DCAMKL-1在小鼠结肠急性及慢性黏膜损伤中的表达

Investigation of Doublecortin and Calcium/Calmodulin-dependent Protein Kinase-like-1-expressing Cells in the Mouse Colon with Acute and Chronic Mucosal Injury

目的探讨微管相关蛋白-1(DCAMKL-1)在结肠上皮的表达,同时分析DCAMKL-1阳性细胞在急性结肠黏膜损伤〔葡聚糖硫酸钠(DSS)诱导的结肠炎〕及慢性结肠黏膜损伤(结肠炎相关结直肠癌)中的表达变化。方法取60只健康雌性C57BL/6J小鼠,其中40只用于DSS结肠炎模型实验,20只用于结肠炎相关结直肠癌模型实验。DSS结肠炎模型实验中小鼠分为4组,每组10只,其中3组饮用2.5%DSS溶液7d,再分别自由饮用自来水不同天数后处死,对照组小鼠仅自由饮用自来水,7d后处死;结肠炎相关结直肠癌模型实验中小鼠分为2组,每组10只,其中实验组小鼠腹腔内注射氧化偶氮甲烷(AOM)后给予3个周期DSS溶液和自来水饮用,对照组小鼠腹腔注射等量生理盐水,始终自由饮用自来水,两组小鼠均61d后处死。采用免疫组化观察DCAMKL-1、Musashi-1、增殖细胞核抗原(PCNA)和β-Catenin的表达,Westem blot分别检测2种模型实验中小鼠结肠上皮DCAMKL-1的表达。结果正常小鼠结肠上皮存在DCAMKL-1的表达,多数位于结肠隐窝的基底部。所有DCAMKL-1阳性细胞均表达Musashi-1。在DSS结肠炎模型中,DCAMKL-1阳性细胞在DSS作用7d后明显减少,停用DSS 3d后逐渐恢复正常;DCAMKL-1在结肠炎相关结直肠癌小鼠结肠上皮中表达明显增加,除隐窝底部以外,部分DCAMKL-1阳性细胞位于隐窝的中部,且部分DCAMKL-1阳性细胞胞浆内出现β-Catenin表达。结论DCAMKL-1可能是结肠干细胞的标志物之一。使用DCAMKL-1作为结肠干细胞的标记物,我们能够描述结肠干细胞在急慢性结肠黏膜损伤中的表达变化情况。

Objective To identify the expression of Doublecortin and calcium/calmodulin-dependent protein kinase-like-1-expressing cells （DCAMKL-l-expressing cells） in the colon epithelium and to analyze their deviation in dextran sulfate sodium （DSS） induced colitis and colitis associated cancer. Methods A total of 60 healthy female C57 BL/6J mice were used, 40 for the DSS induced colitis model group and 20 for colitis associated cancer model group. In the former group, mice were sacrificed at day 7 after DSS administration （B group）, 3 days （C group） and 7 days （D group） after DSS withdraw, separately. The control （A group） mice were sacrificed at day 7. In the latter group, mice were gave three repetitive oral administrations of DSS and regular water after injected intraperitoneally with azoxymethane （AOM）. The control group mice were injected intraperitoneally with physiological saline, and then regular water was given. All the mice were sacrificed 61 days later. DCAMKL-1 expressions were detected by both immunohistochemistry and Western blot. Results There were some DCAMKL- 1-expressing cells in the normal mouse colon epithelium. DCAMKL-l-expressing cells expressed Musashi-1. In Most of them were located in the base of the crypt. All the DSS-induced colitis, the number of DCAMKL-1- expressing cells decreased 7 days after DSS administration and recovered 3 days later. The expression of DCAMKL- 1 increased apparently in the mice with colitis-induced cancer. Except the base of the crypt, some of them were located in the middle portion of the crypt and some exhibited cytoplasm β-Catenin staining. Conclusion DCAMKL- 1 is a putative intestinal stem cell marker. Using DCAMKL-1 as a marker for colon stem cells, we could describe the expression pattern of colon stem cells during acute and chronic mucosal injury.