白细胞介素1β对人牙周膜干细胞和骨髓间充质干细胞骨向分化的影响

doi:10.3877/cma.j.issn.1674-1366.2020.02.005

中华口腔医学研究杂志(电子版) ›› 2020, Vol. 14 ›› Issue (02) : 88 -94. doi: 10.3877/cma.j.issn.1674-1366.2020.02.005

所属专题：文献；

基础研究

白细胞介素1β对人牙周膜干细胞和骨髓间充质干细胞骨向分化的影响

张静¹^,^†(

), 李晨晨², 孟箭³

^1. 徐州市医学科学研究所　221009；徐州医科大学徐州临床学院　221009；徐州市中心医院口腔科　221009
^2. 徐州市中心医院口腔科　221009
^3. 徐州医科大学徐州临床学院　221009；徐州市中心医院口腔科　221009

收稿日期:2019-10-22 出版日期:2020-04-01
通信作者: 张静

The effect of interleukin-1β on the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells

Jing Zhang¹^,^†(), Chenchen Li², Jian Meng³

^1. Xuzhou Institute of Medical Science, Xuzhou 221009, China; Xuzhou Clinical College of Xuzhou Medical University, Xuzhou 221009, China; Department of Stomatology, Xuzhou Central Hospital, Xuzhou 221009, China
^2. Department of Stomatology, Xuzhou Central Hospital, Xuzhou 221009, China
^3. Xuzhou Clinical College of Xuzhou Medical University, Xuzhou 221009, China; Department of Stomatology, Xuzhou Central Hospital, Xuzhou 221009, China

Received:2019-10-22 Published:2020-04-01
Corresponding author: Jing Zhang
About author:
Corresponding author: Zhang Jing, Email: jingz77@163.com
Supported by:
National Natural Science Foundation of China(31700814); Science and Technology Project of Xuzhou(KC18032)

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引用本文：

张静, 李晨晨, 孟箭. 白细胞介素1β对人牙周膜干细胞和骨髓间充质干细胞骨向分化的影响[J]. 中华口腔医学研究杂志(电子版), 2020, 14(02): 88-94.

Jing Zhang, Chenchen Li, Jian Meng. The effect of interleukin-1β on the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2020, 14(02): 88-94.

目的

研究白细胞介素1β（IL-1β）作用下人牙周膜干细胞（PDLSC）和骨髓间充质干细胞（BMSC）骨向分化的差异。

方法

应用茜素红染色、碱性磷酸酶（ALP）活性检测、细胞增殖能力（MTT）法和实时荧光聚合酶链反应（PCR）对PDLSC和BMSC在IL-1β作用下的骨向分化能力进行检测，并比较两者之间的差异。实验组为含有IL-1β的各处理组，对照组内未加IL-1β。采用单因素方差分析和t检验对数据进行统计分析。

结果

随IL-1β浓度的增高，PDLSC形成矿化结节减少，茜素红染色逐渐变浅；而BMSC骨向分化能力未见明显减弱；ALP活性实验结果表明，在7 d时PDLSC各实验组与对照组相比差异具有统计学意义（F = 2361.11，P<0.001），BMSC各实验组与对照组相比ALP活性差异具有统计学意义（F = 240.68，P<0.001）；在14 d时PDLSC随炎症因子浓度增高，ALP活性显著降低，差异具有统计学意义（F = 1519.89，P<0.001），BMSC在IL-1β浓度为0.001、0.01、1 ng/mL的实验组与对照组相比ALP活性差异具有统计学意义（F = 22.52，P<0.001），两种干细胞在IL-1β最大浓度时（1 ng/mL）ALP活性最低；MTT结果表明，IL-1β能抑制干细胞的增殖能力，与PDLSC相比，BMSC增殖能力较低。实时荧光PCR结果显示，与对照组相比，两种干细胞实验组内ALP、Col-1、OCN和Runx2 mRNA表达水平均降低，BMSC内实验组中骨向分化基因表达与PDLSC内实验组相比，差异均有统计学意义（P<0.05）。

结论

在IL-1β作用下，BMSC的成骨能力较PDLSC高。

关键词: 炎症，细胞因子类, 干细胞, 细胞分化, 骨向分化

Objective

To investigate the effect of interleukin-1β (IL-1β) on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and bone marrow mesenchymal stem cells (BMSCs) .

Methods

The osteogenic differentiation ability of PDLSCs and BMSCs was tested by Alizarin Red staining, alkaline phosphatase (ALP) activity, MTT, and real-time PCR with the presence of IL-1β. The PDLSCs or BMSCs cultured with IL-1β were the experimental groups, while the control group was cultured without IL-1β. One-Way ANOVA and t-test were applied to perform the statistical analysis of the data.

Results

Mineralization staining showed that an inhibitory effect of IL-1β on the osteogenic differentiation of PDLSCs with increasing dose. However, BMSCs were more resistant to the inflammatory cytokine compared to PDLSCs in terms of a stronger alizarin red staining. The results of ALP activity test showed that a significant difference between the PDLSCs experimental groups and the control group was found at 7 days (F = 2361.11, P<0.001) ; for BMSCs, a similar result was also observed between the experimental groups and the control group (F = 240.68, P<0.001) . At 14 days, with the increase of inflammatory factor concentration, the activity of ALP in PDLSCs decreased significantly (F = 1519.89, P<0.001) . The ALP activity of the experimental groups with IL-1β concentration of 0.001, 0.01 and 1 ng/mL in BMSCs was significantly different from that of the control group (F = 22.52, P<0.001) . ALP activity of the two kinds of stem cells was the lowest with the presence of the maximum concentration of IL-1β (1 ng/mL) . Proliferation results showed that IL-1β exhibited inhibitory effects on cell proliferation and BMSCs possessed a lower proliferative potential than PDLSCs. Real-time PCR analysis of osteoblast lineage gene expression revealed that the IL-1β suppressed the expression levels of ALP, Col-1, OCN and Runx2 mRNA in PDLSCs and BMSCs at 14 days compared with the control group. The difference of mRNA expression levels was statistically significant between the BMSCs experimental group and the PDLSCs experimental group.

Conclusions

A significant difference on the osteogenesis between PDLSCs and BMSCs was observed. Compared with PDLSCs, BMSCs showed a stronger capacity of modulation in local microenvironment via anti-inflammatory functions.

Key words: Inflammation, cytokines, Stem cells, Cell differentiation, Osteogenic differentiation

表1 实时定量PCR引物序列

基因	引物序列
ALP	正向：5′-GGACCATTCCCACGTCTTCAC-3′
?	反向：5′-CCTTGTAGCCAGGCCCATTG-3′
Col-1	正向：5′-CCAGAAG AACTGGTACATCAGCAA-3′
?	反向：5′-CGCCATACTCGAACTGGAATC-3′
OCN	正向：5′-AGCAAAGGTGCAGCCTTTGT-3′
?	反向：5′-GCG CCTGGGTCTCTTCACT-3′
Runx2	正向：5′-CCCGTGGCCTTCAAGGT-3′
?	反向：5′-CGTTACCCGCCATGACAGTA-3′
β-actin	正向：5′-CAGGCTGTGCTATCCCTGTA-3′
?	反向：5′-CATACCCCTCGTAGATGG GC-3′

表1 实时定量PCR引物序列

基因	引物序列
ALP	正向：5′-GGACCATTCCCACGTCTTCAC-3′
?	反向：5′-CCTTGTAGCCAGGCCCATTG-3′
Col-1	正向：5′-CCAGAAG AACTGGTACATCAGCAA-3′
?	反向：5′-CGCCATACTCGAACTGGAATC-3′
OCN	正向：5′-AGCAAAGGTGCAGCCTTTGT-3′
?	反向：5′-GCG CCTGGGTCTCTTCACT-3′
Runx2	正向：5′-CCCGTGGCCTTCAAGGT-3′
?	反向：5′-CGTTACCCGCCATGACAGTA-3′
β-actin	正向：5′-CAGGCTGTGCTATCCCTGTA-3′
?	反向：5′-CATACCCCTCGTAGATGG GC-3′

图1 牙周膜干细胞（PDLSC）和骨髓间充质干细胞（BMSC）茜素红染色

图2 不同浓度IL-1β处理牙周膜干细胞（PDLSC）和骨髓间充质干细胞（BMSC）后各组相对碱性磷酸酶（ALP）活性统计图　相同时间点上同种干细胞各实验组与对照组（0 ng/mL）相比，^aP<0.001；A：7 d；B：14 d

表2 不同浓度IL-1β对牙周膜干细胞（PDLSC）和骨髓间充质干细胞（BMSC）碱性磷酸酶（ALP）活性的影响（ ± s）

组别		样本量	7d		14 d
组别		样本量	PDLSC	BMSC	PDLSC	BMSC
实验组		9	0.864 ± 0.015	0.664 ± 0.014	1.229 ± 0.023	1.518 ± 0.038
对照组		?	?	?	?	?
?	0.001 ng/mL	9	0.702 ± 0.014^a	0.618 ± 0.012^a	0.894 ± 0.032^a	1.633 ± 0.057^a
?	0.01 ng/mL	9	0.665 ± 0.015^a	0.573 ± 0.014^a	0.819 ± 0.018^a	1.343 ± 0.028^a
?	0.1 ng/mL	9	0.452 ± 0.010^a	0.545 ± 0.014^a	0.582 ± 0.018^a	1.482 ± 0.011
?	1 ng/mL	9	0.193 ± 0.007^a	0.440 ± 0.010^a	0.305 ± 0.010^a	1.312 ± 0.131^a
F值		?	2361.11	240.68	1519.89	22.52
P值		?	<0.001	<0.001	<0.001	<0.001

表2 不同浓度IL-1β对牙周膜干细胞（PDLSC）和骨髓间充质干细胞（BMSC）碱性磷酸酶（ALP）活性的影响（ ± s）

组别		样本量	7d		14 d
组别		样本量	PDLSC	BMSC	PDLSC	BMSC
实验组		9	0.864 ± 0.015	0.664 ± 0.014	1.229 ± 0.023	1.518 ± 0.038
对照组		?	?	?	?	?
?	0.001 ng/mL	9	0.702 ± 0.014^a	0.618 ± 0.012^a	0.894 ± 0.032^a	1.633 ± 0.057^a
?	0.01 ng/mL	9	0.665 ± 0.015^a	0.573 ± 0.014^a	0.819 ± 0.018^a	1.343 ± 0.028^a
?	0.1 ng/mL	9	0.452 ± 0.010^a	0.545 ± 0.014^a	0.582 ± 0.018^a	1.482 ± 0.011
?	1 ng/mL	9	0.193 ± 0.007^a	0.440 ± 0.010^a	0.305 ± 0.010^a	1.312 ± 0.131^a
F值		?	2361.11	240.68	1519.89	22.52
P值		?	<0.001	<0.001	<0.001	<0.001

图3 不同浓度IL-1β处理牙周膜干细胞（PDLSC）和骨髓间充质干细胞（BMSC）后各组相对细胞增殖活性统计图　相同时间点上同种干细胞各实验组与对照组（0 ng/mL）相比，^aP<0.001；A：1 d；B：4 d；C：7 d

表3 不同浓度IL-1β对牙周膜干细胞（PDLSC）和骨髓间充质干细胞（BMSC）细胞增殖活性的影响（ ± s）

组别		样本量	1d		4d		7d
组别		样本量	PDLSC	BMSC	PDLSC	BMSC	PDLSC	BMSC
实验组		9	0.384 ± 0.004	0.271 ± 0.006	0.487 ± 0.006	0.355 ± 0.001	0.773 ± 0.005	0.443 ± 0.009
对照组		?	?	?	?	?	?	?
?	0.001 ng/mL	9	0.337 ± 0.003^a	0.236 ± 0.006^a	0.421 ± 0.004^a	0.323 ± 0.002^a	0.739 ± 0.006^a	0.383 ± 0.006^a
?	0.01 ng/mL	9	0.338 ± 0.003^a	0.232 ± 0.003^a	0.401 ± 0.004^a	0.334 ± 0.002^a	0.715 ± 0.002^a	0.377 ± 0.004^a
?	0.1 ng/mL	9	0.289 ± 0.003^a	0.256 ± 0.004^a	0.393 ± 0.005^a	0.334 ± 0.002^a	0.796 ± 0.008^a	0.387 ± 0.002^a
?	1 ng/mL	9	0.355 ± 0.012^a	0.266 ± 0.011	0.448 ± 0.004^a	0.394 ± 0.002^a	0.632 ± 0.008	0.358 ± 0.006^a
F值		?	86.33	18.48	172.29	538.77	308.15	77.21
P值		?	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

表3 不同浓度IL-1β对牙周膜干细胞（PDLSC）和骨髓间充质干细胞（BMSC）细胞增殖活性的影响（ ± s）

组别		样本量	1d		4d		7d
组别		样本量	PDLSC	BMSC	PDLSC	BMSC	PDLSC	BMSC
实验组		9	0.384 ± 0.004	0.271 ± 0.006	0.487 ± 0.006	0.355 ± 0.001	0.773 ± 0.005	0.443 ± 0.009
对照组		?	?	?	?	?	?	?
?	0.001 ng/mL	9	0.337 ± 0.003^a	0.236 ± 0.006^a	0.421 ± 0.004^a	0.323 ± 0.002^a	0.739 ± 0.006^a	0.383 ± 0.006^a
?	0.01 ng/mL	9	0.338 ± 0.003^a	0.232 ± 0.003^a	0.401 ± 0.004^a	0.334 ± 0.002^a	0.715 ± 0.002^a	0.377 ± 0.004^a
?	0.1 ng/mL	9	0.289 ± 0.003^a	0.256 ± 0.004^a	0.393 ± 0.005^a	0.334 ± 0.002^a	0.796 ± 0.008^a	0.387 ± 0.002^a
?	1 ng/mL	9	0.355 ± 0.012^a	0.266 ± 0.011	0.448 ± 0.004^a	0.394 ± 0.002^a	0.632 ± 0.008	0.358 ± 0.006^a
F值		?	86.33	18.48	172.29	538.77	308.15	77.21
P值		?	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

表4 IL-1β作用下牙周膜干细胞（PDLSC）和骨髓间充质干细胞（BMSC）骨向分化相关基因mRNA表达水平的统计结果（ ± s）

组别	样本量	ALP	Col-1	OCN	Runx2
PDLSC对照组	12	1.000 ± 0.117^a	1.000 ± 0.110^a	1.000 ± 0.124^a	1.000 ± 0.108^a
PDLSC实验组	12	0.053 ± 0.004^b	0.446 ± 0.038^b	0.291 ± 0.052^b	0.116 ± 0.017^b
BMSC对照组	12	13.157 ± 1.517^c	2.235 ± 0.278	4.286 ± 0.570^c	2.142 ± 0.374^c
BMSC实验组	12	3.119 ± 0.744^b	1.870 ± 0.312^b	1.770 ± 0.358^b	0.824 ± 0.257^b

表4 IL-1β作用下牙周膜干细胞（PDLSC）和骨髓间充质干细胞（BMSC）骨向分化相关基因mRNA表达水平的统计结果（ ± s）

组别	样本量	ALP	Col-1	OCN	Runx2
PDLSC对照组	12	1.000 ± 0.117^a	1.000 ± 0.110^a	1.000 ± 0.124^a	1.000 ± 0.108^a
PDLSC实验组	12	0.053 ± 0.004^b	0.446 ± 0.038^b	0.291 ± 0.052^b	0.116 ± 0.017^b
BMSC对照组	12	13.157 ± 1.517^c	2.235 ± 0.278	4.286 ± 0.570^c	2.142 ± 0.374^c
BMSC实验组	12	3.119 ± 0.744^b	1.870 ± 0.312^b	1.770 ± 0.358^b	0.824 ± 0.257^b

图4 14 d时牙周膜干细胞（PDLSC）和骨髓间充质干细胞（BMSC）骨向分化基因的表达（^abcP<0.05）A：ALP；B：Col-1；C：OCN；D：Runx2

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The effect of interleukin-1β on the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells

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The effect of interleukin-1β on the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells