重组腺病毒-p53基因治疗患者基因变异和血清抗体检测及其临床意义

目的检测应用重组人腺病毒-p53（SBN-1）基因抗癌注射液的临床试验病例的机体免疫反应和组织样本p53基因变异状况,以客观评价临床疗效.方法通过对22例SBN-1治疗前后患者肿瘤组织样本的p53基因进行分析,明确用于重组腺病毒p53基因联合放疗二期临床试验的肿瘤标本中p53基因的变异状况并检测癌症患者血清中抗SBN-1抗体水平;用免疫组织化学技术检测p53基因的表达水平;显微切割技术分离微量肿瘤组织制备DNA进行聚合酶链反应扩增;变性高效液相色谱技术（DHPLC）和DNA测序技术检测内源性p53基因突变的分布和类型.采用酶联免疫吸附实验检测血清中抗腺病毒抗体水平、血清中腺病毒IgG和IgM抗体水平.结果有p53突变的病例中完全缓解率较高,通过免疫组织化学检测阳性率为40%（6/15）,DHPLC和DNA测序检测点突变为32%（7/22）.实验结果也表明试验者绝大多数近期未感染过腺病毒,IgM阳性率低于6%（1/16）,接受SBN-1治疗的患者随注射SBN-1次数增加,血清中抗SBN-1特异性抗体水平升高.结论通过对入组病例临床样本p53基因变异和SBN-1抗体水平的检测,为客观评价临床疗效提供了基因和蛋白水平的指标.

Detection of p53 gene change and serum antibody level in phase II clinical trial of ad p53 gene therapy

Objective To determine the alteration of p53 gene in tumor tissues and to monitor the immunoresponsiveness to anti adenovirus in patients following treatment of recombinant adenovirus-p53 (Adp53, SBN-1) combined with radiation. Methods Tumor tissues were collected from 22 patients with malignant tumors at advanced stage before and after treatment with recombinant adenovirus-p53 (Adp53, SBN-1) combined with radiation. Immunohistochemistry was used to detect the p53 expression. Microdissection, PCR, and denaturation high performance liquid chromatography (DHPLC), and DNA sequencing were used to detect the mutation of p53 gene. The levels of the anti-adenovirus antibody SBN-1, IgG and IgM in the serum were detected by using ELISA. Results p53 positive staining was shown in the nuclei of tumor cells in 6 of 15 tumor samples showing the increase of stability of protein caused by gene mutation. DHPLC and DNA sequencing showed point mutation in 7 of 22 tumor samples. Six of the 7 cases with p53 gene mutation showed complete and partial remission at the degree >50%, and the 8 cases without p53 gene mutation only showed partial remission or stability of condition. The IgG positive rate was 50% (8/16), showing that 50% of the patients had been infected by adenovirus (mainly the types 3, 7, and 11) before. The IgM positive rate was 6% (1/16), showing that most of the patients had not been infected by adenovirus (mainly the types 3, 7, and 11) at that time. The anti-SBN-1 antibody was negative in all patients before the rAd-53 therapy and became positive since the 4th week after the beginning of treatment and the intensity of positivity increased along with the time. Conclusion An effective gene diagnosis system for detecting the p53 gene alteration has been developed.