CN105998616A - 古日古木-13促进肝再生的应用及作用机制评价方法 - Google Patents

古日古木-13促进肝再生的应用及作用机制评价方法 Download PDF

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CN105998616A
CN105998616A CN201610447439.8A CN201610447439A CN105998616A CN 105998616 A CN105998616 A CN 105998616A CN 201610447439 A CN201610447439 A CN 201610447439A CN 105998616 A CN105998616 A CN 105998616A
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张海峰
边浩
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Abstract

本发明提供古日古木‑13在促进肝再生上的应用及其作用机制的评价方法，(1)将实验鼠分为分为12组；(2)对PH手术组的小鼠进行70％肝大部切除术，假手术组采用剖腹探查术，空白对照组不进行处理；(3)对所有实验动物按分组给古日古木高、中、低剂量试剂和溶剂，每天1次；(4)灌胃第1、3、5、7、9天分别断头取血，完整切取肝脏称重；(5)取一小部分肝称重，用盐水匀浆，测SOD、MDA、NO，另取部分肝组织福尔马林固定后，HE染色进行组织病理学检测；(6)检测血请中各种生化指标、细胞因子表达情况、计算肝指数、观察小鼠肝脏细胞的增殖、凋亡和分化。本发明的有益效果是丰富了古日古木的药用价值。

Description

古日古木-13促进肝再生的应用及作用机制评价方法
技术领域
本发明属于医药技术领域，尤其是涉及一种古日古木-13促进肝再生的应用及作用机制评价方法。
背景技术
古日古木-13(别名红花清肝十三味丸)是蒙医传统药方，出自《中华人民共和国卫生部药品标准》(蒙药分册)。本药气香，味苦、涩，性凉，由红花、丁香、莲子、麦冬、木香、诃子、川楝子、栀子、紫檀香、麝香、水牛角浓缩粉、牛黄、银朱组成。具有清热、解毒、凉血之功效。该药对各种肝热症、配毒症及肝功减退都有作用，尤以血热引起的眼病为甚，亦对“亚玛病”之肾伤、尿频有效。目前，对蒙药古日古木-13研究主要集中在酒精性肝病、病毒性肝炎、胆汁淤积性肝炎、高血压病、药物性肝损伤、急性肝损伤伤、老年性高血脂等病，而对肝再生的研究鲜有报道。
本发明拟通过对小鼠进行肝大部分切除术从而建立动物模型，分别给予实验动物高、中、低剂量蒙药古日古木-13，一周后检测血中各种生化指标、细胞因子的表达情况、计算肝指数、观察动物肝脏细胞的增殖、凋亡和分化的指标，并对对动物的肝脏进行油红0染色；同时分别用不同剂量的蒙药古日古木-13处理肝癌细胞HepG2，观察其对细胞增殖、分化和凋亡的影响。
发明内容
本发明通过动物实验和细胞实验两个方面，探讨蒙药古日古木-13对肝再生的影响及可能机制，为蒙药古日古木-13的进一步药用开发开拓新的途径，为丰富肝再生的理论及临床上肝衰竭的治疗提供新的思路。
本发明的技术方案是：
古日古木-13在促进肝再生上的应用。
本发明的另一方面还包括古日古木-13影响肝再生作用机制的评价方法，包括以下步骤：动物实验：(1)C57BL/6小鼠60只，雌雄各半，实验室适应性喂养5d后，随机分为12组，每组5只，分为PH手术-古日古木高剂量组，PH手术-古日古木中剂量组，PH手术-古日古木低剂量组，PH手术-溶剂对照组，假手术-古日古木高剂量组，假手术-古日古木中剂量组，假手术-古日古木低剂量组，假手术-溶剂对照组，和空白对照-古日古木高剂量组，空白对照-古日古木中剂量组，空白对照-古日古木低剂量组，空白对照-溶剂对照组；
(2)对PH手术组的小鼠进行经典的70％肝大部切除术，假手术组采用剖腹探查术，空白对照组不进行手术处理；
(3)待小鼠苏醒恢复后对所有实验动物按分组给古日古木高、中、低剂量试剂和溶剂，每天1次；
(4)灌胃第1、3、5、7、9天五个时间点后分别断头取血，并完整切取小鼠肝脏并称重；
(5)取一小部分肝称重，用-4℃的盐水匀浆，按说明书的方法测SOD、MDA、NO。另取部分肝组织用10％福尔马林固定后，HE染色进行组织病理学检测；
(6)检测血请中各种生化指标、细胞因子的表达情况、计算肝指数、观察小鼠肝脏细胞的增殖、凋亡和分化的情况；
细胞实验：同时分别用不同剂量的蒙药古日古木-13处理肝癌细胞HepG2，观察其对细胞增殖、分化和凋亡的影响。
附图说明
图1a-图1g，利用所有实验数据统计ALT，AST，GLU，TG，CHOL，HDL，LDL。
图2a-图2g，除去蓝色底纹(极端值)和黑鼠值后ALT，AST，GLU，TG，CHOL，HDL，LDL的统计结果
具体实施方式
下面结合附图对本发明做详细说明。
1、实验动物分组与处理
C57BL/6小鼠60只，雌雄各半，实验室适应性喂养5d后，随机分为12组，每组5只，分为PH手术-古日古木高剂量组，PH手术-古日古木中剂量组，PH手术-古日古木低剂量组，PH手术-溶剂对照组，假手术-古日古木高剂量组，假手术-古日古木中剂量组，假手术-古日古木低剂量组，假手术-溶剂对照组，和空白对照-古日古木高剂量组，空白对照-古日古木中剂量组，空白对照-古日古木低剂量组，空白对照-溶剂对照组，古日古木高、中、低剂量组分别为1600mg/(kg·d-1)，800mg/(kg·d-1)，400mg/(kg·d-1)。对所有实验动物按分组给古日古木高、中、低剂量试剂和溶剂，每天1次，共灌胃7天。
然后对PH手术组的小鼠进行经典的70％肝大部切除术，假手术组采用剖腹探查术，空白对照组不进行手术处理，待小鼠苏醒恢复后对所有实验动物按分组给古日古木高、中、低剂量试剂和溶剂，每天1次，灌胃第1、3、5、7、9天五个时间点后分别断头取血，并完整切取小鼠肝脏并称重。取一小部分肝称重，用-4℃的盐水匀浆，按说明书的方法测SOD、MDA、NO。另取部分肝组织用10％福尔马林固定后，HE染色进行组织病理学检测。检测血请中各种生化指标、细胞因子的表达情况、计算肝指数、观察小鼠肝脏细胞的增殖、凋亡和分化的情况。
2、肝大部切除术
PH手术组小鼠饥饿过夜，用体重计称重，并标记。用1ml无菌空针抽取4％水合氯醛溶液，按8μl/g在腹腔内注射，小鼠完全麻醉倒后(约1-3min)，采取仰卧位，将其固定于操作台上，注意观察小鼠胸廓起伏情况，以此来判断其呼吸及心跳情况。麻醉后用用小剪刀或脱毛剂去除小鼠胸部至下腹部毛发，安尔碘消毒腹部三次，后一次消毒范围应小于前一次强力碘消毒区域，消毒好的小鼠从剑突下做横行正中切口，切口长约1-2cm，充分显露肝脏，剪开镰状韧带至下腔静脉前方。挤出小鼠肝脏，用棉签抬起左外叶，剪开肝脏脏面的肝胃韧带和部分肝左侧冠状韧带，此时肝左外叶基本游离。取1号丝线置于肝脏脏面，绕过左外叶，在肝脏膈面尽量靠近根部结扎，剪下肝左外叶。同时抬起肝左中叶和右中叶，从脏面绕过1号丝线，在肝脏膈面靠近根部处结扎，剪下这两叶。这样就达到了肝脏大部切除(近70％)。用棉签清除腹腔内出血，查看无活动性出血后，用0号丝线分别缝合腹膜和腹壁。用酒精棉球擦去小鼠切口处血液，防止小鼠咬破切口处缝线。术后将PH实验组小鼠放回鼠笼，并置于灯光下促进小鼠苏醒。保持室内温度于25℃-30℃，并给与自由进食水，观察小鼠的存活状态。
3、剖腹探查假手术组
假手术组(剖腹探查术组)小鼠饥饿过夜，用体重计称重，并标记。用1ml无菌空针抽取4％水合氯醛溶液，按8μl/g在腹腔内注射，小鼠完全麻醉倒后(约1-3min)，采取仰卧位，将其固定于操作台上，注意观察小鼠胸廓起伏情况，以此来判断其呼吸及心跳情况。用小剪刀或脱毛剂去除小鼠胸部至下腹部毛发，安尔碘消毒腹部三次，后一次消毒范围应小于前一次强力碘消毒区域。消毒好的小鼠从剑突下做横行正中切口，切口长约1-2cm，充分显露肝脏，。再将小鼠肝脏重新放回小鼠腹腔内，注意观察有无出血情况的发生。用0号丝线分别缝合腹膜和腹壁。用酒精棉球擦去小鼠切口处血液，防止小鼠咬破切口处缝线。术后将假手术组小鼠放回鼠笼，并置于灯光下促进小鼠苏醒。保持室内温度于25℃-30℃，并给与自由进食水，观察小鼠的存活状态。
4、生化指标的检测
取得的血液在3500r/min，离心10min，血清测ALT，AST，总蛋白和白蛋白；取一小部分肝称重，用-4℃的盐水匀浆，按说明书的方法测SOD、MDA、NO。另取部分肝组织用10％福尔马林固定后，HE染色进行组织病理学检测。
5、统计学分析
如图1a-图1g，图2a-图2g所示，采用SPSS22.0统计软件进行分析。计量资料以均数±标准差表示，ALT，AST，GLU，TG，CHOL，HDL，LDL等定量差异比较采用t检验。P＜0.05为差异有统计学意义。
以上对本发明的一个实施例进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。

Claims (2)

1.古日古木-13在促进肝再生上的应用。
2.古日古木-13影响肝再生作用机制的评价方法，其特征在于：包括以下步骤：动物实验：(1)C57BL/6小鼠60只，雌雄各半，实验室适应性喂养5d后，随机分为12组，每组5只，分为PH手术-古日古木高剂量组，PH手术-古日古木中剂量组，PH手术-古日古木低剂量组，PH手术-溶剂对照组，假手术-古日古木高剂量组，假手术-古日古木中剂量组，假手术-古日古木低剂量组，假手术-溶剂对照组，和空白对照-古日古木高剂量组，空白对照-古日古木中剂量组，空白对照-古日古木低剂量组，空白对照-溶剂对照组；
(2)对PH手术组的小鼠进行经典的70％肝大部切除术，假手术组采用剖腹探查术，空白对照组不进行手术处理；
(3)待小鼠苏醒恢复后对所有实验动物按分组给古日古木高、中、低剂量试剂和溶剂，每天1次；
(4)灌胃第1、3、5、7、9天五个时间点后分别断头取血，并完整切取小鼠肝脏并称重；
(5)取一小部分肝称重，用-4℃的盐水匀浆，按说明书的方法测SOD、MDA、NO，另取部分肝组织用10％福尔马林固定后，HE染色进行组织病理学检测；
(6)检测血请中各种生化指标、细胞因子的表达情况、计算肝指数、观察小鼠肝脏细胞的增殖、凋亡和分化的情况；
细胞实验：同时分别用不同剂量的蒙药古日古木-13处理肝癌细胞HepG2，观察其对细胞增殖、分化和凋亡的影响。
CN201610447439.8A 2016-06-07 2016-06-07 古日古木-13促进肝再生的应用及作用机制评价方法 Pending CN105998616A (zh)

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CN102178772A (zh) * 2011-03-24 2011-09-14 白国荣 一种强筋壮骨、活血化瘀、消炎镇痛的蒙药

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CN102178772A (zh) * 2011-03-24 2011-09-14 白国荣 一种强筋壮骨、活血化瘀、消炎镇痛的蒙药

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李圆: "白藜芦醇对大鼠Egr-1表达及肝细胞再生的影响", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 *
李岚 等: "蒙药古日古木-13对小鼠CCl4肝损伤模型的影响", 《中国民族医药杂志》 *
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