组织中少量细胞的微卫星长度精细分析

Precise Analysis of Microsatellite Length in Small Amounts of Tissue Cells

微卫星是基因组上的特殊短重复序列,微卫星不稳定的程度与一些肿瘤的分型、治疗和预后相关。目前,微卫星长度变化的检测往往是基于大量细胞,检测灵敏度低。本文整合激光显微切割技术,基于多次退火环状循环扩增(multiple annealing and looping-based amplification cycles,MALBAC)的单细胞全基因组放大技术和毛细管电泳长度测量方法,经过关键技术点的改进,建立了一套针对组织中少量细胞的多微卫星位点检测方法。研究结果表明,基于技术改进,HE染色的组织细胞经激光显微切割分选后,可成功用MALBAC技术进行全基因组放大,并在多微卫星位点的检测上,获得了高度的准确性和重复性。利用所建立的方法,发现肠型胃癌早期病变组织-肠上皮化生(intestinal metaplasia,IM)的单个腺体内部出现多种长度变化的微卫星改变,说明该癌前病变组织中DNA错配修复系统已经出现问题。本方法所获得的全基因组放大产物,也可以用于外显子组测序和全基因组测序。另外,该方法适用于任何组织的少量细胞,甚至单细胞的多微卫星位点检测,以及全基因组的研究,为精细研究少量病变细胞的基因组特征以及组织异质性提供了有效的方法。

Microsatellites are special short repetitive DNA sequences of length variation. Microsatellite instability has been linked to the classification and prognosis of cancers. Currently,the method for detecting microsatellite lengths is limited in sensitivity and often requires large number of cells. We integrated laser microdissection,multiple annealing and looping-based amplification cycles（ MALBAC）,and adopted capillary electrophoresis to establish a technology allowing analysis of multiple microsatellite length using small amounts of cells. After optimizing the sample collection and cell lysis processes,the HE stained tissues were isolated with laser micro-dissection,subjected to amplification with MALBAC single cell whole genome assay. The lengths of microsatellite markers were determined with capillaryelectrophoresis. The lengths of microsatellite markers of five sub-segments（ one sub-segment contains about 5 cells） from individual gastric glands indicated that the method satisfied the accuracy and reproducibility. Individual intestinal metaplastic（ IM） glands at pre-cancerous stage were analyzed for variations. The detected microsatellite instability suggested defectives in the DNA mismatch repair system. Our established method is useful for microsatellite studies,and the amplified products can be further analyzed in exon or whole genome sequencing.