肠三叶因子减轻烧伤后内质网应激促进肠上皮细胞谷氨酰胺转运的机制研究

Intestinal trefoil factor(ITF), an important protective molecule on intestine, can facilitate intestinal glutamine (Gln) absorption after burn injury. But its mechanism is unclear. Our previous study has demonstrated that cytoskeleton-associated protein 4 (CKAP4) , an endoplasmic reticulum retention protein, is ITF interaction protein. It is essential to maintain the space structure of endoplasmic reticulum. CKAP4 amino terminal phosphorylation can destroy endoplasmic reticulum connection with microtubules, and induce endoplasmic reticulum collapse. We also found that ITF can activate the Wnt/JNK signal pathway and elevate the activity of Rho and ROCK, thus suppress endoplasmic reticulum stress (ERS). Hence, we deduce the synthesis disorder of intestinal Gln transporter after burn injury may be associated with ERS. ITF can stabilize endoplasmic reticulum structure by combining with CKAP4 and closing its phosphorylation sites. In addition, ITF can suppress endoplasmic reticulum stress by activating Rho and ROCK, as well as promote Gln transporter modification in endoplasmic reticulum and correct location in cell membrane. In order to confirm the hypothesis, we will first verify the combining site of ITF and CKAP4 and inhibit CKAP4 amino terminal phosphorylation by using rite-directed mutagenesis, FRET and co-immunoprecipitation. Subsequently, will observe the influence of ITF to P58IPK and PERK/eIF2α/ATF4 endoplasmic reticulum associated apoptosis pathway. Finally, will detest the change of the synthesis rate and membrane location of Gln transporter (ASCT2 and B0AT1) , and its transport capacity of Gln. This study is expected to partly clarify the mechanisms of ITF promote intestinal Gln transport, and it has important theoretical significance and potential application value.

肠三叶因子（ITF）是肠道重要的保护分子，能促进烧伤后肠道对谷氨酰胺（Gln）的吸收，但其机制不甚清楚。前期研究发现，ITF相互作用蛋白CKAP4对维持内质网空间结构至关重要,同时发现ITF能抑制内质网应激（ERS）。进而提出烧伤后肠道Gln转运载体合成障碍可能与ERS有关，ITF通过与CKAP4结合，封闭其磷酸化位点，稳定内质网结构，降低ERS，从而促进Gln转运载体在内质网的加工和修饰。为此，本项目拟采用定点突变、FRET和免疫共沉淀等手段分析ITF与CKAP4的结合位点，确证ITF能抑制其磷酸化。进而观察ITF对ERS负调控蛋白P58IPK及PERK/eIF2α/ATF4凋亡通路的影响，最后通过检测Gln转运载体ASCT2和B0AT1的合成速率和膜定位情况，以及转运Gln的能力来证实上述假说。本研究有望初步阐明ITF促进肠道Gln转运的分子机制，具有重要的理论意义和潜在的应用价值。

本课题通过四年的研究，着重观察了ITF对烧伤大鼠肠道Gln转运的影响，并深入探讨了其机制。本研究采用钙离子螯合法和镁离子梯度离心法分别提取肠上皮细胞(IECs)及其刷状缘囊泡(BBMVs)，电镜观察BBMVs结构变化，同位素液相闪烁法检测IECs及BBMVs对3H-Gln的转运能力， Western blot观察IECs中Gln转运载体ASCT2和B0AT1及其相关调控蛋白p-AMPK、CHOP、GRP-78、LC3-Ⅱ、p62、PDI表达量的变化，Q-PCR检测ASCT2、B0AT1、CHOP、GRP-78、PDI的mRNA水平，采用多种抑制剂在IEC-6细胞水平上深入研究了ITF促进烧伤后大鼠肠道Gln转运的机制。结果显示，烧伤后ASCT2和B0AT1的蛋白水平降低，肠道对Gln的转运能力下降。给予ITF 能明显提高ASCT2和B0AT1的蛋白水平，促进肠道Gln转运。综合上述研究结果，ITF提高烧伤大鼠肠道Gln转运能力的机制与ITF能减轻烧伤导致的肠上皮细胞损伤，以及ITF能减轻肠上皮细胞内质网应激（ERS）并促进二硫键异构酶（PDI）的合成有关，其最终效应为促进Gln转运载体ASCT2和B0AT1的合成，进而提高肠道对Gln的转运能力。通过本课题的研究，为后期研究ITF与ASCT2、B0AT1的空间结构的关系奠定了基础。项目资助发表核心论文2篇，SCI论文1篇（online），培养研究生2名，均获得硕士学位顺利毕业。