【摘要】：研究表明刺五加在胃腸疾病治療方面有良好效果,且具有有效促進(jìn)哺乳母豬腸道發(fā)育的功能,并能夠促進(jìn)仔豬的生長(zhǎng)[1]。本研究在體外條件下,以仔豬小腸上皮細(xì)胞為模型,研究刺五加主要活性單體之一——刺五加苷B(Eleutheroside B)對(duì)豬小腸上皮細(xì)胞屏障功能的影響。預(yù)試驗(yàn):刺五加苷B作用濃度的確定。本試驗(yàn)在細(xì)胞完全培養(yǎng)液的基礎(chǔ)上分別添加濃度為0、0.05、0.1、0.2 mg/mL的刺五加苷B后培養(yǎng)IPEC-J2細(xì)胞,其中0mg/mL為對(duì)照組。應(yīng)用實(shí)時(shí)細(xì)胞分析儀(RTCA)系統(tǒng)觀察不同濃度刺五加苷B作用下48h的IPEC-J2細(xì)胞的增殖情況。根據(jù)本試驗(yàn)結(jié)果本研究選取0.1mg/mL刺五加苷B為添加濃度。試驗(yàn)一:刺五加苷B對(duì)仔豬小腸上皮細(xì)胞膜通透性的影響。取生長(zhǎng)狀態(tài)相同的豬小腸上皮細(xì)胞為實(shí)驗(yàn)對(duì)象,通過(guò)測(cè)定0.1mg/mL刺五加苷B培養(yǎng)后的細(xì)胞跨上皮電阻(TEER)值和培養(yǎng)上清液的堿性磷酸酶(ALP)活性來(lái)反應(yīng)刺五加苷B對(duì)細(xì)胞膜通透性的影響。結(jié)果顯示細(xì)胞培養(yǎng)液添加刺五加苷B組顯著增加細(xì)胞TEER值,且顯著降低了細(xì)胞培養(yǎng)液中ALP活性,說(shuō)明刺五加苷B顯著降低了IPEC-J2細(xì)胞膜的通透性。試驗(yàn)二:刺五加苷B對(duì)仔豬小腸上皮細(xì)胞緊密連接蛋白及其基因相對(duì)表達(dá)量的影響。本部分試驗(yàn)分別采用實(shí)時(shí)熒光定量PCR及蛋白免疫印跡方法分別測(cè)定0.1mg/mL刺五加苷B對(duì)緊密連接Claudin-3、Occludin以及ZO-1的相對(duì)表達(dá)量的影響。熒光定量PCR結(jié)果顯示刺五加苷B添加組顯著增加Claudin-3、Occludin和ZO-1的mRNA的相對(duì)表達(dá)量;且Western Bolt結(jié)果與其相符合,刺五加苷B組增加了這3種緊密連接的蛋白表達(dá)量。試驗(yàn)三:刺五加苷B對(duì)仔豬小腸上皮細(xì)胞免疫功能的影響。本部分試驗(yàn)采用實(shí)時(shí)熒光定量PCR方法測(cè)定細(xì)胞因子IL-6、IL-10、TNF-α、TGF-β及INF-γ的相對(duì)表達(dá)量變化。試驗(yàn)結(jié)果表明刺五加苷B組增加炎性細(xì)胞因子IL-10和TGF-β的mRNA表達(dá)量,且減弱促炎性細(xì)胞因子IL-6、TNF-α、INF-γ的mRNA表達(dá)量。綜上,0.1mg/mL的刺五加苷B在體外條件下,對(duì)仔豬小腸上皮細(xì)胞的增殖、膜通透性的降低、緊密連接的表達(dá)以及免疫功能均能起到一定的促進(jìn)作用,即刺五加苷B能夠?qū)ψ胸i小腸上皮細(xì)胞屏障功能起到一定保護(hù)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- 偽, TGF- 尾 and INF- 緯 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- 偽 and INF- 緯 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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S828.5

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本文編號(hào)：2292007