its relationship with TNM staging was established(P＜0.05).Furthermore,it was shown that the suwival rate of patients with negative Pokemon expression was significantly higher than that of those with positive Pokemon expression(P=0.004),therefore,the expression of Pokemon is believed to be an independent factor affectinq prognosis (P=0.034).Concluaion Pokemon was over-expressed in NSCLC tissue and the expression of Pokemon might be of clinical significance in non-small cell lung cancer prognostic evaluation。

Abstract：Background RNA interference(RNAi)technology is emerging as a very potent tool to generate a cellular knockdown of a desired gene.The aim of this study was to explore whether RNAi targleting vascular endothelial growth factor-C (VEGF-C)could inhibit colorectal tumor lymphangiogenesis and tumor growth.Methods We used vector-based RNAi to inhibit VEGF-C expression in colon cancer in vitro and in vivo.VEGF-C expression was quantified by real-time polymerase chain reaction and Westen blotting.To establish LoVo cell tumor xenografts in mice,we subcutaneously inoculated 1.0×1 06LoVo cells in nude mice;after injection,tumors were allowed to grow for 4 weeks until the volume reached (75.8±55.8)mm3.The mice were then randomly divided into two groups:(1)the VEGF-C-siRNA group(n=10)received direct injection of"therapeutic"plasmid 50 μg of LoVo-VEGF-C-siRNA into the tumor mass;(2)the control group(n=10)were injected with LoVo-control in 20 μl of sterile 0.9%NaCl solution into the tumor mass.Tumor growth,microlymphatics and microvessels were compared for mice administered either systemic VEGF-C-siRNA or control over 4 weeks.Results The mRNA and protein expression of VEGF-C in the colon tumor cell line,LoVo,stably transfected with a VEGF-C-siRNA vector,were significantly downregulated 45.3%and 35.3%respectively.In vivo,four weeks after injection,the tumor volume were significantly smaller in VEGF-C-siRNA group than in LoVo-control group((314.8±54.8)mm3vs (553.9±90.1)mm3);the incidences of lymph node metastasis(30%)in VEGF-C-siRNA were significantly inhibited compared with LoVo-control group(70%);in VEGF-C-siRNA group,the number of microlymphatics per microscopic field was(5.3±0.7)and the number of microvessels per microscopic field was(24.2±6.5)decreased compared with LoVo-control group(12.5±6.9)and(42.1±7.4)(all P＜0.001).Conclusion Inhibition of VEGF-C expression using siRNA-mediated gene silencing vectors reduced lymphangiogenesis and lymph node metastasis and enhanced suwival。

Abstract：Backgroud Interleukin 1β(IL-1 β)is the principal mediator in the pathogenesis of rheumatoid arthdtis.Continuous injection of interleukin 1β(IL-1β)into the knee articular cavities of anamals can induce models that resemble rheumatoid arthritis.The obiective of this study was to evaluate the feasibility of local recombinant retrovirus viral intedeukin 10(rRV-vIL-10)gene transfer treatment of a rabbit model of arthritis induced by IL-1β.Methods An hIL-1β-induced rabbit rheumatoid arthdtis model was established using the MFG-hIL-1β-neo-HIG-82 cell line,which is capable of continuous secretion of hIL-1a.After transfecting the rabbit synovial fibroblast cell line (MFG-hIL-1β-neo-HIG-82)with rRV-vIL-10,G418 was then added to identify the positive clone.The rRV-vIL-10 positive clone was injected into the established rabbit rheumatoid arthritis model through intra-articular injection.Successful gene transfer was determined by reverse transcription-polymerase chain reaction(RT-PCR)and immunohistochemistry.The levels of IL-1β before and after treatment were determined by enzyme-linked immunosorbent assay.Results Retrovirus vector was an effective vector both to synoviocytes in vitro and synovium tissue in vivo as confirmed by RT-PCR and immunohistochemistry.The rabbit arthritis model treated with rRV-vIL-10 showed a dramatic remission of arthritis and a decline in the level of cytokines such as IL-1β.Conclusions Retrovirus-mediated transfection of vIL-10 successfully transferred the gene into rabbit syrnovium ex vivo and was able to suppress intra-articular inflammation response to IL-1β。

Abstract：Background Hereditary spastic paraplegia(HSP)is a group of inherited neurodegenerative disorders with the shared characteristics of slowly progressive spasticity and weakness of the lower limbs.Thirteen loci for autosomal dominant HSP have been mapped.Methods A Chinese family with HSP was found in the Shandong province and Inner Mongolia Autonomous Region of China and genomic DNA of all 19 family members was isolated.After exclusion of known autosomal dominant loci,a genome wide scan and linkage analysis were performed.Results The known autosomal dominant loci of SPG3A,SPG4,SPG6,SPG8,SPG9,SPG10,SPGl2,SPG13,SPG17,SPG19,SPG29,SPG31 and SPG33 were excluded by linkage analysis.The results of a genome wide scan demonstrated candidate linkage to a locus on chromosome 11p14.1-p11.2,over an 18.88 cM interval between markers D11S1324 and D11S1933.A maximal,two point LOD score of 2.36 for marker D11S935 at a recombination fraction(Φ)of 0 and a multipoint LOD score of 2.36 for markers D11S1776,D11S1751,D11S1392,D11S4203,D11S935,D11S4083,and D11S4148at Φ=0,suggest linkage to this locus.Conclusion The HSP neuropathy in this family may represent a novel genetic entity,which will facilitate discovery of this causative gene。

Abstract：Background Proliferation,cell migration and phenotypic modulation of airway smooth muscle cells(ASMCs)are important features of airway remodelling in asthma.The precise cellular and molecular mechanisms that regulate ASMCs proliferation,migration and phenotypic modulation in the lung remain unknown.Basic fibroblast growth factor(bFGF),a highly specific chemotactic and mitogenic factor for many cell types,appears to be involved in the development of airway remodelling.Our study assessed whether bFGF directly stimulates the proliferation,migration and phenotypic modulation of ASMCs.Methods Confluent and growth arrested human ASMCs were treated with human recombinant FGF.Proliferation was measured by BrdU incorporation and cell counting.Migration was examined using Boyden chamber apparatus.Expressions of smooth muscle(sm)-α-actin and sm-myosin heavy chain(MHC)isoform 1 were determined by RT-PCR and Western blot analysis.Results It was found that hrbFGF(10 ng/ml),when added to ASMCs,induced a significant increase in BrdU uptake and cell number by ASMCS as compared to controls and a significant increase in ASMCs migration with respect to controls.The mRNA and protein expressions of sm-α-actin and sm-MHC in ASMCs that were stimulated with hrbFGF decreased with respect to controls.Conclusion It appears that bFGF can directly stimulate proliferation and migration of ASMCs.however,the expressions of cells'contractive phenotype decreased。

Abstract：Background The balance between vasodilation and vasoconstriction plays a maior role ln maintaining vascular homeostasis.However,the underlying mechanisms are unclear.More and more evidence suggested that there was an interaction in the regulation of vasorelaxation between nitric oxide(NO)and hydrogen sulfide(H2S).We explored the interaction between and effects of NO and H2S on the relaxation of pulmonaw arteries in rats.Methods Seven male Sprague-Dawley rats were anaesthetized with chloral hydrate and the pulmonary arteries of each rat separated for the study of vascular activities.The vasorelaxing activities of pulmonary artery rings in response to different doses of a NO donor,sodium nitroprusside(SNP),or a H2S donor,sodium hydrogensulfide(NaHS),were measured in vitro.When pulmonary artery rings were treated with a cystathionine-y-lyase inhibitor,DL-propargylglycine,in the presence of SNP or a nitric oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester,in the presence of NaHS,the changes in relaxing activities were analyzed.Results The relaxation of pulmonary artery rings was in a dose dependent manner in response to either SNP or NaHS.The relaxation rates of pulmonaw artery rings increased from(30.90±4.62)%10(60.50±8.08)%when the concentration of SNP increased from 1 μmol/L to 3 μmol/L and from(26.13±4.12)%to(53.09±14.01)%when the concentration of NaHS increased from 25 μmol/L to 100 μmol/L.However,when appropriate inhibitor was added.the relaxation responses to SNP and NaHS decreased.Conclusions The results suggested that similarly to NO,H2S acted as a vasorelaxant either independently of,or synerqistically with NO in the regulation of vasorelaxation.The interaction between NO and H2S played an important role in regulating relaxing activities of pulmonary arteries。

Abstract：Background The mechanism of cerebral vasospasm following subarachnoid haemorrhage(SAH)is not understood.Here,we hypothesized that apoptosis of endothelial cells induced by p53 and its target gene em dash p53 upregulated modulator of apoptosis(PUMA)played an important role in development of cerebral vasospasm.We also observed the effects of a p53 inhibitor,pifithrin-α(PFT-α),on reducing the expression of p53 and PUMA,consequently decreasing the apoptosis of endothelial cells and alleviating cerebral vasospasm.Methods Male Sprague-Dawley rats weighing 300-350 g were randomly divided into five groups:a control group (sham surgery),a SAH group,a SAH+dimethyl sulfoxide(DMSO)group,a SAH+PFT-α(0.2 mg/kg)group and a SAH+PFr-α(2.0 mg/kg)group.PFT-α was injected intraperitoneally immediately after SAH.Rats were sacrificed 24hours after SAH.Western blot and immunohistochemical staining were used to detect the levels of p53,PUMA and caspase-3 protein.In addition,mortality and neurological scores were assessed for each group.Statistical significance was assured by analysis of variance performed in one way ANOVA followed by the Tukey test.The neurological and mortality scores were analyzed by Dunn's method and Fisher exact test,respectively.Results After SAH,Western blot and immunohistochemical staining showed the levels of p53,PUMA and caspase-3 in the endothelial cells and the numbers of TdT mediated dUTP nick end labelling(TUNEL)positive endothelial cells were all significantly increased in the basilar arteries (P＜0.05),but significantly reduced by PFT-α(P＜0.05).These changes were accompanied by increasing diameters and declining wall thickness of basilar arteries(P＜0.05),as well as reduced mortality and neurological deficits of the rats(P＜0.05).Conclusions PFT-α could protect cerebral vessels from development of vasospasm and improve neurological outcome as well as reduce the mortality via suppressing apoptosis induced by p53 in the endothelial cells of cerebral vessels。

Prognostic factors are biological or physical characteristics of a oatient or the Datient's cancer that can be used to predict the outcome of the individual.1,2

Vascular calcification is commonly found in atherosclerosis and recognized as a marker of atherosclerotic plaque burden.Many evdiences have demonstrated that vascular calcification is an active process and can be seen in all stages of development and intimately associated with atherosclerosis。

Thymic carcinoma is a malignancy of the anterior mediastinum with a poor prognosis that is thought to be derived from thyrnic epithelium and they represent approximately 6% of primary mediastinal tumors.1Because of the lack of literature discussing the clinical and pathologic features and treatment Of this tumor,thymic carcinoma has been a somewhat controversial disease up to now。

Coronarv artery fistula(CAF), an uncommon congenital heart disease,often results in myocardial ischemia.1-3 In the last two decades,there are some reports about interventional treatment of CAF,4-6but few on transcatheter treatment of multiple CAFS.With different interventional procedures, we treated successfully two cases of percutaneous closure of two multiple CAFs which were confluent at the distal ends.…… Biography：Dr. SHEN Xiang-qian,Department of Cardiology,Second Xiangya Hospital,Central South University,Changsha,Hunan 410011,Chirla(Tel:86-731-5292012.Email:xiangqian19982008@yahoo.com.cn)Correspondence to

To the Editor:As we know.Calculus Boris(Niuhuang in Chinese,the gallstone of Bos taurus domesticus Gmelin)is one of the most precious and commonly-used medicinal materials in China.1