Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (15): 2345-2350.doi: 10.3969/j.issn.2095-4344.2014.15.009
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Wang Li-yuan 1, 2, Liu Da-yong1, Jia Zhi1
Online:
2014-04-09
Published:
2014-04-09
Contact:
Jia Zhi, Dental Hospital, Tianjin Medical University, Tianjin 300070, China
About author:
Wang Li-yuan, Master, Associate chief physician, Dental Hospital, Tianjin Medical University, Tianjin 300070, China; Tianjin Stomatological Hospital, Stomatological Hospital of Nankai University, Tianjin 300041, China
Supported by:
Scientific and Technology Development Project of Universities in Tianjin, No. 20110139
CLC Number:
Wang Li-yuan, Liu Da-yong, Jia Zhi. Dynamic expression of Runx2 gene profile during osteogenesis in stem cells from human exfoliated deciduous teeth[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(15): 2345-2350.
2.1 乳牙干细胞生长情况 人乳牙牙髓组织通过Ⅰ型胶原酶和dispase酶消化处理后,可获得单细胞悬液(图1A)。接种到培养瓶后,在倒置显微镜下观察,呈悬浮、折光性强的圆形细胞。细胞体积较小,胞浆均匀、明亮,边界光滑清晰。原代乳牙牙髓细胞接种约2 h后,即有细胞下沉固定不动,24 h后可见有细胞开始伸展(图1B),48 h基本展开。完全展开的细胞呈多形性,大部分呈梭形,成纤维细胞状,体积较小,轮廓清楚;少数为多角形、纺锤状或椭圆形(图1C)。之后,少数几个细胞迅速增殖,呈巢式或集落状生长,细胞间排列紧密,境界不清,周边细胞呈短梭形,体积较小(图1D),10-12 d细胞达到90%汇合。 苏木精-伊红染色观察可见胞核深染,卵圆形或圆形,胞核体积大,内可见一个或多个核仁,胞浆着色浅(图1E)。随着传代次数的增加,细胞的总数呈对数级上升。细胞明显呈编织状、放射状、漩涡状集落生长,形态趋于变长,呈牙髓成纤维细胞样(图1F)。"
2.2 细胞表面抗原检测结果 STRO-1+占总细胞量的(20.01±1.62)%,CD146+占总细胞量的(92.51±1.34)%。 2.3 成脂诱导分化后油红O染色结果 成脂诱导体系培养4周,在相差显微镜下可见变大、变圆的胞体内有串珠样折光明显的脂滴。油红O染色可见橙红色阳性颗粒(图2A)。 2.4 成骨诱导细胞生长情况 诱导培养前2 d,在倒置显微镜下观察,细胞形态和数量没有明显变化。诱导培养4 d后,倒置显微镜下可观察到部分细胞体积增大,胞浆丰富,由原来的长梭形变为立方形,有些带有数个突起,呈星形、多角形。细胞排列呈一定的极性,少数有细长的突起。随着培养时间的延长,某些局部诱导后的细胞可重叠生长,呈结节状,随着细胞的不断增殖,最终铺满连成一片,可复层生长。 2.5 矿化结节形成能力 矿化诱导体系的作用下,前2 d生长缓慢,随后增殖加快,五六天单层长满皿底。八九天后,换液过程中发现较前阶段有较多的细胞漂浮死亡。10-12 d可观察到个别位置的细胞呈复层生长,透光性变差。15-17 d前后可见有散在的极细小沙粒样杂质贴敷培养皿底细胞表面,换液不能去除。行茜素红染色后,提示未经矿化诱导(0 d)的呈阴性(图2B),经矿化液诱导21 d的呈阳性反应(图2C),细胞内有钙盐沉积。 2.6 PCR检测结果 于成骨诱导不同时间点均可检测到成骨分化特异性转录因子Runx2在琼脂糖凝胶上400-500 bp之间有不同程度表达(图2D)。其在成骨诱导分化过程中的变化趋势可描绘成的一条平滑曲线(图2E)。0 d有微弱表达,0-6 d成明显增强趋势,6-12 d显著下降,12-18 d表达再次上升,以后相对平稳。"
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