【作者】 闫炳智；

【导师】 王洁；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2005， 博士

【摘要】 目的:涎腺腺样囊性癌（salivary adenoid cystic carcinoma,SACC）是较为常见的涎腺恶性肿瘤之一。由于其侵袭性广,尤其是嗜神经并沿其逐渐扩散,易血行转移,术后易于复发。该肿瘤对放疗、化疗不敏感,所以远期疗效不佳。为了探讨p53基因在临床治疗涎腺腺样囊性癌的可行性,本实验以涎腺腺样囊性癌细胞SACC-83为对象观察了腺病毒载体介导的外源性野生型p53基因对其端粒酶活性及转录调节的影响以及对SACC-83细胞生物学特性的作用;检测SACC-83细胞中DNA polβ启动子的活性;并对不同启动子调控的p53基因表达的变化和p53基因在引起DNA损伤的各种刺激因素下的反应做初步的探讨,旨在为开展体内涎腺腺样囊性癌的p53基因治疗提供新的途径及理论依据。方法:将外源性野生型p53基因片段（1.8 kb）重组于腺病毒表达载体pΔE1构建真核表达载体pΔE1-p53;以Tfx^TM-20脂质体法瞬时转染涎腺腺样囊性癌细胞SACC-83,并以转染空载体p?E1及未转染细胞为对照。RT-PCR检测外源性野生型p53基因mRNA的表达,TRAP-PCR-ELISA法（端粒重复序列扩增法-酶联免疫试验）定量分析转染涎腺腺样囊性癌细胞SACC-83端粒酶活性的变化情况;构建含有端粒酶逆转录催化亚单位hTERT启动子核心调控区-586bp～+35bp片段（630bp）的荧光素酶报告重组体pGL2-630,将pΔE1-p53、pGL2-630瞬时共转染SACC-83细胞,转染中以β-半乳糖苷酶表达质粒做共转染,以便校正转染效率,48h后收获细胞,液体闪烁计数仪检测荧光素酶报告基因活性,酶标仪分析β-gal活性,计算荧光素酶活性与β-gal活性的比值,得到相对荧光素酶活性,分析外源性p53基因表达对hTERT转录调节的直接作用。脂质体法将外源性野生型p53基因稳定转染涎腺腺样囊性癌细胞SACC-83,以稳定转染空载体pΔE1的SACC-83细胞及未转染的SACC-83细胞为对照。400μg.ml^-1 G418筛选4周,挑选稳定单个克隆并逐步扩大培养;同样RT-PCR检测外源性野生型p53基因的表达以及p53基因对端粒酶逆转录催化亚单位hTERT mRNA表达的影响;流式细胞术分析更多还原

【Abstract】 Objective: Salivary adenoid cystic carcinoma （SACC） is a very common malignancy of salivary gland. Recurrence and early metastasis, with failure radiotherapy and chemotherapy are its biological properties. To evaluate the inhibitory effect of p53 gene on adenoid cystic carcinoma cells, telomerase activity and the transcriptional activity of hTERT were measured in this experiment. The activity of DNA pol βpromoter in SACC-83 cells and the changes of biological properties of SACC-83 cells were investigated and the expression of p53 under different promoters was studied. Methods: Human wild-type p53 gene was constructed into adenoviral vector pΔE1. Lipofection-mediated gene transfection method was used to transfect pΔE1-p53 into SACC-83 cells transiently. The cells with transfection of empty vector pΔE1 and without transfection were used as controls. The expression of p53 mRNA was assessed by reverse transcriptase polymerase chain reaction （RT-PCR）. The activity of telomerase was examined by TRAP-PCR-ELISA. Then, hTERT gene promoter-luciferase reporter vector pGL2-630 was constructed and transfected into SACC-83 cells with p?E1-p53, and also β-gal was transfected simultaneously to rectify the efficiency of transfection. After 48 hours, the luciferase activity was examined and used to evaluate the effect of p53 on transcriptional activity of hTERT promoter. Human wild-type p53 gene was stably transfected into SACC-83 cells with lipofection-mediated gene transfection method. Controls were the cells with transfection of empty vector pΔE1 and the cells without transfection. G418 (400 μg.ml^-1) was used to select the monoclones in 4 weeks. The expression of p53 mRNA and hTERT mRNA were assessed by reverse transcriptase polymerase chain reaction （RT-PCR）, and the effects of transfected p53 on SACC-83 cells were analyzed by TRAP-PCR-ELISA, luciferase reporter, flow cytometry （FCM）, soft agar assay and tumorigenicity test. In the experiment, 15 BLAB/c nude mice were randomly divided into 3 groups of 5 mice each and were respectively injected with different SACC-83 cell suspension as mentioned above. After 30 days, the sizes of transplanted tumors were measured and the tumorigenicities in nude mice were also accounted. Pathological changes were observed with routine tissue immobilization and dye process. In the whole experiment, x±s and t test were used. DNA pol βpromoter-luciferase reporter vector pGL2-B was transfected into SACC-83 cells. The luciferase activity was examined and used to evaluate the activity of DNA pol βpromoter in SACC-83 cells. Eukaryotic expression plasmids of p53 gene with different promoters （CMV promoter and DNA pol βpromoter） were constructed and stably transfected into SACC-83 cells. G418 (400 μg . ml^-1) was used to select the monoclones. Controls were the cells with transfection of empty vector pcDNA3 and the cells without transfection. After transfection, RT-PCR was used to identify each clone and assess the expression of p53 with different promoters. The SACC-83 cells were subjected to the treatments of H₂O₂, ultraviolet radiation, BLEOCIN, and affected p53 mRNA and protein level in SACC-83 cells were characterized with RT-PCR and Western Blotting. Results: Eukaryotic expression plasmid of p53 gene （p?E1-p53） and hTERT gene promoter-luciferase reporter vector pGL2-630 were constructed. After gene transfection, the level of p53 mRNA was increased and the activity of telomerase was decreased obviously （0.805）. The luciferase activity was also depressed （10.5%） after the co-transfection of p?E1-p53 and pGL2-630. The results proved that foreign p53 gene might depress the activity of telomerase in SACC cells through inhibiting the transcriptional activity of hTERT promoter. Plasmids p?E1-p53 were stably transfected into SACC-83 cells. RT-PCRproved that human wild-type p53 gene were transfected into SACC-83 cells successfully and expressed effectively. But the differences between test group and the controls were obvious. Compared to SACC-83 cells transfected with p?E1 and those untransfected, the expression of hTERT mRNA was decreased obviously in the cells transfected with pΔE1-p53. The percentage of cell distribution in G₁ phase, S phase and G₂ phase was 74.43%, 15.44% and 10.13% respectively in experiment group, but in control groups, the percentage was 54.75%, 34.92%, 10.33% and 53.27%, 35.86%, 10.87% respectively. The cells cycles of transfected SACC-83 were arrested in G1 phase. The cell number increased in G₁ phase, decreased in S phase and had no change in G₂ phase. Compared to the control groups, the cells transfected with p53 grew more slowly in soft agar, the cell colonies appeared late and the number was small. Furthermore, the colony size was different: the colony sizes in the test group were smaller than those in the control groups. The number of cell colonies was 26.6±0.94, 24.7±1.24 and 15.56±0.82, and colony-formation efficiency was 8.9% , 8.2% and 5.2% respectively. The result showed that the introduction of wild-type p53 by adenoviral vector could decrease the ability to form colony in soft agar of SACC cells. The difference between cells transfected with p53, cells transfected with pΔE1 and cells untransfected were obvious （P<0.01）. After the transfection of p53 gene, the growth of the transplanted tumor in nude mice became slowly and late. The tumor volumes of negative group, test group and blank group were 250.60±28.21, 63.00±77.18 and 263.20±38.36, and the weights were 0.25±0.02, 0.06±0.07 and 0.26±0.03. The volume and the weights were all smaller in the control groups than in the p53 transfecting cells group（P<0.01）. Human wild-type p53 gene could inhibit SACC-83 cells growth and decrease the tumorigenicity in nude mice. The statistical results suggested that the introduction of wild-type p53 by adenoviral vector could suppress malignant phenotypes of SACC cells.更多还原