抑制氯通道阻抑鼻咽癌细胞周期和细胞增殖

目的:研究Cl^-通道在鼻咽癌细胞调节性容积回缩(RVD)、增殖及细胞周期分布中的作用。方法:活细胞图像分析低分化鼻咽癌细胞(CNE－2Z)RVD,用台盼蓝拒染法检测细胞存活率,MTT法检测细胞增殖能力。用流式细胞仪测定细胞周期不同时相细胞百分率。结果:Cl^-通道抑制剂硝基苯丙胺基苯甲酸(NPPB)剂量依赖性抑制RVD和细胞增殖,100μmol/LNPPB明显阻抑细胞周期进程,使细胞停滞于G₁期,G₁期细胞百分率从54%提高到71%,但对细胞存活率没有显著性影响。结论:阻抑Cl^-通道可阻滞细胞于G₁期而抑制细胞增殖。提示Cl^-通道和RVD的激活是促进细胞从G₁期进入S期和维持增殖所必需的因素。     

牛磺酸减轻β-甘油磷酸诱导的大鼠血管平滑肌细胞钙化氧化应激在反流物所致食管粘膜损伤中的作用

目的：观察牛磺酸对钙化的形成及逆转作用的影响，探讨牛磺酸在血管钙化发生中的作用。方法：利用β-甘油磷酸制备钙化血管平滑肌细胞（VSMCs），测定细胞钙含量、碱性磷酸酶活性、[⁴⁵Ca]沉积及[³H]-胸腺嘧啶。结果：与对照组相比，钙化细胞的钙含量、ALP活性和[⁴⁵Ca]沉积均明显升高（P<0.05），而牛磺酸与β-甘油磷酸同时孵育呈剂量依赖性地抑制钙化发生。在牛磺酸逆转实验中，钙化细胞的钙含量、ALP活性和[⁴⁵Ca]沉积均较换液前明显降低（P<0.01）；且牛磺酸呈剂量依赖性地逆转已钙化细胞。与对照组相比，钙化细胞的细胞数量和[³H]-TdR掺入量增加（P<0.01），牛磺酸早期干预组显著抑制钙化细胞的增殖，但牛磺酸对已钙化的细胞,增殖抑制效应不明显。结论：牛磺酸可抑制细胞钙化形成且能逆转已形成钙化。     

SC58125对HepG-2细胞增殖和凋亡的影响

目的：探讨SC-58125对HepG-2细胞增殖和凋亡的作用及其分子机理。方法：应用细胞培养、MTT、TUNEL、流式细胞光度术、琼脂糖凝胶电泳及Western blot等方法研究SC-58125对HepG-2细胞增殖和凋亡的作用及其分子机理。结果：SC58125抑制HepG-2细胞的增殖、诱导其凋亡及引起G₀/G₁期阻滞，S期抑制。并使P33^cdk2、P34^cdc2、cyclinB₁、cyclinE、Mpm-2、Rb、PCNA 7种蛋白水平下降。结论：SC58125抑制HepG-2细胞的增殖及诱导其凋亡，可能与P33^cdk2、P34^cdc2、cyclinB₁、cyclinE、Mpm-2、Rb、PCNA 7种蛋白水平的下降有关。     

吗啡抑制大鼠肾上腺嗜铬细胞瘤(PC12)增殖

目的：探讨吗啡对大鼠肾上腺嗜铬细胞瘤(PC12)细胞增殖的影响及作用机制。方法: 应用MTT比色法检测细胞存活率,流式细胞术检测细胞周期。RT-PCR及免疫细胞化学检测增殖细胞核抗原（PCNA）的表达。结果: 10 μmol/L吗啡对PC12细胞的增殖有抑制作用,G₁期细胞增多，S期细胞减少。24 h PCNA mRNA表达开始低于对照组(P<0.05),48 h和72 h明显降低(P<0.01)。免疫细胞化学也显示PCNA表达量降低(P<0.05)。纳洛酮具有逆转吗啡降低PCNA表达的作用。结论:吗啡可能通过μ受体降低PC12细胞PCNA表达，进而抑制细胞增殖。     

丹参酮ⅡA对U251胶质瘤细胞的增殖抑制和原癌基因表达的影响

目的:探讨丹参酮ⅡA对人胶质瘤细胞株U251的增殖抑制作用及其作用机理。方法:应用MTT比色法检测不同浓度丹参酮ⅡA对U251细胞的增殖抑制,应用流式细胞仪观察丹参酮ⅡA对U251细胞周期的影响,应用RT-PCR观察相关基因C-myc表达的变化。结果:丹参酮ⅡA对U251细胞增殖抑制作用呈剂量依赖性,当浓度为0.10 g·L^-1时,对U251细胞增殖的抑制率达到(53.7±6.0)%。流式细胞仪分析显示,用0.10 g·L^-1丹参酮ⅡA培养3 d,细胞周期中G₀/G₁期所占比例增高,S期占细胞周期的比例降低。RT-PCR结果显示,随着丹参酮ⅡA作用剂量的增加,c-myc基因的表达被明显抑制。结论:丹参酮ⅡA对人胶质瘤细胞系U251的增殖具有明显的抑制作用,并对原癌基因c-myc的表达具有抑制作用。     

雌激素对培养的兔平滑肌细胞增殖与移行的影响

目的: 探讨雌激素对培养的兔平滑肌细胞增殖与移行的影响。方法: 分别测定不同浓度雌激素作用下兔血管平滑肌细胞[³H]-TdR掺入量、增殖细胞核抗原(PCNA)的表达、血管平滑肌细胞(VSMC)移行的变化。结果: 低浓度(1 nmol/L)的雌激素组的培养细胞[³H]-TdR掺入量及VSMC平均光密度值、VSMC移行细胞数与对照组相比无明显差异(P>0.05), 较高浓度(10 nmol/L、100 nmol/L)的雌激素可以显著降低培养的[³H]TdR掺入量及培养VSMC平均光密度值, 并能显著减少VSMC移行细胞数目(P<0.01)。结论: 雌激素可抑制血管平滑肌细胞的增殖与移行, 从而发挥抗动脉粥样硬化作用。     

瓜蒌注射液对兔血管平滑肌细胞增殖细胞核抗原表达的影响

目的:观察瓜蒌注射液对血管平滑肌细胞(SMC)增殖细胞核抗原(PCNA)表达的影响。方法:用免疫组织化学LSAB法检测培养的兔主动脉SMC中PCNA的表达,液闪法测定SMC氚-胸腺嘧啶核苷([³H]-TdR)掺入量,同时测定培养液中超氧化物歧化酶(SOD)、脂质过氧化物(LPO)、前列环素(PGI₂)及环磷酸腺苷(cAMP)的含量。结果:瓜蒌注射液能增加SOD活性,降低LPO和升高PGI₂、cAMP水平,抑制SMC的[³H]-TdR掺入量和PCNA的表达(P均＜0.05,0.01)。结论:瓜蒌有抑制SMC增殖的作用。     

神经生长因子通过TrkA信号途径促进肝细胞增殖

目的: 观察神经生长因子(NGF)及其受体(TrkA^NGFR和p75^NTR)在肝细胞中的表达,探讨外源性神经生长因子 β(NGF-β)对肝细胞的生物学作用。方法: 体外培养L02肝细胞,免疫细胞化学和荧光定量PCR法分别检测NGF、TrkA^NGFR和p75^NTR在L02细胞中的表达。XTT法检测外源性NGF-β、anti-NGF、anti-TrkA^NGFR和anti-p75^NTR对L02细胞增殖的作用,流式细胞术检测外源性NGF-β对L02细胞凋亡和细胞周期的影响。结果: L02细胞表达NGF及其受体TrkA^NGFR、p75^NTR,NGF主要位于细胞质和细胞核,TrkA^NGFR和p75^NTR位于细胞质和细胞 膜;外源性NGF-β上调L02细胞表达NGF和TrkA^NGFR。低剂量外源性NGF-β(12.5~200 μg/L) 通过调控L02细胞的S期,促进细胞增殖,抗细胞凋亡,高剂量(>400 μg/L)NGF-β不能促进L02细胞增殖;anti-NGF和anti-TrkA^NGFR抑制NGF-β诱导的L02细胞增殖,anti-p75^NTR并不影响NGF-β诱导的L02细胞增殖。结论: L02细胞表达NGF及其受体TrkA^NGFR、p75^NTR;合适剂量外源性NGF-β可能通过NGF/TrkA^NGFR信号途径促进L02细胞增殖。     

酪氨酸蛋白激酶在自发性高血压大鼠血管平滑肌细胞增殖肥大中的作用

目的: 明确自发性高血压大鼠血管平滑肌细胞(SHR-VSMC)增殖与血小板源生长因子-AA(PDGF-AA)、PDGF-α受体表达的关系及酪氨酸蛋白激酶在其中的作用。方法: 在培养的血管平滑肌细胞中,采用免疫印迹(Westernblot)、[³H]-TdR及[³H]-Leu掺入等方法,观察在不同来源大鼠(SHR/WKY)血管平滑肌细胞中,PDGF-AA、PDGF-α受体及PDGF-β受体表达的差异性;在PDGF-AA刺激下细胞的增殖、肥大反应及酪氨酸激酶抑制剂(genistein)对其的影响。结果: SHR-VSMC中PDGF-AA、PDGF-α受体蛋白表达明显高于WKY-VSMC,而PDGF-β受体蛋白表达在SHR-VSMC与WKY-VSMC无明显差异;在不同浓度PDGF-AA刺激下,PCNA及[³H]掺入率在SHR-VSMC明显增强且呈剂量依赖性;酪氨酸激酶抑制剂(genistein)明显抑制PCNA表达及[³H]掺入。结论: 自发性高血压大鼠VSMCPDGF-A链及其α受体的自发性增高,可能是导致SHR-VSMC异常增殖、肥大,从而触发血管反应性和血管构型变化的重要原因之一;酪氨酸蛋白激酶介导的信号转导在其中发挥重要作用。     

辛伐他汀抑制胎牛血清及PDGF-BB诱导的血管平滑肌细胞增殖及对抑癌基因PTEN表达的影响

目的:观察辛伐他汀(simvastatin)对血清及血小板源生长因子(PDGF-BB)诱导的血管平滑肌细胞(VSMCs)增殖的抑制作用及simvastatin对细胞周期和G₁/S周期转换重要调节基因PTEN蛋白表达的影响。方法:[³H]-胸腺嘧啶核苷酸掺入测定VSMCsDNA合成,流式细胞仪检测细胞周期情况,Western印迹杂交法检测PTEN蛋白表达。结果:Simvastatin以剂量依赖关系抑制血清及PDGF-BB诱导的VSMCs[³H]-胸腺嘧啶核苷酸掺入,使G₀/G₁期细胞比例明显增多,S期细胞比例显著减少,并上调PTEN蛋白表达,而3羟3甲戊二酰辅酶A代谢产物甲羟戊酸能抑制simvastatin上调PTEN的表达。结论:Simvastatin抑制血清及PDGF-BB诱导的VSMCs增殖阻滞细胞周期可能与上调PTEN表达有关,且simvastatin调节PTEN的表达可能通过抑制甲羟戊酸的合成而实现。     

β3整合素在生长因子促血管平滑肌细胞粘附和迁移中的作用

目的:探讨血小板衍生生长因子(PDGF)对β₃整合素表达的影响及β₃整合素在PDGF促血管平滑肌细胞(VSMC)粘附、迁移和增殖中的作用。方法:用抗β₃整合素胞外域抗体封闭VSMCβ₃整合素后,通过-TdR掺入、细胞粘附、细胞迁移分析及RT-PCR等方法,检测PDGF对β₃整合素表达及对VSMC粘附、迁移和增殖的影响。结果:阻断β₃整合素与细胞外基质(ECM)蛋白相互作用后,可在一定程度上抑制PDGF刺激的VSMC增殖,使-TdR掺入率降低39%;在β₃整合素抗体浓度为10mg/L时,PDGF引发的细胞粘附和迁移受到显著抑制(P＜0.05);PDGF作用于VSMC6h,β₃整合素基因表达活性达到峰值,比对照细胞高87%。结论:PDGF可显著诱导β₃整合素在VSMC中表达;β₃整合素与ECM蛋白相互作用在PDGF促VSMC粘附、迁移方面发挥重要作用。     

低分子量肝素抑制血管成形术后平滑肌细胞增生及再狭窄形成

观察血管成形术后平滑肌细胞增生及再狭窄形成过程，探讨低分子量肝素的抑制作用。     

Effects of UVC irradiation on cultured mouse embryonic limb bud cells

Effects of UVC irradiation (UVC) at a dose range from 0 to 30 J/m² were investigated on the cultured embryonic limb bud cells (LBC), isolated from fore- and hindlimbs of day 11 mouse embryos. Although dose-dependent inhibition was found for both cellular proliferation and chondrogenesis, the chondrogenic proteoglycan (PG) synthesis was more sensitive to UVC than cellular proliferation when compared at ID₅₀, the inhibitory dose that reduced assessment value by 50% of the control. No significant difference in induction and repair kinetics of UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts ((6-4)PPs) were found between LBC and NIH3T3 mouse cell line. The fluorescent light (FL) treatment of LBC pre or post UVC irradiation did not affect repair kinetics of CPDs and (6-4)PPs, cellular proliferation, formation of PG-producing nodule and the PG synthesis.     

人参皂苷Re对球囊损伤所致大鼠血管内膜增生及NF-κB p65信号通路的影响

目的:研究人参皂苷Re对大鼠颈动脉球囊损伤所致血管内膜增生的作用及其对NF-κB p65炎症信号通路的影响。方法:40只SD大鼠随机分组为5组:假手术组,模型组,人参皂苷Re低、中、高剂量组。除假手术组外,其余组大鼠均用2F球囊导管建立颈动脉球囊损伤模型,制模后次日灌胃给药,假手术组和模型组给予等体积蒸馏水,人参皂苷低、中、高剂量组大鼠分别给予12.5、25、50 mg/kg人参皂苷Re。连续给药14 d后取损伤段颈动脉,HE染色观察血管形态学改变,并测定血管管腔面积(lumen area,LA)、内膜面积(intima area,IA)、中膜面积(media area,MA),计算内膜/中膜面积比(IA/MA);real-time PCR法检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-1β(interleukin-1β,IL-1β)的mRNA水平;免疫组织化学法检测血管壁增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和核因子κB(nuclear factor-kappa B,NF-κB)p65蛋白的表达。结果:与假手术组相比,模型组大鼠血管腔明显变窄(P0.01),血管壁TNF-α、IL-1β的mRNA水平及PCNA、NF-κB p65蛋白的表达均明显增高(P0.05);与模型组比较,人参皂苷Re中、高剂量组血管内膜增生明显减轻(P0.05),血管壁TNF-α和IL-1β的mRNA水平及PCNA和NF-κB p65的蛋白表达则明显下调(P0.05)。结论:人参皂苷Re能减轻球囊损伤所致大鼠颈动脉内膜增生,其机制可能与抑制NF-κB p65炎症信号通路有关。     

人白细胞介素1β通过caspase途径诱导人黑色素瘤A375-S2细胞凋亡

目的:对interleukin-1β(IL-1β)诱导人黑色素瘤A375-S2细胞凋亡的信号转导途径进行研究。方法:使用倒置显微镜观察细胞形态学变化。通过MTT法测定IL-1β对A375-S2细胞的抑制作用以及细胞内半胱氨酸蛋白酶(caspases)与这种作用的关系。利用乳酸脱氢酶(LDH)测定法对IL-1β作用后细胞的损伤情况进行分析。琼脂糖凝胶电泳法检测IL-1β对细胞DNA降解的影响。结果: IL-1β对A375-S2细胞的抑制作用呈剂量和时间依赖性,在10^-9mol/L作用72 h时达到90%以上。caspase-1、-3、-8、-9和caspase-10的抑制剂能够部分抑制IL-1β早期诱导的细胞凋亡。 LDH活力测定显示,在IL-1β诱导的细胞死亡过程中,凋亡占主导地位,并呈现剂量和时间依赖性 。细胞经过10^-11mol/L IL-1β处理72 h后,出现凋亡典型的DNA梯状条带,与上述结果一致。 结论: IL-1β能够诱导人黑色素瘤A375-S2细胞凋亡,这种作用可能依赖于激活一类介导凋亡的caspase家族蛋白酶。     

Sulindac对人肝癌细胞HepG2增殖及β-catenin表达的影响

目的　观察Sulindac对人肝癌细胞HepG2增殖、凋亡及β-catenin蛋白表达的影响,探讨Sulindac抗肝癌的可能机制。 方法　不同浓度的Sulindac作用HepG2细胞后,采用MTT实验检测细胞增殖抑制作用;采用Hoechst33258染色法检测Sulindac对HepG2细胞凋亡的影响;利用RT-PCR及Western blotting检测HepG2细胞在Sulindac作用后Wint通路中β-catenin的表达变化。 结果　Sulindac对人肝癌细胞HepG2有增殖抑制作用,且呈剂量时间依赖关系;Hoechst33258结果显示,Sulindac作用24 h后HepG2细胞凋亡数目明显增多;随着Sulindac浓度的增加,β-catenin mRNA及蛋白表达量逐渐下降。 结论　Sulindac能够抑制人肝癌细胞HepG2增殖,通过阻断Wnt信号传导通路,降低β-catenin表达,诱导人肝癌细胞HepG2的凋亡。     

GSK-3β抑制剂对结肠癌SW480细胞增殖和凋亡的影响

目的: 探讨糖原合成酶激酶3β(GSK-3β)抑制剂(2'Z,3'E)-6-溴靛'-3'-肟(BIO)对结肠癌SW480细胞β-catenin 、Bcl-2蛋白表达及细胞周期、凋亡的影响。方法: 应用不同浓度BIO作用于结肠癌SW480细胞,采用流式细胞术检测细胞周期及凋亡,Western blotting检测β-catenin 和Bcl-2蛋白表达,免疫细胞化学检测β-catenin 及cyclin D1的表达,HE染色观察细胞形态。结果: 与BIO处理前SW480细胞相比,BIO处理组SW480细胞β-catenin蛋白表达明显上调并出现部分细胞核移位,cyclin D1蛋白表达及S期和G₂/M期细胞不同程度增多,Bcl-2蛋白表达有所下调,细胞凋亡率明显下降,并出现细胞形态改变。结论: GSK-3β抑制剂BIO作用于结肠癌SW480细胞呈现促增殖及抑凋亡作用,其机制主要与β-catenin信号转导通路激活以及与Bcl-2调控通路的平衡有关,其中β-catenin上调可能是影响结肠癌SW480细胞转归的主要因素。     

The direct effect of carbon monoxide on KCa channels in vascular smooth muscle cells

 The vasorelaxation induced by carbon monoxide (CO) has been demonstrated previously. Both a guanosine cyclic monophosphate (cGMP) signalling pathway and cGMP-independent mechanisms have been proposed to be responsible for the vascular action of CO. A direct effect of CO on the activity of calcium-activated K (K_Ca) channels in vascular smooth muscle cells (SMCs) and the underlying mechanisms were investigated in the present study. It was found that CO hyperpolarized single SMCs isolated from rat tail arteries. The whole-cell outward K⁺ channel currents in vascular SMCs, but not in neuroblastoma cells, were enhanced by CO. Extracellularly or intracellularly applied CO increased the open probability of single high-conductance K_Ca channels concentration-dependently without affecting the single channel conductance. Although it did not increase the resting level of intracellular free calcium concentration, CO significantly enhanced the calcium sensitivity of single K_Ca channels in SMCs. Furthermore, the effect of CO on K_Ca channels was not mediated by cGMP or guanine nucleotide-binding proteins (G proteins, G_i/G_o or G_s) in excised membrane patches. Our results suggest that the direct modulation of high-conductance K_Ca channels in vascular SMCs by CO may constitute a novel mechanism for the vascular effect of CO. Received: 9 January 1997 / Received after revision: 21 February 1997 / Accepted: 10 March 1997     

Therapeutic effect of 188Re-MAG3-depreotide on non-small cell lung cancer in vivo and in vitro

Objective: To investigate the in vivo and in vitro therapeutic effect of 188Re-MAG3-depreotide on non-small cell lung cancer (NSCLC). Methods: MTT was done to measure the cell proliferation; flow cytometry to detect cell apoptosis; Transwell invasion assay to determine the invasiveness of NSCLC. In addition, HE staining, TUNEL staining and immunohistochemistry for CD34 were employed to investigate the influence of 188Re-MAG3-depreotide on the growth of NSCLC. Results: 1) Within 2-6 days, the inhibitory effect of 188Re-MAG3-depreotide on the proliferation of A549 cells and SPC-A1 cells increased over time. 2) At 48 h after treatment with 188Re-MAG3-depreotide, the apoptosis rate of A549 cells and SPC-A1 cells was 23.1% and 22.6%, respectively. 3) After 188Re-MAG3-depreotide treatment, the number of invasive A549 cells and SPC-A1 cells was reduced by about 3 times when compared with control group. 4) The cancer in the control group presented with unlimited growth. The cancer growth continued after treatment with 188Re or MAG3-depreotide alone, while the cancer growth was markedly inhibited after 188Re-MAG3-depreotide treatment when compared with control group. Conclusion: 188Re-MAG3-depreotide can inhibit the proliferation and invasion of A549 cells and SPC-A1 cells. Treatment with 7.4MBq 188Re-MAG3-depreotide via tail vein can significantly suppress the in vivo cancer growth and induce the apoptosis of cancer cells. These findings demonstrate that 188Re-MAG3-depreotide can induce the apoptosis of NSCLC cells and directly kill the NSCLC cells, which provide evidence for the radiotherapy of NSCLC.     

The Effects of X-Ray Irradiation on the Proliferation and Apoptosis of MCF-7 Breast Cancer Cells

Abstract

To investigate the effects of X-ray irradiation on the proliferation and apoptosis of MCF-7 breast cancer cells; MCF-7 breast cancer cells were irradiated with X-ray. After irradiation, morphological changes and growth inhibition rate of the irradiated cells were observed under an inverted microscope. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess the proliferation of the irradiated MCF-7 cells. Transmission electron microscope was used to observe the morphology and ultrastructure of the irradiated MCF-7 cells. Western blotting was used to analyze the expression level of apoptosis-related protein caspase-3. Our results showed, at 48?h after the irradiation (0?Gy and 8?Gy), cells oval in shape, cell shrinkage or swelling and partial formation of debris under inverted microscope; as well as cytoplasmic vacuolization or inspissation, increased electron density of cytoplasm, structural damage of organelles, blurred mitochondrial cristae and chromatin margination under transmission electron microscopy; the survival rate of MCF-7 cells in X-ray group was 17.3% lower than that in control group (0?Gy) (p?<?0.001); while caspase-3 expression increased evidently in X-ray group compared with control group (0?Gy) (p?<?0.05). In conclusion, X-ray irradiation can inhibit the proliferation of MCF-7 cells and induce apoptosis through increasing caspase-3 expression.