CN113514648A - 基于胎盘源性间充质干细胞研究治疗卵巢早衰的实验方法 - Google Patents
基于胎盘源性间充质干细胞研究治疗卵巢早衰的实验方法 Download PDFInfo
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Abstract
本发明提供一种基于胎盘源性间充质干细胞研究治疗卵巢早衰的实验方法,包括如下步骤:S1:充质干细胞的提取与分离,所述充质干细胞为胎盘间充质干细胞或者脐带间充质干细胞,所述充质干细胞进行提取与分离后,进行传代。本发明从解决免疫性POF患者自身免疫持续伤害的角度,通过对比不同来源,不同表型,不同比例以及不同代次的间充质干细胞在免疫相关基因位点的表达,体外免疫抑制实验,体内统一的自身免疫性POF小鼠动物模型上的作用的差异化研究,来寻找并验证具有最强免疫负调节/修复功能的细胞来源/制剂,并研究其可能的作用机制,确定针对免疫性卵巢早衰的最佳剂型,然后便于优化工艺流程和使用方法等,找到更好的卵巢早衰的治疗方法。
Description
技术领域
本发明涉及生物干细胞技术领域,具体为基于胎盘源性间充质干细胞研究治疗卵巢早衰的实验方法。
背景技术
卵巢是女性生殖和内分泌器官之一,卵巢的正常功能是保证女性生活质量的重要保障。卵巢早衰(premature ovarian failure,POF)是指女性40岁前由于卵巢内卵泡耗竭或因医源性损伤而发生的卵巢功能衰竭。POF是妇科内分泌领域的常见病,其发病率约为1%~3%,女性40岁以前的发生率为1%,30岁以前的发生率为0.1%。近年来,POF的发病率逐年增高,在女性不孕患者中所占的比例亦有逐年上升的趋势。PO对妇女生殖健康的两大威胁主要是雌激素水平降低及生育能力丧失。雌激素水平降低增加了妇女患骨质疏松和冠心病的危险。而处于生育期的妇女提早闭经,生育能力丧失,会导致一系列心理和社会问题,如易出现抑郁、焦虑、人际交往困难、敌对等情绪、社交方面的心理卫生问题以及婚姻生活质量下降。PO的病因涉及遗传学因素、自身免疫因素、酶学障碍、原始卵巢储备过少、卵泡闭锁或耗竭过快、医源性因素、环境及感染因素、心理因素等。免疫因素是PO常见的原因,相关临床研究显示,大约有20%POF患者是由于自身免疫系统无法识别卵巢组织所导致的,20%~30%的POF伴有其他自身免疫性疾病。
间充质干细胞在卵巢早衰治疗中的应用进展,把MSC对于POF的作用归纳为MSCs可归巢于卵巢,不仅可增加卵巢各级卵泡数,改善内分泌功能,还可增加受孕率。研究表明其主要通过作用于卵巢颗粒细胞、原始生殖细胞及卵巢血管等相关细胞和组织或是发挥其免疫作用和抗氧化应激作用而刺激卵巢功能的恢复。
间充质干细胞(MSC)在卵巢早衰的治疗上展现了可能的美好的前景。但是自身免疫问题不解决,输入的细胞会被迅速清除,持续的免疫伤害会抵消掉干细胞的修复作用,即使短期有效果也难以持续。缺少统一的自身免疫性POF小鼠动物模型上的作用的差异化研究方法,进而去寻找并验证具有最强免疫负调节/修复功能的细胞来源/制剂,并研究其可能的作用机制,确定针对免疫性卵巢早衰的最佳剂型。
发明内容
本发明目的在于提供一种基于胎盘源性间充质干细胞研究治疗卵巢早衰的实验方法,能够方便研究人员寻找并验证具有最强免疫负调节/修复功能的细胞来源/制剂,并研究其作用机制。
为达成上述目的,本发明提出如下技术方案:基于胎盘源性间充质干细胞研究治疗卵巢早衰的实验方法,包括如下步骤:
S1:充质干细胞的提取与分离,所述充质干细胞为胎盘间充质干细胞或者脐带间充质干细胞,所述充质干细胞进行提取与分离后,进行传代;
S2:体外实验,使用流式细胞术分析比较不同来源,不同代次的充质干细胞之间表型的差异,用实时定量PCR检测不同充质干细胞在免疫相关基因表达上的差异,确认不超过3个最强免疫负调节品种,将PHA刺激的人外周血单个核细胞,与充质干细胞共培养,ELISA检测上清中IFN-γ、TNF-α的分泌;
S3:动物实验,利用小鼠透明带多肽3建立自身免疫性卵巢早衰小鼠模型,通过酶联检测定性检测小鼠血清抗透明带抗体筛选小鼠后,部分给予尾静脉注射不同的MSCs悬液,部分给予尾静脉注射生理盐水/PBS0.5 mL处理。
进一步的,在本发明中,所述S1中的胎盘间充质干细胞的提取与分离步骤如下,准备胎盘,分离胎盘,用含三抗的生理盐水反复冲洗,直至绒胎膜组织无血污,变成乳白色,从胎膜中剥离绒毛膜组织,并剪成约2.0mm×2.0mm大小的小块,加入0.2%胶原酶消化60min,用200目金属滤网过滤,用生理盐水洗涤后用Lonza间充质专用培养基悬浮所获细胞,用免疫磁珠法筛选中目的细胞,以9000/cm2的密度接种于六孔板/细胞培养瓶中,置于含5%二氧化碳的37℃细胞培养箱中培养24h后,全量换液去除未贴壁细胞,以后每隔3~4d换液1次,直到细胞融合度达到85%时进行1∶3传代。
进一步的,在本发明中,胎盘间充质干细胞的提取与分离步骤中所述胎盘为新鲜娩出的无菌无钙化的胎盘,使用无菌剪刀将胎盘剪开分离。
进一步的,在本发明中,所述S1中的脐带间充质干细胞的提取与分离步骤如下,取新鲜娩出的脐带组织,用生理盐水反复冲洗直至脐带组织无血污,变成乳白色,用无菌器械从脐带中剥取华通氏胶,剪成2mm*2mm小块,接种于培养皿或者培养瓶中,放入含5%二氧化碳的37℃细胞培养箱中培养30分钟后加入培养基没过组织块,第6天全量换液、第11天半量换液、镜下观察出现大量细胞时,进行传代。
进一步的,在本发明中,定义所述S2中3个最强免疫负调节品种为YRK-101-MSC,YRK-102-MSC,YRK-103-MSC;
定义所述S3中设置若干分组,分为空白对照,脐带间充质干细胞组,胎盘间充质干细胞组各10例,YRK-101-MSC,YRK-102-MSC,YRK-103-MSC组各20例,并使用ELISA法检测小鼠血清血清雌二醇、促卵泡素、抗缪勒管激素、透明带糖蛋白抗体;并进行卵巢组织HE染色,使用ELISA法检测血清CD4+CD25+Treg细胞相关因子γ干扰素、转化生长因子β1(TGF-β1);使用流式细胞术检测分析脾细胞中T细胞亚群变化。
有益效果,本申请的技术方案具备如下技术效果:
本发明从解决免疫性POF患者自身免疫持续伤害的角度,通过对比不同来源,不同表型,不同比例以及不同代次的间充质干细胞在免疫相关基因位点的表达,体外免疫抑制实验,体内统一的自身免疫性POF小鼠动物模型上的作用的差异化研究,来寻找并验证具有最强免疫负调节/修复功能的细胞来源/制剂,并研究其可能的作用机制,确定针对免疫性卵巢早衰的最佳剂型,然后便于优化工艺流程和使用方法等,找到更好的卵巢早衰的治疗方法。
应当理解,前述构思以及在下面更加详细地描述的额外构思的所有组合只要在这样的构思不相互矛盾的情况下都可以被视为本公开的发明主题的一部分。
从下面的描述中可以更加全面地理解本发明教导的前述和其他方面、实施例和特征。本发明的其他附加方面例如示例性实施方式的特征和/或有益效果将在下面的描述中显见,或通过根据本发明教导的具体实施方式的实践中得知。
具体实施方式
为了更了解本发明的技术内容,特举具体实施例说明如下。本公开的实施例不必定意在包括本发明的所有方面。应当理解,本发明所公开的构思和实施例并不限于任何实施方式。另外,本发明公开的一些方面可以单独使用,或者与本发明公开的其他方面的任何适当组合来使用。
本实施例提供一种基于胎盘源性间充质干细胞研究治疗卵巢早衰的实验方法,能够方便研究人员寻找并验证具有最强免疫负调节/修复功能的细胞来源/制剂,并研究其作用机制,实施例如下:
胎盘间充质干细胞(PMSC)的提取与分离:取新鲜娩出的无菌无钙化的胎盘,用无菌剪刀将胎盘剪开,用含三抗的生理盐水反复冲洗,直至绒胎膜组织无血污,变成乳白色,从胎膜中剥离绒毛膜组织,并剪成约2.0mm×2.0mm大小的小块,加入0.2%胶原酶消化60min,用200目金属滤网过滤。用生理盐水洗涤后用Lonza间充质专用培养基悬浮所获细胞,用免疫磁珠法筛选中目的细胞,以9000/cm2的密度接种于六孔板/细胞培养瓶中,置于含5%二氧化碳的37℃细胞培养箱中培养24h后,全量换液去除未贴壁细胞,以后每隔3~4d换液1次。直到细胞融合度达到85%时进行1∶3传代。
脐带间充质干细胞(UMSC)的分离:取新鲜娩出的脐带组织,用生理盐水反复冲洗直至脐带组织无血污,变成乳白色,用无菌器械从脐带中剥取华通氏胶,剪成2mm*2mm小块,接种于培养皿/培养瓶中,放入含5%二氧化碳的37℃细胞培养箱中培养30分钟后加入培养基没过组织块,第6天全量换液、第11天半量换液、镜下观察出现大量细胞时,进行传代。
体外实验:
1. 使用流式细胞术分析比较不同来源,不同代次的MSC之间表型的差异。
2. 用实时定量PCR检测不同MSC在免疫相关基因(COX-2,IL-1α,IL-1β,IL-6和IL-8等)表达上的差异,确认不超过3个最强免疫负调节品种(YRK-101-MSC,YRK-102-MSC,YRK-103-MSC)。
3.将PHA刺激的人外周血单个核细胞(peripheral blood mononuclear cells,PBMC)与各种MSC共培养,ELISA检测上清中IFN-γ(interfern-γ)、TNF-α的分泌。
动物实验:
目的:对比YRK-101-MSC,YRK-102-MSC,YRK-103-MSC与脐带胎盘来源的间充质干细胞移植在改善/恢复自身免疫性卵巢早衰小鼠卵巢功能上是否存在显著差异并探讨导致差异的可能的机理。
分组:空白对照,脐带间充质干细胞组,胎盘间充质干细胞组各10例, YRK-101-MSC,YRK-102-MSC,YRK-103-MSC组各20例。
方法:利用小鼠透明带多肽3(zona pellucida glycoprotein 3,ZP3)建立自身免疫性卵巢早衰小鼠模型,通过酶联检测(enzyme linked immunosorbent assay,ELIS A)定性检测小鼠血清抗透明带抗体(Anti-zona pellucida antibody,AZPAb)筛选小鼠后,部分给予尾静脉注射MSCs悬液1X10^6/0.5mL;或者生理盐水/PBS0.5 mL处理(空白对照)。
观察指标:ELISA法检测小鼠血清血清雌二醇(E2)、促卵泡素(FSH)、抗缪勒管激素(AMH),透明带糖蛋白抗体(AZPAb);卵巢组织HE染色;ELISA法检测血清CD4+CD25+Treg细胞相关因子γ干扰素(IFN-γ)、转化生长因子β1(TGF-β1);流式细胞术检测分析脾细胞中T细胞亚群变化,最优结果命名为YRK-001-MSC。
本实施例还给出了临床研究:目的:检测应用本实施例方法研究改善/恢复自身免疫性卵巢早衰(POF)患者的卵巢功能上是否有显著作用。
方法:选取自身免疫性POF患者300例,PMSC静脉输注+传统激素治疗(HRT)组为实验组(按照1X10^6个PMSC/公斤体重,按照2X10^6个PMSC/公斤体重,按照3X10^6个PMSC/公斤体重每隔一周输注一次共计3次),传统激素治疗组(HRT)为对照组各150例,PMSC治疗前,治疗中每30天,治疗后1,3,6,12,24,36月检测卵巢功能(血清促卵泡激素(FSH)和黄体生成激素(LH)雌二醇),免疫功能(T淋巴细胞亚群/IMF(有条件的话),IFN-γ,IL2,IL6),自体免疫(入组阳性指标)。
入组标准:
1、POF:⑴年龄<40岁。⑵闭经时间≥6个月。⑶两次(间隔1个月以上)血FSH>40mIU/ml。
2、自体免疫抗体指标任一指标阳性:AoAb、ANA、ds-DNA、A-TG(也有推荐StCA+AAA(抗类固醇生成细胞抗体(steroid-cell autoantibody,StCA)和抗肾上腺皮质抗体(anti-adrenocortical autoantibody,AAA)。
3、免疫紊乱/炎性表现指标:T淋巴细胞亚群(CD8+增高|CD4+/CD8+下降)/IMF增高[14]+IFN-γ,IL2,IL6[15-16]+B超、活检。
排除标准:(1)严重感染;(2)肝肾功能严重损伤;(3)有子宫或卵巢切除病史;(4)就诊前近3个月已接受免疫抑制剂或激素治疗;(5)异种蛋白质过敏史/干细胞禁忌症。
终止标准:自然妊娠,细胞输注严重过敏反应。
统计:两组都有的指标(免疫紊乱与卵巢功能)直接用检验结果做方差分析,没有对应指标的(自体免疫指标)治疗前后做T检验。
结果:实验组免疫紊乱/炎性表现指标,自体免疫抗体指标,卵巢功能恢复程度好于对照,自然妊娠率好于对照。
利用本实施例的方法,能够便于研究人员来寻找并验证具有最强免疫负调节/修复功能的细胞来源/制剂,并研究其可能的作用机制,确定针对免疫性卵巢早衰的最佳剂型,然后便于优化工艺流程和使用方法等,找到更好的卵巢早衰的治疗方法。
虽然本发明已以较佳实施例揭露如上,然其并非用以限定本发明。本发明所属技术领域中具有通常知识者,在不脱离本发明的精神和范围内,当可作各种的更动与润饰。因此,本发明的保护范围当视权利要求书所界定者为准。
Claims (5)
1.基于胎盘源性间充质干细胞研究治疗卵巢早衰的实验方法,其特征在于:包括如下步骤:
S1:充质干细胞的提取与分离,所述充质干细胞为胎盘间充质干细胞或者脐带间充质干细胞,所述充质干细胞进行提取与分离后,进行传代;
S2:体外实验,使用流式细胞术分析比较不同来源,不同代次的充质干细胞之间表型的差异,用实时定量PCR检测不同充质干细胞在免疫相关基因表达上的差异,确认不超过3个最强免疫负调节品种,将PHA刺激的人外周血单个核细胞,与充质干细胞共培养,ELISA检测上清中IFN-γ、TNF-α的分泌;
S3:动物实验,利用小鼠透明带多肽3建立自身免疫性卵巢早衰小鼠模型,通过酶联检测定性检测小鼠血清抗透明带抗体筛选小鼠后,部分给予尾静脉注射不同的MSCs悬液,部分给予尾静脉注射生理盐水/PBS0.5 mL处理。
2.根据权利要求1所述的基于胎盘源性间充质干细胞研究治疗卵巢早衰的实验方法,其特征在于:所述S1中的胎盘间充质干细胞的提取与分离步骤如下,准备胎盘,分离胎盘,用含三抗的生理盐水反复冲洗,直至绒胎膜组织无血污,变成乳白色,从胎膜中剥离绒毛膜组织,并剪成约2.0mm×2.0mm大小的小块,加入0.2%胶原酶消化60min,用200目金属滤网过滤,用生理盐水洗涤后用Lonza间充质专用培养基悬浮所获细胞,用免疫磁珠法筛选中目的细胞,以9000/cm2的密度接种于六孔板/细胞培养瓶中,置于含5%二氧化碳的37℃细胞培养箱中培养24h后,全量换液去除未贴壁细胞,以后每隔3~4d换液1次,直到细胞融合度达到85%时进行1∶3传代。
3.根据权利要求2所述的基于胎盘源性间充质干细胞研究治疗卵巢早衰的实验方法,其特征在于:胎盘间充质干细胞的提取与分离步骤中所述胎盘为新鲜娩出的无菌无钙化的胎盘,使用无菌剪刀将胎盘剪开分离。
4.根据权利要求1所述的基于胎盘源性间充质干细胞研究治疗卵巢早衰的实验方法,其特征在于:所述S1中的脐带间充质干细胞的提取与分离步骤如下,取新鲜娩出的脐带组织,用生理盐水反复冲洗直至脐带组织无血污,变成乳白色,用无菌器械从脐带中剥取华通氏胶,剪成2mm*2mm小块,接种于培养皿或者培养瓶中,放入含5%二氧化碳的37℃细胞培养箱中培养30分钟后加入培养基没过组织块,第6天全量换液、第11天半量换液、镜下观察出现大量细胞时,进行传代。
5.根据权利要求1所述的基于胎盘源性间充质干细胞研究治疗卵巢早衰的实验方法,其特征在于:定义所述S2中3个最强免疫负调节品种为YRK-101-MSC,YRK-102-MSC,YRK-103-MSC;
定义所述S3中设置若干分组,分为空白对照,脐带间充质干细胞组,胎盘间充质干细胞组各10例,YRK-101-MSC,YRK-102-MSC,YRK-103-MSC组各20例,并使用ELISA法检测小鼠血清血清雌二醇、促卵泡素、抗缪勒管激素、透明带糖蛋白抗体;并进行卵巢组织HE染色,使用ELISA法检测血清CD4+CD25+Treg细胞相关因子γ干扰素、转化生长因子β1(TGF-β1);使用流式细胞术检测分析脾细胞中T细胞亚群变化。
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