鸢尾素对大鼠骨髓间充质干细胞<i>Sost</i>基因表达及成骨分化的影响

doi:10.3877/cma.j.issn.1674-1366.2019.03.003

目的

探究鸢尾素（Irisin）对大鼠骨髓间充质干细胞（BMSC）Sost基因表达及成骨分化的影响。

方法

设置Irisin不同浓度组（0、80、100、120 ng/mL），细胞计数试剂盒（CCK-8）法选择Irisin的最佳实验浓度。用含有Irisin的培养液培养大鼠BMSC设置为实验组，对照组为单纯培养液培养，3、7、14 d进行茜素红、von Kossa钙盐染色；3、10 d采用反转录聚合酶链反应（RT-PCR）和Western bolt检测成骨相关基因mRNA及蛋白表达。分别采用单因素方差分析和t检验对数据进行统计分析。

结果

（1）培养3 d时，100 ng/mL的Irisin促进大鼠BMSC增殖的效果明显（OD=0.951），与对照组相比差异有统计学意义（F=102.52，P<0.05）；（2）实验组染色深度、钙结节大小和数量都明显高于对照组；（3）与对照组相比，实验组Sost mRNA及其蛋白表达水平明显下降，ALP、Lrp5、BMP2、Smad1的mRNA及其蛋白表达水平明显增高，差异均有统计学意义（P<0.001）。

结论

（1）Irisin可抑制Sost基因的表达；（2）Irisin可在体外促进大鼠BMSC增殖和成骨分化；（3）Wnt和BMP信号通路可能在Irisin促进大鼠BMSC成骨分化中发挥作用。

关键词: 骨髓，间质干细胞, 信号传导, 鸢尾素, 骨硬化蛋白

Objective

To investigate the effect of Irisin on osteogenic differentiation and Sost of rat bone marrow mesenchymal stem cells (BMSCs) .

Methods

Irisin was set to have different concentrations (0, 80, 100, 120 ng/mL) , and the optimal concentration of Irisin was determined by CCK-8 method. The BMSCs cultured with Irisin was the experimental group, while the control group was cultured without Irisin. The alizarin red and von Kossa were stained on the 3rd, 7th and 14th day to observe the positive region of osteogenic differentiation and the expression intensity. The mRNA and protein expression levels of the bone-related genes were detected by RT-PCR and Western bolt at 3rd and 10th days. The One-Way ANOVA and t-test were applied to perform the statistical analysis of the data.

Results

(1) Irisin at a concentration of 100 ng/mL promoted the proliferation of rat BMSCs significantly after cultured three days (OD=0.951; F=102.52, P<0.05) . (2) The depth of staining and the size as well as the number of calcium nodules in the experimental group were significantly higher than those in the control group. (3) Compared with the control group, the mRNA and protein expression levels of the bone-related genes: ALP, Lrp5, BMP2 and Smad1 were significantly increased, but the expression levels of Sost mRNA and protein were significantly decreased (P<0.001) .

Conclusions

(1) Irisin can inhibit the expression of the Sost gene (2) Irisin can promote the proliferation and osteogenic differentiation of rat BMSCs in vitro. (3) Wnt and BMP signaling pathway may play a role in Irisin promoting osteogenic differentiation of rat BMSCs.

Key words: Bone marrow, mesenchymal stem cells, Signal transduction, Irisin, Sclerostin

表1 实时荧光定量聚合酶链反应目的基因的引物序列

基因名称	引物序列
GAPDH	F:5′-GGCACAGTCAAGGCTGAGAATG-3′
GAPDH	R:5′-ATGGTGGTGAAGACGCCAGTA-3′
ALP	F:5′-AACGTGGCCAAGAACATCATCA-3′
ALP	R:5′-TGTCCATCTCCAGCCGTGTC-3′
BMP2	F:5′-CAACACCGTGCTCAGCTTCC-3′
BMP2	R:5′-TTCCCACTCATTTCTGAAAGTTCC-3′
Smad1	F:5′-GGTACCAATGACATTTATTTTCC-3′
Smad1	R:5′-CCTCGAGTTTATACATGAAATAGG-3′
Lrp5	F:5′-CTTCATCCACCGTGCCAACCTG-3′
Lrp5	R:5′-TCTGCCAGTCTGTCCAGTAGAGTG-3′
Sost	F:5′-GCCTTGCCTGCCTGCCTGTAC-3′
Sost	R:5′-CCGGTTCATGGTCTGGTTGTTCTC-3′

表1 实时荧光定量聚合酶链反应目的基因的引物序列

基因名称	引物序列
GAPDH	F:5′-GGCACAGTCAAGGCTGAGAATG-3′
GAPDH	R:5′-ATGGTGGTGAAGACGCCAGTA-3′
ALP	F:5′-AACGTGGCCAAGAACATCATCA-3′
ALP	R:5′-TGTCCATCTCCAGCCGTGTC-3′
BMP2	F:5′-CAACACCGTGCTCAGCTTCC-3′
BMP2	R:5′-TTCCCACTCATTTCTGAAAGTTCC-3′
Smad1	F:5′-GGTACCAATGACATTTATTTTCC-3′
Smad1	R:5′-CCTCGAGTTTATACATGAAATAGG-3′
Lrp5	F:5′-CTTCATCCACCGTGCCAACCTG-3′
Lrp5	R:5′-TCTGCCAGTCTGTCCAGTAGAGTG-3′
Sost	F:5′-GCCTTGCCTGCCTGCCTGTAC-3′
Sost	R:5′-CCGGTTCATGGTCTGGTTGTTCTC-3′

图1 P1、P3和P5代大鼠骨髓间充质干细胞生长曲线

图2 体外培养大鼠骨髓间充质干细胞各代细胞镜下形态（低倍放大）A：P1代细胞，融合度未达到95%，细胞中存在杂细胞；B：P2代细胞，杂细胞数量减少，且细胞形态规律，呈现长梭形；C：P3代细胞，融合度95%以上，且排列出显著的方向性，活力旺盛；D：P5代细胞，细胞出现老化，融合度有所减少

图3 不同浓度Irisin对骨髓间充质干细胞增殖活性的影响

图4 各组大鼠骨髓间充质干细胞（BMSC）茜素红染色结果（低倍放大）A、B、C：对照组分别在3、7和14 d的茜素红染色结果；D、E、F：实验组分别在3、7和14 d的茜素红染色结果

图5 各组大鼠骨髓间充质干细胞（BMSC）von Kossa钙盐染色结果（低倍放大）A、B、C：对照组分别在3、7和14 d的von Kossa钙盐染色结果；D、E、F：实验组分别在3、7和14 d的von Kossa钙盐染色结果

表2 大鼠骨髓间充质干细胞成骨相关基因mRNA表达水平的统计结果

组别	ALP	Lrp5	Sost	BMP2	Smad1
对照组	1.00 ± 0.11	0.98 ± 0.12	1.02 ± 0.19	1.00 ± 0.09	1.86 ± 0.26
实验组	1.75 ± 0.51	1.78 ± 0.23	0.64 ± 0.13	1.41 ± 0.26	1.04 ± 0.34
t值	4.276	9.057	4.886	4.571	6.176
P值	0.001	0.001	0.001	0.001	0.001

表2 大鼠骨髓间充质干细胞成骨相关基因mRNA表达水平的统计结果

组别	ALP	Lrp5	Sost	BMP2	Smad1
对照组	1.00 ± 0.11	0.98 ± 0.12	1.02 ± 0.19	1.00 ± 0.09	1.86 ± 0.26
实验组	1.75 ± 0.51	1.78 ± 0.23	0.64 ± 0.13	1.41 ± 0.26	1.04 ± 0.34
t值	4.276	9.057	4.886	4.571	6.176
P值	0.001	0.001	0.001	0.001	0.001

图6 各组大鼠骨髓间充质干细胞成骨相关基因mRNA表达量　A：ALP（3 d）；B：Lrp5（3 d）；C：Sost（3 d）；D：BMP2（10 d）；E：Smad1（10 d）；与对照组比较，^aP<0.001

图7 各组大鼠骨髓间充质干细胞成骨相关蛋白表达量

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Effect of Irisin on osteogenic differentiation and Sost of rat bone marrow mesenchymal stem cells

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Effect of Irisin on osteogenic differentiation and Sost of rat bone marrow mesenchymal stem cells