真空導入法を用いた3次元配線電極付きマイクロ流体デバイス

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 竹内 昌治, 東京大学教授 岡田 慧, 東京大学教授 金 範埈, 東京大学准教授 高橋 宏知, 東京大学准教授 小穴 英

Solid-phase multiple displacement amplification for multi-locus genotyping of single chromosome molecules

(1) Background: Despite recent innovations in high throughput shotgun sequencing technologies, complex rearrangements in addition to the original dizygotic existence of homologous chromosomes complicate cancer genome sequencing. Thus there is an urgent need for establishing a convenient method to manipulate long stretches of individual chromosomes for analyzing sequence information.(2) Materials and Methods: A novel methodology has been developed to amplify single chromosomes for genotyping. A key feature of this methodology is a solid-phase multiple displacement amplification, that is an enzymatic reaction of Phi29 DNA polymerase, within a solidified agarose gel. It consists of the following seven steps. (I) Lysis of a limited number of cultured cells within a heated agarose gel solution to release chromosome molecules. (II) Careful aliquoting of small volumes of gel solutions containing limited number of chromosome molecules. (III) Solidification of the gel on ice. (IV) Solid-phase multiple displacement amplification of the gel-immobilized individual chromosome molecules. (V) Recovery of the amplification products by heating. (VI) Screening of target chromosomes by real-time QPCR. (VII) Multi-loci SNP typing using newly developed on-plastic chip allele-specific primer extension method (Michikawa et al., Anal. Sci., 2006, 22, 1537-1545). (3) Results: Utilization of agarose gel as a reaction matrix enabled reliable amplification-ready limited dilution of DNA to the level that homologous chromosomes rarely co-localize. Aggregation of chromosomes during the dilution step was dramatically reduced by incubating the gel solution at alkaline pH and at high temperature. Separation of homologous chromosomes by this method provided reliable determination of multi-loci genotypes on each homologous chromosome. Using this methodology, we have successfully determined haplotype of multiple SNPs in human ATM region that span 200 kilobase pairs. (4) Conclusions: The methodology developed in this study is effective for genotyping individual homologous chromosomes. Since amplified materials are easily recovered in a solution as PCR-ready form, this methodology is not restricted for genotyping. Further applications, such as chromosome-wide sequencing, are considerable.The 14th European Cancer Conference, ESTRO26 meeting, European Society for therapeutic radiology and oncolog

Combination effects of distinct cores in 11q13 amplification region on cervical lymph node metastasis of oral squamous cell carcinoma

Lymph node metastasis (LNM) in oral squamous cell carcinoma (OSCC) is known to associate with a significant decrease of 5-year survival. Genetic factors related to the difference of the LNM status in the OSCC have been not fully elucidated. Array-based comparative genomic hybridization (CGH) with individual gene-level resolution and real-time quantitative polymerase chain reaction (QPCR) were conducted using primary tumor materials resected from 54 OSCC patients with (n=22) or without (n=32) cervical LNM. Frequent gain was observed at the 11q13 region exclusively in patientswith cervical LNM, which was confirmed by real-time QPCR experiments using 11 genes (TPCN2, MYEOV, CCND1, ORAOV1, FGF4, TMEM16A, FADD, PPFIA1, CTTN, SHANK2 and DHCR7) in this region. It was revealed that two distinct amplification cores existed, which were separated by a breakpoint between MYEOV and CCND1 in the 11q13 region. The combination of copy number amplification at CTTN (core 2) and/or TPCN2/MYEOV (core 1), selected from each core, was most significantly associated with cervical LNM (P=0.0035). Two amplification cores at the 11q13 regionmay have biological impacts on OSCC cells to spread from the primary site to local lymph nodes. Further study of a larger patient series should be conducted to validate these results

Whole genome-wide screening of cervical lymph node metastasis-associated genetic alterations in oral squamous cell carcinoma of Japanese patients

To precisely identify genetic markers that reflect occurrence of cervical lymph node metastasis (LNM) in Japanese oral squamous cell carcinoma (OSCC) patients, oligonucleotide array-based comparative genomic hybridization (CGH) with individual gene-level resolution and real-time quantitative polymerase chain reaction (QPCR) was conducted using primary tumor materials surgically resected from total of 54 OSCC patients with or without cervical LNM (LNM present, n = 22; LNM absent, n = 32). Frequent gain was observed at 11q13 region exclusively in the patient group with cervical LNM using array-based CGH. This observation was confirmed by real-time QPCR experiments designed for 11 genes (TPCN2, MYEOV, CCND1, ORAOV1, FGF4, TMEM16A, FADD, PPFIA1, CTTN, SHANK2 and DHCR7) in this region. Moreover, we refined a comprehensive physical map of the 11q13 amplification region including 33 kb of DNA sequence covering this region and found a novel breakpoint between MYEOV and CCND1. There were hypothetical two cores in 11q13 region. Combination of copy number amplification at CTTN and/or TPCN2/MYEOV, which were selected from each core separated by novel breakpoint, was most significantly associated with cervical LNM (P = 0.0035). Following detailed assessment of selected loci by real-time QPCR used to be quite adequate strategy for finding out genetic markers in regions with complicated alterations. Further study with larger patient numbers should be conducted to validate this result.The 14th European Cancer Conference, ESTRO26 meeting, European Society for therapeutic radiology and oncolog

Novel condylar repositioning method for 3D-printed models

Abstract Background Along with the advances in technology of three-dimensional (3D) printer, it became a possible to make more precise patient-specific 3D model in the various fields including oral and maxillofacial surgery. When creating 3D models of the mandible and maxilla, it is easier to make a single unit with a fused temporomandibular joint, though this results in poor operability of the model. However, while models created with a separate mandible and maxilla have operability, it can be difficult to fully restore the position of the condylar after simulation. The purpose of this study is to introduce and asses the novel condylar repositioning method in 3D model preoperational simulation. Methods Our novel condylar repositioning method is simple to apply two irregularities in 3D models. Three oral surgeons measured and evaluated one linear distance and two angles in 3D models. Results This study included two patients who underwent sagittal split ramus osteotomy (SSRO) and two benign tumor patients who underwent segmental mandibulectomy and immediate reconstruction. For each SSRO case, the mandibular condyles were designed to be convex and the glenoid cavities were designed to be concave. For the benign tumor cases, the margins on the resection side, including the joint portions, were designed to be convex, and the resection margin was designed to be concave. The distance from the mandibular ramus to the tip of the maxillary canine, the angle created by joining the inferior edge of the orbit to the tip of the maxillary canine and the ramus, the angle created by the lines from the base of the mentum to the endpoint of the condyle, and the angle between the most lateral point of the condyle and the most medial point of the condyle were measured before and after simulations. Near-complete matches were observed for all items measured before and after model simulations of surgery in all jaw deformity and reconstruction cases. Conclusions We demonstrated that 3D models manufactured using our method can be applied to simulations and fully restore the position of the condyle without the need for special devices