CN104546820A - 二苯乙烯二聚体防治造血干细胞放射性损伤的用途 - Google Patents
二苯乙烯二聚体防治造血干细胞放射性损伤的用途 Download PDFInfo
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Abstract
本发明公开了式（Ⅰ）所示的Vam3(Amurensis H)在制备预防和/或治疗造血干细胞放射性损伤疾病药物中的用途。
Description
技术领域
本发明涉及式（Ⅰ）所示的二苯乙烯二聚体Vam3(Amurensis H)的新医药用途，其具体为Vam3在制备防治造血干细胞放射性损伤疾病药物中的应用。
背景技术
随着核技术应用领域的不断扩大，尤其是核电站、辐射加工工艺等的出现，核辐射事故的发生呈现增加趋势，造成了严重的环境放射性污染，人员伤亡和经济损失。同样，由于家用电器的普及应用，特别是对于日常手机、电视、电脑、微波炉等电磁辐射给人体带来的危害，如失眠、焦躁、易疲劳，严重的有再障贫、白血病等，也都在严重危害着人们的身心健康，目前还没有专用的防治用药，同样需要引起我们高度重视。
造血干细胞是造血组织中一类具有自我更新，并且能够分化成外周血液系统中各类成熟的祖细胞。绝大部分的骨髓造血干细胞（Hematopoietic stem cell，HSC）存在于骨髓组织中，少量游离于外周血液循环系统中。日常生活中小剂量的辐射可以增强人体免疫功能，但是大剂量的电离辐射会给机体造成严重的生物学效应，包括生殖细胞遗传物质的改变，造血系统功能障碍，癌症的发生率增高等。近年来大量的研究表明，辐射会造成骨髓造血干细胞DNA结构和功能的改变，基因表达异常，细胞凋亡和活性氧（reactiveoxygen species，ROS）水平的增加等。正常情况下，骨髓造血干细胞及其支持细胞主要定位于骨髓的低氧微环境中，这使得骨髓造血干细胞能够免受氧化引起的损伤，维持骨髓造血干细胞正常的自我更新和分化功能。当细胞内活性氧水平升高时，定向分化产生红细胞系克隆和粒细胞/红细胞/单核细胞/巨噬细胞集落形成单位（FCU-GEMN）的骨髓造血干细胞则定向分化产生更多的髓系克隆而不是红细胞系克隆，进而影响骨髓造血干细胞的自我更新和定向分化。另外，ROS对骨髓造血干细胞细胞周期也会产生影响，如高水平的ROS会降低处于相对静止状态的G0期骨髓造血干细胞的比例，导致骨髓造血干细胞静止期缺陷，使得骨髓造血干细胞被消耗。近年来随着肿瘤放疗及核工业的迅速发展，有目的寻找毒性低，疗效高的防辐射新药成为当今的研究热点。氨基类化合物，蛋白酶抑制剂，天然药物，激素，维生素，金属元素和一些生物制剂等对辐射防护起着很大的作用，但是这些药物一般只能缓解症状，无法达到完全治愈的目的，而且有些药物具有一定的负作用，长期使用不可避免的造成机体器官的损害。
Vam3(Amurensis H)，是发明人从山葡萄根乙醇提取物中首次分离得到，现已可化学合成，结构式如式（Ⅰ）所示，制备方法见“山葡萄根提取物治疗炎症疾病的用途”（申请号03134647.2，公开号CN1600304）。Vam3预防和/或治疗造血干细胞放射性损伤未见报道。
发明内容
本发明的目的在于提供了如式I所述的化合物Vam3(AmurensisH)在制备预防和/或治疗造血干细胞放射性损伤疾病药物中的作用。
骨髓单个核细胞（Bone marrow mononuclear cells，BMMNCs）含有多种干细胞成分及辅助因子，对于维持造血干细胞的自我更新具有重要的作用。此外，这些细胞中还含有一定数量的造血辅助细胞，如造血基质祖细胞、成熟基质细胞等，它们可能产生多种细胞因子，具有支持和促进造血祖细胞增殖和分化功能。因此，骨髓单个核细胞的活力直接影响着骨髓造血系统的正常功能。受到电离辐射的病人常常表现为较低的骨髓单个核细胞活力。在本发明化合物对辐射暴露小鼠BMMNCs活力影响中，Vam3促进小鼠BMMNCs细胞活力增加了30%(P<0.005)，表明本发明化合物具有增强BMMNCs细胞活力的作用，能用于预防和/或治疗造血干细胞放射性损伤疾病。
辐射诱导的ROS可以与DNA反应，导致DNA被氧化，双链断裂；与膜脂质反应，导致细胞膜和细胞核损伤。ROS包括诸如超氧化物和羟基的自由基，还有非自由基，如H2O2。与ROS相关的生物学行为包括大量的自由基对细胞功能和活力是有害的，ROS与NO结合导致亚硝酸盐的产生等。受到电离辐射的病人，BMMNCs内ROS水平升高。高水平的ROS抑制BMMNCs正常功能的发挥，进而影响骨髓造血干细胞长期自我更新与定向分化。在本发明化合物对辐射暴露小鼠BMMNCs细胞内ROS水平的影响中，Vam3促进小鼠BMMNCs细胞ROS水平下降19%(P<0.005)，表明本发明化合物具有抑制BMMNCs细胞ROS水平增高的作用，能用于预防和/或治疗造血干细胞放射性损伤疾病。
骨髓是辐射敏性感极高的组织。辐射损伤后，存活的造血细胞的数量和增殖能力决定着伤后造血功能的状态和预后。BMMNCs克隆形成能力直接影响着骨髓HSC的数目和自我更新及分化能力，进而决定着骨髓HSC正常的造血功能。在本发明化合物对辐射暴露小鼠BMMNCs细胞克隆形成能力的影响中，Vam3促进小鼠BMMNCs细胞克隆形成能力增加增强75%(P<0.05)，表明本发明化合物具有促进BMMNCs细胞的克隆形成能力，能用于预防和/或治疗造血干细胞放射性损伤疾病。
上述实验证明，如式（Ⅰ）所示化合物能用于制备预防和/或治疗造血干细胞放射性损伤疾病的产品。所述的产品包括药物、保健食品。
附图说明：
图1、Vam3对辐射暴露小鼠BMMNCs活力的影响。与ctr（正常组）比较，P<0.001；与IR（辐射组）比较，P<0.001。n=9。
图2、Vam3对辐射暴露小鼠BMMNCs内ROS水平的影响。与ctr（正常组）比较，P<0.001；与IR（辐射组）比较，P=0.0188。n=6。
图3、Vam3对辐射暴露小鼠BMMNCs克隆形成能力影响。与ctr（正常组）比较，P=0.015；与IR（辐射组）比较，P=0.0156。n=6。
具体实施方式
1、给药与照射方法：小鼠灌胃给药，剂量为25mg/kg，给药后1小时小鼠4Gyγ射线全身照射，剂量率0.80Gy/min。
2、小鼠BMMNCs细胞的分离无菌分离小鼠双侧股骨胫骨，去除表面附着肌肉，用2.5ml一次性无菌注射器在Hank's液中将骨髓细胞冲出。将冲出的细胞悬液加入50ml EP管内，按照细胞悬液与Ficoll2：1比例用腰穿针在细胞悬液底部缓慢加入Ficoll，分离时将离心机升速与降速均调至最低。离心完毕取中间絮状层加入至另一50ml EP管内，Hank's液离心洗涤细胞两次，最后用RPMI-1640（加10%胎牛血清）培养基将细胞浓度调为1×106细胞/ml使用。
3、小鼠BMMNCs细胞活力检测实验分为对照组、照射对照组、照射加药组。将1×106细胞/ml浓度细胞悬液与接种于96孔板内，每孔200μl。多功能酶标仪检测细胞活力。
4、小鼠BMMNCs内ROS水平检测实验分为对照组、照射对照组、照射加药组。将1×106细胞/ml浓度细胞悬液1ml加入2ml无菌EP管内，各管按照细胞悬液与活性氧检测探针（DCFH-DA）1000：1的比例加入DCFH-DA，37℃CO2孵箱内孵育30min后离心，弃去上清后用无血清RPMI-1640培养基洗涤三次以洗去探针，最后用无血清去`RPMI-1640培养基将细胞悬起，接种至96孔黑板内，多功能酶标仪检测细胞荧光值。吸收光488nm，发射光522nm。
5、小鼠BMMNCs克隆形成能力检测实验分为对照组、照射对照组、照射加药组。调整细胞浓度为接种浓度的10倍，0Gy接种细胞浓度1×104细胞/ml、4Gy接种细胞浓度1×105细胞/ml。加0.2ml细胞至2ml M3534培养基中，振荡器充分混匀，静置2～5min，待气泡消失。用2.5ml注射器连接16＃平头注射器针头，吸取0.5ml细胞混悬液，加入24孔板，轻摇培养板，使培养基均匀分布。将培养板置入37℃，5％CO2培养箱中培养，第五天低倍观察集落形成情况，细胞数≥30为阳性集落。
实施例1Vam3对辐射暴露小鼠BMMNCs细胞活力影响
小鼠灌胃给药，剂量为25mg/kg，给药后1小时小鼠以4Gyγ射线全身照射，剂量率0.80Gy/min。无菌分离小鼠BMMNCs细胞，用RPMI-1640（加10%胎牛血清）培养基将细胞浓度调为1×106细胞/ml，接种于96孔板内，每孔200μl。
本实验的目的在于考察Vam3对辐射暴露小鼠BMMNCs活力的影响，结果见图1。
结果：受照射小鼠与未经过辐射暴露小鼠比较，BMMNCs活力下降27.4%，经灌胃给药小鼠辐射暴露后BMMNCs活力与未给予Vam3辐射暴露组的小鼠相比，细胞活力提高了30%。
实施例2Vam3对辐射暴露小鼠BMMNCs内ROS水平影响
将1×106细胞/ml浓度的小鼠BMMNCs细胞悬液1ml与活性氧检测探针（DCFH-DA）混合，孵育离心洗涤后，用无血清RPMI-1640培养基将细胞悬起，接种至96孔黑板内，多功能酶标仪检测细胞荧光值。
本实验的目的在于考察Vam3对辐射暴露小鼠BMMNCs内ROS水平的影响，结果见图2。
注：与ctr（正常组）比较，P<0.001；与IR（辐射组）比较，P=0.0188。n=6。
结果：辐射暴露后小鼠与未经过辐射暴露小鼠比较，BMMNCs单细胞内ROS值升高75%，经灌胃给药小鼠辐射暴露后BMMNCs单细胞内ROS水平下降约20%。
实施例3Vam3对辐射暴露小鼠BMMNCs克隆形成能力影响
调整细胞浓度为接种浓度的10倍，0Gy接种细胞浓度1×104细胞/ml、4Gy接种细胞浓度1×105细胞/ml。加0.2ml细胞至2ml M3534，充分混匀，接种于24孔板中，放入培养箱中培养，第五天低倍观察集落形成情况。
本实验的目的在于考察Vam3对辐射暴露小鼠BMMNCs克隆形成能力影响。
结果：辐射暴露后小鼠与未经过辐射暴露小鼠比较，BMMNCs克隆形成能力显著下降，经灌胃给药小鼠辐射暴露后BMMNCs克隆形成能力与未给予Vam3辐射暴露组小鼠相比增加75%。
Claims (2)
1.如式（Ⅰ）所示化合物在制备预防和/或治疗造血干细胞放射性
损伤疾病产品中的应用
2.根据权利要求1的应用，其特征在于，所述的产品包括药物、、保健食品。
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