CN115894629B - 定向诱导间充质干细胞制备的活性肽及其制备方法与在治疗肺癌药物中的应用 - 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治疗肺癌药物中的应用 Download PDFInfo
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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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治疗肺癌药物中的应用。所述活性肽含有如下氨基酸序列:AMGIMNSFVNDIFER。通过实验证实,本发明提供的活性肽,可以提高机体和肿瘤内部的抗氧化能力,从而起到抗肿瘤的作用,促使肿瘤组织中的肿瘤细胞明显凋亡、坏死,同时无毒副作用,为治疗肺癌的药物研发提供了新的方向。
Description
技术领域
本发明涉及生物药物领域,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治疗肺癌药物中的应用。
背景技术
据统计,在发达国家和我国大城市中,肺癌的发病率已居男性各种肿瘤的首位。肺癌病人男女之比为3:1~5:1。根据世界卫生组织发布的数据,无论发病率和死亡率,肺癌均居首位。我国肺癌的发病率也呈上升趋势,在主要城市中已位列恶性肿瘤之首。
肺癌治疗手段有多种,应当根据患者的机体状况、肿瘤、细胞学、病理学类型、侵及范围和发展趋向,采取多学科综合治疗模式,强调个体化治疗。有计划、合理地应用手术、化疗、放疗和生物靶向等治疗手段,以期达到根治或最大限度控制肿瘤,提高治愈率,改善患者生活质量,延长患者生存期目的。其中,药物治疗仍然是最为重要和有效的治疗手段。
目前针对肺癌的治疗药物,包括小分子靶向治疗药物、化学药物治疗以及中药等。其中,小分子靶向治疗药物价格居高不下,并不能对广大患者普遍适用。化学药物在一般的抗肿瘤治疗过程中应用的比较多,然而其毒副作用较大,起效的同时损害免疫系统,最常见的是消化道反应,有恶心、呕吐、食欲降低、厌食,可能还会表现出神经毒性,比如奥沙利铂等会有手足末梢麻木的感觉;此外还会产生比较严重的脱发反应,如紫杉醇和多西他赛这一类药物表现出的脱发反应更加重,甚至产生过敏反应,比如培美曲塞,需要提前口服叶酸片,预防过敏等。中药起效慢,并且需要多种组份协同作用,疗效不明确,依然需要配合化疗药物使用。因此,开发一种新的安全有效的肺癌治疗药物,可以为肺癌治疗提供一个新的方向。
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:AMGIMNSFVNDIFER。
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pH8-9、温度35℃-37℃条件下,加入诱导剂人白介素2,反应时间为30~40min,之后对定向诱导后的人脐带间充质干细胞进行1:2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%胰酶并在pH8.5~9条件下进行裂解40~50min。
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。
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,可以提高机体和肿瘤内部的抗氧化能力,从而起到抗肿瘤的作用,促使肿瘤组织中的肿瘤细胞明显凋亡、坏死,同时无毒副作用,为治疗肺癌的药物研发提供了新的方向。
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-T1的一级质谱图。
图2为本发明实施例2提供的ZHTG-T1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提供的各组小鼠肿瘤体积变化。图中,*表示:与Control组比较,p<0.05;***表示:与Control组比较,p<0.001。
图5为本发明实施例3提供的各组小鼠肿瘤重量。图中,*表示:与Control组比较,p<0.05;***表示:与Control组比较,p<0.001。
图6为本发明实施例3提供的各组小鼠的肿瘤图片。
图7为本发明实施例3提供的ZHTG-T1对LLC荷瘤小鼠血清中SOD的活性影响。图中,*表示:与Control组比较,p<0.05;***表示:与Control组比较,p<0.001。
图8为本发明实施例3提供的ZHTG-T1对LLC荷瘤小鼠血清中MDA的含量影响。图中,*表示:与Control组比较,p<0.05;***表示:与Control组比较,p<0.001。
图9为本发明实施例3提供的ZHTG-T1对LLC荷瘤小鼠肿瘤组织中SOD活性影响。图中,*表示:与Control组比较,p<0.05;***表示:与Control组比较,p<0.001。
图10为本发明实施例3提供的ZHTG-T1对LLC荷瘤小鼠肿瘤组织中MDA含量的影响。图中,*表示:与Control组比较,p<0.05;***表示:与Control组比较,p<0.001。
图11为本发明实施例3提供的ZHTG-T1对小鼠肿瘤组织病理的影响。
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pH8~9、温度35℃~37℃条件下,加入诱导剂人白介素2(IL2),反应时间为30~40min,之后对定向诱导后的hUC-MSCs进行1:2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%胰酶并在pH8.5~9条件下进行裂解40~50min。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3.5mg/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,150mMTrisHCl 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40μLTrypsin 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*
100mm 3um-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(ThermoFinnigan)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,Isolationwindow:2m/z,二级质谱分辨率:17,500at m/z 200,Microscans:1,二级MaximumIT: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1775.81Da,主要区间为887.61-889.21Da。对主成分(m/z888.41)进行MS/MS分析,如图2所示,含有11种氨基酸,丙氨酸(Ala,A)、蛋氨酸(Met,M)、甘氨酸(Gly,G)、异亮氨酸(Lle,I)、天冬酰胺(Asn,N)、丝氨酸(Ser,S)、苯丙氨酸(Phe,F)、缬氨酸(Val,V)、天冬氨酸(Asp,D)、谷氨酸(Glu,E)、精氨酸(Arg,R),氨基酸序列为:AMGIMNSFVNDIFER。命名为ZHTG-T1。
实施例3活性肽对肺癌的治疗效果研究
3.1构建LLC荷瘤小鼠模型
构建小鼠LLC肺癌模型,选用6-8周龄,18g左右的雄性ICR小鼠,第0天予以右腋部皮下接种LLC肺癌细胞悬液(1×106),0.2mL/只。观察小鼠体重和肿瘤体积变化情况,当种瘤体积长到50mm3左右,随机分组,每天给药1次,连续给药12天:
实验分组:
Control:腹腔注射等体积生理盐水。
ZHT G-T1 5mg/mL:腹腔注射5mg/mL浓度的ZHT G-T1,0.1mL/10g小鼠体重。
ZHT G-T1 10mg/mL:腹腔注射10mg/mL浓度的ZHT G-T1,0.1mL/10g小鼠体重。
每3天测量一次肿瘤大小,在第13天,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,取出肿瘤组织,拍照,称重,待用。
3.2绘制肿瘤生长曲线
用游标卡尺分别在给药后第0、1、3、5、7、9、11、13天测量小鼠皮下肿瘤的长径(a)和断经(b),按照公式为:V(cm3)=ab2/2,计算肿瘤体积,绘制肿瘤生长曲线。各组小鼠给药后第13天小鼠处死,完整剥取皮下瘤体并称取质量,绘制肿瘤生长曲线。
3.3肿瘤组织总蛋白的提取
组织匀浆制备:取部分肿瘤组织,再预冷的生理盐水中反复漂洗,除去血液,滤纸拭干,称重,按1:9比例加入组织裂解液。在冰水浴条件下,用组织匀浆器制备组织匀浆。将组织匀浆液3500rpm/min离心15min后吸取上清液分装于EP管中,-80℃保存备用。
3.4丙二醛(MDA)含量检测
检测原理:过氧化脂质降解产物中的MDA可与硫代巴比妥酸缩合,形成红色产物,在532nm处有最大吸收峰。测量MDA含量可反映机体内脂质过氧化的程度,间接地反映出细胞损伤的程度。
将肿瘤组织和血清按照试剂盒说明说进行操作,在532nm出测出吸光度。并按照公式计算出组织和血清中MDA的含量。
3.5超氧化物歧化酶(SOD)活性测定
检测原理:黄嘌呤及黄嘌呤氧化酶反应系统产生超氧阴离子自由基(O2·)O2·能使羟胺氧化形成亚硝酸盐,在显色剂的作用下显紫红色;样品中含有的SOD能抑制其作用,使亚硝酸盐形成量减少,通过检测显色后溶液的吸光度值即可计算被测样品中SOD的活性。
将肿瘤组织和血清按照试剂盒说明书操作,测定组织和血清中SOD的活性。
3.6HE染色
烘片及脱蜡:置于烘片机在60℃烘片2h,依次将切片放入二甲苯ⅠII III各10min,无水乙醇ⅠII III各5min,95%酒精5min,85%酒精5min,75%酒精,蒸馏水洗。
苏木素染细胞核:切片入苏木素染3-8min,自来水洗返蓝。
伊红染细胞质:切片入伊红染液中染色1-3min。
脱水、透明:将切片快速过85%酒精,95%酒精2s,无水乙醇Ⅰ5min,无水乙醇Ⅱ5min,二甲苯Ⅰ10min,二甲苯Ⅱ10min中透明。
封片:滴加适量中性树胶封片。
3.7结果
一般情况变化
Control组小鼠活动明显减弱,精神萎靡,饮水、进食量明显减少、毛色暗淡无光泽,ZHTG-T1两个剂量组小鼠活动略少,精神正常,饮水、进食量明显多于Control组小鼠,毛色正常,无毒副反应发生。
体重变化
在给药第一天开始称量体重,如图3所示,前7天,ZHTG-T1两组小鼠的体重有所减少,是因为小鼠的肿瘤体积在逐渐缩小,7天以后,ZHTG-K1两组小鼠的体重逐渐增加,是由于小鼠的生活状态、饮水饮食都有所改善。由于Control组小鼠的肿瘤体积处于逐渐增长状态,所以导致Control组小鼠体重一直处于升高趋势。
肿瘤变化
观察小鼠肿瘤体积变化,如图4所示,Control组小鼠的肿瘤体积一直处于增长趋势,而ZHTG-T1两个剂量组小鼠的肿瘤体积明显减小,在第13天,与Control组小鼠肿瘤体积比较,明显减小,有显著性差异(p<0.001)。ZHTG-T1高剂量组与低剂量组的小鼠肿瘤体积无显著性差异。肿瘤取出后称重,如图5所示,与Control组比较,ZHTG-T1(5mg/mL)和ZHTG-T1(10mg/mL)组小鼠肿瘤的重量均减少,其中ZHTG-T1(10mg/mL)组小鼠肿瘤重量明显减少,具有显著性差异(p<0.05)。各组小鼠的肿瘤图片如图6所示。
血清及肿瘤组织中SOD和MDA变化
将每组小鼠的冻存血清取出,按试剂盒说明测定每组小鼠血清中SOD的活性和MDA的含量,结果如图7和图8所示,与Control组比较,ZHTG-T1(5mg/mL)和ZHTG-T1(10mg/mL)组小鼠血清中SOD活性均有所升高,MDA含量均明显减少(p<0.001),其中ZHTG-T1(10mg/mL)组小鼠血清中SOD活性显著升高,具有显著性差异(p<0.05)。
将每组小鼠的肿瘤组织处理后,按试剂盒说明测定每组小鼠血清中SOD的活性和MDA的含量,结果如图9合伙图10所示,与Control组比较,ZHTG-T1(5mg/mL)和ZHTG-T1(10mg/mL)组小鼠肿瘤组织中SOD活性均显著升高(p<0.05,p<0.001),MDA含量均减少,其中ZHTG-T1(10mg/mL)组小鼠肿瘤组织中MDA含量明显减少,具有显著性差异(p<0.05)。
以上结果说明,ZHTG-T1可以提高机体和肿瘤内部的抗氧化能力,从而起到抗肿瘤的作用。
组织病理形态学观察
通过HE染色法观察肿瘤组织的病理特征,如图11所示,与Control组相比,ZHTG-T1(5mg/mL)组肿瘤组织显示出明显的肿瘤细胞收缩、稀疏排列、破碎、肿瘤内广泛坏死,ZHTG-T1(10mg/mL)组肿瘤组织中少见肿瘤细胞,坏死出血较多。说明ZHTG-T1促使肿瘤组织中的肿瘤细胞明显凋亡、坏死。
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的活性肽在制备治疗肺癌药物中的应用,其特征在于,所述活性肽的分子量为1775.81 Da,主要区间为887.61-889.21 Da,主成分的氨基酸序列为:AMGIMNSFVNDIFER;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pH8~9、温度35℃~37℃条件下,加入诱导剂人白介素2,反应时间为30~40min,之后对定向诱导后的人脐带间充质干细胞进行1:2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%胰酶并在pH8.5~9条件下进行裂解40~50min,对获得的裂解产物用10 kD超滤膜进行超滤和0.22 μm的滤膜除菌过滤,得到活性肽。
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CN103992394A (zh) * | 2014-05-23 | 2014-08-20 | 哈尔滨医科大学 | 一种人工合成的阳离子肽及其在制备抗肿瘤药物中的用途 |
CN113274485A (zh) * | 2021-05-31 | 2021-08-20 | 南方医科大学 | 蝎毒多肽Smp24在制备抗肿瘤药物的应用 |
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