CN111481666B - 可抑制calb2蛋白表达的抑制剂的应用和肝癌药物 - Google Patents
可抑制calb2蛋白表达的抑制剂的应用和肝癌药物 Download PDFInfo
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Abstract
本发明涉及抑制肝癌转移的方法和试剂。本发明提供了一种可抑制CALB2蛋白表达的抑制剂在制备治疗肝癌转移药物中的应用。肝癌转移是影响肝癌患者预后的重大障碍。本发明提供了一种可抑制肝癌转移的方法和试剂,用于治疗肝癌转移,提高肝癌患者预后效果。
Description
技术领域
本发明涉及肝癌治疗领域,特别地,涉及一种可抑制CALB2蛋白表达的抑制剂在制备治疗肝癌转移药物中的应用。
背景技术
肝脏是人体物质代谢,能量转换及供应的枢纽和主导器官。癌症是人口死亡的第二位原因。肝癌发病率高而且死亡率高,其5年生存率仅5%-6%。目前原发性肝癌已上升为我国第二位癌症杀手。由于原发性肝癌早期一般无任何症状,一旦出现临床表现,病情大多已进入中期或晚期,而且恶性程度高、预后差、侵袭转移性强。临床数据显示,肝癌的高侵袭转移能力和术后高复发转移率是影响肝癌病人长期生存的重要因素。因此,肝癌转移是生命科学需要迫切解决的问题。
肝癌转移分为远处转移和肝内转移。转移进程大体包含肿瘤细胞获得向外侵袭的能力使得细胞脱离原发器官侵入周围组织并穿入淋巴管和血管;随循环系统运行,停留;肿瘤细胞增殖,形成转移灶。染色体水平的变化,癌基因表达增加,抑癌基因的失活,上皮细胞-间充质转化,肿瘤细胞间蛋白水解酶的合成,细胞与基底膜的黏着,免疫系统免疫识别低下,一些细胞因子及受体的改变等因素都可能促进肝癌的转移。细胞迁移与运动能力的提高是引起肿瘤侵入周围组织和发生远处转移的关键因素。但是,调控肝癌细胞运动侵袭能力的分子机制仍不明朗,难以开发出有效的抑制肝癌转移的药物,因此,寻找并发现肝癌转移相关蛋白用于转移和复发研究、药物靶标的设计,这对于实现肝癌病人生存率的提高,具有重要意义。
CALB2是一种钙结合蛋白,在听觉神经元中含量丰富。CALB2蛋白在多种细胞功能中起作用,包括信息靶向和细胞内钙缓冲,参与钙信号传导通路。Ca2+是协调内质网-线粒体的相互作用,调节细胞凋亡的信使。线粒体Ca2+动力学还参与细胞能量代谢的调节以及细胞运动和神经递质释放等过程。CALB2还是一种间皮瘤诊断标志(Doglioni C,etal.Calretinin:A Novel Immunocytochemical Marker for Mesothelioma.AmericanJournal of Surgical Pathology,1996,20(9):1037–1046;Chu A Y,et al,Utility ofD2-40,a novel mesothelial marker,in the diagnosis of malignantmesothelioma.Modern Pathology,2005,18(1):105–110.)。有研究显示,正常结肠上皮细胞中没有CALB2的表达,但在原发性结肠癌细胞中发现有表达(Gotzos V,et al.Selectivedistribution of calretinin in adenocarcinomas of the human colon and adjacenttissues.American Journal of Surgical Pathology,1999,23(6):701–711;Gotzos V,etal.Heterogeneity of expression of the calcium-binding protein calretinin inhuman colonic cancer cell lines.Anticancer Research,1996,16(6B):3491–3498.)。结直肠癌细胞经5-FU治疗后,CALB2可通过内在的线粒体途径参与凋亡诱导(Stevenson L,et al.Calbindin 2(CALB2)Regulates 5-Fluorouracil Sensitivity in ColorectalCancer by Modulating the Intrinsic Apoptotic Pathway.Plos One,2011,6(5):1575-1581.)。还有报道显示CALB2作为间质细胞内的因子,通过PLC-Ca2+-PKC信号通路参与调节类固醇激素的合成,并可能是通过加强PI3K-AKT和ERK l/2信号促进间质细胞增殖(Xu W,et al.Calretinin Participates in Regulating Steroidogenesis by PLC-Ca2+-PKCPathway in Leydig Cells.Scientific Reports,2018,8.)。然而目前,关于CALB2能否调控肝癌细胞的生长,迁移和侵袭以及如何调控,并没有报道。
发明内容
本发明目的在于提供一种可抑制CALB2蛋白表达的抑制剂在制备治疗肝癌转移药物中的应用,以解决现有技术中肝癌患者术后肝癌细胞侵袭转移的技术问题。
为实现上述目的,本发明提供了一种可抑制CALB2蛋白表达的抑制剂在制备治疗肝癌转移药物中的应用。
进一步地,药物含有效剂量的抑制CALB2蛋白的基因的病毒载体。
进一步地,病毒载体为慢病毒载体,采用的慢病毒包装质粒为PSPAX,PVSVG,抑制CALB2蛋白的基因序列有三种中的一种或二种或三种,优选sh1。转染细胞后采用抗生素进行筛选。
CALB2-sh1
sh1 full hairpin
TGCTGTTGACAGTGAGCGCTAACGC
CALB2-sh1
TGATCTTCTTCAGGTTAACCCAACAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCGCTAACGC
sh1 full hairpin
GATCTTCACATTTTATAGTGAAGCCACAGATGTATAAAATGTGAAGATCGCGTTAATGCCTACTGCCTCG
CALB2-sh1
GATCTTCACATTTTATAGTGAAGCCACAGATGTATAAAATGTGAAGATCGCGTTAATGCCTACTGCCTCG
sh1 full hairpin
GA
CALB2-sh1
GAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGAT
sh1 full hairpin
CALB2-sh1
ACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCCCAGCTCTGGAGT
CALB2-sh2
sh2 full hairpin
TGCTGTTGACAGTGAGCGATACGAGAAAAA
CALB2-sh2
TCTTCTTCGGTTACCCAACAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCGATACGAGAAAAA
sh2 full hairpin
CAAAAAGGAATAGTGAAGCCACAGATGTATTCCTTTTTGTTTTTCTCGTACTGCCTACTGCCTCGGA
CALB2-sh2
CAAAAAGGAATAGTGAAGCCACAGATGTATTCCTTTTTGTTTTTCTCGTACTGCCTACTGCCTCGGAATT
sh2 full hairpin
CALB2-sh2 CAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATT
sh2 full hairpin
CALB2-sh2 TTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCGGAATGCGTTTCACTCGG
CALB2-sh3
sh3 full hairpin
TGCTGTTGACAGTGAGCGCTTGAAGGATCTGTAC
CALB2-sh3
CTCAGGTTACCCAACAGAAGGCTCGAGAAGGTATAGTGCTGTTGACAGTGAGCGCTTGAAGGATCTGTAC
sh3 full hairpin GAGAAATAGTGAAGCCACAGATGTATTTCTCGTACAGATCCTTCAAATGCCTACTGCCTCGGA
CALB2-sh3
GAGAAATAGTGAAGCCACAGATGTATTTCTCGTACAGATCCTTCAAATGCCTACTGCCTCGGAATTCAAG
sh3 full hairpin
CALB2-sh3 GGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTA
sh3 full hairpin
CALB2-sh3
CAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCATGACCCTGTTCGACTCTAAGT。
进一步地,一种可抑制CALB2蛋白表达的抑制剂在抑制肝癌细胞侵袭转移的药物中的应用。
本发明通过一系列的功能实验(如CCK-8实验、细胞划痕实验、Transwell实验、动物实验)验证CALB2对肝癌细胞迁移侵袭的调控作用。
本发明还提供了一种肝癌转移的治疗方法,所述方法包括抑制CALB2基因的表达,或抑制CALB2蛋白的表达,或抑制CALB2蛋白的活性。
本发明还提供了一种治疗肝癌转移药物的筛选方法,可以通过在对不同转移能力的肝癌细胞添加筛选药物后或在对实验动物施用筛选药物后检测CALB2基因的表达或蛋白的表达,来初步测定筛选药物的预后效果,当CALB2基因的表达水平或蛋白的表达水平下调时,可初步选择该药物作为治疗肝癌转移的药物。
本发明还提供了一种治疗肝癌转移的药物,所述药物包含CALB2的抑制剂。
本发明所提及的CALB2基因或蛋白水平的抑制剂,不受来源或形式等限制,只要是可以抑制CALB2或涉及CALB2上游或下游途径的物质的表达或活性,且对于治疗肝癌有效的物质即可。
本发明的抑制剂可以单独给药,或者以各种组合形式给药,可以根据需要制备成各种剂型,使用途径及使用剂量不受限制。
本发明具有以下优点:
本发明通过一系列的功能实验验证了CALB2对肝癌细胞迁移侵袭运动的调控作用。本发明首次发现CALB2与肝癌转移具有密切相关性,为治疗肝癌转移的药物开发提供了潜在的药物靶标。
附图说明:
图1.CALB2蛋白在肝细胞及不同转移能力肝癌细胞系中的表达
图2.稳转细胞系表达效果的Western blot验证
图3.稳转细胞系CCK-8实验验证CALB2对肝癌细胞增殖的影响
图4.稳转细胞系划痕实验验证CALB2对肝癌转移的影响
图5.稳转细胞系Transwell实验验证CALB2对肝癌转移的影响(图中标注的HCCLM3-shcontrol均表示HCCLM3对照组稳转细胞系,HCCLM3-shCALB2均表示HCCLM3下调CALB2表达的稳转细胞系)
具体实施方式
以下结合附图对本发明的实施例进行详细说明,但是本发明可以由权利要求限定和覆盖的多种不同方式实施。本发明中的实验操作通常是按照常规实验条件进行,如Sambrook等人著,分子克隆:实验室指南(New York:Cold Spring Harbor LaboratoryPress,1989)中所述条件,也可按照实施例中实验条件操作,试剂使用可按照制造厂商所附说明使用。
实施例1高低转移能力肝癌细胞系中CALB2蛋白表达的差异
生长良好的10cm培养皿的细胞(LO-2,SMMC7721,Bel7402,MHCC97L,MHCC97H和HCCLM3)培养至融合率达80%以上时,6ml 1×PBS(美国Gibco公司)清洗2次,500μL 0.25%胰酶(美国Gibco公司)消化2min,500g离心3min,得到细胞沉淀,加入500μL RIPA裂解液(购买于南京凯基生物科技发展有限公司),冰上裂解后,14000rpm转速下离心15min,收集上清。
总蛋白的浓度采用BCA Protein Assay Kit试剂盒(碧云天,上海)进行测定。测定方法按试剂盒说明进行操作即可。每个蛋白样品上样量80μg,CALB2抗体(Anti-CALB2antibody produced in rabbit,货号HPA007306)购自美国Sigma公司,采用蛋白质印迹法western blot(参考《分子克隆》)检测蛋白质表达水平。CALB2的抗体用溶解了质量浓度5%的脱脂奶粉的TBST缓冲液1:500稀释(V/V)。20×TBST缓冲液用180gNaCl和122gTris(美国Sigma公司)溶解于800ml的水,12mol/L的浓HCL调节pH到7.6,最后定容至1000ml。Biorad电泳仪,Biorad转膜仪(购买于美国Biorad公司)。loading buffer和PVDF膜(购买于南京凯基生物科技发展有限公司)
结果如图1显示CALB2在高转移能力肝癌细胞系HCCLM3和MHCC97H中的表达高于低转移肝癌细胞系MHCC97L,SMMC7721,Bel7402与正常肝细胞系LO-2中的表达,说明CALB2对肝癌转移有促进作用。
实施例2CCK-8实验验证CALB2促进肝癌细胞增殖
首先委托上海亚载生物科技有限公司合成抑制CALB2蛋白表达的质粒CALB2-sh1,其基因序列如下。以慢病毒包装质粒方式转染HCCLM3细胞后加入嘌呤霉素进行筛选来构建稳定转染细胞系,采用的慢病毒包装质粒为PSPAX,PVSVG(参考《分子克隆》),得到的稳定细胞株进行扩大培养。
CALB2-sh1
sh1 full hairpin
TGCTGTTGACAGTGAGCGCTAACGC
CALB2-sh1
TGATCTTCTTCAGGTTAACCCAACAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCGCTAACGC
sh1 full hairpin
GATCTTCACATTTTATAGTGAAGCCACAGATGTATAAAATGTGAAGATCGCGTTAATGCCTACTGCCTCG
CALB2-sh1
GATCTTCACATTTTATAGTGAAGCCACAGATGTATAAAATGTGAAGATCGCGTTAATGCCTACTGCCTCG
sh1 full hairpin
GA
CALB2-sh1
GAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGAT
sh1 full hairpin
CALB2-sh1
ACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCCCAGCTCTGGAGT
得到高转移能力肝癌细胞HCCLM3下调CALB2蛋白表达的HCCLM3-shCALB2稳定转染细胞系及其对照细胞HCCLM3-shcontrol。采用Western-blot实验(参考实施例1中方法)验证稳定转染细胞系的表达效果,结果见图2,相比于对照组,敲低组出现了CALB2的表达下调,说明转染成功。
取生长良好且处于对数期的HCCLM3-shcontrol细胞和HCCLM3-shCALB2细胞,制成均匀的单细胞悬液,稀释后进行细胞计数,制备每100μL含2000个细胞的细胞悬液。在96孔板中接种细胞悬液(100μL/孔)。将培养板放在37℃,5%CO2培养箱中培养。每孔加入10μL/CCK-8试剂(购自上海东仁化学科技有限公司),斜着侧壁加,防止产生气泡,影响OD值读数。将培养板在培养箱内孵育1-4h。酶标仪测定在450nm处的吸光度,进行统计分析。
结果见图3,在高转移能力的HCCLM3细胞中下调CALB2的表达后,与对照组相比,HCCLM3细胞的增殖能力下调,说明CALB2对HCCLM3细胞的增殖具有促进作用。差异具有统计学意义(*p<0.05)。
实施例3划痕实验验证CALB2促进肝癌细胞迁移
首先委托上海亚载生物科技有限公司合成抑制CALB2蛋白表达的质粒CALB2-sh2,其基因序列如下。以慢病毒包装质粒方式转染HCCLM3细胞后加入嘌呤霉素进行筛选来构建稳定转染细胞系,采用的慢病毒包装质粒为PSPAX,PVSVG(参考《分子克隆》),得到的稳定细胞株进行扩大培养。
CALB2-sh2
sh2 full hairpin
TGCTGTTGACAGTGAGCGATACGAGAAAAA
CALB2-sh2
TCTTCTTCGGTTACCCAACAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCGATACGAGAAAAAsh2 full hairpin
CAAAAAGGAATAGTGAAGCCACAGATGTATTCCTTTTTGTTTTTCTCGTACTGCCTACTGCCTCGGA
CALB2-sh2
CAAAAAGGAATAGTGAAGCCACAGATGTATTCCTTTTTGTTTTTCTCGTACTGCCTACTGCCTCGGAATTsh2 full hairpin
CALB2-sh2 CAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTsh2 full hairpin
CALB2-sh2 TTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCGGAATGCGTTTCACTCGG
得到高转移能力肝癌细胞HCCLM3下调CALB2蛋白表达的HCCLM3-shCALB2稳定转染细胞系及其对照细胞HCCLM3-shcontrol。采用Western-blot实验(参考实施例1中方法)验证稳定转染细胞系的表达效果,结果显示相比于对照组,敲低组出现了CALB2的表达下调,说明转染成功。
取生长良好且处于对数期的HCCLM3-shcontrol细胞和HCCLM3-shCALB2细胞提前铺至六孔板,置于37℃,5%CO2培养箱中培养,待细胞汇合率达90%时进行划痕,取200μL枪头,统一划出十字,动作要慢,用力均匀,划完后,PBS清洗2次,换液为无血清的DMEM培养基(美国Gibco公司),排除细胞增殖对迁移的影响,倒置显微镜下观察并拍照记录0h伤痕状态。划痕后24h,PBS清洗一次,换液为无血清的DMEM培养基(美国Gibco公司),拍照记录相同位置伤痕愈合状态。统计分析两组细胞划痕的愈合率。
结果见图4,在高转移能力的HCCLM3细胞中敲低CALB2的表达后,与对照组相比,HCCLM3细胞的迁移能力下调,提示CALB2对HCCLM3细胞的迁移具有促进作用。差异具有统计学意义(*p<0.05)。
实施例4Transwell实验验证CALB2促进肝癌细胞迁移和侵袭
(1)Transwell迁移实验
首先委托上海亚载生物科技有限公司合成抑制CALB2蛋白表达的质粒CALB2-sh3,其基因序列如下。以慢病毒包装质粒方式转染HCCLM3细胞后加入嘌呤霉素进行筛选来构建稳定转染细胞系,采用的慢病毒包装质粒为PSPAX,PVSVG(参考《分子克隆》),得到的稳定细胞株进行扩大培养。
CALB2-sh3
sh3 full hairpin
TGCTGTTGACAGTGAGCGCTTGAAGGATCTGTAC
CALB2-sh3
CTCAGGTTACCCAACAGAAGGCTCGAGAAGGTATAGTGCTGTTGACAGTGAGCGCTTGAAGGATCTGTAC
sh3 full hairpin GAGAAATAGTGAAGCCACAGATGTATTTCTCGTACAGATCCTTCAAATGCCTACTGCCTCGGA
CALB2-sh3
GAGAAATAGTGAAGCCACAGATGTATTTCTCGTACAGATCCTTCAAATGCCTACTGCCTCGGAATTCAAG
sh3 full hairpin
CALB2-sh3 GGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTA
sh3 full hairpin
CALB2-sh3
CAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCATGACCCTGTTCGACTCTAAGT
得到高转移能力肝癌细胞HCCLM3下调CALB2蛋白表达的HCCLM3-shCALB2稳定转染细胞系及其对照细胞HCCLM3-shcontrol。采用Western-blot实验(参考实施例1中方法)验证稳定转染细胞系的表达效果,结果显示相比于对照组,敲低组出现了CALB2的表达下调,说明转染成功。
取生长良好且处于对数期的HCCLM3-shcontrol细胞和HCCLM3-shCALB2细胞,饥饿24h。PBS(美国Gibco公司)洗2次后,胰酶(美国Gibco公司)消化2min,500g离心3min弃去培养基,并用无血清的DMEM培养基(美国Gibco公司)制成单细胞悬液,吹打混匀,计数,并调整细胞浓度到1.0×106cell/ml。将250μL的细胞悬液加入到Transwell上室中。在Transwell下室中加入500μL含体积浓度10%胎牛血清的DMEM培养基。将上室放入下室中,上室和下室的培养基之间避免有气泡,放入培养箱中37℃,5%CO2继续培养48h。取出小室,弃去上室内的液体,放入含PBS的下室中清洗2遍。固定和染色:下室加入200μL质量浓度0.1%的结晶紫(含体积浓度10%甲醇)染色20分钟。用棉棒轻轻刮去上室内侧未迁移的细胞,并用PBS清洗3次。待干后,在倒置显微镜下观察迁移的细胞数,并拍照记录。每组随机取5个视野进行细胞计数,并进行统计学分析。
(2)Transwell侵袭实验
将Transwell Matrigel胶置于室温下30min,加入300μL提前预热的无血清DMEM培养基到上室,放入培养箱孵育1h后备用。水化后去除培养基留大约50μL。将250μL的上述细胞悬液加入到Transwell上室中。在Transwell下室中加入500μL含体积浓度10%胎牛血清的DMEM培养基。将上室放入下室中,上室和下室的培养基之间避免有气泡,放入培养箱中37℃,5%CO2培养48h。取出小室,弃去上室内的液体,放入含PBS的下室中清洗2遍。固定和染色:下室加入200μL质量浓度0.1%的结晶紫(含体积浓度10%甲醇)染色20分钟。用棉棒轻轻刮去上室内侧未迁移的细胞,并用PBS清洗3次。待干后,在倒置显微镜下观察迁移的细胞数,并拍照记录。每组随机取5个视野进行细胞计数,并进行统计学分析。
结果见图5,在高转移能力的HCCLM3细胞中下调CALB2的表达后,与对照组相比,HCCLM3细胞的迁移和侵袭能力下调,说明CALB2对HCCLM3细胞的迁移和侵袭具有促进作用。差异具有统计学意义(*p<0.05)。
实施例5动物实验验证CALB2促进肝癌细胞转移
首先委托上海亚载生物科技有限公司合成抑制CALB2蛋白表达的质粒CALB2-sh1,其基因序列如下。以慢病毒包装质粒方式转染HCCLM3细胞后加入嘌呤霉素进行筛选来构建稳定转染细胞系,采用的慢病毒包装质粒为PSPAX,PVSVG(参考《分子克隆》),得到的稳定细胞株进行扩大培养。
CALB2-sh1
sh1 full hairpin
TGCTGTTGACAGTGAGCGCTAACGC
CALB2-sh1
TGATCTTCTTCAGGTTAACCCAACAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCGCTAACGC
sh1 full hairpin
GATCTTCACATTTTATAGTGAAGCCACAGATGTATAAAATGTGAAGATCGCGTTAATGCCTACTGCCTCG
CALB2-sh1
GATCTTCACATTTTATAGTGAAGCCACAGATGTATAAAATGTGAAGATCGCGTTAATGCCTACTGCCTCG
sh1 full hairpin
GA
CALB2-sh1
GAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGAT
sh1 full hairpin
CALB2-sh1
ACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCCCAGCTCTGGAGT
得到高转移能力肝癌细胞HCCLM3下调CALB2蛋白表达的HCCLM3-shCALB2稳定转染细胞系及其对照细胞HCCLM3-shcontrol。采用Western-blot实验(参照实施例1中方法)验证稳定转染细胞系的表达效果,结果显示相比于对照组,敲低组出现了CALB2的表达下调,说明转染成功。
4-6周龄BALB/C nude雄性裸鼠20只,购买于北京维通利华公司,饲养于SPF级实验动物中心。取生长良好且处于对数期的HCCLM3-shcontrol细胞和HCCLM3-shCALB2细胞,PBS(美国Gibco公司)洗2次后,胰酶(美国Gibco公司)消化2min,500g离心3min弃去培养基,离心后重悬于PBS。细胞计数,稀释至1×106cells/100μl PBS。无菌操作台混匀细胞,裸鼠随机分为2组,每组10只,用酒精棉消毒裸鼠尾巴,使用1ml无菌注射器穿刺尾静脉,回针见回血后,注入100μl细胞悬液。拔出针头后,酒精棉按压止血。注射完成后的裸鼠继续在笼中饲养,定期观察裸鼠状态。种瘤45天后,将裸鼠脱颈椎处死,完整取出裸鼠的肺脏,并依次排开观察并拍照。然后采用福尔马林溶液浸泡所取下来的组织。将裸鼠肺脏经石蜡包埋后,制成厚度4μm的切片,HE染色后镜下观察裸鼠肺脏内有无转移灶并计数。
肉眼观察,HCCLM3-shcontrol组肺表面有癌结节的裸鼠数目为7只,HCCLM3-shCALB2组肺表面有癌结节的裸鼠数目为2只。镜下观察HE染色的切片,HCCLM3-shcontrol组有转移灶的裸鼠数目为8只,HCCLM3-shCALB2组有转移灶的裸鼠数目为3只。与对照组相比,HCCLM3-shCALB2组裸鼠的肺部形成的癌结节显著性减少,转移灶也显著性减少。说明CALB2对肝癌细胞转移具有促进作用。
以上所述仅为本发明的优选实施例,并不用于限制本发明的实施,对于本领域的研究人员来说,本发明可以有各种更改。凡在本发明的精神和原则之内,所作的任何修改、改进、组合等,均应包含在本发明的保护范围之内。
序列信息
CALB2-sh1
--sh1 full hairpin
TGCTGTTGACAGTGAGCGCTAACGCGATCTTCACATTTTATAGTGAAGCCACAGATGTATAAAATGTGAAGATCGCGT
TAATGCCTACTGCCTCGGA
--CALB2-sh1
TGATCTTCTTCAGGTTAACCCAACAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCGCTAACGCGATCTTCACATTTTATAGTGAAGCCACAGATGTATAAAATGTGAAGATCGCGTTAATGCCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCCCAGCTCTGGAGT
CALB2-sh2
--sh2 full hairpin
TGCTGTTGACAGTGAGCGATACGAGAAAAACAAAAAGGAATAGTGAAGCCACAGATGTATTCCTTTTTGTTTTTCTCGTACTGCCTACTGCCTCGGA
--CALB2-sh2
TCTTCTTCGGTTACCCAACAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCGATACGAGAAAAACAAAAAGGAATAGTGAAGCCACAGATGTATTCCTTTTTGTTTTTCTCGTACTGCCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCGGAATGCGTTTCACTCGG
CALB2-sh3
--sh3 full hairpin
TGCTGTTGACAGTGAGCGCTTGAAGGATCTGTACGAGAAATAGTGAAGCCACAGATGTATTTCTCGTACAGATCCTTCAAATGCCTACTGCCTCGGA
--CALB2-sh3
CTCAGGTTACCCAACAGAAGGCTCGAGAAGGTATAGTGCTGTTGACAGTGAGCGCTTGAAGGATCTGTACGAGAAATAGTGAAGCCACAGATGTATTTCTCGTACAGATCCTTCAAATGCCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCATGACCCTGTTCGACTCTAAGT。
Claims (5)
1.一种可抑制 CALB2 蛋白表达的核酸在制备治疗肝癌转移药物中的应用,所述核酸的序列为:
CALB2-sh1
CGATCTTCACATTTTATAGTGAAGCCACAGATGTATAAAATGTGAAGATCGCGTTAATGCCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCCCAGCTCTGGAGT
或CALB2-sh2
ACAAAAAGGAATAGTGAAGCCACAGATGTATTCCTTTTTGTTTTTCTCGTACTGCCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCGGAATGCGTTTCACTCGG
或CALB2-sh3
ACGAGAAATAGTGAAGCCACAGATGTATTTCTCGTACAGATCCTTCAAATGCCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCATGACCCTGTTCGACTCTAAGT。
2.根据权利要求 1 所述的应用,其特征在于,所述治疗肝癌转移为抑制肝癌细胞的侵袭和/或转移。
3.一种治疗肝癌转移的药物,其特征在于,所述药物以可抑制 CALB2 蛋白表达的核酸为活性成分;抑制 CALB2蛋白表达核酸的序列为:
CALB2-sh1
CGATCTTCACATTTTATAGTGAAGCCACAGATGTATAAAATGTGAAGATCGCGTTAATGCCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCCCAGCTCTGGAGT
或CALB2-sh2
ACAAAAAGGAATAGTGAAGCCACAGATGTATTCCTTTTTGTTTTTCTCGTACTGCCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCGGAATGCGTTTCACTCGG
或CALB2-sh3
ACGAGAAATAGTGAAGCCACAGATGTATTTCTCGTACAGATCCTTCAAATGCCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCATGACCCTGTTCGACTCTAAGT。
4.一种治疗肝癌转移的药物,其特征在于,所述药物包含有效剂量的可抑制 CALB2 蛋白表达的核酸的病毒载体;所述核酸序列为:
CALB2-sh1
CGATCTTCACATTTTATAGTGAAGCCACAGATGTATAAAATGTGAAGATCGCGTTAATGCCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCCCAGCTCTGGAGT
或CALB2-sh2
ACAAAAAGGAATAGTGAAGCCACAGATGTATTCCTTTTTGTTTTTCTCGTACTGCCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCGGAATGCGTTTCACTCGG
或CALB2-sh3
ACGAGAAATAGTGAAGCCACAGATGTATTTCTCGTACAGATCCTTCAAATGCCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTTTTGGCCGGCCATGACCCTGTTCGACTCTAAGT。
5.根据权利要求 4 所述的治疗肝癌转移的药物,其特征在于,所述病毒载体为慢病毒载体,采用的慢病毒包括装质粒为 PSPAX或PVSVG。
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