重组野生型p53基因转染人骨肉瘤细胞株体外实验

THE IN VITRO STUDY OF HUMAN OSTEOSARCOMA CELL LINETRANSFECTED BY RECOMBINANT WILD-TYPE p53 PLASMID

目的：将外源性野生型ｐ５３基因导入人骨肉瘤细胞株，研究野生型ｐ５３蛋白在诱导瘤细胞凋亡中的作用。方法：用ＤＮＡ转染技术将７重组野生型ｐ５３基因ＰＭ４７质粒导入人骨肉瘤细胞株ＨＯＳ８６０３中，用ＰＣＲ技术监测外源性ｐ５３基因在瘤细胞中存在的稳定性；用免疫组化方法检测在地塞米松的诱导下瘤细胞中ｐ５３的表达情况；用相差显微镜去观察凋亡瘤细胞的形态特点；用细胞凋亡原位检测技术标记并统计凋亡的瘤细胞数。结果：成功地将重组野性型ｐ５３基因ＰＭ４７质粒导入人骨肉瘤细胞株ＨＯＳ８６０３中，在ｇｐｔ选择培养基中培养两周后，分离生长出阳性细胞克隆；经ＰＣＲ证实外源性ｐ５３基因在瘤细胞内稳定存在并可随瘤细胞分裂而传给子代；当向培养液中加入１μｍｏｌ／Ｌ地塞松（Ｄｅｘ）后，８ｈ即可用免疫组化方法检测到ｐ５３蛋白的表达并可观察到瘤细胞出现凋亡的一系列形态学改变，转染后的瘤细胞凋亡率高达９０％。结论：当人骨肉瘤细胞株中重组野生型ｐ５３基因诱导过表达时，瘤细胞呈现凋亡，本实验为骨肉瘤的基因治疗提供了理论依据。

Objective: Exogenous wild-type p53 gene was transfected into human osteosarcoma cell line to investigate the roles of p53 protein in inducing apoptosis. Methods: DNA transfection techniques were used to introduce recombinant wildtype p53 plasmid, PM 47, into human osteosarcoma cell line HOS8603. PCR was used to monitor the stability of PM 47 in the tumor cells. Under the induction of 1μmol/L dexamethasone, immunohistochemical method was used to detect the overexpression of exogenous wildtype p53 protein. Morphologic features of apoptotic tumor cells were observed under phase contrast microscope. The apoptotic tumor cells were labelled in situ and counted. Results: Exogenous wildtype p53 gene was transfected into human osteosarcoma cell line successfully. After shifting to selective medium for 2 weeks, individual gpt+ cell colonies were isolated. Exogenous wildtype p53 gene was verified in the osteosarcoma cell line and could be transmitted to daugher cells through polymerase chain reaction amplifing the sixth exon of p53 gene. The overexpression of p53 protein in transfected tumor cells was detected by immunohistochemical method under the induction of dexamethasone for 8 hours and a series of morphological features of apoptosis were observed. Apoptotic rates in transfected tumor cells approximately accounted for 90 percent. Conclusion: After inducing overexpression of exogenous wild-type p53 gene in transfected osteosarcoma cell line, the tumor cells demonstrated apoptosis. This provided experimental clues for gene therapy of patients with osteosarcoma.