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目的:为了探讨5-HT损伤断奶仔小鼠应激腹泻肠黏膜屏障的机制,检测了5-HT对断奶仔小鼠肠隐窝干细胞标记基因(Olfm4,Lgr5)的影响。方法:将40只雄性21 d断奶ICR小鼠随机分为4组,分别为正常对照组,应激腹泻组(番泻叶0.4 kg/L按体重15mL/kg灌胃同时后肢束缚应激),CH药物对照组(氢溴酸西酞普兰,是一种高选择性5-HT再摄取抑制剂,抑制5-HT再摄取灭活过程,使5-HT含量增加。10 mg/kg体重溶解于0.1 mL生理盐水,腹腔注射),CH+应激腹泻组(CH药物处理4h后进行应激腹泻处理),常规饲养。处理5d后小鼠脱颈椎死,取十二指肠、空肠、回肠、结肠组织保存于-80℃用作PCR检测。结果:Olfm4是肠上皮隐窝干细胞的标记基因,其表达量高低可能间接反应肠上皮干细胞的增殖状况。应激腹泻组小鼠小肠干细胞表达量均显著升高,十二指肠13.07%(P=0.000),空肠32.96%(P=0.000),回肠15.54%(P=0.000),结肠47.75%(P=0.000)。CH对照组与正常对照组相比十二指肠14.38%(P=0.000),空肠23.94%(P=0.000),回肠12.29%(P=0.011),结肠48.88%(P=0.000)均显著升高。CH+应激腹泻组与应激腹泻组相比,十二指肠29.07%(P=0.000),空肠6.36%(P=0.000),回肠16.80%(P=0.000),结肠47.03%(P=0.000)均显著升高。CH+应激腹泻组与CH对照组相比十二指肠60.63%,空肠25.55%(P=0.000),回肠23.16%(P=0.000)和结肠48.03%(P=0.000)。Lgr5干细胞标记基因表达量显著低于Olfm4,但是各处理组之间差异显著增加。与正常对照组相比应激腹泻组十二指肠,空肠,回肠Lgr5干细胞标记基因表达量均显著升高,分别为十二指肠71.95%(P=0.015),空肠47.11%(P=0.010),回肠1.52倍(P=0.012),CH对照组与正常对照组相比,十二指肠升高3.48倍(P=0.000),空肠1.15倍(P=0.001),回肠1.65倍(P=0.001)。CH+应激腹泻组与应激腹泻组相比,十二指肠、空肠、回肠分别显著升高了2.88倍(P=0.003),1.88倍(P=0.014),3.59倍(P=0.000)。而CH+应激腹泻组与CH对照组相比也呈现显著增加的趋势,Lgr5干细胞标记基因表达量表现为十二指肠升高48.59%(P=0.029),空肠96.54%(P=0.024),回肠3.36倍(P=0.000)。但是在结肠四组之间Lgr5干细胞标记基因表达量呈现逐渐下降的趋势,为应激腹泻组、CH对照组,CH+应激性腹泻组均显著低于正常对照组51.70%(P=0.000),75.56%(P=0.000),84.36%(P=0.000)。CH+应激腹泻组Lgr5干细胞标记基因表达量最低,比应激腹泻组和CH对照组显著降低67.63%(P=0.000)和36.02%(P=0.142)。结论:断奶应激腹泻与CH诱导的肠道5-HT含量升高均使小鼠小肠肠隐窝干细胞标记基因表达量代偿性升高,破坏维持肠隐窝上皮细胞自我更新的稳态。
OBJECTIVE: To investigate the mechanism of 5-HT on the intestinal mucosal barrier of weaned mice induced by 5-HT, we detected the effect of 5-HT on Olfm4 and Lgr5. Methods: Forty male weaned 21-day-old ICR mice were randomly divided into 4 groups: normal control group, stress diarrhea group (senna 0.4 kg / L body weight 15 mL / kg gavage and hind limb restraint stress) CH drug control group (citalopram hydrobromide) is a highly selective 5-HT reuptake inhibitor that inhibits 5-HT reuptake inactivation and increases 5-HT content.10 mg / kg body weight is soluble in 0.1 mL of saline, intraperitoneal injection), CH + stress diarrhea group (CH treatment 4h stress diarrhea treatment), routine feeding. After 5 days of treatment, the mice were killed by cervical spondylosis, duodenum, jejunum and ileum were taken and the colon tissues were stored at -80 ℃ for PCR assay. Results: Olfm4 is a marker gene of crypt epithelial stem cells. The expression level of Olfm4 may indirectly reflect the proliferation of intestinal epithelial stem cells. The expression of small intestine stem cells in stress diarrhea group was significantly higher than that in control group (13.07%, 32.96%, P = 0.000), ileum (15.54%, P = 0.000) = 0.000). Compared with the control group, the CH control group showed significantly higher rates of duodenum 14.38% (P = 0.000), jejunum 23.94% (P = 0.000), ileum 12.29% (P = 0.011) and colon 48.88% high. Compared with stress diarrhea group, CH + stress diarrhea group showed 29.07% (P = 0.000) of duodenum, 6.36% of jejunum (P = 0.000), 16.80% of ileum ) Were significantly increased. Compared with CH control group, CH + stress diarrhea group had 60.63% of duodenum, 25.55% of jejunum (P = 0.000), 23.16% of ileum (P = 0.000) and 48.03% of colon (P = 0.000). Lgr5 stem cell marker gene expression was significantly lower than Olfm4, but the difference between the treatment groups significantly increased. Compared with the normal control group, the expression of marker genes of duodenum, jejunum and ileum of Lgr5 stem cells in stress diarrhea group were significantly increased, 71.95% (P = 0.015), 47.11% (P = 0.010) ) And ileal 1.52-fold (P = 0.012). Compared with the normal control group, the CH control group showed a 3.48-fold increase in duodenum and a 1.15-fold increase in jejunum (P = 0.001) 0.001). Compared with the stress diarrhea group, the duodenum, jejunum and ileum of CH + stress diarrhea group were significantly increased by 2.88 times (P = 0.003), 1.88 times (P = 0.014) and 3.59 times (P = 0.000), respectively. The expression of Lgr5 stem cell marker gene showed 48.59% (P = 0.029) and jejunum 96.54% (P = 0.024) in CH + stress diarrhea group compared with CH control group. Ileum was 3.36 times (P = 0.000). However, the expression of Lgr5 stem cell marker gene showed a decreasing trend in the four groups of colon, which was significantly lower than that of the control group (51.70%, P = 0.000), the control group and CH + stress diarrhea group, 75.56% (P = 0.000), 84.36% (P = 0.000). The expression of Lgr5 stem cell marker gene in CH + stress diarrhea group was the lowest, 67.63% (P = 0.000) and 36.02% (P = 0.142) lower than those in diarrhea control group and CH control group. Conclusion: Weaning stress diarrhea and enteral chemo-induced 5-HT increased the expression of marker gene of small intestine crypts in mice with compensatory increase, destroying the homeostasis of intestinal crypt epithelial cells self-renewal.