TIMP-3-pcDNA3.0转染平滑肌细胞及其在大鼠心肌梗死瘢痕中的存活与表达

目的 探讨基质金属蛋白酶抑制因子3(TIMP-3)基因转染平滑肌细胞的效果及移植的转染细胞能否在大鼠心梗瘢痕中存活并表达目的 蛋白.方法 植块法培养大鼠主动脉中层平滑肌细胞并鉴定;脂质体介导法以"TIMP-3基因质粒转染平滑肌细胞;RT-PCR和Western blot检测转染细胞的基因水平和蛋白表达;通过BrdU移植检测移植细胞的存活与增殖情况.实验大鼠随机分成单纯PBS注射、单纯平滑肌细胞移植和转染平滑肌细胞移植3组(每组7只实验大鼠),将移植物注射人(五点法)心梗大鼠的室壁内.1周后,提取其左心室组织mRNA和蛋白,分别进行RT-PCR和Western blot检测,鉴定其.TIMP-3基闪含量和表达情况.结论 植块法培养可以获得高纯度的平滑肌细胞;脂质体介导法可以使TIMP-3基因转染平滑肌细胞并获得TIMP-3蛋白的表达;移植的转染细胞能够在心梗缀痕中存活并增殖;转染细胞组大鼠左心室组织中TIMP-3 mRNA含量明显多于单纯平滑肌细胞移植组(P<0.01)及单纯PBS注射组(P<0.01);转染细胞移植组大鼠左心室组织中TIMP-3蛋白含量明显多于平滑肌细胞移植组(P<0.01)及单纯PBS注射组(P<0.01).结论TIMP-3基因可以通过脂质体介导法转染平滑肌细胞,且转染细胞可以在大鼠梗死心肌中存活、增殖并表达TIMP-3蛋白.

Transient transfecting TIMP-3 gene into the smooth muscle cells and its Survival and expression in the scar Of the rat with myocardial infarction

Objective To transient transfect TIMP-3 gene into the smooth muscle cells and observe the survival and expression of the transplanted transfected cell in the scar of the rat with myocardial infarction. Methods Smooth muscle cells were cultured from tunica media of rat aorta by sticking little tissue clump and identified;The plasmid containing TIMP-3 gene fragment were transient transfected into smooth muscle cells by Lipofectamine (TM) 2000. TIMP-3 and TIMP-3 gene fragment of the transfected cells were detected through Western blot and RT-PCR respectively; Smooth muscle cells marked by BrdU were transplanted to the sear, and the survival and proliferation were detected through immunohistochemistry. The rats were divided into 3 groups: the first group were injected by PBS, the second were injected by smooth muscle cells and the third were injected by transfected smooth muscle cells. The myocardial infarction model was built by ligating the distal end of the aeteria coronaria of the rat. After 1 week, the left ventricle of the testing rat was got. And mRNA and tissue protein for RT-PCR and Western blot were extracted to identify the existence and expression of the interested gene. Results Highly purified smooth muscle cells were got; TIMP-3 gene were transient transfected into the smooth muscle cells and expressed the interested protein; Immunohistochemistry showed that transplanted transfected cell could survive and proliferate in the scar of the rat with myocardial infarction; Tissues of the rat heart which were injected by transfected smooth muscle cells contented much more TIMP-3 mRNA than those injected by smooth muscle cells(P<0.01), and PBS(P <0.01). Tissues of the rat heart which were injected by transfected smooth muscle cells contented much more TIMP-3 protein than those injected by smooth muscle cells (P <0.01) , and PBS (P < 0.01). Conclusion TIMP-3 gene can be transient transfected into the smooth muscle cells by Lipofectamine(TM) 2000,and the transplanted transfected cell can survive in the scar of rat with myocardial infarction and express interest protein.