【作者】 郑马庆；

【导师】 韩晓；

【作者基本信息】 南京医科大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 目的:1、采用噬菌体展示技术筛选MMP-2抑制性结合肽,观察该短肽体外抑制肿瘤细胞的侵袭作用;2、根据筛选短肽和MMP-2酶活性中心三维立体结构特征,借助生物信息学方法设计并合成系列化合物,经体内、外试验,筛选能够抑制肿瘤生长、转移的化合物。方法:1.采用噬菌体展示技术从噬菌体文库中筛选出对MMP-2具有较强亲和力的噬菌体单克隆;2.用MMP-2的特异性荧光分析药物筛选试剂盒对所筛选的噬菌体单克隆进行再次筛查,找出对MMP-2具有抑制作用的噬菌体单克隆;3.提取噬菌体DNA,测序,进行序列比对,找出噬菌体PⅢ末端表达的、与MMP-2结合的多肽的编码序列;4.体外合成短肽;5.用MMP-2特异性荧光分析药物筛选试剂盒测定该短肽对MMP-2酶活性的抑制作用;6.采用Transwell技术,观察该短肽体外抑制肿瘤细胞侵袭作用;7.根据所筛选短肽结构特点以及MMP-2酶活性中心三维立体结构,借助生物信息学技术设计并合成系列化合物,体外采用MTT法及MMP-2荧光分析药物筛选技术筛选出既可以抑制肿瘤生长又可以抑制MMP-2活性的化合物;8.复制乳腺癌肿瘤动物模型,观察该化合物对荷瘤鼠的治疗作用。结果:1.采用噬菌体展示技术,筛选出22个对MMP-2具有较强亲和力的噬菌体单克隆。为进一步确定该单克隆对MMP-2酶活性抑制作用,将筛选单克隆扩增,用荧光分析药物筛选试剂盒检测筛选单克隆对MMP-2酶活性抑制作用,结果表明,22个单克隆均对MMP-2具有一定的抑制作用。扩增噬菌体单克隆,提取DNA,测序,比对后得到19条对MMP-2具有抑制作用的结合肽。2.根据以上筛选结果,确定并合成对MMP-2抑制能力较强的2条短肽M204C4与M205C4。其多肽编码序列如下:M204C4:HWWQWPSSLQLRGGGSM205C4:HNWTRWLLHPDRGGGS3.采用MMP-2荧光分析药物筛选试剂盒体外对MMP-2抑制作用的研究表明:在一定浓度范围内,M204C4和M205C4对MMP-2酶活性具有不同程度的抑制作用,且随着浓度增加,对MMP-2活性抑制作用逐渐增强,呈现剂量依赖性。M204C4、M205C4对MMP-2酶活性抑制作用的IC₅₀分别为78.0、38.8nmol/L。4.M204C4对胰腺癌细胞系PANC-1、CFPAC-1侵袭转移能力研究表明:在一定浓度范围内,M204C4能显著抑制胰腺癌细胞侵袭,具有一定的剂量效应关系。高剂量200 nmol/L M204C4能显著抑制两种细胞系的侵袭转移,与对照相比,具有统计学差异（P＜0.001）;中剂量60 nmol/LM204C4也具有相似的抑制作用（P＜0.001或0.01）;低剂量20 nmol/LM204C4虽然也可以抑制PANC-1的侵袭转移,但其作用弱于高、中剂量组,与对照组相比亦具有统计学差异（P＜0.01）,在此剂量下,M204C4虽可抑制CFPAC-1的侵袭,但与对照组相比无统计学差异。5.M205C4对胰腺癌细胞系PANC-1、CFPAC-1侵袭转移能力研究表明:高剂量100 nmol/L M205C4能显著抑制PANC-1、CFPAC-1细胞侵袭转移,与对照组相比,具有统计学差异（P＜0.01或0.001）;中剂量30nmol/L M205C4也具有相似的作用效果（P＜0.05或0.01）;低剂量10nmol/L M205C4虽然也可以抑制PANC-1、CFPAC-1的侵袭转移,但与对照组相比无统计学差异（P＞0.05）。6.在筛选短肽结构基础上,结合MMP-2酶活性中心三维立体结构特征,借助生物信息学方法设计并合成48个化合物。采用MTT实验和MMP-2酶活性抑制实验对合成化合物进行筛选,确定WB-1、WB-45、WB-46用于体内实验研究。7.复制乳腺癌细胞系MDA-MB-435裸鼠肿瘤动物模型,并予不同剂量WB-1、WB-45、WB-46灌胃给药。研究结果表明:用药组与对照组所有动物脏器病理切片均未见药物引起的毒性反应。对照组中所有动物均发现有肿瘤肺部转移现象,而WB-1用药组均未见转移灶。WB-1用药组动物体重较对照组有所增加,且肿瘤重量较对照组轻,200mg/kg组瘤重仅为对照组的24.7%,100mg/kg与10mg/kg组肿瘤重量也有所下降,具有一定的剂量效应关系。WB-45、WB-46用药组在动物肺部可观察到大小不等的转移灶。8.采用ELISA法检测血清中VEGF浓度,结果表明,WB-1（100mg/kg和10mg/kg）可以降低乳腺癌模型血清中VEGF浓度,且随着浓度增加,血清中VEGF含量明显减少,100mg/kg组与对照组相比具有统计学差异（P＜0.05）。9.采用酶谱法对血清中的MMP-2/9测定结果表明:WB-1 100mg/kg可以降低血清中的MMP-2的酶原及活化形式表达,与对照组相比,有统计学差异（P＜0.05）;但WB-1对MMP-9表达无影响。结论:1.经噬菌体展示技术筛选后,得到2条能够很好抑制MMP-2活性的多肽:M204C4与M205C4。2.M204C4与M205C4在体外能够抑制MMP-2酶活性,并且能够抑制MMP-2介导的胰腺癌细胞侵袭转移。3.借助生物信息学方法,根据所筛选短肽结构和MMP-2酶活性中心三维立体结构合成的化合物WB-1,在体外能够抑制MMP-2/9酶活性及肿瘤细胞的增殖,在体内能够抑制肿瘤生长及转移,同时能够降低血清中VEGF、MMP-2酶原及活化形式表达。更多还原

【Abstract】 Objectives:1.To screen MMP-2 inhibitory peptides from phage display library and observe their effects on tumor cell invasion.2.To design and synthesis compounds using bioinformatics according to the conformation of the selected peptides and MMP-2 active domain.3.To observe the effects of the compounds on tumor cell proliferation in vitro,and tumor growth,invasion and metastasis in vivo.Methods:1.Phages with high affinity to MMP-2 enzyme were selected from phage display library.2.MMP-2 fluorescent assay kit for drug discovery was used to screen the selected phage clones and determine their effect on MMP-2 activity.3.Extracting single-stranded phage DNA and sequencing.4.Synthesizing the peptides in vitro.5.Determining the synthetic peptide effect on MMP-2 activity using MMP-2 fluorescent assay kit for drug discovery.6.Matrigel-coated transwell inserts were used to observe the effects of synthetic peptides on cancer cell invasion.7.According to the conformation of selected peptides and MMP-2 active domain,a series of compounds were designed using bioinformatics and synthesized.All compounds were assayed with MMP-2 fluorescent assay kit for drug discovery and MTT method to determine their effects on MMP-2 activity and cancer cell proliferation.8.Breast cancer nude mice model was made to observe their treatment effects in vivo.Results:1.22 phage clones with high affinity to MMP-2 enzyme were obtained after screening of phage display library,and all of these clones could inhibit the activity of MMP-2 enzyme.19 peptides were obtained after sequencing.2.Two peptides were obtained after screening of phage display library and MMP-2 enzyme activity inhibitory effect assay.Their sequences were as follows:M204C4:HWWQWPSSLQLRGGGSM205C4:HNWTRWLLHPDRGGGS3.Those two peptides M204C4 and M205C4 inhibited the activity of MMP-2 in the different effects and the inhibitory effects were dose dependent. The median effective inhibiting concentration(IC₅₀)for M204C4 and M205C4 were 78.0 and 38.8 nmoml/L,respectively.4.M204C4 inhibited MMP-2 mediated invasion of the pancreatic cancer cell lines PANC-1 and CFPAC-1 with a dose dependent manner.High concentration（200 nmol/L）of M204C4 significantly inhibited the invasion of these two cell lines than the control（P＜0.001）.The other two concentrations 60 nmol/L and 20 nmol/L of M204C4 had similar inhibitory effects,but milder than high concentration.5.M205C4 inhibited MMP-2 mediated invasion of the pancreatic cancer cell lines PANC-1 and CFPAC-1 with a dose dependent manner.The high and middle concentration（100 nmol/L and 30 nmol/L）of M205C4 could inhibit the pancreatic cancer cell invasion significantly.The low concentration （10nmol/L）could inhibit the pancreatic cancer cell invasion,but no difference was observed compared with the control group（P＞0.05）.6.48 compounds were synthesized.According to the results of MTT assay and MMP-2 enzyme activity inhibitory assay,WB-1,WB-45 and WB-46 were selected to further investigate in vivo.7.No toxic reaction was observed between the control and the drug administration groups.In the control group,breast cancer metastases were observed in the lung pathologic section,but no metastasis in the WB-1 group. The average weight of the animals in WB-1 administration group was higher than the control group,but no difference was observed.The average tumor weight in the 200mg/kg group decreased to 24.7%of the control group,and the same phenomenon was observed between 100mg/kg and 10mg/kg groups. In the WB-45 and WB-46 group,tumor metastases were observed in almost all lung pathologic sections.In other organs,no metastasis was observed.8.The serum level of VEGF in the WB-1 group（100mg/kg and 10mg/kg） were lower than that in the control group,and a statistical difference was observed between 100mg/kg and the control group（P＜0.05）.9.The serum levels of pro- and active MMP-2 were lower in the WB-1 100mg/kg administration group than that in the control group（P＜0.05）,but no difference for pro-MMP-9 expression was observed between the drug administration group and the control group.Conclusions:1.Two peptides M204C4 and M205C4 were obtained after screening the phage display library.2.In vitro,M204C4 and M205C4 inhibited the activity of MMP-2 enzyme significantly,meanwhile inhibited MMP-2 mediated pancreatic cancer cell invasion.3.The mimetic compound WB-1 not only inhibited MMP-2 activity and cancer cell proliferation in vitro,but also inhibited tumor growth and metastasis and decreased the serum levels of VEGF,pro- and active MMP-2 in vivo.更多还原