寡核苷酸YW002对人牙周膜干细胞增殖、周期、凋亡和早期成骨分化的影响

doi:10.13481/j.1671-587x.20190211

摘要/Abstract

摘要： 目的：探讨免疫刺激型寡核苷酸（ODN） YW002对人牙周膜干细胞（hPDLSCs）增殖、周期、凋亡和成骨分化的影响，阐明ODN YW002对hPDLSCs生物学性能的调控作用。方法：原代培养hPDLSCs至3~5代，将细胞分为空白对照组、免疫抑制型ODN MT01组和ODN YW002组，共培养1、2、3和5d后采用MTT法检测各组hPDLSCs的增殖活性，采用流式细胞术检测各组hPDLSCs的细胞周期和凋亡情况，采用磷酸苯二钠微板法检测各组hPDLSCs中碱性磷酸酶（ALP）活性。结果：与空白对照组比较，ODN YW002组在培养1和3d时hPDLSCs增殖活性升高（P<0.01）；培养5d时hPDLSCs细胞增殖活性升高，但差异无统计学意义（P>0.05）。与ODN MT01组比较，ODN YW002组培养1d时hPDLSCs增殖活性下降（P<0.05）；培养3和5d时细胞增殖活性升高，但差异无统计学意义（P>0.05）。与空白对照组和ODN MT01组比较，ODN YW002组培养1 d时G₀/G₁期hPDLSCs百分比降低，但差异无统计学意义（P>0.05）；S期hPDLSCs百分比升高，但差异无统计学意义（P>0.05）。与空白对照组比较，ODN YW002组培养1 d时hPDLSCs早期和晚期细胞凋亡率降低（P<0.05）；培养2 d时早期和晚期细胞凋亡率降低，但差异无统计学意义（P>0.05）。与ODN MT01组比较，ODN YW002组培养2d时hPDLSCs早期和晚期细胞凋亡率降低（P<0.05）。与空白对照组比较，培养1、3和5d时ODN YW002组hPDLSCs中ALP活性升高（P<0.01）；与ODN MT01组比较，ODN YW002组培养1和5d时hPDLSCs中ALP活性升高（P<0.05），培养3d时ALP活性降低（P<0.05）。结论：ODN YW002可通过抑制细胞凋亡以促进hPDLSCs增殖，且诱导ALP活性升高，提示其有潜在的促hPDLSCs成骨分化功能。

关键词: 寡核苷酸, 牙周膜干细胞, 细胞增殖, 成骨分化, 细胞凋亡

Abstract: Objective: To investigate the effects of oligodeoxynucleotide(ODN) YW002 on the proliferation, cell cycle, apoptosis and early osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs), and to clarify the regulation effect of ODN YW002 on the biological properties of hPDLSCs.Methods: The HPDLSCs were cultured until the third to the fifth generations. The cells were divided into PBS blank control group, ODN MT01 group and ODN YW002 group and incubated for 1, 2, 3 and 5 d. The proliferation activities of the hPDLSCs in various groups were detected by MTT assay. The cell cycle and apoptosis of the hPDLSCs in various groups were detected by flow cytomety.The alkaline phosphates(ALP) activities of the hPDLSCs in various groups were detected by alkaline phosphatase colorimetric assay.Results: Compared with blank control group, the proliferation activities of hPDLSCs in ODN YW002 group at 1 and 3 d after culture were increased (P<0.01); the proliferation activity was inceased at 5 d after culture, but there was no significant difference(P>0.05). Compared with ODN MT01 group, the proliferation activity of hPDLSCs in ODN YW002 group at 1 d after culture was decreased (P<0.05); the proliferation activities were increased at 3 and 5 d after culture, but there were no significant differences(P>0.05). Compared with blank control and ODN MT01 group, the percentage of hPDLSCs in G₀/G₁ phase in ODN YW002 group was decreased at 1 d after cultrue,but there was no significant difference(P>0.05); the percentage of hPDLSCs in S phase was increased at 1 d after culture,but there was no significant difference (P>0.05). Compared with blank control group, the early and late apoptotic rates of hPDLSCs in ODN YW002 group were decreased at 1 d after culture (P<0.05);the early and late apoptotic rates were decreased at 2 d after culture, but there were no significant differences(P>0.05).Compared with ODN MT01 group, the early and late apoptotic rates of hPDLSCs in ODN YW002 group were decreased at 2 d after culture (P<0.05). Compared with blank control group, the ALP activities of hPDLSCs in ODN YW002 group were increased at 1, 3 and 5 d after culture(P<0.01).Compared with ODN MT01 group, the ALP activities of hPDLSCs in ODN YW002 group were increased at 1 and 5 d after culture(P<0.05), but the ALP activity was decreased at 3 d after culture (P<0.05).Conclusion: ODN YW002 can promote the proliferation of hPDLSCs by inhibiting the apoptosis, and increase the ALP activity, suggesting that ODN YW002 has the function of promoting the osteogenic differentiation of hPDLSCs.

Key words: oligodeoxynucleotide, periodontal ligament stem cells, cell proliferation, osteogenic differentiation, apoptosis

中图分类号:

R781

毕雪婷, 申玉芹, 徐晓薇, 李文洁, 王卓然, 林崇韬. 寡核苷酸YW002对人牙周膜干细胞增殖、周期、凋亡和早期成骨分化的影响[J]. 吉林大学学报(医学版), 2019, 45(02): 273-279.

BI Xueting, SHEN Yuqin, XU Xiaowei, LI Wenjie, WANG Zhuoran, LIN Chongtao. Effects of oligodeoxynucleotide YW002 on proliferation, cell cycle, apoptosis and early osteogenic differentiation of human periodontal ligament stem cells[J]. Journal of Jilin University(Medicine Edition), 2019, 45(02): 273-279.

参考文献

[1] TOKUNAGA T, YAMAMOTO H, SHIMADA S, et al. Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity[J]. J Natl Cancer Inst, 1984, 72(4):955-962.
[2] LIN T, PAJARINEN J, NABESHIMA A, et al. Orthopaedic wear particle-induced bone loss and exogenous macrophage infiltration is mitigated by local infusion of NF-κB decoy oligodeoxynucleotide[J]. J Biomed Mater Res A, 2017, 105(11):3169-3175.
[3] YU X Q, WANG Y H, LIN J, et al. Lipopolysaccharides-induced suppression of innate-like B cell apoptosis is enhanced by CpG oligodeoxynucleotide and requires toll-like receptors 2 and 4[J]. PLoS ONE, 2016, 11(11):e0165862.
[4] HUANG Q J,YANG J H,LIN Y,et al. Differential regulation of interleukin 1 receptor and toll-like receptor signaling by MEKK3[J]. Nat Immunol, 2004, 5(1):98-103.
[5] HANAGATA N. CpG oligodeoxynucleotide nanomedicines for the prophylaxis or treatment of cancers, infectious diseases, and allergies[J]. Int J Nanomed, 2017, 16(12):515-531.
[6] AMCHESLAVSKY A, HEMMI H, AKIRA S, et al. Differential contribution of osteoclast-and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis[J]. J Bone Miner Res, 2005, 20(9):1692-1699.
[7] AMCHESLAVSKY A, ZOU W, BAR-SHAVIT Z. Toll-like receptor 9 regulates tumor necrosis factor-alpha expression by different mechanisms implications for osteoclastogenesis[J]. J Biol Chem, 2004, 279(52):54039-54045.
[8] SHEN Y, FENG Z, LIN C, et al. An oligodeoxynucleotide that induces differentiation of bone marrow mesenchymal stem cells to osteoblasts in vitro and reduces alveolar bone loss in rats with periodontitis[J]. Int J Mol Sci, 2012, 13(3):2877-2892.
[9] 高涵,申玉芹,刘引,等.MT01对感染状态人成骨样细胞内ALP活性及mRNA表达的影响[J].口腔医学研究,2016,32(6):580-583.
[10] SEO B M, MIURA M, GRONTHOS S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament[J]. Lancet, 2004, 364(9429):149-155.
[11] 潘春玲,赵海礁,谭丽思,等.牙龈卟啉单胞菌感染MG63细胞对细胞周期的影响[J].口腔医学研究,2015,31(9):863-866.
[12] 于海蛟,申玉芹,刘引,等.特定序列寡核苷酸MT01对牙龈卟啉单胞菌感染的成骨细胞的增殖、细胞周期及凋亡的影响[J].华西口腔医学杂志,2015,33(6):617-621.
[13] ZHOU Y, ZHANG H, ZHANG G, et al. Calcitonin gene related peptide reduces Porphyromonas gingivalis LPS induced TNF α release and apoptosis in osteoblasts[J]. Mol Med Rep, 2018, 17(2):3246-3254.
[14] 阳美霞,张虹亮,李蓝祁,等.含CpG-ODN基序重组抑制素质粒的构建及其对小鼠颗粒细胞相关凋亡基因表达的影响[J].畜牧兽医学报,2018,49(8):1625-1632.
[15] HE X Y, LIU B Y, WU J L, et al. A dual macrophage targeting nanovector for delivery of oligodeoxynucleotides to overcome cancer-associated immunosuppression[J]. ACS Appl Mater Interfaces, 2017, 9(49):42566-42576.
[16] GHAZI B, THONNART N, BAGOT M, et al. KIR3DL2/CpG ODN interaction mediates Sezary syndrome malignant T cell apoptosis[J]. J Invest Dermatol, 2015, 135(1):229-237.
[17] 杨锋,孙玉华,刘佃滨,等.骨质疏松患者骨碱性磷酸酶、钙、磷代谢变化及与牙槽骨骨密度的相关性[J].中国骨质疏松志,2017,23(9):1160-1166.
[18] 周岳,申玉芹,高涵,等.MT01对Pg感染的成骨细胞MG63特异性成骨相关因子表达的影响[J].口腔医学研究,2016,32(1):1-4.
[19] ZHENG Y, LIN C T, HOU X, et al. Enhancing the osteogenic capacity of MG63 cells through N-isopropylacrylamide-modified polyethylenimine-mediated oligodeoxynucleotide MT01 delivery[J]. RSC Adv, 2017, 7(43):27121-27127.
[20] LIN T H, SATO T, BARCAY K R, et al. NF-κB decoy oligodeoxynucleotide enhanced osteogenesis in mesenchymal stem cells exposed to polyethylene particle[J]. Tissue Eng Part A, 2015, 21(5/6):875-883.