水华蓝藻微囊藻毒素-RR分离纯化及急性毒性效应

发布时间：2018-05-17 13:20

本文选题：微囊藻毒素-RR + 纯化　；参考：《华中科技大学》2013年硕士论文

【摘要】：一、目的和意义近年来,国内蓝藻水华的爆发频度逐年增加。当蓝藻死亡后，藻细胞破裂释放大量的藻类毒素，其中以微囊藻毒素的存在最为普遍，且与人类健康的关系最为密切，成为目前研究最多的一类藻类毒素。其毒理学、免疫学以及降解和去除等成为研究的热点，需要大量微囊藻毒素纯品。但因其价格昂贵且主要依靠进口，成为困扰研究人员进一步深入开展上述研究的瓶颈。本文旨在开展微囊藻毒素-RR（MC-RR）纯化以及急性毒性的研究，通过选择与优化提取方法、固相萃取、Sephadex LH-20凝胶色谱层析各种参数，获得纯度超过90%的MC-RR。再用纯化的MC-RR作为受试物经腹腔注射和经口两种不同的途径，测定LD50值，初步评价MC-RR对机体的急性毒性效应特征、靶器官、剂量-反应关系，为今后自主提取、纯化、收获藻毒素纯品提供一套较为成熟的方法，并为其他毒性实验的剂量设计和观察指标选择提供参考依据。二、方法 1.化学提取与鉴定以野外采集的蓝藻水华为原料，通过比较反复冻融、水浴法和70%甲醇细胞破碎法提取效率，确定70%甲醇提取法为最优方法后，对后者提取重复次数，每次提取时间及提取温度等参数进行优化。获得的粗提液经旋转蒸发去除甲醇后，经固相萃取初步纯化和浓缩，，上样至Sephadex LH-20凝胶层析柱，以甲醇为流动相，用自动部分收集器收集洗脱液，紫外分光光度计测定每管洗脱液在238nm的吸收值。以A238为纵坐标，以管编号为横坐标绘制洗脱曲线。对上样量，洗脱液流速进行优化，获得最优洗脱条件。用高效液相色谱对峰值组分进行鉴定，并同时利用紫外分光光度计对毒素光谱特征进行鉴定。 2.生物毒性鉴定采用改进寇氏法计算MC-RR经腹腔注射小鼠LD50值。根据预实验的结果，分为400μg/kg、282.84μg/kg、200μg/kg、141.42μg/kg、100μg/kg五个剂量组，对照组为生理盐水组，每组10只昆明小鼠，雌雄各半。染毒后观察急性中毒行为表现，记录小鼠死亡数及死亡时间，并对死亡小鼠及时取心肝肾以计算脏器系数以及进行组织病理学检查。用序贯法测MC-RR经口的LD50值，并根据测定的LD50值染毒，测定小鼠在其开始发生中毒反应时间和平均死亡时间这两个时点的血清酶学、肝脏脏器指数以及病理组织学的改变。三、结果 1.用70%的甲醇溶液溶解35g藻浆，经离心等系列处理，得粗提液，旋转蒸发去除甲醇；经HLB柱固相萃取后，7.5mL洗脱液含4.92mg MC-RR和0.53mg微囊藻毒素-LR(MC-LR)，然后将其浓缩至2mL；再用Sephadex LH-20凝胶过滤层析进一步纯化，通过测定流出组分在238nm处的吸光度值，确定MC-RR在第一个洗脱峰，最终获得3.65mg样品纯度超过90%的MC-RR，产品得率为74.1%。其吸收光谱在238nm处有特征吸收。 2.小鼠经腹腔注射染毒LD50=192.75μg/kg，95%的置信区间为176.59~206.89μg/kg。400μg/kg、282.84μg/kg、200μg/kg、141.42μg/kg、100μg/kg五个剂量组小鼠死亡率分别为10/10,9/10,6/10,1/10,0/10。随着剂量增加，肝脏脏器系数显著增加，组织病理学肝细胞坏死、肝窦充血逐渐加重。经口染毒LD50为12.10mg/kg，95%的置信区间为10.75~13.45mg/kg。以LD50剂量给小鼠染毒后，约45min后开始有中毒反应，平均死亡时间为4h。与对照相比，血清中ALT、AST、ALP及LDH的水平在45min时明显上升，4h后进一步上升。肝脏脏器系数与血清酶学的变化一致，而心脏和肾脏脏器系数未见明显改变。组织病理学变化显示染毒45min后，肝细胞呈点状坏死和肝脏组织少量充血，4h后肝细胞大面积坏死，肝窦大面积充血。四、结论以野外收集的水华蓝藻为原料，采用75%的甲醇溶液提取，经过固相萃取初步浓缩纯化后，再通过Sephadex LH-20凝胶层析，获得纯度超过90%的MC-RR。MC-RR具有明显的肝毒性，在急性毒性中未观察到肾脏和心脏毒性。
[Abstract]:I . Purpose and significance

In recent years , the outbreak frequency of blue - green algae bloom in China increased year by year . When the blue algae died , the algae cells burst and released a large amount of algae toxin , which was the most common type of algae toxin in the present study .

The purpose of this study was to study the purification and acute toxicity of microcystin - RR ( MC - RR ) . By selecting and optimizing extraction method , solid phase extraction and Sephadex LH - 20 gel chromatography , MC - RR was obtained . The acute toxicity effect , target organ , dose - response relationship between MC - RR and MC - RR were determined .

II . Methodology

1 . The raw materials of blue - green algae water collected in the wild were extracted and identified by chemical extraction and identification . After the extraction of methanol with 70 % methanol was carried out by means of comparison of repeated freeze - thaw , water - bath method and 70 % methanol - cell crushing method , the parameters such as the number of repetitions , the time of each extraction and the extraction temperature were optimized .

2 . The LD50 value of MC - RR by intraperitoneal injection of MC - RR was calculated by the improved method . According to the results of pre - experiment , the mice were divided into four groups : 400 渭g / kg , 282.84 渭g / kg , 200渭g / kg , 141.42渭g / kg , 100渭g / kg , and control group were physiological saline group .

III . Results

1 . dissolving 35g of algae slurry with 70 % methanol solution , centrifuging to obtain crude extract , and rotary evaporation to remove methanol ;
After solid - phase extraction with HLB column , 7.5 mL of eluent contained 4.92 mg of MC - RR and 0.53 mg of microcystin - LR ( MC - LR ) , and then concentrated to 2 mL ;
After further purification by Sephadex LH - 20 gel filtration chromatography , the MC - RR was determined by measuring the absorbance of the effluent component at 238 nm , the MC - RR was determined at the first elution peak , finally 3.65 mg of MC - RR was obtained with a purity of over 90 % , and the yield of the product was 74.1 % . Its absorption spectrum was characterized by absorption at 238 nm .

2 . The LD50 = 192.75渭g / kg , 95 % confidence interval was 176.59 - 206.89渭g / kg . 40渭g / kg , 282.84 渭g / kg , 200渭g / kg , 141.42渭g / kg , 100 渭g / kg , respectively .

IV . Conclusions

The MC - RR . MC - RR with purity of more than 90 % was obtained by gel chromatography on Sephadex LH - 20 after preliminary concentration and purification by Sephadex LH - 20 gel chromatography . The renal and cardiotoxicity were not observed in acute toxicity .
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

【参考文献】