CN101234191B - 人重组肝刺激因子增强肝癌细胞抗凋亡的作用及其应用 - Google Patents
人重组肝刺激因子增强肝癌细胞抗凋亡的作用及其应用 Download PDFInfo
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Abstract
本发明涉及人重组肝刺激因子在制备抗肿瘤细胞凋亡的药剂中的用途。所述的人重组肝刺激因子纯化后为分子量是31kD的同源二聚体蛋白,等电点为4.5,在pH4-10和温度≤80℃的环境中结构稳定。本发明还涉及人重组肝刺激因子在抗肿瘤细胞凋亡中的用途。
Description
技术领域
本发明涉及肝刺激因子(HSS)增强肝癌细胞抗凋亡的作用及其在医学领域的应用。
发明背景
肝刺激因子(hepatic stimulator substance,HSS)最初在1975年由LaBrecque等人自肝脏部分切除的大鼠再生肝脏中提取,并发现其能特异性的刺激肝细胞增殖(1.Teir H,Ravanti K.Mitotic activity and growthfactors in the liver of the whole rat.Exp Cell Res,5:500-507,1953)。尔后又从初断乳大鼠肝脏及多种动物肝脏包括人类肝脏中发现了HSS的存在。研究证实,HSS是一种热稳定、带强负电荷的多肽类物质,分子量约为15kD,具有抗酸、耐碱等特点(Blomqvist K.Growth stimulationin the liver and tumor development following intraperitonealinjections of liver homogenates in the rat.Acta Pathol MicrobidScand(suppl),121,1957;LaBrecqueD.R.,Pesch L.A.Preparation andpartial characterization of hepatic regenerative stimulator substance(hss)from rat liver.J Physical,248:273-284,1975)。HSS的功能具有器官特异性,但无种属特异性(LaBrecqueD.R.,Pesch L.A.Preparation and partialcharacterization of hepatic regenerative stimulator substance(hss)fromrat liver.J Physical,248:273-284,1975;唐翰满,贺福初:胞源性肝细胞生长因子研究进展。生理科学进展,24:348-350,1993)。HSS基因在很多组织例如肝、肾等都有表达,但是它只能特异性促进肝细胞或肝源性肿瘤细胞系DNA合成,使其由G0期进入S期,而对体内或体外的非肝源性正常组织细胞及恶性细胞均无刺激增殖作用(唐翰满,贺福初:胞源性肝细胞生长因子研究进展。生理科学进展,24:348-350,1993;贺福初,涂强,邢桂春等:人胎肝细胞生长刺激物poly(A)+mRNA在非洲爪蟾卵母细胞内的翻译。中国应用生理学杂志,6:298-302,1990)。表明HSS对肝脏细胞的促增殖作用是特异性的。
近年研究表明,HSS具有稳定细胞膜,减轻CCl4、半乳糖胺及H2O2对肝细胞的毒性作用,且呈剂量依赖性,即剂量与效应呈正相关(LaBrecque DR,Bachur NR:Hepatic stimulator substance(HSS)-physical-chemical characteristics and specificity.Am J Physical,242(Gadtrointest Liver Physical 5):G281-G288,1982;LaBrecque DR,Steele G,et al:Purification and physical-chemical characterization ofhepatic substance.Hepatology,7:100-106,1987;Fleig WE,Hoss G:Partialpurification of rat hepatic stimulator substance and characterization of itsaction on hepatoma cells and normalhepatocytes.Hepatology,9:240-248,1989;Mei MH,An W,Zhang BH,etal:Hepatic stimulator substance protects against acute liver failure byCCl4 poisoning in mice.Hepatology,17(4):638-44,1993)。此外,HSS还有保护肝细胞膜、抗纤维化、抑制Na+-K+-ATPase抑制因子等作用(Mei MH,An W,Zhang BH,et al:Hepatic stimulator substance protectsagainst acute liver failure by CCl4 poisoning in mice.Hepatology,17(4):638-44,1993;Francavilla A,Barone M,et al:Further steps ofhepatic stimulatory substance purification.Dig Dis Sci,36:674-680,1991;Francavilla A,Ove P,Polimeno L,et al:Extraction and partial purificationof a hepatic stimulatory substance in rats,mice,and dogs.Cancer Res,47:5600-5605,1987)。有人认为,肝再生过程中NK细胞对肝细胞增殖具有明显的抑制作用,而HSS可抑制NK细胞功能从而使肝细胞从束缚中得到解脱(LaBrecque DR:In vitro stimulation of cellgrowth by hepatic stimulator substance(HSS).Am JPhysiol,242(5):G289-G295,1982;An W,Strobel D,Hahn EG,et al:Increased expression of epidermal growth factor receptor mRNA by hepaticstimulatory substance(abstract).Jap J Pathophysiology 4(2):19,1995)。而且,当HSS与EGF合用,两者产生协同作用(synergistic effect),可使肝细胞的DNA合成大大增强,因此也有人将HSS称为肝再生增强因子(augmentor of liver regeneration,ALR)(An W,Strobel D,Hahn EG,et al:Increased expression of epidermal growth factor receptor mRNA byhepatic stimulatory substance(abstract).Jap JPathophysiology,4(2):19,1995;LaBrecque DR:Hepatic regenerativestimulator substance(HSS)-liver specific growth promoter(abstract).Clin.Res,28)。
相关报道称H2O2等超氧化物损伤细胞可诱导凋亡(贺福初,涂强,邢桂春等:人胎肝细胞生长刺激物poly(A)+mRNA在非洲爪蟾卵母细胞内的翻译。中国应用生理学杂志,6:298-302,1990)。
最近,为了探讨HSS在H2O2引起的细胞损伤中抗细胞凋亡的作用,本发明人克隆了人HSS基因,将其构建入真核表达载体,然后转染肝癌细胞。结果发现HSS定位于细胞的线粒体上,可以减弱细胞的凋亡程度。因此,为治疗肝癌肿瘤提供了新的靶点和途径。
发明内容
本发明的目的是提供人重组肝刺激因子(human hepatic stimulatorsubstance,hHSS)在抗肿瘤细胞凋亡的药剂中的用途。所述的人重组肝刺激因子可以增强肿瘤细胞的抗凋亡作用,纯化后的人重组肝刺激因子为分子量是31kD的同源二聚体蛋白,等电点为4.5,人重组肝刺激因子具有强的耐酸耐碱和耐高温能力,在pH4-10和温度≤80℃的环境中结构稳定。
另一方面,本发明还涉及人重组肝刺激因子在抗肿瘤细胞凋亡中的一种新用途。
换言之,本发明涉及人重组肝刺激因子在制备抗肿瘤细胞凋亡的药剂中的用途。所述的人重组肝刺激因子纯化后为分子量是31kD的同源二聚体蛋白,等电点为4.5,人重组肝刺激因子在pH4-10和温度≤80℃的环境中结构稳定。本发明还涉及人重组肝刺激因子在抗肿瘤细胞凋亡中的用途。
附图说明
图1.纯化的hHSS蛋白性质。其中分别为:
(A)Superose 12HR 10/30SEC纯化hHSS时洗脱峰,洗脱液为50mMPB,pH 7.2,150mM NaCl,流速0.5ml/min。
(B)MALDI-TOF spectrum of hHSS,蛋白分子量为31KD。
(C)SDS-PAGE凝胶电泳检测纯化蛋白。其中Lane 1,蛋白分子量标准;lane 2,纯化的hHSS,单体分子量约15kD;lane 3,菌液纯化前.。
(D)Western印迹检测纯化的hHSS,lane 2,与hHSS抗血清呈现阳性反应。
(E)hHSS等电点凝胶电泳。
图2.不同pH溶液(A1)和不同温度中(B1)hHSS的SFS光谱,其中
(A2)不同pH条件下峰值344.0nm处的相对荧光强度。
(B2)不同温度条件下峰值342.4nm处的相对荧光强度。
图3.不同pH溶液(A1,A2)和不同温度中(B)hHSS的Far-UV CD谱。其中C:不同pH条件下的[θ]222,[θ]代表103×deg×cm2×dmol-1。D:不同温度的[θ]222。
图4A:实时荧光定量PCR结果。
图4B:激光共聚焦发现HSS定位于线粒体内的情况。
图4C:Western blot检测结果。
图5A-B:荧光显微镜观察结果。图5C:Philips EM208s电镜观察结果。
图6:为流式细胞检测结果。
图7:Western blot检测结果。
图8:ATP检测结果。
以下将结合附图和实施例对本发明进行详细说明,其中实施例仅仅起说明而非限定的作用。本领域技术人员完全可以在本发明披露的具体实施方案中的技术上做出改进,但是不超过本发明权利要求的范围或者本发明范围之内所做出的改进都将落入本发明的保护范围。
具体实施方式
实施例1:表达人重组肝刺激因子载体的构建
利用鼠HSS基因探针,经RT-PCR扩增人HSS,经DNA序列分析,得出含有开放读码框(open reading frame,ORF)命名为hHSS的基因,将其插入pcDNA3.1质粒(Invitrogen)相应位点,构建成pcDNA3.1-hHSS。
实施例2:细胞培养与转染
将BEL-7402肝癌细胞系在含有15%胎牛血清的DEME培养基中,在37℃,5%CO2条件下培养。
转染:用hHSS基因的转染与筛选按1×104/ml的细胞密度接种BEL-7402肝癌细胞于三个25cm2培养瓶内,以DMEM培养基培养细胞。待细胞密度达到3×105/ml(60-80%细胞汇合)后,去除培养液,以无Ca2+、Mg2+的Dulbecco′s PBS(pH 7.4)洗涤3次,消除残留血清影响。加入无血清DMEM培养基(含胰岛素10μg/ml)。次日进行hHSS基因的转染。准备4只1.5ml无菌Eppendorf管,两两对应标记。取pcDNA3.1-hHSS和pcDNA3.1各5μg,放入其中两只Eppendorf管中,用HBS(HEPES-buffer saline)配成0.1μg/μl。另取DOTAP(N-[2,3-Dioleoyloxyl]-N,N,N-trimethylammonium methylulfate)60μl以HBS稀释至200μl,各取100μl移至两只Eppendorf管中。分别加入pcDNA3.1-hHSS和pcDNA3.1稀释液,以手指轻轻弹匀。室温静置15min,分别将DOTAP/pcDNA3.1-hHSS(或pcDNA3.1)转染液加入两瓶BEL-7402肝癌细胞的培养液中(其中一瓶作为阳性对照)。缓缓晃动培养液,使转染液均匀分散于无血清DMEM培养基中,转染BEL-7402细胞8h,去除培养液,以无Ca2+、Mg2+的Dulbecco′s PBS(pH 7.4)洗涤3次,消除残余转染液对细胞的毒害作用,换以新鲜DMEM培养基继续培养。第二天加G418(终浓度400μg/ml)于DMEM培养液中,筛选转染细胞。培养细胞72h后,显微镜下可见少量死亡细胞。更换新鲜DMEM培养液(G418,400μg/ml)继续筛选。一周后,显微镜下可观察到阳性克隆细胞,更换DMEM培养液(含G418,400μg/ml),直至肉眼可见单个克隆细胞团。去除培养液,以PBS(pH 7.4)洗涤3次,以吸管滴加细胞消化液于克隆细胞团处,显微镜下观察,细胞变圆后,吸出克隆细胞,转入新的培养瓶中,加入DMEM培养液(含G418,200μg/ml)培养单克隆细胞。以实时荧光定量PCR逆转录聚合酶链反应(RT-PCR)鉴定hHSS基因在转染细胞中的表达。(见以下实施例4)。
下述内容的统计学处理采用以X±SD表示实验数值,应用t检验分析,P<0.05为差异有显著性。
实施例3:重组hHSS的理化性质和结构稳定性
在适合重组蛋白表达的条件下,表达如实施例2获得的分别表达人重组肝刺激因子和空载体的单克隆细胞株。回收纯化该人重组肝刺激因子蛋白,并采用同步荧光光谱技术和圆二色光谱技术检测其理化性质和结构稳定性。
参见图1,检测结果表明纯化后的重组hHSS为分子量为31kD的同源二聚体蛋白,等电点为4.5。
参见图2、3,通过同步荧光光谱技术和圆二色光谱技术对重组hHSS的研究发现:hHSS二级结构非常稳定,具有很强的耐酸耐碱和耐高温能力,在pH4-10和温度≤80℃的环境中结构不会发生改变。实施例4.实时荧光定量逆转录聚合酶链反应(RT-PCR)检测HSS/mRNA表达,鉴定hHSS基因在转染细胞中表达。
收集培养的如实施例2获得的分别表达人重组肝刺激因子和空载体的单克隆细胞株细胞以及野生型肝癌细胞,按TRIzol试剂盒(Invitrogen)提供的说明书提取总RNA。以总RNA为模板,OligodT为引物逆转录合成cDNA,SYBR Green荧光染料法进行实时荧光PCR。用GADPH做内参照物。反应条件:50℃2min,95℃预变性10min,95℃变性15s,60℃退火1min,共40个循环。在延伸的过程中搜集荧光信号。每次扩增的同时设置无cDNA的阴性对照。扩增结果采用ABIPrism 7300实时荧光定量PCR仪(Applied Biosystems)自带的实时荧光定量PCR分析程序分子Ct值,然后计算出相应的ΔCt值(ΔCt值=阴性对照Ct值-待测样本Ct值),ΔCt值越大说明基因拷贝数越多。
参见图4A,实时荧光定量PCR结果表明:在转染HSS的肝癌细胞中检测到HSS在mRNA水平的表达较野生型肝癌细胞和转染过空载体细胞高。
同样,参见图4,通过Western blot发现转染HSS的肝癌细胞线粒体蛋白中HSS表达量较另外两种细胞高。表明HSS定位于线粒体。
实施例5:激光共聚焦检测HSS定位于线粒体内
如实施例2获得的分别表达人重组肝刺激因子和空载体的单克隆细胞株细胞在培养后,将其中的培养液弃去,换成37℃预热的复染液(按所需的量向无血清DMEM中加入MitoTracker Red580和Hoechst33342,使其终浓度分别为250nM和0.2g/L),在合适的生长条件下孵育45分钟;将复染液弃去,用PBS漂洗三遍;向细胞中加入新鲜37℃预热的完全培养基,用激光共聚焦观察。
参见图4B,激光共聚焦发现HSS定位于线粒体内。
实施例6:形态学检测细胞凋亡
HE染色和荧光染料Hoechst33342染色观察细胞凋亡
将野生型肝癌细胞及如实施例2获得的分别表达人重组肝刺激因子和空载体的单克隆细胞株分种于25cm2培养皿(培养皿底部铺以0.01%多聚赖氨酸Poly-L-Lysine处理过夜的盖玻片),1×106个细胞/培养皿,无血清DMEM培养12h同步化后分别给予0.6mmol/L H2O2损伤细胞6小时。以甲醇固定10分钟后,用苏木精-伊红染色,蒸馏水冲洗,甘油明胶封片,镜下观察。经HE染色后,细胞核染成深兰色,胞浆染成深浅不等的粉红色。未加损伤组细胞形态、结构完整,核大着色较浅,染色质分布均匀。给予0.6mMH2O2(以PBS稀释)损伤后,野生型细胞表现为大量细胞核明显固缩,染色加深。而转染HSS细胞的胞核多数明显完整,形态正常。提示转染细胞组中凋亡细胞数较对照组明显减少。
另将同样培养的野生型肝癌细胞和H2O2损伤的野生型肝癌细胞和如实施例2获得的表达人重组肝刺激因子的单克隆细胞株细胞以3%多聚甲醛固定15分钟,加入Hoechst 33342荧光染料(终浓度为10μg/ml),室温孵育15分钟。荧光显微镜下观察。以Hoechst33342特异性DNA染料染色发现,野生型细胞核呈弥散均匀荧光。给予H2O2损伤,野生型细胞核内可见大量浓染致密颗粒状荧光,提示细胞由于受到损伤而发生DNA碎裂。而如实施例2获得的表达人重组肝刺激因子的单克隆细胞株细胞核内荧光较均匀,说明转染HSS后,细胞核损伤程度明显减弱(参见图5A-B)。
透射电镜细胞凋亡形态检测
将同样培养的野生型肝癌细胞和如实施例2获得的分别表达人重组肝刺激因子和空载体的单克隆细胞株细胞给予H2O2损伤处理,收集细胞,以PBS洗涤后,多聚甲醛-戊二醛固定液固定2小时,1%锇酸固定2小时,4℃,梯度酒精脱水,环氧丙烷置换,浸透、包埋、修块、切片。Philips EM208s电镜观察。
参见图5C,结果提示野生型组细胞形态结构完整,核大而圆,核染色质均匀,核仁清晰完整,胞质中细胞器结构完整,线粒体丰富,内质网及高尔基体无扩张。通过观察发现给予H2O2损伤后野生型组细胞体积变小,染色质浓缩凝聚成块,边聚于核膜周围,此为明显的调亡现象,提示细胞发生了调亡;转染空载体组细胞损伤后染色质也呈新月状或小帽状聚集于核膜周围;而转染HSS细胞组,只发现染色质有少量的凝集未出现细胞质新月状凝集于核膜周围,表明转染HSS后,由H2O2损伤导致的细胞调亡程度明显减弱。
实施例7:细胞凋亡率检测
给予野生型肝癌细胞和如实施例2获得的分别表达人重组肝刺激因子的单克隆细胞株细胞H2O2损伤处理,收集细胞,以PBS洗涤后,采用异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的Ca2+依赖性磷脂结合蛋白(AnnexinV-FITC)和碘化丙啶(PI)染色(LaBrecqueDR,Bachur NR:Hepatic stimulator substance(HSS)-physical-chemicalcharacteristics and specificity.Am J Physical,242(Gadtrointest LiverPhysical 5):G281-G288,1982,于FCAS-440流式细胞仪上进行细胞凋亡检测。
参见图6,流式细胞检测结果发现,野生型细胞组损伤后的凋亡细胞占总体细胞数的55.64%;而转染HSS细胞组的凋亡细胞数仅为31.39%(图6A)。提示转染HSS的细胞组凋亡细胞出现率明显降低,同样损伤条件下,细胞凋亡率降低了43.58%。说明转染HSS的细胞可以减轻H2O2损伤而造成的细胞凋亡。
实施例8:线粒体跨膜电位检测(MPT)
收集野生型肝癌细胞和如实施例2获得的分别表达人重组肝刺激因子的单克隆细胞株细胞的细胞悬液,用PBS将细胞稀释成1×106/ml,使用MitoProbeTM DiIC1(5)Assay Kit(Molecular Probes)试剂盒检测。将DiIC1(5)加入到培养基中在37℃CO2孵箱中孵育30分钟,于FCAS-440流式细胞仪检测线粒体跨膜电位。
参见图6(A-D),流式细胞检测结果发现,H2O2损伤后三种细胞的跨膜电位都明显降低,转染HSS细胞组和转染空载体组细胞的跨膜电位降低较野生型细胞组明显,提示HSS对于H2O2损伤的细胞有保护作用。(图6B)
实施例9:ATP的检测
野生型肝癌细胞和如实施例2获得的分别表达人重组肝刺激因子的单克隆细胞株细胞受H2O2损伤处理,加入细胞裂解液提取蛋白并定量;采用ATP Assay Kit(Promega),通过荧光素酶检测仪测定ATP含量。
参见图8,ATP检测结果表明,在H2O2损伤后三种细胞的ATP都明显降低,转染HSS细胞组和转染空载体组细胞的跨膜电位降低较野生型细胞组明显,提示HSS对于H2O2损伤的细胞有保护作用。
实施例10:Western blot检测细胞色素c
提取野生型肝癌细胞和如实施例2获得的分别表达人重组肝刺激因子的单克隆细胞株细胞的线粒体蛋白及胞浆蛋白并定量。50μg蛋白于15%SDS-PAGE凝胶分离后,转移至硝酸纤维素膜,过夜封闭(4℃)。次日分别与兔抗人anti-cytochem c单克隆抗体(Santa Cruz)及羊抗小鼠IgG抗体-辣根过氧化物酶(中山公司)各孵育1h。采用增强化学发光法(enhanced chemiluminescene,ECL)检测杂交信号。经GS-700扫描仪(Bio-Rad,美国)进行半定量分析。
参见图7,Western blot结果表明:H2O2损伤后三种细胞中的线粒体蛋白中细胞色素c的表达量均降低,而在胞质蛋白中损伤后的细胞色素c的表达量均升高,表明在细胞受损后细胞色素c从线粒体中释放出进入胞质中,这种改变提示我们HSS对细胞的保护可能与线粒体有关。
Claims (4)
1.人重组肝刺激因子在制备保护细胞线粒体并且抗肿瘤细胞凋亡的试剂中的用途。
2.根据权利要求1中所述的用途,其特征在于人重组肝刺激因子纯化后为分子量是31kD的同源二聚体蛋白,等电点为4.5,人重组肝刺激因子在pH4-10和温度≤80℃的环境中结构稳定。
3.人重组肝刺激因子在保护细胞线粒体并且抗肿瘤细胞凋亡中的用途。
4.根据权利要求3中所述的用途,其特征在于人重组肝刺激因子纯化后为分子量是31kD的同源二聚体蛋白,等电点为4.5,人重组肝刺激因子在pH4-10和温度≤80℃的环境中结构稳定。
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