Expression of TIMP-1 and TIMP-2 in rats with hepatic fibrosis

AIM: To investigate the location and expression of TIMP-1 and TIMP-2 in the liver of normal and experimental hepatic fibrosis in rats. METHODS: The rat models of experimental immunity hepatic fibrosis (n=20) were prepared by the means of immunologic attacking with human serum albumin (HSA),and normal rats (n=10) served as control group. Both immunohistochemistry and in situ hybridization methods were respectively used to detect the TIMP-1 and TIMP-2 mRNA and related antigens in liver. The liver tissue was detected to find out the gene expression of TIMP-1 and TIMP-2 with RT-PCR. RESULTS: The TIMP-1 and TIMP-2 related antigens in livers of experimental group were expressed in myofibroblasts and fibroblasts (TIMP-1: 482±65 vs 60±20; TIMP-2:336±48 vs 50±19, P<0.001). This was the most obvious in portal area and fibrous septum. The positive signals were located in cytoplasm, not in nucleus. Such distribution and location were confirmed bysitu hybridization (TIMP-1/β-actin: 1.86±0.47 vs 0.36±0.08; TIMP-2/β-actin: 1.06±0.22 vs 0.36±0.08,P<0.001). The expression of TIMP-1 and TIMP-2 was seen in the liver of normal rats, but the expression level was very low. However, the expression of TIMP-1 and TIMP-2 in the liver of experimental group was obviously high. CONCLUSION: In the process of hepatic fibrosis, fibroblasts and myofibroblasts are the major cells that express TIMPs.The more serious the hepatic fibrosis is in the injured liver,the higher the level of TIMP-1 and TIMP-2 gene expression.     

Expression and function of classical protein kinase C isoenzymes in gastric cancer cell line and its drug-resistant sublines

AIM：To investigate the expression and function of classical protein kinase C(PKC)isoenzymes in inducing MDR phenotype in gastric cancer cells.METHODS:Two cell lines were used in the study:Gastric cancer cell SGC7901 and its drug-resistant cell SGC7901/VCR stepwise-selected by vincristine 0.3,0.7and 1.0mg.L^-1,respectively.THe expression of classical PKC(cPKC) isoenzymes in SGC7901 cells and SGC7901/VCR cells were detected using immunofluorescent cytochemistry,laser confocal scanning microscope and Western blot .The effects of anti-PKC isoenzymes antibody on adriamycin accumulation in SGC7901/VCR cells were setermined using flow cytometric analysis.RESULTS:(1) SGC7901 cells exhibited positive staining of PKC-αSGC7901／VCR cells exhibited stronger staining of PKC-αthan SGC790-1 cells.The higher dosage vincristine selected,the much stronger staining of PKC-α was observed on SGC7901/VCR cells.(2)BOth SGC7901 and SGC7901/VCR cells exhibited positive staining of PKC-βI and PKC-βⅡwith no significant difference.(3) Compared with SGC7901,SGC7901/VCR cells had decreased adriamycin accumulation and retention.Accumulation of adriamycin in SGC7901 was 5.21±2.56mg.L^-1,in SGC7901/VCR0.3was 0.85±0.29mg.L^-1,in SGC7901/VCR0.7was0.81±0.32mg.L^-1,and in SGC7901/VCR 1.0 was 0.80±0.33mg.L^-1;Retention of adriamycin in SGC 7901 was 2.51±1.23mg.L^-1,in SGC7901/VCR0.3 was 0.47±0.14mg.L^-1,in SGC7901/VCR0.7 was 0.44±0.15mg.L^-1,and in SGC 7901/VCR 1.0 was 0.41±0.11mg.L^-1.(4)Fluorescence intensity presented adriamycin accumulation in SGC7901/VCR cells was increased from 1.14±0.35to 2.71±0.94when cells were co-incubated with antiPKC-αbut not with anti-PKC-βI,PKC-βⅡand PKCγ antibodies. CONCLUSION:PKC-α.but not PKC-βⅠ,PKC-βⅡor PKCγ,may play a role in multidrug resistance of gastric cancer cells SGC7901/VCR.     

Significance of cyclooxygenase—2 expression in human primary hepatocellular carcinoma

AIM：To clarife the significance of cyclooxygenase-2(COX-2)expression in human primary hepatcellular carcinoma(HCC)and adjacent nontumorous tissues.METHODS;TheCOX-2protein and mRNA were investigated in 27HCC tissues with adjacent nontumorous tissues,and 5histologically normal liver tissues,using immunohistochemistry and in situ hybridization.RESULTS:The well-differentiated HCC expressed COX-2protein(5.68±1.19)more strongly than moderated HCC(3.43±1.98)and poor differentiated HCC(3.33±1.50)(P<0.05 respectively),adjacent nontumorous tissues(4.93±1.05)and normal live tissues(3.20±1.92)(P<0.01 respectively);More intensive staining of COX-2in adjacent nontumorous tissues was observed than that in normal liver tissues(P<0.05).There was no significant difference among adjacent nontumorous tissues,moderately differentiated HCC and poorly differentiated HCC(P>0.05).The expression of COX-2mRNA was observed in the cytoplasm of the cells of HCC and of gtthe hepatocytes in adjacent nontumorous tissues in which COX-2 protein was positive.CONCLUSION:The overexpression of COX-2 in well-differentiated HCsuggets that COX-2 may play a role in the early stages of hepatocarcinogensis.     

Overexpression of p28／gankyrin in human hepatocellular carcinoma and its clinical significance

AIM：To investigate the expression of p28/gankyringene and its role in the carcinogenetic process of human hepatocellular carcinoma(HCC).METHODS:64specimens of HCC and para-carcinoma tissues,22specimens of non-tumor liver tissues(7normal,15cirrhosis),10 specimens of normal human tissues and 5hepatoma cell lines were studied for the expression of p28/gankyrinby Northern blot.The expressionof p28/gankyrin protein was detected immunohistochemically by using the specific polyclonal antibody.RESULTS:Northern blot analysis indicated that the expression of p28/gankyrinmRNA was intensively distributed in brain and heart,weakly in lung,spleen and muscle,undetectable in digestive system including liver,pancreas,stomach,small and large intestines.p28/gankyrinmRNAwas absent in normal liver,weakly detected in liver cirrhosis and in 18of 64para-carcinoma liver tissues.In contrast,the expression of p28/gankyrinmRNA was intensitely detected in all5hepatoma cell lines tested,markedly increased in 57of 64and moderately increased in 5of64HCCsamples.In comparison with liver cirrhosis and para-carcinoma liver tissues.the average expression of p28/gankyrinmRNAin HCCwas increased3.6-(2.901±0.507vs0.805±0.252,P<0.05)and5.2-fold(2.901±0.507vs0.557±0.203,p<0.01),respectively.In addition,p28/gankyrinmRNA expression level was higher in HCCwith portal vein tumor thrombus and microscopic hepatic vein involvement(P=0.021and P=0.047,respectively).Theoverexpression of p28/gankyrin protein in HCCwas targeted in hepatic tumor cells,not in bile duct cells and other interstitial cells.Plays an important role and contributes to the metastasis potential in the process of carcinogenesis.p28/gankyrinmay become a specific biological tissue marker for the pathological diagnosis of HCC.     

Preparation of monoclonal antibody against apoptosis—associated antigens of hepatoma cells by subtractive immunization

AIM：To elucidate the expression of the apoptosis-associated molecules in human primary hepatocellular carcinoma(HCC)cells,and prepare the monoclonal antibodies(mAb)against the apoptosis-associated antigens of HCC cells.METHODS:Human HCC cell line HCC-9204 cells were induced apoptosis with 60mL·L^-1 ethanol for 6h and their morphological changes were obserevd by transmission electron microscope,The cell DNA fragmentations were detcted by Terminal Deoxynucleotidyl transferase-mediated dUTPnick end labeling(TUNEL)assay,and the cell DNA contents by flow cytometry.Ten mice were immunized with ethanol-induced apoptoticHCC-9204 cells with the method of subtractive immunization.while the other 10mice used as the control were immunized by the routin procedures.The tail blood of all the mice were prepared after the last immunization.and the produced antibodies were determined by the immunocytochemical ABC staining .The splenic cells of the mice whose tail blood sera-HCC-9204 cells serum reactions were most different between the apoptotic and the non-apoptotic were prepared an fused with the mouse myeloma cell lineSP2/0cells.The positive antibodies were selected by ELISA assay.The fusion rates of hybridona cells and the producing rates of antibodies were calculated.The fused cells that secreted candidate objective antibody were cloned continually with the of limited dilution method.and then selected and analyzed further by the immunocytochemical ABCstaining.The chromosomes of the cloned hybridoma cells that secreted objective mAb and the mAb immunoglobulin(Ig)subtpe of the prepared mAb were also determined.The molecular mass of the mAb associated antigen was analyzed by Western blot assay.RESULTS:HCC-9204 cells treated with60mL·L^-1 ethanod for 6h,manifested obvious apoptotic morphological changes,the majority of the cells wereTUNEL-positive,and the sub-G1 apoptotic peak was evident.There were 2mice in the experimental group whose tail blood serum reacted strongly with the apoptotic HCC-9204 cells,but weakly with their non-apoptotic counterparts,In the fusion rates of hybridoma cells as well as the producing rates of the antibody deseribed above,there did not show significant difference between the experimental and the control group,but weakly with non-apoptoticHCC-9204.However,the total producing rate of antibodies in the experimental group wa significantly lower compared with the control(P<0.01).and so was the producing rate of the Antibodies which racted strongly with both paoptotic and non-apoptotic HCC-9204cells(P<0.01).After cloned continually for several times the cell that produce mAb which reacted strongly with the nuclei of ethanol-induced apoptotic HCC-9204cells,but very weakly with that of non-apoptotic cells was selected out.Chromosme analysis revealed that the selected cell was with the universal characteristics of the monoclonal hybridoma cells which secreted mAb,and the Ig subtype of the prepared mAb was IgG1,The molecular mass of this mAb associated antigen of was about75ku.CONCLUSION :Subtractiv immunization is a useful method to prpar the mAb against the apoptosis-associated antigens of cells,The expression of some molecules increases to some extent in HCC-9204cells in the process of apoptosis induced by low-concentration ethanol.The mAb that may be against ethanol-induced apoptosis-associated antigens of HCC cells was successfully prepared and primarily identified.     

The role of KDR in the interactions between human gastric carcinoma cell and vascular endothelial cell

AIM：To study the interactions between human gastric carcinoma cell(HGCC)and human vascular endothelial cell(HVEC),and the role of KDRin these interactions.METHODS:Antisenes oligodexynucleotide(ASODN)specific to KDR gene was devised and added to the culture medium of HGCC and HIVEG.After the action of ASODN,the proliferation of two cells was mesured by MTTmethod.The role of KDR in regulating the proliferation of two kinds of cells was known through oroliferation of two kinds of cells was known through observing the effect of ASODN on them.The conditioned mediums(CMs)of HGCCand HVEC were prepared.The CMof one kind of cell was added acting on the other kind of cell,then the cell proliferation was measured byMTT,After the action of ASODNor CM,the cellular expression of KDR gene was detected with in situ hybridization(ISH)for mRNA level and with immunohistochemical staining for protein level.ABC-ELISA was used to detect hVEGFin the CMs of two cells.RESULTS:KDR ASODN could specifically inhibit the proliferation of HGCCand HVEC significantly.The growth inhibitory rate amounted to 55.35%and 54.83%,respectively(P<0.01).HGCC and HVECcould secreta certain level of hVEGF(92.06±1.69ng/L.77.70±8.04ng/L).The CMof HGCCcould significantly stimulate the growth(2.70±0.01times)and KDRgene expression of HVEC(P<0.01)while the CM of HVEC could significantly inhibit the growth(52.97±0.01%)and KDRgene expression of HGCC(P<0.01).CONCLUSION:KDR plays a key role in regulating the proliferation of HGCC and HVEC.There exist complicated interactions between HGCCand HVEC.HGCCcan significantly stimulate the growth of HVE while HVEC can significantly inhibit the growth of HGCC.KDRis involved in the interactions between them.     

Vascular endothelial growth factor antisense oligodeoxynucleotides with lipiodol in arterial embolization of liver cancer in rats

AIM:Transcatheter arterial embolization (TAE) of the hepatic artery has been accepted as an effective treatment for unresectable hepatocellular carcinoma (HCC). However,embolized vessel recanalization and collateral circulation formation are the main factors of HCC growth and recurrence and metastasis alter TAE. Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis.This study was to explore the inhibitory effect of VEGF antisense oligodeoxynucleotides (ODNs) on VEGF expression in cultured Walker-256 cells and to observe the anti-tumor effect of intra-arterial infusion of antisense ODNs mixed with lipiodol on rat liver cancer.METHODS: VEGF antisense ODNs and sense ODNs were added to the media of non-serum cultured Walker-256 cells.Forty-eight hours later, VEGF concentrations of supernatants were detected by EUSA. Endothelial cell line ECV-304 cells were cultured in the supernatants. Seventy-two hours later,growth of ECV-304 cells was analyzed by NTT method. Thirty Walker-256 cell implanted rat liver tumor models were divided into 3 groups.0.2 mL lipiodol (LP group, n=10), 3OD antisense ODNs mixed with 0.2 mL lipiodol (LP+ODNs group, n=10) and 0.2 mL normal saline (control group, n=10) were infused into the hepatic artery. Volumes of tumors were measured by MRI before and 7 d alter the treatment.VEGF mRNA in cancerous and peri-cancerous tissues was detected by RT-PCR. Microvessel density (MVD) and VEGF expression were observed by immunohistochemistry.RESULTS: Antisense ODNs inhibited Walker-256 cells‘ VEGF expression, The tumor growth rate was significantly lower in LP+ODNs group than that in LP and control groups (140.1±33.8%, 177.9±64.9% and 403.9±69.4% respectively, F=60.019,P<0.01).VEGF mRNAs in cancerous and peri-cancerous tissues were expressed highest in LP group and lowest in LP+ODNs group. The VEGF positive rates showed no significant difference among LP, control and LP+ODNs groups (90%,70% and 50%, H=3.731, P>0.05).The MVD in LP+ODNs group (53.1±18.4) was significantly less than that in control group (73.2±20.4) and LP group(80.3±18.5) (F=5.44, P<0.05).CONCLUSION: VEGF antisense ODNs can inhibit VEGF expression of Walker-256 cells.It may be an antiangiogenesis therapy agent for malignant tumors. VEGF antisense ODNs mixed with lipiodol embolizing liver cancer is better in inhibiting liver cancer growth, VEGF expression and microvessel density than lipiodol alone.     

Effects of c9,t11-conjugated linoleic acid on adhesion of human gastric carcinoma cell line SGC-7901

AIM: To investigate the effect of c9, t11-conjugated linoleic acid (c9,t11-CLA) on the adhesion of human gastric carcinoma cell line (SGC-7901).METHODS: SGC-7901 cells were at first treated with different concentrations (25, 50, 100, 200 μmol/L) of c9,t111-CLA and 1 mL/L ethanol (as a negative control) for 24 h.Using adhesion assay and Western blot, we investigated the ability of SGC-7901 cells to adhere to intracellular matrix and examined the expression of E-cadherin (ECD), α-catenin,intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in these cells.RESULTS: The attachment rate to laminin of SGC-7901 cells treated with different concentrations of c9,t11-CLA (0,25, 50, 100, and 200 μmol/L) was 100.0±3.3, 95.7±4.0,89.2±4.6, 87.9±6.1, and 65.9±5.8, respectively. The attachment rate to fibronectin was 100.0±4.7, 96.8±3.8,94.5±4.1, 76.5±4.3, and 61.8±4.8, respectively. The attachment rate to Matrigel was 99.9±6.6, 91.4±6.8,85.5±7.4, 79.3±5.6, and 69.6±5.1, respectively. Besides,c9,t11-CLA could increase the level of ECD and α-catenin,and decrease the level of ICAM-1 and VCAM-1 in SGC-7901 cells.CONCLUSION: c9, t11-CLA can reduce the adhesion of human gastric carcinoma cells to laminin, fibronectin and Matrigel. c9,t11-CLA can increase the level of ECD and α-catenin, and decrease the level of ICAM-1 and VCAM-1 in human gastric carcinoma cells.     

Expression of CD14 protein and its gene in liver sinusoidal endothelial cells during endotoxemia

AIM：To observe expression of CD14 protein and CD14 gene in rat liver sinsoidal endothelial cells(LSECs) during endotoxemia,and the role of CD14 protein in the activation of lipopolysaccharide(LPS)-induced LSECs.METHODS:Wistar rat endotoxemia model was established first by injection of a dose of LPS(5mg/kg,Escherichia coli O111:B4)via the tail vein,then sacrificed after 0h,3h,6h,12h,and 24h,respectively,LSECs were isolated from normal and LPS-injected rats by an in situ colagenase perfusion technique.The isolated LSECs were incubated with rabbit anti-rat CD14 polyclonal antibody,then stained with goat anti rabbit IgG conjgated fluorescein isothiocyanate(FITC) and flow cytometric analysis(FCM) was performed.The percentage and mean fluorescence intensity(MFI) of CD14-positive cells were taken as the indexes.LSECs were collected to measure the expression of CD14 mRNA by in situ hybridization analysis.The isolated LSECs from normal rats were incubated firstly with anti-CD14 antibody,then stimulated with different concentrations of LPS,and the supernatants of these cells were then collected for measuring the levels of tumor necrosis factor (TNF)-a and lnterleukin(IL)-6 with ELISA.RESULTS:In rats with endotoxemia,LSECs displayed a strong MFI distinct from that of control rats .CD14 positive cells in rats with endotoxemia were 54.32%,65.83%,85.64%,and 45.65%,at 3h,6h,12h,and 24h respectively there was significant difference when compared to normal group of animals(4.45%)(P<0.01).The expression of CD14 mRNA in isolated LSECs was stronger than that in control rats,In LPS group,the levels of TNF-αand IL-6were 54±6ng.L^-1,85±9ng.L^-1,206±22ng.L^-1,350±41ng.L^-1,366±42ng.L^-1and103±11ng.L^-1,187±20ng.L^-1,244±26ng.L^-1,290±31ng.L^-1,and 299±34ng.L^-1,respectively at different concentration points,In anti-CD14 group,the levels of TNF-αand IL-6 were 56±5ng.L^-1,67±8ng.L^-1,85±10ng.L^-1,113±12ng.L^-1, ,199±22ng.L^-1and104±12ng.L^-1,125±12ng.L^-1,165±19ng.L^-1,185±21ng.L^-1,and222±23ng.L^-1,respectively at different concentration poirts.THere was significant difference between the two groups(P<0.01).CONCLUSION:LSECs can synthesize CD14 protein and espress CD14 gene during endotoxemia .CD14 protein plays an important role in the activation of LPS-induced LSECs.This finding has important implications for the understanding of the mechanisms by which LPS may injure liver sinusoidal endothelial cells during sepsis.     

人肝癌细胞膜上HSP70的表达

用流式细胞仪和Cell-ELISA检测了 4株人肝癌细胞膜上HSP70的表达。结果表明正常情况下人肝癌细胞膜上HSP70的表达较低 ,经热处理后表达增加 (P <0 0 1)并显示出细胞之间的差异 (P <0 0 1)。推测其低表达可能与肝癌细胞逃逸机体免疫监视有关 ,对制定肝癌的防治对策有重要的指导意义。     

黑色素瘤相关抗原A1在肝癌细胞株中的表达与基因甲基化程度的相关性

目的 探讨人肝癌细胞株中黑色素瘤相关抗原A1(MAGE-A1)表达状况与肝癌细胞基因甲基化的关系。方法 提取10种人肝癌细胞株的总RNA,用逆转录聚合酶链反应对细胞株的MAGE-A1基因的表达进行检测;提取10种人肝癌细胞株的基因组DNA,用限制性酶切和Southern印迹杂交对每种细胞的基因组甲基化程度进行定量分析。另外对基因组DNA行HpaⅡ酶切后,用引物CDS21、EDP4和CDS20、EDP4进行聚合酶链反应扩增,然后用特异性探针杂交来检测肝癌细胞株MAGE-A1启动子的甲基化状态。用特异性序列聚合酶链反应方法检测每一种细胞株的人白细胞抗原-A位点型别。结果 QGY-7703、SMMC-7721、HLE、BEL-7402、BEL-7404、BEL-7405肝癌细胞株的MAGE-A1基因表达阳性,且细胞分化程度均为中低分化;HepG2 2．2．15、HepG2、QGY-7701和Huh7肝癌细胞株的MAGE-A1基因表达阴性,且细胞分化程度均为高中分化。通过定量分析表明,MAGE-A1表达的肝癌细胞株与MAGE-A1不表达的肝癌细胞株比较,前者基因组甲基化程度较低(t=2．896,P=0．02)。肝癌细胞株MAGE-A1基因启动子区甲基化分析结果表明HepG2 2．2．15、HepG2、QGY-7701和Huh7为高甲基化,SMMC-7721、HLE、BEL-7402、BEL-7404、BEL-7405为低甲基化。结论 肝癌细胞株MAGE-A1 mRNA表达与其基因甲基化程度有关。     

三氧化二砷选择性诱导人肝癌细胞凋亡及 相关基因的实验研究

目的 明确三氧化二砷对人肝癌细胞的诱导凋亡作用及其分子机制。方法 用四甲基偶氮唑蓝法、AO/EB荧光染色法、透射与扫描电镜观察、DNA电泳、流式细胞术、TUNEL法及免疫组织化学等方法,观察了三氧化二砷对体外培养的人肝癌细胞株QGY-7701、QGY-7703和正常人肝细胞株L-02的生长活力、细胞凋亡、细胞周期及其相关基因表达的影响。结果 三氧化二砷能显著地抑制人肝癌细胞QGY-7701和QGY-7703的生长,用药后诱导其出现了典型的细胞凋亡形态学和生化学改变,并使细胞周期阻滞于S期,且bcl-2基因表达明显下降,bax和Fas基因表达显著增强;三氧化二砷对正常人肝细胞株L-02无明显作用。结论 三氧化二砷对人肝癌细胞有显著的选择性诱导凋亡作用,且受到多种基因调控,这一结论为应用三氧化二砷治疗原发性肝癌提供了可靠的实验依据。     

肝癌细胞株中肿瘤特异性抗原MAGE、GAGE、BAGE基因表达的研究

目的 研究MAGE、GAGE、BAGE基因在肝癌细胞株中的表达情况,评价这些肿瘤特异性抗原作为肿瘤分子标记以及肿瘤免疫治疗特异性靶位的可能性。 方法 用RT-PCR检测国内建株的肝癌细胞株SMMC-7721、QQY-7701、BEL-7402中MAGE-1、MAGE-3、GAGE1-8、GAGE1-2和BAGE基因mRNA的表达,以GAPDH基因作为检测内对照,并与非肿瘤肝穿组织比较。 结果 肝癌细胞株SMMC-7721表达MAGE-1和BAGE基因;QQY-7701表达MAGE-3和BAGE基因;BEL-7402表达MAGE-1和GAGE1-2基因;3株肝癌细胞至少表达其中一个基因。肝硬化病人肝穿刺组织中MAGE、GAGE、BAGE基因表达均为阴性。 结论 MAGE、GAGE、B A G E肿瘤特异性抗原可以作为肝癌早期诊断的分子标记,并具有作为肝癌免疫治疗特异性靶位的潜在价值。     

不同转移潜能人肝癌细胞株趋化因子受体谱差异表达

目的对比观察不同转移潜能人肝癌细胞株趋化因子受体谱差异性表达。方法Pre- mier软件设计18对趋化因子受体引物,RT-PCR分析SMMC-7721、MHCC97-L、MHCC97-H和HCCLM6细胞侵袭转移潜能逐渐增强的人肝癌细胞株趋化因子受体谱。结果4组不同转移潜能细胞株趋化因子受体表达谱存在明显差异（P＜0．01）,其中CCR10、CXCR4、CXCR6表达随转移潜能增加逐渐降低。HCCLM6表达谱中CCR3、CCR4、CCR10、CCR12及XCR1比SMMC-7721表达明显降低甚至缺失（P＜0．01）,而CXCR1（P=0．006）、CXCR5（P=0．003）表达高于低转移潜能组SMMC-7721。MHCC97-H和MHCC97-L比较,除CXCR2、CXCR6、XCR1外差异均有统计学意义,其中CCR1（P=0．002）、CCR2（P=0．004）、CCR5（P=0．046）表达高于MHCC97- L。CXCR4在模板减量时只能在SMMC-7721组检测到。结论高低转移潜能肝癌细胞株趋化因子受体表达在mRNA水平存在差异性表达,与肝癌细胞株差异性转移潜能相关。     

肝癌细胞放射敏感性与survivin蛋白表达的关系

目的 探讨肝癌细胞放射敏感性与survivin表达的关系.方法 肝癌细胞HepG2和SMMC-7721在接受不同剂量γ射线照射后,分别采用克隆形成法、免疫细胞化学法、流式细胞术、比色法等检测细胞存活率、survivin蛋白表达、细胞周期变化和Caspase-3活性.结果 在2Gy照射下HepG2和SMMC-7721细胞的存活分数分别为0.43±0.01与0.70±0.02,SMMC-7721较HepG2放射抗拒.γ射线对SMMC-7721细胞的G2/M期阻滞时间较HepG2细胞长(48 h对24 h),在阻滞峰各剂量点SMMC-7721细胞的G2/M期比例也更高.γ射线可上调两株肝癌细胞survivin蛋白的表达,照射后48～72 h,SMMC-7721细胞的survivin蛋白表达水平显著高于HepG2细胞(t值为2.81～5.20,P值均＜0.05).而Caspase-3的活化水平在放射敏感的HepG2细胞中更高(t值为6.05～6.72,P值均＜0.01).结论 射线诱导的survivin表达上调及survivin对Caspase-3的负调控可能是SMMC-7721细胞较HepG2细胞放射抗拒的原因之一.     

血清和糖皮质激素诱导的蛋白激酶2在肝细胞癌中过表达并介导肝癌细胞中糖原合成酶激酶-3β/β-连环蛋白信号传导

目的探讨血清和糖皮质激素诱导的蛋白激酶(SGK2)在肝癌组织与正常肝脏组织中的表达差异以及介导肝细胞癌(HCC)细胞中糖原合成酶激酶-3β(GSK-3β)/β-连环蛋白(β-catenin)信号传导的相关机制。方法收集配对的HCC及正常组织20对,采用实时荧光定量PCR技术检测SGK2 mRNA表达情况。应用蛋白质印迹法检测人肝癌细胞系Huh-7、SMMC-7721以及正常人肝细胞系L02中SGK2蛋白水平。应用SGK2 siRNA转染人肝癌细胞系SMMC-7721、Huh-7,然后使用蛋白质印迹方法检测上述转染成功细胞系中GSK-3β、β-catenin的蛋白质表达水平。计量资料以均值±标准差(±s)表示,统计学方法采用Student t检验。结果与配对正常肝组织中的表达水平相比,所有20个HCC样品中SGK2 mRNA表达上调。在两种人肝癌细胞系(Huh-7和SMMC-7721)中SGK2蛋白水平显著高于正常人肝细胞系(P<0.01)。在人HCC细胞系SMMC-7721和Huh-7中,SGK2表达下调抑制了未磷酸化GSK-3β表达。另外,在HCC细胞系中SGK2表达下调通过阻止β-catenin蛋白酶体降解来降低β-catenin的去磷酸化。结论SGK2在HCC中过表达并介导HCC细胞中GSK-3β/β-catenin信号传导。     

PITX1和Pan-ras在肝癌细胞中的表达及意义

目的研究抑癌基因PITX1和其下游癌基因Pan-ras在正常胚肝细胞株L02，肝癌细胞株HepG2和SMMC-7721中的表达，探讨其在肝癌发生发展中的作用和关系。方法应用SABC免疫组织化学染色技术和westem blot蛋白质印迹以及半定量RT-PCR检测L02、HepG2和SMMC-7721细胞株中PITX1和Pan-ras基因的表达情况，并分析其意义。结果PITX1在肝癌细胞（HepG2、SMMC-7721）中表达比正常肝细胞L02显著降低，Pan-ras在肝癌细胞（HepG2、SMMC-7721）中的表达与正常肝细胞L02相比显著升高。结论PITX1在肝癌细胞中的低表达，以及Pan-ras的高表达，可能导致肝癌无限增殖，构成了肝癌细胞信号传导网络中的重要一环。     

Docetaxel inhibits SMMC-7721 human hepatocellular carcinoma cells growth and induces apoptosis

AIM:To investigate the in vitro anti-hepatocellular carcinoma(HCC) activity of docetaxel against SMMC-7721 HCC cells and its possible mechanism.METHODS: The HCC cells were given different concentrations of docetaxel and their growth was measured by colony forming assay. Cell cycle and apoptosis were analyzed by flow cytometry and fluorescence microscopy (acridine orange/ethidium bromide double staining, AO/EB), as well as electronic microscopy. The SMMC-7721 HCC cell reactive oxygen species (ROS) and glutathione (GSH) were measured after given docetaxel.RESULTS: Docetaxel inhibited the hepatocellular carcinoma cells growth in a concentration dependent manner with IC50 5&#215;10^-10M. Marked cell apoptosis and G2/M phase arrest were observed after treatment with docetaxel ≥10^-8M.Docetaxel promoted SMMC-7721 HCC cells ROS generation and GSH deletion.CONCLUSION: Docetaxel suppressed the growth of SMMC-7721 HCC cells in vitro by causing apoptosis and G2/M phase arrest of the human hepatoma cells, and ROS and GSH may play a key role in the inhibition of growth and induction of apoptosis.