TIMP-3基因真核表达载体的构建及其对乳腺癌MDA-MB-453细胞生长及侵袭的抑制作用

Construction of eukaryotic expression vector of TIMP-3 gene and its inhibitory effect on growth and invasion of MDA-MB-453 cells

目的:构建组织基质金属蛋白酶抑制剂3(TIMP-3)真核表达载体并转染乳腺癌MDA-MB-453细胞,探讨TIMP-3对MDA-MB-453细胞生长及侵袭的抑制作用。方法:采用RT-PCR法从人新鲜胎盘组织中获得TIMP-3cDNA,连接至pMD18-T载体,EcoRⅠ、XbaⅠ双酶切pMD18-T-TIMP3重组质粒及pcDNA3.1载体并连接,构建pcDNA-TIMP-3真核载体,酶切鉴定、测序。将乳腺癌MDA-MB-453细胞分为正常对照组、pcDNA空载体组(只转染pcDNA空载体)和pcDNA-TIMP-3组(转染pcDNA-TIMP-3)。Western blotting法测定蛋白表达,采用MTT法和Boyden小室侵袭实验测定各组细胞的增殖活性及侵袭能力。结果:重组载体经过EcoRⅠ和XbaⅠ酶切后,产生633bp的TIMP-3目的片段和pcDNA 3.1线性化载体,经自动测序仪测定TIMP-3序列完全正确。转染重组载体后MDA-MB-453细胞稳定表达了TIMP-3,与正常对照组和pcDNA空载体组比较,pcDNA-TIMP-3组细胞增殖活性明显降低(P<0.05),穿透以Ⅰ型胶原(ColⅠ)、层黏连蛋白(LN)、纤维连接蛋白(FN)和玻璃连接蛋白(VN)包被的Matrigel膜的侵袭细胞数明显减少(P<0.05)。结论:成功构建pcDNA3.1-TIMP-3真核表达载体,可在MDA-MB-453细胞中高表达TIMP-3蛋白,并对MDAMB-453细胞生长及侵袭性有抑制作用。

Objective： To construct a mammalian tissue inhibitor of metalloproteinase-3 （TIMP-3） recombinant eukaryotic expression vecor and transfect the breast cancer MDA-MB-453 cells, and to explore the inhibitory effect of TIMP-3 gene on the growth and invasion of MDA-MB-453. Methods： The TIMP-3 cDNA was obtained from the human fresh placenta by RT-PCR. TIMP-3 gene was connected to pMD18-T vector. The recombinant pMD18-T vector and peDNA3.1 were digested by EcoR I and Xba I Then TIMP-3 gene was subcloned into pcDNA3.1 vetor to construct pcDNA-TIMP-3 recombinant vector and enzyme digestion identification and sequencing. The MDA-MB-453 cells were divided into normal control group, pcDNA group （transferted with pcDNA） and pcDNA- TIMP-3 group （transfected with pcDNA3.1-TIMP-3）. The proliferation activities and invasion abilities of the MDA-453 cells in various groups were determined by MTT method and Boyden invasion experiment. Results.. The correct construction of pcDNA-TIMP-3 was identified by means of restriction enzyme EcoR I and Xba I The gene fragment of 633 bp and linearized vector were obtained. The result of TIMP-3 gene sequencing was correct. The Western blotting results showed that the transfected MDA-MB-453 cells expressed TIMP-3. The proliferation activity and invasion ability of MDA-MB-453 cells in pcDNA-TIMP-3 group were reduced significantly compared with normal control group and pcDNA group （P〈0.05）. Conclusion. The pcDNA-TIMP-3 eukaryotic expression vector is constructed successfully and TIMP-3 can be expressed in the MDA-MB-453 cells. The pcDNA-TIMP-3 has inhibitory effect on the growth and invasion of MDA-MB-453 cell lines.