Marselli Lorella, Thorne Jeffrey, Ahn Yu-Bae, Omer Abdulkadir, Sgroi Dennis C, Libermann Towia, Otu Hasan H, Sharma Arun, Bonner-Weir Susan, Weir Gordon C
Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02215, USA.
J Clin Endocrinol Metab. 2008 Mar;93(3):1046-53. doi: 10.1210/jc.2007-0931. Epub 2007 Dec 11.
Human beta-cell gene profiling is a powerful tool for understanding beta-cell biology in normal and pathological conditions. Assessment is complicated when isolated islets are studied because of contamination by non-beta-cells and the trauma of the isolation procedure.
The objective was to use laser capture microdissection (LCM) of human beta-cells from pancreases of cadaver donors and compare their gene expression with that of handpicked isolated islets.
Endogenous autofluorescence of beta-cells facilitated procurement of purified beta-cell tissue from frozen pancreatic sections with LCM. Gene expression profiles of three microdissected beta-cell samples and three isolated islet preparations were obtained. The array data were normalized using DNA-Chip Analyzer software (Harvard School of Public Health, Boston, MA), and the lower confidence bound evaluated differentially expressed genes. Real-time PCR was performed on selected acinar genes and on the duct cell markers, carbonic anhydrase II and keratin 19.
Endogenous autofluorescence facilitates the microdissection of beta-cell rich tissue in human pancreas. When compared with array profiles of purified beta-cell tissue, with lower confidence bound set at 1.2, there were 4560 genes up-regulated and 1226 genes down-regulated in the isolated islets. Among the genes up-regulated in isolated islets were pancreatic acinar and duct genes, chemokine genes, and genes associated with hypoxia, apoptosis, and stress. Quantitative RT-PCR confirmed the differential expression of acinar gene transcripts and the duct marker carbonic anhydrase II in isolated islets.
LCM makes it possible to obtain beta-cell enriched tissue from human pancreas sections without the trauma and ischemia of islet isolation.
人类β细胞基因谱分析是了解正常和病理状态下β细胞生物学特性的有力工具。由于非β细胞的污染以及分离过程造成的损伤，研究分离的胰岛时评估工作变得复杂。
目的是对尸体供者胰腺中的人类β细胞进行激光捕获显微切割（LCM），并将其基因表达与精心挑选的分离胰岛的基因表达进行比较。
β细胞的内源性自发荧光有助于通过LCM从冷冻胰腺切片中获取纯化的β细胞组织。获得了三个显微切割的β细胞样本和三个分离胰岛制剂的基因表达谱。使用DNA芯片分析仪软件（哈佛公共卫生学院，马萨诸塞州波士顿）对阵列数据进行标准化，并评估差异表达基因的较低置信区间。对选定的腺泡基因以及导管细胞标志物碳酸酐酶II和角蛋白19进行实时PCR。
内源性自发荧光有助于对人胰腺中富含β细胞的组织进行显微切割。与纯化的β细胞组织的阵列谱相比，将较低置信区间设定为1.2时，分离的胰岛中有4560个基因上调，1226个基因下调。在分离的胰岛中上调的基因包括胰腺腺泡和导管基因、趋化因子基因以及与缺氧、凋亡和应激相关的基因。定量RT-PCR证实了分离胰岛中腺泡基因转录本和导管标志物碳酸酐酶II的差异表达。
LCM使得从人胰腺切片中获得富含β细胞的组织成为可能，而无需进行胰岛分离的创伤和缺血操作。
