CN113633772A - 一种治疗肝癌的药物及其验证模型构建方法、筛选方法 - Google Patents
一种治疗肝癌的药物及其验证模型构建方法、筛选方法 Download PDFInfo
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Abstract
本发明涉及一种治疗肝癌的药物,其特征在于,所述药物能够提升癌细胞中COMMD10基因的表达水平。本发明还提供该药物的验证模型构建方法、以及筛选方法。本发明的药物,具有够提升肝癌放疗敏感性、降低复发率、提升放疗成功率等优点。
Description
技术领域
本发明涉及药物领域,具体涉及一种治疗肝癌的药物及其验证模型构建方法、筛选方法。
背景技术
癌,又称肿瘤,现在已经成为危害人类健康的重大疾病之一。为了更好的维护患者的健康,实现安全有效的抗肿瘤治疗已经成为医药工作者的重要任务。从现有的医学临床情况来看,化疗(或称放射治疗、放疗)是一种重要的有效治疗手段,但化疗因其作用非特异性,会无差别的对正常细胞进行杀伤,在治疗过程中会产生严重的毒副作用,其临床应用受到了很大的限制。
并且,在放疗手术中,放疗抵抗是肝癌复发或者放疗失败的重要原因,导致患者五年内生存率低下,因此提高肝癌放疗敏感性、进而提升放疗成功率、降低复发率成为医药工作者亟待解决的严峻任务。
发明内容
本发明的目的在于提供一种能够提升肝癌放疗敏感性、降低复发率、提升放疗成功率的药物,同时,本发明还提供该药物的验证模型方法,以及该药物的筛选方法。
为解决上述问题,本发明所采用的技术方案如下:
一种治疗肝癌的药物,所述药物能够提升癌细胞中COMMD10基因的表达水平。
本发明中,进一步优选的方案为,所述治疗肝癌的药物为增强肝癌放疗敏感性的药物。
本发明中,进一步优选的方案为,所述药物能够进入肝癌细胞。
本发明中,进一步优选的方案为,所述药物中包含COMMD基因片段。
本发明中,进一步优选的方案为,所述药物还包括用于搭载COMMD10基因片段的质粒。
本发明中,进一步优选的方案为,所述药物还包括用于包载所述质粒的载体材料。
本发明中,进一步优选的方案为,所述药物还包括肝癌靶向肽。
本发明还提高一种所述治疗肝癌药物的验证模型构建方法,包括步骤A,以及包括步骤B1-B3中的至少一个步骤:
A、实验细胞构建和验证:构建内源性低表达COMMD10基因的肝癌细胞,通过荧光定量PCR技术检测构建后的肝癌细胞的COMMD10的表达量相对构建前是否降低进行验证;
B1、克隆形成实验验证:将步骤A构建得到的内源性低表达COMMD10基因的肝癌细胞和对照组肝癌细胞利用不同剂量X射线照射进行单击多靶对比实验,计算克隆形成率,并根据各肝癌细胞在各剂量照射的克隆形成率拟合存活曲线进行验证;
B2、流式细胞仪检测凋亡验证:构建内源性低表达COMMD10基因的肝癌细胞,然后将步骤A构建得到的内源性低表达COMMD10基因的肝癌细胞、与内源性过表达的肝癌细胞进行培养不低于2天,然后通过流式细胞仪测量各组肝癌细胞凋亡情况进行比较验证;
B3、构建裸鼠皮下瘤放疗模型验证:将步骤A构建得到的内源性低表达 COMMD10基因的肝癌细胞和对照组肝癌细胞注射到裸鼠背部,然后观察肿瘤生长情况,至肿瘤140-160mm3时随机分为照射组和非照射组,照射组利用X 射线进行照射,然后定期测量各裸鼠背部肿瘤生长大小进行比较验证。
本发明还提高上述治疗肝癌药物的筛选方法,包括如下步骤:取待筛选药物加入具有癌细胞的培养基中,同时设置对照组,所述对照组未加入待筛选药物的具有癌细胞的培养基,将各培养基在36-38℃温度下培养不低于8h,然后测量两培养基中肝癌细胞COMMD10基因表达量或者COMMD10基因表达蛋白的含量,筛选能够促进COMMD10基因表达量的药物。
本发明中,进一步优选的方案为,所述待筛选药物为N种,所述具有癌细胞的培养基为N+1个,其中N个培养基中各加入1中待筛选药物,另一个培养基作为对照组。
与现有技术相比,本发明具有如下优点:本发明的药物,能够有效增强肝癌细胞放疗敏感性,进而能够提升肝癌放疗成功率、降低复发率。
下面结合说明书附图和具体实施方式对本发明进行详细说明。
附图说明
图1为实验例1中对照组与测试组的COMMD10基因表达量测试数据图;
图2为实验例1中对照组与测试组的COMMD10基因表达相应蛋白的蛋白免疫印迹侧视图;
图3为实验例1中对照组与测试组的细胞在不同X射线照射剂量存活率数据图;
图4为实验例1中对照组与测试组的克隆形成实验代表图;
图5为实验例1中对照组与测试组经流式细胞仪转染COMMD10siRNA的HepG2 肝癌细胞接受6Gy X-ray照射后进行AnnexinV/Pi染色后的荧光图及数据图;
图6为实验例1中对照组与测试组的经流式细胞仪检测转染COMMD10过表达质粒的SMMC-7721肝癌细胞接受6Gy X-ray照射后进行AnnexinV/Pi染色后的荧光图及测试数据图;
图7为实验例1的裸鼠皮下瘤放疗模型构建示意图;
图8为实验例1中对照组与测试组的裸鼠皮下瘤放疗模型大体图;
图9为实验例1中对照组与测试组的裸鼠皮下瘤肉眼图;
图10为实验例1中对照组与测试组的裸鼠皮下肿瘤体积-时间生长曲线图。
其中,图1-图5、图8-图10中,Vector为对照细胞组,shCOMMD10为内源性稳定低表达组(或称测试组)。
具体实施方式
下面,结合附图以及具体的实施方式对本发明做进一步的描述,需要说明的是,在不相冲突的前提下,以下描述的各实施例之间或各技术特征之间可以任意组合成新的实施例。除特殊说明的之外,具体实施方式中的实施例与实验例,其中的设备和试剂材料均从市场途径获得,具体的实施例是示范性的,仅用于解释本申请,而不能理解作为本申请保护范围的限制。
一种治疗肝癌的药物,所述药物能够提升癌细胞中COMMD10基因的表达水平。进一步的是,所述治疗肝癌的药物为增强肝癌放疗敏感性的药物。
经过研究证实,铜代谢基因COMMD10与肝癌细胞的放射敏感性及凋亡相关,并且通过实验研究发现,铜代谢基因COMMD10能够增强肝癌细胞的放射敏感性;通过本发明的药物,可以使得肝癌细胞中的COMMD10基因表达水平提升,进而增强肝癌细胞的放疗敏感性,降低肝癌细胞的放疗抵抗,提升放疗成功率,降低肝癌复发率;同时COMMD10基因的表达水平提升,能够促进肝癌细胞的凋亡,与放疗进行配合,能够进一步提升对肝癌的疗效。
本发明的药物,能够提升肝癌细胞中的COMMD10基因的表达的即可,可以为传统的化学分子药、中药、或者是生物药,例如:包含COMMD基因片段的药物;为了便于药物进入肝癌细胞,可以将上述基因用质粒进行搭载;为了便于药物的吸收、以及避免药物在进入肝癌细胞前结构遭到破坏,对应的可以在药物中设置包载质粒的载体材料;为了提升药物的靶向性,可以在药物中加入肝癌靶向肽(如肝癌靶向肽SP94)进行修饰,这样,药物会和肝癌细胞进行靶向结合,而不会与其他细胞进行结合,以避免/降低对其他细胞的损害,进而提升药物的安全性和高效性。
本发明的靶向药物中,对于包含COMMD10基因片段的质粒,可以采用现有的质粒包载材料进行包载,为了更好的让药物进入细胞和提高安全性能,可以通过纳米复合材料进行包载,对于用于包载质粒的纳米复合材料,可以选用沸石咪唑酯骨架材料(无毒、生物相容性良好)。
对于本发明的治疗肝癌的药物,可以通过如下方法构建药物的验证模型,该方法包括步骤A,以及包括步骤B1-B3中的至少一个步骤:
A、实验细胞构建和验证:构建内源性低表达COMMD10基因的肝癌细胞,通过荧光定量PCR技术检测构建后的肝癌细胞的COMMD10的表达量相对构建前是否降低进行验证;
B1、克隆形成实验验证:将步骤A构建得到的内源性低表达COMMD10基因的肝癌细胞和对照组肝癌细胞利用不同剂量X射线照射进行单击多靶对比实验,计算克隆形成率,并根据各肝癌细胞在各剂量照射的克隆形成率拟合存活曲线进行验证;
B2、流式细胞仪检测凋亡验证:构建内源性低表达COMMD10基因的肝癌细胞,然后将步骤A构建得到的内源性低表达COMMD10基因的肝癌细胞、与内源性过表达的肝癌细胞进行培养不低于2天,然后通过流式细胞仪测量各组肝癌细胞凋亡情况进行比较验证;
B3、构建裸鼠皮下瘤放疗模型验证:将步骤A构建得到的内源性低表达 COMMD10基因的肝癌细胞和对照组肝癌细胞注射到裸鼠背部,然后观察肿瘤生长情况,至肿瘤140-160mm3时随机分为照射组和非照射组,照射组利用X 射线进行照射,然后定期测量各裸鼠背部肿瘤生长大小进行比较验证。
为了更好的利用COMMD10基因能够增强肝癌细胞放疗敏感性的特点,本发明还提供一种药物筛选方法,以能够选择更多种类的药物应用于肝癌的治疗当中,具体的药物筛选方法包括如下步骤:取待筛选药物加入具有癌细胞的培养基中,同时设置对照组,所述对照组未加入待筛选药物的具有癌细胞的培养基,将各培养基在36-38℃温度下培养不低于8h,然后测量两培养基中肝癌细胞 COMMD10基因表达量或者COMMD10基因表达蛋白的含量,筛选能够促进 COMMD10基因表达量的药物。
其中,所述待筛选药物为N种,所述具有癌细胞的培养基为N+1个,其中 N个培养基中各加入1中待筛选药物,另一个培养基作为对照组;这样就可以同时筛选多种药物,以提升药物研发效率;对应的还可以通过计算机软件辅助药物的筛选和设计。
本发明先在细胞层面证实COMMD10增强肝癌细胞放疗敏感性,且促进肝癌细胞凋亡。
随后构建裸鼠皮下瘤放疗模型,在动物水平证实COMMD10能够延缓裸鼠放疗后皮下瘤生长,增强肝癌细胞放疗敏感性,对COMMD10作为一个调控肝癌放射敏感性的新靶点开展进一步研究。
首先,构建内源性稳定低表达COMMD10 HepG2肝癌细胞(shCOMMD10) 及对照细胞(Vector),利用荧光定量PCR技术,Western blot技术检测肝癌细胞系中COMMD10的相对表达量。结果表明干扰COMMD10表达后HepG2细胞的COMMD10水平明显降低。
第二步,利用克隆形成实验(colon assay)检测感染COMMD10siRNA病毒后HepG2细胞的辐射敏感性情况的变化。HepG2肝癌细胞接受0Gy,2Gy,4Gy, 6Gy,8GyX-ray照射,2周后计算克隆形成率。根据单击多靶模型拟合存活曲线。研究发现,稳定干扰COMMD10表达后诱导了HepG2细胞对X-ray的抵抗,克隆存活率明显增多。
第三步,利用流式细胞仪检测干扰COMMD10表达后的HepG2细胞,以及过表达COMMD10后SMMC-7721细胞的凋亡情况。结果表明干扰COMMD10 后HepG2细胞的凋亡情况明显受到抑制,而过表达COMMD10后促进 SMMC-7721细胞的凋亡。
第四步,构建裸鼠皮下瘤放疗模型,在动物水平验证COMMD10对肝癌细胞的放疗敏感性。结果证实干扰COMMD10表达能够促进裸鼠放疗后皮下瘤生长,诱导肝癌细胞HepG2放疗抵抗。
实验例1
对COMMD10基因表达对肝癌细胞的作用进行测试:
1、构建内源性稳定低表达COMMD10 HepG2肝癌细胞株。
实验材料:HepG2肝癌细胞株,购自中国科学院上海细胞研究所细胞库。
实验方法与结果分析:
1)细胞培养:首先37℃水浴复苏细胞,然后接种于细胞培养瓶中。细胞培养使用含10%胎牛血清的DMEM培养基(FBS,Gibco,澳大利亚)。培养箱条件为37℃,5%CO2。
2)稳定株构建:HepG2细胞复苏后待细胞密度长至70-80%,以2×104/孔接种于24孔板,无抗生素培养基生长过夜。COMMD10低表达及对照组慢病毒各取108TU/ml均用0.5mlENi.S.液稀释,加入8mg/ml Polybrene液,37℃孵育12h。10%血清浓度DMEM培养48h后,加入0.5mg/ml Puromycin持续筛选10天。筛选后用荧光定量PCR和Western blot验证COMMD10表达水平。
2、内源性稳定低表达COMMD10 HepG2肝癌细胞株的验证
1)Realtime-PCR:总RNA通过Trizol(Invitrogen,Carlsbad,美国加州) 试剂提取。COMMD10和GAPDH的逆转录引物购自英潍捷基公司。
COMMD10引物:
正向:5'-GCGGTAGGCGTGTACGGT-3'
反向:5'-ATTGTGGATGAATACTGCC-3'
内参GAPDH的PCR引物序列为:
正向:5'-CCATCAATGACCCCTTCATTGACC-3'
反向:5'-GAAGGCCATGCCAGTGAGCTTCC-3'
根据Premix Ex Taq II(Takara)说明书要求配置PCR反应液(20 μl体系),将样品加入到实时PCR的专用PCR管中后,设置ABI7500(Perkin Elmer/appliedBiosystems)的PCR反应条件为:95℃5s,60℃34s,40cycles。完成实时PCR后对熔解曲线进行分析,并分析PCR产物是否特异。数据按2-ΔΔCt 公式计算出基因的相对表达量。
2)Western blot:收集细胞,通过全蛋白提取试剂盒(杭州弗德生物有限公司)提取总蛋白,使用Bio-Rad公司BCA Protein Assay Reagengt Kit进行蛋白定量,煮沸变性。
3)SDS-PAGE电泳:浓缩胶恒定电压在100V,分离胶恒定电压在120V,至跑出目的蛋白(COMMD10分子量为23KD,GAPDH为37KD)。
4)转膜:Tenon微型电转系统200MA进行转膜,COMMD10约转50min,GAPDH 约转1h。
5)封闭:5%脱脂牛奶室温孵育1h。
6)敷一抗:COMMD10兔多克隆抗体(1:1000,Abcam),GAPDH鼠单克隆抗体,1:5000,Proteintech),4℃孵育过夜。
7)敷二抗:HRP标记的抗兔及抗鼠二抗(1:10000,杭州弗德生物有限公司) 室温孵育1h。
8)ECL超敏化学发光试剂(南京凯基生物科技发展有限公司)对条带进行检测。
结合图1-2,结果发现,构建的内源性稳定低表达COMMD10 HepG2肝癌细胞株(shCOMMD10)的COMMD10基因及相应蛋白的表达水平均比对照组(Vector) 明显降低,相应的稳定株构建成功。
3、COMMD10低表达诱导肝癌HepG2细胞放疗抵抗
1)克隆形成实验:用稳定感染Vector和shCOMMD10病毒的HepG2细胞经消化制成单细胞悬液后,按照预定数量接种于六孔板中,设0,2,4,6,8Gy5个剂量组,每个剂量组3个复孔。照射后将细胞置于37℃,5%CO2饱和湿度培养箱内继续培养10-14天,期间根据培养液PH值变化适时更换新鲜培养液。当培养板孔中出现肉眼可见克隆时,终止培养。PBS清洗2遍,甲醇固定,苏木素染色。显微镜下计数含50个细胞以上的克隆数。计算克隆形成率和存活分数,并用多靶单击模型拟合曲线;
结合图3,结果表明,COMMD10低表达HepG2肝癌细胞的存活分数明显增加,诱导放疗抵抗。
4、COMMD10对肝癌细胞凋亡的影响
1)流式检测凋亡:将细胞均匀地铺在六孔板中,将COMMD10的Vector/siRNA 通过lipo2000与opti-MEMI(约1500μl)共孵育后转入细胞中,转染6h后将细胞培养液换成新鲜培养液。转染48小时后,将收集到的细胞用FITC凋亡试剂盒(南京凯基生物有限公司)染色细胞。用流式细胞计数检测肝癌细胞的凋亡情况。
结合图4-6,结果表明,干扰COMMD10表达后肝癌细胞凋亡明显减少。
5、构建裸鼠皮下瘤放疗模型
把已构建好的稳定感染Vector和shCOMMD10病毒的HepG2肿瘤细胞注射到裸鼠背部。观察肿瘤生长情况,待肿瘤体积长到150mm3大小随机分为照射组 (IR)和非照射组(NoIR)两大组,各大组均包含Vector,shCOMMD10两小组。在第14天和第21天照射组(IR)裸鼠在同时间照射6Gy X射线,实验期间每3 天测量一次肿瘤大小。观察经射线照射后,COMMD10下调对肝癌细胞皮下成瘤的影响。
结合图7-图10,结果表明,其中,图10中,到实验终点41天时,,在非照射组(No IR)中,shCOMMD10组(1351.72±100.13)肿瘤体积较Vector组 (325.11±63.68)明显增大,Vector/shCOMMD10比值为4.18。而在照射组(IR) 中,shCOMMD10组(927.33±91.47)与Vector组(129.87±45.47)肿瘤体积比非照射组(No IR)进一步增大(Vector/shCOMMD10比值为7.14)。说明COMMD10 低表达能够促进裸鼠放疗后皮下瘤生长,诱导肝癌细胞HepG2放疗抵抗;可见,干扰COMMD10表达能够促进裸鼠放疗后皮下瘤生长,诱导肝癌细胞HepG2放疗抵抗。
上述实施方式仅为本发明的优选实施方式,不能以此来限定本发明保护的范围,本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (10)
1.一种治疗肝癌的药物,其特征在于,所述药物能够提升癌细胞中COMMD10基因的表达水平。
2.根据权利要求1所述的药物,其特征在于,所述治疗肝癌的药物为增强肝癌放疗敏感性的药物。
3.根据权利要求2所述的药物,其特征在于,所述药物能够进入肝癌细胞。
4.根据权利要求2所述的药物,其特征在于,所述药物中包含COMMD基因片段。
5.根据权利要求4所述的药物,其特征在于,所述药物还包括用于搭载COMMD10基因片段的质粒。
6.根据权利要求5所述的药物,其特征在于,所述药物还包括用于包载所述质粒的载体材料。
7.根据权利要求4所述的药物,其特征在于,所述药物还包括肝癌靶向肽。
8.一种根据权利要求1-7任一项所述药物的验证模型构建方法,其特征在于包括步骤A,以及包括步骤B1-B3中的至少一个步骤:
A、实验细胞构建和验证:构建内源性低表达COMMD10基因的肝癌细胞,通过荧光定量PCR技术检测构建后的肝癌细胞的COMMD10的表达量相对构建前是否降低进行验证;
B1、克隆形成实验验证:将步骤A构建得到的内源性低表达COMMD10基因的肝癌细胞和对照组肝癌细胞利用不同剂量X射线照射进行单击多靶对比实验,计算克隆形成率,并根据各肝癌细胞在各剂量照射的克隆形成率拟合存活曲线进行验证;
B2、流式细胞仪检测凋亡验证:构建内源性低表达COMMD10基因的肝癌细胞,然后将步骤A构建得到的内源性低表达COMMD10基因的肝癌细胞、与内源性过表达的肝癌细胞进行培养不低于2天,然后通过流式细胞仪测量各组肝癌细胞凋亡情况进行比较验证;
B3、构建裸鼠皮下瘤放疗模型验证:将步骤A构建得到的内源性低表达COMMD10基因的肝癌细胞和对照组肝癌细胞注射到裸鼠背部,然后观察肿瘤生长情况,至肿瘤140-160mm3时随机分为照射组和非照射组,照射组利用X射线进行照射,然后定期测量各裸鼠背部肿瘤生长大小进行比较验证。
9.一种根据权利要求1-7任一项所述药物的筛选方法,其特征在于包括如下步骤:取待筛选药物加入具有癌细胞的培养基中,同时设置对照组,所述对照组未加入待筛选药物的具有癌细胞的培养基,将各培养基在36-38℃温度下培养不低于8h,然后测量两培养基中肝癌细胞COMMD10基因表达量或者COMMD10基因表达蛋白的含量,筛选能够促进COMMD10基因表达量的药物。
10.根据权利要求9所述的筛选方法,其特征在于,所述待筛选药物为N种,所述具有癌细胞的培养基为N+1个,其中N个培养基中各加入1中待筛选药物,另一个培养基作为对照组。
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