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染色体不安定性に着目した大腸癌の抗癌剤耐性機序の解析

以染色体不稳定性为中心的结直肠癌抗癌耐药机制分析

基本信息

批准号：
22H03060
负责人：
高山哲治
金额：
$ 11.23万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2022
资助国家：
日本
起止时间：
2022-04-01 至 2025-03-31
项目状态：
未结题

项目摘要

大腸癌患者よりpatient-derived organoid(PDO)を樹立し、以下の解析を行なった。1) 大腸癌患者からの治療前及び耐性獲得後のPDOの樹立：未治療の進行大腸癌(Stage IV) 58例よりPDOを樹立した。また、１次治療を行い効果判定でPD(進行)が認められた時点で再度内視鏡検査を行い耐性獲得PDOを４例樹立した。2) PDOにおける染色体分析とCIN-scoreの評価：治療前PDO25例と耐性PDO4例を用いて染色体Gバンド分析を行なった。また、全ゲノム解析データを基に染色体不安定性の定量的評価法を行い、Chromosomal Instability (CIN)-scoreを算出した。治療前PDO25例中CIN-high 10例、CIN-mediate 6, CIN-low 9例であった。耐性獲得後のPDO4例ではCIN-low 2例、CIN-mediate 2例であった。3) 治療前及び耐性PDOの染色体不安定性候補遺伝子の抽出：治療前のPDO25例の全ゲノム解析データを用いて、CIN-score highの治療前PDOのゲノム解析データからアミノ酸変異を伴う変異遺伝子を抽出し、CIN-score-lowのPDOの変異遺伝子を差引いて候補遺伝子を56個を絞り込んだ。このうち、文献検索などを行いCIN候補遺伝子を10個に絞り込んだ。4) PDOにおけるα-tubulin/cadherin1の蛍光標識：CIN-score lowの治療前PDOを対象に、染色体分の微小管を構成するα-tubulinをGFP-Puroで、細胞膜のcadherin1をRFP-Puroでtaggingした。高速レーザー共焦点顕微鏡を用いて、細胞分裂を多色タイムラプス観察できることを確認した。念の為、別のより強い蛍光色素を用いてHistoneH2bを標識したPDOも作製した。

The patient-derived organoid (PDO) of the patients with cancer in Dazhou is abnormal, and the following analysis will be performed. The main results were as follows: 1) before and after treatment, 58 patients with major cancer (Stage IV) were diagnosed with PDO before and after treatment: 58 patients with PDO before treatment and 58 patients with PDO without treatment. The results of one-time treatment and one-time treatment were used to determine the time point of PD. 4 cases of PDO were diagnosed by endurance test at the same time. 2) PDO chromosome analysis, CIN-score chromosome analysis: before treatment, PDO25 cases, tolerance PDO4 cases, chromosome G analysis, chromosome analysis. Chromosomal Instability (CIN)-score method for quantitative analysis of chromosomal uneasiness and quantitative analysis. Before treatment, there were 10 cases of CIN-high, 9 cases of CIN-mediate 6 and 9 cases of CIN-low in PDO25. There were 4 cases of PDO, 2 cases of CIN-low and 2 cases of CIN-mediate. 3) pre-treatment and tolerance PDO chromosomal instability phenotypes were extracted: before treatment, the PDO25 cases were completely analyzed. Before the treatment, the PDO samples were analyzed. Before the treatment, the PDO symptoms were analyzed. The acid symptoms were extracted with the cures, and the CIN-score-low samples were extracted from the chromosomal instability cells. 56 samples were collected from the patients before treatment. There are 10 copies of the CIN waiting list in the literature. 4) PDO diagnosis alpha-tubulin/cadherin1 marker: before the treatment of CIN-score low, the PDO image, the chromosomal division of microtubules into alpha-tubulin GFP-Puro, and the membrane cadherin1 RFP-Puro tagging imaging. The high-speed signal is in common focus, and the cell division is polychromatic and multicolor. It is thought that light pigments are not required to use the HistoneH2b label, PDO, as an example.