人间充质干细胞成软骨分化中DNA甲基化调控Ⅹ型胶原表达

doi:10.3877/cma.j.issn.1674-134X.2020.02.007

目的

在人间充质干细胞(hMSCs)诱导成软骨分化过程中，探讨Ⅹ型胶原与其启动子甲基化状态之间的关系，为揭示表观遗传学调控参与成软骨分化的可能机制提供理论基础。

方法

收集6例来源于深圳市第二人民医院脊柱外科患者的腰椎椎体骨髓间充质干细胞为研究对象，运用离心沉淀法进行细胞微球培养，用含有转化生长因子β3(TGF-β3)及2%胎牛血清(FBS)的高糖细胞培养基(DMEM)进行成软骨诱导并作为实验组(3例)；在该诱导液中加入5’-氮杂胞苷(5’-AZA)作为诱导组(3例)；不含TGF-β3的2%胎牛血清的高糖细胞培养基的为对照组(3例)。在分化3 d后进行定量PCR，检测成软骨分化相关基因表达以及Ⅹ型胶原启动子区域甲基化改变情况，采用方差分析及多样本间的均数比较(q检验)分析基因水平表达差异；组间DNA甲基化水平差异采用Kruskal-Wallis检验统计学差异。

结果

通过生物信息学预测Ⅹ型胶原的启动子区域可能存在CpG富集区域，在含有5’-氮杂胞苷的成软骨诱导分化过程中，Ⅹ型胶原表达逐渐上升，与对照组相比差异具有统计学意义(F＝37.526, P<0.001)，而Ⅹ型胶原启动子区域的甲基化水平与对照组相比逐渐下降，差异也具有统计学意义(F＝11.066，P<0.01)。

结论

在体外诱导人间充质干细胞成软骨分化过程中，参与维持Ⅹ型胶原启动子甲基化状态降低，分化能力得到增强；提示表观遗传学调控方式是影响干细胞分化方式之一。

关键词: 表观遗传抑制, 间充质干细胞, 软骨细胞, 胶原Ⅹ型

Objective

To evaluate the relationship between collagen Ⅹ and the methylation status of its promoter during the initial period of human mesenchymal stem cells (hMSCs) chondrogenic differentiation. ]

Methods

The hMSCs were collected from six patients who underwent the intervertebral fusion and informed the ethical agreement. The pellet was cultured by centrifugal precipitation, within 2% fetal bovine serum (FBS) high-glucose Dulbecco's modified Eagle medium containing TGF-β3. Quantitative PCR was performed 3 d after differentiation to detect the expression of cartilage -related genes and methylation changes in the collagen Ⅹ promoter region. The data were analyzed by variance analysis and Kruskal-Wallis test.

Results

Bioinformatics indicated that there might be CpG-rich regions in the promoter region of collagenⅩ(F=37.526, P<0.001). During the process of chondrogenic differentiation, the expression of collagen Ⅹgradually increased, and the difference was statistically significant compared with the control group. Compared with the control group, the methylation level of the collagen Ⅹ promoter region gradually decreased, and the difference was also statistically significant(F=11.066, P<0.01).

Conclusion

In the process of the hMSCs′ chondrogenic differentiation in vitro, the results would suggest that the methylation status of the collagen Ⅹ promoter may involve and enhance the differentiation ability; which implies that epigenetic regulation would affect stem cell differentiation.

Key words: Epigenetic repression, Human mesenchymal stem cells, Chondrocytes, Collagen typeⅩ

表1 q-PCR扩增片段及引物序列

基因名称	正向引物	反向引物
DNMT3A	5’-GGGACGGAAAGAGAGAGACA-3’ (F)	5’-CCTGCTGCTAACTGGGTTCT-3’ (R)
DNMT3B	5’-GAGCGGGCGGCAACG-3’ (F)	5’-CCCTTCATGCTTTCCTGCCG-3’ (R)
DNMT1	5’-GGAGGGCTACCTGGCTAAAG-3’ (F)	5’-TACGGGCTTCACTTCTTGCT-3’ (R)
蛋白聚糖	5’-GCTGCAGTGATCTCAGAAGAAG-3’ (F)	5’-GATGGTGAGGGAAGACCCTA-3’ (R)
胶原蛋白Ⅱa1(col-Ⅱa1)	5’-CCAGGATGCCCGAAAATTAG-3’ (F)	5’-TTCTCCCTTGTCACCACGAT-3’ (R)
胶原蛋白Xa1(col-Xa1)	5’-GCAGCATTACGACCCAAGAT-3’ (F)	5’-CATGATTGCACTCCCTGAAG-3’ (R)
内参18s RNA	5’-CCTGGATACCGCAGCTAGGA-3’ (F)	5’-GCGGCGCAATACGAATGCCCC-3’ (R)

表1 q-PCR扩增片段及引物序列

基因名称	正向引物	反向引物
DNMT3A	5’-GGGACGGAAAGAGAGAGACA-3’ (F)	5’-CCTGCTGCTAACTGGGTTCT-3’ (R)
DNMT3B	5’-GAGCGGGCGGCAACG-3’ (F)	5’-CCCTTCATGCTTTCCTGCCG-3’ (R)
DNMT1	5’-GGAGGGCTACCTGGCTAAAG-3’ (F)	5’-TACGGGCTTCACTTCTTGCT-3’ (R)
蛋白聚糖	5’-GCTGCAGTGATCTCAGAAGAAG-3’ (F)	5’-GATGGTGAGGGAAGACCCTA-3’ (R)
胶原蛋白Ⅱa1(col-Ⅱa1)	5’-CCAGGATGCCCGAAAATTAG-3’ (F)	5’-TTCTCCCTTGTCACCACGAT-3’ (R)
胶原蛋白Xa1(col-Xa1)	5’-GCAGCATTACGACCCAAGAT-3’ (F)	5’-CATGATTGCACTCCCTGAAG-3’ (R)
内参18s RNA	5’-CCTGGATACCGCAGCTAGGA-3’ (F)	5’-GCGGCGCAATACGAATGCCCC-3’ (R)

图1 分离培养hMSCs(人间充质干细胞)及其表面标志物的检测。图A　为抽取术中骨髓血体外培养hMSCs相差显微镜图像，图B　为hMSCs表面标志物的流式细胞检测结果

表2 hMSCs成软骨分化调控基因相对表达量对比(±s)

分组	样本	DNMT1	DNMT3A	DNMT3B
对照组	3	0.78±0.14	0.82±0.13	0.75±0.05
诱导组	3	0.73±0.09	0.94±0.21	1.27±0.25^a
诱导＋5’-AZA组	3	0.61±0.14	0.59±0.04	0.67±0.13^b
F值		1.447	4.485	11.329
P值		>0.05	>0.05	<0.05

表2 hMSCs成软骨分化调控基因相对表达量对比(±s)

分组	样本	DNMT1	DNMT3A	DNMT3B
对照组	3	0.78±0.14	0.82±0.13	0.75±0.05
诱导组	3	0.73±0.09	0.94±0.21	1.27±0.25^a
诱导＋5’-AZA组	3	0.61±0.14	0.59±0.04	0.67±0.13^b
F值		1.447	4.485	11.329
P值		>0.05	>0.05	<0.05

表3 hMSCs成软骨分化关键标志基因表达(±s)

分组	样本	蛋白聚糖	胶原蛋白IIa1	胶原蛋白Xa1
对照组	3	1.01±0.11	5.65±0.18	1.49±0.22
诱导组	3	0.98±0.05	2.22±0.21^a	1.23±0.13^a
诱导＋5’-AZA组	3	1.11±0.24	2.25±0.66^b	2.85±0.34^b
F值		0.528	64.722	37.526
P值		>0.05	<0.05	<0.05

表3 hMSCs成软骨分化关键标志基因表达(±s)

分组	样本	蛋白聚糖	胶原蛋白IIa1	胶原蛋白Xa1
对照组	3	1.01±0.11	5.65±0.18	1.49±0.22
诱导组	3	0.98±0.05	2.22±0.21^a	1.23±0.13^a
诱导＋5’-AZA组	3	1.11±0.24	2.25±0.66^b	2.85±0.34^b
F值		0.528	64.722	37.526
P值		>0.05	<0.05	<0.05

图2 Col-Xa1启动子区域的CpG岛甲基化比率比较。图A　为Col-Xa1启动子上游约1000bp的序列及CpG位点；图B　为30个克隆子经测序显示Col-Xa1启动子区域的CpG岛甲基化程度差异；图C　为比较各组间Col-Xa1启动子区域的CpG岛甲基化率，对照组vs诱导组(F＝11.066, P>0.05)，*-对照组与诱导＋5'-AZA组比较(P<0.05)

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Epigenetic regulation of collagen Ⅹ in human mesenchymal stem cells during chondrogenic differentiation

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Epigenetic regulation of collagen Ⅹ in human mesenchymal stem cells during chondrogenic differentiation