小檗胺对大鼠骨肉瘤UMR-106细胞增殖的抑制作用

目的:探讨小檗胺体外对大鼠骨肉瘤UMR-106细胞增殖的抑制作用及机制.方法:0、2、4、8、16、32mg/L小檗胺作用UMR-106细胞24、48、72 h后,采用四甲基偶氮唑蓝(MTT)比色法检测小檗胺对UMR-106细胞增殖的抑制作用;0、4、8、16 mg/L小檗胺作用UMR-106细胞24 h后,Hoechst33258染色激光共聚焦扫描显微镜观察细胞的形态学变化;Annexin V/碘化丙啶(PI)荧光标记流式细胞术(FCM)检测细胞凋亡率;PI荧光标记FCM检测细胞周期变化.结果:小檗胺以剂量依赖方式显著抑制UMR-106细胞的增殖(P＜0.01),其作用24、48、72 h的半数抑制浓度(IC50)分别为24.69、8.03和3.54 mg/L.小檗胺处理组可见核固缩和凋亡小体.空白对照组和小檗胺4、8、16 mg/L处理组的细胞凋亡率分别为(1.64±0.29)％、(3.58±0.31)％、(6.27±0.47)％和(11.27±1.09)％,与空白对照组比较,差异有统计学意义(P＜0.01);同时,小檗胺可以诱导细胞坏死;此外,与空白对照组比较,小檗胺4、8、16 mg/L处理组UMR-106细胞的G0/G1期细胞比例增高,而S期和G2/M期细胞比例呈降低趋势(P＜0.01).结论:小檗胺体外能够抑制骨肉瘤UMR-106细胞增殖,其机制与诱导细胞凋亡、坏死和G0/G1期阻滞有关.     

姜黄素对骨肉瘤细胞增殖和凋亡的影响

目的研究姜黄素对骨肉瘤细胞增殖和凋亡的影响,并探讨其机制。方法采用姜黄素单体处理人骨肉瘤U2OS细胞,通过台盼蓝拒染法检测细胞增殖活力,采用流式细胞术和DNA Ladder检测凋亡,通过Westernblot法检测p53和caspase-3的表达。结果 4、8、16、32、64μmol/L姜黄素处理U2OS细胞48h,生长抑制率为:(9.21±3.11)%、(16.85±3.95)%、(29.57±5.33)%、(52.30±6.07)%和(79.16±6.78)%。姜黄素(16μmol/L)在24、48和72h均可诱导U2OS细胞凋亡,凋亡率为:(18.8±3.05)%、(29.6±5.22)%和(57.3±6.62)%,姜黄素(16μmol/L)作用72h后,DNA电泳显示梯形条带改变。并且姜黄素作用48h可明显上调p53的表达和caspase-3的活化。结论姜黄素可抑制骨肉瘤U2OS细胞增殖,并通过p53-caspase-3通路诱导凋亡。     

Nocodazole对骨肉瘤MG-63细胞增殖凋亡的影响及机制研究

目的以细胞有丝分裂阻滞剂Nocodazole为诱导物,诱导骨肉瘤MG-63细胞凋亡,并进行生物化学指标的检测。方法首先,观察1nmol/L～10μmol/L递增浓度的Nocodazole对MG-63细胞的作用,然后选取10,50,100,500nmol/L4个浓度诱导凋亡,通过噻唑蓝(MTT)比色实验测定细胞生长曲线;流式细胞技术(FCM)分析细胞周期;免疫细胞化学方法检测PLK1表达情况;用反转录-聚合酶链反应(RT-PCR)分析Nocodazole作用前后MG-63细胞内PLK1基因表达情况。结果不同浓度Nocodazole作用MG-63细胞24h后,对细胞增殖的抑制作用呈浓度依赖性增加。流式细胞仪分析结果显示,Nocodazole作用MG-63细胞后,G0/G1期细胞含量降低,S期细胞比例增加,G2/M期细胞比例则明显增加,且随着处理浓度的增加其G2/M期细胞含量逐渐增加,与对照组比较差异有统计学意义(P<0.05)。Nocodazole作用后,免疫细胞化学检测显示,PLK1表达明显下降。RT-PCR分析显示经Nocodazole作用后的骨肉瘤细胞株MG-63表达PLK1较用药前明显下降。上述结果都有明显的量-效关系。结论 Nocodazole诱导MG-63细胞凋亡、G2/M期阻滞的机制可能是通过抑制PLK1基因的表达实现的。     

苦参碱对骨肉瘤细胞凋亡的影响

目的 探讨苦参碱在MG-63细胞中促凋亡的作用及机制。方法 实验分为0浓度组和10%苦参碱组,分别灌胃生理盐水和10%苦参碱制备大鼠含药血清,干预人成骨肉瘤细胞株MG-63。采用CCK-8法检测细胞增殖,流式细胞术检测细胞凋亡,荧光定量聚合酶链式反应（PCR）检测c-myc、胱天蛋白酶9(caspase-9)基因的表达,采用免疫印迹（Western blot）法检测细胞外信号调节激酶5(ERK5)信号通路中相关蛋白Nur77、丝裂原激活蛋白激酶5(MEK5)及凋亡相关蛋白c-myc,caspase-9的表达水平。结果 与0浓度组相比,10%苦参碱组能显著抑制MG-63细胞增殖,并促进其凋亡;荧光定量PCR实验结果表明,10%苦参碱组能抑制c-myc转录,并促进caspase-9转录;Western blot实验结果表明,在ERK5信号通路中,10%苦参碱组Nur77、MEK5及c-myc蛋白表达下调,而caspase-9蛋白表达上调。结论 苦参碱可能通过干扰ERK5信号通路,调控c-myc,caspase-9等蛋白的表达,从而抑制MG-63细胞增殖,并促进其凋亡。     

小檗胺对血管平滑肌细胞钙动力学影响

本文以Fluo-3/AM 荧光技术和激光扫描共聚焦显微镜检查法, 研究小檗胺 (Ber) 对培养家兔胸主动脉血管平滑肌细胞(VSMC)内游离钙 ([Ca 2 ]i ) 的影响结果表明：在胞外钙 ([Ca 2 ]o ) 为?.0 mmol·L -1 条件下，Ber 抑制60 mmol·L -1 KCL1 mmol·L -1 哇巴因Ouabain 30 mmol·L -1 去甲肾上腺素 ( NE ) 1 mmol·L -1 5-羟色胺5-HT 和30 mmol·L -1 三磷酸腺苷 (ATP)引起的 [Ca 2 ]i 升高，而且荧光强度达峰时间亦延长。在无胞外钙条件下，Ber 对咖啡因 (Caffeine)引起的[Ca 2 ]i 升高没有作用。结论：Ber 抑制电压依赖性钙通道 (VDCC) 和受体调控性钙通道 (ROCC) 激活引起的外钙内流，对Caffeine 引起的内钙释放没有影响。Ber 可抑制Ouabain 诱导的细胞内钙升高。     

依托泊苷诱导骨肉瘤细胞凋亡的实验研究

目的：探讨依托泊苷对骨肉瘤细胞的毒性作用和作用机制。方法：用NTT比色法检测0．02,0．04,0．08,0．16,0．31,0．62,1．25,2．5,5．0mg·L~(-1)浓度梯度依托泊苷作用骨肉瘤细胞24,48,72和96h后的细胞活性。用流式细胞仪检测依托泊苷作用骨肉瘤细胞后细胞凋亡情况。用电镜观察药物作用后骨肉瘤细胞形态改变。结果：依托泊苷对骨肉瘤细胞有明显的生长抑制作用,随着药物浓度和作用时间的增加,细胞凋亡明显增加,依托泊苷可以诱导骨肉瘤细胞凋亡。依托泊苷作用后骨肉瘤细胞出现典型的凋亡改变,透视电镜可见细胞核仁消失,染色质浓缩聚集核膜下。结论：依托泊苷对骨肉瘤细胞有杀伤作用,细胞毒作用与促细胞凋亡有关。     

冬凌草甲素对骨肉瘤细胞的凋亡诱导效应的研究

目的:考察冬凌草甲素是否能诱导MG-63细胞凋亡。方法:用不同浓度的冬凌草甲素作用于MG-63细胞,用Hochest33258染色鉴定MG-63细胞凋亡的形态学特征;用流式细胞仪分析MG-63早期凋亡细胞的百分率;用Caspase-8蛋白印迹分析证明MG-63细胞凋亡发生的分子特征。结果:凋亡细胞形态学鉴定、流式细胞仪检测均有力证明冬凌草甲素能以浓度依赖性方式诱导MG-63细胞凋亡;冬凌草甲素诱导MG-63细胞凋亡伴有Caspase-8蛋白表达上调和裂解激活。结论:冬凌草甲素能以浓度依赖性方式诱导MG-63细胞凋亡。     

低分子量肝素对骨肉瘤细胞生长与凋亡的影响研究

目的 研究低分子量肝素（LMWH）体外抗肿瘤生长及诱导肿瘤细胞凋亡的作用,初步探讨低分子量肝素抗肿瘤作用机制,为该药的抗肿瘤作用开发和临床应用提供试验依据.方法在体外培养的骨肉瘤系（MG-63）细胞培养基中加入不同浓度的LMWH,对照组加完全培养基,用MTT法检测不同浓度LMWH在干预不同时间后对骨肉瘤细胞生长的影响,流式细胞仪检测不同浓度干预组细胞凋亡率的变化.结果LMWH可抑制骨肉瘤细胞系MG-63生长,并具有时间剂量依赖性,在LMWH浓度为200 U&#8226;mL^-1时,对细胞生长有明显的抑制作用. 流式细胞仪检测显示,随着LMWH剂量的增加,MG-63细胞凋亡率明显增加. 结论LMWH在体外具有抗肿瘤活性,其作用可能通过抑制细胞增殖和促进肿瘤细胞凋亡而实现.     

氧化苦参碱诱导骨肉瘤细胞凋亡的实验研究

目的探讨不同浓度的氧化苦参碱对人骨肉瘤细胞的生长抑制和诱导凋亡作用.方法将不同浓度的氧化苦参碱作用于培养的OS732骨肉瘤细胞,通过四氮噻唑蓝(MTT)比色法、流式细胞仪和DNA凝胶电泳、光学显微镜技术观察检测细胞凋亡,研究分析其对骨肉瘤细胞的生长抑制和诱导凋亡作用.结果浓度为1.77,3.55,5.32 mmoL·L-1的氧化苦参碱对OS732骨肉瘤细胞相对抑制率分别为16.4%,29.0%,47.0%;流式细胞技术显示骨肉瘤细胞的凋亡率分别为11.8%,18.6%,27.7%;DNA电泳见DNA"梯形"图谱;显微镜显示细胞的凋亡形态.结论氧化苦参碱能显著抑制OS732细胞增殖、促进其凋亡.     

葛根素对UMR106细胞增殖及碱性磷酸酶活性的影响

目的:研究葛根素对UMR106细胞增殖及碱性磷酸酶活性的影响.方法:UMR106细胞培养于α-MEM培养基中,葛根素处理后光学显微镜下观察其形态学变化,绘制细胞生长曲线,并采用对硝基苯磷酸钠基质动力学法测定UMR106细胞内、外碱性磷酸酶活性.结果:UMR106细胞经葛根素和10-8mol·L-1雌二醇处理后d1,d2和d3,与空白对照组相比,增殖速度均加快,d2差异最显著.与对照组相比,10-8,10-7,10-6mol·L-1葛根素组的增殖率分别为9.3%,23.3%和40.9%,10-8mol·L-1雌二醇组增殖率为49.7%.与对照组相比,细胞经10-6mol·L-1葛根素处理后1 d和2 d时,细胞内ALP活性显著增加(P＜0.01);细胞经10-6mol·L-1葛根素处理1 d后,细胞外ALP活性显著增加(P＜0.05).结论:葛根素可以促进成骨样细胞UMR106细胞合成和分泌ALP,提示其具有促进成骨细胞增殖和分化的作用.     

Staurosporine enhances parathyroid hormone-induced calcium signal in UMR-106 osteoblastic cells

Parathyroid hormone (PTH) treatment of bone and kidney-derived cells not only activates adenylyl cyclase but also increases intracellular free calcium, and translocates protein kinase C (PKC) from cytosol to plasma membranes. We have found that acute phorbol ester pretreatment significantly decreases PTH-induced calcium transients and the effect of phorbol ester was antagonized by staurosporine (ST). Although the major effect of ST in that study was the reversal of the action of phorbol ester, it appeared that ST may also have promoted the effect of PTH directly. To further investigate the observation, we examined the effect of ST on the intracellular calcium transients induced by PTH and alpha-thrombin (alpha-TH). For calcium transient experiments, UMR-106 cells were loaded with 2 mM fluo-acetoxymethylester for 30 min at room temperature. The cells were then washed and suspended in buffer containing 1 mM calcium. Fluorescence was detected at 530 nm, with excitation at 505 nm. ST alone did not cause calcium transients, but enhanced the transients elicited by PTH when added 5 min before the hormone. Another protein kinase inhibitor H-7 likewise enhanced the calcium responses elicited by PTH, while genistein did not affect PTH response. Calcium transients elicited by alpha-TH were also enhanced by ST. The results suggest that there might be tonically activated endogenous protein kinase(s) which inhibit calcium signaling of some calcemic agents.     

Naringin improves bone properties in ovariectomized mice and exerts oestrogen-like activities in rat osteoblast-like (UMR-106) cells

Background and purpose:

Naringin, a flavanone glycoside in citrus fruits, has been recently reported to stimulate bone formation in vitro and in vivo. The present study was designed to determine if naringin could exert oestrogen-like protective actions in bone.

Experimental approach:

Young C57/BL6J mice were ovariectomized (OVX) and treated orally with naringin (0.2 or 0.4 mg·g⁻¹·day⁻¹), 17β-oestradiol (2 µg·g⁻¹·day⁻¹) or its vehicle for 6 weeks. Bone mineral densities (BMD) and polar stress-strain index (SSI) were measured by peripheral quantitative computed tomography. Rat osteoblast-like UMR-106 cells were co-incubated with the oestrogen receptor (ER) antagonist ICI 182780 to determine if the effects of naringin on osteoblastic functions were ER dependent. Functional transactivation of ERα and ERβ as well as ERα phosphorylation by naringin were also studied.

Key results:

Naringin at 0.4 mg·g⁻¹·day⁻¹ increased BMD at trabecular-rich bone in OVX mice. Naringin (at both doses) significantly increased SSI at distal femur and lumbar spine and increased biomechanical strength (ultimate load and energy for breaking) at tibia diaphysis in OVX mice. The stimulatory effects of naringin on osteoblastic functions could be abolished by co-incubation with ICI 182780 in UMR-106 cells. Naringin failed to stimulate ERα- or ERβ-mediated oestrogen response element-dependent luciferase activity but could significantly induce ERα phosphorylation at serine 118, in UMR-106 cells.

Conclusions and implications:

Naringin was effective in protecting against OVX-induced bone loss in mice and its actions might be mediated through ligand-independent activation of ER in osteoblastic cells.     

Fluoride mediates apoptosis in osteosarcoma UMR 106 and its cytotoxicity depends on the pH

 Although an excess intake of fluoride has been reported to cause skeletal fluorosis, very little is known about the mechanism of adverse effects of fluoride on bone. In the present study cytotoxic effects of fluoride were studied using the osteosarcoma cell line, UMR 106. The DNA ladder formation upon agarose electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining revealed that UMR 106 underwent apoptosis following exposure to 5 mM fluoride for 8 h. On the other hand exposure to A23187, a calcium ionophore, caused necrosis while co-exposure to fluoride and A23187 inhibited fluoride-mediated apoptosis in UMR 106. The proliferation of UMR 106 cells cultured for 6 days in the presence of 0.5 mM fluoride was significantly decreased compared to the control culture. The cytotoxic effects of fluoride were modulated by both the cell density and the pH of the culture medium. The fluoride-induced viability loss in UMR 106 was enhanced in culture of high cell-density and inversely correlated with pH of the culture medium. Enhancement of fluoride cytotoxicity at acidic pH was also observed in rat alveolar macrophages and RAW 264, a macrophage cell line. The results suggest that fluoride-mediated apoptosis and culture conditions, including pH of the medium, should be taken into consideration to evaluate toxicity of fluoride in vitro. Received: 13 May 1997 / Accepted: 4 August 1997     

Ipriflavone inhibits phosphoinositide hydrolysis and Ca2+ uptake in the osteoblast-like UMR-106 cells.

The mechanism of action of ipriflavone, an isoflavone derivative, was studied in the osteoblastic-like UMR-106 cell line. Ipriflavone affected both phosphoinositide hydrolysis and 45Ca2+ uptake. A repeated treatment of UMR-106 cells (once a day, for 3 days) with ipriflavone decreased, in a concentration-dependent manner, [3H]inositol monophosphate accumulation. This effect was also achieved after single addition of high concentrations of ipriflavone or 100 nM [Asu1,7]eel-calcitonin, a semi-synthetic analog of eel calcitonin. When repeatedly added to UMR-106 cells, 17 beta-estradiol produced a marked inhibition of [3H]inositol monophosphate accumulation, an effect which appeared significant only at a concentration of 1 microM and which was accompanied by a reduced incorporation of [3H]inositol into membrane phospholipids. A repeated treatment with ipriflavone reduced 45Ca2+ uptake as well. This effect was observed also after a single addition of [Asu1,7]eel-calcitonin but not following single or repeated treatment with 17 beta-estradiol. The present data indicate the osteoblast as a direct and specific target for ipriflavone and suggest that this compound may share intracellular transducing mechanisms with other antiosteoporotic hormones such as estrogen and calcitonin.     

小檗胺对培养的HeLa细胞内游离钙浓度的作用(英文)

目的:研究Ca~(2 )信号传导是否参与Hela细胞的信号传导过程以及小檗胺(Ber)对HeLa细胞内钙浓度([Ca~(2 )]_i)变化的影响。方法:Fluo 3-AM负载HeLa细胞,共聚焦法测定[Ca~(2 )]_i,结果以荧光强度(FI)表示。结果:(1)有外钙时,HeLa细胞静息FI为186±44,KCl、NE、Cal,及咖啡因均升高HeLa细胞的[Ca~(2 )]_i。(2)Ber处理后,静息FI无影响,但抑制KCl、NE和Cal引起的[Ca~(2 )]_i升高(P<0.01),FI变化的速率减慢,达峰值的时间延长。(3)无外钙时,咖啡因诱导的[Ca~(2 )]_i升高不被Ber抑制。(4)Ber的上述作用与Ver的作用相似。结论:HeLa细胞属于非兴奋性细胞,但部分生物学特征与兴奋性细胞相似,Ca~(2 )同样在其信息转导中发挥重要作用。     

吗啡和曲马多对MADB-106乳腺癌细胞增殖和凋亡的影响

目的观察不同浓度吗啡和曲马多对MADB-106乳腺癌细胞增殖和凋亡的影响,并对其机制进行初步研究。方法将不同浓度吗啡和曲马多与MADB-106乳腺癌细胞直接作用24 h后采用流式细胞法检测细胞凋亡,MTT法检测细胞增殖。采用Western blot法检测细胞半胱氨酸蛋白酶-3（caspase-3）和半胱氨酸蛋白酶-8（caspase-8）的表达。结果吗啡和曲马多均可浓度依赖性促进MADB-106乳腺癌细胞的凋亡、抑制细胞生长与增殖;等效镇痛浓度吗啡与曲马多对MADB-106乳腺癌细胞的增殖与凋亡的影响并无差异;吗啡可促进MADB-106乳腺癌细胞caspase-3和caspase-8激活片段表达。而曲马多仅促进caspase-3激活片段表达,caspase-8激活片段无显著变化。结论吗啡和曲马多均可浓度依赖性促进MADB-106乳腺癌细胞的凋亡、抑制肿瘤细胞的生长与增殖;吗啡和曲马多均可激活caspase-3途径诱导MADB-106乳腺癌细胞的凋亡,同时吗啡可激活caspase-8途径,而曲马多无此作用。     

Effects of N-methyl berbamine on delayed outward potassium current in isolated rat hepatocytes

目的：研究Ｎ甲基小檗胺（ＮＭＢ）对大鼠肝细胞外向钾电流的影响．方法：应用膜片箝技术和全细胞记录方法，箝制电位－５０ｍＶ，指令电位＋３０至＋１４０ｍＶ，持续时间９００ｍｓ．结果：ＮＭＢ以浓度依赖方式降低外向钾电流．电流幅值在２０，５０，４００ｎｍｏｌ·Ｌ－１和５０μｍｏｌ·Ｌ－１时分别从４４±１０（ｎ＝４），２５±１８（ｎ＝４），５８±２１（ｎ＝５），４６±１３（ｎ＝６）ｎＡ降到３６±０４（Ｐ＞００５），２１±１６（Ｐ＞００５），３７±１６（Ｐ＜００５），２３±１３（Ｐ＜００１）ｎＡ．抑制率分别为１０％，１５％，３７％，５１％．结论：ＮＭＢ是一种钾通道阻断剂．     

小檗胺对培养的HeLa细胞内游离钙浓度的作用

AIM: To study the involvement of Ca2+ signaling and the effects of berbamine (Ber) on intracellular calcium concentration ([Ca2+]i) elevated in cultured HeLa cells. METHODS: [Ca2+]i was measured by confocal microscopy in single HeLa cell loaded with Fluo 3-AM. The change of [Ca2+]i was represented by fluorescent intensity (FI). RESULTS: (1) In the presence of extracellular Ca2+ 1.3 mmol.L-1, the resting level of FI was 186 +/- 44, n = 49 cells from all control experiments, and KCl, NE, caffeine, and calcimycin (Cal) all induced [Ca2+]i elevations in cultured HeLa cells. (2) The resting level of FI was not affected by pretreatment with Ber. The FI increased by KCl 60 mmol.L-1, NE 100 micromol.L-1, and Cal 30 micromol.L-1 were attenuated (P < 0.05 or P < 0.01), the slope and the time to peak of FI increase were decreased and prolonged. (3) In the absence of extracellular Ca2+, caffeine 80 mmol.L-1-induced [Ca2+]i mobilization was not inhibited by Ber 100 micromol.L-1 pretreatment. (4) These effects of Ber were similar to those of verapamil (Ver) 10 mumol.L-1. CONCLUSION: Although it was derived from cervical cancer, the HeLa cells which were belong to the nonexcitable cell possessed the similar biological properties with excitable cells, and Ca2+ also played a crucial role in signal transduction processes.