增龄对牙周膜干细胞生物学特性的影响

doi:10.24920/J1001-9294.2017.012

摘要/Abstract

摘要：

目的分别获取不同年龄组的人牙周膜干细胞（periodontal ligament stem cells, PDLSC）,对其增殖、分化和凋亡能力进行分析对比,评估增龄因素对其生物学特性的影响。方法选取来源于24位不同患者因正畸减数需要拔除的前磨牙24颗,根据患者年龄分为 A组（年龄 18-20岁）、 B组（年龄 30-35岁）、 C组（年龄 45-50岁）。低密度多克隆法分离培养获得PDLSC。分别检测各组细胞的克隆形成能力、碱性磷酸酶( alkaline phosphatase,ALP)活性,流式细胞仪分析细胞周期和凋亡,衰老相关β半乳糖苷酶(Senescence-Associated β-Galactosidase, SA-βG) 染色分析细胞衰老情况;定量RT-PC检测Runx-2、col-1和ALP 等成骨相关基因的表达水平。结果不同年龄组供体的牙周膜中均分离出PDLSC,其克隆形成率分别为36.67%,22.67%和9.33% (P<0.05),随年龄增加而降低;SA-βG 染色随年龄增加阳性染色细胞数目增多,阳性染色细胞数目的百分比分别为4.14%, 16.39%和 50.38% (P<0.05);成骨分化能力随年龄增加而降低;G2/S期的比例在A,B和C组分别为 38.73%, 29.88%和18.25%(P<0.05),随年龄增加而降低;凋亡早期与晚期的比例在A,B和C组分别为1.57%, 4.56%和5.84% (P<0.05),随年龄增加而升高;成骨诱导7d后ALP染色和成骨相关基因Runx-2、col-1和ALP的表达也显示出年龄增加相关的下降趋势。结论不同年龄组患者的牙周膜中均可分离培养出PDLSC,但其增殖和成骨分化能力随供体年龄的增加而降低。

关键词: 牙周膜干细胞, 增龄, 细胞增殖, 成骨分化

Abstract:

Objective The aim of this study is to investigate the proliferation, differentiation and apoptosis of periodontal ligament stem cells (PDLSC) derived from different aged donors, and to evaluate the effects of aging on the biological characteristics of PDLSC.Methods Periodontal ligament tissues were obtained from 24 surgically extracted human premolars during orthodontics therapy. The specimens were divided into three groups according to the donor’s age. Group A: 18-20 years, group B: 30-35 years, group C: 45-50 years. PDLSC were isolated and cultured using a tissue-block-based enzymolytic method by limiting dilution assay. The colony forming efficiency of PDLSC for three experimental groups was determined. Senescence-Associated β-Galactosidase (SA-β-G) expression in the three groups was examined using β-galactosidase staining working solution. Cell cycle and apoptosis of the PDLSC were examined by the flow cytometry. Alkaline phosphatase (ALP) activity was evaluated by ALP staining. The expression of osteoplastic differentiation related genes Runt-related transcription factor-2 (Runx-2), Collagen Type 1 (col-1), and ALP of PDLSC were examined by quantitative real-time RT-PCR.Results The colony forming efficiency of PDLSC in Group A, B and C was 36.67%, 22.67% and 9.33%, respectively, which decreased with donors’ age (P<0.05). SA-β-G expression of the senescent PDLSC in group A, B and C were 4.14%, 16.39%, 50.38%, respectively (P<0.05). Cells in G2/S phase was 38.73%, 29.88%, 18.25% (P<0.05), and the apoptosis rate was 1.57%, 4.56%, 5.84% (P<0.05), in group A, B and C respectively. The ALP staining in the three groups decreased with the increase of donors’ ages, and the expression of Runx-2, col-1 and ALP decreased gradually from group A to group C (all P<0.05), which indicated the osteogenic differentiation capacity of PDLSC decreased while donor aging.Conclusion Human PDLSC could be successfully isolated from periodontal ligament tissues of different aged donors. However, the proliferation and osteogenic differentiation capacity of PDLSC decreased while donor aging.

Key words: periodontal ligament stem cells, aging, proliferation, osteogenic differentiation

Du Tingting, Liu Na, Gu Bin, Li Ying, Yuan Yifang, Zhang Wei, Zhang Tong. Effects of Aging on the Proliferation and Differentiation Capacity of Human Periodontal Ligament Stem Cells^△[J].Chinese Medical Sciences Journal, 2017, 32(2): 83-91.

图/表 8

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Genes name	Sequences of the primers	bp
GAPDH	F:5'-TCTGCATCATCCAGGAGCTTATT-3' R:5'-TGATACAGAAGGCAGGTTCACAA-3'	238
ALP	F:5'-AGCTTTCGAAGAACAACGGA-3' R:5'-TCTTGAAATGCTTTGGGTCC-3'	239
Runx-2	F:5'-CCCGTGGCCTTCAAGGT-3' R:5'-CGTTACCCGCCATGACAGTA-3'	92
Col-1	F:5'-GCAAGGTGTTGTGCGATGA-3' R:5'-TGGTCGGTGGGTGACTCTG-3'	235

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