人前列腺特异性同源框基因NKX3．1真核表达载体的构建及其对PC3细胞周期与凋亡的影响

The construction of human prostate specific homeobox gene NKX3. 1 eukaryotic expression vector and preliminary study on the biological function of NKX3. 1

目的构建NKX3．1真核表达载体，探讨NKX3．1的基因定位以及其对前列腺癌细胞株PC3细胞周期和凋亡的影响。方法利用PCR技术从正常人前列腺组织中扩增NKX3．1全长读码框（ORF），并将其克隆到T载体。以该载体为模板，将NKX3．1克隆到真核细胞高效表达载体pcDNA3．1（＋）和pEGFPC1，转染入PC3细胞株并观察NKX3．1在细胞内的定位，采用流式细胞术检测细胞周期和凋亡的情况。结果本实验成功构建了NKX3．1真核表达载体。转染NKX3．1inpEGFPC1的荧光主要在细胞核表达，而转染pEGFPC1的荧光则胞浆和胞核均有表达。与转染pcD-NA3．1空载体的PC3细胞株比较，转染NKX3．1的PC3细胞株细胞周期没有明显改变（P〉0．05），但早期凋亡明显增加（P〈0．01）。结论本实验在成功构建了NKX3．1真核表达载体的基础上，证明了NKX3．1是一种胞核蛋白，它不影响细胞周期，但是具有一定促进早期凋亡的作用。

Objective To investigate the basic biological functions of NKX3.1 with construction of a series of eukaryotic expression vector of NKX3.1 to study its effects on cell cycle and apoptosis. Methods The full-length reading frames （ORF） of NKX3.1 was amplified from normal human prostate tissue with polymerase chain reaction （PCR）, the ORF was cloned into T vector, and then NKX3.1 was cloned into eukaryotic expression vector pcDNA3.1 （ ＋ ） and pcDNA3.1（ - ） with this T vector as template. NKX3.1 in pEGFP C1 was transfected into PC3 cell line, and C1 pEFP was taken as control to observe the localization of NKX3. 1 in cells. At the same time, NKX3.1 in pcDNA3.1 and pcDNA3.1 were transfected into PC3 cells and the cells were collected after 24-48 hours to detect cell cycle and apoptosis by flow cytometry. Results The eukaryofic expression vector of NKX3. 1 was constructed successfully. Fluorescent protein was ocated in the nucleus after transfection of NKX3.1 in pEGFP C1, but pEGP C1 was both expressed in nucleus and cytoplasm. There was significant effect on the early apoptosis （P 〈0. 01 ）, but no effect on the cell cycle （P 〉 0. 05） after transfection of NKX3. 1 compared to PC3 cell-transfected with empty vector of pcDNA3.1. Conclusions The result data suggest that NKX3.1 be nuclear protein and have effect on the early apoptosis of PC3 cell line based on the construction of eukaryotic expression vector.