CN105078958B - 淫羊藿素在制备抗nk/t细胞淋巴瘤药物中的用途 - Google Patents
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Abstract
本发明属中药领域，具体涉及淫羊藿素在制备抗NK/T细胞淋巴瘤药物中的用途。本发明经淫羊藿素干预人NK/T细胞淋巴瘤细胞株(SNK‑10、SNT‑8)实验证实，淫羊藿素体外可显著抑制SNK‑10和SNT‑8细胞的增殖，明显促进其凋亡，增加内源性Caspase蛋白和促凋亡蛋白Bax表达，抑制Bcl‑2、pBad表达；可诱导SNK‑10细胞内EB病毒活化，增加肿瘤细胞对抗病毒药物的敏感性。本发明的淫羊藿素可作为药物有效成分制备抗NK/T细胞淋巴瘤药物与药物学上可接受的载体制备抗NK/T细胞淋巴瘤的药物组合物；进一步与抗病毒药物制备抗NK/T细胞淋巴瘤增敏药物。
Description
技术领域
本发明属中药领域，涉及中药有效成分淫羊藿素新的药用用途，具体涉及淫羊藿素在制备抗NK/T细胞淋巴瘤药物中的用途。
背景技术
NK/T细胞淋巴瘤是一种高度侵袭性非霍奇金淋巴瘤，主要发生于淋巴结外的组织。该疾患早期临床表现不典型，病程进展迅速，预后较差，目前临床无特效化疗药物。研究显示，NK/T细胞淋巴瘤其发病与EB病毒(EBV)感染关系密切，EBV在肿瘤细胞中是潜伏感染，因此，为抗病毒治疗带来困难。有研究将EBV诱导进入裂解感染(活化)再联合抗病毒药物更昔洛韦(GCV)靶向抗病毒治疗EBV相关肿瘤。为相关领域提供从天然药物中筛选活性成分诱导EBV激活NK/T细胞淋巴瘤靶向治疗的新方向。
淫羊藿素是淫羊藿属植物提取物淫羊藿苷的水解衍生产物，其化学结构式如下：
淫羊藿素(Icaritin)
目前关于淫羊藿素在制备抗NK/T细胞淋巴瘤药物方面的用途尚未见报道。
发明内容
本发明的目的在于提供淫羊藿素的新的药用用途，具体涉及淫羊藿素在制备抗NK/T细胞淋巴瘤药物中的应用。
本发明的又一目的在于提供淫羊藿素在制备治疗NK/T细胞淋巴瘤曾敏药物中的应用。
本发明所述的淫羊藿素是淫羊藿属植物提取物淫羊藿苷的水解衍生产物，其化学结构式如下：
淫羊藿素(Icaritin)
本发明的目的通过以下技术方案实现，
采用淫羊藿素干预人NK/T细胞淋巴瘤细胞株(SNK-10、SNT-8)，结果显示：1.淫羊藿素体外可显著抑制SNK-10和SNT-8细胞的增殖，明显促进其凋亡，增加内源性Caspase蛋白和促凋亡蛋白Bax表达，抑制Bcl-2、pBad表达；2.淫羊藿素可诱导SNK-10细胞内EB病毒(EBV)活化，进而增加肿瘤细胞对抗病毒药物更昔洛韦(GCV)的敏感性。
本发明经实验证实单独应用淫羊藿素可抑制NK/T细胞淋巴瘤，而作为病毒激活剂的淫羊藿素联合抗病毒药物更昔洛韦(GCV)对NK/T细胞淋巴瘤具有更强的抗肿瘤作用。
本发明的淫羊藿素可作为药物有效成分制备抗NK/T细胞淋巴瘤药物；或，所述的的淫羊藿素可作为药物有效成分与药物学上可接受的载体制备抗NK/T细胞淋巴瘤的药物组合物；
更进一步的，本发明淫羊藿素与抗病毒药物制备抗NK/T细胞淋巴瘤增敏药物，本发明的实施例中，优选淫羊藿素作为药物有效成分与抗病毒药物更昔洛韦(GCV)制备抗NK/T细胞淋巴瘤增敏药物；所述的淫羊藿素可通过激活NK/T细胞淋巴瘤内潜伏感染的EB病毒，增加NK/T细胞淋巴瘤对抗病毒药物的敏感性。
附图说明
图1：淫羊藿素对两种NK/T细胞淋巴瘤细胞株细胞增殖的影响，
其中：MFI为平均荧光强度；图1A为CCK-8法检测各浓度淫羊藿素对SNK-10和SNT-8细胞活力的影响；图1B为CFDASE染色法检测淫羊藿素处理两株细胞48h对细胞增殖的影响，细胞MFI值越高细胞增殖速度越慢；与对照组相比，*P<0.05，#P<0.01；各组实验重复3遍以上。
图2：淫羊藿素对SNK-10和SNT-8细胞凋亡的影响，
其中：图2A为AnnexinV/PI双染法流式检测细胞凋亡率，淫羊藿素干预时间为48h，与对照组相比，*P<0.05，#P<0.01；图2B为Hoechst33258染色法示细胞凋亡形态改变，药物处理组细胞核出现核固缩、核断裂和浓染白色荧光等典型凋亡形态改变，淫羊藿素干预时间为48h。
图3：淫羊藿素对NK/T细胞淋巴瘤细胞株Caspase、Bcl-2家族蛋白表达的影响，
其中：检测方法为Westernblot，淫羊藿素干预时间为48h。
图4：淫羊藿素对SNK-10细胞内EBV相关基因表达的影响，
其中：检测方法为Real-timePCR，淫羊藿素干预时间为48h，与对照组相比，*P<0.05，#P<0.01；实验重复3遍以上。
图5：淫羊藿素对Zta蛋白表达的影响，
其中：检测方法为Westernblot，淫羊藿素干预时间为48h。
图6：淫羊藿素增加SNK-10细胞对GCV的敏感性，
其中：AnnexinV/PI双染法流式检测细胞凋亡率，淫羊藿素干预时间为48h，与对照组相比，*P<0.05，#P<0.01。
具体实施方式
实施例1：淫羊藿素抑制NK/T细胞淋巴瘤细胞增殖、诱导细胞凋亡试验
一、材料与方法
1.细胞株，采用研究NK/T细胞淋巴瘤常用的细胞系：本实施例的人NK/T细胞淋巴瘤细胞株SNK-10和SNT-8购自日本东京医科齿科大学，均为EB病毒阳性，所述细胞株用含有10％灭活人血清及700U/mlIL-2的PRIM-1640培养基在37℃、体积分数为5％CO2、完全饱和湿度条件下常规培养，2～3天传代1次，实验选用对数生长期细胞；
2.细胞活力的检测
分别调整SNK-10和SNT-8细胞株细胞悬液浓度为1×105/ml，加入96孔培养板内，每孔100μl，分别加入等体积不同浓度淫羊藿素(SNK-10细胞所用药物浓度为50μM，30μM，20μM，10μM；SNT-8细胞所用药物浓度为32μM、16μM、8μM、4μM)，设空白对照组和调零组，每组均设3复孔，分别培养24h、48h、72h。每孔加入10μLCCK-8溶液,置37℃、5％CO2孵箱培养4h，在490nm处检测各孔的吸光值；细胞活力＝(加药组OD平均值-调零孔OD平均值)/(空白对照组OD平均值-调零孔OD平均值)×100％。实验重复3遍以上；
2.细胞增殖的检测
取SNK-10细胞4×106个(或SNT-8细胞5×106个)，用1mlCFDASE细胞标记液重悬细胞，置于15ml离心管内。加入1mlCFDASE储存液(2X)，轻轻混匀，37℃孵育10分钟，加入约10ml完全细胞培养基洗涤2次，再加入8ml完全细胞培养液，37℃孵育5分钟，以促进CFDASE在细胞内的驻留及未反应的CFDASE进入完全细胞培养液；1000rpm×5min离心去上清，完成最后一次洗涤，用10ml完全培养基重悬细胞，混匀，将细胞接种于6孔板，每孔2ml，SNK-10细胞密度为4×105/ml，SNT-8细胞密度为5×105/ml；细胞培养孔中分别加入等体积不同浓度淫羊藿素，SNK-10细胞培养孔中淫羊藿素终浓度分别为0μM、10μM、20μM、30μM、50μM，SNT-8细胞培养孔内淫羊藿素终浓度分别为0μM、4μM、8μM、16μM、32μM，置于CO2孵育箱培养48h，PBS洗涤一次后用流式细胞仪检测CFSE荧光，每组实验重复3次，计算均数±标准差；
3.细胞凋亡检测
调整SNK-10细胞悬液浓度为4×105/ml(SNT-8细胞浓度为5×105/ml)，将细胞接种于6孔板，每孔2ml，分别加入等体积不同浓度淫羊藿素(药物浓度同上)，置于37℃、5％CO2孵育箱中培养48h后，收集细胞300g×5min、4℃离心去上清，用冷PBS洗涤细胞两次(300g×5min、4℃离心收集细胞沉淀)，用400μL1×AnnexinV结合液悬浮细胞，调整细胞浓度约为1×106/ml，在细胞悬液中加入2.5μLAnnexinV染色液，轻轻混匀后4℃避光孵育15min，再加入5μLPI染色液，轻轻混匀后于4℃避光孵育5min，立即用流式细胞仪检测细胞凋亡率；
4.细胞凋亡形态检测
调整SNK-10细胞悬液浓度为4×105/ml(SNT-8细胞浓度为5×105/ml)，将细胞接种于6孔板，每孔2ml，分别加入等体积不同浓度淫羊藿素(药物浓度同前)，置于37℃、5％CO2孵育箱中培养48h后，收集细胞1000rpm×5min离心去上清，加入0.5ml固定液，置于4℃冰箱固定10min，1000rpm×5min离心去固定液，用PBS洗两遍，1000rpm×5min离心后吸去大部分液体保留约50μL液体，再缓缓悬起细胞，滴加至载玻片上，尽量使细胞分布均匀，稍晾干后均匀滴上0.5mlHoechst33258染色液，染色5分钟，微晾干后去染色液，用PBS洗两遍，每次3分钟，吸尽液体，滴一滴抗荧光淬灭封片液于载玻片上，盖上一洁净的盖玻片，用荧光显微镜检测细胞核形态；
4.Caspase、Bcl-2家族蛋白表达的检测
调整SNK-10细胞悬液浓度为4×105/ml(SNT-8细胞浓度为5×105/ml)，将细胞接种于6孔板，每孔2ml，分别加入等体积不同浓度淫羊藿素(药物浓度同前)，置于37℃、5％CO2孵育箱中培养48h后，抽提蛋白，定量后行SDS-PAGE电泳分离，待电泳完毕后转印至硝酸纤维素膜，在室温下封闭，加入Caspase、Bcl-2家族蛋白一抗,4℃过夜，加入HRP标记二抗,室温反应1h,增强化学发光法显色,X线底片曝光显影；
二、结果显示
1.淫羊藿素对两种NK/T细胞淋巴瘤细胞株细胞增殖影响的检测结果
本实验采用CCK-8法检测细胞活力与毒性，结果显示，淫羊藿素体外对两种NK/T细胞淋巴瘤细胞株(SNK-10和SNT-8)细胞活力均显示了一定的抑制作用(如表1、表2所示)，且呈浓度和时间依赖性(如附图1A)所示；淫羊藿素对SNT-8细胞活力的抑制作用更显著，CFDASE染色法能够反应细胞增殖的速度，随着细胞分裂次数的增加，细胞内荧光强度逐渐下降，流式检测结果显示SNK-10细胞对照组MFI值为164.3，20、30和50μM淫羊藿素分别将SNK-10细胞的MFI值增加至451.7(p<0.05)、767.3(P<0.01)和1039.3(P<0.01)；与CCK-8法所测结果相似，淫羊藿素能抑制两株细胞的分裂增殖，且对SNT-8的抑制作用更强(如附图1B所示)；
表1SNK-10细胞经淫羊藿素处理后的细胞活力
F＝22.08，p<0.0001
表2SNT-8细胞经淫羊藿素处理后的细胞活力
F＝19.69，p<0.0001
2.淫羊藿素对NK/T细胞淋巴瘤细胞凋亡影响的检测结果
采用AnnexinV/PI双染流式细胞术检测表明，淫羊藿素对NK/T细胞淋巴瘤瘤两株细胞显示了诱导细胞凋亡作用，不同浓度淫羊藿素(0μM、10μM、20μM、30μM、50μM)作用SNK-10细胞48h，其细胞凋亡率由8.92％±1.31％增至31.21％±4.88％，淫羊藿素(0μM、4μM、8μM、16μM、32μM)作用SNT-8细胞48h，细胞凋亡率由10.33％±2.31％增至58.44％±8.29％，与对照组相比均具有差异(P＜0.01)(如附图2A所示)；Hoechst33258染色结果显示，药物处理组细胞出现明显的核固缩、核断裂等典型凋亡形态改变(如附图2B所示)；
3.淫羊藿素对Caspase、Bcl-2家族蛋白表达的影响
Westernblot结果表明，淫羊藿素可增加裂解的Caspase9、Caspase3蛋白和促凋亡蛋白Bax表达，抑制Bcl-2、pBad表达，呈量效关系，对总Bad蛋白表达无影响(如附图3所示)。
实施例2
淫羊藿素诱导SNK-10细胞内EBV活化，并增加其对GCV的敏感性试验一、材料与方法
1.细胞株：人NK/T细胞淋巴瘤细胞株SNK-10，培养方法同上。
2.淫羊藿素对EBV相关基因表达的影响
调整SNK-10细胞悬液浓度为4×105/ml，将细胞接种于6孔板，每孔2ml，分别加入等体积不同浓度淫羊藿素(药物浓度同前)培养48h，抽提RNA，逆转录为cDNA后行Real-timePCR(使用Takara公司SYBRPremixExTaqTM试剂盒)，检测EBV裂解期基因(BZLF1、BRLF1和BMRF1)和潜伏期基因EBNA1表达，以GAPDH为内参，计算各样品的△Ct(目的基因的Ct值-GAPDH基因Ct值)，以表达最低样品作为校正样品，计算△△Ct(样品△Ct-校正品△Ct)，最终计算基因相对表达量RQ＝2-△△Ct；
3.淫羊藿素对Zta蛋白表达影响
调整SNK-10细胞悬液浓度为4×105/ml，将细胞接种于6孔板，每孔2ml，分别加入等体积不同浓度淫羊藿素(药物浓度同前)培养48h，抽提蛋白，定量后行SDS-PAGE电泳分离，待电泳完毕后转印至硝酸纤维素膜，在室温下封闭，加入Zta蛋白一抗，4℃过夜，加入HRP标记二抗，室温反应1h，增强化学发光法显色，X线底片曝光显影；
4.淫羊藿素增加SNK-10细胞对GCV的敏感性
调整SNK-10细胞悬液浓度为4×105/ml，将细胞接种于6孔板中4孔，每孔2ml，分别加入淫羊藿素(50μM)、GCV(25μg/ml)和淫羊藿素(50μM)联合GCV(25μg/ml)，空白组加含0.1％DMSO的培养基，药物作用48h后，收集细胞300g×5min、4℃离心去上清，冷PBS洗涤细胞两次，用400μL1×AnnexinV结合液悬浮细胞，调整细胞浓度约为1×106/ml，在细胞悬液中加入2.5μLAnnexinV染色液，轻轻混匀后4℃避光孵育15min，再加入5μLPI染色液，轻轻混匀后于4℃避光孵育5min，立即用流式细胞仪检测细胞凋亡率；
二、结果显示
1.淫羊藿素对EBV相关基因表达的影响
Real-timePCR结果示，淫羊藿素可使EBV裂解期基因BZLF1、BRLF1和BMRF1表达升高，抑制潜伏期基因EBNA1表达，且呈剂量依赖性(如附图4所示)；
2.淫羊藿素对Zta蛋白表达影响
Zta蛋白是EBV即刻早期基因BZLF1的表达产物，可作为转录因子发挥反式激活作用引起下游裂解期蛋白的瀑布式效应；本实验结果显示，Zta蛋白随淫羊藿素浓度增高表达逐渐增加，50μM淫羊藿素对EBV的激活作用最强(如附图5所示)；
3.淫羊藿素增加SNK-10细胞对GCV的敏感性检测结果
AnnexinV/PI双染法检测细胞凋亡，结果显示，GCV单用组细胞凋亡率与对照组相比无显著差异，而淫羊藿素联合GCV处理组细胞凋亡率为59.99％±3.35％，显著高于单用淫羊藿素组30.19％±2.23％(P<0.01)和单用GCV组10.78％±0.94％(P<0.01)(如附图6所示)。
本发明的实验结果表明，淫羊藿素体外可显著抑制SNK-10和SNT-8细胞的增殖，明显促进其凋亡，增加内源性Caspase蛋白和促凋亡蛋白Bax表达，抑制Bcl-2、pBad表达；淫羊藿素可诱导SNK-10细胞内EB病毒(EBV)活化，进而增加肿瘤细胞对抗病毒药物更昔洛韦(GCV)的敏感性。
Claims (2)
1.淫羊藿素在制备抗NK/T细胞淋巴瘤药物中的应用，所述的淫羊藿素其化学结构式如下：
2.按权利要求1所述的应用，其特征在于，所述的淫羊藿素抑制SNK-10和SNT-8细胞的增殖，促进其凋亡，增加内源性Caspase凋亡蛋白和促凋亡蛋白Bax表达，抑制Bcl-2、pBad表达。
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| 淫羊藿素体外抗淋巴瘤细胞增殖效应;范双翼等;《中国比较医学杂志》;20110630;第21卷(第6期);6-11 * |
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