视网膜干细胞培养上清液诱导骨髓间充质干细胞的分化

doi:10.3969/j.issn.2095-4344.2013.40.001

摘要/Abstract

摘要：

背景：骨髓间充质干细胞向神经细胞诱导分化可为神经系统受损伤后的修复和再生带来了新的希望。
目的：探讨骨髓间充质干细胞在视网膜干细胞培养上清液诱导条件下向神经元细胞分化。
方法：采用全骨髓培养方法，用视网膜干细胞培养上清液诱导骨髓间充质干细胞，通过免疫荧光染色鉴定其分化的结果。
结果与结论：诱导72 h，骨髓间充质干细胞表达神经干细胞的特异性抗体巢蛋白和神经元中的标志性微管相关蛋白微管相关蛋白2。视网膜干细胞培养上清液能够促进骨髓间充质干细胞向神经元样细胞分化，提示视网膜干细胞可能分泌神经生长因子。

关键词: 干细胞, 骨髓干细胞, 骨髓间充质干细胞, 诱导分化, 神经元样细胞, 视网膜干细培养上清液, 巢蛋白, 微管相关蛋白2, 干细胞图片文章

Abstract:

BACKGROUND: Differentiation of bone marrow mesenchymal stem cells into nerve cells has brought a new hope for the repair and regeneration after nervous system injuries.
OBJECTIVE: To study the neuron-like differentiation of bone marrow mesenchymal stem cells induced by the retinal stem cell culture supernatant.
METHODS: The whole bone marrow culture was applied. The differentiation results of bone marrow mesenchymal stem cells induced by retinal stem cell culture supernatants were identified by immunofluorescence method.
RESULTS AND CONCLUSION: After 72 hours of induction, bone marrow mesenchymal stem cells express nestin and microtubule-associated protein 2. Retinal stem cell culture supernatant can promote bone marrow mesenchymal stem cells differentiating into neuron-like cells, suggesting that retinal stem cells may secrete nerve growth factor.

Key words: stem cells, mesenchymal stem cells, neural stem cells, retina, cell differentiation, microtubule proteins

中图分类号:

R394.2

汪卓，王丹，郝冬海，李博. 视网膜干细胞培养上清液诱导骨髓间充质干细胞的分化[J]. 中国组织工程研究, 2013, 17(40): 7027-7027.

Wang Zhuo, Wang Dan, Hao Dong-hai, Li Bo. Retinal stem cell culture supernatant induces differentiation of bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(40): 7027-7027.

图/表（结果） 7

2.1 骨髓间充质干细胞的原代培养 原代培养骨髓间充质干细胞，1 d后镜下可见大量细胞贴壁，仍有部分未贴壁的红细胞，贴壁细胞形态不一，但可见较少的纺锤形细胞；3 d时，贴壁细胞增殖迅速，细胞密度增加，纺锤形细胞呈放射状排列；5-7 d时，细胞增殖明显，铺满瓶底，此时细胞呈梭形纤维样排列，成旋涡状或放射状；10 d时，经多次换液后非目标细胞被逐渐清除，且骨髓间充质干细胞形态均匀一致，成窝轮状，基本不见圆形细胞，见图1。

2.2 骨髓间充质干细胞免疫细胞化学鉴定 对培养细胞进行CD34，CD45，CD90，CD105标记，结果显示培养第3代细胞CD90，CD105均为阳性表达，CD34，CD45均为阴性表达，见图2。

2.3 视网膜干细胞的形态学特点 原代培养的视网膜干细胞多为细胞团，部分为单细胞并呈圆形，边缘发亮，折光性好。采用2%血清培养1 d后，大部分细胞贴壁，形状不一，培养两三天后，可见到数量较少的细胞团(2-8个细胞组成)形成，细胞团内的细胞呈圆形，较大，边界清楚，折光性强，成圆形或桑椹状聚集。细胞分离后第7天进行第1次传代。随后，每隔4-7 d传代1次，见图4。

2.5 视网膜干细胞免疫细胞化学鉴定 对培养细胞进行巢蛋白，微管相关蛋白2标记，结果显示培养第3代细胞巢蛋白、微管相关蛋白2均为阳性细胞，在荧光显微镜下可见细胞核大，呈明显红色强荧光，结果显示来自新生1 d大鼠视网膜细胞大多数表现视网膜干细胞的特性，见图5-10。

2.6 免疫荧光鉴定诱导后的骨髓间充质干细胞 诱导后骨髓间充质干细胞巢蛋白，微管相关蛋白2均有表达，见图11-16。未诱导的骨髓间充质干细胞不表达巢蛋白、微管相关蛋白2。

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引言

材料方法

设计：体外实验，细胞形态学观察。

时间及地点：于2011年9月至2012年8月在辽宁省眼科干细胞组织工程重点实验室完成。

材料：

实验动物：健康成年SD大鼠4只，雌雄兼用，体质量180-200 g，新生1 d鼠6只，购自北京华阜康生物科技股份有限公司，实验动物许可证号：SCXK2010-1001，动物饲养环境为清洁级，实验过程对动物的处置符合相关动物伦理学要求^[8] 。

实验方法：

骨髓间充质干细胞的原代培养：SD大鼠过量麻醉处死后，体积分数75%乙醇消毒，切开双侧后肢皮肤，摘除完整股骨，仔细剔除骨表面的残存肌肉及软组织，无菌条件下用1 mL注射器抽取骨髓，置于DMEM/F12培养基中，离心半径6 cm，1 500 r/min离心5 min，弃去上清将沉淀物用含体积分数10%胎牛血清的DMEM /F12培养基稀释、吹打均匀后，接种于25 mL培养瓶，置于37 ℃，体积分数5% CO₂培养箱中培养^[9-10] 。第3天全量换液，随后两三天换液1次，每日于光镜下观察细胞生长情况。

骨髓间充质干细胞的传代培养：培养细胞达70%融合度时进行传代，弃去培养基，用37 ℃预热PBS冲洗3次，加入37 ℃预热的胰酶替代物1.5 mL，于37 ℃培养箱中消化2-5 min，镜下观察细胞形态变圆并有部分细胞脱壁时，加入含体积分数10%胎牛血清培养基终止消化，玻璃吸管轻柔吹打仍贴壁细胞后将细胞悬液移入15 mL离心管，1 500 r/min离心3 min。含体积分数10%胎牛血清新鲜培养基重悬细胞，计数，以4×10⁷L^-1细胞浓度接种于24孔板，2 mL/wells，置于37 ℃、体积分数5%CO₂培养箱中培养。

视网膜干细胞的原代与传代方法：无菌取出新生1 d SD大鼠完整眼球，PBS冲洗3次，在T式显微镜下，去除多余的组织及视神经，取出视网膜，机械法吹打制成单细胞悬液，计数，调整细胞浓度为(1.0-2.0)×10⁹ L^-1后，接种于含体积分数1% N₂及10 µg/L 碱性成纤维细胞生长因子和表皮生长因子的DMEM/ F12 培养基的25 mL培养瓶中，置于37 ℃，体积分数5%CO₂培养箱中培养，每日于光镜下观察细胞生长情况，2 d后全量换液。5-8 d后，胰酶消化，传代。

骨髓间充质干细胞免疫荧光鉴定：细胞培养至第5代，将细胞以1×10⁸ L^-1细胞浓度接种于24孔板中37 ℃孵育24 h，PBS洗3次，加入40 g/L多聚甲醛固定10 min后弃去，加入5%Triton透化15 min后弃去，加入1% BSA封闭30 min后弃去，加入稀释好的一抗CD105，37 ℃孵育60 min后弃去，加入适量的二抗，37 ℃避光孵育30 min，免疫荧光检测表达。空白对照用PBS代替一抗。

视网膜干细胞免疫细胞化学鉴定：将细胞以1×10⁸ L^-1细胞浓度接种于24孔板中孵育24 h，PBS洗3次，加入40 g/L多聚甲醛固定10 min后弃去，加入5% Triton透化15 min后，1%BSA封闭30 min，加入稀释好的巢蛋白，微管相关蛋白2一抗，37 ℃孵育 60 min，加入适量的二抗，37 ℃孵育30 min，避光操作。免疫荧光检测表达。免疫空白对照用PBS代替一抗。

视网膜干细胞上清液收集：将细胞以1×10⁸ L^-1细胞浓度接种于24孔板中，孵育24 h后，吸取上清液离心后，收集离心后的上清液贮存。

体外诱导骨髓间充质干细胞向神经元样细胞分化：取体外扩增，第2代后的骨髓间充质干细胞，以1×10⁵浓度接种于24孔板内，其中6个孔加入含体积分数10%血清的DMEM/F12培养基进行培养，6孔加入视网膜干细胞的上清液进行培养72 h。

免疫荧光鉴定诱导后的骨髓间充质干细胞：弃上清后PBS洗3次，加入4 g/L多聚甲醛固定10 min后弃去，加入5%Triton透化15 min后弃去，加入1%BSA封闭30 min后弃去，加入稀释好的巢蛋白，微管相关蛋白2一抗，37 ℃孵育60 min后弃去，加入适量的二抗，37 ℃避光孵育30 min，免疫荧光检测表达。空白对照用PBS代替一抗。

主要观察指标：进行间充质干细胞在神经细胞上清液的诱导下分化为神经元样细胞的形态学观察及免疫荧光鉴定。