牵张力对大鼠髁突纤维软骨干细胞成软骨分化的影响

doi:10.12439/kqhm.1005-4979.2025.01.003

摘要/Abstract

摘要：

目的： 观察牵张力对大鼠髁突纤维软骨干细胞（fibrocartilage stem cells，FCSCs）成软骨分化的影响。方法： 提取大鼠髁突软骨表层中的FCSCs，通过三系分化诱导验证大鼠髁突FCSCs的多向分化能力，并通过流式细胞术检测其表面标志物的表达情况。然后对大鼠髁突FCSCs施加10%拉伸幅度的牵张力培养48 h后，通过实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）与蛋白质印迹法检测牵张力对成软骨相关基因表达的影响。此外，建立大鼠下颌前导（mandibular advancement，MA）模型以探究体内牵张力对髁突软骨表层的影响。结果：大鼠髁突FCSCs具有向脂肪、骨、软骨多向分化的能力，其表达间充质标志物CD90与CD29，而不表达髓系标志物CD45与CD11。与对照(Control)组相比，牵张加力(Stretch)组的大鼠髁突FCSCs中SRY相关高迁移率族盒蛋白9（SRY-related high mobility group-box 9，Sox9）、蛋白聚糖4（proteoglycan 4，Prg4）与Ⅰ型胶原(typeⅠ collagen，Col1)的基因表达水平显著上升，而基质金属蛋白酶13（matrix metalloproteinase 13，MMP13）的基因表达水平显著降低。与Control组相比，牵张力可以在体内显著增加大鼠髁突软骨表层的厚度。结论：牵张力可以促进大鼠髁突FCSCs的成软骨分化与髁突软骨表层基质合成。

关键词: 牵张力, 髁突, 纤维软骨干细胞, 成软骨分化

Abstract:

Objective: To investigate the effect of mechanical stretch on the chondrogenic differentiation of rat condylar fibrocartilage stem cells (FCSCs).Methods: Triple-lineage induction was used to verify the multi-lineage differentiation ability of rat condylar FCSCs, and flow cytometry was conducted to examine the surface markers of rat condylar FCSCs. Afterward, the rat condylar FCSCs were stretched with a strength of 10% for 48 h, and the expression of related chondrogenic genes was detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. We also established rat mandibular advancement (MA) model to find out the in vivo effect of mechanical stretch on the superficial layer of the condylar cartilage.Results: The rat condylar FCSCs could differentiate towards adipogenesis, osteogenesis, and chondrogenesis, and they expressed mesenchymal markers CD90 and CD29, while negative for hemopoietic markers CD45 and CD11. Compared with the Control group, the Stretch group expressed a higher level of SRY-related high mobility group-box 9 (Sox9), proteoglycan 4 (Prg4), and typeⅠcollagen (Col1) genes, while a lower level of matrix metalloproteinase 13 (MMP13) gene. In vivo, mechanical stretch could increase the superficial layer thickness in the rat condylar cartilage compared with the Control group.Conclusion: Mechanical stretch can promote the chondrogenic differentiation of rat condylar FCSCs and the cartilage matrix formation in the superficial layer of the condylar cartilage.

Key words: mechanical stretch, condyle, fibrocartilage stem cells, chondrogenic differentiation

中图分类号:

R782

龚吴仪, 米晓晖, 李永明. 牵张力对大鼠髁突纤维软骨干细胞成软骨分化的影响[J]. 《口腔颌面外科杂志》, 2025, 35(1): 14-20.

GONG Wuyi, MI Xiaohui, LI Yongming. Effects of stretch on chondrogenic differentiation of rat condylar fibrocartilage stem cells[J]. 《Journal of Oral and Maxillofacial Surgery》, 2025, 35(1): 14-20.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2025/V35/I1/14

图/表 7

表1 引物序列

Table 1 Primer sequences

基因名称	上游引物序列5'-3'	下游引物序列5'-3'
GAPDH	AGGTCGGTGTGAACGGATTTG	GGGGTCGTTGATGGCAACA
Sox9	TCCCCGCAACAGATCTCCTA	AGCTGTGTGTAGACGGGTTG
蛋白聚糖4（Prg4）	CTGTGGAGAGGGTTCCCAAA	CAGGTATAAGCCATGCAATGGGA
Col1	GGAGAGAGCATGACCGATGG	GGTGGGAGGGAACCAGATTG
基质金属蛋白酶13（MMP13）	TCCATCCCGAGACCTCATGT	CTCAAAGTGAACCGCAGCAC

图1 大鼠髁突FCSCs的形态学观察 A.光镜下P0代大鼠髁突FCSCs（×40）；B—D.荧光显微镜(×400)下CD90在大鼠髁突FCSCs中的表达，红色荧光为CD90，蓝色荧光为DAPI。

Figure 1 Morphological observation of rat condylar FCSCs

图2 大鼠髁突FCSCs的多向分化能力鉴定 A.光镜下大鼠髁突FCSCs油红染色（×200）；B.光镜下大鼠髁突FCSCs茜素红染色（×40）；C.体视显微镜下大鼠髁突FCSCs软骨球（×100）；D.光镜下软骨球阿利新蓝染色（×100）。

Figure 2 Identification of the multi-lineage differentiation of rat condylar FCSCs

图3 大鼠髁突FCSCs的表面标志物鉴定 A—D.依次为大鼠髁突FCSCs的表面标志物CD90、CD29、CD45、CD11的表达情况。

Figure 3 Surface marker identification of rat condylar FCSCs

图4 大鼠髁突FCSCs成软骨相关基因的表达 A—D.依次为大鼠髁突FCSCs中Sox9、Prg4、Col1和MMP13的mRNA相对表达量。*表示P<0.05，与Control组比较，每组n=4。

Figure 4 Expression of chondrogenic genes of rat condylar FCSCs

图5 大鼠髁突FCSCs成软骨相关基因的蛋白表达 A.大鼠髁突FCSCs中Sox9与Col1的蛋白条带图；B.大鼠髁突FCSCs中Sox9与Col1蛋白表达情况的半定量结果，*表示P<0.05，与Control组比较，每组n=3。

Figure 5 Protein expression of chondrogenic genes of rat condylar FCSCs

图6 大鼠髁突软骨表层厚度的变化 A.光镜下大鼠髁突HE染色图（×200）；B.大鼠髁突软骨表层厚度的测量结果，**表示P<0.01，与Control组比较，每组n=3。

Figure 6 Alteration of superficial zone thickness in rat condyles

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