小肠上皮分化相关microRNA的筛选

发布时间：2018-01-06 13:08

本文关键词：小肠上皮分化相关microRNA的筛选　出处：《第三军医大学》2009年硕士论文　论文类型：学位论文

【摘要】：在小肠组织中,单层柱状上皮可向固有层折叠形成众多的隐窝结构,同时也向腔面突起形成指状的绒毛结构。在结肠只存在隐窝结构,大小和细胞组分在不同部位呈现不同。小肠上皮是个典型的干细胞分化系统,多种祖细胞不断分化成不同的细胞子系。关于肠上皮细胞持续的产生、分化的分子机制事关小肠疾病例如癌症的发生、发展,所以已经有广泛研究。多条信号通路包括Notch和Wnt通路被认为在肠上皮细胞的增殖和分化调控中发挥重要作用。同时也提示上皮细胞的定向分化过程在基因表达的严格调控下。微小RNA (MicroRNAs)是一类有20-25个核苷酸的非编码RNA可以在多个物种中对基因的表达进行转录后调控。miRNA可通过和靶基因的mRNA序列部分互补来抑制它们的翻译或者调解它们的稳定性。miRNA可调控多个细胞事件,包括在器官发育和肿瘤形成中的细胞增殖分化事件。在本研究中,我们基于改良的肠上皮细胞分离方法,对肠上皮细胞分化相关的miRNA进行筛选。目的: 1.建立改良的小肠上皮细胞低温快速分离的方法 2.用miRNA芯片和Northern blot的方法筛选到肠上皮细胞分化相关的miRNA。材料和方法: 1.肠道上皮的分离和富集小肠上皮细胞的隐窝单位、绒毛单位以及结肠的隐窝单位在低温条件下应用螯合剂进行洗脱和分离。上皮细胞间连接的保持和与基底膜的完整分离用收集细胞的HE切片来观察。固有层来源的血细胞污染用CD45的免疫荧光染色来鉴定。 2.肠上皮细胞分化miRNA的筛选小肠上皮的隐窝和绒毛细胞被分离和分别富集后进行RNA提取。18到100nt的小RNA从总RNA中进一步纯化。经过荧光标记,小RNA和miRNA芯片杂交得到小肠隐窝和绒毛细胞差异miRNA表达谱。对差异基因进行SAM和cluster分析处理。 3.对miRNA芯片的相关结果进行验证挑选6个隐窝和绒毛细胞间差异表达的miRNA用Northern Blot的方法进行验证。结果: 1.获得了高纯度的小肠上皮绒毛、隐窝细胞和结肠隐窝上皮细胞。小肠上皮绒毛、隐窝细胞和结肠隐窝上皮细胞在地位条件下被洗脱和富集。固有层腔面的基底膜保存完整。在上皮细胞富集物中偶见少量CD45表达阳性的造血系统来源细胞。 2.肠上皮细胞分化相关miRNA的筛选和隐窝细胞比较,35个miRNA在绒毛细胞中差异表达,其中16个上调的miRNA(倍数大于2)和19个下调的miRNA(倍数小于0.7).层次聚类树通过cluster软件生成。 3. miRNA芯片结果用Northern blot方法验证。来源于小肠肠段,结肠肠段,去上皮的小肠肠段,小肠绒毛细胞,小肠隐窝和结肠隐窝细胞的RNA完整性保存良好。这些总RNA用来电泳和检测6个挑选出来的差异表达miRNA。验证结果显示六个中有三个和芯片结果一致。相对于隐窝细胞,绒毛细胞上调miR-183,miR-31,下调miR-19-b。结论: 1.我们成功的建立了改良性的小肠上皮和结肠上皮细胞的分离方法 2.我们获得一系列肠上皮分化相关的miRNA候选分子。 3.我们确认了miR-143, miR-31, miR-22, miR-183, miR-17-5p和miR-19b在小肠和结肠的表达模式
[Abstract]:In the small intestine, columnar epithelium can form a recess structure of numerous folding to the natural layer, but also to the cavity surface protrusions formed to villus structure like. There is only in the colon crypt structure, size and cell components show different in different parts. The intestinal epithelial stem cell differentiation is a typical system, a variety of progenitor the cells continue to differentiate into different cell lines. The son of intestinal epithelial cells continue to produce, the molecular mechanism is related to the differentiation of the intestinal diseases such as cancer, development, so has been widely studied. Several signaling pathways including Notch and Wnt pathways are thought to play an important role in regulating the proliferation and differentiation of intestinal epithelial cells at the same time. Also suggested that strict regulation during directional differentiation of epithelial cells in gene expression.
Micro RNA (MicroRNAs) is a kind of non encoding RNA and 20-25 nucleotide can be in multiple species on gene expression of post transcriptional regulation by.MiRNA and the mRNA of target gene sequences complementary to suppress their translation or stability of.MiRNA they can mediate the regulation of multiple cellular events, including cell proliferation and differentiation events in the formation of organs and tumors. In this study, we modified the intestinal epithelial cells based on the separation method of intestinal epithelial cell differentiation related miRNA were screened.
Objective:
1. to establish a modified method for rapid isolation of small intestinal epithelial cells at low temperature
2. miRNA and Northern blot were used to screen the miRNA. related to the differentiation of intestinal epithelial cells
Materials and methods:
1. isolation and enrichment of intestinal epithelium
Intestinal epithelial cells crypt units, villus units and colon crypt units using chelating agents at low temperature were eluted and separated. The connection between the epithelial cells and maintain the integrity of the basement membrane for separation and collection of cell HE slice to observe. The inherent layer source of blood cells by immunofluorescence staining of CD45 pollution to identify.
Screening of 2. intestinal epithelial cell differentiation miRNA
Small intestinal villus and crypt cells were separated and enriched respectively after extraction of RNA.18 to 100nt RNA from the total RNA. After further purification of fluorescent markers, RNA and miRNA chip hybridization of small intestinal crypt and villus cells. Expression of miRNA spectrum of different gene SAM and cluster analysis.
3. verify the related results of miRNA chip
The differential expression of miRNA between 6 recess and villi cells was selected by Northern Blot.
Result:
1. obtain the high purity of the villi of small intestinal epithelium, crypt cells and colonic crypt epithelial cells. The intestinal epithelial villi, crypt cells and colonic crypt epithelial cells in position conditions were isolated and purified. The inherent layer cavity surface of basement membrane intact. I see in the epithelial cell enrichment in less expression of CD45 in cells of hematopoietic origin..
2., screening of intestinal epithelial differentiation related miRNA and comparison of crypt cells. 35 miRNA expressed differently in villous cells, of which 16 upregulated miRNA (multiple greater than 2) and 19 down regulated miRNA (multiple less than 0.7). Hierarchical clustering tree was generated by cluster software.
3. miRNA chip results using Northern blot method to verify. From the intestinal segment, colon segment, segment of small intestine to epithelium, intestinal villi cells, RNA integrity of the intestinal crypt and colonic crypt cells were well preserved. The expression of miRNA. results showed that three out of six results and chip electrophoresis and detection of the 6 selected consistent out of these differences to total RNA. Compared with the crypt cells, upregulation of villus cells miR-183, miR-31, down miR-19-b.
Conclusion:
1. we successfully established a method of separating the benign small intestinal epithelium from the colon epithelial cells.
2. we have obtained a series of miRNA candidate molecules associated with intestinal epithelial differentiation.
3. we confirmed the expression patterns of miR-143, miR-31, miR-22, miR-183, miR-17-5p, and miR-19b in the small intestine and colon

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

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