食品中3-氯-1，2-丙二醇对小鼠T淋巴细胞抑制作用及机制研究

发布时间：2018-03-22 06:33

本文选题：3-MCPD(+)　切入点：细胞因子　出处：《吉林大学》2014年硕士论文　论文类型：学位论文

【摘要】：本研究为中国博士后科学基金一类项目801120106201。 3-氯-1，2-丙二醇(α-chlorohydrin，3-MCPD)是目前被国际上广为关注的食品污染物—氯丙醇类化合物中最常见的代表物质。在食品工业中，利用酸水解植物蛋白工艺生产氨基酸的过程中，会不可避免地产生氯丙醇类副产物，其中3-MCPD的含量最高。与传统的发酵酿造工业相比，酸水解工艺具有生产时间短，，成本低等优点，因此被广泛应用。利用酸水解蛋白进一步加工得到配制酱油等调味品，因此在配制酱油和相关调味品中3-MCPD的污染较为常见。本研究以3-MCPD(+)为研究对象，以18-22g BALB/c小鼠为实验动物，在非细胞毒剂量范围内，探究3-MCPD(+)对小鼠是否具有体内外的免疫抑制作用，如果具有免疫抑制作用，探索其发生抑制作用的机制。主要取得如下研究结果： (1)通过MTT法筛选出3-MCPD(+)的非细胞毒剂量范围，并进一步确定3-MCPD(+)的实验剂量。在0～4mM范围内，吸光度值与空白值相比没有显著性差异，说明该剂量范围是BALB/c小鼠脾淋巴细胞的非细胞毒剂量范围。进一步通过刀豆蛋白A(ConA)的诱导确定1，2，4mM剂量的3-MCPD(+)对脾淋巴细胞的增殖具有抑制作用。 (2)系统研究了3-MCPD(+)对BALB/c小鼠脾淋巴细胞产生免疫抑制作用的机制。进行具有代表性的免疫学实验如流式细胞术分析、ELISA试剂盒检测、RNA提取和半定量反转录聚合酶链反应、细胞内钙离子的荧光测定，结果确定1，2，4mM剂量的3-MCPD(+)对小鼠脾淋巴细胞具有免疫抑制作用。采用SDS-聚丙烯酰胺凝胶电泳检测分析3-MCPD(+)对ConA激活的信号通路NF-κB、NFAT、AP-1的影响，发现3-MCPD(+)抑制了NF-κB、NFAT2、AP-1代表性蛋白的表达。得出结论：经ConA刺激后，NF-κB、NFAT2、AP-1蛋白的表达量增多，但是随着3-MCPD(+)剂量的不断升高，三种蛋白的表达量逐渐减少，呈剂量依赖关系。3-MCPD(+)对BALB/c小鼠脾淋巴细胞具有免疫抑制作用，这种抑制作用可能是通过抑制NF-B，NFAT及AP-1信号通路来实现的。 (3)构建二硝基氟苯(DNFB)所致的迟发型超敏反应(DTH)小鼠模型。进行外耳廓组织学分析。结果表明：3-MCPD(+)对BALB/c小鼠具有体内的免疫抑制作用。综上所述，食品中氯丙醇类化合物的代表物质3-MCPD对BALB/c小鼠体内外都具有免疫抑制作用，这种抑制作用呈现出剂量依赖性，从而为食品中3-MCPD进一步的安全性评价提供毒理学依据。
[Abstract]:This research is the first class project of China postdoctoral Science Foundation 801120106201. At present, 伪 -chlorohydrin3-MCPDis the most common representative substance in the food pollution-chloropropanol compounds. In the food industry, the production of amino acids by acid-hydrolyzed vegetable protein process. It is inevitable to produce chloropropanol by-products, in which 3-MCPD content is the highest. Compared with the traditional fermentation industry, acid hydrolysis process has the advantages of shorter production time and lower cost. Therefore, it is widely used. Soy sauce and other condiments can be prepared by further processing of acid hydrolysate, so the contamination of 3-MCPD in the preparation of soy sauce and related condiments is more common. In this study, 3-MCPD () was used as research object, and 18-22g BALB/c mice were used as experimental animals. In the range of non-cytotoxic dose, to explore whether 3-MCPD () has immunosuppressive effect in vivo and in vitro, and if it has immunosuppressive effect, to explore the mechanism of its inhibition. The main results are as follows:. (1) the non-cytotoxic dose range of 3-MCPD () was screened by MTT method, and the experimental dose of 3-MCPD () was further determined. In the range of 0~4mM, there was no significant difference between the absorbance value and the blank value. The results indicated that the dose range was the non-cytotoxic dose range of splenic lymphocytes of BALB/c mice, and further confirmed that 3-MCPD () at the dose of 1t2mM could inhibit the proliferation of splenic lymphocytes through the induction of concanavalin Cona (Cona). The mechanism of immunosuppressive effect of 3-MCPD () on splenic lymphocytes of BALB/c mice was systematically studied. The representative immunological experiments such as flow cytometry and Elisa kit were used to detect RNA extraction and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The immunosuppressive effect of 3-MCPD (3 MCPD ()) on mouse splenic lymphocytes was determined by fluorescence assay of intracellular calcium ion. SDS- polyacrylamide gel electrophoresis (SDS-polyacrylamide gel electrophoresis) was used to detect the effect of 3-MCPD (3 MCPD ()) on ConA activated signal pathway NF- 魏 Bn NFATATAP-1. It was found that 3-MCPD () inhibited the expression of NF- 魏 B ~ (2) -NFAT2AP-1. It was concluded that the expression of NF- 魏 B ~ (2 +) -NFAT2 (AP-1) was increased after ConA stimulation, but the expression of these three proteins decreased gradually with the increasing dose of 3-MCPD (). In a dose-dependent manner. 3-MCPD () has immunosuppressive effect on splenic lymphocytes of BALB/c mice, which may be achieved by inhibiting NF-BnFAT and AP-1 signaling pathway. The model of delayed type hypersensitivity (DTH) induced by dinitrofluorobenzene (DNFB) was established. The histological analysis of the external auricle showed that: 3-MCPD () had immunosuppressive effect on BALB/c mice in vivo. In conclusion, 3-MCPD, the representative of chloropropanol compounds in food, has immunosuppressive effect on BALB/c mice in vivo and in vitro, and the inhibitory effect is dose-dependent. So as to provide toxicological basis for further safety evaluation of 3-MCPD in food.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965;TS201.6

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