COMPARATIVE TRANSCRIPTOMIC AND PROTEOMIC ANALYSIS OF LGR5+ve STEM CELLS AND THEIR DAUGHTERS (AFFYMETRIX ARRAYS)

The identification of Lgr5 as an intestinal stem cell marker has made it possible to isolate and study primary intestinal stem cells. Applying quantitative mass spectrometry as well as transcriptomic analysis, we profiled the protein and gene changes between FACS-sorted Lgr5+ve stem cells and their immediate undifferentiated daughter cells. The overall comparison of mRNA and protein levels revealed a high level of correlation, implying that the initial control of intestinal stem cell biology occurs largely at the mRNA level. Taken together, our study presents a valuable resource for the study of intestinal stem cell biology. We used cell fractions of intestines from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter. RNA was isolated from two FACS sorted cell populations, one expressing GFP at high levels (GFPbright) and the other expressing GFP at low levels (GFPdim). The gates set to sort cells for the expression profiling were the same as for the cells used for the mass spectometry analysis. cRNA from GFPbright and GFPdim cells from three different sorts (each combining three to four mice) were hybridized on Affymetrix Mouse HT MG-430 PM plate arrays.将Lgr5鉴定为肠道干细胞标志物，使得分离并研究原代肠道干细胞成为可能。我们采用定量质谱法（quantitative mass spectrometry）与转录组分析（transcriptomic analysis），对经荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS）分离的Lgr5阳性干细胞与其未分化的直接子代细胞之间的蛋白质与基因表达变化开展了表达谱分析。对信使RNA（messenger RNA, mRNA）与蛋白质水平的整体比较显示二者具有高度相关性，这表明肠道干细胞生物学特性的初始调控主要发生在mRNA层面。综上，本研究为肠道干细胞生物学研究提供了极具价值的研究资源。我们使用了来自Lgr5-EGFP-ires-CreERT2小鼠的肠道细胞组分，该模型小鼠的绿色荧光蛋白（Green Fluorescent Protein, GFP）表达受Lgr5启动子调控。我们从两类经FACS分选的细胞群中分离得到RNA：一类为高表达GFP的细胞（GFPbright），另一类为低表达GFP的细胞（GFPdim）。用于表达谱分析的细胞分选门控参数，与质谱分析所用细胞的分选门控参数完全一致。将来自三次独立分选（每次分选均合并3~4只小鼠）的GFPbright与GFPdim细胞的互补RNA（complementary RNA, cRNA），在昂飞（Affymetrix）小鼠HT MG-430 PM板阵列芯片上完成了杂交。