CN114014911A - 一种抗癌脂肽的制备及其在抗肿瘤治疗中的应用 - Google Patents
一种抗癌脂肽的制备及其在抗肿瘤治疗中的应用 Download PDFInfo
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Abstract
本发明涉及一种抗癌脂肽C8H15O‑Asp‑Ser‑Asp‑Val‑Trp‑Trp‑Gly‑Gly‑Arg‑Arg‑Leu‑Leu‑Arg‑Arg‑Leu‑Arg‑Arg‑Leu的制备及其在抗肿瘤治疗中的应用,属于生物医药领域。所述抗癌脂肽基于膜裂解肽的关键生物学特征(正电荷、α‑螺旋结构和两亲性)制备。本发明提供了所述抗癌脂肽的固相合成方法。本抗癌脂肽具有疏水力矩小、溶血活性低以及血清稳定性好的特点,能有效弥补大多数抗癌肽高溶血毒性的缺陷,解决肽类药物体内稳定性差的问题。体外抗癌实验及体内抗肿瘤实验证明本抗癌脂肽具有良好的抗肿瘤效果,有很好的应用前景。
Description
技术领域
本发明属于生物医药领域,具体涉及一种人工合成的具有优异体内外抗癌活性的抗癌脂肽的制备及其在抗肿瘤治疗中的应用。
背景技术
尽管癌症的治疗取得了长足的发展,但它仍然是造成人类死亡最严重的疾病之一。目前主要通过化疗和放疗治疗癌症,然而在治疗的同时往往伴随着严重副作用以及出现多药耐药性。抗癌肽(Anticancer peptides,ACPs)具有选择性高、生物相容性好、肿瘤渗透性高等优点。大多数ACPs是α-螺旋阳离子两亲性肽,其活性主要是由净电荷、疏水性和螺旋度等决定的。相对于正常细胞,癌细胞膜表面携带大量的磷脂酰丝氨酸、磷脂酰肌醇等阴离子成分,使癌细胞表面呈现净负电荷。大多数抗癌肽中含有多个赖氨酸与精氨酸,使其带有大量正电荷,这些带有正电荷的抗癌肽可以通过静电相互作用被带负电荷癌细胞膜吸引,进一步形成α-螺旋构象,插入脂质双分子层中,形成孔道、破坏了膜的完整性从而导致癌细胞死亡。精氨酸可以通过侧链胍基离子与细胞膜表面带负电组分相互作用产生大量氢键,从而实现从水溶到膜溶的转变,同时寡聚精氨酸的穿膜能力随着连续精氨酸残基的数目和多肽浓度的增加而增加。此外多肽疏水性氨基酸的增加可以提高多肽的抗癌活性,然而高疏水性通常会产生高溶血性,通过在α-螺旋两亲性肽的疏水侧引入亲水残基降低了疏水力矩,从而降低溶血性。
肽类药物由于半衰期短、生物利用度低、稳定性差和易被蛋白酶水解等缺点限制了其临床应用。脂肪酸的修饰可以增加肽的膜通透能力,也可以增加抗癌能力,同时脂肪酸修饰的治疗剂(如蛋白质、肽和siRNA)可以延长体内循环时间并避免蛋白酶水解。因此本发明设计合成了一种新颖的富含精氨酸的阳离子两亲性抗癌脂肽,其中精氨酸(Arg)能提供正电荷与癌细胞膜表面大量的负电组分通过静电作用结合,此外引入色氨酸(Trp)、亮氨酸(Leu)等氨基酸增加肽链的疏水性来提高破膜活性,通过正辛酸修饰增加肽链的稳定性、增强抗癌作用。发明人将本发明的抗癌脂肽全序列氨基酸结构经NCBI蛋白质数据库进行搜索对比时,未发现有任何相同多肽。本发明抗癌脂肽具备良好的体内外抗癌活性,可对癌症的治疗提供新思路。
发明内容
本发明的目的是通过固相合成技术合成一种具有优异抗癌活性的抗癌脂肽并将这种抗癌脂肽应用于抗肿瘤治疗。
为了实现本发明的目的,本发明提供了如下技术方案:
本发明抗癌脂肽的氨基酸序列为:C8H15O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu,其技术方案如下:
(1)抗癌脂肽的设计:首先根据阳离子抗癌肽的正电荷性和两亲性,引入带正电荷的精氨酸(Arg)和疏水性的色氨酸(Trp)、亮氨酸(Leu)等氨基酸,并通过氨基酸间的合理的排列组合使肽链的疏水力矩减小以降低溶血毒性,同时在N末端用正辛酸(C8H16O2)修饰,提高多肽的稳定性,最终设计得到阳离子两亲性的抗癌脂肽;
(2)抗癌脂肽通过固相合成法进行制备,其制备方案概括如下:将带有Fmoc保护基和侧链保护基的氨基酸通过脱除Fmoc保护基再使用催化剂催化各氨基酸依次从C端到N端逐个偶联在王树脂(Wang resin)上,然后在N末端偶联正辛酸,最后用三氟乙酸切割树脂并脱除侧链保护基团最终得到抗癌脂肽。本发明抗癌脂肽结构式如下:
(3)采用MTT法和流式细胞术对抗癌脂肽的体外抗癌活性进行检测:通过MTT法测试抗癌脂肽对HepG2细胞的细胞毒性,通过Annexin V-FITC/PI凋亡检测试剂盒用流式细胞仪检测抗癌脂肽对HepG2细胞凋亡的影响;
(4)通过圆二色谱(CD)检测抗癌脂肽的二级结构:用水模拟亲水环境,用SDS溶液来模拟细胞膜等疏水环境,通过圆二色谱检测抗癌脂肽在水溶液和SDS溶液中二级结构的变化情况;
(5)对抗癌脂肽的破膜活性进行研究:将抗癌脂肽与HepG2细胞共培养3h后显微镜下观察抗癌脂肽对细胞膜形态的影响,通过乳酸脱氢酶(LDH)释放试验定量测定抗癌脂肽对癌细胞膜的裂解能力;
(6)对抗癌脂肽溶血和血清稳定性进行研究:预先用胎牛血清(FBS)和抗癌脂肽共培养,然后用MTT检测经FBS预处理后抗癌活性变化来评估其稳定性;通过抗癌脂肽对小鼠红细胞形态变化的影响评估其溶血活性;
(7)抗癌脂肽的体内抗肿瘤活性研究:选用植瘤小鼠作为实验模型,隔天尾静脉注射抗癌脂肽,并对小鼠体重及肿瘤体积进行测量,将小鼠肿瘤组织解剖并进行组织切片和HE染色来检测抗癌脂肽的体内抗肿瘤活性。
附图说明
图1:MTT法检测抗癌脂肽对HepG2细胞的抗癌活性。(a)将不同浓度的抗癌脂肽与HepG2细胞共培养24h的相对细胞存活率。(b)24μM的抗癌脂肽与HepG2细胞共培养不同时间的相对细胞存活率;
图2:抗癌脂肽在纯水和SDS溶液中的CD光谱;
图3:FITC-AnnexinV/PI双染检测细胞凋亡。通过流式细胞术检测空白对照(a)、4μM(b)、8μM(c)、16μM(d)的脂肽对HepG2细胞凋亡数据图;
图4:抗癌脂肽的破膜活性。空白对照(a)、8μM(b)、16μM(c)脂肽与HepG2细胞共孵育3h后荧光显微镜(400×)观察HepG2细胞形态。(d)不同浓度脂肽与HepG2细胞共培养24h后HepG2细胞的乳酸脱氢酶释放率;
图5:抗癌脂肽的溶血活性和血清稳定性。小鼠尾静脉注射生理盐水(a)和2.25mg/mL抗癌脂肽(b)2h后取血,显微镜下观察(400×)红细胞形态。(c)预先将抗癌脂肽分别与含10%血清和不含血清的PBS在37℃下孵育24h和48h后通过MTT法检测抗癌脂肽对HepG2细胞的抗癌活性变化数据图;
图6:抗癌脂肽的体内抗肿瘤活性。(a)植瘤小鼠隔天尾静脉注射抗癌脂肽后体重随时间变化图。(b)植瘤小鼠相对肿瘤体积随时间变化图。(c)隔天注射生理盐水11天后肿瘤组织切片。(d)隔天注射抗癌脂肽11天后肿瘤组织切片(400×)。
具体实施方式
以下给出本发明的具体实施方式,用来对本发明的构成做进一步的说明,但并不认为本发明仅局限于下述的实施方式。
实施例1:抗癌脂肽的制备方法
本发明抗癌脂肽的合成基于Fmoc保护的固相多肽合成法进行。抗癌脂肽的序列为C8H15O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu,具体合成步骤如下:
(1)Fmoc-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-WangResin的合成。
称取一定量负载量为0.535mmol/g的Fmoc-Leu-Wang Resin置于烧杯中,加入无水DMF溶胀树脂30min。取少量上述树脂进行茚三酮检测,若无色则继续。按照DMF:哌啶=4:1的比例加入哌啶溶液,磁力搅拌30min脱除Fmoc基团,反应完成后进行抽滤,并依次用DMF、DCM、DMF各洗涤树脂3次,重新加入无水DMF溶胀30min。加入两倍摩尔量的Fmoc-Arg(Pbf)-OH、2.6倍摩尔量的DCC第、HO页BT和DIEA置于烧杯中,室温下磁力搅拌反应48h以上,反应完成后依次用DMF、DCM、DMF各洗涤树脂5次,最后用无水乙醇多次洗涤,抽滤完成后将样品转移到透析袋内(MW:8000-14000),每隔30min换一次透析液,透析20次以上,将透析完的样品冷冻干燥得Fmoc-Arg(Pbf)-Leu-Wang Resin,重复上述步骤依次偶联氨基酸直至合成Fmoc-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gl y-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin,其中Pbf为Arg的侧链保护基,Boc为Trp侧链保护基,tBu为Ser侧链保护基,OtBu为As p侧链保护基;
(2)C8H15O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu的合成。
首先将Fmoc-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin加入无水DMF溶胀树脂30min,按照DMF:哌啶=4:1的比例加入哌啶,磁力搅拌30min脱除Fmoc基团,反应完成后进行抽滤,并依次用DMF、DCM、DMF各洗涤树脂3次,冷冻干燥得Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin。将2倍摩尔量的C8H16O2、2.5倍摩尔量的NHS和2.5倍摩尔量的EDC加入无水DMF中磁力搅拌5h活化羧基,然后加入Asp(OtBu)-Ser(tB u)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Le u-Arg(Pbf)-Arg(Pbf)-Leu-WangResin反应48h,反应完成后依次用DMF、DCM、DMF各洗涤树脂5次,最后用无水乙醇多次洗涤,抽滤完成后将样品转移到透析袋内(MW:8000-14000),每隔30min换一次透析液,透析20次以上,将透析完的样品冷冻干燥得C8H15O-As p(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pb f)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin,按照三氟乙酸(TFA):水(H2O):三异丙基硅烷(Tis)=95:2.5:2.5的比例加入切割液,磁力搅拌1.5h以脱除树脂和所有侧链保护基,反应完成后抽滤取滤液,旋蒸浓缩,加入冰乙醚产生沉淀,离心并加入乙醚洗涤沉淀5次,冷冻干燥得到脂肽C8H15O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Ar g-Arg-Leu。
实施例2:抗癌脂肽的体外抗癌活性测定
通过MTT法测试抗癌脂肽的体外抗癌活性。选用处于对数生长期的HepG2细胞,将He pG2细胞接种到96孔板中,然后将该96孔板放入5%CO2、37℃的培养箱中进行培养,24h后待细胞铺满孔底,将旧培养基吸出,将脂肽用DMEM培养基配制成不同的浓度,每孔加入含药培养基100μL,为保证实验反映情第况4真实页可靠,每个浓度设置5个平行实验以确保实验的可靠性。培养24h后,每个孔加入20μL(5mg·mL-1)MTT溶液放置在细胞培养箱中培养4h。将上清液弃掉后再在每个孔中加入150μL DMSO以溶解蓝紫色MTT晶体,将96孔板用锡纸包裹避光放置在振荡器上,170r·min-1下振荡7min,使孔内的晶体充分溶解。溶解完成后,转移至酶联免疫检测仪进行检测,波长设置为485nm,测量每个孔的吸光度,计算细胞存活率。
实施例3:抗癌脂肽的二级结构测定
通过圆二色谱仪检测脂肽在水和SDS溶液中的二级结构。将脂肽配制成终浓度为150μM的水溶液和终浓度为150μM的十二烷基硫酸钠(SDS)溶液(30mM),其中水溶液模拟亲水环境,SDS溶液用来模拟细胞膜等疏水环境,放入石英比色皿(光程长度为0.5mm),用圆二色谱仪进行测试,波长范围设为180-260nm,将测得的数据用下列公式将获得的光谱换算为平均残基椭圆度:
θM=θobs×1000/cln
其中θM是平均残基椭圆度;θobs是在给定波长下通过缓冲液校正得到的椭圆度;c表示样品浓度(mM);l表示石英比色皿的光程长度(mm),此处为0.5mm;n是多肽样品的氨基酸残基数。
实施例4:抗癌脂肽对癌细胞凋亡的影响
通过AnnexinV-FITC/PI凋亡检测试剂盒检测细胞凋亡。将状态良好的HepG2细胞用胰蛋白酶消化,在6孔板中每孔加入2mL细胞悬液(每孔5×105细胞),放入CO2培养箱培养不超过24h,待细胞贴壁后,吸除旧培养液,PBS清洗细胞2次,然后加入含有各浓度脂肽溶液的DMEM培养基继续培养24h,然后将各孔旧培养基收集后用胰蛋白酶(不含EDTA)消化,离心吸除上清液,PBS洗涤两次,然后分别与之前收集的旧培养基混合,1000rpm离心5min,弃去上清液,加PBS重悬并离心重复洗涤一次,加入100μL AnnexinV-FITC Binding buffer重悬细胞,加入5μLAnnexinV-FITC和10μLPI室温避光孵育15min,染色完成后冰浴避光放置,并于1h内用流式细胞仪完成检测。AnnexinV-FITC显示绿色荧光,PI显示红色荧光。
实施例5:抗癌脂肽对癌细胞的破膜活性研究
为研究抗癌脂肽的破膜活性,将8μM、16μM的抗癌脂肽与HepeG2细胞共培养3h,显微镜下(400×)观察抗癌脂肽对细胞膜形态的影响。
通过乳酸脱氢酶释放实验检测破膜活性,当细胞膜结构被破坏时,会导致细胞内的乳酸脱氢酶释放,测试乳酸脱氢酶释放量是研究细胞膜裂解情况的重要方法。首先将HepG2细胞接种于96孔板,设置空白阴性对照组、药物组第和阳页性对照组(加入1%TritionX-100代表100%释放),培养24h后加入不同浓度的多肽溶液(不含血清)继续培养24h,阳性对照组提前一小时加入TritionX-100继续培养1h,然后经多孔板离心机400g离心5min,按照LDH cytotoxicity Assay Kit(Beyotime,China)试剂盒每孔加入60μL检测工作液,置于摇床避光孵育30min,490nm处测吸光度,根据以下公式计算LDH释放率。
LDH释放率(%)=(OD实验-OD阴性)/(OD阳性-OD阴性)×100%
实施例6:抗癌脂肽的血清稳定性和溶血活性测试
为了评估抗癌脂肽的血清稳定性,预先用胎牛血清(FBS)和抗癌脂肽共培养,然后用MT T检测经FBS预处理后抗癌脂肽的抗癌活性变化来评估脂肽的稳定性。首先分别用10%FB S和PBS均预先孵育抗癌脂肽24h和48h,HepG2细胞在培养皿中长到90%左右时用胰酶消化细胞,并将细胞接种到96孔板中(每孔约2000个细胞),在细胞培养箱(37℃,5%CO2)中培养24h,吸出旧培养液加入预先孵育好的脂肽继续培养24h,24h后每孔加入20μL(5mg·mL-1)MTT溶液,避光培养4h,然后吸出上清,每孔加150μL DMSO,避光置于摇床(160r·min-1)8min,酶标仪(485nm)检测吸光度,计算HepG2存活率。
在溶血活性检测实验中,随机选择两只雌性小鼠(30-35g),将生理盐水和2.25mg/mL(15mg·kg-1)的脂肽通过尾静脉注射的方法注入小鼠体内,2h后通过眼球取血将两滴新鲜血液滴到抗凝管中,然后用生理盐水稀释,在荧光显微镜下观察红细胞形态。
实施例7:抗癌脂肽的体内抗肿瘤活性
建立体内荷瘤小鼠模型检验脂肽的体内抗肿瘤活性。实验所用小鼠均为(30~40g)雌性昆明小白鼠,并严格按照“动物实验的护理和使用指南”饲养。将1×106个H22细胞皮下注射接种到小鼠的左前肢腋下,当肿瘤体积大约为150-220mm3时,将小鼠随机分为2组,每组5只。分别为生理盐水组和脂肽组。将0.2mL(15mg·kg-1即2.25mg·mL-1)的药物通过尾静脉注射的方式隔天给药,在给药前给小鼠称重,并测量肿瘤大小计算肿瘤体积,共注射5次,实验完成后通过颈椎脱臼法处死小鼠。将各组小鼠肿瘤解剖后浸泡在4%的多聚甲醛溶液中固定24h以上,最后对肿瘤组织切片并进行HE染色。
本发明制备抗癌脂肽,具有良好的生物医学性能:
(1)本抗癌脂肽具有优异的体外抗癌活性
通过MTT法检测抗癌脂肽的体外抗癌活性。由图1a所示随脂肽浓度的增加HepG2细胞的相对细胞存活率逐渐降低,且在16μM时细胞相对存活率仅为44.68%,说明脂肽对癌细胞的杀伤呈浓度依赖性的同时具有较强的体外抗癌效果。如图1b所示,随时间的延长,HepG2细胞的相对存活率在0-4h内迅速下降,说明抗癌脂肽能发挥高效的抗癌能力,所以本发明抗癌脂肽具有优异的体外抗癌活性。
(2)本抗癌脂肽在细胞膜模拟环境中呈α螺旋结构
通过圆二色谱检测抗癌脂肽在水和SDS溶液中的二级结构。如图2所示,实线表示水溶液,模拟多肽遇到的亲水环境,虚线表示SDS溶液,用来模拟细胞膜疏水环境,结果表明抗癌脂肽在SDS溶液中,在195nm处为正峰,208和222nm处出现负峰,呈现明显的α螺旋结构,而在水溶液中抗癌脂肽大部分呈现无规则卷曲结构,其双负峰不明显,说明抗癌脂肽在水中大部分不呈α螺旋结构,然而当遇到疏水环境时会呈现明显的α螺旋结构。因此可以推测当抗癌脂肽遇到细胞膜时会以α螺旋结构插入细胞膜,发挥膜裂解作用。
(3)本抗癌脂肽具有优异的诱导癌细胞凋亡的能力
通过Annexin V-FITC/PI细胞凋亡试剂盒检测不同浓度抗癌脂肽对HepG2细胞凋亡的影响。细胞膜内侧的磷脂酰丝氨酸外翻是细胞早期凋亡的标志,FITC标记的Annexin V可以与细胞膜表面的磷脂酰丝氨酸结合来检测早期凋亡;PI只能进入细胞膜受损的细胞内,晚期凋亡的细胞由于细胞膜被破坏,因此可以同时被FITC-Annexin V和PI所渗透进而检测晚期凋亡。如图3(a-d)所示,随浓度增加晚期凋亡(UR象限)的比率均明显增大,说明癌细胞膜破裂程度随多肽浓度的增加而逐渐增强,另外早期凋亡比率也随浓度的增加而增加。通过早期凋亡和晚期凋亡的加和计算总凋亡率,对照组、4μM、8μM、16μM组凋亡率分别为4.9%、7.1%、20.2%、42.4%,呈剂量依赖性,而且结果与MTT结果一致。因此,抗癌脂肽呈剂量依赖性地诱导细胞凋亡。
(4)本抗癌脂肽具有强效的的癌细胞膜裂解能力
为研究抗癌脂肽的破膜活性,将8μM、16μM的抗癌脂肽与HepeG2细胞共培养3h,显微镜下观察抗癌脂肽对细胞膜形态的影响,如图4a-c所示,未加药组(图4a)细胞膜形态完整,细胞状态良好,8μM抗癌脂肽组(图4b)细胞开始皱缩,少量细胞膜出现裂解,16μM抗癌脂肽组(图4c)大部分细胞收缩变圆、细胞膜受损严重,细胞形态不再规则,因此可见抗癌脂肽呈剂量依赖性地破坏癌细胞膜,进而发挥抗肿瘤效果。
当细胞膜结构被破坏时,会导致细胞内的LDH(乳酸脱氢酶)释放到细胞外,通过LDH释放试验定量测定抗癌脂肽对癌细胞膜的裂解能力。如图4d所示LDH释放量随抗癌脂肽浓度的提高而增加,且在16μM时LDH释放量就达到67.04%,说明本抗癌脂肽拥有较好的癌细胞膜裂解能力。
(5)本抗癌脂肽具有较高的血清稳定性和较低的溶血毒性
多肽的溶血活性是评价其体内安全性的重要指标。通过抗癌脂肽对小鼠红细胞形态的影响来评估抗癌脂肽的溶血活性。如图5a-b所示,生理盐水组与抗癌脂肽组小鼠红细胞形态变化不大,均呈正常的双凹盘形,因此本抗癌第脂肽没页有明显的溶血毒性,具备较高的生物安全性。
大多数多肽体内稳定性差是其临床应用的最大障碍。将24μM的抗癌脂肽预先分别在10%FBS和PBS中均孵育24h、48h,然后通过检测细胞存活率来评价多肽血清稳定性。如图5c所示,抗癌脂肽经10%FBS以及PBS预处理24h后细胞存活率为40%和36%,其抗癌活性变化不大,同时经10%FBS预处理48h的脂肽的抗癌活性仅有小幅降低,因此本抗癌脂肽具有较高的血清稳定性。
(6)本抗癌脂肽具有良好的体内抗肿瘤效果
为了研究抗癌脂肽的体内抗肿瘤活性,建立了荷瘤小鼠模型,在小鼠腋下注射H22细胞(约1×106),肿瘤体积长到150-220mm3时开始隔天注射药物,测量肿瘤体积并称重。实验完成后将小鼠的肿瘤组织进行解剖,经4%多聚甲醛固定后进行组织切片和HE染色。如图6a所示,生理盐水组和抗癌脂肽组荷瘤小鼠的体重随给药次数的增加和时间的延长均变化不大,而抗癌脂肽组小鼠的相对肿瘤体积(图6b)相比于生理盐水组明显减小,说明抗癌脂肽在不影响小鼠体重的情况下能显著抑制肿瘤的生长。为了进一步研究抗癌脂肽的抗肿瘤活性,在实验完成后将小鼠的肿瘤组织进行解剖,经4%多聚甲醛固定后进行组织切片和HE染色。如图6c-d所示,NS组(图6c)肿瘤细胞密集、细胞核形态完整,抗癌脂肽组(图6d)肿瘤细胞密度小于NS组,且坏死细胞比NS组明显增多,因此本抗癌脂肽能发挥显著的体内抗肿瘤作用。
以上所述仅为本发明的优选实例,并不用于限制于上述事实。凡在本发明基础上所作出的非本质的改动、修改、替换等,均应包含在本发明的保护范围内。
序列表
<110> 滨州医学院
<120> 一种抗癌脂肽的制备及其在抗肿瘤治疗中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Asp Ser Asp Val Trp Trp Gly Gly Arg Arg Leu Leu Arg Arg Leu Arg
1 5 10 15
Arg Leu
Claims (3)
2.抗癌脂肽C8H15O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu的制备方法,其特征在于,包括以下步骤:
(1)首先通过固相合成法合成Fmoc-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp-(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin;
称取一定量的Fmoc-Leu-Wang Resin(Fmoc全称为9-芴基甲氧基羰基在此作为氨基保护基)加入DMF(N,N-二甲基甲酰胺)溶胀树脂30min,然后按照DMF:哌啶=4:1的比例加入哌啶,反应30min脱除Fmoc基团,反应完成后进行抽滤,并依次用DMF、DCM(二氯甲烷)、DMF多次洗涤树脂,重新加入无水DMF溶胀30min,加入两倍摩尔量的Fmoc-Arg(Pbf)-OH(Pbf为Arg的侧链保护基)、2.6倍摩尔量的DCC(二环己基碳二亚胺)、HOBT(1-羟基苯并三唑)和DIEA(N,N-二异丙基乙胺),在室温下反应48h,反应完成后依次用DMF、DCM、DMF多次洗涤树脂,然后转移到透析袋内(MW:8000-14000),用无水乙醇透析纯化,将透析完的样品冷冻干燥得Fmoc-Arg(Pbf)-Leu-Wang Resin,重复上述步骤依次偶联氨基酸直至合成Fmoc-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin,其中Boc为Trp侧链保护基,tBu为Ser侧链保护基,OtBu为Asp侧链保护基;
(2)C8H15O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu的合成;
首先将Fmoc-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin按照(1)中步骤脱除Fmoc基团得到Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin;将2倍摩尔量的C8H16O2(正辛酸)、2.5倍摩尔量的NHS(N-羟基琥珀酰亚胺)和2.5倍摩尔量的EDC(1-乙基-(3-二甲基氨基丙基)碳酰二亚胺)加入无水DMF中磁力搅拌5h以活化C8H16O2的羧基,然后加入Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin反应48h,抽滤并转移到透析袋内(MW:8000-14000),用无水乙醇透析纯化,冷冻干燥得C8H15O-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin,按照三氟乙酸(TFA):水(H2O):三异丙基硅烷(Tis)=95:2.5:2.5的比例配制成切割液与冻干后的树脂混合,磁力搅拌1.5h以脱除树脂和所有侧链保护基,反应完成后抽滤取滤液,旋蒸浓缩,加入冰乙醚产生沉淀,离心并加入乙醚多次洗涤沉淀,冷冻干燥得到脂肽C8H15O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu。
3.权利要求1所述抗癌脂肽C8H15O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu在制备抗肿瘤药物中的应用。
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