Effects of Yigan Decoction on proliferation and apoptosis of hepatic stellate cells

AIM：To investigate the effects of Chinese herb Yigan Decoction on proliferation and apoptosis of the hepatic stellate cells(HSC) in vitro .METHODS:The study in vitro was carried out in the Culture of HSC lines.Various comcentration of Yigan Decoction were added and incubated .Cell proliferation was detected with MTT colorimetric assay.Cell apoptosis was detected by electron microscopy,flow cytometry and TUNEL.RESULTS:The proliferation of HSC was inhibited by Yigan Decoction,which depending on dose and time significantly.The HSC proliferation rates of groups at the end concentrations 144 and 72(g.L^-1) were 21.62%and 40.54% respectively,significantly lower than that of normal control group(P<0.01),The HSC proliferation rates of groups at the and concentrations 36,18 and 9(g.L^-1) were 54.05%,45.95%and 51\35% respectively,lower than that of control group(P<0.05).When the end concentration was 4.5g.L^-1,the proliferation rate was 83.78%,which appeared no significant differences compared with control group.At the same concentrations of 18 g.L^-1,the inhibitory effects of Yigan Decoction at 24h,48h and 72h time point were observed,the effects were time-dependent and reached a peak at 72h.Meanwhile,it was showed that the inducing effects of Yigan Decoction on HSC apoptosis were dose dependent and time-dependent,The apoptosis index(AL)was detected by TUNEL.After Yigan Decoction had been incubated for 48h at the end concentration of 18g.L^1,the Al(14.5±31.%),was significantly higher than that of control group(4.3±1.3)^(P<0.01).When visualized under transmission electron microscopy,some apoptotic stellate cells were found ,i.e.dilated endoplasmic reticulum,irregular nuclei,chromatin condensation and heterochromatin ranked along inside of nuclear membrane.By flow cytometry detection,after HSC was treated with Yigan Decoction at different concentrations of 36,18and 9(g.L^-1)for48h,Al(%)were 13.3±3.2,10.7±2.7and 10.1±2.5respectively,which were significantly higher than that of control group(4.1±1.9)(P<0.01),At the same concentration of 18g.L^-1 or 24h,48h and 72h,Al(%)were9.3±1.8,10.7±2.7and 14.6±4.3repectively,which were significantly higher than that of control group(P<0.01).CONCLUSION:Yigan Decoction could significantly inhibit HSC proliferation and increase the apoptosis index of HSC dose-dependently and time-dependently,which may be related to its mechanism of antifibrosis.     

Removing oxygen-derived free radicals stimulates healing of ethanol-induced erosive gastritis in the rat

The effect of the radical removing agents, allopurinol and dimethyl sulphoxide (DMSO), on the healing rate of ethanol (1 ml of 40% solution) induced gastric mucosal injury was studied in the rat. One millilitre of 1, 2 or 5% allopurinol or DMSO were instilled into the stomach 1, 24 and 48 h after giving ethanol by gavage. One hour after administration of ethanol, gastric mucosal injury was produced in all animals (20.4 +/- 1.2 mm2, mean +/- SEM, n = 10). Treatment for 24 h with 2% allopurinol or DMSO significantly (p less than 0.01) reduced the extent of the ethanol injury (11.1 +/- 0.8 and 11.9 +/- 0.9 mm2, respectively, vs. 19.2 +/- 1.1 mm2, n = 10) and this was similarly achieved by the 5% solutions (10.4 +/- 0.9 and 10.2 +/- 0.8 mm2, respectively, vs. 19.2 +/- 1.1 mm2, n = 10). After treatment for 48 h, 30% of animals having 1% allopurinol or DMSO remained with injury significantly (p less than 0.001) less than that seen with ethanol alone (4.1 +/- 0.4 and 3.9 +/- 0.5 mm2, respectively, vs. 12.1 +/- 0.9 mm2, n = 10); however, none treated with 2 or 5% solutions remained with any injury. Healing of this injury was confirmed microscopically and was achieved by regeneration. Thus, removing oxygen-derived free radicals stimulates the healing of ethanol-induced acute gastric mucosal injury in the rat.     

牛磺酸通过调控细胞周期蛋白抑制肝星状细胞增殖

目的进一步研究牛磺酸对肝星状细胞(HSC)增殖抑制作用的机制。方法用四甲基偶氮唑盐法检测细胞增殖;流式细胞仪测定细胞周期;免疫细胞化学和实时荧光定量PCR测定细胞周期调控蛋白Cyclin D1和P21waf1表达。结果牛磺酸对HSC增殖具有抑制作用,在浓度为5、10、20,30、40、50 mmol/L 作用48h时的抑制率分别为6.7%、14.4%、23.3%、32.2%、36.7%和45.6%,t值为2.939～6.369,P<0.05～0.01。流式细胞仪检测发现牛磺酸可阻滞HSC由G0/G1期向S期转换,使G0/G1期细胞增多,S期细胞减少。G0/G1期、S期细胞,牛磺酸浓度为40 mmol/L时,分别为(68.2±1.4)%和(26.2±1.3)%,与对照组分别为(56.2±1.7)%和(38.5±0.8)%,差异有统计学意义,t≥5.422,P<0.01。牛磺酸可抑制Cyclin D1表达、促进P21waf1表达,用免疫细胞化学染色结合数码图像分析系统软件分析发现牛磺酸浓度在40 mmol/L时HSC的Cyclin D1表达的平均吸光度为0.13±0.02,P21waf1为0.19±0.02,对照组分别为0.18±0.02和0.14±0.01,差异有统计学意义,t=6.689和t=6.528,P<0.01。实时荧光定量PCR检测也发现经40 mmol/L牛磺酸处理的HSC的Cyclin D1 mRNA表达量(拷贝数与106磷酸甘油醛脱氢酶比值)降低为5776.7±3345.0,对照组为18 400.6±1374.8,而P21waf1 mRNA表达量(拷贝/106磷酸甘油醛脱氢酶)增多为44 866.7±3910.7,对照组为16 933.3±960.9。结论牛磺酸通过抑制Cyclin D1表达、促进P21waf1表达,使HSC阻滞于G0/G1期,而抑制HSC增殖。     

熊果酸对肝星状细胞增殖与凋亡的影响

目的 体外观察熊果酸对肝星状细胞增殖与凋亡的影响,探讨熊果酸诱导肝星状细胞凋亡的可能作用机制. 方法 将不同浓度熊果酸作用于肝星状细胞HSC-T6及肝细胞L02,分别在药物作用24、48、72 h后用四甲基偶氮唑盐法检测熊果酸对HSC-T6及L02细胞增殖的影响;流式细胞仪检测熊果酸对HSC-T6凋亡的影响;光学显微镜观察熊果酸作用后细胞形态学变化情况;免疫细胞化学法检测HSC-T6中Bcl-2、Bax和Caspase-3蛋白的表达情况. 结果 各种浓度的熊果酸均可抑制HSC-T6细胞的增殖,且呈剂量-时间依赖性;当熊果酸浓度为25、50、75μmol/L时可促进L02细胞增殖,浓度＞75μmol/L则表现为抑制L02细胞增殖.在病理形态学方面,熊果酸作用HSC-T6细胞48 h后,光学显微镜下可见细胞缩小变圆、核浓缩等.25、50、75 μmol/L熊果酸作用HSC-T6细胞48 h后,流式细胞仪检测显示细胞凋亡率分别为10.30%±3.85%、21.87%±4.46%、31.33%±6.18%,比对照组(2.93%±1.60%)明显升高(P＜0.01).免疫细胞化学显示Bax及Caspase-3蛋白表达较对照组升高(P＜0.05),且呈剂量依赖性,而Bcl-2蛋白表达水平与对照组无明显差异(P＞0.05).结论 在体外熊果酸可较明显地抑制HSC-T6细胞增殖,诱导其凋亡;对L02细胞的生长具有双向调节作用.熊果酸诱导HSC-T6细胞凋亡可能与降低Bcl-2/Bax比值、激活Caspase-3蛋白有关.     

Adoption of long-term cultures to evaluate the cryoprotective potential of trehalose for freezing hematopoietic stem cells

Dimethylsulfoxide (DMSO), which is widely used as a cryoprotectant for hematopoietic stem cells (HSC), has considerable toxicity for both the thawed cells and the patient. The aim of this study was to evaluate the cryoprotective potential of trehalose in comparison to DMSO for human HSC. Human bone marrow (BM) and peripheral blood stem cells (PBSC) of volunteer donors were cryopreserved in the presence of different concentrations of trehalose with and without insulin, as well as with DMSO 10%. After thawing to 37 degrees C colony-forming unit (CFU) assays were performed. Long-term marrow-cultures (LTMC) were established and used for the detection of long-term culture-initiating cells (LTCIC). The total amount of CFUs detected was 104+/-134 (mean+/-s.d.) in DMSO-preserved cells and 179+/-34 in trehalose-protected cells. For LTMC the best feeder layer proved to be fresh human BM and the most useful concentration of trehalose was 0.5 M. Using these culture conditions we could detect after 5 weeks LTMC a total of 172+/-28 CFUs for trehalose-protected cells and 170+/-52 for DMSO-preserved cells. In conclusion, trehalose exerts a similar cryoprotective potential for hematopoietic progenitor and stem cells like DMSO and could possibly replace DMSO at least in part as cryoprotectant in the setting of hematopoietic cell transplantation.     

Effect of rat serum containing Biejiajian oral liquid on proliferation of rat hepatic stellate cells

AIM: Liver fibrosis is a common pathological process of chronic liver diseases. Activation of hepatic stellate cells (HSCs) is the key issue in the occurrence of liver fibrosis. In this study, we observed the inhibitory action of rat serum containing Biejiajian oral liquid (BOL), a decoction of turtle shell, on proliferation of rat HSCs, and to explore the anti-hepatofibrotic mechanisms of BOL. METHODS: A rat model of hepatic fibrosis was induced by subcutaneous injection of CCl(4). Serum containing low, medium and high dosages of BOL was prepared respectively. Normal and fibrotic HSCs were isolated and cultured. The effect of sera containing BOL on proliferation of HSCs was determined by (3)H-TdR incorporation. RESULTS: The inhibitory rate of normal rat HSC proliferation caused by 100 mL/mL sera containing medium and high dosages of BOL showed a remarkable difference as compared with that caused by colchicine (medium dosage group: 34.56+/-4.21% vs 29.12+/-2.85%, P<0.01; high dosage group: 37.82+/-1.32% vs 29.12+/-2.85%, P<0.01). The inhibitory rate of fibrotic rat HSC proliferation caused by 100 mL/L serum containing medium and high dosages of BOL showed a remarkable difference as compared with that caused by colchicine (medium dosage group: 51.31+/-3.14% vs 38.32+/-2.65%, P<0.01; high dosage group: 60.15+/-5.36% vs 38.32+/-2.65%, P<0.01). The inhibitory rate of normal rat HSC proliferation caused by 100 mL/L and 200 mL/L sera containing a medium dosage of BOL showed a significant difference as compared with that caused by 50 mL/L (100 mL/L group: 69.02+/-9.96% vs 50.82+/-9.28%, P<0.05; 200 mL/L group: 81.78+/-8.92% vs 50.82+/-9.28%, P<0.01). The inhibitory rate of fibrotic rat HSC proliferation caused by 100 mL/L and 200 mL/L sera containing a medium dosage of BOL showed a significant difference as compared with that caused by 50 mL/L (100 mL/L group: 72.19+/-10.96% vs 61.38+/-7.16%, P<0.05; 200 mL/L group: 87.16+/-8.54% vs 61.38+/-7.16%, P<0.01). CONCLUSION: Rat serum containing BOL can inhibit proliferation of rat HSCs, and the inhibition depends on the dosage and concentration of BOL. The inhibitory effect on HSC proliferation is one of the main anti-hepatofibrotic mechanisms of BOL.     

Transforming growth factor beta and tumor necrosis factor alpha inhibit both apoptosis and proliferation of activated rat hepatic stellate cells.

Transforming growth factor beta (TGF-beta) as well as tumor necrosis factor alpha (TNF-alpha) gene expression are up-regulated in chronically inflamed liver. These cytokines were investigated for their influence on apoptosis and proliferation of activated hepatic stellate cells (HSCs). Spontaneous apoptosis in activated HSC was significantly down-regulated by 53% +/- 8% (P <.01) under the influence of TGF-beta and by 28% +/- 2% (P <.05) under the influence of TNF-alpha. TGF-beta and TNF-alpha significantly reduced expression of CD95L in activated HSCs, whereas CD95 expression remained unchanged. Furthermore, HSC apoptosis induced by CD95-agonistic antibodies was reduced from 96% +/- 2% to 51 +/- 7% (P <.01) by TGF-beta, and from 96% +/- 2% to 58 +/- 2% (P <.01) by TNF-alpha, suggesting that intracellular antiapoptotic mechanisms may also be activated by both cytokines. During activation, HSC cultures showed a reduced portion of cells in the G0/G1 phase and a strong increment of G2-phase cells. This increment was significantly inhibited (G1 arrest) by administration of TGF-beta and/or TNF-alpha to activated cells. In liver sections of chronically damaged rat liver (CCl4 model), using desmin and CD95L as markers for activated HSC, most of these cells did not show apoptotic signs (TUNEL-negative). Taken together, these findings indicate that TGF-beta and/or TNF-alpha both inhibit proliferation and also apoptosis in activated HSC in vitro. Both processes seem to be linked to each other, and their inhibition could represent the mechanism responsible for prolonged survival of activated HSC in chronic liver damage in vivo.     

Effective engraftment but poor mid-term persistence of mononuclear and mesenchymal bone marrow cells in acute and chronic rat myocardial infarction

Bone marrow cells are used with promising results for cell therapy after myocardial infarction (MI). We determined the survival and organ distribution of transplanted mononuclear (MNC) or mesenchymal (MSC) bone marrow cells, and the influence of cell type, cell number and application time. MNC and MSC (male Fischer 344 rats) were injected into the border zone of MI (syngeneic females) immediately or 7 days after LAD ligation (10(5) or 10(6) cells, 50 microl). After 0 h, 48 h, 5 days, 3 weeks and 6 weeks, DNA of heart, lung, liver, spleen, kidney, blood, bone marrow, brain and skeletal muscle was isolated and the number of donor cells determined by quantitative real-time PCR with Y-chromosome specific primers (each n>or=4). The percentage of donor-cells in the heart decreased rapidly from 34-80% of injected cells (0 h) to 0.3-3.5% (6 weeks) independent from cell type, number and application time. The absolute number increased after increasing injected cell number (10(6) vs. 10(5)). In the lung, MNC and MSC were found at 0 h (126+/-48 and 140+/-3 per million organ cells), but in liver and kidney, only few. At 48 h and 6 weeks, an increasing number of MNC, but not MSC, were detected in the spleen (6 weeks, 602+/-173 per million organ cells vs. 95+/-50 in the heart, P=0.02). In all other organs, only few or no grafted cells of either cell type were detected at these times. Organ distribution was independent from injection time. The low survival of grafted cells may limit their therapeutic impact, while their distribution to other organs must be considered in all cell therapy applications.     

Cell cycle arrest and apoptotic cell death in cultured human gastric carcinoma cells mediated by arsenic trioxide

AIM: To investigate the effect of arsenic trioxide on human gastric cancer cell line MKN45 with respect to both cytotoxicity and induction of apoptosis in vitro. METHODS: MKN45 cells were treated with arsenic trioxide (As2O3) at the concentration of 1, 5, and 10 μmol/L, respectively, for three successive days. Cell growth and proliferation were observed by cell counting and trypan blue exclusion. Cytotoxicity of As2O3 was determined by MTT assay. Morphologic changes were studied with light microscopy. Flow cytometry was used to assay cell DNA distribution and apoptotic cells were confirmed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and DNA electrophoresis. RESULTS: The growth of MKN45 cells was significantly inhibited by As2O3 which was confirmed by colony-forming assay. After 7 d of culture with various concentrations of As2O3, colony-forming capacity of MKN45 cells decreased with As2O3 increment in comparison with that of control group. The inhibitory rate of colony-formation was 38.5%, 99.1%, and 99.5% when the concentration of As2O3 was 1, 5, and 10 μmol/L in culture medium, respectively. The cell number of a single colony in drug treatment groups was less than that of control group. The cell-killing rate of As2O3 to MKN45 cells was both dose- and time-dependent with an IC50 of (11.05±0.25) μmol/L After incubation in 10 μmol/L As2O3 for 24 h, the cell-killing rate was 27.1%, and it was close to 50% after 48 h. The results showed that As2O3 induced time- and dose-dependent apoptosis in MKN45 cells, blocked at G2/M phase. The apoptotic peak (sub-G1 phase) appeared and cell apoptotic rate in MKN45 cells was 18.3-32.5% after treatment by 10 umol/L As2O3 for 48 h. The percentage of G2/M cell of the experimental groups was 2.0-5.0 times than that of the control group. Gel electrophoresis of DNA from cells treated with each concentration of As2O3 for 48 h revealed a "ladder" pattern, indicating preferential DNA degradation at the internucleosomal, linker DNA sections. TUNEL also demonstrated strand breaks in DNA of MKN45 cells treated with As2O3, while control cells showed negative labeling. CONCLUSION: As2O3 can induce apoptosis of human gastric carcinoma cells MKN45, which is the basis of its effectiveness. It shows great potential in the treatment of gastric carcinoma.     

Effects of the tyrosine protein kinase inhibitor genistein on the proliferation,activation of cultured rat hepatic stellate cells

AIM：Hepatic stellate cell(HSC)plays a pivotal role in liver fibrosis and is considered as the therapeutic target for the treatment of hepatic fibrosis,Tyrosine protein kinase plays an important role in the proliferation,activation of HSC.The purpose of the study is to investigate the effects of the tyosine protein kinase inhibitor genistein on the proliferation and activation of cultured rat HSC.METHODS:Rat HSC were isolated from Wistar rats by in situ perfusion of collagenase and pronase and single-step density Nycodenz gradient,Culture-activated HSC were serum-starved and incubated with10^-9to10^-5mol/L concentration of genistein for 24,48or 72h,In PDGF-induced HSC proliferation,HSC were stimulated with10μg&#183;L^-1PDGF-BBfo15min,and thentreated with genistein for the same time.Cell proliferation was measured by MПassay and based on flow cytometric analysis of cell cycle.The a-smooth muscle actin(α-SMA）expression in HSC was studied with confocallaser microscopy and flow cytometry.c-fos,c-jun and cyclinD1expression in HSCwas also detected by flow cytometry.RESULTS:Genistein inhibited basal and PDGF-induced proliferation of HSCat the concentration of 10^-8to10^-5mol/L,and treatment with10^-7mol/L concentration of genistein for 48h inhibited the HSCproliferation significantly(the inhibition rate was 70.3%,P&lt;0.05).Immunofluorescence detected by confocal laser microscopy and flow cytometry showed that treatment with10^-7mol/L genistein for48h suppressed the expression of α-SMA significantly in HSC(the specific fluorescence intensity were60.2&#177;21.5vs35.3&#177;11.6and12.8&#177;10.4vs9.54&#177;6.39,respectively,bothP&lt;0.05).The intensity of c-fos,c-jun and cyclinD1 expression of HSCs treated with 10^-7mol/L genistein for 48h was also significantly decreased compared with the controls.CONCLUSION;Genistein influences proliferation of HSC,suppresses the expression of α-SMA in HSC and tinhibits the intensity of c-fos,c-jun and cyclinD1 expression of HCSs,Genistein has therapeutic potential against liver fibrosis.     

转化生长因子β1对不同活化状态肝星状细胞增殖与Ⅰ型胶原表达的影响

目的 观察不同活化状态肝星状细胞(HSC)对外源性转化生长因子-β_1(TGF-β_1)旁分泌刺激的生物学效应作用。方法 原代分离培养大鼠HSC,无包被塑料培养皿上分别培养1、4、7d,细胞处于静止、中间活化与完全活化状态,继以10～500 pmol/L TGF-β_1温育细胞24h,~3H—TdR掺入法测定细胞增殖,western blot法检测细胞α-平滑肌肌动蛋白(α-SMA)与Ⅰ型胶原蛋白表达沉积,~3H-脯氨酸掺入与胶原酶消化法测定细胞总胶原的分泌量。100pmol/L TGF-β_1温育细胞15～90min,northern blot法检测细胞Ⅰ型前胶原mRNA的表达水平。结果 TGF-β_1浓度依赖性抑制培养1d HSC的细胞增殖,10～500 pmol/L TGF-β_1浓度组细胞内~3H—TdR掺入率分别为对照组的52.8％～16.8％,与对照组比较,q值为5.44～10.37,P<0.01。但TGF-β_1对培养4d与7d的细胞增殖无影响。随细胞活化,HSC基础性α-SMA、Ⅰ型胶原蛋白与mRNA水平明显增加,而TGF-β_1刺激各培养时间HSC以上蛋白与基因的表达。培养1、4、7d HSC基础水平与TGF-β_1刺激的总胶原分泌量分别为(804±274)dpm/孔与(1 200±708)dpm/孔;(2 966±1 701)dpm/孔与(6 160±1 123)dpm/孔;(2 580±767)dpm/孔与(4 583±1 467)dpm/孔,后2组组内比较,t值分别为3.84与2.96,P<0.01或P<0.05。以培养4d HSC     

Molecule action mechanisms of NM-3 on human gastric cancer SGC-7901 cells in vivo or in vitro

AIM: To study the molecule action mechanisms of NM-3 on the growth of human gastric cancer SGC-7901 cells in vivo or in vitro. METHODS: SGC-7901 from human non-differentiated gastric cancer cell line was cultured with NM-3 at 100 mg/ml for 24 h. We observed its inhibitory rate and the density of micro-vascular growth in grafted mice with human gastric cancer SGC-7901. The apoptosis of human gastric cancer SGC-7901 was revealed in NM-3 treatment group by using terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate-fluorescene nick end labeling (TUNEL) method and flow cytometry analysis. RESULTS: The growth of SGC-7901 cells was markedly inhibited compared with control growp, which was smaller than that in normal saline control group (4.17 g+/-0.22 g vs 9.45 g+/-1.38 g, P<0.01). The level of apoptosis of human gastric cell line SGC-7901 was obviously increased in NM-3 treatment group at 1 mg.L(-1) for 24 h. NM-3 inducing apoptotic index in NM-3 plus carboplatin group was 3.5 times that of carboplatin control group (TUNEL: 27.98+/-6.12% vs 12.94+/-2.12%, FACScan: 26.86+/-5.69% vs 11.86+/-1.09%, P<0.01). Western blot analysis showed that the apoptotic index of human gastric cancer was elevated for 12, 24 and 36 h with an evident time-effect relationship in groups at 100 mg.L(-1). NM-3 enhanced the inhibitive effects and sensitivity of chemotherapy for human gastric cancer in nude mice. These results suggested that NM-3 played a key inhibitive role in the growth of grafted human gastric cancer in nude mice. CONCLUSION: NM-3 can inhibit the growth of human gastric cancer cell line SGC-7901, and enhance the sensitivity of carboplatin on SGC-7901 and induced its apoptosis.     

Anticancer activity of resveratrol on implanted human primary gastric carcinoma cells in nude mice

AIM: To investigate the apoptosis of implanted primary gastric cancer cells in nude mice induced by resveratrol and the relation between this apoptosis and expression of bcl-2 and bax. METHODS: A transplanted tumor model was established by injecting human primary gastric cancer cells into subcutaneous tissue of nude mice. Resveratrol (500 mg/kg, 1 000 mg/kg and 1 500 mg/kg) was directly injected beside tumor body 6 times at an interval of 2 d. Then changes of tumor volume were measured continuously and tumor inhibition rate of each group was calculated. We observed the morphologic alterations by electron microscope, measured the apoptotic rate by TUNEL staining method, detected the expression of apoptosis-regulated genes bcl-2 and baxby immunohistoch-emical staining and PT-PCR. RESULTS: Resveratrol could significantly inhibit carcinoma growth when it was injected near the carcinoma. An inhibitory effect was observed in all therapeutic groups and the inhibition rate of resveratrol at the dose of 500 mg/kg, 1 000 mg/kg and 1 500 mg/kg was 10.58%, 29.68% and 39.14%, respectively. Resveratrol induced implanted tumor cells to undergo apoptosis with apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation. The inhibition rate of 0.2 mL of normal saline solution, 1 500 mg/kg DMSO, 500 mg/kg resveratrol, 1 000 mg/kg resveratrol, and 1 500 mg/kg resveratrol was 13.68±0.37%, 13.8±0.43%, 48.7±1.07%, 56.44±1.39% and 67±0.96%, respectively. The positive rate of bd-2 protein of each group was 29.48±0.51%, 27.56±1.40%, 11.86±0.97%, 5.7±0.84% and 3.92±0.85%, respectively by immunohistochemical staining. The positive rate of bax protein of each group was 19.34±0.35%, 20.88±0.91%, 40.02±1.20%, 45.72±0.88% and 52.3±1.54%, respectively by immunohistochemical staining. The density of bcl-2 mRNA in 0.2 mL normal saline solution, 1 500 mg/kg DMSO, 500 mg/kg resveratrol, 1 000 mg/kg resveratrol, and 1 500 mg/kg resveratrol decreased progressively and the density of bax mRNA in 0.2 mL normal saline solution, 1 500 mg/kg DMSO, 500 mg/kg resveratrol, 1 000 mg/kg resveratrol, and 1 500 mg/kg increased progressively with elongation of time by RT-PCR. CONCLUSION: Resveratrol is able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated by down-regulating apoptosis-regulated gene bcl-2 and up-regulating the expression of apoptosis-regulated gene bax.     

过氧化物酶体增殖物活化受体γ配体15d-PGJ2对肝星状细胞增殖活化的影响

目的观察过氧化物酶体增殖物活化受体γ(PPARγ)的天然配体15d-PGJ2对HSC增殖及活化的影响,以探讨PPARγ在HSC活化过程中的作用。方法采用MTT法和RT-PCR方法观察5μmol/L及10μmol/L 15d-PGJ2对体外培养的HSC自发活化及血小板衍生生长因子(PDGF)引起的HSC增殖及活化的影响。结果以5μmol/L 15d-PGJ2处理原代HSC 3 d后,可明显抑制HSC活化标志物α-平滑肌肌动蛋白的表达,而PPARγ的表达较未处理组明显增高(0.64±0.03对比0.09±0.01,t=36.0517,P<0.01);15d-PGJ2可剂量依赖性地抑制PDGF引起的HSC增殖;经5μmol/L和10μmol/L 15d-PGJ2预处理后再用PDGF干预,则PPARγ的表达较单用PDGF干预组明显增高(分别为0.03±0.02对比0.60±0.03,t=42.6616,P<0.01;以及0.03±0.02对比0.69±0.04,t=33.83,P<0.01),而HSC的活化指标α-平滑肌肌动蛋白、α1(I)型胶原及单核细胞趋化蛋白-1的表达则受抑制。结论激活PPARγ可调控HSC的促纤维化和促炎症作用,促进PPARγ的表达可能成为抗肝纤维化的新手段。     

PTEN过表达及其突变对体外活化肝星状细胞凋亡的影响

目的 探讨过表达的野生型PTEN及其突变体G129E对体外培养的活化肝星状细胞(HSC)增殖、凋亡的影响及其机制.方法 体外培养活化的HSC,以腺病毒为载体将野生型PTEN基因及其突变体G129E基因瞬时转染HSC;四甲基偶氮唑盐(MTT)法检测HSC增殖;末端转移酶标记技术(TUNEL)及流式细胞术测定HSC凋亡;Western印迹及实时荧光定量PCR方法 检测HSC PTEN表达;Western印迹测定HSC Bcl-2及Bax表达.结果 外源性野生型PTEN基因及G129E基因成功转染体外活化HSC,并引起HSC的Bax表达增加,Bcl-2表达下降(P<0.01).过表达的野生型PTEN及G129E明显抑制HSC增殖,在转染HSC后48 h、72 h的增殖抑制率分别为14.03%、23.12%和9.52%、12.63%.野生型PTEN基因及G129E基因转染HSC后72 h,HSC凋亡率均显著增加(P<0.01).在上述作用中野生型PTEN均明显强于其突变体G129E.结论 过表达的野生型PTEN及其突变体G129E通过降低Bcl-2/Bax途径诱导体外活化HSC凋亡,并抑制其增殖;并且,野生型PTEN的作用明显强于G129E.     

Effect of endotoxin pretreatment on hepatic stellate cell response to ethanol and acetaldehyde

BACKGROUND AND AIM: The role of endotoxin in alcohol-induced liver damage is well recognized. How pre-exposure to endotoxin might affect alcohol injury is not known. We herein studied the effect of endotoxin pretreatment on hepatic stellate cell (HSC) response to ethanol and acetaldehyde. METHODS: Rat HSC (CFSC-2G) were exposed to media supplemented with 1 microg/mL lipopolysaccharide (LPS). This was followed by a 24 h exposure to media containing LPS plus 50 mmol/L ethanol or 175 micromol/L acetaldehyde. Lipid peroxidation, collagen, and tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6 and transforming growth factor (TGF)-beta1 secretion were determined at the end of both periods of exposure. RESULTS: Lipopolysaccharide pretreatment did not modify lipid peroxidation induced by ethanol or acetaldehyde alone. Glutathione (GSH) content decreased to 4.2 +/- 0.5 and 16.3 +/- 0.8 nmol protein after exposure to ethanol or acetaldehyde alone, and decreased further with LPS pretreatment (2.4 +/- 0.2 and 2.7 +/- 0.3 nmol/mg protein, respectively). Oxidized GSH (GSSG) content increased in ethanol and acetaldehyde LPS-pretreated cells only. Collagen secretion increased to 988 +/- 82 and 1169 +/- 91 microg/10(6) cells after exposure to acetaldehyde or LPS alone. Lipopolysaccharide pretreatment enhanced collagen secretion significantly in both ethanol- and acetaldehyde-treated cells (969 +/- 56 and 1360 +/- 72 microg/10(6) cells, respectively). Interleukin-6 production increased to 288 +/- 48, 1195 +/- 86 and 247 +/- 35 pg/mL per 10(6) cells after ethanol, acetaldehyde and LPS exposure, and increased further with LPS pretreatment in ethanol-exposed cells (680 +/- 23 pg/mL 10(6) cells). CONCLUSION: Lipopolysaccharide pretreatment of HSC adds to the damage produced by ethanol and acetaldehyde by diminishing GSH content and increasing GSSG content, collagen and IL-6 secretion.     

抗结缔组织生长因子小干扰RNA对肝星状细胞合成分泌细胞外基质的影响

目的 研究化学合成抗结缔组织生长因子(CTGF)小干扰RNA(siRNA)对肝星状细胞(HSC)CTGF基因表达及合成分泌细胞外基质(ECM)的影响。 方法 将化学合成抗CTGF siRNA以Oligofectamine包裹,转染HSC T6细胞,设空白对照,抽提孵育24、48、72 h细胞总RNA及蛋白质,并收集培养上清液,应用western blot和(或)逆转录聚合酶链反直检测HSC T6细胞CTGF及Ⅰ、Ⅲ型胶原基因表达,应用放射免疫法检测培养上清液中透明质酸及Ⅲ型前胶原含量。 结果 与空白对照相比,转染siRNA的HSC T6细胞CTGF及Ⅰ、Ⅲ型胶原基因表达水平显著下调,培养上清液中透明质酸及Ⅲ型前胶原含量24、48、72h分别降低46％±7％,52％±7％,53％±7％(F=157.45,P=0.0001)和29％±18％,29％±7％,27％±5％(F=10.77,P=0.0079)。 结论 化学合成抗CTGF siRNA能高效抑制HSC CTGF基因表达,显著减少HSC Ⅰ、Ⅲ型胶原及透明质酸等ECM的合成与分泌,抑制效应可持续72 h,提示化学合成CTGF siRNA具有预防及治疗肝纤维化的潜力并具有极好的开发前景。     

Helicobacter pylori infection induces apoptosis in gastric cancer cells through the mitochondrial pathway

BACKGROUND AND AIMS: To clarify the role of the mitochondrial pathway in apoptosis induced by H. pylori infection in gastric epithelial cells. METHODS: Cells of a gastric adenocarcinoma cell line SGC-7,901 were co-cultured with H. pylori NCTC 11,637, with or without preincubation with the inhibitors of caspases -3, -8, and -9. Apoptosis was determined by flow cytometry. RT-PCR was used to determine the expression of Bid, Bax, and Bcl-2 mRNA, and Western blotting was used to determine the expression of Bid, Bax, and Bcl-2 proteins, and the activation of caspases -3 and -9. RESULTS: H. pylori directly induced apoptosis in SGC-7,901 cells. Apoptotic indices (AIs) were 6.30 +/- 0.40%, 11.57 +/- 0.78%, 8.63 +/- 0.67%, and 7.22 +/- 0.97%, respectively, at 6, 12, 24, and 48 h after SGC-7,901 cells were co-cultured with H. pylori. H. pylori up-regulated the expression of Bid and Bax at both protein and mRNA levels, and induced a time-dependent activation of caspases -3 and -9. Apoptosis was inhibited significantly by the preincubation of SGC-7,901 cells with the inhibitors of caspase-3 (AIs were 1.72 +/- 0.59%, 2.97 +/- 0.55%, 4.38 +/- 1.56%, and 3.29 +/- 0.83%, respectively, at 6, 12, 24, and 48 h), and caspase -9 (AIs were 2.47 +/- 0.53%, 6.68 +/- 0.47%, 5.97 +/- 0.46%, and 5.43 +/- 0.15%, respectively, at 6, 12, 24, and 48 h). The caspase-8 inhibitor also reduced H. pylori-induced apoptosis by 20%. CONCLUSIONS: H. pylori infection induces apoptosis and the activation of caspases -3 and -9 in gastric cancer cells. Moreover, the caspase inhibitors significantly suppress H. pylori-induced apoptosis. These findings suggest that the mitochondrial pathway may be the major pathway in H. pylori-induced apoptosis in gastric epithelial cells.     

Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker

Hematopoietic tissues in acute myeloid leukemia (AML) patients contain both leukemia stem cells (LSC) and residual normal hematopoietic stem cells (HSC). The ability to prospectively separate residual HSC from LSC would enable important scientific and clinical investigation including the possibility of purged autologous hematopoietic cell transplants. We report here the identification of TIM3 as an AML stem cell surface marker more highly expressed on multiple specimens of AML LSC than on normal bone marrow HSC. TIM3 expression was detected in all cytogenetic subgroups of AML, but was significantly higher in AML-associated with core binding factor translocations or mutations in CEBPA. By assessing engraftment in NOD/SCID/IL2Rγ-null mice, we determined that HSC function resides predominantly in the TIM3-negative fraction of normal bone marrow, whereas LSC function from multiple AML specimens resides predominantly in the TIM3-positive compartment. Significantly, differential TIM3 expression enabled the prospective separation of HSC from LSC in the majority of AML specimens with detectable residual HSC function.     

Apoptosis mechanisms of human gastric cancer cell line MKN-45 infected with human mutant p27

AIM: To explore the inducing effect of human mutant p27 gene on the apoptosis of the human gastric cancer cell line MKN-45 and its associated mechanisms. METHODS: The recombinant adenovirus Ad-p27mt was constructed to infect the human gastric cancer cell line MKN-45. Using flow cytometry, TUNEL assay and DNA fragment analysis, we measured the apoptotic effect of Ad-p27mt on the human gastric cancer cells. RESULTS: Ad-p27mt was successfully constructed and the infection efficiency reached 100%. After 18 h of infection, we observed an apoptotic hypodiploid peak on the flow cytometer before G1-S and apoptotic characteristic bands in the DNA electrophoresis. The apoptotic rate detected by TUNEL method was significantly higher in the Ad-p27mt group (89.4±3.12%) compared to the control group (3.12±0.13%, P < 0.01). CONCLUSION: Human mutant p27 can induce apoptosis of the human gastric cancer cells in vitro.