CN116747223A - 铁死亡诱导剂rsl3在制备预防和/或治疗增生性瘢痕的产品中的应用 - Google Patents
铁死亡诱导剂rsl3在制备预防和/或治疗增生性瘢痕的产品中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,具体涉及铁死亡诱导剂RSL3在制备预防和/或治疗增生性瘢痕的产品中的应用。本发明以铁死亡诱导剂RSL3为活性成分刺激增生性瘢痕成纤维细胞24h,发现0.2μmol/L铁死亡诱导剂RSL3对成纤维细胞的抑制率已经达到50%以上;并且还通过检测细胞内蛋白表达以及ROS的水平,发现铁死亡诱导剂RSL3处理后,成纤维细胞内GPX4、SLC7A11和PCNA蛋白表达显著降低,ACSL4和p27蛋白表达增加,且成纤维细胞内的ROS水平呈明显上升,引发铁死亡,从而抑制成纤维细胞的增殖。铁死亡诱导剂RSL3可以作为预防和/或治疗增生性瘢痕的活性成分。
Description
技术领域
本发明属于生物医药技术领域,具体涉及铁死亡诱导剂RSL3在制备预防和/或治疗增生性瘢痕的产品中的应用。
背景技术
增生性瘢痕(Hypertrophic scarHS)是一种以持续性真皮纤维化为特征的病理性瘢痕,是烧伤和创伤的常见并发症。当HS发生在面部、手臂或腿部时,会导致患者外观不良,导致精神创伤、身体疼痛,并给患者带来沉重的经济负担。全世界每年大约有1000万新的烧伤患者,烧伤后瘢痕的发生率为91.4%。瘢痕给患者和社会带来了很高的医疗负担,每年用于瘢痕治疗的费用预计至少为40亿美元。瘢痕组织可以恶性转化为瘢痕癌,成纤维细胞是HS形成的主要细胞类型,HS成纤维细胞(HSFs)表现出增强的增殖能力。但是,由于HS形成机制复杂,临床治疗效果并不理想,仍缺乏特异性有效的抗瘢痕药物,迫切需要一种强有力和可靠的治疗HS的方法。
铁死亡是近年来发现的一种新型细胞死亡,由于细胞膜上的谷胱甘肽过氧化物酶(GPX4)失效以及多不饱和脂肪酸磷脂(PUFA-PL)在铁催化下发生过氧化,当超过防御系统的缓冲能力时,导致脂质过氧化物在细胞膜上积累,导致膜破裂。近年来的研究表明,铁死亡与肿瘤、神经系统疾病、缺血-再灌注损伤、肾损伤、血液疾病等多种疾病的病理生理过程密切相关,且铁死亡可以抑制肿瘤细胞的增殖。某些细胞已被证实对于铁死亡有天然的易感性,因此诱导细胞发生铁死亡可能成为预防和/或治疗疾病的新策略。RSL3是一种谷胱甘肽过氧化物酶4(GPX4)的抑制剂,可直接抑制GPX4的表达从而抑制半胱氨酸和谷氨酸酯转运蛋白,诱导细胞发生铁死亡,但是现有技术中并未有关于RSL3可以预防和/或治疗增生性瘢痕的记载。
发明内容
本发明的目的在于为临床上预防和/或治疗增生性瘢痕提供新的药物,有效预防和/或治疗增生性瘢痕。
为了实现上述目的,本发明提供了铁死亡诱导剂RSL3在制备预防和/或治疗增生性瘢痕的产品中的应用。
优选的,所述产品包括抑制成纤维细胞增殖能力的产品。
优选的,所述产品中铁死亡诱导剂RSL3的浓度>0,且≤1.6μmol/L。
优选的,所述产品包括药物。
本发明还提供了一种预防和/或治疗增生性瘢痕的药物,所述药物包括铁死亡诱导剂RSL3和药学上可接受的辅料。
优选的,所述药物中铁死亡诱导剂RSL3的浓度>0,且≤1.6μmol/L。
有益效果:
本发明以铁死亡诱导剂RSL3为活性成分,利用不同浓度的铁死亡诱导剂RSL3刺激增生性瘢痕成纤维细胞24h后,利用CCK8法检测成纤维细胞的活力,并计算抑制率,发现0.1~1.6μmol/L铁死亡诱导剂RSL3对成纤维细胞活力有不同程度的抑制,其中0.2μmol/L铁死亡诱导剂RSL3对成纤维细胞的抑制率已经达到50%以上;并且还通过检测细胞内GPX4、SLC7A11和ACSL4蛋白表达以及ROS的水平,发现铁死亡诱导剂RSL3处理后,成纤维细胞内GPX4和SLC7A11蛋白表达显著降低,ACSL4蛋白表达增加,且成纤维细胞内的ROS水平呈明显上升,引发铁死亡,从而抑制成纤维细胞的增殖。铁死亡诱导剂RSL3可以作为预防和/或治疗增生性瘢痕的活性成分,为增生性瘢痕的有效预防和/或治疗提供了新的方向。
附图说明
为了更清楚地说明本发明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。
图1为实施例1利用CCK8法检测不同处理方式处理成纤维细胞后细胞活力情况;
图2为实施例2步骤(1)不同处理方式成纤维细胞内ROS水平;
图3为实施例2步骤(2)利用Westernblot检测不同处理方式处理成纤维细胞后细胞内GPX4、SLC7A11和ACSL4蛋白的表达情况;
图4为实施例2步骤(2)不同处理方式处理成纤维细胞后细胞内GPX4、SLC7A11和ACSL4蛋白的表达情况;其中*p<0.05,**p<0.01;
图5为实施例2步骤(2)利用Westernblot检测不同处理方式处理成纤维细胞后细胞相关蛋白PCNA和p27的mRNA表达情况;
图6为实施例2步骤(2)不同处理方式处理成纤维细胞后细胞内PCNA和p27的蛋白的表达情况;其中**p<0.01;
图7位实施例2步骤(2)qRT-PCR检测不同处理方式处理成纤维细胞后细胞相关蛋白PCNA和p27的mRNA表达情况;其中,*p<0.05,**p<0.01。
具体实施方式
本发明提供了铁死亡诱导剂RSL3在制备预防和/或治疗增生性瘢痕的产品中的应用。
在本发明中,所述铁死亡诱导剂RSL3的结构式如下:
在本发明中,所述产品优选包括抑制成纤维细胞增殖能力的产品。
在本发明中,所述产品中铁死亡诱导剂RSL3的浓度优选>0,且≤1.6μmol/L,进一步优选为0.1~1.6μmol/L,进一步优选为0.2~0.8μmol/L,更优选为0.2~0.4μmol/L。本发明优选在0.1~1.6μmol/L的范围内任意取值,例如0.1μmol/L、0.15μmol/L、0.18μmol/L、0.2μmol/L、0.24μmol/L、0.25μmol/L、0.28μmol/L、0.3μmol/L、0.35μmol/L、0.36μmol/L、0.4μmol/L、0.45μmol/L、0.48μmol/L、0.5μmol/L、0.55μmol/L、0.6μmol/L、0.7μmol/L、0.8μmol/L、0.9μmol/L、1.0μmol/L、1.1μmol/L、1.2μmol/L、1.4μmol/L、1.5μmol/L和1.6μmol/L。
在本发明中,所述产品优选包括药物。
本发明还提供了一种预防和/或治疗增生性瘢痕的药物,所述药物包括铁死亡诱导剂RSL3和药学上可接受的辅料。
在本发明中,所述药物中铁死亡诱导剂RSL3的浓度优选>0,且≤1.6μmol/L,进一步优选为0.1~1.6μmol/L,进一步优选为0.2~0.8μmol/L,更优选为0.2~0.4μmol/L。本发明优选在0.1~1.6μmol/L的范围内任意取值,例如0.1μmol/L、0.15μmol/L、0.18μmol/L、0.2μmol/L、0.24μmol/L、0.25μmol/L、0.28μmol/L、0.3μmol/L、0.35μmol/L、0.36μmol/L、0.4μmol/L、0.45μmol/L、0.48μmol/L、0.5μmol/L、0.55μmol/L、0.6μmol/L、0.7μmol/L、0.8μmol/L、0.9μmol/L、1.0μmol/L、1.1μmol/L、1.2μmol/L、1.4μmol/L、1.5μmol/L和1.6μmol/L。本发明对所述药学上可接受的辅料没有严格要求,常规选择即可。
本发明以铁死亡诱导剂RSL3为活性成分,利用不同浓度的铁死亡诱导剂RSL3刺激增生性瘢痕成纤维细胞24h后,发现0.1~1.6μmol/L铁死亡诱导剂RSL3对成纤维细胞活力有不同程度的抑制,其中0.2μmol/L铁死亡诱导剂RSL3对成纤维细胞的抑制率已经达到50%以上;检测细胞内GPX4、SLC7A11和ACSL4蛋白表达以及ROS的水平,发现铁死亡诱导剂RSL3处理后,成纤维细胞内GPX4和SLC7A11蛋白表达显著降低,ACSL4蛋白表达增加,且成纤维细胞内的ROS水平呈明显上升,引发铁死亡,从而抑制成纤维细胞的增殖,达到预防和/或治疗增生性瘢痕的作用。
为了进一步说明本发明,下面结合附图和实施例对本发明提供的铁死亡诱导剂RSL3在制备预防和/或治疗增生性瘢痕的产品中的应用进行详细地描述,但不能将它们理解为对本发明保护范围的限定。
在本发明实施例中,使用Prism5.0统计软件对结果进行统计学分析和处理,以均数±标准差(Mean±SD)表示,两样本均数间比较采用Student’s t检验,多个样本均数间比较采用One-way ANOVA检验,组间比较采用Student-Newan-Keuls检验,以P≤0.05具有统计学意义。
实施例1
1.材料
1.1材料与试剂:DMEM-F12培养基(Hyclone,中国);胎牛血清(BI,以色列);PBS(G-CLONE,中国);PCNA、p27引物(生工,上海);CCK-8试剂盒(APExBIO,美国);蛋白提取试剂盒(凯基,江苏);anti-GPX4 antibody、anti-SLC7A11 antibody、anti-ACSL4 antibody、anti-GPX4 antibody、anti-PCNA antibody和anti-p27 KIP 1antibody(abcam,英国);β-actin抗体(ABclonal,中国);GoatAnti-Mouse IgG(H+L)HRP(ABclonal,中国);GoatAnti-Rabbit IgG(H+L)HRP(ABclonal,中国);DCFH-DA探针(UElandy,中国);铁死亡诱导剂RSL3(Abmole,美国)。
1.2仪器与设备:负压吸引器(ROCKER 300,中国);手持式涡旋振荡器(Kylin-bell,中国);微量移液器(Eppendorf,德国);双温水浴摇床(HLD,江苏);紫外超净工作台(安泰,中国);台式离心机(Eppendorf,德国);手持式便携离心机(SCILOGEX,美国);生物安全柜(苏净安泰,中国);匀浆仪(FLUKO,德国);Biotek酶标仪(德泉兴业,北京);6孔板、96孔板(康宁,美国);水平摇床(Kylin-bell,中国);荧光定量PCR仪(analytik jena,德国);LUNA细胞计数仪(东胜,北京);凝胶成像仪(BIO-RAD,美国)。
2.实验样品
(1)将刚切下来的增生性瘢痕组织及其瘢痕旁正常组织迅速放入液氮中运输回实验室;
(2)在细胞间超净台(以下步骤均在细胞间超净台完成)内将增生性瘢痕及其瘢痕旁正常组织切成小块;
(3)加入PBS、双抗和庆大霉素摇动清洗组织小块30min,重复3次,每次15min;
(4)把组织小块放入含有消化酶的溶液中,即0.125%TE溶液;然后放入4℃冰箱过夜;
(5)第二天把组织切成肉糜样;
(6)将肉糜样组织放入盛有CoA胶原酶(胶原I型蛋白)的皿中,然后放入37℃的细胞培养箱过夜;
(7)第三天加入PBS清洗组织块后过200目筛网过滤;
(8)过滤后的细胞悬液转移入新的1.5mL EP管中,4℃/1000r离心10min;
(9)弃上清后,时刻在显微镜下观察原代细胞活动情况,2-4天后,用PBS清洗2次;
(10)隔天观察细胞状况,定期换液,取第3-5代细胞(以下记为成纤维细胞)用于实验。
实施例2
CCK8细胞活力测定
1.在96孔板中每孔接种5000个左右的成纤维细胞,随机分为实验1~6组和对照组,同时设置不添加接种成纤维细胞,只添加培养基的空白组,其中每个实验组实验孔、对照组的对照孔和空白组的空白孔均至少3个复孔;
实验1组实验孔:不转染铁死亡诱导剂RSL3,向实验1组实验孔加入DMSO,使DMSO的体积比为0.01%;
实验2组实验孔:利用DMSO溶解铁死亡诱导剂RSL3后,加入实验2组实验孔,使铁死亡诱导剂RSL3的终浓度为0.1μmol/L,DMSO的体积比为0.01%;
实验3组实验孔:利用DMSO溶解铁死亡诱导剂RSL3后,加入实验3组实验孔,使铁死亡诱导剂RSL3的终浓度为0.2μmol/L,DMSO的体积比为0.02%;
实验4组实验孔:利用DMSO溶解铁死亡诱导剂RSL3后,加入实验4组实验孔,使铁死亡诱导剂RSL3的终浓度为0.4μmol/L,DMSO的体积比为0.04%;
实验5组实验孔:利用DMSO溶解铁死亡诱导剂RSL3后,加入实验5组实验孔,使铁死亡诱导剂RSL3的终浓度为0.8μmol/L,DMSO的体积比为0.08%;
实验6组实验孔:利用DMSO溶解铁死亡诱导剂RSL3后,加入实验6组实验孔,使铁死亡诱导剂RSL3的终浓度为1.6μmol/L,DMSO的体积比为0.16%;
实验1~6组转染铁死亡诱导剂RSL3刺激24h后,向对照组、实验1~6组和空白组中加入10μL/孔的CCK8溶液,37℃下孵育2小时。取出96孔板后,避光在摇床上轻轻摇1min,使用酶标仪检测波长为450nm的OD值,求OD值的平均值,通过下述公式计算细胞抑制率;
细胞抑制率(%)=[(Ac-As)/(Ac-Ab)]×100
其中,As=实验组吸光度;Ab=空白组吸光度;Ac=对照组吸光度
2.重复步骤1中分组-实验-检测的步骤两次,即进行3次重复试验,最终取3次重复的平均值,计算平均细胞抑制率,结果如表1和图1所示。
表1不同处理方式下细胞活力检测结果
根据表1和图1记载的内容可以看出,不同浓度铁死亡诱导剂RSL3刺激成纤维细胞24h后,对成纤维细胞活力有不同程度的抑制,其中0.2μmol/L RSL3(实验3组)对成纤维细胞的抑制率已经达到50%以上。
实施例2
共聚焦小皿中增生性瘢痕成纤维细胞密度达75%左右时,随机分为实验组(RSL3)和对照组(control),对照组不进行任何处理,实验组添加铁死亡诱导剂RSL3,使铁死亡诱导剂RSL3的终浓度为0.2μmol/L;
(1)DCFH-DA探针检测活性氧(ROS)水平
实验组铁死亡诱导剂RSL3刺激24h后,取出实验组和对照组的培养基,向细胞加入PBS稀释的DCFH-DA 1mL,在37℃、5%CO2培养箱中避光孵育30min,PBS洗涤两次后在激光共聚焦荧光显微镜下观察(激发光波长:488nm,发射光波长:524nm),结果如图2所示。
根据图2可以看出,与Control组相比,铁死亡诱导剂RSL3组的增生性瘢痕成纤维细胞ROS荧光强度显著增加。
(2)免疫印迹法检测GPX4、SLC7A11和ACSL4表达
实验组铁死亡诱导剂RSL3刺激24h后,将PMSF与NP-40裂解液(购买自碧云天)按1:100的体积比混合,配制成蛋白裂解液;按照5×106个细胞:500μL蛋白裂解液的用量比将原代成纤维细胞和蛋白裂解液混合,置于4℃摇床30min,12000rpm、4℃离心20min后,转移蛋白上清液至新的EP管中,按照蛋白与缓冲液(loadingbuffer,为蛋白免疫印迹通用试剂)4:1的比例,向EP管中加入loadingbuffer,上样进行SDS-PAGE电泳,将胶转印至0.22μm PVDF膜上,5wt.%BSA室温封闭2h,TBST洗膜3次,每次10min,分别加入GPX4、SLC7A11、ACSL4和β-actin一抗4℃过夜,其中β-actin为内参,TBST室温洗膜3次,每次10min,辣根过氧化物酶标记二抗(HRP标记羊抗兔IgG)室温孵育2h后,TBST室温洗膜3次,每次10min,曝光,并计算灰度值,计算GPX4、SLC7A11和ACSL4的相对表达量,结果如表2和图3~4所示。
表2GPX4、SLC7A11和ACSL4的表达情况
注:表2中RSL3组GPX4重复3数值和重复1和2的数值相差较大,属于离群值,去除该值取平均;RSL3组SLC7A11重复1数值和重复1和3的数值相差较大,属于离群值,去除该值取平均。
根据表2和图3~4可以看出,与Control组相比,铁死亡诱导剂RSL3组的增生性瘢痕成纤维细胞GPX4、SLC7A11的表达下降,而ACSL4表达明显增加。
(3)免疫印迹法检测PCNA和p27蛋白表达
实验组铁死亡诱导剂RSL3刺激24h后,将PMSF与NP-40裂解液(购买自碧云天)按1:100的体积比混合,配制成蛋白裂解液;按照5×106个细胞:500μL蛋白裂解液的用量比将原代成纤维细胞和蛋白裂解液混合,置于4℃摇床30min,12000rpm、4℃离心20min后,转移蛋白上清液至新的EP管中,按照蛋白与缓冲液(loadingbuffer,为蛋白免疫印迹通用试剂)4:1的比例,向EP管中加入loadingbuffer,上样进行SDS-PAGE电泳,将胶转印至0.22μm PVDF膜上,5wt.%BSA室温封闭2h,TBST洗膜3次,每次10min,分别加入PCNA、p27和β-actin一抗4℃过夜,其中β-actin为内参,TBST室温洗膜3次,每次10min,辣根过氧化物酶标记二抗(HRP标记羊抗兔IgG)室温孵育2h后,TBST室温洗膜3次,每次10min,曝光,并计算灰度值,计算PCNA和p27表达情况,结果如表3和图5~6所示。
表3PCNA和p27的表达情况
注:表3中RSL3组p27重复2数值和重复1和3的数值相差较大,属于离群值,去除该值取平均。
根据表3和图5~6可以看出,与Control组相比,铁死亡诱导剂RSL3组的增生性瘢痕成纤维细胞PCNA的表达下降,而p27表达明显增加。
(4)qRT-PCR检测PCNA和p27表达
实验组铁死亡诱导剂RSL3刺激24h后,按TakaraRNA试剂盒的说明书操作提取实验组原代成纤维细胞的总RNA(核糖核酸),依照逆转录试剂盒说明书步骤进行逆转录。RT-qPCR(实时定量荧光PCR)采用Takara Real-Time PCR MasterMix(绿色荧光染料标记的实时PCR混合物)试剂盒对PCNA、p27表达量用耶拿PCR仪进行检测分析。GAPDH被用作为对照,实验结果按照如下公式进行计算:检测目的基因的相对表达量=2-△△Ct,其中,ΔΔCt=[CtGI(检测样品)-CtGAPDH(检测样品)]-[CtGI(校正样本)–CtGAPDH(校正样本)]。GI是指所测目的基因,Ct是指检测到的反应体系中荧光信号强度,校正样本指的是所有被选做能够代表1倍所测目的基因表达量的样本,本实施例以GAPDH作为校正样本,检测结果如表4和图7所示。
表4不同处理方式下PCNA和p27表达情况
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注:表4中RSL3组p27重复1中检测3数值、重复2中检测1和3数值、重复3中检测1数值和其他数值相差较大,属于离群值,去除这些数值后取平均。
根据表4和图7可以看出,与Control组相比,铁死亡诱导剂RSL3组的增生性瘢痕成纤维细胞PCNA的表达下降,而p27表达明显增加。
综合步骤(1)~(4)的结论可以看出,铁死亡诱导剂RSL3能够抑制增生性瘢痕成纤维细胞GPX4、SLC7A11、PCNA蛋白并促进ACSL4和p27蛋白的表达,促进细胞内ROS和脂质过氧化物的合成,诱导铁死亡,抑制成纤维细胞的增殖,从而参与增生性瘢痕的进展。
根据上述内容可以看出,铁死亡诱导剂RSL3能够抑制增生性瘢痕成纤维细胞的增殖,可作为预防和/或治疗增生性瘢痕的一种抑制剂,达到预防和/或治疗增生性瘢痕的作用,具有较好的应用价值和前景。
尽管上述实施例对本发明做出了详尽的描述,但它仅仅是本发明一部分实施例,而不是全部实施例,人们还可以根据本实施例在不经创造性前提下获得其他实施例,这些实施例都属于本发明保护范围。
Claims (6)
1.铁死亡诱导剂RSL3在制备预防和/或治疗增生性瘢痕的产品中的应用。
2.根据权利要求1所述的应用,其特征在于,所述产品包括抑制成纤维细胞增殖能力的产品。
3.根据权利要求1所述的应用,其特征在于,所述产品中铁死亡诱导剂RSL3的浓度>0,且≤1.6μmol/L。
4.根据权利要求1~3任一项所述的应用,其特征在于,所述产品包括药物。
5.一种预防和/或治疗增生性瘢痕的药物,其特征在于,所述药物包括铁死亡诱导剂RSL3和药学上可接受的辅料。
6.根据权利要求5所述的药物,其特征在于,所述药物中铁死亡诱导剂RSL3的浓度>0,且≤1.6μmol/L。
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