CN114432386A - 一种马归液在非可控性炎症中的用途及验证方法 - Google Patents
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Abstract
本发明属于中医药技术领域,公开了一种马归液在非可控性炎症中的用途及验证方法,所述马归液在非可控性炎症中的用途,包括马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达具有干预作用;所述验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法包括:细胞培养;免疫荧光法检测CRL‑9520 SV‑HUC‑1细胞p‑HER2;Western Blotting方法检测p‑HER2在CRL‑9520细胞中表达;统计学分析。结果表明,LPS刺激SV‑HUC‑1细胞HER2磷酸化;马归液能较好控制LPS致炎细胞HER2磷酸化,通过降低致炎细胞内HSP90蛋白的表达抑制HER2的磷酸化。
Description
技术领域
本发明属于中医药技术领域,尤其涉及一种马归液在非可控性炎症中的用途及验证方法。
背景技术
目前,膀胱癌(Bladder Cancer,BC)是全球第九大常见癌症,男性高发。在美国,BC是男性第四大常见癌症。膀胱癌的主要发病机制尚未完全清楚,但血吸虫、感染及机械损伤等导致的慢性炎症,可能诱发膀胱癌的产生。慢性炎症导致诸多细胞因子释放,形成炎症微环境,如HSP90等因子,而HSP90可与癌基因HER2相互作用。
通过上述分析,现有技术存在的问题及缺陷为:膀胱癌的主要发病机制尚未完全清楚,在分子生物学层面,肿瘤细胞表现出特有的异质性,目前尚不能确定其发生过程中的效应子及分子标志物。
解决以上问题及缺陷的难度为:综合目前的国内外研究,尚没有阐明膀胱癌的主要发病机制,其发生发展的机制错综复杂,主要涉及以下8个方面:①癌基因激活及过度表达;②抑癌基因突变及缺失;③与修复功能相关的基因功能丧失;④在基因组中出现了异常的核苷酸串联重复;⑤调控信号的传导通路功能紊乱;⑥细胞端粒酶过度表达;⑦细胞凋亡机制发生障碍;⑧出现浸润转移等相关分子事件。从分子生物学的角度来看,参与这些过程的效应子主要可以分为癌相关基因以及包括Micro-RNA在内的非编码RNA,然而,目前尚没有确定的分子能全部解释膀胱癌的发生发展及转归情况,因此,在庞大的人类全基因组序列中,以及肿瘤细胞具备异质特性,不断的寻找膀胱癌发病机制中的相关效应子和分子标志物十分重要和具备一定的挑战。
解决以上问题及缺陷的意义为:膀胱癌是严重威胁人类健康及生活质量的疾病,其发病机制尚不明确,阐明其发病机制有望预测膀胱癌的预后及转归,为新的靶向药物开发提供理论依据,以及有望“攻克”癌症。
发明内容
针对现有技术存在的问题,本发明提供了一种马归液在非可控性炎症中的用途及验证方法。
本发明是这样实现的,一种马归液(50mg/ml)(无辅助药物)在非可控性炎症中的用途,所述马归液在非可控性炎症中的用途,包括马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达具有干预作用。
本发明的另一目的在于提供一种验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法,所述验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法包括以下步骤:
步骤一,进行细胞培养和实验细胞分组;
步骤二,免疫荧光法检测CRL-9520 SV-HUC-1细胞p-HER2;
步骤三,Western Blotting方法检测p-HER2在CRL-9520细胞中表达;
步骤四,统计学分析:采用SPSS19.0软件对结果进行数据分析。
进一步,步骤一中,所述细胞培养,包括:
CRL-9520 SV-HUC-1细胞培养在含10%FBS、100U/ml青霉素和100μg/ml链霉素的DMEM/F12完全培养基中,保持37℃5%CO2不变;细胞密度1×105个/ml接种在各规格培养板或培养皿,当细胞生长80%融合时,按预实验结果,选择小鼠抗人的EGF抗体50ng/ml,母液10μg/ml;小鼠抗人TGFα抗体5ng/ml,母液5μg/ml;37℃,30min后,在培养基中加LPS 10μg/mL,培养96h。
进一步,步骤一中,所述实验细胞分组,包括:
正常细胞对照组;加入等量的培养液代替LPS;
LPS模型组:加入所述LPS溶液;
马归液组:在加入LPS前加入50mg/ml的马归液;
抗内毒素抗体组:于加入LPS前加入等量的抗内毒素抗体;
HSP90抑制剂组:加入10ng/mL浓度的17-AAG溶液后,再加入LPS溶液。
进一步,步骤二中,所述免疫荧光法检测CRL-9520 SV-HUC-1细胞p-HER2,包括:
(1)盖玻片清洗,过酸过夜,蒸馏水及去离子水清洗后,擦干,60℃处理2h备用;处理的盖玻片平放六孔板内,将密度为10×104/mL细胞滴加于盖玻片上,37℃,50%CO2培养96h;将培养好的细胞进行镜下观察,并用4℃ PBS轻轻洗2遍;4%多聚甲醛4℃固定10min;4℃ PBS温柔漂洗3遍,5min/次;
(2)用含0.25%TrixtonX-100的PBS作用10min,透化细胞膜;4℃ PBS温柔漂洗3遍,5min/次;5%BSA封闭1h;室温下直接用10%正常的山羊血清进行封闭,按1:100的比例加入兔抗人的p-HER2抗体,置湿盒4℃孵育过夜;
(3)第二天取出复温20min,4℃PBS温柔冲洗3遍,洗净一抗;加入荧光二抗,37℃与羊抗兔二抗和羊抗小鼠二抗联合孵育1h,50~100μL/孔,室温孵育2h;4℃PBS温柔冲洗3遍,洗净二抗;细胞核用显核剂染色,在免疫荧光显微镜下采集图像,并用ImageJ计算平均光密度。
进一步,步骤(1)中,培养过程中,细胞培养24h换细胞生长维持液。
进一步,步骤三中,所述Western Blotting方法检测p-HER2在CRL-9520细胞中表达,包括:
(1)蛋白样品制备
单层贴壁细胞核蛋白的提取:①选择长满单层的,无污染的CRL-9520细胞,吸弃去培养液,将细胞瓶倒扣在吸水纸上,吸干培养液;②每瓶细胞加2mL预冷的PBS 1min弃去PBS液,反复3次;③加蛋白酶抑制剂,磷酸酶抑制剂各100μL/孔作用10min后,冰上加RIPA裂解液0.5mL,细胞培养瓶置冰上,加入400μL裂解液,冰上30min,来回摇动细胞瓶,使细胞充分裂解;④无菌细胞刮棒将细胞刮于培养瓶的一侧,超声裂解细胞;⑤4℃ 16000g离心5min,取上清-80℃保存备用。
BCA法检测样品蛋白浓度:①称取0.5g牛血清白蛋白,溶于蒸馏水中并定容至100ml,制成5mg/ml的溶液;用时稀释十倍;②绘制标准曲线:取96孔酶标板,加入试剂;③吸取20μl样品溶液于酶标板孔中,加入BCA试剂200μl,轻摇,37℃ 50min,室温10min,以空白为对照,在酶标仪562nm处测定吸光值OD,以牛血清白蛋白含量为横坐标,以吸光值为纵坐标,绘制标准曲线;以标准曲线空白为对照,根据样品的吸光值从标准曲线上查出样品的蛋白质含量。
以BSA各级工作液对应的蛋白质浓度蛋白质浓度Y,mg/mL,对OD值x作图;Y=0.32.36X-0.07,相关系数r=0.964。
(2)SDS--PAGE电泳
①灌胶与上样:玻璃板对齐后放入夹中卡紧,垂直卡在架子上准备灌胶;制10%分离胶:称取一定量的琼脂糖于三角瓶中,加入1xTBE液,微波炉加热3min,使琼脂糖完全溶解,待溶解的琼脂糖温度下降至60℃时,加入终浓度0.1pg/ml的GeneFinder液,充分混匀后倒板,待凝固,小心移去梳子,将凝胶板置入电泳槽,向电泳槽中倒入1xTBE,使之没过胶面2mm;配好的10%分离胶加入TEMED后立即摇匀,灌胶;配8%的浓缩胶,加入TEMED后立即摇匀灌胶,剩余空间灌满浓缩胶后,将梳子水平插入浓缩胶中;待到浓缩胶凝固后,两手分别捏住梳子的两边垂直向上轻慢将其拔出;用无离子水冲洗浓缩胶,将其放入电泳槽中。
测完蛋白含量后,计算出20μL蛋白的溶液体积作为上样量;吸取样品至200μL的EP管中,加入5xSDS上样缓冲液至终浓度为l×;上样前将样品于沸水中煮5min使蛋白变性;加足够的电泳液后,即电泳液漫过内测的小玻璃板,用微量进样器挨壁吸取样品,将加样器针头插至加样孔中缓慢加入样品。
②电泳:浓缩胶时用60V跑,到分离胶以后用120V跑,电泳时间2h;电泳至溴酚兰刚跑出即可终止电泳,进行转膜。
③转膜:准备6张7.0~8.3cm的滤纸和1张7.3~8.6cm的硝酸纤维素膜;切好的硝酸纤维素膜用镊子捏住一边轻轻置于有超纯水的平皿里,使膜浮于水上浸1h;在加有转移液的盘里放入转膜用的夹子、两块海绵垫、一支玻棒、滤纸和浸过的膜;用夹子打开使黑的一面保持水平;在上面垫一张海绵垫,用玻棒来回擀走里面的气泡;再垫上垫三层滤纸,一手固定滤纸一手持玻棒擀去气泡;将玻璃板轻柔撬掉剥胶,除去小玻璃板后,将浓缩胶切去,小心剥下分离胶盖于滤纸上,使其与滤纸对齐,擀去气泡;将膜盖盖满整个胶,除掉气泡;再在膜上盖3张滤纸并除去气泡,盖上另一个海绵垫合起夹子;将夹子放入转移槽槽中,使夹的黑面对槽的黑面,夹的白面对槽的红面;50V转移2.5h;转膜完成后,用1x丽春红染液染膜5min,于脱色用摇床摇;用水冲洗掉没染上的染液观察膜上有无蛋白。
⑤抗体孵育:将膜用TBS从下向上浸湿后,移至含有封闭液的平皿中,室温下脱色,摇床上摇动封闭2h;依次加入相应一抗;冰箱孵育一抗过夜;次日用PBST清洗一抗,选用合适种属的荧光二抗在室温下按照1:5000内参的稀释度进一步孵育;再次PBST冲洗条带,用LI-COR-Odyssey红外成像系统观察其发光信号;通过软件Image-J计算灰度值。
进一步,步骤(1)中,所述裂解液的浓度为:每1ml裂解液含10μL 100mM的PMSF。
进一步,步骤(2)中,所述一抗为:HSP90 1:1000;IL-6 1:1000;HER2 1:1000;p-HER2 1:500;β-actin 1:3000。
进一步,步骤四中,所述统计学分析,包括:
采用SPSS19.0软件对结果进行数据分析,实验结果是均数±标准差表示;选用单因素方差分析ANOVA比较各实验组与对照组之间的统计学差异,组间的比较可以采用Dunnett’s T3检验;两独立样本间比较采用t检验;p<0.05作为差异有统计学意义判断水准。
结合上述的所有技术方案(表皮生长因子受体(EGFR)家族成员主要有EGFR、HER2、HER3和HER4,其中HER2在肿瘤发生中发挥重要作用,被称为癌基因。其过度激活,可能导致上皮性肿瘤的发展[Lai H W,Chien S Y,Kuo S J,et al.The Potential Utility ofCurcumin in the Treatment of HER-2-Overexpressed Breast Cancer:An In Vitroand In Vivo Comparison Study with Herceptin[J].Evid Based Complement AlternatMed,2012,2012:486568],而且往往与患者的生存不良相关。在过去十年中获得的数据表明,HER2起着最为重要的作用[Dittrich A,Gautrey H,Browell D,et al.The HER2Signaling Network in Breast Cancer--Like a Spider in its Web[J].J MammaryGland Biol Neoplasia,2014,19(3-4):253-270]。HER2活化后,可激活多条与细胞增殖、生存、侵润和血管发生有关的信号通路,在促进乳腺癌、膀胱癌、胃癌、肺癌等上皮癌的发生、发展、浸润和转移中发挥作用。在HER2二聚体形成和活化过程中,热休克蛋白HSP90可与HER2细胞质激酶区域交互作用,使HER2及其下游信号蛋白AKT、Raf-1、K-Ras等更稳定,发挥更持久的促癌作用[Chakrabarty A,Surendran S,Bhola N E,et al.The H1047R PIK3CAoncogene induces a senescence-like state,pleiotropy and acute HSP90dependency in HER2+mammary epithelial cells[J].Carcinogenesis,2019.]。HSP90普遍存在于真核细胞中,定位于细胞膜、细胞质、细胞核等几乎所有的细胞内区域,并发挥多种生物学功能[Trepel J,Mollapour M,Giaccone G,et al.Targeting the dynamicHSP90 complex in cancer[J].Nat Rev Cancer,2010,10(8):537-549.]是细胞内最丰富的蛋白,维持组织正常稳态所必需。HSP90有特殊的分子伴侣功能,大量原癌蛋白依赖HSP90分子伴侣获得活性构象和功能,它可维持客户蛋白,如HER2、HER3、Raf、AKT、Cyclin、CDK等表达,保护这些癌蛋白免于降解,在调节信号通路活化和细胞增殖中起重要作用[Park KS,Hong Y S,Choi J,et al.HSP90 inhibitor,AUY922,debilitates intrinsic andacquired lapatinib-resistant HER2-positive gastric cancer cells[J].BMB Rep,2018,51(12):660-665]。在几种恶性肿瘤中HPS90表达升高,包括膀胱癌[Karkoulis P K,Stravopodis D J,Konstantakou E G,et al.Targeted inhibition of heat shockprotein 90 disrupts multiple oncogenic signaling pathways,thus inducing cellcycle arrest and programmed cell death in human urinary bladder cancer celllines[J].Cancer Cell Int,2013,13(1):11.]。炎症环境应激下,细胞内的HSP90能分泌到细胞外,成为e HSP90。HSP90抑制剂能够诱导蛋白酶体介导的HER2降解,可用于抗癌的治疗。体外研究证实,IL-6可减少膀胱癌细胞的增殖、迁移和侵润[Tsui K H,Wang S W,ChungL C,et al.Mechanisms by which interleukin-6 attenuates cell invasion andtumorigenesis in human bladder carcinoma cells[J].Biomed Res Int,2013,2013:791212.],并可上调细胞生长转移抑制因子(NDRG1)的表达。本发明的目的为研究马归液对致炎膀胱上皮细胞相关细胞因子及癌基因HER2活化的影响,为临床应用中药防治非可控性炎症,预防肿瘤的发生发展提供依据。),本发明所具备的优点及积极效果为:本发明提供的马归液在非可控性炎症中的用途,旨在验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用。前期研究发现内毒素能诱导膀胱上皮细胞炎症因子的表达,而中药制剂“马归液”在一定程度上能抑制炎症因子的表达。此次实验则是进一步验证内毒素对膀胱上皮细胞中HER2及其相关蛋白HSP90的表达影响,及马归液对其的干预作用。
本发明通过采用免疫荧光技术、Western Blotting实验,验证分别经马归液、LPS、抗内毒素、抗HSP90处理的膀胱上皮细胞中HER2磷酸化水平及HSP90表达水平。结果表明,与正常细胞对照组相比,LPS模型组细胞p-HER2水平显著升高;与正常对照组、与抗内毒素抗体相比,马归液组细胞p-HER2增高(p<0.05;p<0.05);马归液组、抗内毒素抗体组细胞p-HER2均明显低于LPS模型组(p<0.05);马归液能降低内毒素刺激的SV-HUC-1细胞内HSP90蛋白的表达,与模型组比较差别有统计学意义(p<0.05);马归液组HSP90蛋白高于17-AAG抑制剂组差别有显著性(p<0.05);17-AAG抑制剂组与模型组比较HSP90蛋白表达显著降低(p<0.05)。因此,LPS刺激SV-HUC-1细胞HER2磷酸化;马归液能较好控制LPS致炎细胞HER2磷酸化。马归液可能通过降低致炎细胞内HSP90蛋白的表达,抑制HER2的磷酸化。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对本发明实施例中所需要使用的附图做简单的介绍,显而易见地,下面所描述的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下还可以根据这些附图获得其他的附图。
图1是本发明实施例提供的验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法流程图。
图2是本发明实施例提供的标准蛋白质浓度吸光值曲线图。
图3是本发明实施例提供的各组致炎细胞HER2蛋白的荧光表达示意图。
图4是本发明实施例提供的免疫荧光检测各组CRL-9520SV-HUC-1细胞p-HER2表达的示意图。
图5是本发明实施例提供的WB法检测CRL-925SV-HUC-1细胞P-HER2蛋白的示意图。
图6是本发明实施例提供的各组细胞p-HER2蛋白比较(WB法)示意图。
图7是本发明实施例提供的各组致炎细胞HSP90蛋白表达(WB法)示意图。
图8是本发明实施例提供的WB法测定各组细胞HSP90蛋白表达示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
针对现有技术存在的问题,本发明提供了一种马归液在非可控性炎症中的用途及验证方法,下面结合附图对本发明作详细的描述。
如图1所示,本发明实施例提供的验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法包括以下步骤:
S101,进行细胞培养和实验细胞分组;
S102,免疫荧光法检测CRL-9520 SV-HUC-1细胞p-HER2;
S103,Western Blotting方法检测p-HER2在CRL-9520细胞中表达;
S104,统计学分析:采用SPSS19.0软件对结果进行数据分析。
下面结合具体实施例对本发明的技术方案作进一步描述。
本发明实施例:马归液对内毒素诱导的膀胱上皮细胞HER2磷酸化及HSP90的影响。
目的:验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用。方法:采用免疫荧光技术、Western Blotting实验,验证分别经马归液、LPS、抗内毒素、抗HSP90处理的膀胱上皮细胞中HER2磷酸化水平及HSP90表达水平。结果:与正常细胞对照组相比,LPS模型组细胞p-HER2水平显著升高。与正常对照组、与抗内毒素抗体相比,马归液组细胞p-HER2增高(p<0.05;p<0.05);马归液组、抗内毒素抗体组细胞p-HER2均明显低于LPS模型组(p<0.05)。马归液能降低内毒素刺激的SV-HUC-1细胞内HSP90蛋白的表达,与模型组比较差别有统计学意义(p<0.05);马归液组HSP90蛋白高于17-AAG抑制剂组差别有显著性(p<0.05);17-AAG抑制剂组与模型组比较HSP90蛋白表达显著降低(p<0.05)。结论:LPS刺激SV-HUC-1细胞HER2磷酸化;马归液能较好控制LPS致炎细胞HER2磷酸化。马归液可能通过降低致炎细胞内HSP90蛋白的表达,抑制HER2的磷酸化。
本课题组前期研究发现内毒素能诱导膀胱上皮细胞炎症因子的表达,而中药制剂“马归液”在一定程度上能抑制炎症因子的表达。此次实验则是进一步验证内毒素对膀胱上皮细胞中HER2及其相关蛋白HSP90的表达影响,及马归液对其的干预作用。
1.材料与方法
1.1材料:
1.1.1细胞:人正常膀胱上皮细胞(CRL-9520 SV-HUC-1)(武汉普诺赛生命科技有限公司)。
1.1.2药物:马归液湖南中医药大学附属一医院制剂室提供1.8g/mL。
制备工艺:将当归20g冷浸0.5小时,用水蒸气蒸馏提取挥发油,蒸馏后的水溶液另器保存,药渣与马鞭草30g、赤芍20g、黄芪20g加水煎煮三次,加水量分别为药材量的10倍、8倍、8倍,第1次2小时,第2、3次各1.5小时,合并煎液,滤过,滤液与上述水溶液合并,静置24小时,滤过,浓缩至近50ml,加入防腐剂适量与当归挥发油,调整总量至50ml,搅匀,灌封,灭菌,即得。药物浓度1.8g/ml。使用时用培养液几何比稀释成不同浓度。
1.1.3试剂及其配制:
DMEM/F12培养基(美国Hyelone);胎牛血清(FBS)(美国Gibco);青霉素、链霉素溶液(美国Gibco);胰蛋白酶(美国Gibco);内毒素(Lipopolysaccharides,LPS)(北京索莱宝科技有限公司);抗内毒素单克隆抗体(Anti-Endotoxin-Gram Negative-Library PackMonoclonal Antibody)货号:153101,规格:9x100μg(购于世联博研北京科技有限公司)。0.25%胰蛋白酶50mL/瓶(美国Gibco)。蛋白酶抑制剂(冻干粉)。磷酸酶抑制剂Cocktail(冻干粉)(北京蓝博斯特生物技术有限公司);坦螺旋霉素(tanespimycin,17-AAG)为HSP90抑制剂(美国sellek产品);高效RIPA组织/细胞裂解液,规格:100mL/瓶(北京索莱宝生物有限公司)。Anti-EGF抗体(ab259398)1mg/支(美国abcom);BCA蛋白测定试剂盒(ab102536)(美国abcom);TritonX-100规格:100mL/瓶(中国上海索莱宝生物公司);RIPA裂解液及ECL发光液(美国BD公司);Genefinder(核酸染料)规格:10000x浓度500ul货号:204001(杭州联科美讯生物医药技术有限公司);TBE(Tris-硼酸-EDTA缓冲溶液)规格:10xTBS 100mL(上海昊鑫生物科技股份公司);TEMED(四甲基乙二胺)(上海叶源科技有限公司)。DAPI,规格:10mg/支(美国Sigma-Aldrich);电泳缓冲液、转印缓冲液,自行配制(中国上海国药集团);Formantas PAGE Ruler以及ECL发光液(美国BD公司);PMSF蛋白去污剂(中国北京索莱宝生物公司);Tfixton X-100(中国上海索莱宝生物公司);96孔培养板(美国Coming)
4%多聚甲醛:4%多聚甲醛(Sinopharm Corp,Shanghai,China)加入PBS至100ml,65℃,24小时,调整PH:7.2~7.4,4℃保存。
SDS-PAGE凝胶电泳配制用溶液:riffs-HCI(pH 6.8,pH 8.8)、10%SDS、TEMED、10%AP,均为自行配制(中国上海国药集团);
细胞生长培养完全培养基:l000mL DMEM/F12培养液中加入10%胎牛血清,青霉素钠和链霉素各100U/mL。
细胞生长维持液:1000mL DMEM/F12培养液中加入2%胎牛血清,青霉素钠和链霉素各100U/mL。
1.1.4仪器:
超净工作台(江苏苏净集团公司)、湿转转膜仪(美国Bio-rad)、YM-1000Y超声波细胞粉碎机(上海豫明仪器有限公司)、垂直电泳仪(北京六一生物科技有限公司产品)、二氧化碳培养箱(美国Thermo Scientific)、高速冷冻离心机(湖南湘仪科技公司)、孔板振荡器(美国Thermo Scientific)、微量样品定量仪(美国Thermo)、荧光显微镜Leica DMIRE2(德国Leica Microsystems Ltd)。
1.2方法与步骤
1.2.1细胞培养
CRL-9520SV-HUC-1细胞培养在含10%FBS、100U/ml青霉素和100μg/ml链霉素的DMEM/F12完全培养基中,保持37℃5%CO2不变。细胞密度1x105个/ml接种在各规格培养板或培养皿,当细胞生长80%融合时,按预实验结果,选择小鼠抗人的EGF抗体50ng/ml(母液10μg/ml);小鼠抗人TGFα抗体5ng/ml(母液:5μg/ml);37℃,30min后,在培养基中加LPS 10μg/mL,培养96h。
实验细胞分组:
正常细胞对照组;加入等量的培养液代替LPS。
LPS模型组:加入上述LPS溶液。
马归液组:在加入LPS前加入50mg/ml(前期研究发现马归液对CRL-9520 SV-HUC-1细胞的半抑制浓度为45.86mg/mL,为了实验便于控制取药,整个实验过程取药液浓度为50mg/mL作为实验用药浓度)的马归液;
抗内毒素抗体组:于加入LPS前加入等量的抗内毒素抗体;
HSP90抑制剂组:加入10ng/mL浓度的17-AAG溶液后,再加入LPS溶液。
1.2.2免疫荧光法检测CRL-9520 SV-HUC-1细胞p-HER2
(1)盖玻片清洗,过酸过夜,蒸馏水及去离子水清洗后,擦干,60℃处理2h备用。(2)处理的盖玻片平放六孔板内,将密度为10×104/mL细胞滴加于盖玻片上,37℃,50%CO2培养96小时(细胞培养24h换细胞生长维持液)。(3)培养好的细胞镜下观察形态良好,无污染,用4℃PBS轻轻洗2遍。(4)4%多聚甲醛(4℃)固定10分钟。4℃PBS温柔漂洗3遍,5min/次。(5)含0.25%TrixtonX-100的PBS作用10分钟,透化细胞膜。4℃PBS温柔漂洗3遍,5min/次。(6)5%BSA封闭1小时。室温下直接用10%正常的山羊血清进行封闭,加入兔抗人的p-HER2抗体(1:100),置湿盒4℃孵育过夜。(7)第二天取出复温20min,4℃ PBS温柔冲洗3遍,洗净一抗。(8)加入荧光二抗(37℃与羊抗兔二抗和羊抗小鼠二抗联合孵育1h),50-100μL/孔,室温孵育2小时。(9)4℃PBS温柔冲洗3遍,洗净二抗。(10)细胞核用显核剂染色。在免疫荧光显微镜下采集图像。用ImageJ计算平均光密度。
1.2.3 Western Blotting方法检测p-HER2在CRL-9520细胞中表达
(1)蛋白样品制备
单层贴壁细胞核蛋白的提取:①选择长满单层的,无污染的CRL-9520细胞,吸弃去培养液,将细胞瓶倒扣在吸水纸上,吸干培养液。②每瓶细胞加2mL预冷的PBS 1min弃去PBS液,反复3次。③加蛋白酶抑制剂,磷酸酶抑制剂各100μL/孔作用10min后,冰上加RIPA裂解液0.5mL,细胞培养瓶置冰上,加入400μL裂解液(浓度:1ml裂解液含10μL PMSF(100mM)),冰上30min,来回摇动细胞瓶,使细胞充分裂解。④无菌细胞刮棒将细胞刮于培养瓶的一侧,超声裂解细胞。⑤4℃16000g离心5min,取上清-80℃保存备用。
BCA法检测样品蛋白浓度:①称取0.5g牛血清白蛋白,溶于蒸馏水中并定容至100ml,制成5mg/ml的溶液。用时稀释十倍。②绘制标准曲线:取96孔酶标板,按表1加入试剂。
表1加入试剂
③吸取20μl样品溶液于酶标板孔中,加入BCA试剂200μl,轻摇,于37℃50min,室温10min,以空白为对照,在酶标仪562nm处测定吸光值(OD值),以牛血清白蛋白含量为横坐标,以吸光值为纵坐标,绘制标准曲线。以标准曲线空白为对照,根据样品的吸光值从标准曲线上查出样品的蛋白质含量。
以BSA各级工作液对应的蛋白质浓度蛋白质浓度(Y,mg/mL),对其OD值(x)作图。Y=0.32.36X-0.07,相关系数r=0.964。见图2。
(2)SDS--PAGE电泳
①灌胶与上样:玻璃板对齐后放入夹中卡紧。然后垂直卡在架子上准备灌胶。制10%分离胶:称取一定量的琼脂糖于三角瓶中,加入1xTBE液,微波炉加热3min,使琼脂糖完全溶解,待溶解的琼脂糖温度下降至60℃时,加入终浓度0.1pg/ml的GeneFinder液,充分混匀后倒板,待凝固,小心移去梳子,将凝胶板置入电泳槽,向电泳槽中倒入1xTBE,使之没过胶面2mm。配好的10%分离胶加入TEMED后立即摇匀,灌胶。配8%的浓缩胶,加入TEMED后立即摇匀灌胶,剩余空间灌满浓缩胶后,将梳子水平插入浓缩胶中。待到浓缩胶凝固后,两手分别捏住梳子的两边垂直向上轻慢将其拔出。用无离子水冲洗浓缩胶,将其放入电泳槽中。
测完蛋白含量后,计算出20μL蛋白的溶液体积作为上样量。吸取样品至200μL的EP管中,加入5xSDS上样缓冲液至终浓度为l×。上样前将样品于沸水中煮5min使蛋白变性。加足够的电泳液后(电泳液漫过内测的小玻璃板),用微量进样器挨壁吸取样品,将加样器针头插至加样孔中缓慢加入样品。
②电泳:浓缩胶时用60V跑,到分离胶以后用120V跑,电泳时间约2h左右。电泳至溴酚兰刚跑出即可终止电泳,进行转膜。
③转膜:准备6张7.0~8.3cm的滤纸和1张7.3~8.6cm的硝酸纤维素膜。切好的硝酸纤维素膜用镊子捏住一边轻轻置于有超纯水的平皿里,使膜浮于水上浸1h。在加有转移液的盘里放入转膜用的夹子、两块海绵垫、一支玻棒、滤纸和浸过的膜。用夹子打开使黑的一面保持水平。在上面垫一张海绵垫,用玻棒来回擀走里面的气泡。再垫上垫三层滤纸,一手固定滤纸一手持玻棒擀去气泡。将玻璃板轻柔撬掉剥胶,除去小玻璃板后,将浓缩胶切去,小心剥下分离胶盖于滤纸上,使其与滤纸对齐,擀去气泡。将膜盖盖满整个胶,除掉气泡。再在膜上盖3张滤纸并除去气泡。最后盖上另一个海绵垫合起夹子。将夹子放入转移槽槽中,使夹的黑面对槽的黑面,夹的白面对槽的红面。50V转移2.5h。转膜完成后,用1x丽春红染液染膜5min,于脱色用摇床摇。用水冲洗掉没染上的染液观察膜上有无蛋白。
⑤抗体孵育:将膜用TBS从下向上浸湿后,移至含有封闭液的平皿中,室温下脱色,摇床上摇动封闭2h。依次加入相应一抗(HSP90 1:1000;IL-6 1:1000;HER2 1:1000;p-HER21:500;β-actin 1:3000)。冰箱孵育一抗过夜。次日,首先PBST清洗一抗,选用合适种属的荧光二抗在室温下按照1:5000内参的稀释度进一步孵育。再次PBST冲洗条带,用LI-COR-Odyssey红外成像系统观察其发光信号。软件Image-J可用来计算灰度值。
1.3统计学方法:
采用SPSS19.0软件对结果进行数据分析,实验结果是均数±标准差表示。选用单因素方差分析(ANOVA)来比较各实验组与对照组之间的统计学差异,组间的比较可以采用Dunnett’s T3检验。两独立样本间比较采用t检验。p<0.05作为差异有统计学意义判断水准。
2.结果:
2.1马归液对LPS致炎CRL-9520 SV-HUC-1细胞HER2表达及磷酸化的影响
为探讨马归液对非可控性炎症的治疗作用,通过用低浓度的LPS致炎CRL-9520SV-HUC-1细胞,观察致炎细胞HER2活化,特将培养细胞与LPS、药液与细胞维持液共培养时间延长至96h,以确定马归液对LPS致炎细胞HER2的作用。如图3所示,通过Western blot分析,与正常细胞对照组相比,LPS模型组细胞p-HER2水平显著升高。免疫荧光显示:与正常对照组、与抗内毒素抗体相比,马归液组细胞p-HER2增高(p<0.05;p<0.05);马归液组、抗内毒素抗体组细胞p-HER2均明显低于与LPS模型组(p<0.05)。同样蛋白表达结果也显示:LPS刺激SV-HUC-1细胞HER2磷酸化;马归液能较好控制LPS致炎细胞HER2磷酸化(见表2、图3~6)。
表2各组致炎细胞p-HER2蛋白积分光密度(n=3)
2.2马归液对致炎SV-HUC-1细胞内HSP90蛋白的影响
实验中发现马归液能降低内毒素刺激的SV-HUC-1细胞内HSP90蛋白的表达,与模型组比较差别有统计学意义(p<0.05);马归液组HSP90蛋白高于17-AAG抑制剂组差别有显著性(p<0.05);17-AAG抑制剂组与模型组比较HSP90蛋白表达显著降低(p<0.05)。提示马归液可能通过降低致炎细胞内HSP90蛋白的表达,抑制HER2的磷酸化(见图7~8)。
3.结果
长期的慢性炎症可导致炎症因子的释放,微环境改变,损伤细胞DNA或者DNA修复不及时,引起DNA突变,从而导致肿瘤的发生、发展、转移以及复发,因此及时的解除引起炎症的因素、控制炎症能有效地抑制肿瘤的发生。
中医中药在治疗膀胱慢性炎症方面具有独特的优势。“马归液”的来源是在中医“扶正祛邪”理论指导下,结合多年的临床经验,总结得出来得方剂,经过长期的临床实践、观察,发现其能有效得治疗腺性膀胱炎。而在细胞分子水平上,“马归液”能调控腺性膀胱炎细胞中的转化生长因子-β1(transforming growth factor-β1,TGF-β1)、表皮生长因子-β(epidermal growth factor-β,EGF-β)、Survivin、PTEN、Bcl-2和Bax的表达。在此基础上,本发明考虑“马归液”是否也对其他的膀胱炎症及膀胱癌具有一定的作用呢?
前期预实验发现LPS能诱导膀胱上皮细胞产生大量的炎症因子(IL-1β和TNF-α),抑制IL-6的表达,而“马归液”则能抑制LPS的致炎作用,使IL-1β和TNF-α表达抑制,IL-6表达上调。说明“马归液”具有改善细胞炎症的作用。在此基础上,本发明更进一步,研究LPS对膀胱上皮细胞中HER2及HSP90表达的影响及“马归液”对其的干预作用。
HER2与膀胱癌的发生发展具有密切的联系。HER2为表皮生长因子受体(EGFR)家族成员之一,在肿瘤发生中发挥重要作用,被称为癌基因,其过度激活,可能导致上皮性肿瘤的发展,而且往往与患者的生存不良相关。在过去十年中获得的数据表明,HER2起着最为重要的作用。在膀胱癌中,HER2在癌组织中呈现过表达,与膀胱癌的分级分期、淋巴结转移、复发以及预后均有一定的关系。
在HER2二聚体形成和活化过程中,热休克蛋白HSP90可与HER2细胞质激酶区域交互作用,使HER2及其下游信号蛋白AKT、Raf-1、K-Ras等更稳定,发挥更持久的促癌作用。HSP90普遍存在于真核细胞中,定位于细胞膜、细胞质、细胞核等几乎所有的细胞内区域,并发挥多种生物学功能,大量原癌基因蛋白依赖HSP90分子伴侣获得活性构象和功能,保护这些癌蛋白免于降解,在调节信号通路活化和细胞增殖中起重要作用,在膀胱癌中,HPS90表达升高。HSP90抑制剂能够诱导蛋白酶体介导的HER2降解,可用于抗癌的治疗。在炎症微环境下,细胞内的HSP90能分泌到细胞外,成为eHSP90。
在实验中,通过用低浓度的LPS致炎CRL-9520 SV-HUC-1细胞,观察致炎细胞HER2活化,结果显示LPS能促进HSP90的表达,促使HER2的磷酸化。而加入“马归液”后,HER2磷酸化水平降低,与之同步降低的还有HSP90,HSP90能使HER2更加稳定,减少降解。说明:①LPS能促使致炎细胞内的癌基因HER2磷酸化,马归液能下调致炎细胞HER2磷酸化水平;②LPS可能促使致炎细胞内的HSP90表达从而促进HER2磷酸化,“马归液”能抑制HSP90的表达。
虽然“马归液”抑制HER2磷酸化及HSP90的效果弱于抗内毒素组及17-AAG抑制剂组,但“马归液”对其仍具有一定的抑制作用。说明“马归液”对LPS诱导的炎症细胞具有确切的治疗作用,并具有一定的抗肿瘤作用。说明了中药在炎症及肿瘤中巨大的作用前景,为临床应用中药防治炎症,预防肿瘤的发生发展提供依据。因此,“马归液”能抑制HSP90的表达及HER2磷酸化。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,都应涵盖在本发明的保护范围之内。
Claims (10)
1.一种马归液在非可控性炎症中的用途,其特征在于,所述马归液在非可控性炎症中的用途,包括马归液50mg/ml对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达具有干预作用。
2.一种验证如权利要求1所述马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法,其特征在于,所述验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法包括以下步骤:
步骤一,进行细胞培养和实验细胞分组;
步骤二,免疫荧光法检测CRL-9520SV-HUC-1细胞p-HER2;
步骤三,WesternBlotting方法检测p-HER2在CRL-9520细胞中表达;
步骤四,统计学分析:采用SPSS19.0软件对结果进行数据分析。
3.如权利要求2所述验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法,其特征在于,步骤一中,所述细胞培养,包括:
CRL-9520SV-HUC-1细胞培养在含10%FBS、100U/ml青霉素和100μg/ml链霉素的DMEM/F12完全培养基中,保持37℃5%CO2不变;细胞密度1×105个/ml接种在各规格培养板或培养皿,当细胞生长80%融合时,按预实验结果,选择小鼠抗人的EGF抗体50ng/ml,母液10μg/ml;小鼠抗人TGFα抗体5ng/ml,母液5μg/ml;37℃,30min后,在培养基中加LPS 10μg/mL,培养96h。
4.如权利要求2所述验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法,其特征在于,步骤一中,所述实验细胞分组,包括:
正常细胞对照组;加入等量的培养液代替LPS;
LPS模型组:加入所述LPS溶液;
马归液组:在加入LPS前加入50mg/ml的马归液;
抗内毒素抗体组:于加入LPS前加入等量的抗内毒素抗体;
HSP90抑制剂组:加入10ng/mL浓度的17-AAG溶液后,再加入LPS溶液。
5.如权利要求2所述验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法,其特征在于,步骤二中,所述免疫荧光法检测CRL-9520SV-HUC-1细胞p-HER2,包括:
(1)盖玻片清洗,过酸过夜,蒸馏水及去离子水清洗后,擦干,60℃处理2h备用;处理的盖玻片平放六孔板内,将密度为10×104/mL细胞滴加于盖玻片上,37℃,50%CO2培养96h;将培养好的细胞进行镜下观察,并用4℃PBS轻轻洗2遍;4%多聚甲醛4℃固定10min;4℃PBS温柔漂洗3遍,5min/次;
(2)用含0.25%TrixtonX-100的PBS作用10min,透化细胞膜;4℃PBS温柔漂洗3遍,5min/次;5%BSA封闭1h;室温下直接用10%正常的山羊血清进行封闭,按1:100的比例加入兔抗人的p-HER2抗体,置湿盒4℃孵育过夜;
(3)第二天取出复温20min,4℃PBS温柔冲洗3遍,洗净一抗;加入荧光二抗,37℃与羊抗兔二抗和羊抗小鼠二抗联合孵育1h,50~100μL/孔,室温孵育2h;4℃PBS温柔冲洗3遍,洗净二抗;细胞核用显核剂染色,在免疫荧光显微镜下采集图像,并用ImageJ计算平均光密度。
6.如权利要求5所述验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法,其特征在于,步骤(1)中,培养过程中,细胞培养24h换细胞生长维持液。
7.如权利要求2所述验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法,其特征在于,步骤三中,所述Western Blotting方法检测p-HER2在CRL-9520细胞中表达,包括:
(1)蛋白样品制备
单层贴壁细胞核蛋白的提取:①选择长满单层的,无污染的CRL-9520细胞,吸弃去培养液,将细胞瓶倒扣在吸水纸上,吸干培养液;②每瓶细胞加2mL预冷的PBS 1min弃去PBS液,反复3次;③加蛋白酶抑制剂,磷酸酶抑制剂各100μL/孔作用10min后,冰上加RIPA裂解液0.5mL,细胞培养瓶置冰上,加入400μL裂解液,冰上30min,来回摇动细胞瓶,使细胞充分裂解;④无菌细胞刮棒将细胞刮于培养瓶的一侧,超声裂解细胞;⑤4℃16000g离心5min,取上清-80℃保存备用;
BCA法检测样品蛋白浓度:①称取0.5g牛血清白蛋白,溶于蒸馏水中并定容至100ml,制成5mg/ml的溶液;用时稀释十倍;②绘制标准曲线:取96孔酶标板,加入试剂;③吸取20μl样品溶液于酶标板孔中,加入BCA试剂200μl,轻摇,37℃50min,室温10min,以空白为对照,在酶标仪562nm处测定吸光值OD,以牛血清白蛋白含量为横坐标,以吸光值为纵坐标,绘制标准曲线;以标准曲线空白为对照,根据样品的吸光值从标准曲线上查出样品的蛋白质含量;
以BSA各级工作液对应的蛋白质浓度蛋白质浓度Y,mg/mL,对OD值x作图;Y=0.32.36X-0.07,相关系数r=0.964;
(2)SDS--PAGE电泳
①灌胶与上样:玻璃板对齐后放入夹中卡紧,垂直卡在架子上准备灌胶;制10%分离胶:称取一定量的琼脂糖于三角瓶中,加入1xTBE液,微波炉加热3min,使琼脂糖完全溶解,待溶解的琼脂糖温度下降至60℃时,加入终浓度0.1pg/ml的GeneFinder液,充分混匀后倒板,待凝固,小心移去梳子,将凝胶板置入电泳槽,向电泳槽中倒入1xTBE,使之没过胶面2mm;配好的10%分离胶加入TEMED后立即摇匀,灌胶;配8%的浓缩胶,加入TEMED后立即摇匀灌胶,剩余空间灌满浓缩胶后,将梳子水平插入浓缩胶中;待到浓缩胶凝固后,两手分别捏住梳子的两边垂直向上轻慢将其拔出;用无离子水冲洗浓缩胶,将其放入电泳槽中;
测完蛋白含量后,计算出20μL蛋白的溶液体积作为上样量;吸取样品至200μL的EP管中,加入5xSDS上样缓冲液至终浓度为l×;上样前将样品于沸水中煮5min使蛋白变性;加足够的电泳液后,即电泳液漫过内测的小玻璃板,用微量进样器挨壁吸取样品,将加样器针头插至加样孔中缓慢加入样品;
②电泳:浓缩胶时用60V跑,到分离胶以后用120V跑,电泳时间2h;电泳至溴酚兰刚跑出即可终止电泳,进行转膜;
③转膜:准备6张7.0~8.3cm的滤纸和1张7.3~8.6cm的硝酸纤维素膜;切好的硝酸纤维素膜用镊子捏住一边轻轻置于有超纯水的平皿里,使膜浮于水上浸1h;在加有转移液的盘里放入转膜用的夹子、两块海绵垫、一支玻棒、滤纸和浸过的膜;用夹子打开使黑的一面保持水平;在上面垫一张海绵垫,用玻棒来回擀走里面的气泡;再垫上垫三层滤纸,一手固定滤纸一手持玻棒擀去气泡;将玻璃板轻柔撬掉剥胶,除去小玻璃板后,将浓缩胶切去,小心剥下分离胶盖于滤纸上,使其与滤纸对齐,擀去气泡;将膜盖盖满整个胶,除掉气泡;再在膜上盖3张滤纸并除去气泡,盖上另一个海绵垫合起夹子;将夹子放入转移槽槽中,使夹的黑面对槽的黑面,夹的白面对槽的红面;50V转移2.5h;转膜完成后,用1x丽春红染液染膜5min,于脱色用摇床摇;用水冲洗掉没染上的染液观察膜上有无蛋白;
⑤抗体孵育:将膜用TBS从下向上浸湿后,移至含有封闭液的平皿中,室温下脱色,摇床上摇动封闭2h;依次加入相应一抗;冰箱孵育一抗过夜;次日用PBST清洗一抗,选用合适种属的荧光二抗在室温下按照1:5000内参的稀释度进一步孵育;再次PBST冲洗条带,用LI-COR-Odyssey红外成像系统观察其发光信号;通过软件Image-J计算灰度值。
8.如权利要求7所述验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法,其特征在于,步骤(1)中,所述裂解液的浓度为:每1ml裂解液含10μL100mM的PMSF。
9.如权利要求7所述验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法,其特征在于,步骤(2)中,所述一抗为:HSP901:1000;IL-61:1000;HER21:1000;p-HER21:500;β-actin 1:3000。
10.如权利要求2所述验证马归液对内毒素致膀胱上皮细胞中HER2磷酸化及HSP90表达的干预作用的方法,其特征在于,步骤四中,所述统计学分析,包括:
采用SPSS19.0软件对结果进行数据分析,实验结果是均数±标准差表示;选用单因素方差分析ANOVA比较各实验组与对照组之间的统计学差异,组间的比较可以采用Dunnett’sT3检验;两独立样本间比较采用t检验;p<0.05作为差异有统计学意义判断水准。
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