CN107158004B - 一种6-苄氨基嘌呤的应用 - Google Patents
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Abstract
本发明公开了一种6‑苄氨基嘌呤(6‑BA)的应用，6‑苄氨基嘌呤成分明确，在酸碱中稳定，来源广、成分明确、纯度高。本发明证实了6‑苄氨基嘌呤可以作为抗小肠局部缺血再灌注损伤的活性物质，并且6‑苄氨基嘌呤能有效防护小肠缺血再灌注引起的氧化损伤和小肠细胞凋亡。将其添加到药物、食品或保健品当中，抑制小肠组织损伤，延缓衰老抗疲劳，增强小肠组织抗缺血再灌注损伤的能力，开发了6‑BA的新用途，具有重要的经济应用价值。
Description
技术领域
本发明属于生物医学技术领域，具体涉及一种6-苄氨基嘌呤的应用。
背景技术
当组织器官血液供应不足时，常导致细胞机能障碍甚至发生坏死，然而血流的迅速恢复会进一步加重器官的损伤程度。肠是机体内细菌和内毒素的最大储存库，当局部缺血再灌注损伤引发肠屏障功能破坏后会引起一系列疾病的发生，如脓毒症、全身炎症反应综合症、大量器官机能障碍(肝、肺、肾等)，因此肠被认为是引发机体器官系统异常的“马达”。
小肠细胞极易受到局部缺血的影响，再灌注后会进一步导致黏膜受损。在急性肠系膜缺血，出血性、外伤性、感染性休克或者严重烧伤以及一些手术操作如小肠移植术、腹主动脉术等过程中常伴有局部缺血再灌注损伤发生。
目前，市场上有许多抗小肠损伤的物质，但是大多数都有不易提取获得、纯度不高、有效成分不明确等缺点，给实际应用带来了很大的麻烦。因此，开发一种来源广，纯度高，成分明确，效果好，安全无毒无害的抗小肠损伤的物质将是非常迫切且具有重要意义的任务。
6-苄氨基嘌呤(6-BA)成分明确，在酸碱中稳定，来源广、成分明确、纯度高，可以作为植物生长调节剂使用，对动物体肝脏和脑组织也具有抗损伤效果，但是对小肠损伤的保护作用还未见报道，开发其在制备抗小肠组织损伤制品中的新应用具有重要的经济价值。
发明内容
本发明提供了一种6-苄氨基嘌呤的应用，6-苄氨基嘌呤(6-BA)成分明确，在酸碱中稳定，来源广、成分明确、纯度高，在制备抗小肠缺血再灌注损伤制品中的新应用具有重要的经济价值。
本发明的第一个目的是提供一种6-苄氨基嘌呤在制备抗小肠组织损伤药物中的应用。
本发明的第二个目的是提供一种6-苄氨基嘌呤在制备抗小肠局部缺血再灌注引起的小肠细胞氧化损伤药物中的应用。
本发明的第三个目的是提供一种6-苄氨基嘌呤在制备抗小肠局部缺血再灌注引起的小肠细胞凋亡药物中的应用。
本发明的第四个目的是提供一种6-苄氨基嘌呤在制备抗小肠组织损伤食品中的应用。
本发明的第五个目的是提供一种6-苄氨基嘌呤在制备抗小肠局部缺血再灌注引起的小肠细胞氧化损伤食品中的应用。
本发明的第六个目的是提供一种6-苄氨基嘌呤在制备抗小肠局部缺血再灌注引起的小肠细胞凋亡食品中的应用。
本发明的第七个目的是提供一种6-苄氨基嘌呤在制备抗小肠组织损伤保健品中的应用。
本发明的第八个目的是提供一种6-苄氨基嘌呤在制备抗小肠局部缺血再灌注引起的小肠细胞氧化损伤保健品中的应用。
本发明的第九个目的是提供一种6-苄氨基嘌呤在制备抗小肠局部缺血再灌注引起的小肠细胞凋亡保健品中的应用。
与现有技术相比，本发明提供的一种6-苄氨基嘌呤的应用，具有以下有益效果：
本发明证实了6-苄氨基嘌呤可以作为抗小肠局部缺血再灌注损伤的活性物质，并且6-苄氨基嘌呤能有效防护小肠缺血再灌注引起的氧化损伤和小肠细胞凋亡，将其添加到药物、食品或保健品当中，抑制小肠组织损伤，延缓衰老抗疲劳，增强小肠组织抗缺血再灌注损伤的能力，具有重要的经济应用价值。
附图说明
图1是不同组大鼠小肠细胞DNA凝胶电泳结果(×200)；
其中，图1A为对照组，图1B为I/R损伤模型组，图1C为低剂量6-BA保护组，图1D为高剂量6-BA保护组；
图2是不同组大鼠小肠细胞Caspase-3表达情况(×400)；
其中，图2A为对照组，图2B为I/R损伤模型组，图2C为低剂量6-BA保护组，图2D为高剂量6-BA保护组。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。下述实施例中的实验方法，如无特殊说明，均为常规方法，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例
1.药品与试剂
6-苄氨基嘌呤(6-BA)，购于厦门星隆达化学试剂有限公司，由美国Sanland公司生产，用0.06mol/L的盐酸配制成1000mg/L、2000mg/L的储备液；丙二醛(MDA)检测试剂盒、谷胱甘肽过氧化物酶(GSH-Px)检测试剂盒、总超氧化物歧化酶(T-SOD)检测试剂盒和考马斯亮兰试剂盒，均为南京建成生物工程研究所生产。低熔点琼脂糖，Sigma；兔抗大鼠Caspase-3单克隆抗体，Abcam；免疫组化试剂盒，武汉博士德生物工程有限公司。
2.动物分组
雄性SD大鼠80只，购自郑州大学实验动物中心。适应环境7d后，随机分为4组，每组20只。依次为对照组、I/R损伤模型组(按照常规方法获得I/R损伤模型组，此处不再详述)、低剂量6-BA保护组和高剂量6-BA保护组。低剂量6-BA保护组于术前3周连续灌胃6-BA，每天一次按照10mg/kg大鼠的量灌胃一次；高剂量6-BA保护组于术前3周连续灌胃6-BA，每天一次按照20mg/kg大鼠的量灌胃一次；对照组和I/R损伤模型组给予同体积的生理盐水灌胃。
动物术前12h禁食，不禁水，以3％戊巴比妥钠(1mL/kg)腹腔注射麻醉。固定后取腹正中切口，对照组在开腹显露肠系膜上动脉后不作阻断；I/R损伤模型组、低剂量6-BA保护组和高剂量6-BA保护组则均用无创动脉夹阻断其系膜血管30min后再灌注60min。实验结束后，立即剪取空肠标本待用。
3.6-BA对大鼠小肠I/R损伤后T-SOD、GSH-Px活性和MDA含量的影响
3.1 T-SOD活力、GSH-Px活力和MDA含量测定方法
制作10g/100ml小肠组织匀浆，用生理盐水稀释成1g/100ml小肠组织匀浆液后参照总超氧化物歧化酶(T-SOD)检测试剂盒说明操作，最后用紫外可见分光光度计在550nm处比色，测定吸光度，计算T-SOD活力。
取10g/100ml小肠组织匀浆用生理盐水稀释成0.8g/100ml的小肠组织匀浆液后，参照谷胱甘肽过氧化物酶(GSH-Px)检测试剂盒说明操作，最后用紫外可见分光光度计在412nm处比色，测定吸光度，计算GSH-Px活力。
取10g/100ml小肠组织匀浆用生理盐水稀释成5g/100ml匀浆液后通过TBA法参照丙二醛(MDA)检测试剂盒说明操作，最后用紫外可见分光光度计在532nm处比色，测定吸光度，计算MDA含量。
3.2 6-BA对大鼠小肠I/R损伤后T-SOD活力、GSH-Px活力和MDA含量的影响
各组T-SOD、GSH-Px活力和MDA含量的测定结果见表1。与对照组相比，I/R损伤模型组大鼠T-SOD、GSH-Px的活力显著降低(P<0.05)，MDA含量显著升高(P<0.05)，证实I/R损伤模型复制成功。低剂量6-BA保护组和高剂量6-BA保护组大鼠小肠组织T-SOD活力和GSH-Px活力显著高于I/R损伤模型组(P<0.05)，MDA含量显著低于I/R损伤模型组(P<0.05)。高剂量6-BA组的MDA含量与对照组间无显著差异。
表1结果显示，6-BA可以显著修复小肠组织损伤造成的T-SOD活力、GSH-Px活力下降问题，可以修复小肠组织损伤造成的MDA含量升高的问题，且高剂量的6-BA修复效果高于低剂量的6-BA。
表1 6-BA对大鼠小肠I/R损伤后T-SOD、GSH-Px活性和MDA含量的影响(mean±SD，n＝7)
注：同列数据间标有不同字母表示差异显著(P<0.05)
4.6-BA对大鼠小肠I/R损伤后DNA损伤的影响
4.1单细胞凝胶电泳法检测大鼠小肠DNA损伤
分别取对照组、I/R损伤模型组、低剂量6-BA保护组和高剂量6-BA保护组的空肠，制作小肠单细胞悬液，保存备用。50℃、0.75％的正常熔点的琼脂糖100μl铺于全磨砂载玻片上，放入37℃烘箱过夜烘干后作为底层胶；再取上述50℃、0.75％琼脂糖110μl加在底胶上，水平放于4℃冰箱中保存20min至凝固，作为第二层胶；吸取70μl制备好的小肠单细胞悬液和140μl在37℃水浴中保温的0.65％的低熔点琼脂糖混匀，吸取混匀液110μl滴加在第二层胶上，水平放于4℃冰箱中保存20min至凝固，作为第三层胶。将制备好的胶板浸入细胞裂解液，4℃裂解1h，然后置于电泳槽中加入电泳缓冲液，4℃解旋25min，电泳20min，电泳条件为20V，200mA。电泳结束后加入中和液中和15min，然后滴加10mg/L的EB水溶液40μl，染色10min。在暗室中用荧光显微镜观察并拍照。每组随机选取3张胶片，每张显微镜下随机选取8个视野，将彗星尾长>35μm视为拖尾，采用CASP软件测量各组细胞彗星尾部DNA含量、彗星尾长并统计拖尾率。
其中，每升细胞裂解液的配方是：2.5mol/LNaCl、100nmol/LNa2EDTA、10nmol/LTris，10g/L肌氨酸钠，临用前加体积分数为1％的TritonX-100，10％DMSO，溶剂为超纯水，pH＝10。每升电泳缓冲液的配方是：lmmol/LNa2EDTA，300mmol/LNaOH，pH＝13，溶剂为超纯水，4℃预冷备用。每升中和液的配方是：0.4mol/L的TriS-HCl，溶剂为超纯水，pH＝7.5。
各组大鼠小肠细胞DNA损伤程度见图1，其中图1A为对照组，图1B为I/R损伤模型组，图1C为低剂量6-BA保护组，图1D为高剂量6-BA保护组，由图1可知，对照组细胞核大小比较均一，为圆形荧光团，荧光强度均匀，边缘光滑，未出现明显彗星状拖尾；I/R损伤模型组小肠DNA损伤严重、彗星头部较其它组变小，出现明显彗星状尾巴；低剂量6-BA保护组和高剂量6-BA保护组小肠细胞DNA拖尾减轻，尾长减小，且高剂量6-BA保护组的小肠细胞DNA拖尾减轻幅度更大，尾长明显减小。
各组细胞尾部DNA含量和拖尾率见表2。由表2可知，与对照组相比，I/R损伤模型组细胞尾部DNA含量和拖尾率显著升高(P<0.05)。与模型组相比，低剂量6-BA保护组和高剂量6-BA保护组显著降低尾部DNA含量和拖尾率(P<0.05)，且高剂量6-BA保护组降低幅度更大。上述结果表明，6-BA可以显著修复大鼠小肠I/R引发的DNA损伤，较少小肠细胞凋亡。
表2 6-BA对大鼠小肠I/R损伤后DNA损伤的影响(mean±SD，n＝5)
注：同列数据间标有不同字母表示差异显著(P<0.05)
5.6-BA对大鼠小肠I/R损伤后Caspase-3表达的影响
5.1免疫组化法检测大鼠小肠凋亡相关蛋白Caspase-3的表达水平
采用免疫组化法检测大鼠小肠组织切片中Caspase-3(1:200)蛋白表达。参照免疫组化试剂盒，严格按照步骤进行。一抗4℃过夜，以PBS做阴性对照。DAB染色，苏木精复染，常规脱水、透明、干燥。光学显微镜下观察到组织细胞中出现黄色颗粒为阳性。
各组大鼠小肠I/R损伤后Caspase-3表达情况见图2，其中，图2A为对照组，图2B为I/R损伤模型组，图2C为低剂量6-BA保护组，图2D为高剂量6-BA保护组。与对照组相比，I/R损伤模型组大鼠小肠I/R损伤后Caspase-3免疫反应阳性细胞数量显著增多；而低剂量6-BA保护组和高剂量6-BA保护组Caspase-3阳性率明显弱于I/R损伤模型组，证实6-BA明显改善了小肠缺血再灌注诱发的小肠组织损伤。
I/R损伤是由缺血组织或者器官在恢复血流灌注后，产生的组织或器官损伤加重的现象，I/R损伤主要是由于再灌注期间，自由基的大量生成、炎症反应、凋亡等引起的。上述实验证明大量实验证实了6-苄氨基嘌呤可以作为抗小肠局部缺血再灌注损伤的活性物质，并且6-苄氨基嘌呤能有效防护小肠缺血再灌注引起的氧化损伤和小肠细胞凋亡，将其添加到药物、食品或保健品当中，抑制小肠组织损伤，延缓衰老抗疲劳，增强小肠组织抗缺血再灌注损伤的能力，具有重要的经济应用价值。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (2)
1.一种6-苄氨基嘌呤在制备抗小肠局部缺血再灌注引起的小肠细胞氧化损伤药物中的应用。
2.一种6-苄氨基嘌呤在制备抗小肠局部缺血再灌注引起的小肠细胞凋亡药物中的应用。
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