TGF-a、TNF-a和VEGF对人嗜铬细胞瘤细胞增殖和凋亡的影响

目的 研究转化生长因子a(TGF-a)、肿瘤坏死因子a(TNF-a)和血管内皮细胞生长因子(VEGF)对原代培养的人嗜铬细胞瘤细胞增殖和凋亡的影响。方法 用噻唑兰(MTT)法观察TGF-a、TNF-a和VEGF对原代培养的人嗜铬细胞瘤细胞增殖的作用；用PI染色流式细胞分析、Hochest33342染色的方法检测上述细胞因子对人嗜铬细胞瘤细胞凋亡的影响。结果 0．1～100ug／L的TGF-a、TNF-a和VEGF均促进入嗜铬细胞瘤细胞增殖。无血清培养72h，人嗜铬细胞瘤细胞的凋亡率为3．98％，100ug／L的TGF-a、TNF-a和VEGF对其凋亡无明显影响。结论 TGF-a、TNF-a和VEGF刺激原代培养的人嗜铬细胞瘤细胞增殖，但对其凋亡无明显影响。     

TGF-α、TNF-α和VEGF对人嗜铬细胞瘤细胞增殖和凋亡的影响

目的研究转化生长因子α(TGFα)、肿瘤坏死因子α(TNFα)和血管内皮细胞生长因子(VEGF)对原代培养的人嗜铬细胞瘤细胞增殖和凋亡的影响。方法用噻唑兰(MTT)法观察TGFα、TNFα和VEGF对原代培养的人嗜铬细胞瘤细胞增殖的作用;用PI染色流式细胞分析、Hochest33342染色的方法检测上述细胞因子对人嗜铬细胞瘤细胞凋亡的影响。结果0.1～100μg/L的TGFα、TNFα和VEGF均促进人嗜铬细胞瘤细胞增殖。无血清培养72h,人嗜铬细胞瘤细胞的凋亡率为3.98%,100μg/L的TGFα、TNFα和VEGF对其凋亡无明显影响。结论TGFα、TNFα和VEGF刺激原代培养的人嗜铬细胞瘤细胞增殖,但对其凋亡无明显影响。     

紫杉醇对耐阿霉素膀胱癌细胞株T24/ADM增殖的影响及机制

目的观察紫杉醇对耐阿霉素（ADM）人膀胱癌细胞株（T24/ADM）增殖的影响,并探讨其机制。方法用阿霉素浓度梯度递增法诱导培养人膀胱癌T24/ADM多药耐药细胞。用1、10、102、103nmol/L紫杉醇培养T24/ADM细胞12、24、48、72 h,用MTT法测算T24/ADM细胞增殖抑制率,用流式细胞仪PI单染法测定培养24、48 h时T24/ADM细胞凋亡率,并进行形态学观察。结果紫杉醇可抑制T24/ADM细胞生长,且在一定剂量范围内具有时间和浓度依赖性。实验组细胞凋亡率较对照组高,且随作用时间的延长,细胞凋亡率明显增高。紫杉醇组T24/ADM细胞镜下可见凋亡。结论紫杉醇可抑制膀胱癌T24/ADM细胞株生长,这种作用呈浓度时间依赖性,其机制可能与紫杉醇诱导T24/ADM细胞凋亡有关。     

人工合成FGL多肽对大鼠PC-12细胞增殖和凋亡的影响

目的探讨人工合成FGL多肽对大鼠肾上腺嗜铬细胞瘤PC-12细胞增殖和凋亡的影响。方法设计并人工合成FGL多肽。培养PC-12细胞,取生长状态良好者,分为对照组和实验组,实验组中加入FGL多肽溶液。对照组预先用多聚赖氨酸包被培养板。分别于培养1、3、5、7、9 d采用细胞活性检测试剂盒（CCK-8）检测两组细胞增殖情况。取PC-12,待细胞贴壁融合80%后,换无血清的培养基继续培养16 h。对照组加入H2O2刺激16 h。实验组先加入FGL多肽预孵育30 min,再加H2O2刺激16 h。流式细胞仪检测两组细胞凋亡情况;荧光定量PCR法检测PC-12中的核因子（NF）-κB mRNA。结果实验组培养1、3、5、7、9 d PC-12增殖数量均高于对照组;H2O2处理后,实验组PC-12凋亡数量及其中NF-κB mRNA的表达量均低于对照组（P均〈0.05）。结论人工合成FGL多肽可促进PC-12细胞增殖,并抑制其凋亡。     

PC12细胞相关分子研究近况

PC12细胞是一种来自大鼠肾上腺髓质嗜铬细胞瘤的克隆细胞系。在正常情况下培养,PC12细胞不仅在形态上,而且在生理及生化的许多方面都与肾上腺髓质细胞相似,加入神经生长因子(NGF)后,PC12细胞在形态上向交感神经元分化,同时伴有生理及生化方面的变化,最终导致PC12细胞呈现神经元     

肌肽对过氧化氢诱导PC12细胞损伤后NF-κB P65表达的影响

目的探讨L-肌肽对H2O2诱导PC12细胞(大鼠肾上腺嗜铬细胞瘤细胞)凋亡的保护机制。方法在H2O2诱导PC12细胞凋亡模型的基础上,加入肌肽作用于细胞,采用MTT比色法检测肌肽对PC12细胞的增殖抑制作用;RT-PCR法检测凋亡相关基因NF-κB P65 mRNA的表达变化;免疫组化SABC法检测凋亡相关蛋白Caspase-3和NF-κB P65的表达,流式细胞仪(FCM)检测细胞凋亡。结果不同浓度的肌肽对H2O2损伤的PC12细胞的存活率有显著的提高作用,20 mmol/L浓度时达最大值(P<0.05)。20 mmol/L的肌肽作用于PC12细胞可降低NF-κB P65的mR-NA表达,降低NF-κB P65蛋白的表达,流式细胞仪检测显示肌肽可抑制细胞的早期和晚期凋亡率。结论肌肽对H2O2损伤的PC12细胞有保护作用,机制可能是通过抑制NF-κB P65的表达来抑制PC12细胞的凋亡而实现的。     

豨莶草提取液对MPP~+诱导的PC12细胞损伤的保护作用

目的探讨豨莶草提取液对1-甲基-4-苯基吡啶离子(MPP~+)诱导的大鼠肾上腺嗜铬细胞瘤细胞(PC12细胞)损伤的保护作用及机制。方法在生长状态良好的PC12细胞中加入终浓度为400μmol/L的MPP~+,造成帕金森病(PD)的离体实验模型,并观察豨莶草提取液对MPP~+诱导的PC12细胞损伤的保护作用。倒置显微镜下观察细胞形态,MTT法检测细胞活力,通过DCFH-DA荧光探针检测细胞内活性氧(ROS)水平。结果 400μmol/L MPP~+作用PC12细胞能明显抑制细胞生长,造成细胞发生损伤,ROS水平增加。同时给予不同浓度豨莶草提取液,PC12细胞存活率明显增加,ROS水平显著降低。结论豨莶草提取液能有效改善MPP~+诱导的PC12细胞的损伤,其作用机制可能与降低ROS水平有关。     

真核细胞翻译延伸因子2参与神经生长因子诱导的大鼠嗜铬细胞瘤细胞的分化

目的研究神经生长因子(NGF)诱导大鼠嗜铬细胞瘤细胞系PC12细胞分化途径中,细胞突触生长的起始和延伸步骤中的关键蛋白质,寻找其中可能的药物靶点。方法 50 ng/ml NGF诱导PC12细胞6 h和12 h,显微镜下观察PC12细胞的形态变化;应用二维凝胶电泳、De Cyder软件分析及基质辅助激光解析/电离-飞行时间质谱,研究其差异蛋白质组学变化。结果 NGF作用于PC12细胞6 h后,细胞出现神经突起;12 h,神经突起进一步生长,蛋白质分析提示真核细胞翻译延伸因子(e EF)2含量减少,α微管蛋白和β肌动蛋白表达增加。结论 NGF诱导PC12细胞发生神经突触生长,e EF-2的减少与细胞骨架蛋白增加与细胞的形态学变化一致,提示突触生长过程中,e EF-2可能起到关键性作用。     

星形胶质细胞条件培养液对鱼藤酮染毒神经元的保护作用

目的 观察星形胶质细胞条件培养液(ACM)对鱼藤酮染毒嗜铬细胞瘤细胞(PC12)的神经保护作用.方法 将新生SD大鼠中脑星形胶质细胞分离、纯化并培养至第三代后,收集星形胶质细胞条件培养液加入到鱼藤酮染毒PC12细胞中,观察其对染毒神经元形态、活力及其合成与释放一氧化氮(NO)、乳酸脱氢酶(LDH)和胞膜ATP酶(ATPase)的影响.结果 20%ACM可明显改善染毒神经元的形态和活力;与对照组比较,20%ACM还有效减少了染毒神经元NO、LDH的合成和释放,保护和提高了ATPase活性.结论 ACM明显促进鱼藤酮染毒PC12细胞的活力,其机制可能与减少NO、LDH合成释放及保护细胞膜ATPase的活性有关.     

Presenilin-1突变对全反式维甲酸诱导的PC12细胞生长和凋亡的影响

目的　观察Presenilin 1(PS 1)突变型L2 86V对全反式维甲酸 (RA)诱导的PC12细胞生长和凋亡的影响。　方法　运用脂质体介导的基因转染技术建立稳定表达PS 1WT和突变型L2 86V基因的细胞克隆 ,采用MTT法、流式细胞仪检测细胞生长曲线、生长周期及细胞凋亡情况。　结果　 (1)PS 1突变型L2 86V对RA诱导的PC12细胞生长具有抑制作用 ;主要表现为G1期细胞增多、S期细胞减少 ,细胞增殖指数降低 ;(2 )在正常培养条件下 ,对照组细胞凋亡率 (% )为 0 31±0 0 9、野生型组为 0 4 3± 0 11、L2 86V组为 0 6 2± 0 2 7;无血清培养 1、2、3d ,对照组细胞凋亡率 (% )分别为 0 5 5± 0 0 7、1 2 3± 0 4 7、5 5 9± 2 91;野生型组为 1 0 9± 0 73、3 2 1± 1 4 1、7 79± 2 78;L2 86V组为 4 6 5± 1 0 4、10 5 4± 3 18、14 4 7± 3 5 3;PS 1突变型L2 86V对RA诱导的PC12细胞均具有促进细胞凋亡的作用 ,以无血清培养时表现得更为明显。　结论　PS 1突变型L2 86V对RA诱导的PC12细胞生长具有抑制作用 ;在有和无血清培养条件下均具有促进细胞凋亡的作用 ,以无血清培养时表现得更为明显。     

Galanin decreases proliferation of PC12 cells and induces apoptosis via its subtype 2 receptor (GalR2)

Galanin is a neuropeptide with a wide range of effects in the nervous and endocrine systems, mediated through three G protein-coupled receptor subtypes (GalR1-3). Interestingly, galanin and its receptors are also expressed in certain tumors. Here we studied the effects of galanin in rat pheochromocytoma (PC12) cells stably transfected with GFP-tagged GalR2. Galanin at 100 nM inhibited cell proliferation in both nontransfected and transfected cells. Conversly, both galanin and the GalR2(R3)-agonist AR-M1896 induced caspase-dependent apoptotic cell death only in GalR2-transfected cells. Western-blot analyses of downstream mediators of the G(q/11)-type G protein showed down-regulation of pAkt and pBad in galanin-exposed transfected cells. Also, the specific PI3 kinase inhibitor LY-294002 increased the level of pBad and decreased activation of caspases. In addition, p21(cip1) levels were up-regulated in galanin-exposed PC12 cells and down-regulated in galanin-exposed GalR2-transfected cells. In agreement, FACS analyses of galanin exposed cells showed occurrence of cell cycle arrest in PC12 cells and cell death in transfected cells. Finally, as shown with real-time PCR, galanin and its receptors were expressed at very high levels in human pheochromocytoma tissues as compared with normal adrenal medulla. These findings point to GalR2 as a possible target for therapeuthic interventions in pheochromocytoma.     

Insulin and insulin-like growth factors stimulate deoxyribonucleic acid synthesis in PC12 pheochromocytoma cells

The effects of insulin and insulin-like growth factors (IGFs) on the replication of PC12 pheochromocytoma cells were investigated. Incubation of PC12 cells for 2-3 days in low (0.3%) serum medium decreased [3H]thymidine incorporation into PC12 cell DNA to approximately 30% of that in control (15% serum) medium. Incubation of the cells in low serum medium also slowed the growth of the cultures and increased the percentage of cells in the G0/G1 phase of the cell cycle. Addition of insulin to cells in low serum medium increased [3H]thymidine incorporation into the cells, increased the number of cells in PC12 cultures, and decreased the percentage of cells in the G0/G1 phase of the cell cycle. IGF-I and IGF-II also increased [3H]thymidine incorporation into PC12 cells incubated in low serum medium. IGF-I (EC50, approximately 0.3 nM) was a more potent stimulus of [3H]thymidine incorporation than was insulin (EC50, approximately 3.5 nM). These data suggest that insulin and IGFs are growth factors for PC12 cells, and that the growth-promoting effects of these agents may be mediated by a type I IGF receptor on PC12 cells.     

In vitro and in vivo angiogenesis in PC12 pheochromocytoma cells is mediated by vascular endothelial growth factor.

Angiogenesis, the formation of new blood vessels from existing vascular endothelium, is essential for tumor growth. Vascular endothelial growth factor (VEGF) is an endotheliumspecific mitogen and regulator of angiogenesis. Angiogenesis has been associated to the malignant phenotype of pheochromocytomas and is readily observed in experimental pheochromocytomas. Although VEGF gene expression has already been demonstrated in the rat PC12 cell line, the detailed mechanisms of action are not known. We have, therefore, studied angiogenesis in the rat PC12 pheochromocytoma cell line in vitro and in vivo. VEGF gene expression and accumulation of VEGF protein in cytoplasm and conditioned medium of PC12 cells was found. Conditioned medium from PC12 cells significantly increased proliferation of VEGF-dependent endothelial cells from human umbilical veins, and this effect reversed upon addition of a neutralizing anti-VEGF antibody. Dexamethasone and nerve growth factor (NGF) increased VEGF mRNA expression and accumulation of VEGF protein of PC12 subclones with established metastatic activity in vivo. PC12 cells xenotransplanted to nude mice had marked VEGF expression and induced host angiogenesis, confirmed by the presence of CD34-positive endothelial cells in the experimental PC12 tumors. When NGF-primed PC12 cells were immobilized in Matrigel supplemented with rising concentrations of the growth factor and xenotransplanted, increasing NGF resulted in tumors with smaller areas of necrosis and increased vital tumor volume. These results suggest that VEGF is a mediator of angiogenesis in the PC12 pheochromocytoma cell line, and that dexamethasone and NGF affect VEGF expression. Our data further suggest that NGF may contribute to angiogenesis in experimental pheochromocytoma.     

The effects of glutamate can be attenuated by estradiol via estrogen receptor dependent pathway in rat adrenal pheochromocytoma cells

Estrogens have been suggested to exhibit neuroprotective activities against several insults including beta-amyloid and glutamate, one of the excitatory neurotransmitters in the central nervous system. In the present study, we showed that exposure to glutamate not only inhibited the cell growth of exponentially growing rat pheochromocytoma PC12 cells in a time- and dose- dependent manner, but also influenced cell adherence capacity. Glutamate-induced growth inhibition was significantly attenuated by the co-administration of estradiol in PC12 cells. Pre-exposure of the PC12 cells to the estradiol was not required for protection against glutamate-induced growth inhibition. Administration of anti-estrogen ICI182,780 efficiently blocked the neuroprotective effects of estradiol. Glutamate-induced changes in cell adherence, on the other hand, were not significantly affected by estradiol. These data indicate that the neuroprotective effects of estradiol against glutamate-induced insults in PC12 cells, at least in part, involve estrogen receptor-dependent pathways.     

Evaluation of PC12 Cells’ Proliferation,Adhesion and Migration with the Use of an Extracellular Matrix (CorMatrix) for Application in Neural Tissue Engineering

The use of extracellular matrix (ECM) biomaterials for soft tissue repair has proved extremely successful in animal models and in some clinical settings. The aim of the study was to investigate the effect of the commercially obtained CorMatrix bioscaffold on the viability, proliferation and migration of rat pheochromocytoma cell line PC12. PC12 cells were plated directly onto a CorMatrix flake or the well surface of a 12-well plate and cultured in RPMI-1640 medium and a medium supplemented with the nerve growth factor (NGF). The surface of the culture plates was modified with collagen type I (Col I). The number of PC12 cells was counted at four time points and then analysed for apoptosis using a staining kit containing annexin V conjugate with fluorescein and propidium iodide (PI). The effect of CorMatrix bioscaffold on the proliferation and migration of PC12 cells was tested by staining the cells with Hoechst 33258 solution for analysis using fluorescence microscopy. The research showed that the percentage of apoptotic and necrotic cells was low (less than 7%). CorMatrix stimulates the proliferation and possibly migration of PC12 cells that populate all levels of the three-dimensional architecture of the biomaterial. Further research on the mechanical and biochemical capabilities of CorMatrix offers prospects for the use of this material in neuro-regenerative applications.     

Effect of extracellular matrix on PC 12 cell shape and dopamine processing

Substrates of various origins can affect the morphology, growth and functional properties of many cell types. PC 12 cells (a clonal line of rat pheochromocytoma) synthesize, store and secrete dopamine as well as other transmitters. These cells are rounded and loosely attached when cultured on plastic, but flatten and spread extensively when cultured on an extracellular matrix secreted by bovine corneal endothelial cells. We determined that spontaneous dopamine release into culture medium was significantly higher from cells which were flattened on matrix, and that the cellular content of dopamine was less than in cells which were maintained on plastic. The higher levels of medium dopamine in matrix cultures were not due to an increase in cellular attachment or growth as determined by [3H]thymidine incorporation. A variant of PC 12 cells which is flat on plastic showed no change in cell shape or dopamine release when plated on matrix. These experiments suggest that extracellular matrix promotes a change in cell shape and that this change in the physical arrangement of these cells alters their release and storage of dopamine.     

A potential pathogenetic mechanism for multiple endocrine neoplasia type 2 syndromes involves ret-induced impairment of terminal differentiation of neuroepithelial cells.

Germ-line missense mutations of the receptor-like tyrosine kinase ret are the causative genetic event of the multiple endocrine neoplasia (MEN) type 2A and type 2B syndromes and of the familial medullary thyroid carcinoma. We have used the rat pheochromocytoma cell line, PC12, as a model system to investigate the mechanism or mechanisms by which expression of activated ret alleles contributes to the neoplastic phenotype in neuroendocrine cells. Here we show that stable expression of ret mutants (MEN2A and MEN2B alleles) in PC12 cells causes a dramatic conversion from a round to a flat morphology, accompanied by the induction of genes belonging to the early as well as the delayed response to nerve growth factor. However, in the transfected PC12 cells, the continuous expression of neuronal specific genes is not associated with the suppression of cell proliferation. Furthermore, expression of ret mutants renders PC12 cells unresponsive to nerve growth factor-induced inhibition of proliferation. These results suggest that induction of an aberrant pattern of differentiation, accompanied by unresponsiveness to growth-inhibitory physiological signals, may be part of the mechanism of action of activated ret alleles in the pathogenesis of neuroendocrine tumors associated with MEN2 syndromes.     

过氧化氢对PC12细胞活性的调控及脑活素的抗氧化效能

目的　探讨活性氧对神经细胞活性的调控及脑活素的抗氧化功效。方法　以过氧化氢 (H2 O2 )作为活性氧 ,应用四甲基偶氮唑 (MTT)比色法 ,观察对培养的PC12细胞生长活性的调控和脑活素的抗氧化活性。结果　以 0 .0 3mmol LH2 O2 对PC12细胞具有促进生长的作用 ( P <0 .0 5 ) ,0 .1～ 1.0mmol LH2 O2 可导致PC12细胞不同程度的氧化损伤 ;脑活素浓度在 0～ 10g L对细胞的生长无明显影响 ,当浓度超过 2 0g L后 ,可导致细胞生长受抑制 (P <0 .0 1) ,10～ 2 0g L脑活素可有效保护PC12细胞免受氧化损伤。结论　活性氧对神经细胞的生长具有双重作用 ,脑活素具有保护PC12细胞免受氧化损伤的效能。     

大黄酚对大鼠肾上腺髓质嗜铬细胞瘤分化株细胞缺氧损伤的保护作用

目的观察大黄酚对缺氧引起的大鼠肾上腺髓质嗜铬细胞瘤分化株（PC12）细胞损伤的保护作用。方法培养PC12细胞,采用自制的密闭三气培养箱建立缺氧所致的PC12细胞损伤模型,根据不同条件,分为对照组、模型组、大黄酚组（包括1μg/ml组和5μg/ml组）。于缺氧处理前及处理后1、2、6、12、24h,每个时间点取6个复孔。采用四甲基偶氮唑盐法（MTT）检测PC12细胞增殖活性,高倍显微镜下观察PC12细胞核形态,测定各组上清液中超氧化物歧化酶（SOD）含量,免疫组化法测定各组线虫抗凋亡基因的人类同源基因表达蛋白（BCL-2）的表达,实时定量PCR测定p38丝裂原活化蛋白激酶（MAPK）和含半胱氨酸天冬氨酸蛋白酶3（Caspase-3）mRNA的表达。结果①缺氧处理后12 h,MTT法测定大黄酚1μg/ml组的细胞活力高于模型组;缺氧处理24 h,大黄酚1μg/ml组和5μg/ml组的细胞活力均高于模型组,差异均有统计学意义（P〈0.05）。②从缺氧处理后1 h开始,大黄酚1μg/ml组和5μg/ml组的SOD值高于模型组,差异有统计学意义（P〈0.05）。③从缺氧后2 h开始,大黄酚1μg/ml组和5μg/ml的BCL-2阳性率均高于模型组。④从缺氧处理2h开始,模型组p38MAPK mRNA表达量高于大黄酚1μg/ml组和5μg/ml组,差异有统计学意义（P〈0.05）。从缺氧处理6 h开始,模型组Caspase-3mRNA表达量高于大黄酚1μg/ml组和5μg/ml组,差异有统计学意义（P〈0.05）。⑤缺氧处理后12、24 h,模型组细胞的细胞核皱缩、形态不清,内见颗粒样物质形成增多;大黄酚组的细胞核形态较清楚,少见颗粒样物质形成。结论大黄酚可以减轻缺氧所致PC12细胞的损伤,对PC12细胞有保护作用。     

Enhanced therapeutic effects of combined chemotherapeutic drugs and midkine antisense oligonucleotides for hepatocellular carcinoma

AIM： To evaluate the effect of combined antisense oligonucleotides targeting midkine （MK-AS） and chemotherapeutic drugs [cisplatin（DDP）, 5-fluorouracil （5-FU） and adriamycin （ADM）] on inhibition of HepG2 cell proliferation, and to analyze the efficacy of MK-AS used in combined ADM in in situ human hepatocellular carcinoma （HCC） model. METHODS： HepG2 cells were treated with MK-AS and/or chemotherapeutic drugs mediated by Lipofectin, and cell growth activity was determined by MTS assay. An in situ HCC model was used in this experiment. MK- AS, ADM and MK-AS ＋ ADM were given intravenously for 20 d, respectively. The animal body weight and their tumor weight were measured to assess the effect of the combined therapy in vivo. RESULTS： Combined treatment with MK-AS reduced the IC50 of DDP, 5-FU and ADM in HepG2 cells. MK-AS significantly increased the inhibition rate of DDP, 5-FU and ADM. Additionally, synergism （Q 1.15） occurred at a lower concentration of ADM, 5-FU and DDP with combined MK-AS. Combined treatment with MK-AS and ADM resulted in the more growth inhibition on in situ human HCC model compared with treatment with chemotherapeutic drugs alone. CONCLUSION： MK-AS increases the chemosensitivity in HepG2 cells and in situ human HCC model, and the combination of MK-AS and ADM has a much better in vitro and in vivo synergism.