关键词： 肺肿瘤；细胞系；p53基因；转染；基因治疗

【摘要】目的观察外源性野生型p53基因对有p53基因突变的人肺癌细胞系生长的影响。方法用多聚酶链反应-单链构象多态性及DNA测序,选择p53基因突变的人肺巨细胞癌系801-D为受体细胞。构建野生型p53表达质粒PZIPneoSV-p53。用基因枪介导外源基因。建立转染细胞系801-D-p53。用聚合酶链反应检测外源基因，观察转染细胞恶性生长的变化。结果转染细胞系801-D-p53体外长期传代有neo基因及外源p53基因存在，转染细胞生长明显受到抑制，克隆形成抑制率达96％，裸鼠异种移植致瘤性降低，肿瘤生长明显缓慢。结论外源性野生型p53经基因枪导入有p53基因突变的人肺癌细胞后可长期存在于转染细胞中，且明显抑制转染细胞的恶性生长。

Effects of exogenous wild type p53 on malignant growth of human lung cancer cell line

Wang Hui, Lai Baitang, Li Jinzhao, et al. Cellular and Molecular Biology Lab, Beijing Thoracic Tumor Research Institute. Beijing 101149

【Abstract】ObjectiveTo study the effects of exogenous wild type p53 suppressor gene on malignant growths of human lung cancer cell line with mutant type p53 gene.MethodFour human lung cancer cell line were screened for mutations in the exon 5 through exon 8 of the p53 tumor suppressor gene with immunohistochemistry, polymerase chain reaction (PCR)/single strand conformation polymorphism (SSCP) and DNA sequence analysis. The recombinent plasmid PZIPneoSV-p53 was constructed, which express wild type p53 gene. A transfected cell line, 801-D-p53, was obtained after transferred the plasmid into 801-D cell line by gene gun mediated and selected by G418. The exogenous p53 in the transfected cell line 801-D-p53 were inspected with PCR, and the alteration of growths of the transfected cell line in vitro and in vivo was observed. ResultThe point mutation were CGG to CTT transversion at codon 248 in exon 7 was found in human lung cancer cell line 801-D by PCR-SSCP and DNA sequence analysis and nuclear accumulation of the p53 protein was observed. The neo gene and exogenous wild type p53 gene were detected in the transfected cell line 801-D-p53. The cell growth experiment in vitro showed that the parent cell line 801-D growth was very fast, from 1×10⁵/ml to 2.5×10⁵/ml within 6 days, the transfected PZIP-neo-SV cell line (plasmid without p53) growth as fast as 801-D, but the transfected cell line 801-D-p53 growth was inhibited seriously. The clonogenic formation rate of the parent cell line 801-D, transfected cell line 801-D-PZIP and the transfected cell line 801-D-p53 were 11.2%, 11.4% and 0.46% respectively. The clononogenic formation inhibition rate of the transfected 801-D-p53 was 96% comparing with the parent 801-D cell line. In the experiment of xenogenic tumor transplantation, each cell line was injected subcutaneously into four mice and the growth of xenogenic tumor transplant in nude mice was observed. xenograft growth in all 4 mice in both 801-D and 801-D-PZIP groups, but only 1 xenograft growth among 4 mice of 801-D-p53 group during 2 months. The average volume of xenogenic tumor transplant of 801-D and 801-D-PZIP groups were 6.500cm³ and 2.231 cm³ respectively and the only xenograft volume of 801-D-p53 group was 0.940 cm³. The tumorigenicity of 801-D-p53 in nude mice was significantly suppressed. ConclusionExogenous wild type p53 gene may stably exist in the human lung cancer cell line with mutant type p53 after plasmid transfection and suppressed the malignant growth of the transfected cell line 801-D-p53 in vitro and in vivo. These results indicate that the recombinent plasmid expressing wile type p53 may be useful for gene therapy of human lung cancer.

【Key words】Lung neoplasmCell linep53 geneTransfectionGene therapy

p53基因具有重要的抑癌功能^［1～3］，现已证实在多种肿瘤中有p53基因突变、缺失或两者兼有的异常。p53基因异常与肺癌尤为相关，大约50％的非小细胞肺癌、70％以上的小细胞肺癌均有p53基因的突变。因此，将外源野生型p53基因导入肿瘤细胞，以补偿或替代其功能，是一种可能逆转或抑制肿瘤细胞恶性生长的治疗癌症的新方法。该方面的研究国际上已有大量报道^［4～6］。我们用基因枪介导含外源性野生型p53基因的表达质粒于有p53突变的人肺癌细胞，并观察外源性野生型p53基因对癌细胞恶性生长的抑制。探讨用外源野生型p53基因治疗肺癌的可能性。

材料与方法

1.受体细胞的选择：人肺巨细胞癌系801和克隆系801-D由解放军总医院建立，人肺腺癌LTEPα₁、α₂，均由我室建立。生长培养基为二甲基亚砜(DMEM)＋15％新生牛血清。按常规方法提取细胞DNA。(1)多聚酶链反应-单链构象多态性(PCR-SSCP)：用扩增p53基因第5,6,7,8外显子的引物(美国Fox Chase癌症中心提供)，扩增上述4个细胞系和正常肺组织的DNA，按常规反应体系，经PCR(PE仪)扩增。扩增产物碱变性，5％聚丙酰胺凝胶电泳，溴化乙锭染色。电泳上有单链异常移动带为p53突变，以选择突变细胞及确定p53的突变外显子区。(2)DNA测序：用有p53突变的外显子区的单链DNA，经电洗脱回收纯化。采用fmol Tag DNA测序试剂盒(Promega公司产)，直接测序。凝胶于－70℃放射自显影后，所获结果与正常p53基因相应外显子区DNA序列比较，确定突变位点。(3)免疫组织化学染色：抗p53突变蛋白的单克隆抗体(Ab-2,Oncogene Science公司提供)。收集上述细胞悬液，制片。镜检细胞核浆成深黄色为阳性。检测转染细胞有无p53突变蛋白改变，用同样方法。

2.构建含野生型p53基因的重组质粒：含1.8 kb p53 C-DNA的pC53-SN质粒(美国Fox chase癌症中心提供)和缺陷型逆转录病毒PZIPneo-SV(北京市肿瘤所提供)，经用内切酶BamHI分别消化后，获得1.8 kb p53 C-DNA,并连接10kbPZIPneo-SV,筛选重组质粒。为了证明p53基因正向连接，用内切酶XhoI,BamHI，ACCI消化PZIPneo-SV和重组质粒PZIP-p53，经电泳酶切图分析确定。

3.基因枪介导外源基因：基因枪由清华大学生物工程系自制。质粒PZIPneo-SV和PZIP-p53(PZIPneo-SV-p53)分别加入到钨粉中，经超声分散轰击受体细胞以后，加入生长培养基，于CO₂暖箱培养。24小时后，加入每毫升800微克新霉素(geneticin G418)，待耐G418集落生长后，混合传代。

4.外源p53基因对人肺癌细胞恶性生长的影响：(1)生长曲线:分别接种3×10⁵母系细胞801-D和PZIP及PZIP-p53转染的细胞于培养瓶，每瓶加有3毫升培养液，每日各取三瓶计数。按平均数绘制细胞生长曲线。(2)集落形成试验:分别接种500个母系和转染的细胞(PZIPneo-SV及PZIP-p53)于平皿，CO₂暖箱培养2周，Giemsa染色，计数集落(每个集落至少含50个细胞)，计算集落形成率(集落形成率＝平均集落数／500×100％)和抑制率(抑制率＝对照组集落形成率－试验组集落形成率／对照组集落形成率)。(3)裸鼠异种移植试验：将5×10⁶的母系细胞和转染PZIP-p53细胞及PZIPneo-SV转染细胞分别接种于裸鼠皮下，各接种4只，从第1周起每周测量肿瘤直径，按DDCHMAHI方法计算肿瘤体积(体积＝πØ3／6，Ø为肿瘤直径)。

5.转染细胞中外源基因的检测：用PCR检测转染细胞中的neo基因及p53 c-DNA。(neo基因及p53 C-DNA引物由上海Sangon公司合成)。p53 C-DNA引物为5＇-CAT ATG TGT AAC AGT TCC TGC ATG-3＇，5＇-CG GGA TCC CCC AGC CTG GGC ATC CTT GAG-3＇可扩增人源p53 C-DNA含1318-1765基因区域的461bp基因片段。用以上引物分别对30代转染细胞和母系细胞的DNA进行PCR检测行聚丙酰胺凝胶电泳。

结果

一、受体细胞p53基因检测

经PCR-SSCP检测4个人肺癌细胞系和正常肺组织。结果显示人肺巨细胞癌系801及克隆系801-D、p53基因仅第7外显子出现单链构象多态性改变(图1)，用p53第7外显子扩增产物直接测序，发现248氨基酸密码有碱基突变，由CGG发生CTT颠换(图2)。免疫组化染色801-D细胞P53突变蛋白呈阳性反应。正常肺组织及其它两个人肺腺癌细胞系的p53,5,6,7,8外显子均未发现有突变。故选用有p53基因突变的801-D细胞为本研究转基因的受体细胞。用以观察外源野生型p53介导入该细胞后，能否影响有突变的细胞的恶性生长。

图1PCR/SSCP分析人肺癌细胞系p53基因突变(1)人肺腺癌细胞系L