Comparison of expression of miR-145-5p in osteogenic differentiation of bone marrow mesenchymal stem cells derived from normal versus type 2 diabetic rats
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摘要:
背景 糖尿病会导致骨髓间充质干细胞(bone marrow mesenchymal stem cells，BMMSCs)的成骨分化能力降低， miRNA在此过程中发挥重要作用，其中miR-145-5p对细胞成骨成软骨分化有重要的调节作用。然而miR-145-5p对糖尿病源BMMSCs成骨分化的影响尚不清楚。 目的 比较正常大鼠与2型糖尿病大鼠BMMSCs成骨分化能力，初步探讨在成骨分化中miR-145-5p及其靶基因SEMA3A和Wnt通路关键蛋白β-catenin表达的差异。 方法 12只GK大鼠采用高糖高脂饲料喂养构建2型糖尿病大鼠模型，12只Wistar大鼠常规饲养作为对照组。无菌条件下分离大鼠股骨，全骨髓培养法培养正常大鼠和2型糖尿病大鼠BMMSCs (分别对应WT-BMMSCs和GK-BMMSCs)；CCK-8检测细胞增殖能力；结晶紫染色检测细胞集落形成能力；碱性磷酸酶染色及半定量分析检测碱性磷酸酶活性；茜素红染色检测矿化基质形成；qRT-PCR和Western blot检测成骨相关标志物、miRNA-145-5p、SEMA3A和β-catenin的表达。 结果 干细胞培养4 ~ 9 d时，GK-BMMSCs的增殖水平低于WT-BMMSCs，差异有统计学意义(P＜0.01)；10 d时，其集落形成率显著低于WT-BMMSCs (P＜0.01)；成骨诱导7 d时，GK-BMMSCs碱性磷酸酶活性(P＜0.001)、成骨相关标志物碱性磷酸酶基因(P＜0.01)和蛋白(P＜0.001)表达、骨钙素基因(P＜0.001)和蛋白(P＜0.001)表达均显著低于WT-BMMSCs，1型胶原蛋白(P＜0.01)基因的表达显著低于WT-BMMSCs，而Runt相关转录因子2(P＜0.001)蛋白表达显著高于WT-BMMSCs。在WT-BMMSCs成骨分化中，miR-145-5p表达下调，SEMA3A表达上调，而在GK-BMMSCs成骨分化中，miR-145-5p和SEMA3A表达均上调，β-catenin (P＜0.001)在GK-BMMSCs中的表达显著降低。成骨诱导21 d时，WT-BMMSCs矿化基质染色较深。 结论 2型糖尿病大鼠BMMSCs细胞增殖、集落形成及成骨分化能力降低，推测在正常大鼠BMMSCs成骨分化中miR-145-5p起抑制作用，在2型糖尿病大鼠BMMSCs成骨分化中miR-145-5p起促进作用。 -
关键词:
- 2型糖尿病 /
- microRNA-145-5p /
- 骨髓间充质干细胞 /
- 成骨分化 /
- 大鼠
Abstract:Background Diabetes reduces the osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMMSCs), and miRNAs are essential in this process, during which miR-145-5p plays a vital role in osteogenic and chondrogenic differentiation of cells. However, the effect of miR-145-5p on the osteogenic differentiation of diabetic BMMSCs remains unclear. Objective To compare the osteogenic differentiation ability of BMMSCs derived from normal and type 2 diabetic rats respectively, and preliminarily explore the differences in the expression of miR-145-5p, its potential target gene SEMA3A and the key protein β-catenin of Wnt pathway in osteogenic differentiation. Methods Twelve GK rats were fed on high-fat and high-sugar diet to establish type 2 diabetes rat model as the experimental group. Twelve Wistar rats were fed on standard diet and served as the control group. Rat femur was isolated under aseptic condition. BMMSCs derived from normal and type 2 diabetic rats (WT-BMMSCs and GK-BMMSCs) were cultured by whole bone marrow culture method. CCK-8 was used to detect the cell proliferation. Colony formation ability was detected by the crystal violet stain assay. Alkaline phosphatase activity was detected by alkaline phosphatase staining and semi-quantitative analysis. Alizarin red staining was used to detect the formation of the mineralized matrix. The expression levels of bone-related markers, miRNA-145-5p, SEMA3A and β-catenin were detected by qRT-PCR and Western blot. Results When the stem cells were cultured for 4-9 days, the proliferation of GK-BMMSCs was significantly lower than that of WT-BMMSCs (P<0.01). On day 10, colony formation rate was significantly lower than that of WT-BMMSCs (P<0.01). On day 7 of osteogenic induction, alkaline phosphatase activity of GK-BMMSCs (P<0.001), osteogenesis-related markers alkaline phosphatase (ALP) gene (P<0.01) and protein (P<0.001) expressions, osteocalcin (OCN) gene (P<0.001) and protein (P<0.001) expressions were significantly lower than those of WT-BMMSCs, type 1 collagen (COL1) gene expression level was significantly lower than that of WT-BMMSCs (P<0.01), and Runt-related transcription factor-2 (RUNX2) protein expression level was significantly higher than that of WT-BMMSCs (P<0.001). While miR-145-5p and SEMA3A were up-regulated during the osteogenic differentiation of WT-BMMSCs, miR-145-5p and SEMA3A were down-regulated during the osteogenic differentiation of GK-BMMSCs, and the expression level of β-catenin (P<0.001) was considerably reduced in GK-BMMSCs. On day 21 of osteogenesis induction, WT BMMSCs had darker staining of the mineralized matrix. Conclusion The proliferation, colony formation and osteogenic differentiation of BMMSCs decrease in type 2 diabetic rats. It is speculated that miR-145-5p plays an inhibitory role in osteogenic differentiation of BMMSCs in normal rats while a promoting role in osteogenic differentiation of BMMSCs in type 2 diabetic rats. -
图 1 8 ~ 13周Wistar与GK大鼠体质量(n=12；aP＜0.001，vs GK)
Figure 1. Changes in body weight of Wistar and GK rats from 8 to 13 weeks (n=12; aP＜0.001, vs GK)
图 2 8 ~ 13周Wistar与GK大鼠血糖(n=12；aP＜0.001，vs GK)
Figure 2. Changes in blood glucose of Wistar and GK rats feom 8 to 13 weeks (n=12; aP＜0.001, vs GK)
图 3 倒置显微镜观察原代(P0)与第3代(P3)正常和糖尿病大鼠BMMSCs (标尺=100 μm)
Figure 3. Primary and the third generation BMMSCs derived from normal and type 2 diabetic rats (scale bar=100 μm)
图 4 流式细胞学鉴定BMMSCs表面抗原
Figure 4. Identification of BMMSCs surface antigens by flow cytometry
图 5 CCK-8试剂盒检测正常和糖尿病大鼠股骨BMMSCs生长曲线(n=6；aP＜0.01，vs GK)
Figure 5. Detection of growth curve of BMMSCs derived from normal and type 2 diabetic rats by CCK-8 kit (n=6; aP＜0.01, vs GK)
图 6 正常和糖尿病大鼠股骨BMMSCs集落形成实验
A：结晶紫染色检测正常和糖尿病大鼠10 d时股骨BMMSCs集落形成能力(标尺=100 μm)；B：正常和糖尿病大鼠股骨BMMSCs集落形成率(n=6)
Figure 6. Colony formation experiment of femoral BMMSCs in normal rats and type 2 diabetic rats
A: Crystal violet staining were performed on day 10 (scale bar=100 μm); B: Colony forming efficiency of the two group (n=6)
图 7 正常与2型糖尿病大鼠BMMSCs成骨分化能力比较
A：成骨诱导7 d碱性磷酸酶染色(标尺=100 μm)；B：成骨诱导7 d ALP半定量分析 (n=6)；C：成骨诱导21 d茜素红染色显示钙化结节形成(标尺=100 μm)
Figure 7. Comparison of osteogenic differentiation capacity of BMMSCs derived from normal rats and type 2 diabetic rats
A: ALP staining was performed on day 7 after osteogenic differentiation (scale bar=100μm); B: Quantification of ALP activity was shown (n=6); C: ARS staining was performed on day 21 after osteogenic differentiation to show the formation of calcified nodules (scale bar=100μm)
图 8 成骨诱导7 d正常与2型糖尿病大鼠BMMSCs ALP、OCN 、RUNX2、COL1的mRNA表达(n=3)
Figure 8. Expression level of the osteoblast differentiation-related genes ALP, OCN, RUNX2, COL1 on day 7 after osteogenic differentiation by real-time PCR (n=3)
图 9 成骨诱导7 d正常与2型糖尿病大鼠BMMSCs ALP、OCN、RUNX2蛋白水平的表达
A： ALP、OCN、RUNX2蛋白的表达； B：ALP、OCN 、RUNX2蛋白水平表达的定量分析(n=3)
Figure 9. Western blot analysis of ALP, OCN, RUNX2 on day 7 after osteogenic differentiation
A：Western blotting of ALP, OCN, RUNX2；B：Protein levels of ALP, OCN, RUNX2 quantified by densitometry (n=3)
图 10 成骨诱导7 d正常与2型糖尿病大鼠BMMSCs miR-145-5p、SEMA3A、β-catenin的表达(n=3)
Figure 10. Expression levels of miR-145-5p, SEMA3A, β-catenin on day 7 after osteogenic differentiation by real-time PCR (n=3)
图 11 成骨诱导7 d正常与2型糖尿病大鼠BMMSCs SEMA3A的蛋白表达
A：SEMA3A蛋白的表达；B：SEMA3A蛋白水平表达的定量分析(n=3)
Figure 11. Western blot analysis of SEMA3A on day 7 after osteogenic differentiation
A: Western blotting of SEMA3A; B: Protein levels of SEMA3A quantified by densitometry (n=3)
表 1 引物序列
Table 1. Primer sequences
基因 引物序列 SEMA3A F-CTTGCTCGGGACCCTTATTG R-AGGCTCTCTGTGACTTCGGACT β-catenin F-TGCCATCTGTGCTCTTCGTC R-CAATCCAACAGTTGCCTTTATCAG ALP F-TGGTGAGTGACACGGACAAGAA R-GCCTGGTAGTTGTTGTGAGCAT COL1 F-CGTGGAAACCTGATGTATGCTTG R-CCTATGACTTCTGCGTCTGGTGA OCN F-TGACAAAGCCTTCATGTCCAA R-CTCCAAGTCCATTGTTGAGGTAG RUNX2 F-TACCCAGGCGTATTTCAGATGAT R-TGTAAGTGAAGGTGGCTGGATAGT GAPDH F-CTGGAGAAACCTGCCAAGTATG R-GGTGGAAGAATGGGAGTTGCT U6 F-CTCGCTTCGGCAGCACA R-AACGCTTCACGAATTTGCGT 
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