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

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

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-puroR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]．以无血清培养堆培养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基因的导入叮延缓细胞增殖，并抑制细胞进入增殖周期.

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 293ToAmphotropic 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 semisolid 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） ×10^9TU/L. The K562 and HL60 transfection efficiency reached 82. 3% ± 3.70% and 62.9% ±4.94%, respectively （ n = 3 ）. After 3d 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 G0 -G1, 49.7% ±3.38% vs43.9% ±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% vs54.5%±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.