Ndst4基因剔除小鼠之表現型分析

Phenotypic analysis of Ndst4 knockout mice

李雅玲(Ya-Lin Li)

指導教授 ： 楊雅倩

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.00319

摘要

大腸直腸癌主要是由於上皮細胞不正常增值，並累積基因變異，包含致癌基因過度活化與抑癌基因失去功能。先前本實驗室利用微衛星標記，於人類第四號染色體4q26區域篩選出N-deacetylase/N-sulfotransferase (NDST4)為大腸直腸癌相關之抑癌基因。NDST4參與Heparan sulfate proteoglycans (HSPGs)合成過程，主要的功能為去乙醯化作用(N-deacetylation)和硫酸化作用(N-sulfation)，HSPGs在發育、生長、腫瘤發生、炎症反應、微生物侵襲等不同生理和病理機制皆扮演重要角色。為了探究NDST4的功能，本實驗室已製造Ndst4基因剔除小鼠，本論文即進行表現型分析。首先，我們觀察到Ndst4基因剔除並不影響其發育與繁衍。接著利用Modified-SHIRPA v1.、全血球計數、血清生化檢驗與組織學檢查進行研究。在外觀和行為分析上，野生型與Ndst4基因剔除小鼠並無顯著差異；而統計全血球計數，發現於Ndst4基因剔除母鼠，其嗜酸性白血球數目和其百分比顯著高於野生型小鼠；在血清生化檢驗結果顯示： Ndst4基因剔除公鼠之高密度脂蛋白顯著低於野生型小鼠。此外，於組織學檢查，以H&E染色觀察大腸組織並計量大腸隱窩(crypt)長度，並無顯著差異，但發現Ndst4基因剔除小鼠之大腸杯狀細胞有增加的現象，進一步以Alcian blue and Periodic Acid Schiff Staining分析，證實Ndst4基因剔除小鼠之近端與中段大腸，杯狀細胞數量皆顯著高於野生型小鼠。接著以Carbonic anhydrase I 免疫組織化學染色法檢測大腸吸收細胞，結果顯示：Ndst4基因剔除小鼠之近端與中段大腸的大腸吸收細胞皆顯著下降。進一步分析大腸道上皮之腸幹細胞與前驅細胞數量，以細胞增生標記ki-67和 5-bromo-2-deoxyuridine進行標定，則無顯著差異。此外，Ndst4基因剔除小鼠之近端與中段大腸隱窩頂端的凋亡小體顯著增加，此現象並以活化態caspase 3染色進行確認。然而，檢查其它組織和器官之形態，並未發現顯著變異。此外，我們發現Ndst4基因剔除小鼠之近端大腸Muc2基因mRNA表現量顯著增高，此結果與組織學檢查之杯狀細胞增加一致。最後，我們也檢測大腸之Ndst家族其他成員表現量，發現Ndst4基因剔除並未使Ndst1-Ndst3基因表現量顯著變化，推測Ndst4於大腸之HSPG生合成過程具有其特定功能，並在大腸道分化與生理功能扮演重要的角色。

關鍵字

大腸直腸癌 ； NDST4 ； 基因剔除小鼠 ； 表現型分析

並列摘要

Colorectal cancer (CRC) originates from abnormal proliferation of epithelial cells and accumulation of many genetic aberration, including activation of oncogenes and inactivation of tumor suppressor genes. Our previous study seaked for CRC-associated tumor suppressor genes on chromosome 4 by loss of heterozygosity study. A putative tumor suppressor gene, namely N-deacetylase/N-sulfotransferase 4 (NDST4) was found at chromosome 4q26. NDST4 participates in biosynthesis of Heparan sulfate proteoglycans (HSPGs), resulting in N-acetylglucosamine (GlcNAc) N-deacetylation and N-sulfation. HSPGs play many important roles in development, growth, tumorigenesis, inflammation and microbial invasion. To study the physiological function of NDST4, we had produced a Ndst4 knockout (Ndst4-/-) mouse strain and aimed to phenotypic characterization in the study. First, the Ndst4-/- mice were fertile and developed normally. To compare the phenotypes between Ndst4-/- and WT mice, we conducted the modified-SHIRPA v1., complete blood count, biochemistry and histological analysis. There was no significant difference in modified-SHIRPA v1. analysis. Nevertheless, the number and percentage of eosinophils were significantly increased in Ndst4-/- female mice, and the concentration of high-density lipoprotein was significantly decreased in Ndst4-/- male mice when compared with their gender-matched wild type (WT) littermates. As for histological examination, the goblet cell density is significantly higher in the proximal and middle colon of Ndst4-/- mice. Consistantly, the colonocyte density was significantly lower in the proximal and middle colon of Ndst4-/- mice. However, there was no difference in length of crypt and intestinal progenitor cell with ki-67 and 5-bromo-2-deoxyuridine staining. Moreover, Ndst4 deficiency increased the basal level of apoptosis in the colonic mucosa. Whereae, there was no obvious aberration found in other organs and tissues by histological examination. Using real-time RT-PCR analyses, Muc2 expression was increased in the proximal colon of Ndst4-/- mice compared with WT mice. The result is compatible with the histological finding of increased goblet cell. On the other hand, the gene expression levels of Ndst1-Ndst3 were not significantly different between WT and Ndst4-/- mice. Taken together, the results in the study indicate that Ndst4 deficiency may influence HS chain modification of specific HSPGs that play a pivotal role in colonic epithelial differentiation and function.

並列關鍵字

Colorectal cancer ； NDST4 ； Gene knockout mouse ； Phenotyping

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