【作者】 于立博；

【导师】 刘继文；

【作者基本信息】 新疆医科大学 ， 劳动卫生与环境卫生学， 2013， 博士

【摘要】 目的：鹰嘴豆是新疆地产药食兼用植物，也是维吾尔族居民喜爱食用的食物同时又是用于防治疾病的药物。木垒县是新疆鹰嘴豆的主产区，由于其得天独厚的原始自然环境，使得当地生产的鹰嘴豆品质优良，属天然绿色食品。为了提升鹰嘴豆的附加值以期对其进行功能性食品开发和药物资源利用，我们对其中的营养素及生物活性物金属硫蛋白进行了提取测定。在此基础上利用鹰嘴豆中提取的金属硫蛋白对铅毒性进行干预研究，从体内和体外观察了其防治铅毒性的效果，以期为铅防治提供一种新的方法。方法：1）采用原子吸收测定法对木垒县当年产鹰嘴豆籽粒中的金属元素进行了检测；采用氨基酸测定仪对鹰嘴豆中的氨基酸和蛋白质进行了检测；用硫酸锌水溶液对鹰嘴豆芽进行水培法孵育诱导豆芽中金属硫蛋白的生成；采用低温高速离心法和镉-血红蛋白包合法对鹰嘴豆籽粒及豆芽中的金属硫蛋白进行分离提取并测定含量。用高效液相色谱法对金属硫蛋白提取液进行初步提纯。对提纯的金属硫蛋白采用聚丙烯酰胺凝胶电泳并结合MALDI-TOF-MS质谱分析法对其氨基酸序列进行分析。2）选择昆明种幼鼠，建立铅中毒模型，用鹰嘴豆-金属硫蛋白进行干预研究，通过HE染色观察肝、肾组织的病理改变，使用透射电镜观察了肾脏组织的超微结构改变，用化学显色法及原子吸收法检测了肝、肾、血清中SOD、GSH-Px、MDA的含量及肝、肾、脑、骨骼组织中铅离子的含量，用酶联免疫法检测了组织中金属硫蛋白含量改变。并通过RT-PCR观察了外源性金属硫蛋白对MT1-EmRNA和MT2AmRNA表达的影响。采用单细胞凝胶电泳法观察了外源性金属硫蛋白对铅所致淋巴细胞DNA损伤的修复作用。3）体外培养淋巴细胞、海马神经元细胞和骨髓间充质细胞，通过预实验所得半数抑制浓度，确定染铅浓度，建立细胞铅中毒模型后给予外源性金属硫蛋白干预，观察鹰嘴豆-金属硫蛋白体外拮抗铅毒性的效果。采用酶联免疫法测定了三种细胞培养液中乳酸脱氢酶含量；采用原子吸收法测定了细胞内钾离子，钙离子和铅离子的含量；采用BCA法测定了细胞中总蛋白含量；采用流式细胞术检测了淋巴细胞、海马神经元细胞和骨髓基质细胞凋亡情况；采用Hoechest33258荧光染色观察了淋巴细胞凋亡情况；采用透射电镜观察了淋巴细胞的超微结构改变；采用荧光免疫方法观察了海马神经元细胞和骨髓基质细胞的Bcl-2和Bax表达情况。结果：1）木垒县产鹰嘴豆中含有17种氨基酸，氨基酸总和达到18.8%，其中人体必需氨基酸全部包含在内。含有人体所需的多种矿物质：钾、铬、钙、铁、锌、铜、镁、铬等，其中钾、镁、钙的含量最高，分别达到了9256.5mg/kg、1289mg/kg和1074.0mg/kg，重金属铅含量未检出，镉含量为5.9ug/kg，总汞含量为2.58ug/kg，总砷含量为4.1ug/kg。鹰嘴豆籽粒中金属硫蛋白含量为：0.16mg/g，鹰嘴豆芽中金属硫蛋白含量为：0.356mg/g。鹰嘴豆-金属硫蛋白的分子量约为10KD。对基质辅助激光解吸-飞行时间质谱（MALDI-TOF-MS）分析结果在NCBI BLAST中进行检索，共检索出有匹配肽段的蛋白96个，根据等电点和分子质量范围筛选出符合条件的蛋白两个gi|330253730和gi|355496648，与这两个蛋白质中匹配上的肽段共有17条。2）动物实验结果显示，染铅后幼鼠的进食量、饮水量及活动量明显减少。HE染色后光镜下观察可见，染铅后小鼠肝、肾组织细胞排列紊乱，组织细胞水肿，给予金属硫蛋白干预后明显好转。透射电镜下观察，可见阳性染铅组小鼠肾小管上皮细胞核固缩，细胞边缘化，细胞内线粒体大量空泡变，微绒毛排列紊乱，内质网及溶酶体增加，线粒体基质膜肿胀，干预组尤其是高剂量干预组细胞损伤状况减轻，细胞内空泡减少，微绒毛排列相对整齐。肝脏、肾脏、血清过氧化指标检测结果显示，阳性对照组的GSH-Px和SOD含量降低，干预后GSH-Px和SOD含量升高，差别有统计学意义（P＜0.05）；阳性对照组的MDA含量上升，各干预组MDA含量降低，其差别有统计学意义（P＜0.05）；与阳性对照组比较，高剂量干预组巯基含量增加，其差别有统计学意义（P＜0.05）；与阳性对照组比较，高、中剂量组的肝脏、肾脏、脑、骨骼、全血组织中铅离子含量降低，其差别有统计学意义（P＜0.05）；与阴性对照组比较，各干预组金属硫蛋白含量升高，其差别具有统计学意义（P＜0.05）；单细胞凝胶电泳结果显示，鹰嘴豆-金属硫蛋白对铅所致淋巴细胞DNA损伤有修复作用，使细胞DNA损伤程度减轻，产生彗星细胞数目减少；RT-PCR结果显示，染铅后阳性组小鼠肝脏MT1-EmRNA和MT2AmRNA表达量增加，给予金属硫蛋白干预后各组MT1-EmRNA和MT2AmRNA表达量降低（P＜0.05）；染铅后阳性组小鼠肾脏MT1-EmRNA和MT2AmRNA表达量增加，给予金属硫蛋白干预后各组MT1-EmRNA和MT2AmRNA表达量降低（P＜0.05）。3）细胞实验结果显示，染铅后淋巴细胞、海马神经元细胞和骨髓基质细胞增殖率降低，给予鹰嘴豆-金属硫蛋白干预后，各组细胞的增值率升高。染铅后阳性组淋巴细胞、海马神经元细胞和骨髓基质细胞中铅离子含量增加，给予外源性金属硫蛋白干预后，高、中剂量组细胞中铅离子含量显著降低，差别有统计学意义（P＜0.05）；24小时、48小时和72小时淋巴细胞、海马神经元细胞和骨髓基质细胞的染铅阳性组细胞中钾离子含量升高，给予外源性金属硫蛋白干预后，高剂量干预组细胞中的钾离子含量降低，差别有统计学差异（P＜0.05）；24小时、48小时和72小时淋巴细胞、海马神经元细胞和骨髓基质细胞的染铅阳性组细胞中钙离子含量升高，给予外源性金属硫蛋白干预后，48小时和72小时高、中剂量组细胞的钙离子含量降低，有统计学差异（P＜0.05）；24小时、48小时和72小时染铅后各组细胞培养液中的乳酸脱氢酶含量显著升高，给予金属硫蛋白干预后高、中剂量组细胞中LDH含量降低（P＜0.05）；Hoechest33258荧光染色结果显示染铅阳性组淋巴细胞凋亡率高于各干预组；透射电镜下观察到淋巴细胞染铅后，出现了凋亡小体，细胞间接触消失，细胞表面绒毛消失，当干预后尤其是高剂量干预组细胞损伤程度减轻，细胞表面绒毛又出现，细胞膜结构完整；染铅后海马神经元细胞和骨髓基质细胞的Bax表达量增加，给予金属硫蛋白干预后Bax表达量下降；染铅阳性组细胞的Bcl-2含量降低，给予外源性金属硫蛋白干预后Bcl-2含量上升。海马神经元细胞和骨髓基质细胞染铅阳性组bcl-2/bax降低，高剂量干预组bcl-2/bax升高（P＜0.05）。流式细胞术结果显示，染铅后海马神经元细胞和骨髓基质细胞凋亡率增加，给予金属硫蛋白干预后，细胞凋亡率降低。结论：新疆木垒县产鹰嘴豆中含有人体必需的氨基酸和矿物质，不含有重金属铅，总汞含量远远低于国家规定的食品容许量（0.02mg/kg），镉含量远远低于日本规定的糙米容许含量（1.0mg/kg）和德国规定的酒中镉容许量（0.1mg/kg），总砷含量远远低于国家规定的原粮容许含量（0.7mg/kg）；采用从鹰嘴豆中提取的金属硫蛋白对染铅幼鼠和染铅细胞进行干预实验，综合评价鹰嘴豆-金属硫蛋白拮抗小鼠铅毒性效果，证明鹰嘴豆-金属硫蛋白无论是在体内状态还是体外状态都能够拮抗铅毒性；通过本次研究，为今后进行鹰嘴豆的保健食品及功能食品开发提供了理论依据，同时为防治儿童铅中毒提供了一种新方法。更多还原

【Abstract】 Objective: Chickpea is a kind of plant grown in Xinjiang，it is Uighur residents’favorite food. Uighur residents has used chickpeas as drug to treat with disease. Muleicount is a main chickpea-producing area, because of its unique original naturalenvironment, the local production of chickpeas has good quality and is thought as naturalgreen food. In order to enhance the added value of chickpeas and develop functional foodand drug resource, we detected nutrients and bioactive substances in chickpea andextracted metallothionein from chickpea. On the basis of previous research, we usemetallothionein extract from chickpea to prevent and treat plumbum toxicity in vivo andin vitro conditions. We hope to find a new approach for the prevention of plumbumpoisoning in children. Methods:1) Atomic absorption spectrometer (AAS) was used todetect metal elements and metallothionein in chickpea. Amino acid analyzer was used todetect content of amino acids and protein in chickpea. Centrifugation and cadmium-hemoglobin method was used to extract metallothionein from chickpea. Highperformance liquid chromatography (HPLC) was used to purify protein. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-Page) and MALDI-TOF-MS was usedto analyse amino acid sequence of the metallothionein from chickpea.2) Kunming micewas treated as animal model of plumbum poisoning was established, metallothioneinextracted from chickpea was used to treat mice against plumbum poisoning. Pathologicalchange of liver and kidney tissues was observed under a litht microscope, transmissionelectron microscopy was used to observe ultrastructure changes of kidney tissue. Contentchange of the SOD, GSH-Px and MDA in liver, kidney and blood serum was detected.Plumbum ion content changes in tissues of liver, kidney, brain and bone was detected byAAS method. ELISA was used to detected content of the MT in liver, kidney, brain andbone tissues. RT-PCR was used to detected expression of the MT1-E mRNA and MT2A mRNA in liver and kidney tissues. Single cell gel electrophoresis (SCGE) was used todetected lymphocytic DNA damage.3) Lymphocyte, hippocampal nerve cell and bonemarrow mesenchymal cell were cultured in vitro as cell model of plumbum poisoningwas established. According to the half inhibitory concentration (IC50) in pre-experimentto determine plumbum concentrations. Metallothionein extracted from chickpea was usedto treat against the toxicity of plumbum in different cell models. ELISA was used todetected content of the LDH in three cell models. The AAS method was used to detectedthe content of the Ca2+and K+and Pb2+in lymphocyte, hippocampal nerve cells and bonemarrow mesenchymal cells at24h,48h and72h time points. Total protein content in threecell models was detected by BCA method. Flow cytometry was used to observe theapoptosis of lymphatic cells, hippocampus nerve cells and bone marrow stromal cells.Hoechest33258fluorescence was used to detected the lymphocyte apoptosis.Transmission electron microscopy was used to observe ultrastructure changes oflymphatic cells. Immune fluorescence assay was used to detected the expression of Bcl-2and Bax in hippocampus nerve cells and bone marrow stromal cells. Results:1) Thechickpeas grow in Mulei count contains17kinds of amino acids which included all thehuman body essential amino acids and total content of amino acid reached18.8%. Thechickpeas contains many kinds of minerals that human body needs, such as potassium,sodium, calcium, iron, zinc, copper, magnesium, chromium, etc. Among these minerals,the content of Kaliun and magnesium and calcium is higher and reach to9256mg/kg and1289mg/kg and1074mg/kg respectively. Plumbum has not been detected, Cadmiumcontent is5.9μg/kg, total mercury content is2.58μg/kg, total arsenic content is4.1μg/kg,metallothionein content in chickpea is0.16mg/g; the molecular weight of the chickpea-metallothionein is about10Da. The results of MALDI-TOF-MS show that there were96protein matched from NCBI BLAST. Based on isoelectric point and molecular weightrange, two qualified protein (gi|330253730and gi|355496648) were selected anddisplayed17protein peptides matched from NCBI BLAST.2）As animal experimentalresults display, the level of eating, drinking and activity has decreased significantly intreated mice. Under optical microscope,we observed tissue edema and disorderedarrangement cells of liver and kidney tissue stained by HE in mice. Exogenousmetallothionein from chickpeas was given to mice to treat tissue damage caused byplumbum, poisoning symptoms were improved significantly after the intervention. Undertransmission electron microscopy, we observed that contraction of renal tubular epithelialcells, a large number of vacuoles in mitochondria, disordered arrangement of microvilli and mitochondrial membrane swelling in positive control group mice. Cell damageconditions was reduced in negative control group mice when metallothionein was givento prevent plumbum toxicity. In positive control mice group, the content of GSH-Px andSOD in liver, kidney and serum decreased while content of MDA increased. Comparedwith positive group, content of GSH-Px and SOD increased while content of MDAdecreased in intervention group, the difference was statistically significant (P＜0.05); thecontent of sulfydryl in high dose of intervention group increased while the content ofplumbum in liver, kidney, brain, bone and whole blood decreased in high and middledose groups after the intervention, the difference was statistically significant (P＜0.05);Compared with negative group, the content of metallothionein increased in interventiongroup, the difference is statistically significant (P＜0.05); As shown in single cell gelelectrophoresis result, metallothionein treating caused lymphocytic DNA damagerepairing, DNA damage degree reducing and comet cell number decreasing; As RT-PCRresult display that expression of MT1-E mRNA and MT2A mRNA increased in livertissue of positive group mice while the expression decreased in intervention groups (P＜0.05); In kidney tissue, the expression of MT1-E mRNA and MT2A mRNA increased inpositive group mice while the expression decreased in intervention groups (P＜0.05)；3）As shown in cells experimental results, proliferation rate of lymphocytes, hippocampalneuronal cells and bone marrow stromal cells reduced when given the plumbum,proliferation rate of cells elevated after given the chickpea–metallothionein. Comparedwith positive control group, the content of Pb2+and LDH in three cell models reducedsignificantly in high and middle dose group at24h,48h and72h time point, the differencewas statistically significant (P＜0.05); the content of K+reduced significantly in highdose group at24h,48h and72h while the content of Ca2+reduced significantly in highand middle dose group at48h and72h, the difference was statistically significant (P＜0.05); As shown in Hoechest33258fluorescent dye result, lymphocytes apoptosis ratedecreased in intenvention group which given chickpea–metallothionein treatmentcompared with positive control group; Under transmission electron microscopy, theapoptotic body were observed and cell surface cilia and intercellular contact disappearedwhile those cell damage symptoms reduced in intervention groups. Compared withpositive control group, the expression of Bax decreased while the expression of Bcl-2increased in intervention groups; the ratio of bcl-2/bax increased in high doseintervention group (P＜0.05). Flow cytometry results showed that the apoptosis rate ofcells in positive group increased, then the apoptosis rate of cells increased when chickpea-metallothionein was used to prevent plumbum toxicity. Conclusion: Chickpeawhich grow in Mulei count in Xinjiang contains essential amino acids and minerals, doesnot contain plumbum metals, it’s total mercury content is much lower than the standardsstipulated by the chinese state in food (0.02mg/kg), cadmium content is much lower thanthe standards stipulated by the Japanese state in brown rice (1.0mg/kg) and the Germanstate in white wine (0.1mg/kg), total arsenic content is much lower than the standardsstipulated by the chinese state in grain (0.7mg/kg); In this experiment, metallothioneinextracted from chickpeas was used to prevent and control the toxic effects of plumbum,results show that chickpeas-metallothionein has antagonism to plumbum toxicity eitherin vivo or in vitro state. The study results provide the basis for health food and functionfood development in the future; at the same time, it provides a new method for preven-tion and treatment of children’s plumbum poisoning.更多还原