人基质金属蛋白酶-2的表达、纯化和性质鉴定

PCR方法扩增人基质金属蛋白酶 2 (MMP 2 )不含信号肽的表达序列 ,酶切和测序鉴定正确后 ,构建酵母重组表达质粒pPIC9 MMP 2 ,电击法转化毕赤酵母 (Pichiapastoris)细胞得到阳性克隆 ,甲醇诱导获得含大量基质金属蛋白酶 2的培养上清 ,经SephacrylS 2 0 0纯化后 ,纯度达到电泳纯。明胶酶谱和SDS PAGE分析说明重组MMP 2能够降解明胶和IV型胶原 ,表明重组蛋白具有与天然MMP 2相似的底物特异性。糖基化分析和SDS PAGE表明 ,表达产物的分子量约为 5 0kD ,重组MMP 2的C 末段可能发生了降解。     

基质金属蛋白酶抑制剂-4在大肠杆菌中的克隆与表达

基质金属蛋白酶抑制剂4(tissueinhibitorofmetalloproteinase4,TIMP4)为心脏特异性表达,在心脏代谢和肿瘤转移、侵袭过程中具有重要作用。为进一步研究其功能和作用机理,以PCR方法扩增人基质金属蛋白酶抑制剂4不含信号肽的表达序列,测序鉴定正确后,构建原核重组表达质粒pMALc2TIMP4,转化大肠杆菌,IPTG诱导阳性克隆,大量表达重组蛋白。细菌经超声破碎后,其上清中的重组蛋白经SDSPAGE初步鉴定分子量大小与理论值一致,为以后基质金属蛋白酶抑制剂4的研究创造了条件 。     

基质金属蛋白酶及其组织抑制剂研究进展

基质金属蛋白酶家族是细胞外基质降解过程中的重要酶类,组织金属蛋白酶抑制剂是基质金属蛋白酶的天然抑制物。研究证实,细胞外基质中基质金属蛋白酶及其组织抑制剂的失衡与多种病理机制有关,尤其与肿瘤的侵袭和转移密切相关。本就基质金属蛋白酶及其组织抑制剂的性质、结构以及功能进行了综述。     

MMP-2结合肽-蜂毒素杂合基因的表达纯化及活性测定

为开发抗癌导向多肽药物,用从噬菌体十二肽库中筛选的与MMP-2具有高度亲和性的十二肽与蜂毒素连接,采用基因工程方法在大肠杆菌中进行了高效表达;并分别通过亲和层析、肠激酶酶切、凝胶层析获得高纯度的多肽(相对分子质量45000),经体外抑瘤作用测定该融合蛋白具有明显抑制肿瘤细胞的生长。该研究对肿瘤的导向治疗和临床应用提供了一定的帮助。     

基质金属蛋白酶-2在酵母系统中表达的初步研究

基质金属蛋白酶-2（明胶酶A）在肿瘤侵袭和转移中具有重要作用,为进一步研究其作用机制,在酵母系统中表达了金属蛋白酶-2通过PCR方法获得明胶酶A的表达序列,酶切和测序结果证明序列正确后,构建明胶酶A的表达载体pPIC9/GelA,电击法转化酵母得到阳性克隆.明胶酶谱分析说明表达产物具有明胶底物特异性,SDS-聚丙烯酰胺凝胶电泳表明,表达产物的分子质量为66 ku.     

基质金属蛋白酶组织抑制剂-2基因的人工合成与克隆

根据GenBank报道的基质金属蛋白酶组织抑制剂-2(TIMP-2)氨基酸序列和毕赤酵母偏爱密码子设计,通过化学方法合成得到适合在毕赤酵母中表达的目的TIMP-2基因序列,并将其克隆到质粒pPIC9中,构建了pPIC9-T2表达载体,PCR鉴定及测序结果表明得到了正确的TIMP-2基因序列。     

基质金属蛋白酶及其抑制剂

基质金属蛋白酶（matrix metalloproteinases,MMP)是降解细胞外基质成分的主要酶类,参与很多重要的生理和病理过程。MMP具有典型的多结构组成,MMP活性在基因转录,酶原激活和内源性抑制等方面受到严重调控,调控的紊乱将导致肿瘤扩散和转移,风湿性关节炎等重大疾病的发生,因此MMP成为治疗这些疾病的理想的药物作用靶点,寻找高亲和性,高选择以及高生物利用度的MMP抑制剂成为一项有意义的工作。     

基质金属蛋白酶和组织金属蛋白酶抑制剂在肾细胞癌组织中的表达

目的:基质金属蛋白酶及组织金属蛋白酶抑制剂在肾细胞癌转移中占有重要的作用,研究肾细胞癌组织中MMP-2、MMP-9、TIMP-1和TIMP-2的表达情况,为肾癌转移的治疗提供理论依据。方法:选取36例肾细胞癌肾组织标本,从相同的肾细胞癌组织及癌旁肾组织获得对照样本,均进行根治性肾切除手术切除。肿瘤分期按TNM分期标准。为了统计评估,肿瘤1期和2期为低级,3期以上为高级。RT-PCR检测肿瘤和正常组织中的MMP-2、MMP-9、TIMP-1和TIMP-2的表达。结果:不同样本MMPs和TIMPs表达水平各不相同。肾细胞癌组织中MMP-2、MMP-9、TIMP-1、TIMP-2在肾细胞癌中的表达明显高于正常肾组织(P0.05)。在肾细胞癌的肿瘤分期方面,MMP-2与MMP-9和肿瘤的分期显著相关,随着肿瘤分期的增加,MMP-2与MMP-9的表达明显升高(P0.05),而TIMP-1与TIMP-2与肿瘤的分期无关。结论:肾细胞癌组织中TIMP-2、MMP-2,MMP-9,TIMP-1的mRNA表达显著高于正常肾组织,抑制MMPS的表达将成为治疗肾细胞癌转移的新的方向。     

基质金属蛋白酶-9及其抑制剂mRNA在吸烟大鼠肺组织中的表达

目的 研究吸烟大鼠肺组织和肺泡巨噬细胞中基质金属蛋白酶9(MMP-9)和金属蛋白酶组织抑制剂-1(TIMP-1)基因的表达,探讨其在细胞外基质重塑中的作用。方法 建立吸烟大鼠模型,随机分为对照组和吸烟1、2、3、4、5及6月组(每组10只),用原位杂交技术检测肺组织和肺泡巨噬细胞MMP-9和TIMP-1 mRNA表达,用免疫组织化学技术观察Ⅳ型胶原在肺内的表达。结果 吸烟组肺组织和肺泡巨噬细胞MMP-9 mRNA的表达逐渐上升,至吸烟6月时均达高峰;而TIMP-1 mRNA的表达渐上升,至吸烟3～4月时达高峰,后逐步下降;肺组织Ⅳ型胶原的表达在吸烟3月时达高峰,然后渐降。结论 MMP-9／TIMP-1的动态平衡在吸烟大鼠肺气肿模型肺组织的细胞外基质重塑中有重要作用。     

基质金属蛋白酶及其抑制剂在乳腺癌中的表达

目的:研究基质金属蛋白酶2(Matrix Metalloproteinase-2,MMP-2),基质金属蛋白酶7(MMP-7),基质金属蛋白酶9(MMP-9),膜型基质金属蛋白酶(Membrane Type-1 Matrix Metalloproteinase,MT1-MMP),金属蛋白酶组织抑制剂1(Tissue Inhibitor of Metalloproteinase,TIMP-1),金属蛋白酶组织抑制剂2(TIMP-2)在乳腺癌组织中mRNA的表达,及与临床病理变量之间的关联。方法:采用150例乳腺癌患者的组织样本。使用半定量逆转录-聚合酶链反应(RT-PCR)法来测定肿瘤组织和正常乳腺组织中MMP-2,MMP-7,MMP-9,MT1-MMP,TIMP-1和TIMP-2的mRNA表达。结果:MMP-2,MMP-7,MMP-9,MT1-MMP,TIMP-1和TIMP-2在乳腺癌中的mRNA表达显著高于正常组织。结论:MMP-2,MMP-7,MMP-9,和MTI-MMP的表达增加和临床病理参数之间的关联,可以用来预测乳腺癌的侵害行为。     

Identification,purification and partial characterization of tissue inhibitor of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle

Bovine pulmonary artery smooth muscle possesses the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) as revealed by Western immunoblot study of its cytosol fraction with bovine polyclonal TIMP-2 antibody. This potent polypeptide inhibitor of matrix metalloproteinases (MMPs) was purified to homogeneity from cytosol fraction of bovine pulmonary artery smooth muscle. This inhibitor was purified by ammonium sulfate precipitation followed by gelatin sepharose and lentil lectin sepharose affinity chromatography and continuous elution electrophoresis by Prep Cell Model 491 (Bio-Rad, USA). SDS-PAGE revealed that the inhibitor has an apparent molecular mass of 21 kDa and was confirmed as TIMP-2 by (i) Western immunoblot assay using bovine polyclonal TIMP-2 antibody; and also by (ii) amino terminal amino acid sequence analysis of the purified inhibitor is found to be identical with TIMP-2 obtained from other sources. The purified 21 kDa inhibitor was found to be active against matrix metalloproteinase-2 (MMP-2, 72 kDa gelatinase) and matrix metalloproteinase-9 (MMP-9, 92 kDa gelatinase), the ambient MMPs in the pulmonary artery smooth muscle. The inhibitor was also found to be sensitive to the activated 72 kDa gelatinase-TIMP-2 complex and also active human interstitial collagenase. By contrast, it was found to be insensitive to the serine proteases: trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation. Treatment of the inhibitor with hydrogen peroxide, superoxide generating system (hypoxanthine plus xanthine oxidase) and peroxynitrite inactivated the inhibitor.     

Identification,purification and partial characterization of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in bovine pulmonary artery smooth muscle

Bovine pulmonary artery smooth muscle tissue possesses the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as revealed by immunoblot studies of the cytosolic fraction with polyclonal TIMP-1 antibody. In this report, we described the purification and partial characterization of the inhibitor from the cytosolic fraction of the smooth muscle. This inhibitor was purified by a series of anion-exchange, gel filtration and affinity chromatographic procedure. The purified inhibitor showed an apparent molecular mass of 30 kDa in SDS-PAGE. Amino terminal sequence analysis for the first 22 amino acids of the purified inhibitor was also found to be identical to bovine TIMP-1. This glycosylated inhibitor was found to be active against matrix metallorpoteinase-9 (MMP-9, gelatinase B), the ambient matrix metalloproteinase in the pulmonary smooth muscle. The purified TIMP-1 was also found to be sensitive to pure rabbit and human fibroblast collagenase and type IV collagenase. In contrast, it had minimum inhibitory activity against bacterial collagenase. It was also found to be inactive against the serine proteases trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation.     

Cloning and expressing a recombinant human tissue inhibitor of metalloproteinase-2 (TIMP-2) in methylotrophic yeast Pichia pastoris and its characterizations

To obtain human tissue inhibitor of metalloproteinase-2 (TIMP-2)cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris,we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique,which was composed of frequently used codons in the highly expressed Pichia pastoris genes.Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing.The verified gene of TIMP-2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2.The plasmid was transformed into GS115 cells of the methylotrophic yeast,Pichia pastoris by electroporation,and we got the expression cell through phenotype selection and induction with methanol.Separation,purification,and bioactivity analysis of the expressed products were performed.     

Modified synthetic siRNA targeting tissue inhibitor of metalloproteinase-2 inhibits hepatic fibrogenesis in rats

BACKGROUND/AIMS: Fibrosis occurs in most chronic liver injuries and results from changes in the balance between synthesis and degradation of extracellular matrix (ECM) components. Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) are known to regulate the ECM turnover. We investigate the effect of modified synthetic small interfering RNA (siRNA) targeting TIMP-2 in rat model of liver fibrosis. METHODS: Rat hepatic fibrosis was induced by CCl4 for 8 weeks. After the 2-week CCl4 injection period, rats in the three siRNA groups simultaneously received a different dosage (0.05, 0.1 and 0.2 mg.kg(-1), respectively) of modified synthetic siRNA targeting TIMP-2 via the tail vein every 3 days for 6 weeks. The pathological changes in liver tissues were observed by light microscopy and transmission electron microscopy. Portal vein pressure and proliferating cell nuclear antigen were measured. Expression of TIMP-2, MMP-2, MT1-MMP, MMP-13, hepatocyte growth factor, collagen type I, collagen type III and alpha-SMA were evaluated by quantitative real-time polymerase chain reaction or Western blotting or gelatin zymography. RESULTS: Modified synthetic siRNA targeting TIMP-2 induced a dose-dependent inhibition of the TIMP-2 expression in the rat model of liver fibrosis with a similar trend in MMP-2 and MT1-MMP, but an increase in MMP-13. Rats administered siRNA targeting TIMP-2 showed promotion of ECM degradation, reduction in activated hepatic stellate cells and enhancement of hepatocyte regeneration. Furthermore, portal hypertension was also ameliorated after treatment with siRNA targeting TIMP-2. CONCLUSIONS: Knock-down of TIMP-2 expression attenuates CCl4-induced liver fibrosis and is a potential pharmacological target for gene therapy in liver fibrosis.     

Matrix metalloproteinase-9,-3 and tissue inhibitor of matrix metalloproteinase-1 in colorectal cancer: relationship to clinicopathological variables

The balance between matrix metalloproteinases (MMPs) and their physiological tissue inhibitors of matrix metalloproteinases (TIMPs) is crucial in tumour invasion and progression. The aim of this study was to investigate the levels of MMP-9, MMP-3 and TIMP-1 in colorectal cancer (CRC) and to evaluate these proteinases and their inhibitor with respect to clinicopathological variables. Activities of pro- and active MMP-9 were measured in paired tumour and distant normal tissue specimens from 43 patients with CRC using gelatin zymography. ELISA was employed for the determination of MMP-9, MMP-3 and TIMP-1 protein expressions. The activity levels of pro- and active MMP-9 and protein expression levels of MMP-9, MMP-3 and TIMP-1 were higher in tumour tissues than in the corresponding normal tissues; the differences being significant for all (p < 0.05), except TIMP-1. Similarly, active MMP-9/proMMP-9 and the ratio of protein expression level of MMP-9-TIMP-1 were found to be significantly higher in tumour tissues ( p < 0.01). Among all the clinicopathological variables investigated, significant correlations were found between MMP-9 and presence of perineural invasion, MMP-3 and lymph node status, TIMP-1 and tumour differentiation, MMP-9/TIMP-1 ratio and histological types ( p < 0.05). In conclusion, MMP-3 was not as notably increased as MMP-9 in tumour tissues. However, different roles may be attributed to MMP-9 and MMP-3 in CRC development and progression. Additionally, assessment of TIMP-1 in relation to MMPs appeared to be crucial in CRC studies to provide a basis for the re-evaluation of the clinical usefulness of TIMP-1 in colorectal cancer.     

Cloning and expressing a recombinant human tissue inhibitor of metalloproteinase-2 (TIMP-2) in methylotrophic yeast Pichia pastoris and its characterizations

To obtain human tissue inhibitor of metalloproteinase-2 (TIMP-2) cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris, we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique, which was composed of frequently used codons in the highly expressed Pichia pastoris genes. Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing. The verified gene of TIMP-2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2. The plasmid was transformed into GS115 cells of the methylotrophic yeast, Pichia pastoris by electroporation, and we got the expression cell through phenotype selection and induction with methanol. Separation, purification, and bioactivity analysis of the expressed products were performed. __________ Translated from Microbiology, 2006, 33(1): 1–6 [译自: 微生物学通报]     

小鼠cofilin2蛋白的原核表达及纯化

[目的]构建小鼠cofilin2原核表达载体并纯化表达产物。[方法]以胚胎期小鼠心脏组织的c DNA为模板PCR扩增cofilin2基因,经酶切连接表达载体PGEX-4T-1后转化入大肠杆菌E. coli BL21感受态细胞中,利用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)进行诱导表达及优化,利用SDS-PAGE凝胶电泳,考马斯亮蓝R-250进行染色,检测GST-cofilin2的表达情况。通过谷胱甘肽树脂(Glutathione Resin)亲和层析进行纯化,最后通过Western Blot进行验证。[结果]PCR成功扩增cofilin2基因,双酶切及测序结果表明p GEX-4T-1-cofilin2原核表达载体构建成功,SDS-PAGE鉴定表明,在22℃、200μmol/L的IPTG能诱导出大量可溶性的GST-cofilin2蛋白,分子量为43 k Da。Western Blot验证得到纯化的GST-cofilin2。[结论]成功构建小鼠cofilin2原核表达载体,纯化得到的重组小鼠cofilin2蛋白可用于后续的生物学研究。     

Influence of organosulphur compounds from garlic on the secretion of matrix metalloproteinases and their inhibitor TIMP-1 by cultured HUVEC cells

Organosulphur compounds from garlic, especially diallyl disulphide (DADS) at non-toxic concentrations, affected production and secretion of some matrix metalloproteinases (MMPs) and of tissue inhibitor of metalloproteinase-1 (TIMP-1), one of their inhibitors, by human umbilical vein endothelial cells. Addition of DADS to the culture medium resulted in a concentration-dependent reduction of secreted MMP-2 protein and activity as well as TIMP-1 protein. In the presence of inducers (phorbol 12-myristate 13-acetate, forskolin and tumor necrosis factor alpha) addition of DADS caused a distinct concentration-dependent decrease of MMP-9 and TIMP-1 secretion, while not affecting MMP-9 mRNA levels. Intracellular protein levels remained low and were not affected. Other organosulphur compounds like allyl mercaptan and S-allylcysteine showed no or less clear effects on MMP-secretion or TIMP-1-secretion. These results suggest that DADS may mediate some of the biological effects ascribed to garlic preparations through affecting MMP-TIMP balance.     

人基质金属蛋白酶2催化区的克隆与表达

为从随机肽库中寻找具有基质金属蛋白酶 2 (MMP 2 )抑制活性的新型小肽抑制剂 ,应用PCR法从含有人MMP 2基因的质粒中扩增了人MMP 2的催化区 .序列分析结果表明无氨基酸突变 .然后构建人MMP 2催化区的表达载体pET MCD ,转化大肠杆菌BL2 1(DE3 ) ,经IPTG诱导表达人MMP 2催化区 .经包涵体分离、变性、金属螯合层析纯化和复性等过程 ,复性后的人MMP 2催化区具有较好的明胶水解活性 .