CN114507638B - 一种抑制间充质干细胞衰老的优化培养基及其应用 - Google Patents
一种抑制间充质干细胞衰老的优化培养基及其应用 Download PDFInfo
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- C12N2501/135—Platelet-derived growth factor [PDGF]
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Abstract
本发明属于生物技术领域,具体提供一种抑制间充质干细胞衰老的优化培养基及其应用。通过改进和优化间充质干细胞的培养基,从而延缓甚至抑制脂肪来源间充质干细胞衰老,进而提高其治疗潜能的方法。本发明的优点有:(1)所提供的优化培养基能更好地促进AMSCPro增殖,提高细胞免疫干预能力,降低细胞甲基化程度,并抵抗复制、化疗药物、辐射等多种因素诱导的细胞衰老;(2)可纠正老年个体或代谢性疾病患者脂肪组织来源AMSC的细胞衰老表型(3)所述的AMSCPro及其所制备的肝病治疗制剂或药物可消除衰老相关的AMSC细胞功能损伤,具有更好的肝脏疾病治疗效果。
Description
技术领域
本发明属于生物技术领域,涉及抑制间充质干细胞衰老的方法,具体提供一种抑制间充质干细胞衰老的优化培养基及其应用。通过改进和优化间充质干细胞的培养基,从而延缓甚至抑制脂肪来源间充质干细胞衰老,进而提高其治疗潜能的方法。
背景技术
间充质干细胞(Mesenchymal stem cell,MSC)已被证实具有强大的分化潜能、自我更新能力、免疫调节作用以及靶向治疗功能。MSC细胞移植已在多种退行性疾病和组织急慢性损伤治疗中显示出明确疗效。动物水平的研究显示,移植MSC能缓解小鼠急、慢性肝损伤模型的肝脏炎症、促进肝细胞再生。并且临床实验研究也显示,重肝患者输注MSC的耐受性良好,可显著改善肝脏功能、降低Child-Pugh和MELD评分、减少腹水及总死亡率。因此,对于目前临床上尚无有效药物和治疗手段的重大疾病,如肝衰竭等,MSC移植有望作为一种极具前景的细胞治疗新策略,用以补充甚或替代因血浆及肝源短缺而受限的目前临床上肝衰竭治疗的两种主要方式——人工肝系统和肝移植。从而有效改善该类患者预后,降低其病死率。
虽然MSC在再生医学及肝病治疗中的作用已备受肯定,但其疗效仍存在局限性。其中增殖、分化和归巢能力差等许多缺陷,可能与其细胞衰老有关。这是由于为了满足治疗所需的细胞量,在MSC移植前往往需要进行体外扩增,这就为复制所诱导的细胞衰老提供了机会。此外,从老年人及代谢性疾病患者身上获取的MSCs也往往具有更高的衰老负荷。因此,缓解甚至抑制MSC衰老,消除衰老相关的细胞功能损伤,有望显著提高MSC移植后的治疗效果。
我们在前期研究已成功建立了从脂肪组织分离培养MSC的技术。脂肪来源的MSC(AMSC)较之骨髓来源的MSC,其来源更为丰富,获取更为方便,扩增效率更高,因此在临床上具有良好的应用前景。但是我们在小鼠(mAMSC)及人的AMSCs(hAMSC)的体外培养扩增过程中均发现,随着体外传代次数的增加,AMSC表现出明显的衰老特征,如SA-β-gal染色增加,胞内p21和p16的蛋白表达水平升高,细胞和上清中细胞衰老相关分泌表型(SASP)相关因子水平也显著增加,并伴随着细胞增殖速率降低,细胞显著增大并呈扁平化以及细胞核的空泡化。同时,细胞线粒体也出现融合增大,线粒体ROS水平上调。另外,采用出现衰老表型的AMSC进行小鼠肝损伤及肝衰竭模型的治疗,发现其疗效亦显著降低,在抑制肝脏炎症、修复肝脏损伤、恢复肝功能等多个方面均与未衰老(状态良好)AMSC的疗效存在显著差异,甚至较之溶剂对照组无明确疗效。
目前已有一些通过基因修饰来抵抗MSC衰老并提高其疾病治疗潜能的方法,如采用c-myc修饰以提高MSC的干性和增殖能力,但该修饰所带来的MSC无限扩增性也可能导致MSC移植后的成瘤性风险。并且基因编辑或修饰也会给MSC的临床应用带来额外的伦理和安全性问题。因此,本发明旨在提供一种通过改进和优化的间充质干细胞的培养基,从而缓解甚或抑制AMSC衰老,进而提高AMSC在肝衰竭等重大肝脏疾病中的治疗潜能,并进一步提升相关AMSC细胞制剂的效能。
发明内容
本发明的目的是要提供一种缓解甚或抑制AMSC衰老的体外培养方法,具体涉及通过优化AMSC的培养基,在AMSC的改良培养体系中额外添加25-羟基胆固醇(25HC)来抵抗培养传代或化药、辐照等因素诱导的AMSC衰老,并纠正老年个体或代谢性疾病个体来源的AMSC的细胞衰老表型的特殊培养方法;以及采用该特殊培养方法所获得的AMSC(AMSCPro)在制备肝病治疗生物制剂中的用途。
本发明通过以下技术方案实现:
本发明提供一种抑制间充质干细胞衰老的优化培养基,在AMSC的培养体系中添加25-羟基胆固醇25HC。
优选的,包含50%DMEM/F12+48%MCDB-201、2%MSC适宜胎牛血清、5μM 25HC、1×胰岛素-转铁蛋白-硒ITS-G、1×MEM非必需氨基酸,10ng/ml EGF、10ng/ml PDGF,50μMβ巯基乙醇,2mM L-谷氨酰胺,100μg/ml青霉素和100U/ml硫酸链霉素的培养液。
另外,本发明保护优化培养基的应用。
抑制间充质干细胞衰老的优化培养基用于培养用于制备用于MSC细胞移植的生物制剂的应用。
抑制间充质干细胞衰老的优化培养基联合IFN-α干预纠正AMSC的衰老表型的生物制剂的应用。
其中,所提供的优化培养基促进AMSCPro增殖,提高细胞免疫干预能力,降低细胞甲基化程度,并抵抗复制、化疗药物或辐射诱导的细胞衰老;纠正老年个体或代谢性疾病患者脂肪组织来源AMSC的细胞衰老表型,提高AMSCPro的治疗潜能。
本发明另外提供了优化培养基的使用方法。
抑制间充质干细胞衰老的优化培养基的使用方法,包括如下步骤:
1)脂肪来源的间充质干细胞(AMSC)的制备及常规培养:
小鼠AMSC(mAMSC)和成人AMSC(hAMSC)按常规方法分离,mAMSC和hAMSC分别采用STEMCELLTM公司的小鼠和人MesenCultTM扩增试剂盒(货号:#05513,#05411)再添加2mM L-谷氨酰胺,100μg/ml青霉素和100U/ml硫酸链霉素作为完全培养基进行培养扩增;AMSC增殖接近75%融合时,进行消化、传代;
2)AMSC的优化培养:
常规培养至第2代的mAMSC细胞和hAMSC细胞,换用优化培养基继续进行传代培养;该优化培养基为:50%DMEM/F12+48%MCDB-201、2%MSC适宜胎牛血清(FBS)、5μM 25HC、1×胰岛素-转铁蛋白-硒(ITS-G)、1×MEM非必需氨基酸(NEAA),10ng/ml EGF、10ng/ml PDGF,50μMβ巯基乙醇,2mM L-谷氨酰胺,100μg/ml青霉素和100U/ml硫酸链霉素的培养液;采用该优化培养基培养传代的细胞标记为AMSCPro;以仍采用前述STEMCELLTM公司的小鼠和人MesenCultTM培养基进行扩增传代的AMSC标记为AMSC;采用不含25HC组分的优化培养基进行培养传代的细胞标记为AMSCCtrl。
还提供了采用本申请优选培养基及其培养方法所获得的脂肪来源间充质干细胞AMSCPro。
AMSCPro在制备肝病治疗生物制剂中的用途。所述的AMSCPro及其所制备的肝病治疗制剂或药物可消除衰老相关的AMSC细胞功能损伤,具有更好的肝脏疾病治疗效果。
本发明提供一种干预脂肪来源间充质干细胞(AMSC)衰老的优化培养体系及相关细胞制剂的制备和应用。通过在AMSC的改良培养体系中额外添加25HC来抵抗培养传代或化药、辐照等因素诱导的AMSC衰老,或采用优化培养基联合IFN-α干预来纠正老年个体或代谢性疾病个体来源的AMSC的细胞衰老表型。并在细胞水平通过WB、CCK8细胞增殖分析、SA-β-gal染色和细胞及线粒体形态观察等研究手段来明确优化培养基对于AMSC衰老表型的纠正和抑制作用,以及对AMSC的免疫干预能力和甲基化水平的调控作用;并通过小鼠肝损伤模型等体内实验进一步明确AMSCPro较之AMSC和AMSCCtrl的肝病疗效优势。本发明提供的制备肝病治疗的生物制剂,具有更快的制备周期,并且可消除AMSC衰老相关细胞功能损伤带来的不良效应,进而提升该细胞制剂在肝病临床治疗中的应用潜能。
本发明还公开了AMSCPro细胞制剂在治疗肝脏疾病中的作用及优势,具备以下作用中的任一项或多项:较之复制性衰老、老年个体或代谢性疾病个体来源的AMSC或AMSCCtrl,AMSCPro可更为有效地(1)发挥对肝衰竭等肝脏疾病的疗效;(2)降低血清ALT、AST水平;(3)缓解肝脏炎症,减少肝组织坏死程度;(4)降低炎症因子风暴;(5)避免衰老细胞所致不良效应。
优选地,所述肝脏疾病包括但不限于药物或内毒素诱导的急性肝损伤、肝炎、肝衰竭、肝纤维化。
所述方法可以是体内的。也可以体外的,例如仅用于科学研究。
本发明的优点有:(1)所提供的优化培养基能更好地促进AMSCPro增殖,提高细胞免疫干预能力,降低细胞甲基化程度,并抵抗复制、化疗药物、辐射等多种因素诱导的细胞衰老;(2)可纠正老年个体或代谢性疾病患者脂肪组织来源AMSC的细胞衰老表型,提高AMSCPro的治疗潜能,为患者自体AMSC移植治疗相关疾病提供可行性;(3)所述的AMSCPro及其所制备的肝病治疗制剂或药物可消除衰老相关的AMSC细胞功能损伤,具有更好的肝脏疾病治疗效果。
附图说明
图1:AMSCPro抵抗细胞复制性衰老:A.体外传代培养至第25代的成人脂肪来源AMSC细胞的SA-β-gal染色情况分析。B.Western Blot分析上述细胞中p21和p16蛋白表达水平。**p<0.01。
图2:AMSCPro部分抵抗TNF-α诱导的细胞衰老:成人脂肪来源AMSC细胞建立TNF-α诱导的细胞衰老模型,在该模型中分析AMSC细胞中的p21和p16蛋白表达水平。U.T代表未经TNF-α处理的正常培养的AMSC。
图3:AMSCPro纠正老年小鼠AMSC的衰老表型:采用Western Blot方法检测yAMSC、oAMSC、oAMSCCtrl和oAMSCPro细胞中p21和p16蛋白表达水平。Image J软件灰度比值分析显示,yAMSC细胞中p21和p16蛋白表达水平显著低于oAMSC;而oAMSCPro的p21和p16蛋白表达水平亦显著低于oAMSC和oAMSCCtrl。**p<0.01。
图4:细胞DNA甲基转移酶表达分析:采用Western Blot方法检测AMSC、AMSCCtrl和AMSCPro细胞中DNA甲基转移酶DNMT1、DNMT3a、DNMT3b的蛋白表达水平。Image J软件灰度比值分析显示,AMSCPro的DNMT1、DNMT3a和DNMT3b的蛋白表达水平均明显低于AMSCCtrl和AMSC。**p<0.01。
具体实施方式
本发明结合附图和实施例作进一步的说明。
1.脂肪来源的间充质干细胞(AMSC)的制备及常规培养:
小鼠AMSC(mAMSC)和成人AMSC(hAMSC)按常规方法分离,mAMSC和hAMSC分别采用STEMCELLTM公司的小鼠和人MesenCultTM扩增试剂盒(货号:#05513,#05411)再添加2mM L-谷氨酰胺,100μg/ml青霉素和100U/ml硫酸链霉素作为完全培养基进行培养扩增。AMSC增殖接近75%融合时,进行消化、传代。
2.AMSC的优化培养:
常规培养至第2代的mAMSC细胞和hAMSC细胞,换用优化培养基继续进行传代培养。该优化培养基为:50%DMEM/F12+48%MCDB-201、2%MSC适宜胎牛血清(FBS)、5μM 25HC、1×胰岛素-转铁蛋白-硒(ITS-G)、1×MEM非必需氨基酸(NEAA),10ng/ml EGF、10ng/ml PDGF,50μMβ巯基乙醇,2mM L-谷氨酰胺,100μg/ml青霉素和100U/ml硫酸链霉素的培养液。采用该优化培养基培养传代的细胞标记为AMSCPro。以仍采用前述STEMCELLTM公司的小鼠和人MesenCultTM培养基进行扩增传代的AMSC标记为AMSC。采用不含25HC组分的优化培养基进行培养传代的细胞标记为AMSCCtrl。
3.在体外培养传代诱导的细胞复制衰老模型中分析AMSC、AMSCCtrl和AMSCPro的细胞衰老表型:
成人脂肪组织来源的天然hAMSC、hAMSCCtrl和hAMSCPro细胞分别传至第25代;小鼠脂肪来源的天然mAMSC、mAMSCCtrl和mAMSCPro细胞分别传至第9代。光镜下观察可见,hAMSC细胞显著增大并呈扁平化,细胞核变大,且出现空泡化;电镜下观察hAMSC中的线粒体也明显融合增大,具有典型的衰老细胞特征。虽然hAMSCCtrl的细胞状态略好于hAMSC,但仍出现了较为显著的衰老特征。而hAMSCPro细胞则仍能维持经典的AMSC细胞的长梭型特征,细胞核及线粒体形态较之传代早期的细胞(如第2、3代细胞)也无明显改变。同样的,小鼠脂肪来源的mAMSC在上述复制衰老模型中也具有类似表现。表明该优化培养基可有效避免AMSC细胞复制性衰老。
3.1.进一步采用细胞衰老β-半乳糖苷酶(SA-β-gal)染色试剂盒(C0602,碧云天)比较AMSC、AMSCCtrl和AMSCPro细胞的染色情况。按试剂盒步骤进行SA-β-gal染色,染色后于普通光学显微镜下观察,并取5个视野计数分析SA-β-gal蓝染阳性率。
结果显示,采用该优化培养基可显著降低hAMSC和mAMSC复制性衰老所致的SA-β-gal蓝染阳性率增加。
3.2.采用Western Blot方法检测天然AMSC、AMSCCtrl和AMSCPro细胞中p21和p16蛋白表达水平。
采用Western Blot方法检测天然AMSC、AMSCCtrl和AMSCPro细胞中FAM60A的蛋白表达水平。具体步骤如下:
1)消化离心收集细胞,于细胞沉淀中加入适量含蛋白酶抑制剂的RIPI裂解液(普利莱,货号:C1053),并充分混匀,12000g,4℃,离心5分钟,收集上清。
2)采用BCA试剂盒(Thermo,货号:23228)进行蛋白定量。
3)上样前吹走4-20%梯度浓度预制胶(GenScript)每孔中的甘油,细胞蛋白样本按40ug/空的蛋白量上样,电压140V跑胶约1小时,然后用自动转膜机转膜(GenScript,Trans-Blot Turbo)约15min。
4)再在5%BSA(Servicebio,货号:G5001-100G)中封闭约1小时,然后孵育p21一抗(Abcam,货号ab109199,1:1000稀释)和p16一抗(Abcam,人细胞样本采用ab108349,小鼠细胞样本采用ab211542,1:1000稀释),摇床4℃过夜。
5)TBST洗膜3次,每次10分钟分钟;然后用HRP标记的抗兔二抗孵育1小时,再用TBST洗膜3次,每次10分钟。
6)最后用ECL(BI,货号20-500-120)曝光。条带采用ChemiScope Western Blot成像系统(Clinx Science Instruments Co.,Ltd)扫描后,再用Image J软件(RawakSoftware,Inc.Germany)进行灰度比值分析。
结果显示,采用该优化培养基可显著降低hAMSC和mAMSC的p21和p16蛋白表达水平。较之AMSC和AMSCCtrl细胞,AMSCPro细胞的p21和p16蛋白水平可分别降低约65%和50%以上。
4.优化培养基联合IFN-α干预纠正老年小鼠AMSC的衰老表型:
分离培养得到的16月龄老年小鼠AMSC(oAMSC),培养至第2代后,换用上述优化培养基,并额外添加500U IFN-α继续传代培养,传至第4代(oAMSCPro)。以仍采用前述STEMCELLTM公司的MesenCultTM培养基进行扩增传代的AMSC标记为oAMSC。采用不含25HC和IFN-α组分的优化培养基进行培养传代的细胞标记为oAMSCCtrl。并以6周龄年轻小鼠AMSC(yAMSC)采用前述STEMCELLTM公司的MesenCultTM培养基进行扩增传代的第4代细胞作为比较。
IFN-α可采用200U~1000U的剂量,优选500U。
采用如前所述的SA-β-gal染色法比较yAMSC、oAMSC、oAMSCCtrl和oAMSCPro细胞的染色情况。结果显示,yAMSC细胞的蓝染阳性率显著低于oAMSC;oAMSCCtrl细胞的蓝染阳性率虽低于oAMSC,但较之oAMSCPro细胞仍显著增加;oAMSCPro的蓝染阳性率显著低于oAMSC和oAMSCCtrl,与yAMSC细胞中的水平相近。
采用如前所述的Western Blot方法检测天然yAMSC、oAMSC、oAMSCCtrl和oAMSC25HC细胞中p21和p16蛋白表达水平。结果显示,yAMSC细胞中p21和p16蛋白表达水平显著低于oAMSC;oAMSCCtrl细胞的p21和p16蛋白表达水平虽低于oAMSC,但较之oAMSCPro细胞仍显著增加;oAMSCPro细胞的p21和p16蛋白表达水平与yAMSC细胞中的水平相近。
5.优化培养基联合IFN-α干预纠正代谢性疾病小鼠来源AMSC的衰老表型:
分离代谢性疾病小鼠(由高脂饮食和STZ处理诱导2型糖尿病合并脂肪肝模型小鼠,mdAMSC),培养至第2代后,换用上述优化培养基,并额外添加500U IFN-α继续传代培养,传至第4代(mdAMSCPro)。以仍采用前述STEMCELLTM公司的MesenCultTM培养基进行扩增传代的AMSC标记为mdAMSC。采用不含25HC和IFN-α组分的优化培养基进行培养传代的细胞标记为mdAMSCCtrl。并以同周龄正常小鼠来源AMSC(ncAMSC)采用前述STEMCELLTM公司的MesenCultTM培养基进行扩增传代的第4代细胞作为比较。
采用如前所述的SA-β-gal染色法和Western Blot方法分别检测ncAMSC、mdAMSC、mdAMSCCtrl和mdAMSCPro细胞中β-gal染色的蓝染情况及p21和p16蛋白表达水平。同样表明,优化培养基联合IFN-α干预可纠正代谢性疾病小鼠来源AMSC的衰老表型。
实施例1 AMSCPro可抵抗培养传代诱导的细胞复制性衰老:
小鼠及成人脂肪组织来源的AMSC、AMSCCtrl和AMSCPro细胞分别传至第9代(mAMSC)和第25代(hAMSC),建立培养传代诱导的细胞复制衰老模型。
采用SA-β-gal染色法比较AMSC、AMSCCtrl和AMSCPro细胞的染色情况。光学显微镜计数5个视野的染色情况,并进行统计分析。结果显示,较之AMSC和AMSCCtrl细胞,AMSCPro细胞的蓝染阳性率显著降低(图1A)。
采用Western Blot方法检测AMSC、AMSCCtrl和AMSCPro细胞中p21和p16蛋白表达水平。结果显示,AMSCPro的p21和p16蛋白表达水平明显低于AMSCCtrl和AMSC(图1B)。
采用相对定量Real-time PCR法分析天然AMSC、AMSCCtrl和AMSCPro细胞中衰老相关分泌表型MIP1a、MCP2、CXCL9、MMP3、MMP13、PAI-1、TNF-ɑ和IL-6的表达水平。结果显示,AMSCPro的SASP相关分子表达水平亦明显低于AMSCCtrl和AMSC。
线粒体融合增大及ROS水平增加是MSC衰老的一个重要标志之一。电镜观察细胞线粒体形态,MitoSOXTM Red染色(Thermofisher,M36008)分析线粒体ROS水平。结果显示,AMSCPro的线粒体融合增大程度明显低于AMSCCtrl和AMSC;并且,AMSCPro的MitoSOXTM红色荧光水平亦明显低于AMSCCtrl和AMSC。
采用CCK8法比较hAMSC、hAMSCCtrl和hAMSCPro细胞的增殖情况。结果显示,hAMSCPro细胞的增殖速度明显高于hAMSC和hAMSCCtrl细胞,尤其在多次传代后,细胞增殖速度的差异更为显著。第15代(P15)hAMSCPro细胞较之hAMSC和hAMSCCtrl细胞,其增殖速度分别提高2.2倍和1.6倍;第25代(P25)hAMSCPro细胞较之hAMSC和hAMSCCtrl细胞,其增殖速度分别提高3.3倍和2.5倍。
实施例2 AMSCPro可抵抗TNF-α等诱导的细胞衰老:
建立TNF-α诱导的细胞衰老模型。hAMSC铺6孔板过夜培养后,加入50nM TNF-α处理48小时,然后更换为普通培养基,其中1组采用STEMCELLTM公司的人MesenCultTM扩增培养基,为hAMSC组;1组采用优化培养基培养,为hAMSCPro组;1组采用不含25HC组分的优化培养基进行培养,为hAMSCCtrl组。再培养6天后收集细胞,采用Western Blot方法检测天然hAMSC、hAMSCCtrl和hAMSCPro细胞中p21和p16蛋白表达水平。WB条带采用ChemiScopeWestern Blot成像系统扫描后,再用Image J软件进行灰度比值分析。
如图2显示,AMSCPro可部分抵抗TNF-α诱导的p21和p16蛋白表达升高。另外,在多柔比星、5-氟尿嘧啶等化疗药物,和H2O2和D-半乳糖等诱导的细胞衰老模型中,以及137Csγ射线诱导的辐照细胞衰老模型中也具有类似现象。表明AMSCPro对多种药物、氧化应激和辐照诱导的细胞衰老均有一定抵抗作用。
实施例3优化培养基联合IFN-α干预纠正老年小鼠AMSC的衰老表型:
按前述内容分别制备并培养扩增yAMSC、oAMSC、oAMSCCtrl和oAMSCPro细胞,然后采用SA-β-gal染色法比较这些细胞的染色情况。结果显示,yAMSC细胞的蓝染阳性率显著低于oAMSC(10.1±3.3%v.s.45.1±6.5%,p<0.01);而oAMSCPro细胞的蓝染阳性率显著低于oAMSCCtrl(12.2±4.5%v.s.31.2±5.7%,p<0.01),与yAMSC细胞中的水平相近(12.2±4.5%v.s.10.1±3.3%,p>0.05)。
采用Western Blot方法检测yAMSC、oAMSC、oAMSCCtrl和oAMSCPro细胞中p21和p16蛋白表达水平。结果显示,yAMSC细胞中p21和p16蛋白表达水平显著低于oAMSC;而oAMSCPro的p21和p16蛋白表达水平亦显著低于oAMSC和oAMSCCtrl,与yAMSC细胞中的水平相近(图3)。
实施例4优化培养基联合IFN-α干预纠正代谢性疾病小鼠来源AMSC的衰老表型:
按前述内容分别制备并培养扩增ncAMSC、mdAMSC、mdAMSCCtrl和mdAMSCPro细胞,然后采用SA-β-gal染色法比较这些细胞的染色情况。结果显示,ncAMSC细胞的蓝染阳性率显著低于mdAMSC(14.3±3.6%v.s.50.4±7.5%,p<0.01);而mdAMSCPro细胞的蓝染阳性率显著低于对照组mdAMSCCtrl(17.2±4.6%v.s.38.2±4.7%,p<0.01),与ncAMSC细胞中的水平相近(17.2±4.6%v.s.14.3±3.6%,p>0.05)。
并采用Western Blot方法检测天然ncAMSC、mdAMSC、mdAMSCCtrl和mdAMSCPro细胞中p21和p16蛋白表达水平。结果显示,ncAMSC细胞中p21和p16蛋白表达水平显著低于mdAMSC;而mdAMSCPro的p21和p16蛋白表达水平显著低于mdAMSC和mdAMSCCtrl,与ncAMSC细胞中的水平相近。
另外,通过MitoSOXTM Red染色(Thermofisher,M36008)分析线粒体ROS水平。结果显示,ncAMSC细胞中线粒体ROS水平(根据红色荧光值分析)显著低于mdAMSC;而mdAMSCPro的线粒体ROS水平显著低于mdAMSC和mdAMSCCtrl l,与ncAMSC细胞中的水平相近。
实施例5 AMSCPro具有更好的免疫干预能力:
PBMC(外周血单个核细胞)用PBS重悬至5×106/mL,加入CFSE(BioLegend,终浓度为1μM)后混匀,室温避光孵育10分钟。然后加入4-5倍体积预冷的完全培养基于冰上孵育5分钟,再以完全培养基洗涤细胞3次。第25代(P25)的hAMSC、hAMSCCtrl和hAMSCPro细胞分别以250μg/mL丝裂霉素C处理30分钟,以抑制细胞分裂增殖。将上述处理后的PBMC和hAMSC按5:1比例进行共培养(采用无血清培养基),并加入anti-CD3/CD28进行诱导激活。以不添加hAMSC的PBMC细胞作为对照。细胞共培养3天后,收集PBMC进行CFSE流式检测。
结果显示,与不添加hAMSC的PBMC细胞相比,P25的hAMSC和hAMSCCtrl对PBMC增殖并无显著抑制,而P25 hAMSCPro则能有效抑制PBMC增殖,抑制率达75.5%以上。
同样的,在老年小鼠来源AMSC或高代数mAMSC与小鼠脾细胞的共培养实验中,也观察到类似现象。表明该优化培养基可显著提升老年个体来源AMSC及高代数AMSC的免疫抑制能力。
实施例6 AMSCPro细胞甲基化水平下调
采用Western Blot方法检测第15代AMSC、AMSCCtrl和AMSCPro细胞中DNA甲基转移酶1(DNMT1)和DNMT3a、DNMT3b的蛋白表达水平。结果显示,AMSCPro的DNMT1、DNMT3a和DNMT3b的蛋白表达水平均明显低于AMSCCtrl和AMSC(图4)。另外,采用Abcam的Global DNAMethylation Assay试剂盒分析细胞甲基化水平,也显示AMSCPro具有较低的DNA甲基化程度。
实施例7 AMSCPro具有更好的肝病疗效:
C57小鼠腹腔注射300mg/kg的APAP建立药物诱导的急性肝损伤(DILI)模型。造模后每只小鼠即刻尾静脉输注1×106的第4代yAMSC、oAMSC、oAMSCCtrl和oAMSCPro细胞,并尾静脉注射对应体积的PBS作为对照组(Vehicle)。造模24h后,处死小鼠并收集血清、肝组织等进行肝功能、肝脏病理检测等。
结果显示,年轻小鼠AMSC(yAMSC)对小鼠DILI的疗效显著;而老年小鼠AMSC(oAMSC)较之Vehicle组,肝脏病理反而加重,血清ALT、AST水平也更加升高;oAMSCCtrl较之Vehicle组,对肝脏病理及血清肝功能指标等无显著影响;oAMSCPro则可消除oAMSC输注的不良效应,同样发挥对小鼠DILI模型的疗效,有效降低血清ALT、AST水平,缓解肝脏炎症,减少肝组织坏死灶。
还比较了ncAMSC、mdAMSC、mdAMSCCtrl和mdAMSCPro对上述DILI模型的疗效。结果显示,mdAMSC细胞输注后和Vehicle组相比,非但不能有效改善肝脏病理,反而进一步导致血清ALT、AST水平的升高;mdAMSCCtrl细胞输注后也无明显疗效;而mdAMSCPro细胞输注后则能明显改善肝脏病理和肝功能,对肝脏炎症亦有明确的控制作用。
在LPS/DalN、酒精、ConA、CCl4等诱导的小鼠急性肝损伤模型中,同样显示优化培养基可消除老年小鼠及代谢性疾病来源AMSC对肝损伤小鼠模型的不良效应,因而AMSCPro具有更好的肝病治疗潜能。
实施例8 AMSCPro可在较低剂量时即发挥明确的肝病疗效:
C57小鼠腹腔注射LPS/GalN(10μg/kg的LPS+500mg/kg的D-GalN)建立急性肝衰竭模型。造模后每只小鼠即刻尾静脉输注第6代(P6)AMSC、AMSCCtrl和AMSCPro细胞,各细胞分别采用2×105和1×106两种输注剂量,并尾静脉注射对应体积的PBS作为对照组(Vehicle)。造模6h后,处死小鼠并收集血清、肝组织等进行肝功能、肝脏病理检测等。
结果显示,P6 mAMSC和P6 mAMSCCtrl细胞仅在1×106/只的输注量时显示出一定疗效;而P6 AMSCPro细胞在2×105/只的输注量时,即显示出明确的疗效,有效改善了肝脏病理,降低了血清ALT、AST、TBiL水平(表2)。
在LPS/DalN、酒精、ConA、CCl4等诱导的小鼠急性肝损伤模型中,同样显示AMSCPro在较低剂量时即发挥明确的肝病疗效,具有更好的治疗潜能。
表1
ALT(U/L) | AST(U/L) | TBiL(mg/dl) | |
正常对照 | 30.9±3.2 | 85.2±5.2 | 0.3±0.1 |
Vehicle组 | 3201.3±503.1 | 1886.5±285.5 | 8.3±1.8 |
P6 AMSC(2×105) | 2699.3±420.2 | 1801.6±238.4 | 7.5±1.5 |
P6 AMSCCtrl(2×105) | 2553.5±357.2 | 1690.4±263.7 | 7.1±2.1 |
P6 AMSCPro(2×105) | 586.3±84.3b | 485.3±71.5b | 3.8±0.8b |
P6 AMSC(1×106) | 2313.5±350.7a | 1399.4±161.8a | 5.9±1.1a |
P6 AMSCCtrl(1×106) | 1883.3±401.8a | 1283.1±213.9a | 5.3±0.9a |
P6 AMSCPro(1×106) | 134.5±20.5b | 218.6±31.8b | 1.7±0.5b |
注:数值为MEAN±SD;a—细胞治疗组与Vehicle相比有统计学差异(p<0.05);b—细胞治疗组与Vehicle相比有统计学差异(p<0.01)。
Claims (7)
1.一种抑制间充质干细胞衰老的优化培养基,其特征在于:在AMSC的培养体系中添加25-羟基胆固醇25HC,包含50% DMEM/F12 + 48% MCDB-201、2% MSC适宜胎牛血清、5 μM25HC、1×胰岛素-转铁蛋白-硒ITS -G、1×MEM 非必需氨基酸,10ng/ml EGF、10ng/mlPDGF,50μM β巯基乙醇,2mM L-谷氨酰胺,100μg/ml 青霉素和100U/ml 硫酸链霉素的培养液。
2.权利要求1所述的抑制间充质干细胞衰老的优化培养基用于培养MSC细胞移植生物制剂的应用,所述应用为非疾病治疗目的。
3.权利要求1所述的抑制间充质干细胞衰老的优化培养基联合IFN-α干预纠正AMSC的衰老表型的生物制剂培养的应用,所述应用为非疾病治疗目的。
4.权利要求1所述的抑制间充质干细胞衰老的优化培养基的非疾病治疗目的使用方法,包括如下步骤:
1)脂肪来源的间充质干细胞的制备及常规培养:
小鼠AMSC和成人AMSC按常规方法分离,mAMSC和hAMSC分别采用STEMCELL™公司的小鼠和人MesenCult™扩增试剂盒再添加2mM L-谷氨酰胺,100μg/ml 青霉素和100U/ml 硫酸链霉素作为完全培养基进行培养扩增;AMSC增殖接近75%融合时,进行消化、传代;
2)AMSC的优化培养:
常规培养至第2代的mAMSC细胞和hAMSC细胞,换用优化培养基继续进行传代培养;该优化培养基为:50% DMEM/F12 + 48% MCDB-201、2% MSC适宜胎牛血清、5 μM 25HC、1×胰岛素-转铁蛋白-硒、1×MEM 非必需氨基酸,10ng/ml EGF、10ng/ml PDGF,50μM β巯基乙醇,2mM L-谷氨酰胺,100μg/ml 青霉素和100U/ml 硫酸链霉素的培养液;采用该优化培养基培养传代的细胞标记为AMSCPro;以仍采用前述STEMCELL™公司的小鼠和人MesenCult™培养基进行扩增传代的AMSC标记为AMSC;采用不含25HC组分的优化培养基进行培养传代的细胞标记为AMSCCtrl。
5.权利要求1所述的抑制间充质干细胞衰老的优化培养基用于制备促进权利要求4所述的AMSCPro增殖,提高细胞免疫干预能力,降低细胞甲基化程度试剂的应用。
6.权利要求1所述的抑制间充质干细胞衰老的优化培养基用于制备抵抗复制的AMSC细胞衰老生物制剂的应用。
7.权利要求1所述的抑制间充质干细胞衰老的优化培养基用于制备纠正老年个体或代谢性疾病患者脂肪组织来源AMSC的细胞衰老表型,提高权利要求4所述的AMSCPro的治疗潜能的生物制剂的应用。
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25-Hydroxycholesterol Inhibits Adipogenic Differentiation of C3H10T1/2 Pluripotent Stromal Cells;Dorothy Moseti等;《Int. J. Mol. Sci》;第21卷(第2期);摘要,第2.3小节,第4.2小节;图3-图10 * |
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