CN116059374A - Lcat基因、纯化蛋白在制备预防、缓解和/或治疗原发性肝细胞癌的药物中的应用 - Google Patents
Lcat基因、纯化蛋白在制备预防、缓解和/或治疗原发性肝细胞癌的药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种LCAT基因、纯化蛋白在制备预防、缓解和/或治疗原发性肝细胞癌的药物中的应用,涉及生物医药技术领域。利用睡美人转座子系统在小鼠肝细胞过表达LCAT基因,证实体内LCAT基因肝脏过表达有效减缓小鼠原发性肝细胞癌进展。肝癌细胞系LCAT基因低表达得到与体内实验一致结果;进一步证实LCAT基因抑制肝细胞癌细胞增殖是依赖于其分泌功能。裸鼠皮下注射HCCLM9肝癌细胞异体成瘤,发现LCAT纯化蛋白瘤内注射可以显著抑制体内肝癌细胞的增殖。本发明首次从体内证实肝细胞LCAT基因表达水平与原发性肝细胞癌的恶性增殖过程直接相关,并且LCAT基因过表达抑制肝癌细胞增殖依赖其分泌蛋白特性;而LCAT纯化蛋白可以成为干预原发性肝细胞癌的有效工具和重要研究靶点。
Description
技术领域
本发明基因的功能与应用领域,具体涉及一种LCAT基因、纯化蛋白在制备预防、缓解和/或治疗原发性肝细胞癌的药物中的应用。
背景技术
截止2020年,原发性肝细胞癌是全球发病率第六的癌症,其造成死亡数量在癌症造成死亡中排名第三;每年新增病例约906000例,造成死亡830000人。病毒感染、酗酒、非酒精性脂肪肝和遗传因素是原发性肝细胞癌的重要危险因素,但这些危险因素尚未被转化为有效的治疗靶点。而晚期原发性肝细胞癌的治疗药物通常效果有限,如多激酶的抑制剂如索拉菲尼和仑法替尼。因此,高发病率高致病性的特点,结合目前临床上缺乏有效的治疗手段,原发性肝细胞癌亟需发展出更加有效的预防、缓解和/或治疗靶点。
卵磷脂胆固醇脂酰转移酶(phosphatidylcholine-sterol acyltransferase,LCAT)是由主要由肝脏和小肠合成释放入血液,以游离或与脂蛋白结合的形式存在,在血浆中起催化作用的酶,其作用是将高密度脂蛋白的卵磷脂的C2位不饱和脂肪酸转移给游离胆固醇,生成溶血卵磷脂和胆固醇酯。血浆胆固醇几乎70%-80%是胆固醇酯,均是LCAT蛋白催化生成所致。LCAT基因缺陷会导致脂质在人体内的异位堆积,并最终降低患者的寿命。有证据提示,LCAT基因转录水平可以作为良好的肝癌恶性评估指标。但目前缺少直接在体内验证LCAT表达水平与肝癌进展的直接证据,而细胞水平实验并不能简单替代肝脏内复杂的病变情况。因此尚未见根本性证据,去证实肿瘤发生过程中LCAT基因表达水平与原发性肝细胞癌进展程度的关系以及LCAT基因作为肝细胞癌药物研发靶点的潜力。
发明内容
为解决现有治疗手段的缺陷和不足,本发明通过构建肝脏LCAT基因过表达小鼠及体外细胞生物学实验,首次于体内证实肝脏LCAT基因表达水平与原发性肝细胞癌进展的关系,并且LCAT蛋白抑制原发性肝细胞癌增殖是依赖其分泌能力。裸鼠皮下成瘤后,瘤内注射LCAT纯化蛋白可以有效抑制皮下成瘤的增殖。提供LCAT纯化蛋白用于缓解原发性肝细胞癌的新用途。本发明首次在动物体内证实肝脏LCAT基因表达与原发性肝细胞癌发展程度的关系,并且纯化LCAT蛋白可以抑制原发性肝细胞癌的增殖,为原发性肝细胞癌及相关疾病的预防和/或治疗找出新的分子靶点。
本发明的目的通过以下技术方案实现:
第一方面,本发明提供一种LCAT基因作为药物靶标在制备预防、缓解和/或治疗原发性肝细胞癌的药物中的应用。
作为优选方案,所述药物是在体内提高肝细胞LCAT基因表达的化学药物和生物分子,实现抑制肝癌细胞的增殖、迁移或侵袭。
第二方面,本发明提供一种LCAT纯化蛋白在制备预防、缓解和/或治疗原发性肝细胞癌的药物中的应用,其特征在于:
LCAT全长蛋白及LCAT蛋白结构域的外源表达载体作为药物靶标以预防和/或缓解原发性肝细胞癌;或者,
LCAT蛋白激动剂作为药物靶标以预防和/或缓解原发性肝细胞癌。
作为优选方案,所述外源表达载体,包括以DNA、RNA及病毒表达载体实现LCAT全长蛋白及LCAT蛋白结构域表达的外源表达载体,最终导致在体内抑制肝癌细胞的增殖、迁移或侵袭。
进一步地,所述药物与LCAT蛋白具有明确的结合特性,并且提高LCAT蛋白活性的药物,药物包含化学药物与生物药物,最终实现在体内抑制肝癌细胞的增殖、迁移或侵袭。
结合发明机理,具体展开来说:
本发明旨在在体内提高肝脏LCAT基因表达方法,实现抑制原发性肝细胞癌的应用。
上述方案包括一种体内改变原发性肝细胞癌增殖能力的方法,其包括利用睡美人转座子系统结合尾静脉高压注射技术实现肝细胞内整合外源基因并实现过表达的技术细节。但本实验体系内实现肝脏内表达LCAT蛋白的技术并不局限于利用睡美人转座子系统和尾静脉高压的手段;任何不违背此处实验精神的表达方式由本领域技术人员按照实际自由决定。
上述应用方法应用于实现预防、缓解和/或治疗原发性肝细胞癌,所述改变为提高肝脏内LCAT蛋白表达。
以上方法包括但不仅限于此处使用的利用睡美人转座子系统实现肝脏LCAT蛋白表达提高方法,外源表达载体(如DNA、RNA、病毒载体)实现肝细胞LCAT蛋白表达提高与提高LCAT蛋白转录活性和/或蛋白稳定性的小分子化合物等处理亦不违背此处科学精神。
上述方案中还包括一种向体外递送以LCAT全长蛋白和/或LCAT蛋白结构域在改变原发性肝细胞癌增殖的应用。
上述方案中,还提供一种体外改变原发性肝细胞癌增殖能力的技术,其包括利用His标签纯化LCAT蛋白并处理肝癌细胞系,实现抑制肝癌细胞增殖的技术。但本实验体系下LCAT蛋白的纯化并不局限于His标签纯化,也并非局限于LCAT蛋白全长;任何不违背此处实验精神的表达及纯化细节由本领域技术人员按照实际自由决定。
上述方案中还包括向体内递送以LCAT全长蛋白和/或LCAT蛋白结构域的为主要功能成分的药物在预防和/或缓解原发性肝细胞癌的应用。
上述方案中,还提供一种体内药物使用缓解原发性肝细胞癌的技术,其包括利用纯化LCAT蛋白进行瘤内注射缓解原发性肝细胞癌。但本实验体系下LCAT蛋白给药时间点、给药方式并不局限于此处提供的实验细节;任何不违背此处实验精神的给药时间点、给药方式由本领域技术人员按照实际自由决定。所述药物应用于预防和/或缓解原发性肝细胞癌,所述药物是指以LCAT全长蛋白和/或LCAT蛋白结构域的为主要功能成分的药物。
本发明的技术原理及研究过程简述如下:
睡美人转座子系统过表达AKT和NRAS基因诱导小鼠原发性肝细胞癌是常用的小鼠快速诱导原发性肝细胞癌的方法。本发明以对照组小鼠(下文称之为NTG小鼠)与LCAT基因过表达小鼠(下文称之为HTG小鼠)为实验对象,结合睡美人转座子系统过表达AKT和NRAS基因诱导小鼠原发性肝细胞癌模型研究LCAT基因的功能。原发性肝细胞癌造模后,与NTG小鼠对比,HTG小鼠表现出肝细胞癌特征减轻,HTG小鼠的肝脏重量、肝癌结节数量均显著低于对照组小鼠。血清谷草转氨酶和谷丙转氨酶酶活检测结果显示,HTG小鼠的肝脏损伤程度明显低于对照组小鼠。病理学结果显示出HTG小鼠肝脏病理形态基本没有发生病变,与大体照中观察不到明显结节的结果吻合;而NTG小鼠则出现明显的“牛眼状”肝癌细胞和假小叶等病理学特征。小鼠肝肝脏重量、肝癌结节数量及肝脏/体重比、血清学结果、肝脏大体照以及病理染色结果说明LCAT基因过表达条件下原发性肝细胞癌病变减轻。这表明LCAT基因在动物体内肝细胞过表达可以迟缓原发性肝细胞癌的进展、改善原发性肝细胞癌的状态。
在体外实验条件下,利用短发卡RNA干扰LCAT基因表达的shLCAT细胞系比较起对照的shCtrl细胞系,则展现出更强的肝细胞癌细胞系增殖和克隆形成能力,这与体内过表达LCAT基因保护肝细胞癌的结论是契合的,此处一致的结果说明以上细胞系可以用来在体外验证LCAT基因发挥功能是否依赖其分泌蛋白特性。使用His标签纯化的LCAT蛋白处理shLCAT细胞系和对照的shCtrl细胞系后,可以将二者的增殖能力和克隆形成能力抑制到没有显著性差异的水平。此实验结果说明改变肝细胞内LCAT表达影响肝细胞癌的增殖是依赖其分泌蛋白特性。
进一步,基于LCAT基因发挥功能是依赖其分泌蛋白特性,本发明利用裸鼠皮下成瘤造模后瘤内注射LCAT纯化蛋白,以评估LCAT纯化蛋白缓解原发性肝细胞癌的潜力。研究发现,LCAT纯化蛋白注射后,皮下成瘤的增殖能力相较于注射生理盐水组有显著的滞后,证明LCAT蛋白使用有抑制肝细胞癌增殖的作用,直接证实LCAT蛋白缓解肝细胞癌的应用潜力。
基于以上实验证据,本发明发现LCAT蛋白可作为药物靶点。针对LCAT基因功能,以LCAT蛋白为靶点设计小分子化合物激活剂和生物药物(如提高LCAT基因转录活性的转录因子和/或提高LCAT蛋白活性和稳定性为基础开发的小分子药物和生物药物),实现预防和/或缓解原发性肝细胞癌的目的。针对LCAT基因功能,LCAT基因也可作为基因治疗中的靶基因,设计并制备表达LCAT蛋白或者LCAT蛋白结构域的外源表达载体,实现预防和/或缓解原发性肝细胞癌的目的。结合LCAT蛋白分泌蛋白的特性,将经过物理和/或化学修饰的纯化LCAT全长蛋白或LCAT蛋白部分结构域通过静脉和/或瘤内注射的方式给药,实现预防和/或缓解原发性肝细胞癌的目的。
相较于现有技术,本发明具有如下的优点及效果:
相较于现有技术,本发明的技术方案更加凸显出体内实验结果可以更好地反映实际情况;LCAT功能的分泌蛋白依赖特性;LCAT分泌蛋白特性赋予的治疗潜力及效果评估,具体优点如下:
1、本发明首次于动物体内证实提高肝脏LCAT基因在表达水平具有缓解原发性肝细胞癌的作用。
2、本发明首次证实通过改变LCAT基因表达水平来影响原发性肝细胞癌的增殖是依赖其分泌蛋白特性。
3、本发明首次于裸鼠体内证实纯化LCAT蛋白可用于制备预防和/或缓解原发性肝细胞癌的药物。
4、本发明显著优点在于LCAT基因具有明确的肝脏表达特异性与分泌蛋白特性,并且蛋白质结构明确,方便针对LCAT靶点设计小分子药物和生物药物。
附图说明
图1是NTG和HTG小鼠β-actin和Flag-HTG蛋白Western Blot鉴定图。
图2是NTG和HTG小鼠的体重、肝重和肝重体重比结果图(**:p<0.01vs HTG组,n.s.:p>0.05vs HTG组)。
图3是NTG和HTG小鼠血清谷丙转氨酶和谷草转氨酶效价图(**:p<0.01vs HTG组)。
图4是NTG和HTG小鼠的肝脏大体照及肝癌结节计数图(**:p<0.01vs HTG组)。
图5是NTG和HTG小鼠的肝脏HE染色图。
图6是LCAT蛋白洗脱液考马斯亮蓝染色图。
图7是LCAT蛋白洗脱液Western Blot鉴定图。
图8是shCtrl和shLCAT细胞系相对LCAT mRNA检测水平图。
图9shCtrl+Saline,shLCAT+Saline,shCtrl+LCAT,shLCAT+LCAT组的细胞计数试剂-8结果(**:p<0.01,*:0.01<=p<0.05shCtrl+Saline vs shLCAT+Saline组,##:p<0.01,shCtrl+Saline vs shCtrl+LCAT组,$$:p<0.01,shCtrl+Saline vs shLCAT+LCAT组,n.s:p>0.05)
图10是shCtrl+Saline,shLCAT+Saline,shCtrl+LCAT,shLCAT+LCAT组的克隆形成实验结果(**:p<0.01,n.s.:p>0.05)
图11是LCAT纯化蛋白瘤内注射干预肝细胞癌路线图。
图12是皮下成瘤的肿瘤体积变化图(**:p<0.01,*:0.01<p<0.05,n.s.:p>0.05)。
图13是生理盐水组和LCAT蛋白组小鼠皮下成瘤大体照。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1小鼠原发性肝细胞癌模型诱导
1、基础饲养条件:购入6周龄雄性C57BL/6小鼠,体重在20-22克左右,C57BL/6小鼠购自江苏集萃药康生物科技有限公司,小鼠生产许可证号为SCXK(苏)-2018-0008。饲养用垫料、饮水、全价颗粒饲料及其他与动物接触的物品均经高压灭菌处理。实验和饲养条件严格按照SPF级规范要求。所有动物实验均经武汉大学生命科学学院动物关爱与利用委员会批准。
2、尾静脉高压注射诱导原发性肝细胞癌模型:NTG小鼠注射2mL生理盐水,包含AKT和NRAS表达质粒和SB100转座酶表达质粒;HTG小鼠注射2mL生理盐水,含LCAT、AKT和NRAS表达质粒和SB100转座酶表达质粒。通过尾静脉注射,注射过程在5-10秒完成。注射整合效率利用Western Blot方法鉴定。注射后每两周记录观察小鼠体重和精神状态,至最终NTG小鼠体重不再增加,且精神状态萎靡,按压胸腹部可以感受到明显肝脏肿大,一般质粒注射后6-8周诱导肝癌模型成功。
实施例2小鼠取材检测及病理染色分析
1、终末取材
(1)小鼠称重后,眼球采血0.6-1mL。仰卧固定小鼠,用蒸馏水将小鼠胸部,腹部毛发润湿。
(2)用镊子钳夹小鼠腹部正中皮肤,沿腹部正中向头部剪开皮肤至剑突下,向尾端剪开皮肤,逐层暴露皮下筋膜,肌肉等,打开腹腔,充分暴露各脏器。
(3)迅速找到并取下小鼠的肝脏,将取下的肝脏标本置于灭菌纱布上,拭干肝脏表面上残留血液,将肝脏置于无菌培养皿中,迅速拍照,称重。
(4)收集鲜样本:切取肝脏上癌与癌旁部分,置于冻存管内,迅速放入液氮急冻。后取出,于-80℃长期保存。
(5)石蜡标本:切取部分肝脏置于10%中性福尔马林中固定。
(6)血清收集:小鼠眼球采血置于常温非抗凝管内凝血1小时,3000转每分钟离心10分钟,上清即为血清。收集上清保存于-80℃。血清相关指标检测于武汉惠康动物检测有限公司。
2、肝脏组织处理及病理染色相关实验
(1)肝脏脱水,透明,浸蜡
切取10%中性福尔马林中固定好的部分肝叶组织于标记的包埋框内,在小流量流水冲洗30分钟以上。按照以下流程在机器上设置以下程序,①脱水:75%酒精(45分钟)→75%酒精(45分钟)→85%酒精(45分钟)→85%酒精(45分钟)→95%酒精(45分钟)→95%酒精(45分钟)→无水酒精(1小时)→无水酒精(1小时);②透明:二甲苯(1小时)→二甲苯(1小时);③浸腊(65℃):石蜡(1小时)→石蜡(1小时)。待组织冲洗完毕后,将包含组织的包埋框装进机器篮筐内,启动上述程序。上述程序完成后,取出组织包埋框送病理室包埋组织,同时清洗机器备用。
(2)肝脏组织切片
使用切片机切片(切片厚度5μm)。
(3)肝脏组织苏木精-伊红(HE)染色
将肝脏组织石蜡切片放入65℃烘箱(30分钟)→二甲苯中(5分钟×3次)→100%酒精(1分钟)→90%酒精(1分钟)→70%酒精(1分钟)→蒸馏水洗→苏木素(5分钟)→自来水洗去切片上的浮色→1%盐酸酒精(1至3秒)→自来水洗数下→Scott促蓝液(碳酸氢钠0.35g,硫酸镁2g,蒸馏水100mL)(1分钟)→自来水洗数下→伊红(1分钟)→蒸馏水洗去切片上的浮色→70%酒精一下→90%酒精一下→100%酒精(30秒×3次)→二甲苯(2分钟×3次)→在二甲苯未干时封片,拍照。
实施例3肝脏蛋白提取及Western Blot检测
1、组织研磨:配制好组织裂解液后4℃保存,装有组织的冻存管置于干冰中。先将5颗小钢珠放入1.5mL离心管中并在管壁写好样本编号,称取组织净重,按1:10质量体积加入裂解液,扣好离心管盖后放入研磨适配器中,妥善固定后开始研磨。充分将组织打碎后在冰上静置10min。
2、12000rpm转速4℃离心30min后,将上清液收集到新的1.5ml离心管中并记录体积。
3、蛋白变性:将盛有样本蛋白的离心管在72℃水浴锅中加热10~15分钟,每2min上下颠倒一次以保证受热均匀,加热完成后分装,置于-20℃保存。
4、制胶:按照10% SDS-PAGE胶配方配制
5、电泳:初始电压设置为80V,电泳约0.5小时后加压至120V;待蛋白marker全部分开后停止电泳,约为1个小时。
6、转膜:PVDF膜在甲醇中浸泡活化。以每个电转槽200mA的恒流水平转膜100分钟,该过程中将电转槽置于4℃冰箱内或部分埋于冰中。
7、封闭、抗体孵育:电转膜结束后取出PVDF膜并将其在TBS中稍作浸泡漂洗,然后放入封闭液中于摇床上封闭1小时,最后TBST洗膜3次,每次5分钟。加入一抗4℃孵育过夜,
8、次日取出PVDF膜,TBST洗膜3次,每次五分钟;对应二抗室温孵育1小时,TBST洗膜3次,每次5分钟。
9、显影
如图1所示,在注射LCAT表达质粒后8周,仍能够在小鼠肝脏内检测到外源表达的Flag-LCAT蛋白,说明LCAT在肝脏内基因组整合并过表达是成功的。这样的结果为本发明在后续分析LCAT在原发性肝细胞癌中的作用提供现实基础。
如图2所示,在经历8周的原发性肝细胞癌诱导后,NTG小鼠与HTG小鼠体重并无明显差异,但是NTG小鼠的肝脏重量和肝重体重比数值明显大于HTG组。结合图3所示,小鼠血清谷丙转氨酶和谷草转氨酶效价说明NTG组小鼠的肝脏损伤要更甚于HTG组小鼠。图4具体展示了肝脏的形态,NTG组小鼠的肝脏整体外观表现为更多的肝癌结节和纤维化病变(肝脏发白)。图5更加说明NTG小鼠在经历肝癌造模后,出现了诸如癌细胞特有的大核仁、假小叶等病理学特征,而对应的HTG小鼠肝脏HE染色结果则显示出现以上病理学特征。
上述结果说明LCAT基因过表达小鼠的原发性肝细胞癌进程明显缓解,LCAT基因对于原发性肝细胞癌具有预防作用。
实施例4LCAT基因敲低细胞系构建
1、载体构建
(1)PLKO.1质粒利用AgeI(FD1464,Thermo)和EcorI(FD0274,Thermo)酶在37℃水浴锅内进行酶切线性化。PHAGE-3xFlag质粒用BamHI(FD0054,Thermo)和XhoI(FD0694,Thermo)酶在37℃水浴锅内进行酶切线性化
(2)合成寡聚核苷酸,序列为:
SEQ1(CCGGGTGCTCTATGAGGATGGTGATCT-CGAGATCACCATCCTCATAGAGCACTTTTTG);
SEQ2(AATTCAAAAAGTGCTCTATGAG-GATGGTGATCTCGAGATCACCATCCTCATAGAGCAC);
SEQ3(TCGGGTTTAAACGGAT-CCATGGGGCCGCCCGGCTCCCC);
SEQ4(GGGCCCTCTAGACTCGAGTTAGTGGTGG-TGGTGGTGGTGTTCAGGAGGCGG)。
(3)寡聚核苷酸退火并连接
将寡聚核苷酸SEQ1与SEQ2溶解到浓度100nM,SEQ1与SEQ2等体积混合。95℃加热5分钟后,利用梯度降温程序退火使双链重新结合。退火后双链DNA与线性化的PLKO.1连接。连接体系为2.5μL的ligation high(LGK-100,Toyobo)加上100ng线性化PLKO.1载体和1μL退火后双链DNA,最终加水补齐至5μL。反应在16℃条件下连接4小时。
(4)连接产物转化
连接产物加入30μL感受态细胞,冰上静置15分钟,42℃热激90秒后,继续冰上静置恢复5分钟,加入LB培养基在37℃180转每分钟摇床上恢复45分钟后,涂布于氨苄抗性的LB固体平板上。待第二日克隆形成后,挑取菌落并测序鉴定。
2、慢病毒包装及病毒感染
(1)种1x106 293T细胞于六孔板内,培养过夜。至细胞密度约为60%时准备转染。
(2)利用PEI(24765,Polyscience)转染包装质粒及慢病毒质粒入293T细胞内,转染后6-8小时换新鲜培养基
(3)转染后48小时收取培养基上清,用0.45μm孔径滤头过滤,为慢病毒
(4)铺对数期生长Huh7细胞于六孔板内,感染病毒并加8μg/mL的polybrene(C0351,Beyotime)。
(5)病毒感染后36小时加入终浓度2μg/mL的puromycin(ST551,Beyotime)筛选未整合细胞,至对照空细胞完全死亡。
(6)细胞系内LCAT表达水平利用Western Blot检测。
实施例5His标签LCAT蛋白纯化
1、His标签LCAT蛋白表达载体构建
(1)PHAGE-3xFlag质粒用BamHI(FD0054,Thermo)和XhoI(FD0694,Thermo)酶在37℃水浴锅内进行酶切线性化
(2)合成寡聚核苷酸,序列为:
SEQ3(TCGGGTTTAAACGGATCCATGGGGCCG-CCCGGCTCCCC),
和SEQ4(GGGCCCTCTAGACTCGAGTTAGTGGTGGTGGTGGTGGT-GTTCAGGAGGCGG)。
(3)将寡聚核苷酸SEQ3与SEQ4稀释到10nM。利用Phanta EVO Super-Fidelity DNAPolymerase(P503-d1,Vazyme)从质粒内扩增LCAT蛋白编码DNA序列。DNA片段用Gelextraction kit(CW2302,CWBIO)从琼脂糖凝胶内回收。DNA片段与线性化PHAGE-3xflag质粒按照ClonExpress II One Step Cloning Kit(C112-01,Vazyme)说明进行重组连接。
(4)连接产物加入30μL感受态细胞,冰上静置15分钟,42℃热激90秒后,继续冰上静置恢复5分钟,加入LB培养基在37℃180转每分钟摇床上恢复45分钟后,涂布于氨苄抗性的LB固体平板上。待第二日克隆形成后,挑取菌落并测序鉴定。
2、His标签LCAT蛋白纯化
(1)扩增50盘100mm dish的293T细胞,待细胞密度至60%情况下,转染His标签LCAT蛋白表达质粒10μg,转染后6小时换液。
(2)转染后48小时用细胞铲刮下细胞并收集培养基。离心收集细胞后,用50mL含蛋白酶抑制剂的Lysis buffer(NaH2PO4·2H2O 50mM,NaCl 300mM,Imidazole 10mM,pH8.0)重悬细胞,冰浴条件下超声破细胞8s,间隔8s,80次,频率为200Hz。
(3)向上清中添加BeyoGoldTMHis-tag Purification Resin(P2233,Beyotime),于4℃缓慢旋转结合过夜(beads预处理:剪去枪头,吸取700μL Resin于1.5mL EP管中,用PBS洗涤3次);将清洗后的BeyoGoldTMHis-tag Purification Resin填入填料柱内,将收集的培养基结合BeyoGoldTMHis-tag Purification Resin过夜。
(4)用100mLWash buffer(5.165g Na2HPO4·12H2O,1.650g NaH2PO4·2H2O,8.775gNaCl and 0.177g Imidazole in 500mL distilled water)洗涤杂蛋白;
(5)洗脱目的蛋白。加入20ml Elusion buffer(5.165g Na2HPO4·12H2O,1.650gNaH2PO4·2H2O,8.775g NaCl and 5.1g Imidazole in 500mL distilled water),悬浮BeyoGoldTMHis-tag Purification Resin,于4℃冰箱快速旋转10min洗脱目的蛋白。最终利用考马斯亮蓝染色和Western Blot鉴定条带,BCA试剂盒(NCI3225CH,Thermo)分析蛋白浓度。
实施例6细胞计数试剂-8
1、收集对数期细胞,利用血球计数板计数,96孔板每孔加入100μL含3000个细胞的培养液。边缘孔用灭菌PBS溶液填充。5%CO2,37℃条件下培养细胞至细胞贴壁并形态舒展。LCAT组加入终浓度为5μg/mL LCAT蛋白溶液,Saline组加入等体积的生理盐水。
2、加入10μL细胞计数试剂-8(A311-01,Vazyme),5%CO2,37℃条件下孵育2小时。在酶标仪上测得450nm处吸光值。以上操作在细胞形态舒展后0、24、48、72小时时间点各重复一次。
3、统计不同时间点、不同组别对应的吸光值并计算其统计学差异。
实施例7克隆形成
1、取对数生长期的各组细胞,分别用0.25%胰蛋白酶消化并吹打成单个细胞,并把细胞悬浮在培养基中计数后备用。
2、梯度稀释细胞悬液至5000个细胞每毫升。LCAT组加入终浓度为5μg/mL LCAT蛋白溶液,Saline组加入等体积的生理盐水。
3、六孔板内接种500个细胞,置37℃5% CO2及饱和湿度的细胞培养箱中培养1周,中间每2-3天换一次新鲜培养基。LCAT组加入终浓度为5μg/mL LCAT蛋白溶液,Saline组加入等体积的生理盐水。
4、当培养皿内出现肉眼可见的克隆后,终止培养。弃去上清液,用PBS小心浸洗2次。加入4%多聚甲醛室温固定30分钟。
5、去固定液,加适量0.5%结晶紫染液染5-10分钟,用流水缓慢洗去染色液,空气干燥。
6、用扫描仪拍照后,克隆数量用ImageJ软件计数并计算克隆生成率及统计学差异。
结合图6和图7综合分析,细胞裂解液纯化得到的LCAT蛋白具有两个条带,并且考马斯亮蓝染色结果显示纯化得到的LCAT蛋白混杂有杂蛋白,可能干扰后续实验评估;而从培养基内纯化得到的LCAT蛋白则仅有单一条带。因而本发明采用从培养基内纯化的分泌出细胞外的LCAT蛋白。
LCAT敲低效率利用实时荧光定量PCR进行验证。根据图9和图10结果显示,添加生理盐水条件下,LCAT敲低细胞系增殖速率和克隆形成能力相较于对照细胞均有明显提高;而向培养基内添加终浓度为5μg/mL LCAT蛋白后,发现LCAT敲低和对照细胞系增殖速率和克隆形成能力均显著下降,且两组之间不再具有显著性差异。这样的结果证明,改变LCAT基因去干预肝癌细胞增殖速率是依赖于其分泌蛋白特性的。
实施例8裸鼠皮下成瘤及瘤内注射
1、基础饲养条件:购入4周龄雄性BALB/c-nude小鼠,体重在14克左右,BALB/c-nude小鼠购自北京华阜康生物科技股份有限公司,小鼠生产许可证号为SYXK(鄂)-2019-0050。饲养用垫料、饮水、全价颗粒饲料及其他与动物接触的物品均经高压灭菌处理。实验和饲养条件严格按照SPF级规范要求。所有动物实验均经武汉大学生命科学学院动物关爱与利用委员会批准。
2、皮下成瘤造模:4周龄小鼠经过一周适应性饲养后进行皮下成瘤注射。每只小鼠注射两侧腋下皮肤内,单侧注射体积为200μL,包含7.5x106个HCCLM9细胞,其中细胞悬液和基质胶(0827045,ABW)按1:1混合。
3、记录肿瘤体积:皮下成瘤造模形成肿瘤多呈椭圆球体。注射后五天待基质胶被基本吸收后,每隔两天测量肿瘤体积。
4、瘤内注射:待肿瘤大小达到约200mm3的体积后,每隔两天注射一次纯化蛋白。共注射四次,注射体积为50μL,生理盐水组注射生理盐水,LCAT蛋白组注射含20μg LCAT蛋白的生理盐水。
5、终末取材:用镊子钳夹小鼠肿瘤处皮肤,剪开皮肤,取出肿瘤,置于冰上预冷PBS溶液内。全部肿瘤取材结束后,将肿瘤统一放置拍照记录。切取部分肿瘤,置于冻存管内,迅速放入液氮急冻。后取出,于-80℃长期保存,为鲜样本。
本发明设计图11的评估方案,验证LCAT蛋白缓解肝细胞癌恶性增殖的潜力。在皮下注射后四天开始测量,每隔两天测量一次;在皮下注射后十四天开始瘤内注射。如图12显示,4-14天,Saline组和LCAT蛋白组肿瘤体积显著增长,且两组之间没有显著差异;在药物注射后,LCAT蛋白组肿瘤体积增长速度开始放缓,LCAT蛋白组肿瘤体积显著小于Saline组。最终于皮下成瘤注射后20天终末取材,如图13显示,Saline组的肿瘤体积明显大于LCAT蛋白组。这样的结果提示,LCAT蛋白的瘤内注射具有良好的抑制肝细胞癌恶性增殖的能力。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (5)
1.LCAT基因作为药物靶标在制备预防、缓解和/或治疗原发性肝细胞癌的药物中的应用。
2.根据权利要求1所述的应用,其特征在于:所述药物是在体内提高肝细胞LCAT基因表达的化学药物和生物分子,实现抑制肝癌细胞的增殖、迁移或侵袭。
3.LCAT纯化蛋白在制备预防、缓解和/或治疗原发性肝细胞癌的药物中的应用,其特征在于:
LCAT全长蛋白及LCAT蛋白结构域的外源表达载体作为药物靶标以预防和/或缓解原发性肝细胞癌;或者,
LCAT蛋白激动剂作为药物靶标以预防和/或缓解原发性肝细胞癌。
4.根据权利要求3所述的应用,其特征在于:所述外源表达载体,包括以DNA、RNA及病毒表达载体实现LCAT全长蛋白及LCAT蛋白结构域表达的外源表达载体,最终导致在体内抑制肝癌细胞的增殖、迁移或侵袭。
5.根据权利要求3或4所述的应用,其特征在于:所述药物与LCAT蛋白具有明确的结合特性,并且提高LCAT蛋白活性的药物,药物包含化学药物与生物药物,最终实现在体内抑制肝癌细胞的增殖、迁移或侵袭。
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CN117334325B (zh) * | 2023-09-26 | 2024-04-16 | 中山大学肿瘤防治中心(中山大学附属肿瘤医院、中山大学肿瘤研究所) | 一种lcat在肝细胞癌诊断、治疗和预测复发的应用 |
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