噻莱昔布(celecoxib)对人骨肉瘤细胞MG-63增殖与凋亡的影响

目的:研究环氧化酶2(cyclooxygenase2,COX2)抑制剂celecoxib对人骨肉瘤细胞MG63抑制增殖及诱导凋亡的作用。方法:MTT比色观察celecoxib的细胞毒性作用及其浓度依赖性;Hoechst33258荧光染色、透射电子显微镜和TUNEL观察细胞凋亡的形态学变化;流式细胞仪检测细胞凋亡率及其时间依赖性。结果:celecoxib分别以10、20、40、80μmol/L作用后,细胞生长受到不同程度抑制;荧光显微镜、电镜和TUNEL观察到细胞胞浆浓缩、核凝聚、核碎裂和凋亡小体形成。流式细胞仪显示celecoxib40μmol/L作用24、48、72h后,细胞凋亡率分别与对照组比较有非常显著性差异(P<0.01)。结论:celecoxib能抑制人MG63细胞增殖,诱导细胞凋亡。     

Survivin-siRNA对骨肉瘤MG-63细胞株增殖抑制的影响

我们应用化学合成的特异性Survivis-siRNA在脂质体的介导下转染骨肉瘤MG-63细胞，观察其对骨肉瘤细胞增殖抑制的影响。[第一段]     

不同转移特性人成骨肉瘤MG-63细胞亚系的建立及特征

目的：建立高低不同转移特性人成骨肉瘤MG-63细胞亚系并研究其生物学特性。方法：通过体外克隆技术和体内移植，筛选并建立了6个细胞亚系，测量其细胞电泳率，细胞增殖率，利用琼脂克隆形成实验和裸鼠体内成瘤实验，比较各细胞亚系的转移特性。结果：A2、A3、A16亚系的细胞电泳率、细胞增殖率、琼脂克隆形成能力和肺转移灶形成率均明显高于A1、A6、A20亚系。结论：通过体外克隆技术和体内移植的方法可建立不同转移特性的MG-63细胞亚系，有助于骨瘤转移机理的进一步研究。     

不同转移特性人成骨肉瘤MG-63细胞亚系的建立及特征

目的:建立高低不同转移特性人成骨肉瘤MG-63细胞亚系并研究其生物学特性.方法:通过体外克隆技术和体内移植,筛选并建立了6个细胞亚系,测量其细胞电泳率、细胞增殖率,利用琼脂克隆形成实验和裸鼠体内成瘤实验,比较各细胞亚系的转移特性.结果:A2、A3、A16亚系的细胞电泳率、细胞增殖率、琼脂克隆形成能力和肺转移灶形成率均明显高于A1、A6、A20亚系.结论:通过体外克隆技术和体内移植的方法可建立不同转移特性的MG-63细胞亚系,有助于骨肉瘤转移机理的进一步研究.     

新型钌(Ⅱ)配合物抑制骨肉瘤MG-63细胞增殖并诱导其凋亡

目的 通过体外实验探讨新型钌(Ⅱ)多吡啶配合物△-[Ru(phen)2MCMIP]2+(△-1)对人骨肉瘤细胞MG-63株增殖及凋亡作用. 方法 CCK-8法检测经0.00、12.50、25.00、50.00、100.00、150.00 μmol/L△-1作用24、48、72 h后,MG-63细胞的增殖情况;流式细胞术检测经0.00、25.00、50.00、100.00 μmol/L△-1作用24h后,MG-63细胞的凋亡及周期变化. 结果 △-1可明显抑制人骨肉瘤MG-63细胞增殖,并呈明显剂量和时间依赖性;24、48、72 h IC5o分别为57.80 μmol/L、45.27 μmol/L、32.51 μmol/L;流式细胞术检测得不同浓度△-1作用24h后,细胞早期凋亡比例从(1.06±0.12)％上升至(37.10±1.25)％,细胞周期被阻滞在Go/G1期. 结论 △-1能抑制MG-63细胞增殖,诱导细胞凋亡,并将细胞周期阻滞在Go/G1期.     

人参二醇组皂苷对人前列腺癌DU145细胞增殖与凋亡的影响

目的 观察人参二醇组皂苷(panoxadiol saponin PDS)体外对DU145前列腺癌细胞增殖及凋亡的影响.方法 采用MTT比色法检测不同浓度PDS对DU145细胞增殖活力的影响.吖啶橙染色观察PDS诱导细胞凋亡情况;流式细胞仪分析细胞周期及凋亡,并计算细胞平均凋亡率;细胞免疫化学和Western blot技术观察细胞内激活的caspase3的表达.结果 经中(100mg/L)、高(200mg/L)剂量的PDS处理后,DU145细胞生长受到不同程度抑制,且呈现剂量依赖性;吖啶橙荧光染色可见经中、高剂量PDS处理后的细胞呈明显凋亡形态,并随药物剂量增加,凋亡细胞数增多;流式细胞术结果显示,PDS可明显提高细胞的凋亡率,呈剂量依赖关系;免疫细胞化学染色和Western blot结果,随着PDS剂量增加,细胞内激活的caspase3的表达上调.结论 PDS体外对DU145细胞增殖具有明显的抑制作用,其机制可能与诱导细胞凋亡有关.     

细胞凋亡与骨肉瘤

细胞凋亡又称程序性细胞死亡（programmed cell death,PCD）,是由基因控制的、不同于细胞坏死的细胞自主有序的死亡。正常生理状态下,机体可通过细胞凋亡清除衰老或异常的细胞,并维持内环境稳态。而在病理状态下,细胞凋亡调控失调会破坏细胞增殖与凋亡之间的平衡,对机体产生破坏性影响,如凋亡过度会引起老年性痴呆、     

人参二醇组皂苷增强顺铂对人前列腺癌DU145细胞凋亡的效应

目的 探讨人参二醇组皂苷(panoxadiol saponin PDS)与顺铂(cisplatin,DDP)联合应用对DU145前列腺癌细胞增殖及凋亡的影响.方法 采用MTT比色法检测PDS与DDP联合应用对DUl45细胞增殖活力的影响;吖啶橙染色观察诱导细胞凋亡情况;流式细胞仪分析细胞周期及凋亡,并计算细胞平均凋亡率;免疫化学和Western blot技术观察细胞内激活的caspase3的表达.结果 100mg/L,PDS与0.2 u mol/L DDP联合给药:(1)48h后可使单独应用0.2 1.tmol/L DDP组其肿瘤细胞生长抑制率由16.35%提高到47.13%;(2)吖啶橙荧光染色可见细胞呈现明显凋亡形态,其凋亡率与2μmol/L DDP组相当:(3)流式细胞术结果显示,可使单独应用0.2μmol/L DDP组其诱导DU145细胞凋亡率从5.53%提高到19.39%,相当于其10倍剂量即2.0 μmol/L DDP的诱导凋亡效应(21.05%),明显高于PDS单独应用的凋亡率:(4)免疫化学和Western blot结果显示,可使单独应用O.2 μmol/L DDP组细胞内激活的caspase3阳性细胞率与2 μmol/L DDP组相当.结论 PDS能增强DDP对DU145前列腺癌细胞的致凋亡效应.     

直接原位RT-PCR检测人成骨肉瘤MG-63细胞株雌激素受体亚型表达

目的观察人成骨肉瘤MG63细胞株雌激素受体亚型mRNA表达特征。方法用直接原位RTPCR及激光共聚焦显微镜观察MG63细胞株在培养第6及第9天ERα、ERβmRNA表达。结果各检测点均见一定量的荧光弥散于细胞内。阳性对照点荧光较其他两点强,阴性对照点荧光明显减弱,仅见少量背景荧光。ERα的荧光强度强于ERβ。结论(1)MG63细胞株作为成骨细胞模型,存在ERα和ERβ基因的mRNA表达。(2)骨基质成熟早期阶段MG63细胞株的ERα表达比ERβ丰富,这表明两者在功能上存在差异。     

Survivin基因干扰RNA对骨肉瘤MG-63细胞系增殖和凋亡的影响

[目的]观察Survivin基因siRNA表达载体对骨肉瘤细胞增殖和凋亡的影响。[方法]体外构建Survivin shRNA表达载体pSilence2．1-neo-Survivin，转染骨肉瘤细胞系MG-63，筛选稳定表达的克隆，倒置显微镜观察各组细胞形态学变化，RT-PCR、Weston-blot方法检测转染后Survivin mRNA及蛋白的表达，噻唑蓝（MTT）法、克隆形成实验检测细胞的增殖情况，吖啶橙荧光染色法观察细胞凋亡情况。[结果]转染后MG-63细胞Survivin基因mRNA水平和蛋白表达显著下降，其抑制率分别为85．08％和81．14％；细胞增殖受到显著抑制，培养48h后与空白组比较，抑制率达63．4l％；吖啶橙染色显示转染组细胞出现明显核碎裂等凋亡改变，转染组凋亡率为24．54％。[结论]靶向Survivin基因的siRNA表达载体可以显著抑制骨肉瘤细胞增殖，促进细胞凋亡。     

Are MG-63 and HOS TE85 human osteosarcoma cell lines representative models of the osteoblastic phenotype?

The aim of this study was to perform a systematic comparison of two widely used osteosarcoma cell lines and ascertain their relevance as experimental models for investigating osteoblast function. We have therefore compared growth, differentiated cell function, integral expression and adhesive profiles of MG-63, HOS TE85, and human bone derived cells. Both osteosarcoma cell lines proliferated more rapidly than osteoblast-like cells with HOS cells exhibiting the shortest doubling time. HOS cells expressed higher levels of alkaline phosphatase than MG-63 cells under basal conditions but only MG-63 cells showed the increased enzyme activity following 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) administration, which is characteristic of bone derived cells. Osteocalcin was not detected in supernatants from any cells under basal conditions but levels produced by MG-63 cells on addition of 1,25(OH)₂D₃ were comparable with those of osteoblast-like cells. ∝1, ∝2, ∝3, ∝5, ∝V, and β1 integrin subunits were detected on all cells and there was no staining for ∝L, ∝M, β2, and β3. ∝3 and β1 were the major subunits detected on MG-63, HOS, and bone derived cells but relative concentrations of other ∝ subunits were dependent on cell type; ∝4 and ∝6 subunits could only be detected on osteosarcoma cell lines. Short term, serum-free cell adhesion assays showed that the three cell types adhered in a saturable manner to collagen I, fibronectin, and laminin. Maximal adhesion of osteosarcoma cell lines and human bone derived cells was observed after 2 hours and similar extracellular matrix protein coating concentrations were required by the three cell types to achieve optimum binding: maximal adhesion to collagen I and fibronectin occurred at 0.1–0.5 μg/cm², and maximal adhesion to laminin occurred at 5–10 μg/cm². These studies indicate that both osteosarcoma cell lines provide appropriate models for studying integrin subunit expression and cell adhesion. In addition, MG-63 cells are also suitable for investigating regulation and production of osteocalcin by human osteoblast-like cells. Proliferation and alkaline phosphatase activity exhibited by both MG-63 and HOS TE85 cells were not very representative of bone cell cultures and therefore neither cell line is ideally suited to studying these aspects of osteoblast function.     

成人成骨细胞和MG-63细胞株孕激素受体的表达

目的 观察成人成骨细胞和MG-63细胞株孕激素受体(progesterone receptor，PR)亚型表达的情况。方法 用改良胶原酶消化法从正常成人松质骨中分离成骨细胞，观察细胞形态，钙钴法行碱性磷酸酶染色，Van Gieson法行Ⅰ型胶原染色，茜素红行矿化结节染色；半定量RT-PCR检测骨钙素和PR亚型mRNA表达；Western blot测定PR蛋白质表达。结果 所分离培养的细胞具有成骨细胞的形态特征，保持了其在体内的功能；人成骨细胞和MG-63细胞株均表达PRA、PRB mRNA和蛋白质。结论 人成骨细胞和MG-63细胞株可能受孕激素的影响。     

Osteocalcin secretion by the human osteosarcoma cell line MG-63

The human osteosarcoma cell line MG-63 has been used to study the production of the bone-specific protein, osteocalcin. In the absence of any stimuli, MG-63 cells secreted very low levels of osteocalcin. The secretion of osteocalcin started after a lag time of 10-12 h upon 1,25-(OH)2D3 treatment. Osteocalcin secretion was measured at doses as low as 0.03 nM (fourfold increase, p less than 0.05), and this activity increased further with higher doses of 1,25-(OH)2D3 to reach a plateau at 50 nM. The secretion increased transiently from very low levels in sparse cell cultures to peak values in subconfluent cultures (+/- 40%), two- to threefold above values obtained for confluent cells. Values for confluent cells average 55.9 +/- 2.0 ng/ml protein per 48 h. A similar behavior is observed for 1,25-(OH)2D3 receptor concentration under similar experimental conditions. Bmax increased transiently from sparse to subconfluent cell cultures (40-60% confluent) and reached values 50% lower in confluent cells. However, the receptor affinity was not affected by cell density. MG-63 cells also possessed an alkaline phosphatase isoenzyme of the bone-liver-kidney type that was stimulated by 1,25-(OH)2D3 treatment (two- to threefold) and inhibited by parathyroid hormone (40 nM, -25%, p less than 0.025). PTH and PGE2 increased cAMP production in a dose-dependent manner, but the cells were irresponsive to salmon calcitonin. Basal and PTH-responsive cyclic AMP production were also modulated by cell density. Dexamethasone pretreatment (100 nM, 48 h) stimulated the PTH-dependent cAMP production but failed to influence the response to PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

siRNA下调THBS4对人骨肉瘤MG63增殖的影响

目的探讨小干扰RNA(small interfering RNA,siRNA)抑制血小板反应蛋白4(thrombospondin 4,THBS4)基因的表达对骨肉瘤MG-63细胞增殖的影响及其分子机制。方法将合成的THBS4 siRNA转染到人成骨细胞株MG63,CCK-8法观察对细胞增殖的影响,荧光定量PCR检测THBS4及TGF-beta1的mRNA表达,Western-blotting检测THBS4及TGF-beta1的蛋白表达。结果 Thbs4 siRNA转染后实验组48 h,72 h OD值高于阴性对照组(P0.05),差异具有显著性;Thbs4 mRNA和蛋白的表达水平明显低于阴性对照组和空白对照组(P0.01);Thbs4 siRNA转染后MG63细胞中TGF-beta1 mRNA及蛋白的表达水平提高(P0.01)。结论 THBS4 siRNA转染MG63后能够促进细胞增殖,其机制可能是激活TGF-beta1信号通路。     

Melatonin inhibits the proliferation of human osteosarcoma cell line MG-63

It seems established that the onset of osteosarcoma and the reduction in melatonin production run in parallel; this suggests that the decline in the cancer-inhibiting agent, melatonin, may contribute to the occurrence of osteosarcoma and that melatonin supplementation may have promise for preventing the development and progression of this condition. There is, however, no direct evidence regarding an antiproliferative effect of melatonin in osteosarcoma cells. In the current study, we examined whether melatonin inhibits the proliferation of human osteosarcoma cell line MG-63. MTT staining showed that at 4 mM–10 mM concentrations, melatonin significantly reduced the MG-63 cell proliferation in a dose-dependent and time-dependent manner. Flow cytometry documented that 4 mM melatonin significantly increased the fraction of cells in the G₀/G₁ phase of the cell cycle, while simultaneously reducing the proportion in the S and G₂/M phases. Western blot and real-time PCR analyses further confirmed that melatonin's inhibitory effect was possibly because of downregulation of cyclin D1 and CDK4, related to the G₁ phase, and of cyclin B1 and CDK1, related to the G₂/M phase. There was no downregulation of cyclin E, CDK2, and cyclin A, which are related to G₁/S transition and S phase. These findings provide evidence that melatonin may significantly inhibit human osteosarcoma cell proliferation in a dose-dependent and time-dependent manner and this inhibition involves the downregulation of cyclin D1, CDK4, cyclin B1 and CDK1.     

氨甲喋呤联合咖啡因对骨肉瘤细胞株的作用观察

[目的]观察氨甲喋呤联合咖啡因对骨肉瘤细胞株的作用.[方法]以国人骨肉瘤细胞株OS-732细胞为研究对象,根据析因试验设计原因,设1无药组(阴性对照组),2咖啡因组,3氨甲喋呤组,4咖啡因、氨甲喋呤联合用药组(混合用药组).培养一段时间后,分别采用MTY法细胞毒性试验和流式细胞仪(FCM)分析,观察3组药物对细胞的毒性(用残存细胞吸光度A值表示)及细胞周期的影响.[结果]FCM分析各组于48 h G2/M期细胞比例(%)为1组(23.210±0.416),2组为(23.120±0.440),3组为(28.770±0.531),4组(23.267±0.319),1组与2组间差异无统计学意义,其余各组间差异有统计学意义(P＜0.01).各组残存细胞吸光度A值为1组(0.411±0.006),2组(0.401±0.006);3组(0.304±0.007),4组(0.105±0.002),组间差异及交互作用有统计学意义(P＜0.01).[结论]氨甲喋呤与咖啡因具有协同作用,可增加氨甲喋呤对骨肉瘤细胞株的抑制率.     

高低不同分化特性人成骨肉瘤MG-63克隆细胞株的建立

目的：建立分化程度高低不同的骨肉瘤细胞株。方法：用有限稀释法将人成骨肉瘤MG-63细胞系单克隆化，并作如下鉴定：(1)细胞增殖；(2)细胞形态学；(3)软琼脂克隆形成率；(4)染色体分析；(5)细胞周期时相分析；(6)碱性磷酸酶的活性；(7)细胞运动能力；(8)裸鼠成瘤实验。结果：共得到23个克隆，命名为MG-63-1～MG-63-23。其中的5、18号细胞株具有明显差异，18号为大核长梭形细胞，无接触抑制，克隆形成率高，体内成瘤时间短，代表低分化的骨肉瘤细胞株。5号以短梭形为主，核质比例变小，有一定的接触抑制，致瘤能力较低，为高分化的骨肉瘤细胞株。结论：通过对骨肉瘤细胞系MG-63的单克隆化，建立了具有高、低不同分化程度的两个人骨肉瘤细胞株。     

降钙素基因相关肽/细胞外调节激酶信号通路对人MG-63细胞增殖的实验研究

目的 研究外源性降钙素基因相关肽(CGRP)对人MG-63细胞增殖的影响及其可能涉及的信号通路.方法 采用甲基噻唑基四唑(MTT)法和细胞计数法观察对比不同浓度外源性CGRP及其受体拮抗剂CGRP8-37与细胞外调解激酶(ERK)抑制剂PD98059对体外培养的人MG-63细胞增殖的影响.根据不同分组将体外培养的人MG-63细胞用CGRP、CGRP8-37及PD98059作用24 h后,通过免疫细胞化学方法观察细胞骨保护蛋白(OPG)、护骨素配体(RANKL)、ERK表达强度的变化.结果 不同浓度CGRP均可促进MG-63细胞的增殖,促进作用随浓度增高而增强,CGRP8-37、PD98059可减弱此作用,与对照组比较差异有统计学意义(P<0.05).CGRP呈剂量依赖性的上调成骨细胞OPG和ERK的表达,同时下调RANKL的表达,CGRP-37和PD98059可减弱此作用,与对照组比较差异有统计学意义(P<0.05).结论 CGRP可通过ERK信号通路改变OPG/RANKL的比值来调控成骨细胞增殖.

Abstract：

Objective To explore the effect of calcitonin gene-related peptide (CGRP) on proliferation and differentiation of MG-63 cells through extracellular signal-regulated kinase (ERK) pathway in vitro. Methods To study the effects of ERK inhibitor PD98059 and CGRP inhibitor CGRP8-37, 2 groups of MG-63 cells were treated with PD98059 and CGRP8-37 for one hour prior to serum treatment. The other 4 groups were cultured with serum of different doses (high, middle, low and blank). Proliferation and apoptosis of MG-63 cells were observed by methyl thiazolyl tetrazolium (MTT) method and cell-counting.Expressions of osteoprotegerin (OPG), receptor activator of nuclear factor kappaβ ligand (RANKL) and ERK were observed through immunocytochemical and image analysis. Results MTT showed that CGRP promoted proliferation of MG-63 cells and expressions of OPG and ERK but not expression of RANKL The effect was dose-dependent, and significantly inhibited by either CGRP 8-37 or PD98059 (P <0.05) . Conclusion CGRP can stimulate proliferation of MG-63 cells via CGRP receptor and ERK pathway to regulate the ratio of OPG/RANKL.