| 引用本文： |
-
李琳,蔡宇艺,何江峰,等.血小板衍生生长因子-BB对骨髓间充质干细胞多向分化及自噬相关蛋白表达的影响[J].同济大学学报（医学版）,2022,43(2):165-173. [点击复制]
- LI Lin,CAI Yuyi,HE Jiangfeng,et al.Effects of PDGF-BB on multidirectional differentiation and autophagy related protein expression of BMSCs[J].同济大学学报（医学版）,2022,43(2):165-173. [点击复制]
|
|
| 摘要: |
| 目的观察血小板衍生生长因子-BB（platelet derived growth factor-BB， PDGF-BB）对骨髓间充质干细胞(bone mesenchymal stem cells， BMSCs）多向诱导分化以及自噬相关蛋白表达的影响；建立异位牙髓再生模型，探讨PDGF-BB对再生牙髓样组织中自噬相关蛋白表达的影响。方法体外分离鉴定大鼠BMSCs，随机分为4组，每组培养基中分别加入0、10、50、100 ng/mL PDGF-BB，通过CCK-8、Transwell、qRT-PCR及细胞免疫荧光染色技术，检测不同浓度PDGF-BB对BMSCs增殖、迁移、多向诱导分化及自噬相关蛋白表达的影响，确定PDGF-BB趋化BMSCs细胞归巢的最佳工作浓度范围。依据是否放入PDGF-BB，将试验大鼠随机分为两组： 对照组（Collagen Type Ⅰ），PDGF-BB诱导组（50 ng/mL PDGF-BB+Collagen Type Ⅰ），将PDGF-BB联合Collagen Type Ⅰ支架移植入经过根管预备后的离体牙根管内，并移植于大鼠腹部皮下。分别于术后2、4个月，采用免疫组化染色技术观察异位再生牙髓样组织的再生效果及其自噬相关蛋白ATG5和LC3的表达。结果CCK-8结果显示，BMSCs增殖活性随PDGF-BB浓度升高递增（P<0.05）；Transwell结果显示，细胞迁移数随PDGF-BB浓度升高递增（P<0.05）；qRT-PCR结果显示，OPN、Runx2、MAP2、β-Ⅲ-tubulin mRNA表达量随PDGF-BB浓度升高递增（P<0.05），当PDGF-BB浓度达到100 ng/mL时，mRNA表达量达到最大值；细胞免疫荧光染色结果显示，ATG5和LC3阳性细胞数量随PDGF-BB浓度升高递增，50 ng/mL PDGF-BB诱导后，ATG5和LC3阳性细胞数最大；术后4个月PDGF-BB诱导组得到的再生牙髓样组织形态与天然人牙髓组织相近，其ATG5、LC3蛋白表达高于对照组（P<0.05）。结论PDGF-BB能够促进大鼠BMSCs增殖，对BMSCs具有显著趋化作用。PDGF-BB能够促进大鼠BMSCs向成骨细胞及神经细胞分化，并促进自噬相关蛋白的表达。联合使用PDGF-BB可以有效获得内源性BMSCs迁移，促进大鼠体内牙髓样组织异位再生，及自噬相关蛋白表达。 |
| 关键词: 牙髓再生 血小板衍生生长因子-BB 多向分化 细胞自噬 细胞归巢 |
| DOI：10.12289/j.issn.1008-0392.21228 |
| 投稿时间：2021-06-02 |
| 基金项目: |
|
| Effects of PDGF-BB on multidirectional differentiation and autophagy related protein expression of BMSCs |
| LI Lin,CAI Yuyi,HE Jiangfeng,HU Ziyue,GE Jianping |
| (Department of Endodontics, School & Hospital of Stomatology, Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China) |
| Abstract: |
| ObjectiveTo investigate the effects of platelet derived growth factor-BB(PDGF-BB) on multidirectional differentiation and autophagy related protein expression of bone mesenchymal stem cells(BMSCs). MethodsRat BMSCswere isolated and identified in vitro. BMSCswere treated with PDGF-BB at concentrations of 0, 10, 50 and 100 ng/mL, respectively. Cell proliferation, migration, multidirectional differentiation and autophagy related protein expression were detected by CCK-8, Transwell assay, qRT-PCR and cellular immunofluorescence staining, respectively. The optimal working concentration range of PDGF-BB for BMSCs homing was determined. The root canal of isolated teeth was treated with collagen type Ⅰ(control group) or 50 ng/mL PDGF-BB+collagen type Ⅰ (study group). The treated teeth were subcutaneously transplanted in the abdomen of male SD rats. After 2 and 4 months, the grafted teeth were examined with immunohistochemical staining to observe the regeneration effect of ectopic dental pulp like tissue and the expression of autophagy related proteins ATG5 and LC3. ResultsCCK 8 results showed that the proliferative activity of BMSCs increased with the increase of PDGF-BB concentration（P<0.05）. Transwell results showed that the number of cell migration increased with the increase of PDGF-BB concentration（P<0.05）. qRT-PCR results showed that mRNA expression of OPN, Runx2, MAP2, β-Ⅲ-tubulin increased with the increase of PDGF-BB concentration（P<0.05）. When using 100 ng/mL PDGF-BB to induce, the mRNA expression reached the maximum. The results of cellular immunofluorescence staining showed that the number of ATG5 and LC3 positive cells increased with the increase of PDGF-BB concentration. When 50 ng/mL PDGF-BB was used to induce, the number of ATG5 and LC3 positive cells was the largest. Four months after transplantation, the morphology of regenerated pulp like tissue obtained by induction was similar to natural human dental pulp, and the expressions of ATG5 and LC3 proteins were higher than those in the control group（P<0.05）. ConclusionPDGF-BB can promote the proliferation of rat BMSCs and has a significant chemotactic effect, it can induce the differentiation of rat BMSCs into osteoblasts and neurons, enhance the expression of autophagy related proteins, and induce the ectopic regeneration of dental pulp like tissue in rats. |
| Key words: dental pulp regeneration PDGF-BB multidirectional differentiation cell autophagy cell homing |
