Effect of type 2 diabetes mellitus on biological characteristics of jaw bone marrow mesenchymal stem cells in rats and its related mechanisms
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摘要:
背景 2型糖尿病患者骨代谢异常，颌骨骨质和骨量都发生明显改变，是牙周炎的全身促进因素之一。Wnt信号通路可以促进干细胞矿化并修复骨组织缺损。然而2型糖尿病影响下Wnt信号通路是否参与了对颌骨骨髓间充质干细胞(jaw bone marrow mesenchymal stem cells，JBMMSCs)的成骨分化调控仍未可知。 目的 研究2型糖尿病对大鼠JBMMSCs增殖及分化等生物学特性的影响，并对其机制进行初步探索。 方法 选取连续两周随机血糖≥16.7mmoL/L的13周龄GK大鼠为2型糖尿病组，相同周龄的Wistar大鼠作为对照组，两组各10只；无菌条件下采用骨髓冲洗法与骨片消化法相结合的方法分离培养两组JBMMSCs作为研究对象。CCK-8法检测并比较两组细胞增殖能力，流式细胞术检测细胞被诱导凋亡的能力。对JBMMSCs进行成骨、成脂诱导后，qRT-PCR评估成骨、成脂及Wnt信号通路相关基因的表达改变，碱性磷酸酶(alkaline phosphatase，ALP)染色检测成骨诱导后ALP的表达变化，茜素红染色比较钙结节形成能力，油红O染色检测成脂诱导后脂滴形成能力。 结果 与对照组相比，2型糖尿病组JBMMSCs的增殖及克隆形成能力降低，凋亡早期与晚期的细胞比例增加(P＜0.05)。两组JBMMSCs成骨诱导后成骨相关基因ALP、OCN和 Runx2 mRNA表达升高，但2型糖尿病组低于对照组(P＜0.05)，2型糖尿病组钙结节形成能力和ALP染色面积及密度较对照组低(P＜0.05)。成脂诱导后，成脂相关基因的表达和脂滴的形成较对照组减少(P＜0.05)。2型糖尿病组JBMMSCs成骨诱导7 d后Wnt信号通路相关分子Wnt4、Wnt5a、Wnt7b的mRNA表达较对照组升高，β-catenin的表达水平较对照组低。 结论 2型糖尿病影响大鼠颌骨骨髓间充质干细胞的增殖和克隆、抑制成骨及成脂分化能力，其成骨能力的降低可能与Wnt信号通路相关。 -
关键词:
- 2型糖尿病 /
- 颌骨骨髓间充质干细胞 /
- 成骨分化 /
- Wnt信号通路 /
- GK大鼠
Abstract:Background Patients with type 2 diabetes have abnormal bone metabolism and marked alterations in both bone quality and bone mass of the jaw, which is one of the systemic contributors to periodontitis. Wnt signaling pathway can promote stem cells mineralization and repair bone tissue defects. However, it is still unknown whether the Wnt signaling pathway is involved in the regulation of osteogenic differentiation of bone marrow mesenchymal stem cells in the jaws under the influence of type 2 diabetes. Objective To investigate the effects of type 2 diabetes on the proliferation and differentiation of jaw bone marrow mesenchymal stem cells (JBMMSCs) in rats and the possible mechanisms. Methods The 13-week-old GK rats with random blood glucose≥16.7 mmol/L for two consecutive weeks were selected as the type 2 diabetes group, and Wistar rats of the same weeks of age served as the control group, with 10 rats in each group. Two groups of JBMMSCs were isolated and cultured as the research object by the combination of bone marrow flashing and bone slice digestion under sterile conditions. CCK-8 method was used to detect and analyze the cell’s proliferative ability. The apoptosis ability of cells was assessed by flow cytometry. The expression of genes related to osteogenesis, adipogenesis and Wnt signaling pathway was assessed by qRT-PCR, alkaline phosphatase (ALP) expression was detected by alkaline phosphatase staining, and the difference in calcium nodule formation was compared by alizarin red staining. Oil Red O staining was used to detect lipid droplet formation differences after lipogenesis induction. Results Compared with the control group, the proliferation and clonogenic ability of JBMMSCs in the type 2 diabetes group decreased, and the proportion of cells of early and late apoptosis increased (P<0.05). Osteogenesis-related genes ALP, OCN and Runx2 mRNA expression elevated in both groups after JBMMSCs osteogenic induction, but the expression in the type 2 diabetes group was lower than that in the control group (P<0.05), and calcium nodule forming ability and ALP staining area and density in the type 2 diabetes group were lower than those in the control group (P<0.05). After adipogenic induction, the expression level of adipogenesis-related genes and lipid droplet formation in the type 2 diabetes group reduced compared with the control group (P<0.05). The mRNA expression levels of Wnt4, Wnt5a and Wnt7b related to the Wnt signal pathway in the type 2 diabetes group were higher than those in the control group at 7 days after JBMMSCs osteogenesis induction, while the expression level of β-catenin was lower than that in the control group. Conclusion Type 2 diabetes inhibits the proliferation, cloning, osteogenic and adipogenic differentiation of JBMMSCs, and the reduction of their osteogenic capacity may be related to the Wnt signaling pathway. -
图 1 对照组和2型糖尿病组随机血糖和体质量
Figure 1. Randomized blood glucose and weight in control and T2DM groups
图 2 对照组和2型糖尿病组JBMMSCs镜下形态观察
A：原代培养第3天；B：原代培养第5天；C：第1代(标尺=100 µm)
Figure 2. Morphological observation of JBMMSCs in control and T2DM groups (bar=100 µm)
A: P0 passage on day3; B:P0 passage on day5; C: P1 passage
图 3 2型糖尿病对JBMMSCs增殖、克隆及凋亡的影响
A：CCK-8法检测细胞的增殖能力；B：细胞克隆形成能力；C：细胞凋亡检测(aP＜0.05，bP＜0.001，vs 2型糖尿病组)
Figure 3. Effect of type 2 diabetes on the proliferation, clone formation and apoptosis ability of JBMMSCs
A: Proliferation ability by CCK-8; B: Clone-forming ability; C: Detection of apoptosis (aP＜0.05, bP＜0.001, vs T2DM group)
图 4 两组JBMMSCs成骨分化能力比较
A：qRT-PCR检测正常培养和成骨诱导7 d后对照组和2型糖尿病组中成骨相关基因的mRNA表达水平的变化(aP＜0.05，vs 对照组-未成骨诱导；bP＜0.05，vs 对照组-成骨诱导；cP＜0.05，vs 2型糖尿病组-未成骨诱导)；B：ALP染色；C：茜素红染色
Figure 4. Comparison of osteogenic differentiation ability of JBMMSCs between the two groups
A: Changes in mRNA expression levels of osteogenesis-related genes in the control group and the T2DM group after 7 days of normal culture and osteogenesis induction by qRT-PCR (aP＜0.05, vs control-NC group; bP＜0.05, vs control-OS group; cP＜0.05, vs T2DM-NC group); B: ALP staining; C: Alizarin red staining
图 5 两组JBMMSCs成脂分化能力比较
A：qRT-PCR检测正常培养和成脂诱导7 d后对照组和2型糖尿病组中成骨相关基因的mRNA表达水平的变化(aP＜0.05，vs 对照组-未成脂诱导；bP＜0.05，vs 对照组-成脂诱导；cP＜0.05，vs 2型糖尿病组-未成脂诱导)；B：油红O染色(标尺=100 µm)
Figure 5. Comparison of adipogenic differentiation ability of JBMMSCs between the two groups
A: Changes in mRNA expression levels of adipogenesis-related genes in the control group and the T2DM group after 7 days of normal culture and adipogenic differentiation induction by qRT-PCR (aP＜0.05, vs control-NC group; bP＜0.05, vs control-AD group; cP＜0.05, vs T2MD-NC group); B: Oil Red O staining (bar=100 µm)
图 6 Wnt信号通路相关分子mRNA水平在两组JBMMSCs成骨诱导过程中的变化(aP＜0.05，vs 对照组-未成骨诱导；bP＜0.05，vs对照组-成骨诱导；cP＜0.05，vs 2型糖尿病组-未成骨诱导)
Figure 6. Changes in mRNA levels of Wnt signaling pathway-related molecules during osteogenesis induction (aP＜0.05, vs control-NC group; bP＜0.05, vs control-OS group; cP＜0.05, vs T2DM-NC group)
表 1 相关基因引物序列
Table 1. Primer sequences of related genes
基因 序列 (5'-3') GAPDH F: ACCCAGAAGACTGTGGATGG R: CACATTGGGGGTAGGAACAC ALP F：CACGTTGACTGTGGTTACTGCTGA R：CCTTGTAACCAGGCCCGTTG OCN F：GGTGGTGAATAGACTCCGGC R：GCAACACATGCCCTAAACGG Runx2 F：GCACCCAGCCCATAATAGA R：TTGGAGCAAGGAGAACCC LPL F：GGAGTTTGGCTCCAGAGTTT R：AAGGTTTTGCTGCTGTGGTTG PPARγ F：ACCGCCCAGGCTTGCTGAAC R：TGGAGCACCTTGGCGAACAGC β-catenin F：AAGTTCTTGGCTATTACGACA R：ACAGCACCTTCAGCACTCT Wnt4 F: TCAGGTTGGCCACGCACTAAAGGAG R: AGTCTGGACTTGGCTCCAGGTACAC Wnt5a F：GCGGGACTTTCTCAAGGACA R：CGGCTGCCTATTTGCATCAC Wnt7b F：CTGGGAGCCAACATCATCTG R：TGCCCAAAGACGGTCTTCTC 
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