Molecular cloning of the genes related to the early stage of the peripheral type of lung adenocarcinomas

在本研究中，我们试图通过早期肺癌鉴别筛查，寻找一些可能与肺腺癌发生发展相关的功能分子。近年来，周围性肺腺癌的早期标本已可用于内镜下肺癌切除术。我们收集了40例肺小肿瘤及其未受影响的正常肺组织。详细的组织学分析显示，这些样本分为细支气管肺泡腺癌和浸润性腺癌。提取总RNA，用罗丹明标记的oligo-d(T)进行逆转录，与24种正向引物联合进行PCR。组合总数为216。样品在测序凝胶上电泳。用荧光生物图像分析仪（FMBIO-II Multi View, Takara, Kyoto）鉴定感兴趣的条带，并从凝胶中剪切。从凝胶中分离cDNA，进行纯化并直接测序。第一步克隆完成后，共有323个克隆被应用于直接测序。最后对255个无性系进行进一步分析，其中163个无性系具有poly(A)^+， 92个无性系不具有poly(A)^+。现在我们利用BLAST分析方法在这些克隆中寻找感兴趣的候选克隆。在本研究中，我们还分析了11l -10基因在一些非小细胞肺癌（NSCLC）中的表达。我们认为IL-10的表达与NSCLC患者的临床预后有关，也与血管生成素系统（ang1, ang2, angiopoietin Receptor）的基因表达有关。

In this study, we tried to identify some functioning molecules which would be related to carcinogenesis and development of lung adenocarcinomas by way of differential screening of early lung cancer. Recently, the specimens of the early stage of peripheral lung adenocarcinomas have been available as endoscopic resection of lung cancer are performed. We collected 40 cases of small lung tumors and their unaffected normal lung tissues. Detail histological analysis revealed that these samples are categorized as bronchioloalveolar adenocarcinomas and invasive adenocarcioma. Total RNA was extracted, reverse-transcribed with Rhodamine-labeled oligo-d(T) and applied for PCR in combination with 24 kinds of forward primers. Total number of combination is 216. The samples were electrophoresed on a sequencing gel. The interested bands were identified by fluorescent bioimage analyzer (FMBIO-II Multi View, Takara, Kyoto) and cut out from the gel. The cDNA was isolated from the gel, purified and directly sequenced. After the first step of cloning, total 323 clones were applied for direct sequencing. Finally 255 clones(163 clones possess poly(A)^+ and 92 clones do not possess poly(A)^+) were taken for the further analysis. Now we are searching the interested candidates in these clones by using BLAST analysis method. In this study we also analyze 1L-10 gene expression in some non-small cell lung cancers (NSCLC). We concluded that IL-10 expression was related to the clinical prognosis of NSCLC patients and also related to the gene expression of angiopoietin system (Ang-1, Ang-2, Angiopoietin Receptor)