刺五加苷B对仔猪小肠上皮细胞屏障功能的影响研究
发布时间：2018-10-24 17:06
【摘要】：研究表明刺五加在胃肠疾病治疗方面有良好效果,且具有有效促进哺乳母猪肠道发育的功能,并能够促进仔猪的生长[1]。本研究在体外条件下,以仔猪小肠上皮细胞为模型,研究刺五加主要活性单体之一——刺五加苷B(Eleutheroside B)对猪小肠上皮细胞屏障功能的影响。预试验:刺五加苷B作用浓度的确定。本试验在细胞完全培养液的基础上分别添加浓度为0、0.05、0.1、0.2 mg/mL的刺五加苷B后培养IPEC-J2细胞,其中0mg/mL为对照组。应用实时细胞分析仪(RTCA)系统观察不同浓度刺五加苷B作用下48h的IPEC-J2细胞的增殖情况。根据本试验结果本研究选取0.1mg/mL刺五加苷B为添加浓度。试验一:刺五加苷B对仔猪小肠上皮细胞膜通透性的影响。取生长状态相同的猪小肠上皮细胞为实验对象,通过测定0.1mg/mL刺五加苷B培养后的细胞跨上皮电阻(TEER)值和培养上清液的碱性磷酸酶(ALP)活性来反应刺五加苷B对细胞膜通透性的影响。结果显示细胞培养液添加刺五加苷B组显著增加细胞TEER值,且显著降低了细胞培养液中ALP活性,说明刺五加苷B显著降低了IPEC-J2细胞膜的通透性。试验二:刺五加苷B对仔猪小肠上皮细胞紧密连接蛋白及其基因相对表达量的影响。本部分试验分别采用实时荧光定量PCR及蛋白免疫印迹方法分别测定0.1mg/mL刺五加苷B对紧密连接Claudin-3、Occludin以及ZO-1的相对表达量的影响。荧光定量PCR结果显示刺五加苷B添加组显著增加Claudin-3、Occludin和ZO-1的mRNA的相对表达量;且Western Bolt结果与其相符合,刺五加苷B组增加了这3种紧密连接的蛋白表达量。试验三:刺五加苷B对仔猪小肠上皮细胞免疫功能的影响。本部分试验采用实时荧光定量PCR方法测定细胞因子IL-6、IL-10、TNF-α、TGF-β及INF-γ的相对表达量变化。试验结果表明刺五加苷B组增加炎性细胞因子IL-10和TGF-β的mRNA表达量,且减弱促炎性细胞因子IL-6、TNF-α、INF-γ的mRNA表达量。综上,0.1mg/mL的刺五加苷B在体外条件下,对仔猪小肠上皮细胞的增殖、膜通透性的降低、紧密连接的表达以及免疫功能均能起到一定的促进作用,即刺五加苷B能够对仔猪小肠上皮细胞屏障功能起到一定保护作用。
[Abstract]:The results show that Acanthopanax senticosus has a good effect in the treatment of gastrointestinal diseases and has the function of promoting the intestinal development of lactating sows and promoting the growth of piglets [1]. The effects of Acanthopanax senticosus (Acanthopanax senticosus) B (Eleutheroside B), one of the main active monomers of Acanthopanax senticosus (Acanthopanax senticosus), on the barrier function of intestinal epithelial cells of piglets were studied in vitro. Pre-test: determination of the action concentration of Acanthopanax senticosus B. Acanthopanax senticosus B (Acanthopanax senticosus) cells were cultured on the basis of complete cell culture medium with 0 0. 05 mg/mL of Acanthopanax senticosus B, in which 0mg/mL was used as control group. The proliferation of IPEC-J2 cells at different concentrations of Acanthopanax senticosus B for 48 h was observed by (RTCA) system. According to the results of this study, 0.1mg/mL Acanthopanax senticosus B was selected as the added concentration. Experiment 1: effects of Acanthopanax senticosus B on membrane permeability of small intestinal epithelium in piglets. Pig intestinal epithelial cells with the same growth state were selected as experimental objects. The effect of Acanthopanax senticosus B on cell membrane permeability was studied by measuring the (TEER) value of the transdermal resistance of 0.1mg/mL Acanthopanax senticosus B and the activity of alkaline phosphatase (ALP) in the supernatant of Acanthopanax senticosus (Acanthopanax senticosus B). The results showed that the addition of Acanthopanax senticosus B to the cell culture medium significantly increased the cell TEER value and significantly decreased the ALP activity in the cell culture medium, indicating that Acanthopanax senticosus B significantly decreased the permeability of IPEC-J2 cell membrane. Experiment 2: the effect of Acanthopanax senticosus B on the relative expression of tight junction protein and its gene in small intestinal epithelial cells of piglets. The effects of 0.1mg/mL Acanthopanax senticosus B on the relative expression of tightly connected Claudin-3,Occludin and ZO-1 were determined by real-time fluorescent quantitative PCR and Western blot respectively. The results of fluorescence quantitative PCR showed that Acanthopanax senticosus B added Acanthopanax senticosus B significantly increased the relative expression of Claudin-3,Occludin and ZO-1 mRNA, and the Western Bolt results were consistent with the results. Acanthopanax senticosus B group increased the expression of these three tightly connected proteins. Experiment 3: effect of Acanthopanax senticosus B on immune function of intestinal epithelial cells in piglets. The relative expressions of cytokines IL-6,IL-10,TNF- 伪, TGF- 尾 and INF- 纬 were measured by real-time fluorescence quantitative PCR. The results showed that Acanthopanax senticosus B increased the mRNA expression of inflammatory cytokines IL-10 and TGF- 尾, and decreased the mRNA expression of IL-6,TNF- 伪 and INF- 纬 in Acanthopanax senticosus group. In conclusion, Acanthopanax senticosus B of 0.1mg/mL can promote the proliferation, membrane permeability, tight junction expression and immune function of small intestinal epithelial cells of piglets in vitro. Acanthopanax senticosus B can protect the barrier function of intestinal epithelial cells in piglets.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828.5
本文编号：2292007
[Abstract]:The results show that Acanthopanax senticosus has a good effect in the treatment of gastrointestinal diseases and has the function of promoting the intestinal development of lactating sows and promoting the growth of piglets [1]. The effects of Acanthopanax senticosus (Acanthopanax senticosus) B (Eleutheroside B), one of the main active monomers of Acanthopanax senticosus (Acanthopanax senticosus), on the barrier function of intestinal epithelial cells of piglets were studied in vitro. Pre-test: determination of the action concentration of Acanthopanax senticosus B. Acanthopanax senticosus B (Acanthopanax senticosus) cells were cultured on the basis of complete cell culture medium with 0 0. 05 mg/mL of Acanthopanax senticosus B, in which 0mg/mL was used as control group. The proliferation of IPEC-J2 cells at different concentrations of Acanthopanax senticosus B for 48 h was observed by (RTCA) system. According to the results of this study, 0.1mg/mL Acanthopanax senticosus B was selected as the added concentration. Experiment 1: effects of Acanthopanax senticosus B on membrane permeability of small intestinal epithelium in piglets. Pig intestinal epithelial cells with the same growth state were selected as experimental objects. The effect of Acanthopanax senticosus B on cell membrane permeability was studied by measuring the (TEER) value of the transdermal resistance of 0.1mg/mL Acanthopanax senticosus B and the activity of alkaline phosphatase (ALP) in the supernatant of Acanthopanax senticosus (Acanthopanax senticosus B). The results showed that the addition of Acanthopanax senticosus B to the cell culture medium significantly increased the cell TEER value and significantly decreased the ALP activity in the cell culture medium, indicating that Acanthopanax senticosus B significantly decreased the permeability of IPEC-J2 cell membrane. Experiment 2: the effect of Acanthopanax senticosus B on the relative expression of tight junction protein and its gene in small intestinal epithelial cells of piglets. The effects of 0.1mg/mL Acanthopanax senticosus B on the relative expression of tightly connected Claudin-3,Occludin and ZO-1 were determined by real-time fluorescent quantitative PCR and Western blot respectively. The results of fluorescence quantitative PCR showed that Acanthopanax senticosus B added Acanthopanax senticosus B significantly increased the relative expression of Claudin-3,Occludin and ZO-1 mRNA, and the Western Bolt results were consistent with the results. Acanthopanax senticosus B group increased the expression of these three tightly connected proteins. Experiment 3: effect of Acanthopanax senticosus B on immune function of intestinal epithelial cells in piglets. The relative expressions of cytokines IL-6,IL-10,TNF- 伪, TGF- 尾 and INF- 纬 were measured by real-time fluorescence quantitative PCR. The results showed that Acanthopanax senticosus B increased the mRNA expression of inflammatory cytokines IL-10 and TGF- 尾, and decreased the mRNA expression of IL-6,TNF- 伪 and INF- 纬 in Acanthopanax senticosus group. In conclusion, Acanthopanax senticosus B of 0.1mg/mL can promote the proliferation, membrane permeability, tight junction expression and immune function of small intestinal epithelial cells of piglets in vitro. Acanthopanax senticosus B can protect the barrier function of intestinal epithelial cells in piglets.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828.5
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