Intestinal genetic inactivation of caspase-8 diminishes migration of enterocytes.
Intestinal genetic inactivation of caspase-8 diminishes migration of enterocytes.
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DOI：
10.3748/wjg.v21.i15.4499
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发表时间：
2015-04
影响因子：
4.3
通讯作者：
E. Kaemmerer;Paula Kuhn;Ursula Schneider;M. Jeon;Christina Klaus;M. Schiffer;D. Weisner;C. Liedtke;J. Jäkel;L. Kennes;R. Hilgers;N. Wagner;N. Gassler
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作者：
E. Kaemmerer;Paula Kuhn;Ursula Schneider;M. Jeon;Christina Klaus;M. Schiffer;D. Weisner;C. Liedtke;J. Jäkel;L. Kennes;R. Hilgers;N. Wagner;N. Gassler
目的验证caspase-8在肠上皮细胞迁移中的重要作用。方法采用Caspase 8沉默的Caco 2细胞进行迁移实验。此外，通过将在鼠Casp 8基因的内含子2和4中携带loxP重组位点的遗传修饰小鼠与在纯C57/BL 6遗传背景中在绒毛蛋白启动子控制下表达cre-转基因的转基因动物杂交，产生肠细胞特异性Casp 8杂合（Casp 8（+/Casint））或纯合敲除小鼠（Casp 8（Casint））。在进行形态测定研究前4 h、20 h或40 h，在雄性Casp 8（+/casinint）和Casp 8（casinint）动物中腹膜内注射核苷类似物BrdU。对6个组织解剖学上不同的肠粘膜区域的至少50个半隐窝中抗BrdU免疫染色细胞（cell（max））的位置进行编号并提取用于统计程序。对于小鼠队列（n = 28），从隐窝（n = 57）、平台（n = 19）和绒毛（n = 172）位置内的细胞（最大值）评价肠上皮细胞的行走距离，共得到6838个观察结果。通过将三水平混合效应模型拟合到数据来进行数据分析。结果在Caco 2细胞培养实验中，经Western blotting检测，RNA干扰介导的Casp 8基因敲低效率为80%。在划痕试验中，与对照组（Casp 8（cascramble）和Caco 2）相比，Casp 8缺失的Caco 2细胞的迁移显著减少。在BrdU标记的Casp 8（cre-阴性）小鼠中，发现细胞（max）位置沿半隐窝沿着位于比Casp 8（+/cre-阴性）或对照（cre-阴性）动物更低的位置。三水平混合效应模型的统计数据分析显示，在六个不同的肠道位置（小肠和大肠的不同部分），三个小鼠组之间的细胞运动差异很大。尤其是在十二指肠半隐窝，肠上皮细胞的运动在各组之间存在差异。在20 h时，十二指肠细胞（max）位置在Casp 8（+/+ int）（25.67 ± 2.49）中显著低于Casp 8（+/+ int）（35.67 ± 4.78; P < 0.05）或对照同窝仔（44.33 ± 0.94; P < 0.01）。结论肠上皮细胞依赖Casp 8的迁移可能参与了肠道生理和炎症相关的病理生理过程。
AIM To verify the hypothesis that caspase-8 (Casp8), which regulates cellular apoptosis and necroptosis, is critically involved in enterocyte migration. METHODS Casp8-silenced Caco2 cells were used in migration assays. In addition, enterocyte-specific Casp8 heterozygous (Casp8(+/∆int)) or homozygous knockout mice (Casp8(∆int)) were generated by crossing genetically modified mice carrying loxP recombination sites in intron 2 and 4 of the murine Casp8 gene with transgenic animals expressing a cre-transgene under control of the villin promoter in a pure C57/BL6 genetic background. The nucleoside analog BrdU was injected i.p. in male Casp8(+/∆int) and Casp8(∆int) animals 4 h, 20 h, or 40 h before performing morphometric studies. Locations of anti-BrdU-immunostained cells (cell(max)) in at least 50 hemi-crypts of 6 histoanatomically distinct intestinal mucosal regions were numbered and extracted for statistical procedures. For the mice cohort (n = 28), the walking distance of enterocytes was evaluated from cell(max) within crypt (n = 57), plateau (n = 19), and villus (n = 172) positions, resulting in a total of 6838 observations. Data analysis was performed by fitting a three-level mixed effects model to the data. RESULTS In cell culture experiments with Caco2 cells, Casp8 knockdown efficiency mediated by RNA interference on Casp8 transcripts was 80% controlled as determined by Western blotting. In the scratch assay, migration of Casp8-deleted Caco2 cells was significantly diminished when compared with controls (Casp8(∆scramble) and Caco2). In BrdU-labeled Casp8(∆int) mice, cell(max) locations were found along the hemi-crypts in a lower position than it was for Casp8(+/∆int) or control (cre-negative) animals. Statistical data analysis with a three-level mixed effects model revealed that in the six different intestinal locations (distinct segments of the small and large intestine), cell movement between the three mice groups differed widely. Especially in duodenal hemi-crypts, enterocyte movement was different between the groups. At 20 h, duodenal cell(max) location was significantly lower in Casp8(∆int) (25.67 ± 2.49) than in Casp8(+/∆int) (35.67 ± 4.78; P < 0.05) or control littermates (44.33 ± 0.94; P < 0.01). CONCLUSION Casp8-dependent migration of enterocytes is likely involved in intestinal physiology and inflammation-related pathophysiology.