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

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-3 cDNA,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pcDNA3.1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(Col Ⅰ)、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MDA-MB-453细胞生长及侵袭性有抑制作用。

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

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 pcDNA3.1 were digested by EcoR Ⅰ and XbaⅠ. 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 EcoRⅠ and XbaⅠ. 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.