發(fā)布時(shí)間：2018-03-22 06:33

  本文選題：3-MCPD(+)　切入點(diǎn)：細(xì)胞因子　出處：《吉林大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：本研究為中國博士后科學(xué)基金一類項(xiàng)目801120106201。 3-氯-1，2-丙二醇(α-chlorohydrin，3-MCPD)是目前被國際上廣為關(guān)注的食品污染物—氯丙醇類化合物中最常見的代表物質(zhì)。在食品工業(yè)中，利用酸水解植物蛋白工藝生產(chǎn)氨基酸的過程中，會不可避免地產(chǎn)生氯丙醇類副產(chǎn)物，其中3-MCPD的含量最高。與傳統(tǒng)的發(fā)酵釀造工業(yè)相比，酸水解工藝具有生產(chǎn)時(shí)間短，，成本低等優(yōu)點(diǎn)，因此被廣泛應(yīng)用。利用酸水解蛋白進(jìn)一步加工得到配制醬油等調(diào)味品，因此在配制醬油和相關(guān)調(diào)味品中3-MCPD的污染較為常見。本研究以3-MCPD(+)為研究對象，以18-22g BALB/c小鼠為實(shí)驗(yàn)動物，在非細(xì)胞毒劑量范圍內(nèi)，探究3-MCPD(+)對小鼠是否具有體內(nèi)外的免疫抑制作用，如果具有免疫抑制作用，探索其發(fā)生抑制作用的機(jī)制。主要取得如下研究結(jié)果： (1)通過MTT法篩選出3-MCPD(+)的非細(xì)胞毒劑量范圍，并進(jìn)一步確定3-MCPD(+)的實(shí)驗(yàn)劑量。在0～4mM范圍內(nèi)，吸光度值與空白值相比沒有顯著性差異，說明該劑量范圍是BALB/c小鼠脾淋巴細(xì)胞的非細(xì)胞毒劑量范圍。進(jìn)一步通過刀豆蛋白A(ConA)的誘導(dǎo)確定1，2，4mM劑量的3-MCPD(+)對脾淋巴細(xì)胞的增殖具有抑制作用。 (2)系統(tǒng)研究了3-MCPD(+)對BALB/c小鼠脾淋巴細(xì)胞產(chǎn)生免疫抑制作用的機(jī)制。進(jìn)行具有代表性的免疫學(xué)實(shí)驗(yàn)如流式細(xì)胞術(shù)分析、ELISA試劑盒檢測、RNA提取和半定量反轉(zhuǎn)錄聚合酶鏈反應(yīng)、細(xì)胞內(nèi)鈣離子的熒光測定，結(jié)果確定1，2，4mM劑量的3-MCPD(+)對小鼠脾淋巴細(xì)胞具有免疫抑制作用。采用SDS-聚丙烯酰胺凝膠電泳檢測分析3-MCPD(+)對ConA激活的信號通路NF-κB、NFAT、AP-1的影響，發(fā)現(xiàn)3-MCPD(+)抑制了NF-κB、NFAT2、AP-1代表性蛋白的表達(dá)。得出結(jié)論：經(jīng)ConA刺激后，NF-κB、NFAT2、AP-1蛋白的表達(dá)量增多，但是隨著3-MCPD(+)劑量的不斷升高，三種蛋白的表達(dá)量逐漸減少，呈劑量依賴關(guān)系。3-MCPD(+)對BALB/c小鼠脾淋巴細(xì)胞具有免疫抑制作用，這種抑制作用可能是通過抑制NF-B，NFAT及AP-1信號通路來實(shí)現(xiàn)的。 (3)構(gòu)建二硝基氟苯(DNFB)所致的遲發(fā)型超敏反應(yīng)(DTH)小鼠模型。進(jìn)行外耳廓組織學(xué)分析。結(jié)果表明：3-MCPD(+)對BALB/c小鼠具有體內(nèi)的免疫抑制作用。 綜上所述，食品中氯丙醇類化合物的代表物質(zhì)3-MCPD對BALB/c小鼠體內(nèi)外都具有免疫抑制作用，這種抑制作用呈現(xiàn)出劑量依賴性，從而為食品中3-MCPD進(jìn)一步的安全性評價(jià)提供毒理學(xué)依據(jù)。
[Abstract]:This research is the first class project of China postdoctoral Science Foundation 801120106201. At present, 偽 -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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R965;TS201.6

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