CN111135195A - 一种可延缓女性阴道肌肉功能退化的干细胞活性因子组合物的制备方法 - Google Patents
一种可延缓女性阴道肌肉功能退化的干细胞活性因子组合物的制备方法 Download PDFInfo
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Abstract
本发明涉及医用、保健用组合物技术领域,特别涉及一种可延缓女性阴道肌肉功能退化的干细胞活性因子组合物的制备方法,通过获取自体或异体具有活跃再生能力的人体肌肉干细胞,通过体外培养大量扩增,建立长期应用的细胞系。通过在低温条件下超声波破碎细胞,离心后收集上清,过滤以及浓缩得到肌肉干细胞提取物,合并以干细胞培养液的提取物,加以活性稳定剂、防腐剂、增稠剂,再混合以渗透剂,得到活性因子组合物凝胶。本发明提供的有益效果是:细胞衰老情况改善显著,有更强的修复效果。
Description
技术领域
本发明涉及医用、保健用组合物技术领域,特别涉及一种可延缓女性阴道肌肉功能退化的干细胞活性因子组合物的制备方法。
背景技术
妊娠、分娩是妇女特殊的生理过程,在这个过程中,妇女体内的激素水平、代谢等均要经历巨大的变化,这种变化也对阴道的肌肉造成影响。妊娠时激素的变化,及由于子宫及其内的胎儿、羊水的重量逐渐增加,加大了盆底肌肉尤其是进行等位收缩的Ⅰ类肌纤维的前负荷,而长时间前负荷的增加使盆底肌肉群的功能下降,表现为Ⅰ、Ⅱ类肌纤维的功能下降,且作为起支撑作用的Ⅰ类肌纤维下降更为明显。初产妇阴道分娩后,盆底组织经过极度的牵拉、扩张,易出现损伤,使盆底功能下降,出现盆底功能障碍性疾病的可能性增加。阴道肌肉的老化,可导致阴道松弛,严重影响夫妻间生活和谐,所以改善阴道肌肉的老化具有重要的意义。
骨骼肌是机体中最重要的肌肉之一,负责为多种机体功能提供支持。不同于所有其他组织骨骼肌肌肉组织专门控制主动运动,而个体的激烈活动,运动锻炼,甚至日常行动都在不断制造程度不同的肌肉组织和细胞的物理微损伤。肌肉自身的损伤修复是必须通过肌肉干细胞实现的。人体内的相当数量的肌肉干细胞则在不断的生长分化来执行修复活动。用以修复肌肉微损伤。年轻个体的肌肉组织有强大的修复能力。随着人体的老化,肌肉的功能和修复能力会发生显著降低。目前,进程性的肌无力和再生障碍,困扰着广大的老龄人群。各种肌肉萎缩症,例如杜氏肌营养不良症,也会在年幼和年轻的病人身上发生。肌肉萎缩症和再生障碍和人体肌肉干细胞的数量和功能的衰竭直接相关。随年龄增长或一些疾病的发生,肌肉干细胞的衰竭持续发生。衰老个体的肌肉干细胞的再生性修复能力随着其老化也逐渐减退直至消失。然而这个过程并非不可逆。体外的培养和刺激可以促进衰老的肌肉干细胞的再生能力的恢复。
肌肉干细胞的再生能力和体外培养传代数很高。由于具有较强的端粒酶活性,肌肉干细胞的增生能力很强。从少量组织提取细胞开始培养就能得到稳定细胞系,可以体外连续培养至少30代还保持干细胞的活跃再生活力。肌肉干细胞的活跃再生能力主要体现在强大的再生能力,抗氧化危机能力,和大量活性因子的产生或分泌。强大再生能力体现在肌肉干细胞有强大的多功能分化能力,已有的证据表明肌肉干细胞不仅可分化成肌肉细胞,还可分化成成骨细胞,成软骨细胞,心肌细胞,神经细胞等等。肌肉干细胞已被证明可很好的促进骨,软骨,筋膜,心肌和神经组织损伤的修复。
肌肉干细胞的活跃再生能力也体现在肌肉干细胞在正常生理条件下处于更活化的状态。肌肉干细胞由于需要修复肌肉微损伤,相较于其他一些干细胞例如脂肪干细胞,肌肉干细胞是相对被激活的状态。肌肉干细胞也具有较强的自我修复的功能系统。肌肉干细胞的强大抗氧化能力体现在高表达水平的乙醛脱氢酶(ALDH),超氧化物歧化酶(SOD),谷胱甘肽(GSH)等。
综合相关的研究成果可知,肌肉干细胞产生的活性因子至少包括以下4类:(I)常见的细胞生长因子。目前已知的如成纤维细胞生长因子(FGF),胰岛素样生长因子-1(IGF-1),神经生长因子(NGF),血管内皮细胞生长因子(VEGF),血小板衍生的生长因子(PDGF),表皮生长因子(EGF),肝细胞生长因子(HGF),抗炎症类的白细胞介素类生长因子(Interleukin,IL),等等。(II)肌肉细胞和肌纤维生长特需的因子。肌纤维细胞生长因子又称肌细胞因子(MGF),是具有刺激肌纤维细胞生长活性的细胞因子。肌细胞细胞生长因子(MGF)通过与特异的、高亲和的细胞膜受体结合,是调节细胞生长与其他细胞功能等多效应的多肽类物。(III)抗衰老和炎症的活性蛋白。目前已知关键的如Sirt1,Notch,Klotho,progranulin等等。这些活性蛋白与促进细胞生长、增生,以及伤口的愈合和制止长期发炎作用有重要关联。(IV)具有修复功能的囊泡体(exosome)所包含的因子。肌肉干细胞可分泌大量的囊泡体以作用于其他的细胞。囊泡体包含有各种蛋白,多肽,核酸和其他活性因子。
肌肉组织占人体很大比重,而体育运动和肌肉的锻炼可以直接或间接地提升人体整体健康状态。而且因为从肌肉干细胞分泌大量良性因子,肌肉组织会直接影响体内的其他组织,比如骨骼,血管,和粘膜等等。即使开始锻炼的年纪较晚,运动也能逆转人们的肌肉以及其他器官的老化。
肌肉干细胞的来源和血管壁细胞紧密相关。体育锻炼可增加血管健康,增加毛细血管数量和血管壁细胞数量。从而改善肌肉干细胞的生存微环境和功能。因此健康肌肉包含更多的肌肉干细胞。粘膜包括外表皮层,皮层,和皮下组织层。皮下组织层的下面是通常是肌肉组织层。肌肉细胞和粘膜细胞的相互影响很密切,健康肌肉和健康粘膜也有直接关系。健康肌纤维细胞及肌肉干细胞所分泌的活性因子会直接影响着粘膜细胞的生长微环境和功能。体育运动或者肌肉损伤后,肌纤维细胞生长因子(MGF)的表达会大量升高。肌纤维细胞生长因子对肌肉的生长和损伤修复有特异的促进作用。肌纤维细胞生长因子(MGF)促进细胞内脂肪类能量的代谢。在增加肌肉生长的同时,对减少体内脂肪有正面作用。类似于运动后肌纤维细胞生长因子(MGF)对肌肉的直接功效,肌纤维细胞生长因子(MGF)也可以直接作用于粘膜细胞。促进粘膜血管新生,使粘膜萎缩,堵塞或消失的毛细血管内皮细胞新生,粘膜血管网增多,血流增加,粘膜营养充足,从而使粘膜充满活力。人体中肌纤维细胞生长因子(MGF)不仅作用于肌纤维,还克作用于粘膜细胞,而且具有修复表皮、抗衰老、平复皱纹之功效。人体中肌纤维细胞生长因子(MGF)的含量多少直接决定着粘膜的年轻程度。肌纤维细胞生长因子(MGF)促使肌肉纤维细胞分裂增殖,分泌胶原纤维,网状纤维和弹性纤维,保持肌肉弹性和厚度,同时调节粘膜基质中胶原酶、透明质酸的含量和活性,平衡粘膜水分和电解质代谢,改善萎缩细胞的缺水状态,使粘膜滋润光泽。保持粘膜弹性。由于肌纤维细胞生长因子(MGF)是由正常细胞分泌,既无药物类毒性,也无免疫反应,因此在研究其生理作用机制同时,已试用于临床治疗。用于人烧伤、创伤、静脉曲张性粘膜溃疡和角膜损伤,可促进伤口愈合。肌肉干细胞的总提取物还可能包含其他更多的未知的类似于肌纤维细胞生长因子(MGF)的蛋白多肽因子。
肌肉干细胞和成纤维化细胞(fibroblast)在肌肉损伤后再生过程中的作用是互相调节的。肌肉干细胞可分泌因子抑制成纤维化细胞向激活状态的转化(myofibroblast),从而抑制肌肉内的疤痕组织的生成。在肌肉干细胞在数量和活性上有缺失时,成纤维化细胞会掌控肌肉再生过程从而导致组织纤维化和疤痕的产生。肌肉干细胞的提取物在体外可促进肌肉干细胞的生长分化,抑制成纤维化细胞,且降低细胞纤维化分化的因子--转化生长因子(TGF-beta1)的表达。如上所述,体外培养的干细胞会向培养液内分泌各种活性因子,例如各种细胞生长因子,肌纤维细胞生长因子,和囊泡体(exosome)及所携带的因子。这些分泌到细胞外的因子和从细胞内提出的因子可以互为补充,有更强的修复效果。
发明内容
为了解决现有技术的问题,本发明提供了一种可延缓女性阴道肌肉功能退化的干细胞活性因子组合物的制备方法。
本发明所述技术方案如下:
一种可延缓女性阴道肌肉功能退化的干细胞活性因子组合物的制备方法,具体步骤如下:
(1)取新鲜的肌肉组织或受创3-7天内的肌肉,用无菌、无热源的磷酸盐缓冲液(PBS)对肌肉进行冲洗,冲洗后的肌肉组织依次进行组织绞碎,直至组织的颗粒粒径不大于1mm,得到肌肉组织匀浆;
(2)、肌肉组织匀浆按照酶底物浓度(其中优选的,终浓度比为0.6%w/w)加入胶原酶(collagenase)和中性蛋白酶(dispase)的混合液来降解消化,得到单个的肌肉干细胞,于37℃下温育1小时,再经灭酶活后得到肌肉组织酶解液;
(3)肌肉组织酶解液通过2000转/分离心得到细胞,用磷酸缓冲液(PBS)进行冲洗,再离心得到清洗的细胞;
(4)把细胞悬浮于细胞生长培养液,用70微米孔径滤膜进行过滤,滤去杂质,通过滤膜的细胞被收集,加入带胶原蛋白铺层的细胞培养板中,用预板培养法(pre-platetechnique)分离培养出有活跃再生能力的肌肉干细胞(需2-3周);
(5)体外培养的肌肉干细胞每20-22小时增生一倍,肌肉干细胞用干细胞生长培养液培养至78-82%密集度时,移出培养液,用无菌、无热源的PBS清洗细胞3次,加入无血清培养液继续培养48小时,然后收集无血清培养液,将收集的无血清培养液进行过滤去处细胞碎片和杂质,然后移入浓缩管中浓缩,再进行冷冻干燥处理,得到冻干粉,未被收集的细胞利用胰酶(trypsin)消化液消化后,用PBS对细胞清洗3次,离心得到细胞团,把细胞悬浮于磷酸盐缓冲液中(约1毫升每1千万细胞),置于冰上,用超声细胞破碎仪进行超声裂解,得到细胞裂解液,再经过滤,既得到干细胞提取液;
(6)将细胞无血清培养液的提取物的冻干粉加入到得到的干细胞提取液中,得到肌肉干细胞混合提取物;
(7)肌肉干细胞混合提取物内再加入海藻酸钠、黄原胶蛋白活性稳定剂与增稠剂以保持活性因子的活性,加入纳他霉素、壳聚糖防腐剂和氮酮溶液,即得干细胞活性因子组合物凝胶,将干细胞活性因子组合物凝胶置于2℃-8℃条件下备用。
所述的海藻酸钠的质量比为1.0%-10%、黄原胶的质量比为1.0%-10%、纳他霉素的浓度为0.002mg/ml-0.01mg/ml、壳聚糖的浓度为0.001mg/ml-0.01mg/ml、氮酮的浓度为0.005g/ml-0.2g/ml。
所述的细胞生长培养液包括DMEM培养基、体积分数为10%的胎牛血清、浓度为0.2ng/mL-0.8ng/mL的干细胞生长因子(SCF),浓度为10ng/mL-80ng/mL的粒细胞刺激因子(G-CSF)。
使用方法:将细胞因子组合物凝胶以直接推注的方式作用于女性阴道,每日一次,分别于10日后观察阴道肌肉的生理状态。
本发明提供的技术方案带来的有益效果是:
通过获取自体或异体具有活跃再生能力的人体肌肉干细胞,通过体外培养大量扩增,建立长期应用的细胞系。通过在低温条件下超声波破碎细胞,离心后收集上清,过滤以及浓缩得到肌肉干细胞提取物,合并以干细胞培养液的提取物,加以活性稳定剂、防腐剂、增稠剂,再混合以渗透剂,得到活性因子组合物凝胶,体外培养的干细胞会向培养液内分泌各种活性因子,这些分泌到细胞外的因子和从细胞内提出的因子可以互为补充,有更强的修复效果。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述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的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是HE染色观察阴道粘膜状况的图片:图A为对照组粘膜组织;图B为经过干细胞活性因子组合物作用的实验组粘膜组织。
图2是染色观察阴道粘膜组织的图片:图A为对照组粘膜组织;图B为经过干细胞活性因子组合物作用的实验组粘膜组织。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明实施方式作进一步地详细描述。
实施例
一种可延缓女性阴道肌肉功能退化的干细胞活性因子组合物的制备方法,具体如下:
肌肉干细胞的分离和培养方法,包括以下步骤:
1.1取年轻个体或病人自身的三角肌5g。所需肌肉组织可以不限于正常肌肉,可以是受创损伤的病理肌肉。受创损伤的肌肉通常在受创3-7天内包含更活化状态的干细胞,具有更快的生长速度和更强大的再生能力;
1.2用无菌、无热源的磷酸盐缓冲液缓冲液(PBS)对步骤1.1得到的肌肉进行冲洗;
1.3将步骤1.2得到的肌肉组织依次进行组织绞碎,直至组织的颗粒粒径不大于1mm,得到肌肉组织匀浆;
1.5在步骤1.4得到的酶解液通过低速离心得到细胞(2000转每分)。用磷酸缓冲液(PBS)进行冲洗,再离心得到清洗的细胞;
1.6把细胞悬浮于细胞生长培养液,进行过滤(70微米孔径)滤去杂质。通过滤膜的细胞被收集,加入细胞培养板(带胶原蛋白铺层)中;
1.7用预板培养法(pre-plate technique)分离培养出有活跃再生能力的肌肉干细胞(需2-3周)。
上所述,体外培养的干细胞会向培养液内分泌各种活性因子,例如各种细胞生长因子,肌纤维细胞生长因子,和囊泡体(exosome)及所携带的因子。这些分泌到细胞外的因子和从细胞内提出的因子可以互为补充,有更强的修复效果。
2:肌肉干细胞提取物和干细胞培养液提取物的混合物的制备方法包括以下步骤:
2.1建立细胞系后,体外培养的肌肉干细胞可大约每20-22小时增生一倍;
2.2肌肉干细胞用干细胞生长培养液培养至大约80%密集度时,移出培养液,用无菌、无热源的PBS清洗细胞3次;
2.3加入无血清培养液继续培养48小时,然后收集无血清培养液;
2.4将收集的无血清培养液进行过滤去处细胞碎片和杂质,然后移入浓缩管中浓缩,再进行冷冻干燥处理,得到冻干粉;
2.5未被收集的细胞利用用胰酶(trypsin)消化液消化后,用PBS对细胞清洗3次,离心得到细胞团;
2.6把细胞悬浮于小体积的磷酸盐缓冲液中(约1毫升每1千万细胞),置于冰上,用超声细胞破碎仪进行超声裂解,得到细胞裂解液。再经过滤,既得到干细胞提取液;
2.7将细胞无血清培养液的提取物的冻干粉按一定比率加入到上步得到的干细胞提取液中,得到肌肉干细胞混合提取物;
2.8如需要长期保存,干细胞提取液也可进行冷冻干燥处理得到冻干粉,与无血清培养液的冻干粉一起混合;
2.9肌肉干细胞混合提取物内再加入海藻酸钠、黄原胶等蛋白活性稳定剂与增稠剂以保持活性因子的活性。加入纳他霉素、壳聚糖等防腐剂和一定比例的氮酮溶液。其中海藻酸钠最终质量比为5.0%、黄原胶最终质量比为5.0%、纳他霉素最终浓度为0.005mg/mL、壳聚糖最终浓度为0.005mg/mL、氮酮的最终浓度为0.1g/ml,将以上步骤得到的细胞因子组合物凝胶置于2℃-8℃条件下备用;
2.10将以上步骤得到的干细胞活性因子组合物以涂抹的方式作用于经产绵羊阴道,分别以对照组和实验组进行实验,10d后将结果得到的组织进行HE染色,观察肌肉组织老化情况。
实验分析:HE染色步骤
3.1将得到的肌肉组织与阴道粘膜组织进行石蜡切片,继而进行HE染色;
3.2二甲苯脱蜡10min、无水乙醇Ⅰ浸泡5min、无水乙醇Ⅱ浸泡5min、80%乙醇5min、70%乙醇5min、蒸馏水5min、苏木精染色液5min、1%盐酸乙醇30s、水洗30s、0.5%伊红液染色3min、蒸馏水稍洗30s、80%乙醇稍洗30s、95%乙醇1min、无水乙醇10min、二甲苯5min、中性树胶封片,将切片置于显微镜下观察拍照。
实验结果:
(1)通过观察HE染色观察阴道粘膜状况,对照组粘膜组织,粘膜褶皱明显、数量多,皮下脂肪较少,且细胞不规则;经过干细胞活性因子组合物作用的实验组粘膜组织,细胞形态规则,粘膜褶皱减少,皮下脂肪干细胞明显增多,细胞衰老情况改善显著,如图1所示。
(2)通过染色观察阴道粘膜组织,对照组粘膜组织,老化细胞数量多,且细胞不规则,肌肉组织纤维化严重;经过干细胞活性因子组合物作用的实验组粘膜组织,细胞形态规则,肌肉中纤维化组织减少,细胞衰老情况改善显著,如图2所示。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (3)
1.一种可延缓女性阴道肌肉功能退化的干细胞活性因子组合物的制备方法,其特征在于,具体步骤如下:
(1)取新鲜的肌肉组织或受创3-7天内的肌肉,用无菌、无热源的磷酸盐缓冲液对肌肉进行冲洗,冲洗后的肌肉组织依次进行组织绞碎,直至组织的颗粒粒径不大于1mm,得到肌肉组织匀浆;
(3)肌肉组织酶解液通过2000转/分离心得到细胞,用磷酸缓冲液(PBS)进行冲洗,再离心得到清洗的细胞;
(4)把细胞悬浮于细胞生长培养液,用70微米孔径滤膜进行过滤,滤去杂质,通过滤膜的细胞被收集,加入带胶原蛋白铺层的细胞培养板中,用预板培养法分离培养出有活跃再生能力的肌肉干细胞;
(5)体外培养的肌肉干细胞每20-22小时增生一倍,肌肉干细胞用干细胞生长培养液培养至78-82%密集度时,移出培养液,用无菌、无热源的PBS清洗细胞3次,加入无血清培养液继续培养48小时,然后收集无血清培养液,将收集的无血清培养液进行过滤去处细胞碎片和杂质,然后移入浓缩管中浓缩,再进行冷冻干燥处理,得到冻干粉,未被收集的细胞利用胰酶消化液消化后,用PBS对细胞清洗3次,离心得到细胞团,把细胞悬浮于磷酸盐缓冲液中,置于冰上,用超声细胞破碎仪进行超声裂解,得到细胞裂解液,再经过滤,既得到干细胞提取液;
(6)将细胞无血清培养液的提取物的冻干粉加入到得到的干细胞提取液中,得到肌肉干细胞混合提取物;
(7)肌肉干细胞混合提取物内再加入海藻酸钠、黄原胶蛋白活性稳定剂与增稠剂以保持活性因子的活性,加入纳他霉素、壳聚糖防腐剂和氮酮溶液,即得干细胞活性因子组合物凝胶,将干细胞活性因子组合物凝胶置于2℃-8℃条件下备用。
2.根据权利要求1所述的一种可延缓女性阴道肌肉功能退化的干细胞活性因子组合物的制备方法,其特征在于,所述的海藻酸钠的质量比为1.0%-10%、黄原胶的质量比为1.0%-10%、纳他霉素的浓度为0.002mg/ml-0.01mg/ml、壳聚糖的浓度为0.001mg/ml-0.01mg/ml、氮酮的浓度为0.005g/ml-0.2g/ml。
3.根据权利要求1所述的一种可延缓女性阴道肌肉功能退化的干细胞活性因子组合物的制备方法,其特征在于,所述的细胞生长培养液包括DMEM培养基、体积分数为10%的胎牛血清、浓度为0.2ng/mL-0.8ng/mL的干细胞生长因子,浓度为10ng/mL-80ng/mL的粒细胞刺激因子。
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