Tautomycetin inhibits growth of colorectal cancer cells through p21cip/WAF1 induction via the extracellular signal-regulated kinase pathway

Tautomycetin is an antifungal antibiotic retaining potent immunosuppressive function. We have identified the roles of tautomycetin on cellular proliferation and transformation of colorectal cancer cells. The proliferation and anchorage-independent growth of HCT-15, HT-29, and DLD-1 colorectal cancer cells were efficiently inhibited without induction of apoptosis by 150 nmol tautomycetin. These growth inhibitory effects were dependent on p21Cip/WAF induction via the extracellular signal-regulated kinase pathway, and the tautomycetin effects were abolished in HCT-116 colon cells and eight other types of cells that did not induce p21Cip/WAF by 150 nmol tautomycetin. The crucial role of p21Cip/WAF1 in the extracellular signal-regulated kinase pathway-dependent antiproliferative responses by tautomycetin was confirmed by using p21Cip/WAF1 gene-deleted HCT-116 cells. The growth inhibitory effect of tautomycetin was acquired by regulation of Raf-1 activity through inhibition of protein phosphatase type 1 and protein phosphatase type 2A with high preference toward protein phosphatase type 1. Tautomycetin could be a potential drug for colorectal cancer.     

骨形态发生蛋白2诱导C3H10T1/2细胞成骨分化中长链非编码RNA的作用

背景：人类长链非编码RNA是现今的研究热点,已有研究报道其在肿瘤发生中的作用机制,但其在成骨分化方面的机制并不明确。目的：观察人类长链非编码RNA在经骨形态发生蛋白2诱导小鼠C3H10T1/2细胞成骨分化中的作用,并探讨其分子机制。方法：首先进行碱性磷酸酶染色和成骨指标基因检测。对C3H10T1/2细胞在骨形态发生蛋白2诱导下的成骨分化过程长链非编码RNA表达变化进行芯片分析。采用高通量测序比较骨形态发生蛋白2诱导组和未经骨形态发生蛋白2诱导组的表达变化,筛选出表达下降的基因。过表达相应长链非编码RNA后观察对骨形态发生蛋白2诱导成骨分化的影响。结果与结论：骨形态发生蛋白2诱导C3H10T1/2细胞导致碱性磷酸酶活性增加。骨形态发生蛋白2诱导72 h后,碱性磷酸酶、Id1、骨钙素、Runx2、sp7表达上升（P〈0.05）。未诱导C3H10T1/2细胞与骨形态发生蛋白2诱导细胞芯片杂交后结果比较,下降达1.5倍的长链非编码RNAs有24条,其中只有AK035085有内含子。与未过表达AK035085的对照组相比,骨形态发生蛋白2诱导72 h后AK035085过表达的C3H10T1/2细胞碱性磷酸酶、Id1、骨钙素、Runx2、sp7表达均下降（P〈0.05）。提示骨形态发生蛋白2可刺激C3H10T1/2细胞发生成骨分化,AK035085可能对C3H10T1/2细胞的成骨分化存在抑制作用。     

Brg1调控重组人骨形态发生蛋白2诱导成骨细胞分化

背景:Brg1是依赖ATP的染色质改变复合物的核心催化亚基,该亚基在基因的转录调控、复制、重组,骨骼肌的分化、抑制肿瘤的发生等活动中起着重要的作用.目的:探索Brg1基因在骨形态发生蛋白2诱导成骨细胞分化过程中的调控机制.方法:采用胶原酶消化法进行小鼠颅骨成骨细胞的原代培养;分别用0,50,200 μg/L的重组人骨形态发生蛋白2诱导原代培养的成骨细胞的分化,摸索骨形态发生蛋白2的最佳作用剂量;实时荧光定量PCR和Western blot进行骨形态发生蛋白2对Brg1的作用时间的动力学分析;实时荧光定量PCR和钙钴染色法检测敲除Brg1对骨形态发生蛋白2诱导的成骨分化的影响;构建Dlx5腺病毒重组表达载体,实时荧光定量PCR和钙钴染色法检测Brg1在骨形态发生蛋白2诱导的成骨分化过程中对Dlx5的调控作用.结果与结论:用自行合成的重组人骨形态发生蛋白2可诱导原代培养小鼠成骨细胞分化,200 μg/L剂量有着较好的诱导分化效果;重组人骨形态发生蛋白2可诱导Brg1基因转录水平和翻译水平表达水平上调;敲除Brg1可抑制重组人骨形态发生蛋白2诱导的成骨分化;Brg1能够调控Dlx5的表达水平.说明Brg1通过调控Dlx5的表达水平调控重组人骨形态发生蛋白2诱导的小鼠成骨细胞的分化.     

Asiatic acid, a triterpene, induces apoptosis and cell cycle arrest through activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways in human breast cancer cells

This study first investigates the anticancer effect of asiatic acid in two human breast cancer cell lines, MCF-7 and MDA-MB-231. Asiatic acid exhibited effective cell growth inhibition by inducing cancer cells to undergo S-G2/M phase arrest and apoptosis. Blockade of cell cycle was associated with increased p21/WAF1 levels and reduced amounts of cyclinB1, cyclinA, Cdc2, and Cdc25C in a p53-independent manner. Asiatic acid also reduced Cdc2 function by increasing the association of p21/WAF1/Cdc2 complex and the level of inactivated phospho-Cdc2 and phospho-Cdc25C. Asiatic acid treatment triggered the mitochondrial apoptotic pathway indicated by changing Bax/Bcl-2 ratios, cytochrome c release, and caspase-9 activation, but it did not act on Fas/Fas ligand pathways and the activation of caspase-8. We also found that mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK1/2), and p38, but not c-Jun NH2-terminal kinase (JNK), are critical mediators in asiatic acid-induced cell growth inhibition. U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole], specific inhibitors of mitogen-activated protein kinase kinase and p38 kinase activities, significantly decreased or delayed apoptosis. Asiatic acid was likely to confine the breast cancer cells in the S-G2/M phase mainly through the p38 pathway, because both SB203580 and p38 small interfering RNA (siRNA) inhibition significantly attenuated the accumulation of inactive phospho-Cdc2 and phospho-Cdc25C proteins and the cell numbers of S-G2/M phase. Moreover, U0126 and ERK siRNA inhibition completely suppressed asiatic acid-induced Bcl-2 phosphorylation and Bax up-regulation, and caspase-9 activation. Together, these results imply a critical role for ERK1/2 and p38 but not JNK, p53, and Fas/Fas ligand in asiatic acid-induced S-G2/M arrest and apoptosis of human breast cancer cells.     

骨髓基质干细胞成骨分化过程中Wnt、骨形态发生蛋白2信号通路相关因子与辛伐他汀的作用

背景：辛伐他汀可促进体外培养的人或鼠骨髓基质干细胞向成骨细胞分化,但作用机制尚不清楚。目的：观察辛伐他汀对大鼠骨髓基质干细胞向成骨细胞分化过程中Wnt与骨形态发生蛋白2信号途径中相关因子表达的影响。方法：取6周龄雌性SD大鼠双侧股骨、胫骨全骨髓进行体外成骨细胞诱导培养。实验分为对照组及SIM组。SIM组加入浓度为10-7mol/L辛伐他汀,对照组加入等量无水乙醇和PBS。培养14d,行碱性磷酸酶染色,28d时,行vonKossa染色观察细胞外基质矿化情况;培养14,21d,免疫荧光细胞化学染色观察成骨细胞中β-catenin,Smad1/5,Cbfa1的表达及分布。结果与结论：大鼠骨髓基质干细胞经体外诱导后可分化为具有碱性磷酸酶活性和矿化细胞外基质能力的成熟成骨细胞。辛伐他汀可显著上调骨髓基质干细胞成骨分化过程中碱性磷酸酶的表达。同时,与对照组比较,SIM组β-catenin,Smad1/5,Cbfa1表达明显增多（P〈0.05）,且呈现明显的核内聚集趋势。说明辛伐他汀促进骨髓基质干细胞向成骨细胞分化的作用可能与调控Wnt与骨形态发生蛋白2信号通路中相关因子的表达及细胞内分布有关。     

骨髓基质干细胞成骨分化过程中Wnt、骨形态发生蛋白2信号通路相关因子与辛伐他汀的作用

背景:辛伐他汀可促进体外培养的人或鼠骨髓基质干细胞向成骨细胞分化,但作用机制尚不清楚。目的:观察辛伐他汀对大鼠骨髓基质干细胞向成骨细胞分化过程中Wnt与骨形态发生蛋白2信号途径中相关因子表达的影响。方法:取6周龄雌性SD大鼠双侧股骨、胫骨全骨髓进行体外成骨细胞诱导培养。实验分为对照组及SIM组。SIM组加入浓度为10-7mol/L辛伐他汀,对照组加入等量无水乙醇和PBS。培养14d,行碱性磷酸酶染色,28d时,行vonKossa染色观察细胞外基质矿化情况;培养14,21d,免疫荧光细胞化学染色观察成骨细胞中β-catenin,Smad1/5,Cbfa1的表达及分布。结果与结论:大鼠骨髓基质干细胞经体外诱导后可分化为具有碱性磷酸酶活性和矿化细胞外基质能力的成熟成骨细胞。辛伐他汀可显著上调骨髓基质干细胞成骨分化过程中碱性磷酸酶的表达。同时,与对照组比较,SIM组β-catenin,Smad1/5,Cbfa1表达明显增多(P<0.05),且呈现明显的核内聚集趋势。说明辛伐他汀促进骨髓基质干细胞向成骨细胞分化的作用可能与调控Wnt与骨形态发生蛋白2信号通路中相关因子的表达及细胞内分布有关。     

Mechanisms linking adenosine A1 receptors and extracellular signal-regulated kinase 1/2 activation in human trabecular meshwork cells

This study was designed to evaluate the signaling pathways coupling adenosine A1 receptors and extracellular signal-regulated kinase (ERK) 1 and 2 in human trabecular meshwork (HTM) cells. Studies were conducted using cultures of primary HTM cells and the HTM-3 cell line. Activation of ERK1/2, location of protein kinase C (PKC) isoforms, and matrix metalloproteinase (MMP) secretion were determined by Western blotting. In primary HTM cells and the HTM-3 cell line, administration of the A1 agonist N6-cyclohexyladenosine (CHA) produced a concentration-dependent increase in ERK1/2 activation. This CHA-induced ERK activation was blocked by pretreatment with the A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine or pertussis toxin. Transfection with dominant negative N17 Ras produced only a small (31%) decline in CHA-induced ERK activation, and the response was not altered by pretreatment with the Src tyrosine kinase inhibitor, PP2 [3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-D] pyrimidin-4-amine], the phosphoinositide kinase-3 inhibitor, LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], or the A3 receptor antagonist, MRS-1191 [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate]. Administration of CHA also induced the translocation of PKCalpha from the cytosol to the membrane, and pretreatment with the phospholipase C (PLC) inhibitor, U73122 [1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]-hexyl]-1H-pyrrole-2,5-dione], blocked ERK1/2 activation induced by CHA. Transfection of short interfering RNA targeting PKCalpha blocked the CHA-induced ERK1/2 activation and the secretion of MMP-2. These results confirm the existence of functional adenosine A1 receptors in the trabecular meshwork cells. These receptors are coupled to the activation of ERK1/2 through G(i/o) proteins and dependent upon the upstream activation of PLC and PKCalpha. These studies provide evidence that adenosine A1 receptor agonists increase outflow facility through sequential activation of G(i/o) > PLC > PKCalpha > c-Raf > mitogen-activated protein kinase kinase > ERK1/2, leading to secretion of MMP-2.     

骨缺损修复的研究-重组人骨形成蛋白-2对骨髓基质细胞增殖分化作用的研究

目的探讨基因重组人骨形成蛋白-2(rhBMP-2)的诱骨活性及对骨髓基质细胞增殖分化的影响,了解其在体外作用的最佳浓度及其可能机制。方法采用大鼠BMP异位成骨试验模型,进行rhBMP-2的诱骨活性评价;采用MTT法研究不同浓度的rhBMP-2对培养兔骨髓基质细胞增殖的影响;采用碱性磷酸酶(ALP)活性检测试剂盒和考马斯亮蓝染色分别研究不同浓度的rhBMP-2对骨髓基质细胞ALP活性及细胞总蛋白含量的影响。结果rhBMP-2植入肌袋内,术后2周即可见大量骨母细胞形成,术后4周可见成熟新生骨组织。MTT结果显示在100ng/ml时,OD值达最高(0.170±0.033)。ALP活性和细胞蛋白含量结果显示各实验组与对照组的OD值差异均有显著性(P<0.05或<0.01),且呈剂量-效应依赖关系。结论rhBMP-2具有良好的诱骨活性,具有明显的促进骨髓基质细胞增殖和分化的作用,其作用呈剂量-效应依赖关系,有望应用于骨组织工程及临床骨缺损修复的研究中。     

Acetoacetate activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in primary cultured rat hepatocytes: role of oxidative stress

Diabetes is characterized by elevated levels of ketone bodies acetoacetate (AA) and 3-hydroxybutyrate (3HB). High levels of ketone bodies have been implicated in generation of cellular oxidative stress. Ketone body activation of cellular signaling pathways associated with oxidative stress, however, has not been established. Thus, ketone body effects on kinase activation in primary cultured rat hepatocytes have been examined. Treatment with AA increased the phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinase (MAPK), maximally by approximately 2.5- and 4-fold, respectively. AA failed to activate c-Jun NH(2)-terminal kinase. AA-mediated Erk1/2 and p38 MAPK activation was detectable at 3 h post-treatment with maximal activation occurring at 12 h. In contrast, 3HB failed to activate any of these kinases. Elevated phosphorylation of Raf and MKK3/6 also occurred in response to AA. Bisindolylmaleimide, a generalized protein kinase C (PKC) inhibitor, and B581, a Ras farnesylation inhibitor, inhibited AA-mediated activation of Erk1/2 and p38 MAPK, suggesting a role for PKC and Ras in mediating such activation. Interestingly, the tyrosine kinase inhibitor genistein prevented the AA-mediated phosphorylation of Erk1/2, but not p38 MAPK. AA treatment resulted in the generation of reactive oxygen species (ROS) and the depletion of cellular glutathione levels, which was ameliorated by the antioxidants N-Acetyl-l-cysteine (NAC) and Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid). NAC and Trolox also ameliorated AA-mediated Erk1/2 and p38 MAPK activation, suggesting that this activation is associated with ROS and oxidative stress.     

Heme oxygenase-1 inhibits the proliferation of pancreatic stellate cells by repression of the extracellular signal-regulated kinase1/2 pathway

Activation of pancreatic stellate cells (PSCs) is the key process in the development of pancreatic fibrosis, a common feature of chronic pancreatitis and pancreatic cancer. In recent studies, curcumin has been shown to inhibit PSC proliferation via an extracellular signal-regulated kinase (ERK)1/2-dependent mechanism. In addition, curcumin is a potent inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1) in other cell types. Therefore, the aims of this study were to 1) characterize the effect of curcumin on HO-1 gene expression in PSCs, 2) explore whether HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation, and 3) clarify the involvement of the mitogen-activated protein kinase (MAPK) family in this context. Cultured rat PSCs were incubated with curcumin and assessed for HO-1 up-regulation by Northern blot analysis, immunoblotting, and activity assays. The effect of HO-1 on platelet-derived growth factor (PDGF)-induced PSC proliferation and MAPK activation was determined by immunoblotting, cell proliferation assays, and cell count analyses. Curcumin induced HO-1 gene expression in PSCs in a time- and dose-dependent manner and inhibited PDGF-mediated ERK1/2 phosphorylation and PSC proliferation. These effects were blocked by treatment of PSCs with tin protoporphyrin IX, an HO inhibitor, or transfection of HO-1 small interfering RNA. Our data provide evidence that HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation. Therefore, therapeutic up-regulation of HO-1 could represent a mode for inhibition of PSC proliferation and thus may provide a novel strategy in the prevention of pancreatic fibrosis.     

Activation of nonsteroidal anti-inflammatory drug-activated gene-1 via extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase revealed a isochaihulactone-triggered apoptotic pathway in human lung cancer A549 cells

The novel lignan isochaihulactone inhibits cell proliferation and is an effective inducer of apoptosis in a variety of carcinoma cell lines. To determine the mechanisms underlying these effects, we examined isochaihulactone-induced changes in gene expression using oligodeoxynucleotide-based microarray screening of a human lung carcinoma cell line, A549. Isochaihulactone-inducible genes included the early growth response gene-1 (EGR-1) and nonsteroidal anti-inflammatory drug-activated gene (NAG-1). Isochaihulactone increased EGR-1 and then NAG-1 mRNA and protein expression. Pure isochaihulactone induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Isochaihulactone-induced increases in EGR-1 and NAG-1 expression were reduced by the mitogen-activated protein kinase kinase 1/2 inhibitor 2'-amino-3'-methoxyflavone (PD98059), and this effect was not blocked by the phosphatidylinositol 3-kinase/protein kinase B pathway inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002). Inhibition of isochaihulactone-induced NAG-1 expression by EGR-1 small interfering RNA blocked isochaihulactone-induced apoptosis in A549 cells, suggesting that induction of EGR-1 expression decreases survival of A549 cells. RNA interference using double-stranded RNA specific for NAG-1 also inhibited isochaihulactone-induced apoptosis, and cells transfected to increased NAG-1 expression had a greater apoptotic response to isochaihulactone and reduced colony formation efficiency. In addition, treatment of nude mice with isochaihulactone increased the in vivo NAG-1 expression as examined by immunohistochemistry from tumor biopsy. Isochaihulactone treatment increased the luciferase activity of NAG-1 in A549 cells transfected with the NAG-1 promoter construct. This induction increased expression of NAG-1 that was p53-independent and Sp1-dependent. Our findings suggest that NAG-1 expression is up-regulated by isochaihulactone through an ERK-dependent pathway involving the activation of EGR-1.     

Activation of the extracellular signal-regulated kinase 1/2 pathway by AAV gene transfer protects retinal ganglion cells in glaucoma.

Glaucoma is the second leading cause of blindness in the world. Loss of vision in glaucomatous optic neuropathy is caused by the selective degeneration of retinal ganglion cells (RGCs). Ocular hypertension is a major risk factor in glaucoma, but visual field defects continue to progress in some patients despite the use of drugs that lower intraocular pressure. At present, there are no effective neuroprotective strategies for the treatment of this disease. The extracellular signal-regulated kinase (Erk) 1/2 pathway is an evolutionarily conserved mechanism used by several peptide factors to promote cell survival. Here we tested if selective activation of Erk1/2 protected RGCs in a rat model of experimental glaucoma. We used recombinant adeno-associated virus to transduce RGCs with genes encoding constitutively active or wild-type MEK1 (approved gene symbol MAP2K1), the upstream activator of Erk1/2. MEK1 gene transfer into RGCs markedly increased neuronal survival: 1366 +/- 70 RGCs/mm(2) (mean +/- SEM) were alive in the dorsal retina at 5 weeks after ocular hypertension surgery, a time when only 680 +/- 86 RGCs/mm(2) of these neurons remained in control eyes. We conclude that the Erk1/2 pathway plays a key role in the protection of RGCs from ocular hypertensive damage. This study identifies a novel gene therapy strategy in which selective activation of the Erk1/2 signaling pathway effectively slows cell death in glaucoma.     

纳米羟基磷灰石复合骨形态发生蛋白2与骨髓基质干细胞的体外培养

背景：纳米级羟基磷灰石支架材料复合骨形态发生蛋白2因子组织构建模式组合后对骨髓基质干细胞的作用是叠加促进或抑制作用以及其生物相容性如何？目的：验证纳米羟基磷灰石复合骨形态发生蛋白2与骨髓基质干细胞在体外共同培养下的生物相容性和成骨性。方法：将兔骨髓基质干细胞体外培养、传代和扩增,经鉴定后将第3代细胞分别接种在对照组、骨形态发生蛋白2组、纳米羟基磷灰石组、纳米羟基磷灰石/骨形态发生蛋白2组4种培养液中。结果与结论：对照组和纳米羟基磷灰石组的细胞倍增时间较骨形态发生蛋白2组、纳米羟基磷灰石/骨形态发生蛋白2组两组长;骨形态发生蛋白2组和纳米羟基磷灰石/骨形态发生蛋白2组细胞增殖能力及碱性磷酸酶测量值明显大于对照组和纳米羟基磷灰石组（P〈0.01）。提示纳米羟基磷灰石复合骨形态发生蛋白2的组织构架不仅与骨髓基质干细胞之间具有良好的生物相容性,还可发挥骨形态发生蛋白2促进骨髓基质干细胞增殖和定向分化成骨的作用。     

依达拉奉对大鼠重型弥漫性脑创伤后细胞外信号调节激酶1/2信号通路的影响

目的 探讨依达拉奉对重型弥漫性脑创伤(TBI)的保护作用及其机制.方法 273只雄性SD大鼠按随机数字表法分为对照组(45只)、模型组(88只)及依达拉奉低剂量组(72只)、高剂量组(68只).采用重物撞击致大鼠TBI模型.伤后1、6、24、48和72 h,在光镜和电镜下观察脑组织病理变化;用蛋白质免疫印迹法(Western blotting)检测磷酸化细胞外信号调节激酶1/2(ERK1/2)表达;用免疫组化法和原位末端缺刻标记法(TUNEL)检测神经细胞凋亡情况.伤后7～10 d应用水迷宫对大鼠学习记忆能力进行评定.结果 与对照组比较,伤后6、24、48、72 h海马区部分神经细胞出现变性、坏死,1、6、24、48 h磷酸化ERK1/2表达水平(pg/U)显著增高(分别为:2.05±0.40、4.40±0.96、6.70±0.87、3.67±0.28比0.40±0.04、0.41±0.05、0.43±0.06、0.40±0.03),6、24、48、72 h神经细胞凋亡数(个)明显增多(分别为:9.60±2.69、12.68±2.99、16.94±3.92、25.82±4.61比2.42±0.38、2.58±0.57、2.74±0.56、2.61±0.58),7～10 d大鼠搜索安全岛潜伏期(s)延长(分别为:119.8±25.0、105.6±24.5、98.5±21.8、92.0±19.5比49.5±7.5、32.7±6.3、25.8±6.5、24.8±5.5,均P<0.05).应用依达拉奉干预后,脑组织损伤程度、磷酸化ERK1/2表达水平降低,神经细胞凋亡数回降,大鼠搜索安全岛潜伏期缩短(依达拉奉低剂量组磷酸化ERK1/2表达6、24、48 h分别为:2.46±0.22、4.00±0.84、2.38±0.32,高剂量组分别为:1.67±0.15、1.86±0.38、1.27±0.28;依达拉奉低剂量组凋亡细胞数6、24、48、72 h分别为:5.20±1.23、7.10±1.72、9.54±1.36、14.12±3.19,高剂量组分别为:3.40±0.49、4.39±0.73、5.02±1.12、8.78±2.16;依达拉奉低剂量组潜伏期7～10 d分别为:94.8±22.8、65.2±19.0、62.0±16.7、59.5±15.6,高剂量组分别为:81.5±20.7、55.4±18.5、40.0±12.3、32.2±11.0,均P<0.05);其中依达拉奉高剂量更为显著(均P<0.05).结论 依达拉奉对TBI有保护作用,其机制与对伤后ERK1/2、神经细胞凋亡通路的调控有关.     

Inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase enhances chemotherapeutic effects on H460 human non-small cell lung cancer cells through activation of apoptosis

The effects of Dox (Dox), paclitaxel (Taxol), and serum starvation on the regulation of XIAP (X-linked inhibitor of apoptosis), Bcl-2 phosphorylation, and apoptosis were evaluated in human H460 non-small cell lung cancer cells. Protein kinases that responded to these treatments as prosurvival elements in signal transduction were identified by simultaneously screening phosphorylation of protein kinases in H460 cells cultured in serum-free medium or treated with Dox. We demonstrated that Dox and Taxol induced apoptosis through down-regulation of XIAP and phosphorylation of Bcl-2 in a concentration-dependent manner without changing expression of Bcl-xL in H460 cells. These effects were paralleled by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase protein. We identified that serum starvation and Dox reduced phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK), protein kinase C (PKC) alpha/beta and c-Jun NH(2)-terminal kinase. The MEK-specific inhibitor U0126 or PKC inhibitor staurosporine (STP) also down-regulated XIAP expression and induced apoptosis. Thus, our data suggest that apoptosis and down-regulation of XIAP induced by Dox exposure or serum starvation may be mediated through inactivation of the MEK/ERK and PKCalpha/beta pathways. In support of this we demonstrated that the cytotoxic effects of Dox when combined with U0126 or STP were enhanced, i.e., synergistic cytotoxic activities were demonstrated. The synergistic interaction of U0126 or STP with Dox was sequence- and concentration-dependent.     

腺病毒介导人骨形态发生蛋白2基因在兔脂肪干细胞的表达与成骨分化效应

目的:采用腺病毒介导人骨形态发生蛋白2基因转染兔脂肪干细胞,观察目的基因在脂肪干细胞中的表达效率及向成骨细胞定向分化的能力。方法:实验于2005-12/2006-09在武汉大学医学院中心实验室和三峡大学医学院病理实验室进行。①实验材料:4月龄健康新西兰大耳白兔2只。质粒pAd-BMP-2由美国哈佛医学院分子骨科中心Oliver博士惠赠;携带β-半乳糖酐酶基因的腺病毒对照载体由李康博士惠赠;大肠杆菌DH5a以及293细胞由本实验室保存。②实验方法:新西兰兔肌注麻醉后,完整取出双侧腹股沟脂肪垫,清除外包膜、明显的结缔组织和小血管,剪碎,离心,体外分离培养脂肪干细胞。表达载体pAd-hBMP-2经293细胞包装重组腺病毒后,取第3代生长良好的脂肪干细胞,按5×105接种于60mm培养皿,将Ad-hBMP-2病毒以感染复数10～20感染细胞。将成功转染骨形态发生蛋白2的脂肪干细胞作为转染组,以转染携带β-半乳糖酐酶基因腺病毒载体的脂肪干细胞作为对照组。两组均置入不含骨形态发生蛋白2的无血清成骨诱导培养基中进行诱导分化。③实验评估:定期观察细胞的形态学变化,MTT法绘制生长曲线,计算增殖时间;成骨诱导培养后测定碱性磷酸酶活性,光倒置显微镜下观察钙化结节形成情况;RT-PCR、免疫组织化学法、Westernblot法检测目的基因人骨形态发生蛋白2和成骨细胞标志性蛋白Ⅰ型胶原、骨钙素的表达。结果:①脂肪干细胞转染前后形态学变化、生长曲线及倍增时间:Ad-hBMP-2基因修饰的脂肪干细胞经诱导培养后形态规则,多角型细胞增多,转染组的细胞生长曲线倍增时间明显短于对照组。②碱性磷酸酶活性及矿化结节形成:转染组脂肪干细胞碱性磷酸酶活性呈增加趋势,成骨诱导培养7,10,14d均显著高于未转染组(P<0.01)。转染组成骨细胞四环素标记显示圆形矿化结节呈金黄色,数量多,结节大。③脂肪干细胞人骨形态发生蛋白2与Ⅰ型胶原、骨钙素的表达:成骨诱导培养7,14d,RT-PCR、免疫组织化学法、Western blot法检测结果显示转染组目的基因人骨形态发生蛋白2、成骨标志性蛋白Ⅰ型胶原、骨钙素的分泌表达均明显强于对照组。结论:经Ad-hBMP-2基因修饰的兔脂肪干细胞在体外能定向分化为成骨细胞,且分化增殖能力强;目的基因骨形态发生蛋白2表达高、持续时间长。     

缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导骨形成

背景:重组人骨形态发生蛋白2在体内半衰期短、易降解代谢,达不到理想的骨再生效果。目的:制备缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料,并观察其缓释性能、骨诱导活性。方法:将重组人骨形态发生蛋白2与壳聚糖混合制备壳聚糖膜,涂覆于生物骨修复材料表面,ELISA方法检测其体外释药性能。茜素红染色检测缓释型人骨形态发生蛋白2/壳聚糖生物骨材料、重组人骨形态发生蛋白2生物骨材料、单纯骨填充材料诱导C2C12细胞骨钙蛋白的形成,观察其诱导成骨细胞能力。同时将3种骨修复材料植入清洁级KM小鼠股部肌袋内,2周后检测新生骨Ca2+离子含量,评价其异位骨诱导能力。结果与结论:材料表面的壳聚糖膜分布均匀,负载的重组人骨形态发生蛋白2呈团簇状。重组人骨形态发生蛋白2/壳聚糖生物骨修复材料体外释药存在突释,前4d释放量达总药量的50%,持续至12d,释药量达到90%,第18天时释放完全。与单纯骨填充材料、重组人骨形态发生蛋白2生物骨材料相比,缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导C2C12细胞向成骨晚期分化能力与异位骨形成能力显著增强(P<0.05)。结果提示缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料缓释性能好,促进骨形成能力强。     

缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导骨形成

背景：重组人骨形态发生蛋白2在体内半衰期短、易降解代谢,达不到理想的骨再生效果。目的：制备缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料,并观察其缓释性能、骨诱导活性。方法：将重组人骨形态发生蛋白2与壳聚糖混合制备壳聚糖膜,涂覆于生物骨修复材料表面,ELISA方法检测其体外释药性能。茜素红染色检测缓释型人骨形态发生蛋白2/壳聚糖生物骨材料、重组人骨形态发生蛋白2生物骨材料、单纯骨填充材料诱导C2C12细胞骨钙蛋白的形成,观察其诱导成骨细胞能力。同时将3种骨修复材料植入清洁级KM小鼠股部肌袋内,2周后检测新生骨Ca2＋离子含量,评价其异位骨诱导能力。结果与结论：材料表面的壳聚糖膜分布均匀,负载的重组人骨形态发生蛋白2呈团簇状。重组人骨形态发生蛋白2/壳聚糖生物骨修复材料体外释药存在突释,前4d释放量达总药量的50%,持续至12d,释药量达到90%,第18天时释放完全。与单纯骨填充材料、重组人骨形态发生蛋白2生物骨材料相比,缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导C2C12细胞向成骨晚期分化能力与异位骨形成能力显著增强（P〈0.05）。结果提示缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料缓释性能好,促进骨形成能力强。     

肠三叶因子激活ERK1/2信号通路促进胃黏膜上皮细胞增殖和迁移

目的：研究ITF对胃黏膜上皮细胞增殖和迁移的影响,及ERK1/2信号通路在其中的作用。方法：以GES-1细胞为研究对象,用Western blot检测ITF对ERK1/2信号通路的作用,以不同浓度ITF及ERK1/2信号通路抑制剂U0126处理GES-1细胞,用CCK-8检测细胞增殖,用细胞穿孔实验观察细胞迁移。比较不同浓度ITF对GES-1细胞增殖和迁移的影响,及ERK1/2信号通路在其中的作用。结果：ITF提高了pERK1/2蛋白的表达水平,U0126抑制了ITF激活的pERK1/2蛋白的表达。与对照组比较,在细胞培养的24h后,ITF以剂量依赖的方式促进了GES-1细胞的增殖和迁移。U0126抑制了ITF对GES-1细胞的促增殖和迁移作用。结论：肠三叶因子通过激活ERK1/2信号通路促进胃黏膜上皮细胞增殖和迁移。     

重组人骨形态发生蛋白2缓释体影响细胞外基质金属蛋白酶诱导因子的表达

背景:目前,重组人骨形态发生蛋白2是否可以抑制炎性细胞因子的产生及活性,从而抑制溶骨反应的分子生物学机制仍不清楚.目的:观察重组人骨形态发生蛋白2缓释体在大鼠皮下气囊中的分子生物学反应,探讨人骨形态发生蛋白2抑制骨溶解生物活性的可能机制.方法:于鼠背部皮下注射过滤后的空气3 mL,每2 d 1次,共6次.1周后,20只SD大鼠按注射成分的不同随机分为2组,每组10只.分别于囊腔内注射人骨形态发生蛋白2缓释体悬液(实验组)和生理盐水(对照组).分别于1,2周后取囊腔组织进行光镜下观察,测定血清碱性磷酸酶水平:real-time PCR法检测细胞外基质金属蛋白酶诱导因子(EMMPRIN)的mRNA水平;Westem blotting法测定EMMPRIN的蛋白表达.结果与结论:光镜下实验组与对照组囊腔组织均未见明显组织细胞反应.实验组第1周血清碱性磷酸酶水平与实验组第2周及对照组相比,差异具有显著性意义(P<0.05),实验组第2周较对照组略低,但差异无显著性意义(P>0.05).实验组EMMPRIN的mRNA及蛋白水平明显低于对照组,而且第2周小于第1周.提示重组人骨形态发生蛋白2缓释体抑制了溶骨性细胞因子的产生及活性,从而抑制溶骨反应.