端粒酶反义寡核苷酸对膀胱癌T24细胞的作用

目的 探讨全硫代修饰的端粒酶反义寡核苷酸（ASODN-t)抑制人膀胱癌T24细胞增殖并诱导其凋亡的可能性。为膀胱癌基因治疗提供新靶点。方法 采用光镜、电镜、四唑盐比色（MTT）法及流式细胞术等检测不同浓度（2-10umol/L)ASODN-t对T24细胞生长、细胞周期和诱导其凋亡的作用。结果 不同浓度ASODN-t对T24细胞生长有抑制作用。且具有序列特异性及剂量依赖性,6umol/lASODN-t作用细胞72h,电镜观察可见典型凋亡特征,流式细胞仪检测到凋亡峰,凋亡率为10.4%,而无关寡核苷酸（N-ODN）则无此作用。结论 ASODN-t对T24细胞生长有抑制作用,可诱导其发生凋亡,说明端粒酶与T24细胞恶性增殖有关。抑制端粒酶活性可能成为膀胱癌基因治疗的新靶点。     

五羟黄酮调节肝癌HepG2细胞bax基因表达诱导细胞凋亡的实验研究

目的:探讨三磷酸肌醇( IP3) 和bax基因表达变化在五羟黄酮(Quercetin)诱导肝癌细胞凋亡中的作用。方法:以肝癌HepG2 细胞培养72 h为对照,以20，40，60，80μmol/L Quercetin作用于 HepG2 细胞72 h 时和60μmol/L Quercetin 作用于 HepG2 细胞 6，12，24，48，72 h，应用同位素试剂盒检测细胞IP3含量,RT-PCR分析bax mRNA表达，Western blotting 分析细胞bax蛋白表达,流式细胞仪检测细胞凋亡率。结果:各浓度的Quercetin作用于肝癌HepG2 细胞72 h，IP3含量显著低于对照组(P<0.01)，bax mRNA和bax蛋白表达显著高于对照组，细胞凋亡率显著高于对照组(P<0.01); 60 μmol/L Quercetin 作用于肝癌HepG2 细胞6，12，24，48，72 h,各时相IP3含量显著低于对照组(P<0.01）;12 h后bax mRNA和bax蛋白表达显著高于对照组，24 h后各时相细胞凋亡率为显著高于对照组（P<0.01）。结论:Quercetin能减少IP3生成,上调bax基因表达,诱导肝癌细胞凋亡。     

重组转化生长因子α-绿脓杆菌外毒素融合蛋白对人膀胱癌细胞生长的抑制作用

目的 探讨重组转化生长因子α-绿脓杆菌外毒素融合蛋白(TP40)对膀胱癌细胞生长的抑制作用。方法 采用蛋白印迹(Western blot)法检测人膀胱癌124细胞表皮生长因子受体(EGFR)的表达。用浓度为5～1000μg／L的TP40处理T24细胞,甲基噻唑基四唑(MTY)法检测细胞生长24、48、72和96h时的生长情况。氚-胸腺嘧啶核苷([^3H]-TdR)掺入法检测TP40对T24细胞蛋白质合成的抑制作用。用浓度为1～7500μg／L的表皮生长因子(EGF)竞争拮抗TP40的细胞毒作用。结果 T24细胞表达EGFR强阳性。浓度为5、50、100、250、500、750和1000μg／L的TP40处理T24细胞96h,细胞生长抑制率分别为10％、19％、27％、41％、47％、53％和6l％。用750μg／L的TP40处理T24细胞2,4、48、72和96h,[^3H]-TdR掺入率分别为80％,69％,48％和51％。浓度为1～7500μg／L的EGF对TP40有竞争抑制作用。结论 人膀胱癌T24细胞能高水平地表达EGFR;重组TP40对T24细胞的生长具有剂量、时间依赖性抑制作用,该作用是通过EGFR进行的。     

胡椒碱对人膀胱癌 T24细胞增殖及侵袭作用的研究

目的：探讨不同浓度胡椒碱对膀胱癌 T24细胞增殖和侵袭作用的影响。方法用不同浓度的胡椒碱（5、10、20、40、80、160μmol／L）处理体外培养的膀胱癌 T24细胞,然后通过 MTT实验、Transwell 实验、Western blot 等方法检测 bax 和 bcl-2的表达以及 T24细胞的增殖和凋亡情况。结果胡椒碱作用于 T24细胞24 h 后,IC50值为38．73μmol／L,并且呈剂量依赖性。随着胡椒碱浓度的增加,T24细胞活性被抑制作用明显增加,且抑凋亡蛋白 bcl-2的表达量减少,促凋亡蛋白 bax 的表达量增加。结论胡椒碱对膀胱癌 T24细胞具有抑制增殖及促进其凋亡的作用。     

紫杉醇联合大蒜素对胃癌细胞的增殖抑制及促凋亡作用

目的：探讨紫杉醇联合大蒜素对胃癌细胞MGC-803的增殖抑制和促凋亡作用及其分子机制。方法：分别及联合应用两药后以MTT法测定MGC-803细胞的增殖，用流式细胞仪检测细胞凋亡，用RT-PCR法观察基因bax和bcl-2 mRNA的表达，用Western blot观察其蛋白表达。 结果：15～60μg/mL大蒜素和4～32μg /mL紫杉醇单用24h对MGC-803细胞均有明显抑制作用，呈剂量依赖性。低剂量大蒜素（9μg /mL）联合紫杉醇（1～16μg/mL）比单用紫杉醇作用更强（P<0.05）。15μg/mL大蒜素,12μg /mL紫杉醇和两药联用24h凋亡率分别为10.7%，30.4%和84.7%（P<0.01 ）。单用大蒜素和紫杉醇对胃癌细胞bax和bcl-2表达无明显影响，两药联用bax基因mRNA和蛋白表达增加，而bcl-2mRNA和蛋白的表达减少。结论：大蒜素可增强紫杉醇对胃癌细胞的增殖抑制作用，两药联用可减少紫杉醇的剂量;其机制可能是通过增强MGC-803细胞的凋亡，后者与bax和bcl-2基因表达有关。     

比较雷帕霉素和吡柔比星抑制膀胱癌T24细胞生长活性的实验研究

目的对单独使用雷帕霉素（RPM）和吡柔比星（THP）抑制膀胱癌T24细胞生长活性的作用进行比较,探究雷帕霉素在膀胱肿瘤患者中的应用前景。方法建立空白对照组,将雷帕霉素和吡柔比星分别调整至10μg/mL、0.8μg/100μL,作用于膀胱癌T24细胞,处理24h后分别使用MTT法、流式细胞仪、RT-PCR和划痕法探索比较在此浓度下两种药物对膀胱癌T24细胞的影响;在体内实验,BALB/c裸鼠种植转移性人膀胱癌构建荷瘤模型。随机分为RPM（2mg/kg）、THP（0.015mg/cm2）组和空白对照组（生理盐水）,观察RPM和TPH对肿瘤生长及转移的影响。结果 RPM和THP分别在10μg/mL、0.8μg/100μL质量浓度下均对膀胱癌T24细胞生长活性具有明显抑制作用,同时此浓度下RPM和THP抑制T24细胞增殖的作用无明显差异（P〉0.05）;RPM组下调血管生长因子（VEGF）的作用明显强于THP组（P〈0.05）;体内实验中,RPM和THP均显著抑制肿瘤的生长及转移。结论雷帕霉素（RPM）和吡柔比星（THP）均具有良好的抑制T24细胞生长的作用,但在此浓度下THP的细胞毒性较雷帕霉素更强,并且雷帕霉素（RPM）明显抑制膀胱癌T24细胞的生长活性的作用为治疗膀胱癌治疗提供了实验参考,表现出良好的临床应用前景。     

赫赛汀联合丝裂霉素C对人膀胱癌T24细胞生长的抑制作用

目的 探讨赫赛汀(Herceptin,HER)联合丝裂霉素C(MMC)对HER-2/neu基因高表达人膀胱癌细胞株生长的抑制作用.方法 应用免疫细胞化学法、逆转录-聚合酶链反应(RT-PCR)法检测膀胱癌T24细胞株HER-2/neu的表达;采用噻唑蓝(MTT)比色法测定不同浓度HER(10、20、40、80、160μg/L)、MMC(4、8、16、32、64μg/L)及联合用药对人膀胱癌T24细胞株体外生长的抑制率并进行比较.结果 应用免疫细胞化学方法及RT-PCR法检测证实HER-2/neu在膀胱尿路上皮癌T24细胞株高表达;单用HER于72 h出现细胞抑制(72 h时各浓度组分别与48 h时比较,均P<0.05);HER和MMC联用在24、48、72 h基本上均表现为协同作用(q值1.264～3.473),在96 h呈相加作用(q值0.913～1.138).结论 HER-2/neu在膀胱尿路上皮癌T24细胞株高表达;HER单药对T24细胞有轻度抑制作用,且起效慢(72 h);与MMC联合应用能协同抑制T24细胞生长.     

硒化麒麟菜多糖对膀胱癌细胞生长、凋亡的作用及Bax、Bcl-2、caspase-3蛋白表达的影响

目的研究硒化麒麟菜多糖对膀胱癌T24细胞生长、凋亡的作用及Bax、Bcl-2、caspase-3蛋白表达的影响。方法体外培养T24膀胱癌细胞,分别利用0、0.125、0.25、0.5、1 mg/mL的硒化麒麟菜多糖处理T24细胞24h、48h、72h,利用噻唑蓝(MTT)检测各组细胞增殖情况,0.25、0.5、1mg/mL的硒化麒麟菜多糖处理细胞48h后,流式细胞仪检测T24细胞周期分布及细胞凋亡,Western-blot检测T24细胞Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤/白血病-2(Bcl-2)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved caspase-3)蛋白表达。结果与0mg/mL处理相比,0.25、0.5、1mg/mL的硒化麒麟菜多糖处理抑制T24细胞增殖,差异有非常显著性统计学意义(P0.01);使用0.125mg/mL硒化麒麟菜多糖处理,作用24h时对细胞增殖抑制不显著(P0.05),作用48h、72h时对细胞增殖具有较为明显抑制作用(P0.05)。1mg/mL的硒化麒麟菜多糖作用后细胞凋亡率高达(23.56±0.96)%。与0 mg/mL处理相比,0.25、0.5、1 mg/mL的硒化麒麟菜多糖作用后,处于G0/G1期的细胞从(52.12±1.74)%分别增加至(64.32±2.42)%、(70.72±1.71)%、(76.09±2.28)%。处于G2/M期和S细胞明显减少(P0.05),Bcl-2蛋白的表达明显降低(P0.01),Bax、Cleaved-caspase-3蛋白的表达明显升高(P0.01)。结论硒化麒麟菜多糖可能通过调控Bax、Bcl-2、Cleaved-caspase-3蛋白表达抑制T24细胞增殖,阻滞细胞周期进程,促进细胞凋亡。     

蒲公英甾醇调控膀胱癌T24 细胞迁移与侵袭能力的作用及机制研究

目的:探索蒲公英甾醇对膀胱癌T24细胞迁移与侵袭能力的作用及机制。方法:体外培养膀胱癌T24细胞,在其中加入不同浓度(10、20、40μg/mL)蒲公英甾醇进行干预。采用划痕实验和Transwell小室法分别观察膀胱癌细胞迁移能力及侵袭能力;Western blotting实验考察细胞中SDF-1/CXCR4信号通路相关蛋白的变化;ELISA试剂盒检测细胞上清中基质金属蛋白酶(MMP)-2/9的水平。结果:蒲公英甾醇可抑制膀胱癌T24细胞的迁移与侵袭能力(P<0.05,P<0.01),降低细胞分泌MMP-2及MMP-9的能力(均P<0.05,P<0.01)。同时蒲公英甾醇能有效抑制膀胱癌T24细胞内基质细胞衍生因子-1(SDF-1)、CXCR4、磷酸化蛋白激酶B(p-Akt)及磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)的表达水平(P<0.05,P<0.01)。结论:蒲公英甾醇通过干扰SDF-1/CXCR4信号通过,下调Akt/mTOR的传导,削弱了细胞分泌MMP-2及MMP-9的能力,从而有效抑制膀胱癌T24细胞的迁移与侵袭。     

靶向沉默Notch1基因对人膀胱癌细胞生物学行为的影响

目的 探讨沉默Notch1基因对人膀胱癌细胞生物学行为的影响. 方法 将靶向Notch1的siRNA真核表达载体psiRNA Notch1-1转染膀胱癌细胞株T24和BIU-87,噻唑盐法、流式细胞术检测沉默Notch1后膀胱癌细胞生长、细胞周期和凋亡情况.RT-PCR和蛋白质印迹法检测转染前后Notch1基因mRNA和蛋白表达的变化. 结果 转染后72 h,T24和BIU-87细胞G0/G1期细胞比例明显增加,分别为(80.13±2.69)%和(69.44±2.41)%,与T24对照组(23.89±1.32)%和BIU-87对照组(24.63±1.68)%比较差异有统计学意义(P值均<0.05).转染后72 h,T24和BIU-87细胞凋亡率分别为(13.75±1.23)%和(8.72±1.01)%,与T24对照组(1.28±0.14)%和BIU-87对照组(1.01±0.27)%比较差异均有统计学意义(P值均<0.05).转染后24 h细胞生长明显受到抑制,该抑制作用持续到转染后96 h.转染后72 h,T24和BIU-87细胞中Notch1基因mRNA和蛋白表达明显下调(P<0.05). 结论 Notch1在膀胱癌细胞株中可能起致癌作用.沉默Notch1基因能够抑制膀胱癌细胞的增殖.     

粉防己碱对激素非依赖性人前列腺癌细胞株PC-3增殖和凋亡影响的研究

目的 探讨粉防己碱(TET)对激素非依赖性人前列腺癌细胞株PC-3细胞增殖抑制和凋亡作用及其机制。方法 应用CCK法观察不同浓度TET（1、2、4、8 μg/mL）对人前列腺癌细胞株PC-3生长的抑制作用;用Annexin-V与PI双染法流式细胞术分析TET对PC-3细胞凋亡的影响;RT-PCR检测TET对人前列腺癌细胞株PC-3 IL-8表达水平的影响。结果 TET对前列腺癌PC-3细胞株增殖有抑制作用,其抑制效应呈剂量依赖性。不同浓度TET作用48 h后,前列腺癌PC-3细胞株的凋亡率随着浓度的增高而升高,差异具有统计学意义（P<0.01）。不同浓度TET作用48 h后IL-8 mRNA表达水平随着浓度的增加而降低,差异具有统计学意义（P<0.01）。结论 TET能抑制人前列腺癌细胞株PC-3细胞增殖,并促进人前列腺癌细胞株PC-3的细胞凋亡,其作用机制可能与其下调IL-8 mRNA表达有关。     

Effect of heat shock protein 47 on epithelial-mesenchymal transdifferentiation induced by transforming growth factor β1 in the renal tubular epithelial cells

Objective To detect the expression of heat shock protein 47(HSP47) in renal proximal epithelial cell lines (HK-2) and to investigate the role of HSP47 in the progress of transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transdifferentiation (EMT) in HK-2 cells. Methods HK-2 cells were exposed to TGF-β1 (0, 2.5, 5, 10 μg/L) for different time (0, 12, 24, 48 h). The expression of HSP47 was examined by Western blotting. Then HK-2 cells were exposed to 10 μg/L TGF-β1, the expressions of vimentin, zona occludens-1 (ZO-1) were examined by Western blotting and real-time PCR. Furthermore, the expressions of p-Smad3 and Smad3 were examined by Western blotting. HK-2 cells were transfected with HSP47 siRNA and siRNA negative control before exposing to TGF-β1. Then the expressions of vimentin, ZO-1 were detected by Western blotting and real-time PCR, meanwhile Western blotting for HSP47, p-Smad3 and Smad3. Results Stimulating HK-2 with TGF-β1resulted in a significant increased expression of HSP47 in time-and concentration-dependent manner (P＜0.05). Meanwhile, TGF-β1up-regulated the protein and mRNA expression of vimentin (P＜0.05), and down-regulated the protein and mRNA expression of ZO-1 (P＜0.05), all in time-dependent manner. Stimulating HK-2 with TGF-β1 resulted in phosphorylation of Smad3, which was peaked at 30 min, slightly decreased at 1 h, and then increased again between 24 and 48 h (P＜0.05). Compared to the TGF-β1group, inhibition of HSP47 expression in HK-2 up-regulated the protein and mRNA expression of ZO-1, down-regulated the protein and mRNA expression ofvimentin (P＜0.05) and down-regulated the ratio of p-Smad3/Smad3. HSP47 siRNA negative control had no significant effect on the expressions of ZO-1, vimentin and p-Smad3/Smad3 (P＞0.05). Conclusion HSP47 can promote the EMT of renal tubular epithelial cell which is possibly via the TGF-β1-Smad3 pathway.     

Overexpression of decorin induces apoptosis and cell growth arrest in cultured rat mesangial cells in vitro

Background: Decorin (DCN) is a small leucine-rich proteoglycan that plays an important role in the regulation of intercellular contact, cell migration and proliferation. DCN suppresses cell growth and induces apoptosis in various tumour cells. The aim of this study was to investigate whether overexpression of DCN could induce apoptosis and cell growth arrest in mesangial cells (MsCs) in vitro. Methods: PcDNA3.1A-DCN plasmid was transfected into cultured rat MsCs, and positive clones stably expressing DCN (MsC/DCN) were selected. SiRNA was used for blocking DCN expression in MsC/DCN. Apoptosis and cell growth of MsCs were assayed by flow cytometry. Hoechst staining was used for observing apoptotic cells. Expressions of active Caspase-3, epidermal growth factor receptor (EGFR), P21 and transforming growth factor-β (TGF-β1) were analyzed using Western blot. Results: Overexpression of DCN in MsCs induced apoptosis and arrested cells in the G₀/G₁ phase. The protein level of active Caspase-3 was significantly elevated in MsC/DCN (P < 0.01). DCN transfection induced downregulation of EGFR and up-expression of P21. In addition, the expression of TGF-β1 was significantly inhibited. DCN-siRNA transfection remarkably blocked the expression of DCN and reversed the downregulatory effects of DCN on MsC's proliferation. Conclusion: Overexpression of DCN could inhibit MsCs proliferation by inducing apoptosis and cell growth arrest in vitro and it also downregulates expression of TGF-β1. These results suggest novel strategies for regulating the proliferation of MsC in glomerular diseases.     

饰胶蛋白聚糖与转化生长因子β1对大鼠肾系膜细胞生长及相关信号途径的影响

目的探讨饰胶蛋白聚糖（DCN）与转化生长因子β1（TGF-β1）对大鼠肾系膜细胞（MsC）生长及相关信号途径的影响。方法经脂质体介导将DCN基因转染体外培养的大鼠肾MsC,经筛选和鉴定后,收集阳性细胞克隆的培养上清（DCN上清）。采用流式细胞仪检测经TGF-β1（2μg/L）和（或）DCN上清作用后MsC细胞周期的变化。采用Western印迹法分别检测两者单独或联合作用后,MsC磷酸化丝裂原活化蛋白激酶（p-MAPK）、磷酸化Smad2 （p-Smad2）和p21蛋白表达情况。采用免疫共沉淀方法检测阳性细胞克隆上清中DCN与TGF-β1的结合情况。结果TGF-β1或DCN上清单独作用时均可抑制MsC的增殖,G_2-M期细胞数分别为对照组的81%及86.5%,而两者共同作用时G_2-M期细胞数与对照组差异无统计学意义。经TGF-β1和DCN上清单独作用12 h后,p-ERK1/2表达为对照组的4.4、3.7倍和3.2、3.7倍;p-SAPK/JNK表达为对照组的2.0、1.5倍和1.9、1.5倍;而两者共同作用组p-ERK1/2和p-SAPK/JNK表达与对照组差异无统计学意义。TGF-β1单独作用12 h后,MsC p-Smad2的表达为对照组的2.6倍,而DCN单独作用组及2者联合作用组其表达与对照组差异无统计学意义。DCN单独作用12 h后p21蛋白的表达为对照组的2.6倍,而TGF-β1单独作用及2者联合作用组其表达与对照组差异无统计学意义。阳性细胞克隆上清中DCN可以直接与TGF-β1结合。结论TGF-β1与DCN单独作用均可抑制MsC增殖及激活MAPK信号途径,但2者共同作用时其作用相互抵消。TGF-β1激活Smad信号途径作用可被DCN阻断;DCN上调p21蛋白作用可被TGF-β1阻断。2者通过不同信号分子抑制细胞生长,其作用可能与两者相互结合有关。     

原花青素对人结肠癌细胞SW620增殖和凋亡的影响

目的:观察葡萄籽提取物原花青素对人结肠癌细胞株SW620细胞增殖和凋亡的影响。方法:SW620细胞与20、40、60、80μg/mL的原花青素共同孵育24h～8d,采用噻唑蓝(MTT)比色法检测细胞增殖抑制率,流式细胞术分析细胞凋亡情况,分光光度法检测Caspase-3酶活性。结果:不同浓度(20～80μg/mL)的原花青素对SW620细胞的生长有明显的抑制作用,呈现出一定的剂量依赖性;含原花青素20、40、80μg/mL的观察组细胞凋亡发生率分别为6.9%、18.6%及22.9%,不含药的对照组细胞凋亡发生率为1.3%。SW620细胞与含药(40μg/mL)或不含药的培养基共同培养48h或72h,对照组细胞几乎无法检出Caspase-3酶活性,而给药组细胞活性显著增加。结论:原花青素在体外可呈浓度依赖性抑制人结肠癌细胞株SW620的增殖并通过Caspase-3通路促进其凋亡。     

饰胶蛋白聚糖对大鼠肾系膜细胞生长的抑制作用及其信号转导途径的研究

目的 探讨饰胶蛋白聚糖(DCN)对大鼠系膜细胞(MsC)生长的抑制作用及其信号转导分子MAPKs和p21表达的影响&#65377;方法 经脂质体介导将DCN基因转染体外培养的大鼠MsC,筛选和鉴定后,收集阳性细胞克隆的培养上清(DCN上清),将其加入正常MsC的培养液中, 采用流式细胞仪检测细胞周期&#65377;用Western 印迹法分别检测MAPKs,包括细胞外调节激酶(ERK)1/2&#65380;应激活化蛋白激酶(SAPK)/氨基末端激酶(JNK)和p38和p21蛋白表达;用免疫荧光法观察p21在细胞中的表达&#65377;结果 DCN上清明显抑制正常MsC的增殖, G2-M期细胞数明显减少,仅为对照组的35%(P < 0.05); 磷酸化ERK1/2及SAPK/JNK表达增强, 分别为对照组的2.2倍&#65380; 1.4倍及1.7倍&#65380; 1.8倍;磷酸化p38无明显变化&#65377;DCN抗体呈浓度依赖性抑制磷酸化ERK1/2&#65380; SAPK/JNK的表达上调&#65377;DCN上清还可使细胞p21的表达明显增强,而DCN抗体也同样呈浓度依赖性地抑制其上调表达的作用, ERK1/2 及SAPK/JNK通路抑制剂U0126和circumin均可抑制其上调表达的作用,分别为对照组的64%和61%;而p38通路抑制剂SB203580则对其无影响&#65377;结论 DCN对肾MsC的生长有抑制作用,其机制可能经ERK1/2&#65380;SAPK/JNK和p21蛋白介导&#65377;     

Distinct functions for Ras GTPases in the control of proliferation and apoptosis in mouse and human mesangial cells

In previous work, we have demonstrated that Ras GTPases regulate proliferation in a range of human renal cells. The present work compares human and mouse mesangial cell (HMC and MMC) responses to specific knockdown of Ras genes with antisense oligonucleotides (AS-oligos), and examines the role of the p21 (cip1) and p27 (kip1) cyclin-dependent kinase inhibitors in these responses in mouse cells. HMC and MMC were lipofectin transfected with ras-targeted AS-oligo at 200-400 nM for 18 h followed by growth of cells in 20% serum for 18-72 h. Cell proliferation was assessed with an MTS assay and bromodeoxyuridine (BrdU) uptake. Apoptosis was quantified using nuclear stain with Hoechst 33342 dye. In MMC, Ha-ras AS-oligo caused an increase in apoptosis from <2% to 10-15% of cells after 18 h in serum (P<0.01). Control, Ki-ras and N-ras AS-oligos had minimal effects on apoptosis. BrdU uptake studies showed that BrdU+ve MMC were increased by 20-40% (P<0.05) after Ha-ras AS-oligo at 24 h; other ras AS-oligos were inactive. HMC number was reduced by 40-80% (P<0.01) at 48-72 h by both Ha-ras and Ki-ras AS-oligos. These actions were associated with reductions in BrdU+ve cells. In HMC, the ras AS-oligos did not induce apoptosis. p21(-,-) MMC showed exaggerated apoptotic responses to Ha-Ras AS-oligo. In mouse cells, Ha-Ras expression appears necessary to prevent apoptotic cell death; Ras expression does not appear necessary for cells to progress through the cell cycle. In human cells, Ras does not appear necessary to prevent apoptosis but Ha-Ras and Ki-Ras appear to be required for cell cycle progression.     

Prostaglandin E2 receptor subtype 3-siRNA reduces the mesangial cell damage induced by TGF-β1through inhibiting MAPK pathway in mice

Objective To investigate the effects and mechanisms of prostaglandin E2 receptor subtype 3 (EP3) on transforming growth factor β1 (TGF-β1)-induced mouse mesangial cells damage. Methods Primary mouse mesangial cells were separated and cultured. Three siRNAs were synthesized and transfected into mesangial cells for silencing EP3 by LipofectamineTM 2000 and the best one was chosen. MCs were grouped into: (1)control group; (2)TGF-β1 (10 μg/L) group; (3)NC-siRNA plus TGF-β1 (10 μg/L) group; (4) EP3-siRNA group; (5)EP3-siRNA plus TGF-β1 (10 μg/L). Then the proliferation of MCs was evaluated by CCK-8 assay. The expression of PGE2 and cAMP in cell supernatant were detected by ELISA. The mRNA and protein expression of fibronectin (FN), connective tissue growth factor (CTGF), cyclooxygenase-2 (COX2), membrane-bound prostaglandin E2 synthase 1 (mPGES1) were detected by real-time quantitative PCR and Western blotting. The phosphorylation of p38 MAPK and ERK1/2 was decected by Western blotting. Results Compared with control group, the cell proliferation induced by TGF-β1 was increased (P＜0.05), the expression of PGE2 and cAMP were improved, mRNA and protein expression of FN, CTGF, COX2 and mPGES1 were up-regulated (all P＜0.05). Compared with TGF-β1 group, the cell proliferation in EP3-siRNA plus TGF-β1 group was reduced, the expression of FN, CTGF, COX2 and mPGES1 mRNA and protein were downregulated (all P＜0.05), the phosphorylation of ERK1/2, p38 MAPK were also declined (P＜0.05). Conclusion EP3-siRNA may reduce TGF-β1-induced cell damage through upregulating the expression of cAMP, repressing the activity of ERK1/2 and p38 MAPK, inhibiting the expression of COX2 mPGES1 and PGE2 by feedback, then decreased the expression of FN and CTGF.     

核心蛋白聚糖蛋白对人结肠癌HCT-8细胞的生物学作用

为探讨核心蛋白聚糖（decorin,DCN）蛋白对人结肠癌HCT8细胞的生物学作用,本实验将重组载体PcDNA3．1DCN／His（MDCN）稳定转染入HCT一8细胞,并将携带有目的基因的细胞进行扩增培养,然后应用RT—PCR及WesternBlot法对M—DCN在细胞mRNA及蛋白水平的表达分别进行鉴定,应用MagExtractor方法提取携带有M—DCN基因组HCT-8细胞（HCT-8-DCN）中的DCN蛋白,应用Bradford法测定提取蛋白的含量;同时,按DCN蛋白30μg／ml对HCT-8细胞进行培养,应用MTT法检测细胞增殖活力,细胞培养96h后用流式细胞术分析细胞周期。结果显示,RT—PCR及WesternBlot实验均可见到目的条带,证实MDCN转染HCT8细胞成功并实现稳定表达。实现了针对6个组氨酸标签使用MagExtractor方法对DCN蛋白的提取。加入DCN蛋白培养HCT-8细胞组细胞生长曲线呈现为生长抑制;流式细胞术结果提示G1期细胞显著增多。结果表明,DCN蛋白具有抑制HCT8细胞增殖的作用。     

核心蛋白聚糖抑制淋巴细胞增殖及皮肤移植免疫排斥反应的研究

目的 观察核心蛋白聚糖(DCN)对小鼠皮肤移植模型术后免疫排斥反应的影响.方法 以Ficoll密度梯度离心法提取C57BL/6J脾脏淋巴细胞,96孔板接种后,各孔加入25μg刀豆蛋白A.将细胞分为5组,分别加入0、25、50、75、100μg/L的DCN,同时设立仅含细胞悬液阴性对照组和培养液空白对照组.各组细胞培养24、48、72、96、120 h后,以噻唑蓝(MTT)比色法检测各组淋巴细胞增殖率,并以流式细胞仪检测各组细胞凋亡率.以24只健康成年BALB/C小鼠为供体,30只健康成年C57BL/6J小鼠为受体,将受体随机分为5组,建立背部皮肤移植模型.A组为阴性对照组,术后腹腔注射生理盐水1 ml;B组、C、D组受体小鼠于术后分别腹腔注射50、100、150μg的DCN.同时设立E组,为C57BL/6J对C57BL/6J的同种同基因移植对照组.观察各组受体移植皮片的存活时间和急性排斥反应发生时间.术后第7天,每组各取1只受体小鼠的移植皮片行病理学检查.结果 各组不同浓度DCN对小鼠脾脏淋巴细胞体外增殖均有抑制作用,并可诱导淋巴细胞凋亡,随DCN浓度升高对淋巴细胞增殖抑制作用更强,100μg/L组抑制作用最明显,细胞凋亡率为77.6%,与其他各组比较差异有统计学意义(P＜0.05);DCN具有抑制皮肤移植免疫排斥反应及延长移植皮片存活时间,150μg组皮片存活时间最长,平均为(16.6±2.4)d,与其他各组比较差异有统计学意义(P＜0.05).且随剂量增大抑制作用明显,移植皮肤急性排斥反应轻.结论 DCN具有免疫抑制作用,可能通过诱导活化的淋巴细胞凋亡抑制小鼠皮肤移植术后急性排斥反应发生,并延长移植皮片的存活时间.

Abstract：

Objective To study the inhibitory effect of decorin (DCN) on immunological rejection in skin transplantation.Methods Spleen lymphocytes were obtained from C57BL/6J mice,inoculated in a 96-well plate,and every well was added with 25 μg CoA.These cells were divided into 5 groups and cultured with DCN in different concentrations (0,25,50,75,100 μg/L) for different time (24,48,72,96 and 120 h).Negative control group (lymphocytes only) and blank control group (culture medium only)were set up.Cell proliferation rate was measured by methylthiazol tetrazolium (MTT) assay,and the percentage of cell apoptosis was analyzed by flow cytometry.Twenty-four healthy BALB/C mice served as donors.Thirty C57BL/6J mice served as recipients and divided into 5 groups.Full-thickness-skin was transplanted through back to back from donor BALB/C to recipient C57BL/6J.Postoperatively,the recipients in group A (negative control) were intraperitoneally injected with 0.9% NaCl,and those in groups B,C and D with DCN (50,100 and 150 μg/L respectively).In group F skin transplantation was performed between two C57BL/6J mice.After skin transplantation,the survival time of skin allografts and the time of acute rejection were observed.Skin specimens were collected from one recipient of every group 7 days after operation and examined pathologically.Results DCN obviously depressed mice spleen lymphocyte proliferation in vitro and inducef lymphocyte apoptosis in vitro.In 100 μg/L DCN group ( group C ),there were most significant inhibitory effects,and the apoptosis rate was 77.6%.There were significant difference between group C and other groups ( P＜0.05 ).DCN could inhibit immunological rejection of skin transplantation and prolong the survival time of skin allografts.Among all the groups,survival time in group D was longest with the average survival time being ( 16.6 ±2.4) days ( P ＜0.05 ).The inhibitory effect of DCN was increased and the acute rejection was alleviated with the increase of DCN dosage.Conclusion DCN has the inhibitory effect.It can induce lymphocyte apoptosis and inhibit acute immunological rejection in skin transplantation.It can prolong the survival time of skin grafts.