Chinese Journal of Tissue Engineering Research ›› 2013, Vol. 17 ›› Issue (6): 1116-1121.doi: 10.3969/j.issn.2095-4344.2013.06.028
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Li Li-chun1, He Zhi2, Li Jian-ping1
Received:
2012-04-13
Revised:
2012-06-18
Online:
2013-02-05
Published:
2013-02-05
Contact:
Li Jian-ping, Chief physician, Liaoning Blood Center, Shenyang 110044, Liaoning Province, China
ljp_63@yahoo.com
About author:
Li Li-chun★, Master, Physician, Liaoning Blood Center, Shenyang 110044, Liaoning Province, China lichunli2009@yahoo.cn
CLC Number:
Li Li-chun, He Zhi, Li Jian-ping. In vitro induced differentiation of placenta-derived mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(6): 1116-1121.
2.1 胎盘间充质干细胞的生物学特性 2.1.1 形态学特点 胎盘间充质干细胞的生物学特性与骨髓间充质干细胞相似,均呈平行排列或漩涡样生长,形态类似成纤维细胞。传代细胞三四天可增殖1倍,培养细胞可稳定生长传代,细胞增殖活跃,具有较强的自我更新和多系分化潜能[1]。崔晶等[2]研究以体外培养人胎盘间充质干细胞为研究对象,分为6组,不加尿酸的对照组,以及尿酸0.1,0.2,0.4,0.6,0.8 mmol/L组,结果显示同一时间内,随着尿酸浓度增加,细胞数目逐渐增加,尤其是 0.8 mmol/L尿酸组;同一浓度尿酸,随着培养时间的延长(时间< 5 d),细胞数目也逐渐增加,第5天的时候细胞数目达到高峰,提示尿酸具有呈时间及浓度依赖性的促进胎盘间充质干细胞增殖的作用,尤其是0.8 mmol/L尿酸在培养第5天的时候促进作用最明显。 2.1.2 胎盘间充质干细胞的表面标志 目前大多数学者认为间充质干细胞是属于中胚层的一类多能干细胞,多种来源的间充质干细胞均无统一的特异性表面标志。而目前的研究倾向于认为胎盘间充质干细胞的表面标志与骨髓间充质干细胞的表面标志相类似,如间质干细胞标志SH2、CD105、SH3.4/CD73、CD90/THY-1、CD166、CD106;主要组织相容性复合物HLA-A,B,C;整合蛋白家族CD49e、CD29;透明质酸盐受体CD44等,不表达造血干细胞表面标志CD34、CD45、CD14、HLA-DR和内皮细胞表面标志VWF、Flk-1、CD31、KDR等,除此之外胎盘间充质干细胞不表达共刺激分子CD80、CD86、CD401等[3-8],因此胎盘间充质干细胞的免疫原性低,并且移植入受者体内时,胎盘间充质干细胞可以抑制受者T淋巴细胞的活性功能,减轻移植物抗宿主反应[9]。也有人认为胎盘间充质干细胞可表达SSEA-4、TRA-1-61、Oct-4等胚胎干细胞的表面标志[5,10],说明胎盘间充质干细胞应该是一群极其原始的多能干细胞,较其他来源的成体干细胞具有强大的增殖能力和多向分化的潜能。Fukuchi等[4]采用RT-PCR的方法检测胎盘来源间充质干细胞mRNA的表达,发现了干细胞的标志性基因Oct-4、Rex-1和造血相关的基因HOXB4、GATA-2、CBFβ、fit-1以及器官特异性基因renin、nestin、GFAP等的表达。张睿婷等[11]采用短串联重复序列分析证明所得到的胎盘绒毛膜来源的间充质干细胞生长呈典型的成纤维细胞形态,细胞表达常见的间充质干细胞表面标记CD90、CD73、CD105、CD44,不表达CD45、CD11b和CD34。同时,细胞也表达Nestin和Sox-2,在不同的条件培养基培养状态下,细胞可向成骨、成脂方向分化,这些绒毛膜间充质干细胞能抑制植物血凝素刺激的人外周血单个核细胞分泌γ-干扰素,可见绒毛膜来源的间充质干细胞具有和传统的骨髓来源间充质干细胞相似的生物学特性。 2.1.3 胎盘间充质干细胞的体外分离培养传代的方法 胎盘间充质干细胞的原代培养:取足月剖腹产胎儿的胎盘(按医院规定,产妇及家属知情同意),取胎盘的胎儿面蜕膜组织,用PBS冲洗3-5次,去除血迹,用剪刀将其剪成1.0-2.0 mm3碎片,加入1%Ⅳ型胶原酶,置于37 ℃水浴锅中消化30 min,用DMEM中止胰酶消化,并充分均匀吹打,100目筛网研磨过滤,收集过滤后的细胞悬液,以1 200 r/min离心5 min,加入完全培养基(含低糖DMEM,体积分数为10%胎牛血清,1%双抗)10 mL均匀吹打,血细胞计数板计数,调整细胞浓度,以(2-5)×108 L-1的细胞浓度接种细胞到细胞培养瓶中,置于37 ℃恒温、体积分数为5%CO2、体积分数为21%O2饱和湿度的培养箱培养,每三四天进行换液1次,待细胞生长铺满至瓶底80%-90%时进行传代,以后按常规方法传代,冻存,培养3代以后的细胞用于实验。 胎盘间充质干细胞的传代培养:细胞生长融合至近80%-90%时,弃培养液,加入PBS 5-10 mL冲洗2遍以清除残留培养液,滴加0.05%胰蛋白酶1.0- 2.0 mL,在倒置显微镜下观察细胞形态,1.0-3.0 min后贴壁细胞皱缩逐渐脱落悬浮,加入完全培养液(DMEM,体积分数为10%胎牛血清,1%双抗)中止胰酶消化,反复吹打直至几乎全部细胞脱落悬浮,以 1 200 r/min离心5 min,弃上清,再加培养液吹打混匀,进行细胞计数,将细胞悬液平均分至3个培养皿中,补足培养基10 mL,置于37 ℃恒温、体积分数为5%CO2、体积分数为21%O2、饱和湿度的培养箱中继续培养。 胎盘间充质干细胞的冻存:收集无污染处于对数生长期的细胞(24 h前换液1次),观察细胞状态是否良好,弃培养液,加入PBS 5-10 mL冲洗2遍,以清除残留培养液,用0.05%胰蛋白酶消化成悬浮细胞,加入新的完全培养液吹打均匀,调整细胞数为1×107左右,以1 200 r/min离心5 min弃上清,用0.5 mL胎牛血清吹打均匀细胞,加入用含20%二甲基亚砜的无血清培养基配制的冻存液0.5 mL缓慢滴入细胞悬液中,用吸管轻轻吹打均匀并转入冻存管中,旋紧冻存管盖,用封口膜封口,标记好名称及日期,置于4 ℃冰箱1 h,再转移到-80 ℃冰箱,1周内放入液氮罐中长期存储。 胎盘间充质干细胞的复苏:预先调配水浴锅水温为37-39 ℃,从液氮中或-80 ℃冰箱中取出细胞冻存管,立即置于水浴锅中迅速晃动,直至冻存液充分完全溶解成液体状,用吸管将细胞冻存悬液转移到无菌离心管中,加入5-10 mL的培养液,混匀吹打15-30次,将细胞悬液经1 000-1 200 r/min离心,弃去上清,向沉淀细胞内加入5-10 mL完全培养液,轻轻吹打混匀,将细胞悬液转移到无菌培养皿内,补足培养液进行培养,24 h内换液以去除残余二甲基亚砜,继续培养,传代。 2.2 胎盘来源间充质干细胞的多向分化潜能 间充质干细胞来源于发育早期的中胚层和外胚层,而胎盘的主要部分为羊膜及绒毛膜中胚层,理论上证实了胎盘中含有间充质干细胞的可能性[12-14]。 2.2.1 胎盘间充质干细胞诱导分化为成骨细胞和软骨细胞[15-19] 诱导间充质干细胞分化为软骨时,先将间充质干细胞低速离心,使其形成细胞团块,然后用含转化生长因子β3的无血清培养基培养,组织学显示细胞团块随培养时间的增长而出现富含糖蛋白的细胞外基质。在10-14 d时表达关节软骨特异性的Ⅱ型胶原蛋白。将其在体外培养体系中培养3个月后,组织学显示软骨细胞样陷窝非常明显,细胞外聚集大量的细胞外基质和Ⅱ型胶原蛋白。杜莉莉等[20]采用组织块培养法分离获取人胎盘间充质干细胞,培养的人胎盘间充质干细胞均一表达CD29和CD44,而CD31、CD34、CD45和HLA-DR呈阴性,用含转化生长因子β3、维生素C、胰岛素、地塞米松、转铁蛋白的诱导培养基处理14 d后,甲苯胺蓝染色及Ⅱ型胶原染色阳性,说明该细胞经诱导液作用可定向分化为软骨细胞。 2.2.2 胎盘间充质干细胞诱导分化为成脂肪细胞 取培养3代以后的细胞种于6孔培养板中,密度为1×105孔,待长至40%-50%时,吸去完全培养基,加入诱导体系包括低糖DMEM,体积分数为10%胎牛血清,胰岛素1 mg/L,地塞米松0.1 μmol/L,吲哚美辛200 μmol/L,IBMX 50 mmol/L,进行成脂肪细胞诱导分化,每3 d进行半量换液,10-15 d后大量细胞出现成脂肪细胞形态,10 d后油红O染色检测出现脂肪滴。 2.2.3 胎盘间充质干细胞诱导分化成神经细胞[16,21-22] 取第2代培养细胞,接种于6孔板,生长密度达到60%时加入神经细胞诱导培养基,包括DMEM、2%二甲基亚砜和100 μmol/L丁羟基茴香醚。37 ℃、体积分数为5%CO2、饱和湿度条件下静置培养诱导分化24 h后,经免疫荧光染色鉴定胎盘间充质干细胞诱导分化后神经元特异性的烯醇化酶、胶质纤维酸性蛋白呈阳性表达。 2.2.4 胎盘间充质干细胞诱导分化为类肝样细胞 在红景天苷和细胞因子的作用下,经过21 d的体外培养,胎盘间充质干细胞由长梭形变为多边形、类圆形,并具有肝细胞特异的表型和功能,免疫荧光可检测到诱导后细胞内CK18表达,培养上清液中有白蛋白的分泌,说明胎盘间充质干细胞在红景天苷和成纤维细胞生长因子4作用下在体外能转化为肝细胞样细胞[23]。王晓萃等[24]取孕20 d的大鼠胎盘,经胶原酶消化、密度离心、贴壁筛选法分离培养胎盘源间充质干细胞,在体外培养体系中加入胎肝滤液,模拟体内肝脏微环境,诱导大鼠胎盘间充质干细胞向肝细胞定向分化,结果显示在体外培养条件下,大鼠胎盘间充质干细胞贴壁生长为成纤维样细胞,CD44表面标志物检测阳性;大鼠胎盘间充质干细胞经胎肝滤液诱导14 d时细胞呈现圆形、卵圆形的特征性改变,甲胎蛋白、CK19表达阳性,说明胎肝滤液能够诱导大鼠胎盘间充质干细胞定向分化为肝细胞样细胞。 2.2.5 胎盘间充质干细胞诱导分化为心源性细胞[25] 取对数生长期的细胞,胰酶消化并吹打成单个细胞后,制成2.5×107 L-1细胞悬液,加入IMDM培养液,诱导体系中含有0.1 mmol/L非必需氨基酸、2 mmol/L L-谷氨酰胺、100 U/mL青链霉素、0.1 mmol/L β-巯基乙醇、体积分数为20%胎牛血清。细胞悬液以20 μL/滴接种于直径10 cm的培养皿盖上,培养皿内放入 10 mL无菌PBS,盖上培养皿盖,置于37 ℃、体积分数为5%CO2的培养箱中,3 d后,从单个细胞悬滴中吸出形成的悬浮拟胚体,转入至预先以0.1%明胶涂抹的24孔细胞培养板中。免疫细胞化学和免疫荧光方法证实胎盘间充质干细胞能被诱导分化为心源性细胞。 2.2.6 胎盘间充质干细胞诱导分化为胰岛素分泌细胞取培养至第3代生长旺盛的细胞以胰蛋白酶消化后,体积分数为10%胎牛血清终止消化,PBS洗2遍,悬浮培养于6孔培养液中,加入诱导培养基,诱导体系包含低糖DMEM、1%非必需氨基酸、0.1 mmol/L β-巯基乙醇、1 mmol/L谷氨酰胺、5 μg/L人碱性成纤维生长因子。诱导培养7 d和14 d后,采用双硫腙染色、免疫细胞化学染色和ELISA等方法检测到诱导分化的细胞中有胰岛素的生成与分泌,用RT-PCR方法检测诱导分化的细胞中有PDX-1、Insulin1、Insulin2和Glut2等相关基因mRNA的表达。 2.2.7 胎盘间充质干细胞诱导分化为表皮细胞胎盘间充质干细胞的体外定向诱导模型不仅有助于组织工程研究,同时也对阐明胎盘间充质干细胞增殖分化机制及相关疾病的发病机制具有重要意义。万振洲等[26]分离培养胎盘间充质干细胞,用含体积分数为2%胎牛血清以及20 μg/L表皮生长因子条件培养基诱导,诱导后细胞表面抗原CD29、CD44、CD105阳性,D34、CD45、CD106以及HLA-DR阴性,诱导后细胞形态明显改变,表皮细胞标志物CK19以及CK10免疫荧光染色阳性,提示胎盘间充质干细胞在体外具有分化为表皮细胞的能力,可以作为皮肤组织工程和干细胞研究的一种有效来源。 2.3 胎盘间充质干细胞的应用 2.3.1 造血支持 因脐血移植物中造血干细胞数量相对少而造成造血恢复和免疫重建延迟,并且在移植中存在免疫排斥反应,胎盘间充质干细胞正逐渐发挥其造血支持的作用[27]。胎盘间充质干细胞作为饲养层与脐血造血干祖细胞共培养后,可见胎盘间充质干细胞可显著扩增培养体系中脐血造血干细胞的数量,而且胎盘可以抑制细胞间相互刺激引起的T淋巴细胞增殖的现象,因此胎盘间充质干细胞是脐血来源的造血干细胞体外培养的最佳饲养层细胞,对其具有肯定的体外支持和扩增作用,而且胎盘间充质干细胞的造血支持作用优于骨髓间充质干细胞[3]。何津等[28]采用酶消化法分离培养人胎盘组织中贴壁细胞,其具有间充质干细胞特性,表达多种造血相关因子,包括干细胞因子、粒细胞集落刺激因子、粒细胞-巨噬细胞集落刺激因子、巨噬细胞集落刺激因子和白细胞介素6,说明人胎盘贴壁细胞可为脐血CD34+细胞体外扩增提供潜在滋养层。 2.3.2 组织修复 卢文宁等[29]用酶消化法自人胎盘组织中分离培养胎盘间充质干细胞,移植到心肌梗死模型大鼠4周后超声心动图检测大鼠心脏功能明显改善。人羊膜上皮细胞体外定向分化的肝细胞可以表达至少30多种人成体肝脏所表达的基因,这说明人羊膜上皮细胞来源的肝细胞可以对肝脏疾病进行有效的治疗[30]。李洪秋等[31]研究表明脊髓损伤后,人胎盘间充质干细胞移植能促进脊髓损伤大鼠后肢运动功能改善,移植14 d即可观察到明显改善,28 d BBB评分有明显提高,与对照组大鼠相比,移植术后7,14和28 d,人胎盘间充质干细胞移植能增加损区域伤中碱性成纤维细胞生长因子的表达(P < 0.05),结果说明脊髓损伤后,人胎盘间充质干细胞移植对神经功能恢复有促进作用,其机制可能与其能在宿主脊髓内产生大量碱性成纤维细胞生长因子从而促进脊髓损伤的修复有关。胎盘间充质干细胞诱导生成的胰岛素分泌细胞可移植于糖尿病小鼠体内并能在短期内降低糖尿病小鼠的血糖,改善糖尿病小鼠的一般状况,在一定的时间内起到替代治疗的作用[32]。"
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