目的：构建人Polo样激酶1（Plk1）活性缺失突变体及结构域突变体的真核表达载体，并在293细胞中表达。方法：用二次PCR方法扩增Plk1基因并点突变，将82位赖氨酸突变为精氨酸，定向克隆到pcDNA3-Flag载体中；用普通PCR方法扩增Plk1激酶区域及Polo盒区域（PBD）基因，定向克隆到pcDNA3-Flag载体中；将上述质粒转染293细胞进行瞬时表达，Western印迹检测Plk1蛋白的表达。结果：构建了Flag—Plk1（K82R）、Flag—Plk1KD、Flag—Plk1PBD真核表达质粒，在293细胞中均可有效表达，蛋白相对分子质量分别为68×10^3、45×10^3、31×10^3。结论：在293细胞中表达了Flaz—Plk1（K82R）、Flaz—Plk1KD、Flaz—Plk1PBD蛋白．有助于进-步探究Plk1对底物的功能。

Objective: To construct eukaryotic expression vector of Polo-like kinase1(Plk1) kinase dead mutant and domain, and express them in 293 cells. Methods: Plk1 gene was amplified by two steps PCR and inserted in-to eukaryotic expression vector pcDNA3-Flag to constructed Plk1 kinase dead mutant. Plk1 kinase domain gene and Polo box domain(PBD) gene were amplified by PCR, and then they were inserted into pcDNA3-Flag vector to construct Plk1 kinase domain and PBD domain eukaryotic expression vector respectively. Those recombinant ex-pression vector were transiently transfected into 293 cells, followed by Western blotting identification. Results: The Flag-Plk1(K82R) and Flag-Plk1KD, Flag-Plk1PBD eukaryotic expression plasmids were constructed, and ex-pressed in 293 cells, the protein band of 68, 45 and 31 kD were visualized by Western blotting respectively. Con-clusion: Flag-Plk1(K82R) and Flag-Plk1KD, Flag-Plk1PBD were expressed successfully in 293 cells, based on which, function of Plk1 on i

目的：克隆并在293细胞中表达人Polo样激酶1（Plk1）基因，探索Plk1对非受体型酪氨酸激酶c-Abl表达水平的影响。方法：利用PCR法扩增Plk1基因，分别定向克隆至pcDNA3-Flag及pCMV-Myc真核表达载体，将上述质粒分别转染293细胞进行瞬时表达，Western印迹检测Plk1蛋白的表达；将上述质粒分别与c-Abl表达质粒共转293细胞，检测带有不同标签的Flag-Plk1或Myc-Plk1对细胞中c-Abl激酶表达的影响。结果：构建了Flag-Plk1和Myc-Plk1真核表达质粒，其在293细胞中均可表达，蛋白的相对分子质量为68×103；与其共转的c-Abl激酶表达水平显著下调。结论：在293细胞中表达了Flag-Plk1和Myc-Plk1蛋白，且初步发现Plk1可以抑制c-Abl的表达。

Objective: To clone and express human Polo-like kinase-1(Plk1) gene in 293 cell lines, and study its effect on the expression of non-receptor tyrosine kinase c-Abl. Methods: Plk1 gene was amplified by PCR, and then inserted into eukaryotic expression vector pcDNA3-Flag and pCMV-Myc respectively. Those recombinant expression plasmids were transiently transfected into 293 cell lines, and the expression of Plk1 was identified by Western blotting. Different tagged Plk1 and c-Abl were cotransfected into 293 cells to identify the role of Plk1 on the expression of c-Abl kinase. Results: The Flag-Plk1 and Myc-Plk1 eukaryotic expression plasmids were con?structed, and expressed in 293 cell lines with the molecular weight of 68 kD. The expression of c-Abl reduced significantly when contrandfected with tagged Plk1 or c-Abl plasmids into 293 cells. Conclusion: Flag-Plk1 and Myc-Plk1 have been expressed in 293 cells, and it was found that Plk1 can inhibit expression of c-Abl.

目的探讨胃癌组织中Nek2、Plk1和Cdk1的表达及意义。方法采用免疫组化SP法检测80例胃癌组织中Nek2、Plk1和Cdk1的表达。结果 80例胃癌组织中Nek2、Plk1和Cdk1的阳性率分别为64%（51/80）、79%（63/80）、85%（68/80）,正常胃黏膜组织中Nek2、Plk1和Cdk1的阳性率分别为15%（3/20）、10%（2/20）、20%（4/20）,胃癌组织中Nek2、Plk1和Cdk1的表达水平明显高于正常胃黏膜组织,差异有统计学意义（P〈0.01）。Nek2表达与胃癌分化程度、浸润深度、淋巴结转移及TNM分期显著相关（P〈0.05）,与患者年龄、性别、肿瘤大小无明显相关性（P〉0.05）。Plk1、Cdk1表达与肿瘤大小、分化程度、浸润深度、淋巴结转移及TNM分期呈显著相关性（P〈0.05）,与患者年龄、性别无明显相关性（P〉0.05）。Nek2与Plk1、Plk1与Cdk1、Nek2与Cdk1的表达呈显著正相关（P〈0.001）。结论 Nek2、Plk1和Cdk1异常表达与胃癌的发生、发展密切相关,联合检测三者对胃癌的诊断、治疗及判断预后具有重要意义。

Purpose To detect the expression of Nek2, Plk1 and Cdk1 in gastric carcinoma and to explore their correlation with clini-copathological factors. Methods We used immunohistochemistry to detect the protein expression of Nek2, Plk1 and Cdk1 in 80 cases of gastric carcinoma. Results The positive rates of Nek2, Plk1 and Cdk1 expressions in 80 cases of gastric carcinoma were 64%, 79% and 85% respectively. While the positive rates of Nek2, Plk1 and Cdk1 expressions in 20 cases normal gastric tissue were 15%, 10% and 20% respectively,with statistical significance (P 0. 05). The expression of Plk1 and Cdk1 had a correlation with tumor size, differentiation of gastric cancer, invasion depth, lymph node metastasis and TNM stage (P 0. 05). There were significant associations between Nek2 and Plk1, Plk1 and Cdk1, Nek2 and Cdk1 expression (P <0. 01). Conclusions Abnormal expression of Nek2, Plk1 and Cdk1 might be one of the mechanisms of tumorigenesis, especially of abnormal tumour prolife

Plk1是一类高度保守的丝氨酸/苏氨酸蛋白激酶，在细胞周期调控中起重要作用。 Plk1在多种肿瘤类型中高表达，是肿瘤的生物标志物和肿瘤治疗靶点。 Plk1与一些重要的肿瘤相关蛋白相互作用并调节其功能，进而影响肿瘤的恶性行为，如肿瘤的增殖及侵袭转移等，所以与肿瘤的预后不良密切相关。目前已报道了 Plk1调控网络中下游信号通路及上游调控。 Plk1有望成为肿瘤免疫治疗的有效靶点。

Plk1 belongs to a class of highly conserved serine /threonine protein kinases that play crucial roles in cell cycle progression.It is overexpressed in a wide spectrum of cancer types and is a tumor marker and therapeutic target in oncology .Plk1 interacts with some important tumor-associated protein, regulates their function and influences their malignant behavior such as its effect on proliferation and invasion /metastasis of cancer cells.Therefore, it is closely related with poor prognosis.In recent years, the signaling pathways downstream and upstream regulation included in the Regulatory network of Plk1 has been reported.Plkl may possibly become the effective medical treatment for oncotherapy and immunotherapy.

目的检测POLO样蛋白激酶1(PLK1)和P53蛋白在卵巢上皮性癌中的表达,探讨其与患者预后的关系。方法采用免疫组织化学SP法检测20例正常卵巢组织、19例卵巢良性上皮性肿瘤组织和52例卵巢上皮性癌组织中PLK1和P53蛋白的表达情况,分析其与卵巢癌临床病理指标的关系及二者在卵巢癌组织中表达的相关性,单因素和多因素Logistic回归分析影响卵巢癌患者预后的危险因素,Kaplan-Meier法分析PLK1和P53表达与卵巢癌患者预后的关系。结果 PLK1和P53在卵巢上皮性癌组织中的表达分别为38.5%和67.3%,显著高于良性肿瘤和正常卵巢组织(P0.05)。在卵巢上皮性癌中,PLK1和P53异常高表达与临床分期和组织分化相关,临床分期越晚、组织分化越差的癌组织中PLK1和P53蛋白表达越高(P0.05);PLK1蛋白的表达与P53蛋白的表达呈负相关(r=-0.629,P=0.000)。单因素Logistic回归分析显示PLK1、P53、临床分期、组织分化和淋巴结转移是影响卵巢癌患者预后的因素,多因素Logistic回归分析显示仅PLK1是影响卵巢癌患者预后的独立因素(OR=2.288,P=0.025,95%CI:0.105~50.050)。与其他患者相比,PLK1表达阳性同时P53表达阳性的卵巢癌患者,生存期最短(?2=17.246,P=0.037)。结论 PLK1和P53共同参与了卵巢癌的发生发展,PLK1可作为判断卵巢癌患者预后的标志物。

Objective To detect the expression of PLK1 and P53 proteins in epithelial ovarian cancer, investigate their relationships with the progression of ovarian cancer. Methods The expression of PLK1 and P53 in 20 specimens of normal ovarian tissues, 19 specimens of benign epithelial ovarian tumor and 52 specimens of epithelial ovarian cancer was detected by immunohistochemistry SP method. Their correlations to the clinicopathologic characteristics of epithelial ovarian cancer and their interrelationships were analyzed. Risk factors of progression were discussed by Univariate and multivariate Logistic regression analysis, the relations between PLK1 and P53 expression and the prognosis were measured by Kaplan-Meier analysis. Results The positive rates of PLK1 and P53 proteins in epithelial ovarian cancer were 38.5% and 67.3%, significant differences were noted between epithelial ovarian cancer and normal ovarian tissues, benign ovarian tumors (P<0.05). In epithelial ovarian cancer, up-regulati