人细胞增殖相关激酶plk3基因逆转录病毒载体的构建及对细胞增殖的影响

目的构建人细胞增殖相关激酶基因plk3的逆转录病毒载体(RV),观察该基因对细胞增殖和细胞周期的影响。方法将plk3基因亚克隆至pMSCV-puroR载体中,经酶切和测序鉴定,制备高滴度的RV,以流动感染法高效感染,半固体集落培养法测定基因导入率。用嘌呤霉素筛选后,获得K562-plk3-puroR和HL60-plk3-puroR细胞。以野生型细胞和导入空载体的K562-puroR和HL60-puroR细胞作为对照,用PCR鉴定基因的导入,并测定细胞周期及凋亡率等,研究plk3基因导入对细胞的影响。结果获得pMSCV-plk3-puroR质粒并经酶切和测序证实。对K562和HL60细胞的基因导入率,分别达82.3%±3.70%和62.9%±4.94%(n=3)。经嘌呤霉素筛选后,转基因细胞的阳性率>99%。K562-plk3-pu-roR和HL-60-plk3-puroR转基因细胞的增殖曲线比对照细胞降低(P<0.01);而导入空载体的K562-puroR和HL60-puroR细胞的增殖曲线与野生型细胞相比较无显著差异。与K562细胞相比较,常规培养时,处于G0~G1期的K562-plk3-puroR细胞增多(49.7±3.38)%vs(43.9±2.34)%,P=0.03]。以无血清培养基培养10h后,进入S期的K562-plk3-puroR细胞减少(43.6±3.74)%vs(54.5±1.52)%,P=0.02],凋亡率降低(1.3±0.31)%vs(3.4±0.37)%,P=0.01]。结论构建了plk3基因的逆转录病毒载体,发现plk3基因的导入可延缓细胞增殖,并抑制细胞进入增殖周期。

Construction of pMSCV recombinant retroviral vector containing polo-like kinase 3(plk3)cDNA and its effects on cell proliferation

AIM: To construct pMSCV retroviral vector (RV) containing Polo-like kinase 3(plk3) cDNA, a new member of Ser/Thr protein kinase family, and to study the function of plk3 gene by introducing it into human K562 and HL60 cells. METHODS: plk3 cDNA was sub-cloned into retroviral vector pMSCV-puroR to generate pMSCV-plk3-puroR, pMSCV-puroR being used as the control. RVs were generated by introducing RV vectors into 293T-Amphotropic packaging cells. Titers of RV were measured by NIH3T3 cells with Large-scale-real-time titration method (LaSRT). K562 and HL60 cells were infected with pMSCV-plk3-puroR or pMSCV-puroR viral supernatant by flow-through transduction method, respectively. Following 3-day selection with 3 mg/L puromycin, K562-plk3-puroR, HL60-plk3-puroR, K562-puroR and HL60-puroR cells were obtained. PCR was used to identify the integration of plk3 gene in K562-plk3-puroR and HL60-plk3-puroR cells. Transduction rates were determined by clone-forming ability against puromycin in semi-solid culture medium. Cell growth curve was measured by MTT colorimetry, the cell cycle and apoptotic analysis were detected by flow cytometry, etc. RESULTS: Virus titers generated was (1.31+/-0.65)x10(9)TU/L. The K562 and HL60 transfection efficiency reached 82.3%+/-3.70% and 62.9%+/-4.94%, respectively (n=3). After 3 d puromycin selection, purified K562-plk3-puroR and HL60-plk3-puroR cell were obtained. The proliferation of K562 and HL60 cells was slowed down by plk3 gene transduction. Compared with wild type K562 cells: (1) when cultured in complete DMEM medium, more K562-plk3-puroR cells were observed in G(0)-G(1), 49.7%+/-3.38% vs 43.9%+/-2.34% (P=0.03); (2) when cultured in serum-free DMEM medium for 10 h, fewer K562-plk3-puroR cells were observed in S phase, 43.6%+/-3.74% vs 54.5%+/-1.52% (P=0.02) with lower apoptosis rate, 3.4%+/-0.37% vs 1.3%+/-0.31% (P=0.01). CONCLUSION: Introduction of plk3 gene into tumor cells with RV vectors can slow down cell proliferation, prevent cells into cell cycle and protect cells from apoptosis in serum-free culture.