CN101591643B - 一种人羊膜组织来源间充质细胞提取及传代培养方法 - Google Patents
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Abstract
本发明涉及“一种人羊膜组织来源间充质细胞提取及传代培养方法”属于再生医学工程领域。羊膜组织来源间充质细胞hAMCs的提取方法，包括去除羊膜上皮细胞和分离纯化间充质细胞步骤，其特征在于：所述分离纯化间充质细胞步骤中是采用0.1％胶原酶III、IV和V进行消化处理；间充质细胞hAMCs的体外传代培养方法，采用DMEM/F12培养基，所述该培养基中加入有生长因子和羊膜上皮细胞培液的上清液。本发明的提取方法，其分离程度高，能够从30ml体积的羊膜组织中最大限度获取1×107/cells数量的细胞，细胞成活率大于90％。本发明的传代培养方法，使间充质细胞的增殖又快又好，在培养了35天还有增殖趋向。
Description
技术领域
本发明涉及再生医学工程领域，特别是人羊膜间充质细胞(Human amniotic mesenchymalcells，hAMCs)作为细胞移植和组织工程组织构建的细胞来源，为组织修复和组织再造提供新途径。
背景技术
间充质干细胞(mesenchymal stem cells，MSCs)作为一种多潜能细胞，由于其在体内所定居的组织不同，微环境中的细胞因子、生长因子等各种调控物质不同，可促进MSCs向不同的谱系分化。体外培养时，在不同的诱导条件下MSCs能够向不同的组织分化，不仅可分化为成骨细胞、脂肪细胞、软骨细胞和肌肉细胞等，而且还可以跨胚层分化，如可以分化为外胚层的神经元、神经胶质细胞及内胚层的肝细胞等。
人羊膜间充质细胞(Human amniotic mesenchymal cells，hAMCs)是间充质细胞的一类，Gianandrea等(Gianandrea Pasquinelli，Pierluigi Tazzari，Francesca Ricci，et al。UltrastructuralCharacteristics of Human Mesenchymal Stromal(Stem)Cells Derived from Bone Marrow andTerm Placenta。Ultrastructural Pathology，2007，31：23-31)通过应用透射电镜对hAMCs的超微结构进行研究，发现hAMCs表现为具有上皮和间质特征的超微结构，上皮样特征包括非肠腔型表面微绒毛、细胞间连接，间质特征包括：rEF轮廓、脂滴等，这些特点从结构上阐述了其具有多项分化潜能。
Sakuragawa等(Sakuragawa N，Kakinuma K，Kikuchi A，Okano H，Uchida S，Kamo I，et al.Human amnion mesenchyme cells express phenotypes of neuroglial progenitor cells.J NeurosciRes.2004；78：208-214.Erratum in：J Neurosci Res，2005；79：72)发现未分化hAMCs表达神经前体细胞表型，87.7％的表达巢蛋白，93.1％表达musashi 1，用神经细胞诱导剂后出现双极或多极突起，巢蛋白和musashi 1表达程度增加。hAMCs还可以被抗-Tuj 1、抗-NF-M和抗-GFAP染色，并且诱导后阳性染色细胞数将明显增加，Tuj 1、NF-M和GFAP均是神经细胞的特异性标志，这些结果表明在合适的培养条件下，hAMCs可以分化为神经细胞。
有人(Zhao P，Ise H，Hongo M，Ota M，Konishi I，Nikaido T.Human amniotic mesenchymalcells have some characteristics of cardiomyocytes.Transplantation.2005，79：528-535)把hAMCs与新生鼠心脏分离块共同培养并植入发生心肌梗死的鼠心脏，hAMCs表达心脏特异性转录因子GATA4、心脏特异性基因、L-型心脏特异性钙通道α单位(α1c)和一过性外向性钾离子通道(Kv4.3)，同时发现经b-FGF诱导后，hAMCs表达心肌特异性转录因子Nkx2.5、心脏特异性标志心钠素，经activinA诱导后表达心脏特异性基因α肌球蛋白重链(α-MHC)。hAMCs在心肌梗死的瘢痕组织中能存活至少2个月，能与心肌组织整合并分化成为心肌样细胞。
Tomoharu(Tomoharu TAMAGAWA，Satoshi OI，Isamu ISHIWATA，et al。Differentiation ofmesenchymal cells derived from human amniotic membranes into hepatocyte-like cells in vitro。Human Cell，2007；20：77-84)发现hAMCs在诱导分化之后表达葡萄糖-6-磷酸酶(G6Pase)、鸟氨酸氨氨甲酰基转移酶(OTC)和肝细胞核移植4α(HNF-4α)的mRNA，并且具有储存糖原的功能，而该功能是肝细胞的特异性功能之一，说明hAMCs可以分化成为肝细胞样细胞。
hAMCs还可以分化成为软骨细胞(Yash M.Kolambkar，Alexandra Peister，Shay Soker，et al。Chondrogenic differentiation of amniotic fluid-derived stem cells。J Mol Hist，2007，38：405-413)，并表达软骨相关基因。此外来源于羊膜的MCs在体外可以自发分化形成血管内皮细胞，加入血管内皮生长因子(VEGF)可以促进这一过程。
综上所述，hAMCs具有多向分化潜能，因此在组织损伤再生和修复重建中具有广阔的应用前景。
鉴于羊膜组织来源间充质细胞(hAMCs)不表达端粒酶活性，尚不能确认其为间充质干细胞，但此类细胞表达胚胎干细胞、间质干细胞以及神经干细胞特异性标记蛋白，具有低分化原始细胞功能，在特定培养条件下具有多分化功能，为此我们可以利用其治疗器官缺损及功能丧失等退行性疾病，如骨和软骨缺损、心肌梗塞、缺血性脑损伤和急性肝功能衰竭等。其原理初步断定，由组织营养和细胞替代作用达到组织整合与器官修复的目的。
发明内容
针对羊膜组织来源间充质细胞(hAMCs)的上述特点和优点，本发明提供一种hAMCs提取方法，其分离程度高，同时还提供一种传代培养方法，使间充质细胞的增殖又快又好。
羊膜组织来源间充质细胞hAMCs的提取方法，包括去除羊膜上皮细胞和分离纯化间充质细胞步骤，其特征在于：所述分离纯化间充质细胞步骤中是采用0.1％胶原酶III、IV和V进行消化处理。
所述胶原酶III、IV和V的重量比为1∶1∶1.5。
所述消化处理方法为37℃震摇30min，转速400～600rpm。
间充质细胞hAMCs的体外传代培养方法，采用DMEM/F12培养基，所述该培养基中加入有生长因子和羊膜上皮细胞培液的上清液。
所述生长因子的加入量为30-100ug/L，羊膜上皮细胞培养液的上清液的加入量为5-20％(v/v)。
所述培养条件为：38.5℃，5％CO2，饱和湿度的CO2培养箱中培养，隔天换液。
所述培养基中还添加有生物活性因子。
所述生物活性因子为EGF、bFGF、TGF-β或/和IGF。
人羊膜是一种天然高分子生物材料，有厚的基底膜，含III、IV、V型胶原、糖蛋白、蛋白多糖、整合素和层粘连蛋白等多种成分，任何组织包括羊膜组织的细胞外基质成分包裹于细胞周围，在分离hAMCs过程中如何消化细胞外基质成分是影响细胞分离质量和数量的关键问题。也就是说在对组织进行处理时，既要破坏组织让细胞释放出来，又要保证细胞的活性，利用蛋白酶能够很好处理羊膜组织中的蛋白质成分，但包裹于羊膜间充质细胞外的胶原成分较其它组织复杂，其中III、IV、V型胶原是构成羊膜的主要基质成分，组织结构致密，有韧性，是影响羊膜间充质细胞分离的主要屏障，为此我们利用不同比例配制的III、IV、V型胶原酶混合液，进行羊膜间充质细胞分离，能够从羊膜组织中最大限度获取hAMCs，能够从30ml体积的羊膜组织中最大限度获取1×107/cells数量的细胞，细胞成活率大于90％。
其鉴于羊膜间充质干细胞不表达端粒酶活性，为能够维持其体外传代培养，也发明了体外培养hAMCs的特定无血清条件培养基。
羊膜上皮细胞具有三种胚原基层细胞的分化潜能，内胚层(肝、胰)、中胚层(心肌细胞)和外胚层(神经细胞)(Miki T.Lehmann T，Cai H，et al.：Stem Cell Characteristics of AmnioticEpithelial Cells.Stem Cells.2005 Aug 4；[Epub ahead of print])。羊膜上皮细胞具有合成释放儿茶酚胺(Elwan MA.：Synthesis of dopamine from L-3，4-dihydroxyphenylalanine by humanamniotic epithelial cells.Eur J Pharmacol.1998 Jul 31；354(1)：R1-2；Elwan MA，Ishii T，Sakuragawa N.et al.：Characterization of the dopamine transporter gene expression and bindingsites in cultured human amniotic epithelial cells.Neurosci Lett.2003 May 15；342(1-2)：61-4.)、乙酰胆碱(Horikoshi T，Fujii T.Kawashima K，et al.：Acetylcholine increase in amniotic fluid ofexperimental rats for intrauterine growth retardation.Life Sci.2003 Mar 28；72(18-19)：2145-9；Uchida S，Suzuki Y，Araie M，Kashiwagi K，et al.：Factors secreted by human amniotic epithelialcells promote the survival of rat retinal ganglion cells.Neurosci Lett.2003 Apr 24；341(1)：1-4.)神经递质的神经生物学功能，还可以分泌神经营养因子等生物活性物质，具有神经营养功能。
目前已检测到脑源性神经营养因子(brain-derived neurotrophic factor，BDNF)、NT-3、神经生长因子(nerve growth factor，NGF)和睫状神经营养因子(ciliary neurotrophic factor，CNTF)(Uchida S，Suzuki Y，Araie M，Kashiwagi K，et al.：Factors secreted by human amniotic epithelialcells promote the survival of rat retinal ganglion cells.Neurosci Lett.2003 Apr 24；341(1)：1-4；Marvin KW，Keelan JA，Eykholt RL，et al.：Expression of angiogenic and neurotrophic factors inthe human amnion and choriodecidua.Am J Obstet Gynecol.2002 Sep；187(3)：728-34.)，提示羊膜组织通过分泌释放神经营养因子进入羊水，对胚胎神经发育的早期阶段具有重要调节作用(Uchida S，Inanaga Y.Kobayashi M，et al.：Neurotrophic function of conditioned mediumfrom human amniotic epithelial cells.J Neurosci Res.2000 Nov 15；62(4)：585-90.)。此外，羊膜上皮细胞还可以分泌合成TGF、EGF、bFGF和HGF等生长因子，因此应该可以运用去细胞羊膜基质作为滋养层促进体外培养胚胎干细胞的分化，运用羊膜组织提取物促进未成熟卵细胞体外成熟。因此本发明人在进行间充质细胞的体外培养中加入了羊膜上皮细胞培养液的上清液，发现比常规的培养基，其细胞增殖快，在培养至35天后，细胞还有增殖的倾向。
在该培养基中还可以加入EGF、bFGF、TGF-β、IGF等生物活性因子，对体外培养hAMCs的增殖、传代有促进作用，提高细胞增殖活性。
附图说明
图1 Brud渗入培养7天后间充质细胞增殖情况照片图
图2细胞增殖曲线图
具体实施方案
实施例1：人羊膜间充质细胞提取步骤：
一、去除羊膜上皮细胞：
1.将用生理盐水浸泡的新鲜羊膜取回实验室后，迅速用4℃的0.9％NaCl(含有三抗：青霉素、链霉素和庆大霉素)进行清洗。直至羊膜两面无任何杂质、血渍、然后用0.9％NaCl进行冲洗后放入无菌盐水瓶，送入无菌间。将羊膜放入三抗液中洗三遍后放置其中浸泡2h。浸泡后用D-hank’s洗3-5遍。
2、羊膜剪成碎片约1mm3
3、转入50ml离心管或合适的容器，加入D-hank’s液稀释的等体积0.25％Trypsin(胰酶)(终浓度为0.125％)
4、混匀后放置生物振荡器内，37℃震摇15min，转速100rpm
5、离心弃上清液
6、加入D-hank’s液稀释的等体积0.25％Trypsin(终浓度为0.125％)并混匀
7、37℃震摇30min，转速400～600rpm
8、弃混悬液，收集羊膜组织
9、重复6～8步骤2次，完全去除羊膜上皮细胞。
二、分离纯化羊膜间充质细胞
10、将羊膜组织用D-hank’s液漂洗干净；
11、组织碎片以配好的0.1％胶原酶III、IV和V(1∶1∶1.5)混合液；
12、37℃震摇30min，转速400～600rpm；
13、细胞筛过滤混悬液；
14、离心收集羊膜间质细胞并计数，视实验情况重复步骤12-13；
15、按适当浓度接种间质细胞，进行培养。
实施例2：羊膜组织来源间充质细胞的培养传代
1、羊膜组织来源间充质细胞的特定培养液为DMEM/F12+(50ug/L)生长因子+(15％占培养液的总体积)羊膜上皮细胞培养液的上清液，38.5℃，5％CO2，饱和湿度的CO2培养箱中培养，隔天换液。
2、待羊膜间充质细胞长到70％～90％时，加入0.025％胰酶进行消化处理4min，加入含10％FBS的DMEM/F12终止消化。
3、终止消化后，将贴壁的间充质细胞小心吹打，形成细胞混悬液。
4、将细胞混悬液进行离心处理，1200r/min，3min。
5、弃去离心后上清液，加入常规培养液进行培养或进行冷冻保存。
实验例：
1、BrdU渗入检测：将小部分实施例2的步骤3中的细胞悬混液于6孔培养板内培养，培养基为DMEM/F12，内含1mol/L的Brud，连续培养7天，每3天换液一次。
Brud渗入培养7天后，离心制成细胞涂片，进行抗Brud免疫酶细胞化学染色，检测细胞增殖情况。见图1
2、MTT实验检测：取一部分实施例2的步骤3的细胞悬浮液于细胞计数器中进行计数。
(1)接种：以每孔103-104个细胞接种于96孔板中，96孔板培养基液同实施例2的步骤1培养基，每孔200ul；37度CO2箱陪养24h。
(2)培养细胞：条件同上一步骤
(3)呈色：培养3天后，开始定时检测细胞增殖状态，检测标本每孔加MTT溶液(5mg/ml用PBS <；ph＝7.4>；配)20ul.加入继续孵育4小时，终止培养，小心吸弃孔内培养上清液，对于悬浮细胞需要离心后再吸弃孔内培养上清液。每孔加150ul DMSO，振荡10分钟，使结晶物充分融解。
(4)比色：选择492nm波长，在酶联免疫监测仪上测定各孔光吸收值，记录结果，以时间为横坐标，吸光值为纵坐标绘制细胞生长曲线。见图2。
Brdu掺入实验和MTT实验，分别从形态学和细胞生物学角度证实了hAEC的增殖活性。人羊膜间充质细胞在含有BrdU的培养基中孵育7d后，免疫荧光化学染色显示培养hAMCs部分呈红色荧光标记的BrdU阳性(见图1)，表明培养人hAMCs中有增殖活性细胞的存在。从细胞增殖曲线图(图2)看，细胞体外培养35天仍然具有增殖趋向。
Claims (2)
1.羊膜组织来源间充质细胞hAMCs的提取方法，包括去除羊膜上皮细胞和分离纯化间充质细胞步骤，其特征在于：所述分离纯化间充质细胞步骤中是采用0.1％胶原酶III、IV和V进行消化处理，所述胶原酶III、IV和V的重量比为1∶1∶1.5。
2.根据权利要求1所述的羊膜组织来源间充质细胞的提取方法，所述消化处理方法为37℃震摇30min，转速400～600rpm。
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Effective date of registration: 20171122 Address after: 100195 East 1000 meters of the former Sha Jian village, Sujia Town, Haidian District, Beijing Patentee after: Beijing Kerun Vitech Bio & Technology Co., Ltd. Address before: 100049 Beijing city Haidian District Liao Gong Zhuang Sijiqing Town, No. 166 Patentee before: Li Rongqi |
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