CN115746098B - 定向诱导间充质干细胞制备的活性肽及其制备方法与在化疗心脏保护剂中的应用 - Google Patents
定向诱导间充质干细胞制备的活性肽及其制备方法与在化疗心脏保护剂中的应用 Download PDFInfo
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Abstract
本发明公开了一种定向诱导间充质干细胞制备的活性肽及其制备方法与在化疗心脏保护剂中的应用。所述活性肽含有如下氨基酸序列:FGIVEGLMTTVHSYTATQK。通过实验证实,本发明提供的活性肽,可以显著降低化疗过程血清中的CK、LDH和AST以及心脏组织中的MDA含量,同时显著升高血清中的GSH‑PX活性和心脏组织中的SOD活性,保护心脏组织不受化疗毒性损伤,同时不产生毒副作用,为化疗过程中保护心脏药物的开发提供了新的方向。
Description
技术领域
本发明涉及生物制剂领域,尤其涉及一种定向诱导间充质干细胞制备的活性肽及其制备方法与在化疗心脏保护剂中的应用。
背景技术
化疗保护剂是用于预防化疗毒性的药物,针对某类化疗药物,特异性保护某种器官的细胞,预防毒性反应发生。因此CSFs,亚叶酸钙及止吐药物均不能被当作化疗保护剂。理想的化疗保护剂应该是:(1)保护组织不受化疗毒性损伤;(2)不影响抗肿瘤效果;(3)不产生毒性。
不同化疗药物对心脏的毒性不一样,对心脏影响最大的是蒽环类化疗药,比如阿霉素、表阿霉素。除此之外,其他的化疗药像紫杉类、氟尿嘧啶类等,都对心脏有一定的毒副反应。另外,还有靶向药物,比如曲妥珠单抗,对心脏也有副反应,它跟高毒性的化疗药物联用容易加重心脏的危险。因此,开发出应用于化疗过程的心脏保护剂来减少化疗药物对心脏的毒性,对于提高癌症患者的生存期和生存质量具有重要作用。
现有的针对心脏的化疗保护剂,右雷佐生(右丙亚胺)是依地酸的衍生物,是位点特异的心脏保护药物,可以有效地对抗蒽环类抗癌药(如阿霉素、柔红霉素、表阿霉素等)所引起的心脏损伤。右丙亚胺(DZR)是螯合剂EDTA的类似物,具强大的铁螯合作用,容易穿透细胞膜并在细胞内发生酶催化和非酶催化水解反应,终产物与一些中间体均有铁螯合作用,不仅可以与游离态铁离子螯合,而且可以从Fe3+-蒽环类螯合物中夺取Fe3+,从而抑制Fe3 +-蒽环类螯合物诱导的自由基的产生,进而抑制蒽环类药物的心脏毒性。即使在无铁无酶的情况下,DZR本身就具有清除自由基和抗氧化的作用。是目前唯一可以有效预防蒽环类药物所致心脏毒性的药物。然而其作为化学合成药物,可引起严重粒细胞减少、血小板减少等不良反应。因此,需要针对心脏开发出一种新型的化疗保护剂,避免心脏组织不受化疗毒性损伤的同时不产生毒副作用。
发明内容
有鉴于此,本发明的目的之一是提供一种定向诱导间充质干细胞制备的活性肽,其含有如下氨基酸序列:FGIVEGLMTTVHSYTATQK。
本发明的目的之二是提供一种药物组合物,其包含上述的活性肽和一种以上药学可接受的赋形剂、载体和/或稀释剂。
本发明的目的之三是提供一种定向诱导间充质干细胞制备上述的活性肽的方法,包括如下步骤:取生长状态良好的人脐带间充质干细胞进行定向诱导,在pH7~8、温度28℃~33℃条件下,加入诱导剂血管内皮细胞生长因子,反应时间为40~50min,之后对定向诱导后的间充质干细胞进行1:3增殖培养,细胞长至90%以上汇合时,1000r/min低速离心收集细胞并加入等量的血浆进行保护;然后加入3.5%胰酶并在pH7.0-7.5条件下进行裂解50~60min。
本发明的目的之四是提供上述活性肽在治疗肺癌药物中的应用。尤其是在针对阿霉素的心脏保护剂中的应用。
与现有技术相比,本发明的技术效果:
本发明提供的活性肽,通过定向诱导间充质干细胞制备,包含特定的氨基酸序列,通过实验证实,可以显著降低化疗过程血清中的CK、LDH和AST以及心脏组织中的MDA含量,同时显著升高血清中的GSH-PX活性和心脏组织中的SOD活性,保护心脏组织不受化疗毒性损伤,同时本身是生物活性肽类,不产生毒副作用,为化疗过程中保护心脏药物的开发提供了新的方向。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明中记载的一些实施例,对于本领域普通技术人员来讲,还可以根据这些附图获得其他的附图。
图1为本发明实施例2提供的ZHTG-CT的一级质谱图。
图2为本发明实施例2提供的ZHTG-CT的二级质谱图。
图3为本发明实施例3提供的各组小鼠体重变化。
图4为本发明实施例3提供的各组小鼠心脏系数变化。图中,**表示:与Control组比较,p<0.01;***表示:与Control组比较,p<0.001;##表示:与DOX组比较,p<0.01。
图5为本发明实施例3提供的ZHTG-CT对小鼠血清中CK活性的影响。*表示:与Control组比较,p<0.05;**表示:与Control组比较,p<0.01;***表示:与Control组比较,p<0.001;#表示:与DOX组比较,p<0.05;###表示:与DOX组比较,p<0.001。
图6为本发明实施例3提供的ZHTG-CT对小鼠血清中GSH-PX活性的影响。*表示:与Control组比较,p<0.05;**表示:与Control组比较,p<0.01;***表示:与Control组比较,p<0.001;#表示:与DOX组比较,p<0.05;###表示:与DOX组比较,p<0.001。
图7为本发明实施例3提供的ZHTG-CT对小鼠血清中LDH活性的影响。*表示:与Control组比较,p<0.05;**表示:与Control组比较,p<0.01;***表示:与Control组比较,p<0.001;#表示:与DOX组比较,p<0.05;###表示:与DOX组比较,p<0.001。
图8为本发明实施例3提供的ZHTG-CT对小鼠血清中AST活性的影响。*表示:与Control组比较,p<0.05;**表示:与Control组比较,p<0.01;***表示:与Control组比较,p<0.001;#表示:与DOX组比较,p<0.05;###表示:与DOX组比较,p<0.001。
图9为本发明实施例3提供的ZHTG-CT对小鼠心脏组织中SOD活性的影响。*表示:与Control组比较,p<0.05;#表示:与DOX组比较,p<0.05;###表示:与DOX组比较,p<0.001。
图10为本发明实施例3提供的ZHTG-CT对小鼠心脏组织中MDA含量的影响。*表示:与Control组比较,p<0.05;#表示:与DOX组比较,p<0.05;###表示:与DOX组比较,p<0.001。
具体实施方式
为了使本领域的技术人员更好地理解本发明的技术方案,下面将结合实施例和附图对本发明作进一步的详细介绍。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法。
下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。
实施例1人脐带间充质干细胞的定向诱导制备活性肽
本发明使用容易获取、来源充足、无道德伦理问题和安全有效的新生儿脐带间充质干细胞作为诱导对象。取生长状态良好的脐带间充质干细胞(hUC-MSCs)进行定向诱导,在pH7~8、温度28℃~33℃条件下,加入诱导剂血管内皮细胞生长因子(vascularendothelial growth factor,VEGF),反应时间为40~50min,之后对定向诱导后的间充质干细胞进行1:3增殖培养,细胞长至90%以上汇合时,1000r/min低速离心收集细胞并加入等量的血浆进行保护;然后加入3.5%胰酶并在pH7.0-7.5条件下进行裂解50~60min。对获得的裂解产物用10kD超滤膜进行超滤和0.22μm的滤膜除菌过滤。利用lorry法检测并最后配置成活性肽浓度为2mg/mL成品液,再经100℃30min的流通蒸汽分装等工艺,制成成品备用。
实施例2液相色谱-质谱联用(LC-MS/MS)测定活性肽氨基酸序列
2.1样品处理及SDS-PAGE电泳
取200mL用10kd超滤管进行超滤浓缩,6000g离心30min浓缩至500μL左右,收集滤液约200mL。加入2Ml 25mM NH4HCO3置换缓冲3次。收集浓缩样品。取约20μL浓缩后样品进行SDS-PAGE检测。滤液肽段经C18SPE脱盐冻干,复溶于2000mL 1%FA。
2.2FASP酶解
取200μL的浓缩后样品,加入200μL UA buffer8M Urea,150mM TrisHCl pH8.0),加入DTT至终浓度为100mM,37℃孵育1.5h,冷却至室温。离心14000g 15min。加入200μL UAbuffer离心14000g 15min,弃滤液。加入100μL IAA(50mM IAA in UA),600rpm振荡l min,避光室温30min,离心14000g 10min。加入100μL UA buffer,离心14000g 10min重复2次。加入100μL(NH4)2CO3 buffer,离心14000g10 min重复2次。加入40μL Trypsin bufer(4μgTrypsin in 40μL(NH4)2CO3 buffer),600rpm振荡1min,37℃16-18h。换新收集管,离心14000g 10min,收集滤液。OD280肽段定量。
2.3毛细管高效液相色谱
采用纳升流速HPLC液相系统Easy nLC进行分离。缓冲液:A液为0.1%甲酸水溶液,B液为0.1%甲酸乙腈水溶液(乙腈为84%)。色谱柱以95%的A液平衡。样品由自动进样器上样到上样Thermo scientific EASY column(2cm*100μm 5μm-C18),再经分析柱Thermoscientific EASY column(75um*100mm3um-C18)分离,流速为250nl/min。相关液相梯度如下:0分钟-50分钟,B液线性梯度从0%到50%;50分钟-54分钟,B液线性梯度从50%到100%;54分钟-60分钟,B液维持在100%。
2.4ESI质谱鉴定
样品经毛细管高效液相色谱分离后用Q-Exactive质谱仪(Thermo Finnigan)进行质谱分析。分析时长:60min,检测方式:正离子,母离子扫描范围:300-1800m/z,一级质谱分辩率:70,000at m/z 200,AGC target:3e6,一级MaximumIT:10ms,Number of scanranges:1,Dynamic exclusion:15.0s。多肽和多肽的碎片的质量电荷比按照下列方法采集:每次全扫描(full scan)后采集10个碎片图谱(MS2 scan),MS2 Activation Type:HCD,Isolation window:2m/z,二级质谱分辨率:17,500at m/z 200,Microscans:1,二级Maximum IT:60ms,Normalized cllision energy:27eV,Underfill ratio:0.1%。
2.5ESI质谱数据分析
原始文件通过Potcome Disoverere1.4提交至Mascot搜索引擎进行数据库检索。数据库为uniprot_Gallus_gllus_24080_20150630。搜库参数设定如下:enzyme为Trpsin(滤液肽段为:None):missed cleavage设为2:静态修饰设定Cartamidomehy C;动态修饰设定Oxidation M。FDR<0.1。
2.6结果
质谱仪自动对来自样品的洗脱组分交替进行一级质谱(MS)和二级质谱(MS/MS)处理,结果如图1所示,分子量为2083.06Da,主要区间为1041.73-1043.33Da,对主成分(m/z1042.03)进行MS/MS分析,如图2所示,含有15种氨基酸,苯丙氨酸(Phe,F)、甘氨酸(Gly,G)、异亮氨酸(Lle,I)、缬氨酸(Val,V)、谷氨酸(Glu,E)、亮氨酸(Leu,L)、蛋氨酸(Met,M)、苏氨酸(Thr,T)、缬氨酸(Val,V)、组氨酸(His,H)、丝氨酸(Ser,S)、酪氨酸(Tyr,Y)、丙氨酸(Ala,A)、谷氨酰胺(Gln,Q)、赖氨酸(Lys,K),氨基酸序列为:FGIVEGLMTTVHSYTATQK。命名为ZHTG-CT。
实施例3活性肽对化疗过程中心脏保护的效果研究
3.1阿霉素诱导小鼠心脏损伤模型的建立
将雄性ICR小鼠(18-22g)随机分为4组:
实验分组:
Control:腹腔注射等体积生理盐水。
阿霉素给药组(DOX):腹腔注射DOX 1.5mg/mL,0.1mL/10g小鼠体重,在第5天给一次。
ZHTG-CT(4mg/mL):尾静脉注射ZHTG-CT(4mg/mL)0.2mL/只,每天一次,共给6天;在第5天腹腔注射DOX 1.5mg/mL,0.1ml/10g小鼠体重。
ZHTG-CT(8mg/mL):尾静脉注射ZHTG-CT(8mg/mL)0.2mL/只,每天一次,共给6天;在第5天腹腔注射DOX 1.5mg/mL,0.1ml/10g小鼠体重。
第7d时,眼眶取血0.5-1mL,4℃3000r/min,离心5min,提取上清液,-20℃冻存待检。然后利用脊椎脱臼法处死小鼠,取出心脏组织,称重,待用。
3.2肌酸激酶同工酶(CK)、天门冬氨酸氨基转移酶(AST)、谷光氨肽过氧化物酶(GSH-PX)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)和丙二醛(MDA)的检测
取小鼠心脏组织和血清样本,按照CK、AST、GSH-PX、LDH、SOD和MDA检测试剂盒步骤进行检测,根据标准曲线计算小鼠血清中CK、AST、GSH-PX和LDH的活性,心脏组织中SOD的活性和MDA的含量。
3.3结果
小鼠一般情况变化
试验开始前,各组小鼠均精神状态良好,具体表现为饮水、吃食正常,毛柔顺光滑。与Control组比较,DOX组陆续出现饮水量和吃食量减少,精神萎靡,毛凌乱脱毛。与DOX组比较,ZHTG-CT两个剂量组的小鼠毛较柔顺光滑,饮水与吃食无显著性差异,精神正常,无毒副反应发生。
体重与脏器系数变化
每天称量小鼠体重,如图3所示,第7天处死小鼠前体重,与Control组比较,其他三组小鼠的体重明显降低,具有显著性差异(p<0.01,p<0.001)。与DOX组比较,ZHTG-CT两个剂量组的小鼠体重显著升高(p<0.01)。
根据心脏重量和小鼠体重,计算出心脏系数,如图4所示,各组无显著性差异。
血清中CK、GSH-PX、LDH和AST的变化
如图5-8所示,与Control组比较,DOX组血清中的CK、LDH和AST活性均显著性升高(p<0.05,p<0.01,p<0.001),GSH-PX活性显著下降(p<0.05)。与DOX组比较,ZHTG-CT(8mg/mL)组CK、LDH和AST均显著降低(p<0.05,p<0.001),GSH-PX活性显著升高(p<0.05)。
结果表明ZHTG-CT与DOX联用,能够改善氧化应激损伤。
心脏组织中SOD活性和MDA含量的变化
如图9-10所示,与Control组比较,DOX组心脏组织中SOD活性明显降低(p<0.05),而MDA含量显著升高(p<0.05)。与DOX组比较,ZHTG-CT(8mg/mL)组心脏组织中的SOD活性明显升高(p<0.001),而MDA含量显著降低(p<0.05)。
以上只通过说明的方式描述了本发明的某些示范性实施例,毋庸置疑,对于本领域的普通技术人员,在不偏离本发明的精神和范围的情况下,可以用各种不同的方式对所描述的实施例进行修正。因此,上述附图和描述在本质上是说明性的,不应理解为对本发明权利要求保护范围的限制。
Claims (1)
1.一种定向诱导间充质干细胞制备的活性肽在制备针对阿霉素化疗的心脏保护剂中的应用,其特征在于,所述活性肽的分子量为2083.06 Da,主要区间为1041.73-1043.33Da,主成分的氨基酸序列为:FGIVEGLMTTVHSYTATQK;所述活性肽通过以下方法制备:
取生长状态良好的人脐带间充质干细胞进行定向诱导,在pH7~8、温度28℃~33℃条件下,加入诱导剂血管内皮细胞生长因子,反应时间为40~50min,之后对定向诱导后的人脐带间充质干细胞进行1:3增殖培养,细胞长至90%以上汇合时,1000r/min低速离心收集细胞并加入等量的血浆进行保护;然后加入3.5%胰酶并在pH7.0-7.5条件下进行裂解50~60min,对获得的裂解产物用 10kD 超滤膜进行超滤和0.22μm 的滤膜除菌过滤,得到活性肽。
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