正义Tankyrase转染大鼠海绵体平滑肌细胞的作用研究

Effects of sense Tankyrase transfection on smooth muscle cells of Corpus Cavernosum in rat

目的构建正义Tankyrase真核表达载体,探讨其转染大鼠海绵体平滑肌细胞的作用。方法(1)构建及鉴定正义Tankyrase真核表达载体；(2)正义Tankyrase重组质粒(pcDNA-TNKS)转染大鼠海绵体平滑肌细胞,进行DNA水平、RNA水平鉴定；(3)端粒酶活性的定量检测(TRAP-ELISA法)、端粒长度的检测(Southern blot法)及生长曲线(MTT法)的检测。结果(1)成功构建正义Tankyrase真核表达载体；(2)正义Tankyrase重组质粒成功转染大鼠海绵体平滑肌细胞：(3)TNKS转染细胞(SMC-TANKS)、空载转染细胞(SMC-Zeo)及未转染细胞(SMC)的端粒酶活性无显著性差异(P＞0．05)；(4)SMC-TANKS的端粒长度较SMC-Zeo及SMC长(P＜0．01)(5)SMC-TANKS的光密度(OD)值显著高于SMC-Zeo及SMC(P＜0．01)。结论正义Tanks重组质粒转染有助于改变海绵体平滑肌细胞的端粒长度而延长细胞寿命。

Objective To construct the eukaryotic expression vectors for sense Tankyrase and to investigate the effects of Tankyrase transfection on smooth muscle ceils of Corpus Cavernosum in rat. Methods （1） The eukaryotic expression vectors for sense Tankyrase were constructed and identified; （2） Smooth muscle cells of rat Corpus Cavemosum were transfected with the recombinant plasmids of sense Tankyrase （pcDNA-TNKS） and, then, the levels of DNA and RNA were evaluated; （3） Measurement of telomerase activity was conducted by TRAP-ELISA assay, the length of telomere by Southern blot and the growth curve by MTT assay. Results （1） The eukaryotic expression vectors for sense Tankyrase were constructed successfully; （2） The recombinant plasmids of sense Tankyrase were transfected into smooth muscle cells of rat Corpus Cavemosum successfully; （3）No significant differences in telomerase activity were observed between TNKS-transfected cells （SMC-TANKS）, zero-load- transfected cells （SMC- Zeo） and non-transfected cells （SMC）（P〉0.05）; （4） The length of telomere in SMC-TANKS was longer than that in SMC- Zeo or SMC （P〈0.01）; （5）The optical density（OD） value of TNKS-transfected cell was significantly higher than that of the non-transfected cells （P〈0.01）. Conclusion Transfection of recombinant plasmids of sense Tankyrase helps change the telomerase length of smooth muscle cells of Corpus Cavemosum and extend cell life span.