Abstract: Objective More and more attention has been given to the presumption that glioma stem cells (GSCs) originate from neural stem cells (NSCs) with gene mutation, however, there is no enough evidence by now. This paper aims to get evidence of this area. Methods Glioma stem/progenitor cells (GSPCs) and neural stem/progenitor cells (NSPCs) were cultivated in vitro, and were identified before the following studies. Total RNA was isolated and then reverse-transcribed into cDNA, with primers specific to phosphatase and tensin homolog deleted onchromosome ten (PTEN), high-fidelity Taq polymerase was used for the polymerase chain reaction (PCR) to avoid the incorporation of pseudomutation. After amplification, 10 μl of the reaction mixture was electrophoresed through 1.5% agarose gel, and the rest of the reaction mixture was used for sequencing in both directions. The procedures for the isolation of total RNA to PCR and sequencing were repeated twice, and the sequencing results of both DNA and PTEN peptide chain were analysed with DNAssist 1.0 software and compared to the sequence of wild-type Homo Sapiens PTEN in GenBank. Results No mutation happened in the PTEN of NSPCs, but there were many base mutations in the mRNA of PTEN of GSPCs compared with the wild-type Homo Sapiens PTEN. Though most of these mutations were same sense mutation, still several mutations were not, including the normal DNA bases of PTEN bases 22 to 42 "ATCGTTAGCAGAAACAAAAGG" in first exon mutated into "CTACGATTGATTTGCATCTTT", base 712 "T" in exon 7 mutated into "C", and base 1192 "A" in exon 9 mutated into "T". Accordingly, for the amino acids (AA) sequence in the peptide chain of PTEN, the mutation included AA from the 8th to the 14th (from "IVSRNKR" to "LRLICIF"), the 238th AA (from "F" to "L"), and the 398th AA (from "T" to "S"). These mutated regions were involved in membrane interaction, particularly the combination with phosphatidylinositol 4, 5-biphosphate (PIP2) and maintaining the protein stability of PTEN. Therefore, these mutations not only lead to the rapid degradation of PTEN, but also hinder the cellular function of PTEN to down-regulate phosphoinostide 3-kinase (PI3K) signaling. Conclusion The mutation of PTEN occurs even in the early stage of malignant transformation, which is probably an initiating agent for the tumorigenesis of gliomas.

Key words: Glioma, Neoplastic stem cells, Genes, tumor suppressor, Mutation, Polymerase chain reaction

摘要： 目的   为胶质瘤干/祖细胞来自神经干/祖细胞基因突变的推测寻找实验证据，以阐明二者之间的关联性。方法   体外培养的胶质瘤干/祖细胞和神经干/祖细胞，经免疫细胞化学法鉴定后分别进行RNA 提取、逆转录生成cDNA，以及针对PTEN 的逆转录-聚合酶链反应；扩增产物进行正反双向测序并与野生型PTEN 基因序列比较肽链序列的异同。结果   神经干/祖细胞PTEN mRNA 序列无变异，胶质瘤干/祖细胞存在大量同义突变和少量异义突变，主要包括第1 号外显子第22 ~ 42 位碱基突变、第7 号外显子第712位碱基突变和第9号外显子第1192位碱基突变。致使PTEN 肽链上第8 ~ 14位氨基酸由原来的“IVSRNKR”突变为“LRLICIF”，第238 位的“苯丙氨酸（F）”突变为“亮氨酸（L）”，第398 位的“苏氨酸（T）”突变为“丝氨酸（S）”。其中第8 ~ 14 位和第238 位氨基酸突变，使PTEN 与细胞膜的结合能力下降，细胞外信号难以转导进入细胞内；第398 位氨基酸突变则使PTEN 稳定性破坏，易被降解。结论   PTEN基因在胶质瘤恶性转化的早期即已失活，可能是胶质瘤形成的始动因素之一。

关键词: 神经胶质瘤, 肿瘤干细胞, 基因, 肿瘤抑制, 突变, 聚合酶链反应