抑制骨桥蛋白表达降低肺癌侵袭和增殖的机制

目的 探讨抑制骨桥蛋白(OPN)表达降低肺癌细胞株A549侵袭、增殖的机制.方法 构建针对人OPN mRNA干扰质粒pENTRTM/U6-INF(pINF-1)及对照质粒pENTRTM/U6-CTR(pCTR),将其转染A549细胞,Western blot测定OPN、基质金属蛋白酶(MMP)和促分裂素原活化蛋白激酶(MAPK)信号通路相关蛋白的表达;明胶酶谱检测MMP的表达.结果 与空白对照组(1.20±0.15)比较,转染72 h后pINF-1组OPN蛋白表达(0.15±0.04)下降87%,与对照组比差异有统计学意义(P＜0.05).Western blot的结果显示,pINF-1组细胞磷酸化细胞外信号调节激酶1/2(pERK1/2)、磷酸化丝裂原细胞外激酶(pMEK)和MMP-2的表达明显下降,差异有统计学意义(P＜0.01);而MMP-9表达差异无统计学意义(P＞0.05).明胶酶谱结果同样表明pINF-1组MMP-2的表达明显下降(P＜0.01).结论 抑制OPN的表达降低肺癌细胞株A549侵袭力和增殖的机制可能与抑制MAPK信号通路和MMP-2的表达有关.

Abstract：

Objective To explore the mechanism of invasion and proliferation of lung cancer cell line A549 mediated by osteopontin. Methods One double-stranded DNA vectors pENTRTM/U6-INF ( pINF-1 ) targeting the mRNA of human osteopontin ( OPN), and the control vector pENTRTM/U6-CTR (pCTR) mismatching with mRNA of OPN were constructed, and then they were transfected into human A549 cells with high metastatic potentials. Western blotting was used to quantify the protein level of OPN,matrix metalloproteinases (MMPs) and mitogen-activated protein kinases (MAPK). The activity of MMPs was detected by gelatin zymography. Parallel experiments were performed in sextuplicate. Results As compared with control group only transfected with LipofectamineTM 2000, OPN protein level in pINF-1 group was decreased by 87% 72 h after transfection (P ＜0. 05), and no significant difference was found in the group transfected with pCTR. Moreover, the expression levels of phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2), phosphorylated MAPK/ERK1/2 kinase (pMEK) and MMP-2 protein were also decreased in pINF-1 group (P ＜ 0. 01 ). The activity of MMP-2 but not MMP-9 was decreased significantly in pINF-1 group (P ＜ 0. 01 ). Conclusion OPN plays an important role in metastasis as well as tumor growth of lung cancer cells probably through activation of the MAPK pathways and MMP-2.     

gax基因转染对血管平滑肌细胞中基质金属蛋白酶2和骨桥蛋白转录的影响

目的:探讨腺病毒介导gax基因转染血管平滑肌细胞(VSMC)后,VSMC中基质金属蛋白酶2(MMP2)和骨桥蛋白基因转录的变化。方法:以携带大鼠gax基因表达序列的复制缺陷型5型腺病毒载体(AdCMV-gax)转染VSMC后,应用免疫细胞化学染色检NGax蛋白表达的变化,以RT-PCR技术检测VSMC中MMP2 mRNA和骨桥蛋白mRNA的变化。结果:AdCMV-gax转染后:(1)VSMC中Gax蛋白的表达比转染前显著增高(P<0.01); (2)VSMC中MMP2 mRNA和骨桥蛋白mRNA 比未转染组均显著降低(P<0.05)。结论:增强gax基因表达可抑制MMP2和骨桥蛋白的转录。     

MIC-1、OPN及CA19-9联合检测在诊断胰腺癌患者中的应用价值

目的 探讨血清巨噬细胞抑制因子1(MIC-1)、骨桥蛋白(OPN)及糖类抗原19-9(CA19-9)联合检测在诊断胰腺癌患者中的应用价值.方法 纳入60例健康者为对照组与63例胰腺癌患者为观察组进行研究,均收集血清进行MIC-1、OPN及CA19-9水平检测.结果 对两组的血清MIC-1、OPN、CA19-9水平进行检测和比较,对照组的上述指标水平分别为(485.12±75.23) pg/ml、(35.12±15.12) ng/ml、(26.34±2.47) U/ml,均显著低于观察组的(1 559.15±225.16)pg/ml、(115.25±35.23)ng/ml、(195.12±18.25)U/ml,(t=15.21、8.15、9.12,P＜ 0.05).3种指标单独检测,以MIC-1检测的敏感性最高,显著高于其他2项指标(x2=5.12、6.03,P＜0.05),其他2项指标差异无统计学意义(P＞0.05).CA19-9检测的特异性最高,显著高于其他2项指标(x 2=5.88、5.75,P＜0.05),其他2项指标差异无统计学意义(P＞0.05).3种检测方法的准确性基本相当,差异均无统计学意义(均P＞ 0.05),MIC-1+OPN+CA19-9检测的敏感性和特异性以及准确性均显著高于3项指标单独检测的水平,差异均有统计学意义(均P＜0.05).结论 胰腺癌诊断中实施MIC-1、OPN及CA19-9联合检测可以获得更好的检测结果,显著提高检测的准确性等,具有一定的临床应用价值.     

骨桥蛋白在胆固醇结石患者胆汁中的表达及成核作用研究

目的 探讨骨桥蛋白在人胆固醇结石形成中的作用.方法 36例胆固醇结石患者为结石组,19例肝移植供体为正常组,分别测定骨桥蛋白、钙离子及脂质成分在胆囊胆汁中的含量,用成核时间法研究骨桥蛋白在胆汁中的成核作用.结果 骨桥蛋白呈剂量依赖性地抑制胆固醇自发成核.50 μg/ml的骨桥蛋白可分别使结石组和正常组胆汁的成核时间延缓48.90％和17.07％,100 μg/ml的骨桥蛋白可使成核时间延缓91.51％和32.93％;并可抑制钙离子诱导的促成核作用.50μg/ml骨桥蛋白+钙离子可分别使结石组和正常组胆汁成核时间延缓75.78％和33.96％,而100μg/ml骨桥蛋白+钙离子则使成核时间分别延缓125.90％和62.26％.结石组胆汁中胆固醇、磷脂、胆汁酸及胆固醇饱和指数均显著高于正常组胆汁(P＜0.05),而骨桥蛋白和钙离子含量则明显低于正常组胆汁中的含量(P＜0.05).结论 骨桥蛋白可抑制胆固醇成核,并参与胆结石的发生发展.     

Bone phosphoprotein of newborn rat calvaria detected histochemically with the cationic carbocyanine dye “Stains-all”

Abstruct The originally mineralized area of a fixed and demineralized specimen of newborn rat calvarium was stained blue with Stains-all, which stained phosphoprotiens, glycoproteins and Ca-binding proteins blue. The material stained blue in the histological sections was solubilized by an acidic solution and found to be the protein (Stains-all positive protein = SAPP). This SAPP was found to consist of two different types of phosphoproteins, which had the same amino acid sequence at the N-terminal region and a quite similar amino acid composition to each other. The minor one was equal to the 44kD phosphoprotein which has previously been reported by Prince et al (J. Biol. Chem., 262, 2900, 1987). The major one was a 66kD phosphoprotein, it's molecular weight being determined by SDS electrophoresis on a 15% polyacrylamide gel. The result of the amino acid analysis and peptide mapping analysis after CNBr cleavage contained no Met. Based on its solubility characteristics at sequential extractions with three acidic solutions (pH 5.5, pH 3.5 and pH 2.6), the histochemically discovered SAPP was believed to be bound to the matrix by a weak ionic bond and covered with mineral. It is suggested that the Met free phosphoprotein is a new protein in the mineralized phase of newborn rat calvaria and participates in the induction of bone mineralization.     

骨桥蛋白在低钙和燃煤污染型氟中毒大鼠肾组织中的表达

目的 观察骨桥蛋白(OPN)在低钙和氟中毒大鼠肾组织中的表达,探讨OPN与氟中毒肾损害的关系.方法 1月龄Wistar大鼠48只,雌雄各半,体质量80～100 g.按2×2析因设计将大鼠按质量随机分为4组:对照组、高氟组、低钙组、低钙高氟组,每组12只,雌雄各半.采用合成饲料喂养,高氟组和低钙高氟组饲料中的玉米来自燃煤污染型氟中毒病区,含氟量为100 mg/kg,对照组和低钙组饲料中的玉米来自于非病区,含氟量为5 mg/kg.喂养16周后处死大鼠,观察各组大鼠牙齿变化,计算大鼠氟斑牙检出率.取大鼠肾脏,采用RT-PCR技术及免疫组化技术检测大鼠肾脏OPN蛋白和OPN mRNA表达.结果 对照组和低钙组牙齿生长良好,高氟组、低钙高氟组大鼠均出现明显的氟斑牙,氟斑牙检出率为100%.免疫组化结果表明,OPN主要定位于肾组织的肾小管上皮细胞中.对照组和低钙组肾组织OPN阳性细胞淡染、散在分布,高氟组与低钙高氟组OPN阳性细胞着色较深,广泛分布在肾小管上皮细胞中.OPN蛋白表达显示,高氟组(168.64±13.21)和低钙高氟组(169.26±8.92)高于对照组(145.78±10.26,P均<0.01)和低钙组(149.60±16.84,P均<0.01);OPN mRNA表达显示,高氟组(1.89±0.37)和低钙高氟组(1.94±0.22)高于对照组(1.32±0.26,P均<0.05)和低钙组(1.30±0.18,P均<0.05).高氟影响OPN蛋白和OPN mRNA表达(F=13.821、4.24,P均<0.05),低钙不影响OPN蛋白和OPN mRNA表达(F=2.164、0.58,P均>0.05),但高氟和低钙联合作用时,二者对OPN蛋白和OPN mRNA表达有交互作用(F=6.257、4.32,P均<0.05).结论 过量氟可促使大鼠肾组织的OPN表达增加,提示OPN与氟中毒肾损害程度密切相关,可考虑作为一种氟中毒肾损害的标志,并且,高氟与低钙同时存在时,大鼠肾损害会进一步加重,提示低钙是氟化物损害肾脏的重要环节.     

Osteopontin upregulation in rotavirus-induced murine biliary atresia requires replicating virus but is not necessary for development of biliary atresia

Biliary atresia (BA) is a progressive fibro-inflammatory pediatric liver disease in which osteopontin (OPN), a glycoprotein with inflammatory and fibrogenic activity, may play a pathogenic role. The current studies were conducted in a mouse model of rotavirus-induced BA to test the hypotheses that live but not inactivated rotavirus causes antigenemia, upregulation of hepatic OPN expression, and induction of BA and fibrosis; and that OPN is necessary for development of BA. Prolonged or transient antigenemia developed in mice inoculated with live or inactivated virus, respectively, but only live virus upregulated hepatic OPN and caused BA and fibrosis. OPN was expressed in intra- and extrahepatic bile ducts in healthy mice and in mice with BA. OPN-deficient mice, similar to WT mice, developed BA. Together, these data show that live but not inactivated rotavirus causes upregulation of hepatic OPN expression and BA but that OPN is not necessary for development of BA.     

骨桥蛋白在肝细胞癌中的表达及其临床病理意义

C中OPN表达评分明显高于高分化HCC(P<0.05).存在转移的HCC中OPN表达评分明显高于无转移者(2.65±1.83比1.30±1.71,P<0.01).结论 HCC中OPN的表达与其临床病理有关,提示OPN可能是HCC转移复发的一个潜在指标.