鞘脂激活肽真核表达载体的构建及其对前列腺癌细胞增殖的影响

目的：构建含有鞘脂激活蛋白原神经营养序列短肽－鞘脂激活肽NP（neurotrophic peptide, NP）的真核表达载体以及NP与TAT的融合蛋白真核表达载体(TAT NP)，探讨NP在前列腺癌细胞中的作用。方法：根据NP和TAT的氨基酸序列合成其DNA序列，利用基因克隆技术构建含有TAT和NP读码框架的真核表达载体pcDNA-TAT-NP和pcDNA-NP。在前列腺癌PC3、LNCaP细胞中转染上述载体后通过RT-PCR、MTT和流式细胞术，分别检测TAT-NP、NP的mRNA的表达，NP对前列腺癌细胞的增殖情况以及对细胞周期的影响。结果：经测序鉴定，成功构建pcDNA-TAT-NP和pcDNA-NP真核表达载体。RT-PCR结果显示，TAT-NP、NP能够在前列腺癌细胞中表达。MTT结果表明，TAT-NP、NP均对前列腺癌细胞增殖具有促进作用。流式细胞结果显示，TAT-NP、NP具有促进细胞进入S和G₂/M期的作用。结论：外源表达的鞘脂激活肽NP能够促进前列腺癌细胞的增殖，并能影响细胞周期各时相的变化。

Effect of ectopic expressed Neurotrophic Peptide on proliferation of human prostate cancer cells

Objective: Based on neurotrophic domain sequence(NP) of saposin C,eukaryotic expression vectors containing NP sequence and NP fused to TAT transduction domain were respectively constructed.The TAT transmembrane signal was expected to help NP go through cell membranes and have effects on prostate cancer cells.Methods: The eukaryotic expression vectors pcDNA-TAT-NP and pcDNA-NP were constructed by using gene cloning technology.After transient transfection of pcDNA-TAT-NP and pcDNA-NP into the prostate cancer cells PC3 and LNCaP,RT-PCR,MTT and flow cytometry were used to respectively detect the expression of TAT-NP and NP in the level of mRNA,prostate cancer cell growth and the cell cycle changes.Results: The eukaryotic expression vectors pcDNA-TAT-NP and pcDNA-NP were constructed and confirmed by sequencing.The result obtained from RT-PCR indicated both pcDNA-TAT-NP and pcDNA-NP were expressed in prostate cancer cells.MTT analysis showed that expressions of TAT-NP and NP had a stimulating effect on prostate cancer cell proliferation,and promoted the cells to enter S and G_2/M phases.Conclusion: Ectopic expression of NP could enhance the prostate cancer cell growth and affect the cell cycle.