光甘草定诱导小鼠黑色素瘤B16F10细胞凋亡的机制研究

通过体外和体内模型研究光甘草定对小鼠黑色素瘤B16F10细胞增殖的抑制作用及其分子机制。B16F10细胞经光甘草定处理后可抑制其增殖,且具有浓度依赖性;诱导B16F10细胞凋亡,观察到明显的细胞凋亡状态,细胞内凋亡相关基因及蛋白Bax表达显著上升,Bcl-2则表达下调。进一步的研究发现,经光甘草定处理后的B16F10细胞中,培养基中葡萄糖含量升高,而ATP含量、乳酸生成均降低;细胞内糖酵解相关基因及蛋白HK2、Ldha表达下调。同时,经光甘草定处理后,移植瘤小鼠的肿瘤组织生长受到明显抑制,肿瘤组织内细胞凋亡率显著升高,Bax表达明显上升,而Bcl-2、HK2和Ldha则表达均下调。说明,光甘草定在一定浓度范围内抑制了小鼠黑色素瘤B16F10细胞的增殖,诱导细胞凋亡,而其诱导细胞凋亡的机制可能与调控糖酵解相关基因的表达相关。     

RORα高表达对二烯丙基二硫抑制人胃癌细胞增殖与迁移侵袭的影响

目的观察高表达RORα对二烯丙基二硫(DADS)抑制人胃癌MGC803细胞增殖、迁移与侵袭的影响。方法集落形成实验与流式细胞术检测细胞增殖与细胞周期;细胞划痕和Transwell实验分别检测细胞迁移与侵袭。RT-PCR与Western blot分别检测RORα、MMP-9和TIMP3 mRNA与蛋白表达水平。结果RT-PCR与Western blot检测显示,RORα高表达与DADS处理较对照组与空载体组RORαmRNA与蛋白表达明显上调,DADS+RORα高表达组上调更为显著(P<0.05)。与对照组和空载体组比较,RORα高表达与DADS处理组MMP-9表达下调,TIMP3表达上调,DADS+RORα高表达组改变最为显著。集落形成实验显示,RORα高表达与DADS处理组较对照组与空载体组的集落形成率明显降低。流式细胞术显示,与对照组和空载体组比较,RORα高表达与DADS处理组G2/M期细胞比率明显升高。细胞划痕和Transwell实验显示,RORα高表达与DADS处理组细胞迁移与侵袭能力明显降低。结论RORα高表达可通过上调TIMP3与下调MMP-9促进DADS阻滞MGC803细胞G2/M期和抑制增殖与迁移侵袭。     

重组天花粉蛋白与-16联合用药抑制黑色素瘤细胞增殖及成瘤

获得人源天花粉蛋白并研究其与YH-16联合用药对小鼠黑色素瘤细胞B16-F10增殖及成瘤的作用。以大肠杆菌BL21重组表达天花粉蛋白r TCS并以亲和层析法纯化r TCS;以MTT法检测r TCS、YH-16、r TCS+YH-16对B16-F10细胞生长的影响;体内实验检测r TCS、r TCS+YH-16对瘤体生长的作用。结果表明,获得了电泳纯重组天花粉蛋白r TCS;r TCS可显著抑制B16-F10细胞增殖,并且抑制率显著高于YH-16组,但是r TCS与YH-16联合处理组并未比r TCS组有显著差异;r TCS与YH-16联合用药明显抑制B16-F10细胞在小鼠体内成瘤。重组天花粉蛋白r TCS联合YH-16用药对黑色素瘤细胞B16-F10的增殖及成瘤有显著抑制作用。联合策略将为治疗黑色素瘤患者提供新的契机。     

siRNA干扰MMP-9和FAK双基因抑制小鼠黑色素瘤生长和在体迁移

目的:观察干扰MMP-9和FAK双基因对恶性黑色素瘤高转移细胞B16F10体内转移的影响。方法:构建PGV102-MMP9-siRNA、PGV102-FAK-siRNA重组质粒载体,脂质体^TM2000介导转染小鼠黑色素瘤B16F10细胞,RT-PCR检测基因的干扰效果;建立C57BL/6小鼠皮下移植瘤模型观察细胞在体成瘤和肿瘤的生长情况,常规组织切片,H&E染色观察肿瘤组织病理学特征;经C57BL/6小鼠尾静脉注射细胞5×10⁵个/只,24天后计数小鼠肺转移结节数评价肿瘤细胞在体迁移能力。结果:RT-PCR结果表明,重组质粒转染细胞组的MMP-9和FAK的mRNA水平显著低于正常细胞组(P<0.01),转染细胞组C57BL/6小鼠皮下成瘤的肿瘤生长速率、黑色素瘤肺转移结节数明显低于正常细胞组(P<0.01)。结论:干扰B16F10细胞MMP-9和FAK双基因可明显抑制小鼠体内恶性肿瘤的生长和迁移。     

RORα高表达抑制人胃癌MGC803细胞Wnt/β-catenin信号通路靶基因表达

该文探讨了RORα(retinoid acid receptor related orphan receptorα)高表达对人胃癌MGC803细胞Wnt/β-catenin信号通路靶基因的作用。采用MTT检测了MGC803细胞增殖。采用RT-PCR、Western blot与免疫共沉淀检测了Wnt/β-catenin信号通路相关分子与靶基因表达。荧光素酶报告基因方法检测c-Myc基因启动子活性。MTT结果显示,RORα高表达人胃癌MGC803细胞的增殖能力较对照组明显减弱(P0.05)。RT-PCR与Western blot结果显示,RORα高表达组Wnt1m RNA与蛋白质水平较对照组下调(P0.05),而β-catenin m RNA与蛋白质水平无差异(P0.05)。免疫共沉淀结果显示,RORα高表达组RORα与β-catenin结合明显增加(P0.05)。RORα高表达可显著下调核内β-catenin水平(P0.05),同时可显著下调TCF-4(T cell factor-4)蛋白质水平(P0.05)。RORα高表达可显著下调Axin、c-Myc、c-Jun m RNA与蛋白质水平(P0.05)。荧光素酶报告基因实验结果显示,RORα高表达c-Myc启动子活性明显降低(P0.05)。以上结果表明,RORα高表达可通过调控Wnt/β-catenin信号通路相关分子基因表达来抑制人胃癌细胞增殖。     

内皮抑素基因抗黑色素瘤细胞高转移的动物研究

目的:验证转内皮抑素基因抑制高转移性黑色素瘤细胞的转移能力.方法:将 pcDNA3.1-Endo 真核表达载体转染小鼠黑色素瘤高转移细胞株B16F10,运用RT-PCR和Western-blot验证Endo的表达后,进行瘤细胞的粘附实验、体外侵袭和运动实验以及C57BL/6小鼠的皮下种植瘤和肺转移瘤试验,并进一步统计分析.结果:内皮抑素基因能明显抑制B16F10黑色素瘤细胞的粘附、体外侵袭和运动、体内成瘤以及肺转移能力,其中粘附抑制率36.4%,体外侵袭抑制率48.4%,细胞运动功能抑制率52.1%,肺转移抑制率为67.3%.结论:转内皮抑素基因能明显抑制黑色素瘤细胞的高转移能力.     

siRNA联合沉默MMP-9和FAK基因对小鼠黑色素瘤高转移细胞B16F10体外侵袭和迁移的影响

目的： 观察双基因联合干扰MMP-9和FAK对小鼠黑色素瘤高转移细胞B16F10体外侵袭、迁移能力的影响。方法：分别构建pGV102-MMP9-siRNA,pGV102-FAK-siRNA重组质粒载体,脂质体^TM2000介导转染小鼠黑色素瘤B16F10细胞,实验分为空白对照组、Anti-MMP-9组,Anti-FAK组、Anti-MMP-9 &FAK组、阴性对照组。经G418筛选GFP+克隆,流式细胞仪分析阳性率,激光共聚焦观察转染后细胞形态,半定量RT-PCR检测各组B16F10细胞MMP-9和FAK基因的mRNA转录水平,Transwell侵袭、迁移实验测定各组B16F10细胞体外侵袭、迁移能力。结果： 经G418筛选,3个转染组阳性率分别为92.41±1.64%,95.72±0.21%,91.52±0.11%,且转染后细胞形态良好;与空白对照组相比,3个转染组的MMP-9,FAK mRNA转录水平下降明显（P<0.01）,迁移、侵袭能力明显降低（P<0.01）,但Anti-MMP-9 &FAK组细胞侵袭迁移能力显著低于Anti-MMP-9 组和Anti-FAK组（P<0.01）。结论： 相比单独沉默MMP-9 或FAK,联合沉默MMP-9 和FAK可明显降低小鼠黑色素瘤B16F10细胞体外迁移、侵袭能力。     

TGM2对胃癌细胞BGC-823增殖、迁移和侵袭能力的影响

目的:转谷氨酰胺酶-2(transglutaminase-2,TGM2)是一种多功能的蛋白,在乳腺癌,胰腺癌,黑色素瘤和前列腺癌等肿瘤中高表达,而且和肿瘤的增殖和转移密切相关。本研究旨在探索TGM2对胃癌细胞BGC-823的增殖、迁移和侵袭能力的影响。方法:q RT-PCR、蛋白质印迹及免疫组化检测TGM2在胃癌细胞及组织中的表达情况;慢病毒转染胃癌细胞BGC-823以干扰TGM2的表达,荧光显微镜下观察转染效率,蛋白质印迹检测TGM2表达干扰效果;CCK-8法检测TGM2下调对BGC-823增殖能力的影响;Transwell法检测TGM2下调对BGC-823的迁移和侵袭能力影响;蛋白质印迹检测TGM2下调对转移和凋亡相关蛋白表达的影响。结果:TGM2在胃癌细胞和组织中均高表达,P0.01(q RT-PCR),P0.001(免疫组化);转染慢病毒后,荧光显微镜下观察结果显示转染效率超过90%,蛋白质印迹结果显示实验组sh TGM2-1的干扰效果最好,TGM2明显下调,差异具有统计学意义,P0.0001;CCK-8结果表明TGM2下调后,从细胞铺板第2d开始,实验组(sh TGM2-1)的细胞增殖倍数明显下降,P0.05(2d),P0.01(3d),P0.001(4d),P0.01(5d);Transwell结果显示,TGM2下调后,BGC-823的迁移和侵袭能力明显减弱,P0.001(迁移),P0.01(侵袭);蛋白质印迹结果显示,TGM2下调后,NF-κB和Bcl-2表达下调,而Bax表达上调,P0.05(NF-κB)、P0.001(Bcl-2)和P0.0001(Bax)。结论:TGM2下调能抑制胃癌细胞BGC-823的增殖、迁移和侵袭能力,并和NF-κB、Bcl-2的下调以及和Bax的表达水平上调有关,为胃癌的临床治疗提供了新靶点。     

ROR1激活AKT/FOXO1信号介导非小细胞肺癌吉非替尼耐药

EGFR-TKI靶向治疗在非小细胞肺癌（non-small cell lung cancer, NSCLC）综合治疗中显示出重要作用;然而,耐药性却极大限制其临床治疗效果。受体酪氨酸激酶样孤儿受体（receptor tyrosine kinase-like orphan receptor 1, ROR1）是I型受体酪氨酸激酶家族中的成员,在肿瘤发生发展中发挥重要作用。本研究拟探讨ROR1介导非小细胞肺癌吉非替尼耐药的作用及机制。采用吉非替尼反复诱导非小细胞肺癌HCC827细胞,建立吉非替尼耐药细胞株HCC827/GR。应用荧光定量PCR和Western 印迹检测HCC827/GR内ROR1的表达。采用shRNA的方法体外检测ROR1敲除前后HCC827/GR对吉非替尼耐药的变化,采用体外检测ROR1过表达前后HCC827对吉非替尼耐药的变化。体内检测ROR1敲除前后HCC827/GR对吉非替尼耐药的变化。Western 印迹检测HCC827/GR内ROR1下游信号分子的活化。实时荧光定量PCR及Western 印迹结果显示,HCC827/GR耐药细胞中的ROR1 mRNA和蛋白质表达水平显著高于HCC827敏感细胞。体外干扰ROR1表达,可明显增强HCC827/GR耐药细胞对吉非替尼的敏感性 (IC50 15.3±3.69 vs. 4.2±1.38),增加吉非替尼诱导的细胞凋亡 (20.5±2.52 vs. 41.8±3.74)。体外过表达ROR1显著增强HCC827敏感细胞对吉非替尼的耐药性(IC50 0.8±0.52 vs. 2.2±0.87)。体内裸鼠移植瘤实验同样发现,干扰ROR1能增强HCC827/GR移植瘤对吉非替尼的敏感性。进一步研究发现,AKT/FOXO1信号在HCC827/GR耐药细胞中异常活化,而干扰ROR1能够抑制AKT的磷酸化,并上调FOXO1的表达。上述结果表明,ROR1参与非小细胞肺癌吉非替尼耐药,抑制ROR1能够逆转吉非替尼耐药,其机制与ROR1调控AKT/FOXO1信号有关。     

RGS16基因抑制肺癌发病进展的功能研究

[目的]研究RGS16基因对人肺癌发病及进展的作用。[方法]构建过表达慢病毒载体Lenti-RGS16,感染A549细胞,以CCK-8实验、平板克隆形成实验、划痕实验、FITC/PI染色法检测A549细胞行为包括增殖、克隆形成、迁移、凋亡率变化,以裸鼠成瘤实验检测A549细胞体内生长。[结果]A549细胞中RGS16基因表达量提高3. 6倍,细胞生长、增殖及迁移能力分别降低73%、62. 8%、88%,细胞凋亡率增加3倍;在动物成瘤实验终点时,RGS16过表达组的肿瘤体积比对照组显著减少(240 vs 1090 mm3),瘤体重量也显著降低(0. 33 vs 0. 95 g)。经分析TCGA数据库发现RGS16基因在肺癌组织中表达比正常对照组织中减少52. 8%。[结论] RGS16基因抑制肺癌A549细胞生长、增殖及迁移,促进细胞凋亡。     

细胞受体与疾病的相关性研究进展

受体是存在于效应细胞中的重要功能蛋白，它和配体具有高亲和力，受体异常亦称受体病，拟就近年来细胞受体和受体病的研究进展作一综述。     

Novel receptor partners and function of receptor activity-modifying proteins

The receptor activity-modifying proteins (RAMPs) comprise a family of three accessory proteins that heterodimerize with the calcitonin receptor-like receptor (CL receptor) or with the calcitonin receptor (CTR) to generate different receptor phenotypes. However, RAMPs are more widely distributed across cell and tissue types than the CTR and CL receptor, suggesting additional roles for RAMPs in cellular processes. We have investigated the potential for RAMP interaction with a number of Class II G protein-coupled receptors (GPCRs) in addition to the CL receptor and the CTR. Using immunofluorescence confocal microscopy, we demonstrate, for the first time, that RAMPs interact with at least four additional receptors, the VPAC1 vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor with all three RAMPs; the glucagon and PTH1 parathyroid hormone receptors with RAMP2; and the PTH2 receptor with RAMP3. Unlike the interaction of RAMPs with the CL receptor or the CTR, VPAC1R-RAMP complexes do not show altered phenotypic behavior compared with the VPAC1R alone, as determined using radioligand binding in COS-7 cells. However, the VPAC1R-RAMP2 heterodimer displays a significant enhancement of agonist-mediated phosphoinositide hydrolysis with no change in cAMP stimulation compared with the VPAC1R alone. Our findings identify a new functional consequence of RAMP-receptor interaction, suggesting that RAMPs play a more general role in modulating cell signaling through other GPCRs than is currently appreciated.     

Acetylcholine receptor: effects of proteolysis on receptor metabolism

Previous studies (Miskin, R., T. G. Easton, and E. Reich, 1970, Cell. 15:1301-1312) have shown that sarcoma virus transformation and tumor promoters reduced the cell surface concentration of acetylcholine receptors (AChR) in differentiating chick embryo myogenic cultures. Both of these agents also induced high rates of plasminogen activator (PA) synthesis in myogenic cultures (Miskin, R., T. G. Easton, A. Maelicke, and E. Reich, 1978, Cell. 15:1287-1300), and the present work was performed to establish whether proteolysis might significantly affect receptor metabolism. Proteolysis in myogenic cultures was modulated by one or more of the following: stimulation of PA synthesis, direct addition of plasmin, removal of plasminogen, or addition of plasmin inhibitors. The results were: (a) When the rates of proteolysis were raised either by addition of plasmin or by stimulating PA synthesis in the presence of plasminogen, both the steady-state concentration and the half-life of surface AChR decreased, but the rate of receptor synthesis was unaffected. (b) The magnitude of these effects, and their dependence on added plasminogen, indicated that proteolysis initiated by plasminogen activation could account almost entirely for the reduction in receptor half-life produced by sarcoma virus transformation and phorbol ester. (c) The rate of receptor synthesis, which is also reduced by viral transformation and tumor promoters, was not modified by proteolysis; hence plasmin action may be responsible for a large part, but not all of the change in surface receptor under these conditions. (d) The plasmin catalysed changes in receptor parameters appear to occur in response to modified membrane metabolism resulting from proteolysis of surface components other than AChR itself.     

Coupling of agonist-induced AMPA receptor internalization with receptor recycling

Excitatory post-synaptic currents in the CNS are primarily mediated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors in response to glutamate. Internalization of cell-surface receptors has been shown to be one mechanism by which to control receptor function. To test for agonist control of AMPA receptor plasma membrane expression we used biochemical assays to study AMPA receptor internalization and insertion processes. In heterologous cells, we observed a slow constitutive internalization and a rapid agonist-induced internalization of AMPA receptors. To our surprise, however, agonist treatment had no effect on the steady-state levels of AMPA receptors on the cell surface. To examine whether this could be explained by an agonist-induced increase in the insertion rate of AMPA receptors into the plasma membrane we developed an assay to independently measure receptor insertion. Remarkably, agonist treatment of cells also dramatically increased AMPA receptor plasma membrane insertion rates. In addition, using an assay to measure recycling of internalized pools we found that internalized receptors are rapidly recycled to the cell surface. These results suggest that agonist-induced receptor internalization is coupled to increases in receptor recycling. This increase in receptor flux through intracellular pools may allow for rapid changes in receptor surface expression by independent regulatory control of internalization and insertion.     

Phosphorylation of brain muscarinic receptor: evidence of receptor regulation

Muscarinic receptor, from porcine synaptic membrane, was purified by affinity chromatography. Molecular weight analysis by SDS-gel electrophoresis revealed one major peptide with an apparent Mr of 68 +/- 2 Kda. The purified receptor was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase resulting in a concomitant loss in specific binding, and this loss was reversed by calcineurin.     

Control of epidermal growth factor receptor endocytosis by receptor dimerization, rather than receptor kinase activation

Given that ligand binding is essential for the rapid internalization of epidermal growth factor receptor (EGFR), the events induced by ligand binding probably contribute to the regulation of EGFR internalization. These events include receptor dimerization, activation of intrinsic tyrosine kinase activity and autophosphorylation. Whereas the initial results are controversial regarding the role of EGFR kinase activity in EGFR internalization, more recent data suggest that EGFR kinase activation is essential for EGFR internalization. However, we have shown here that inhibition of EGFR kinase activation by mutation or by chemical inhibitors did not block EGF-induced EGFR internalization. Instead, proper EGFR dimerization is necessary and sufficient to stimulate EGFR internalization. We conclude that EGFR internalization is controlled by EGFR dimerization, rather than EGFR kinase activation. Our results also define a new role for EGFR dimerization: by itself it can drive EGFR internalization, independent of its role in the activation of EGFR kinase.     

Deorphanization of Dresden G protein-coupled receptor for an odorant receptor

Dresden G protein-coupled receptor (D-GPCR) is one of orphan G protein-coupled receptors (GPCR). Here we report the identification of the ligands and the characterization of D-GPCR. We investigated over 5000 compounds to evoke the response mediated by D-GPCR and identified 3-methyl-valeric acid and 4-methyl-valeric acid as agonists using a cAMP assay. It is of interest that they dramatically enhanced the intracellular cAMP accumulation and the CRE-luciferase activity in CHO-K1 cells and HEK293 cells expressing the chimeric protein of D-GPCR with a rhodopsin-tag at its N-terminus. Our results established new characteristics of D-GPCR as an olfactory receptor. First, agonists of D-GPCR belong to odorants. Second, D-GPCR mRNA is expressed in the olfactory bulb. In addition, D-GPCR was reported to have similar sequences and its genome locus nearby other olfactory receptors. These results suggest D-GPCR is an olfactory receptor.     

G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor

We have shown previously that phosphoinositide 3-kinase in the retina is activated in vivo through light-induced tyrosine phosphorylation of the insulin receptor (IR). The light effect is localized to photoreceptor neurons and is independent of insulin secretion (Rajala, R. V., McClellan, M. E., Ash, J. D., and Anderson, R. E. (2002) J. Biol. Chem. 277, 43319-43326). These results suggest that there exists a cross-talk between phototransduction and other signal transduction pathways. In this study, we examined the stage of phototransduction that is coupled to the activation of the IR. We studied IR phosphorylation in mice lacking the rod-specific alpha-subunit of transducin to determine if phototransduction events are required for IR activation. To confirm that light-induced tyrosine phosphorylation of the IR is signaled through bleachable rhodopsin, we examined IR activation in retinas from RPE65(-/-) mice that are deficient in opsin chromophore. We observed that IR phosphorylation requires the photobleaching of rhodopsin but not transducin signaling. To determine whether the light-dependent activation of IR is mediated through the rod or cone transduction pathway, we studied the IR activation in mice lacking opsin, a mouse model of pure cone function. No light-dependent activation of the IR was found in the retinas of these mice. We provide evidence for the existence of a light-mediated IR pathway in the retina that is different from the known insulin-mediated pathway in nonneuronal tissues. These results suggest that IR phosphorylation in rod photoreceptors is signaled through the G-protein-coupled receptor rhodopsin. This is the first study demonstrating that rhodopsin can initiate signaling pathway(s) in addition to its classical phototransduction.