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目的探讨静水压联合IGF-1对体外单层培养的山羊颞下颌关节盘细胞丝状肌动蛋白(filamentous actin,F-actin)的影响,分析静水压、IGF-1与F-actin的关系变化对细胞生物学行为改变的影响。方法取4只1月龄山羊双侧颞下颌关节盘,采用酶消化法分离培养颞下颌关节盘细胞,以Ⅰ、Ⅱ型胶原免疫组织化学染色鉴定。取第2、3代细胞根据干预方法不同分为4组:A组以完全培养基培养,作为对照;B组以强度0.2 MPa、频率1 Hz的静水压作用3 h;C组以含10 ng/mL IGF-1的完全培养基培养;D组采用静水压与IGF-1联合培养,方法同B、C组。于干预后24、72 h行免疫荧光染色观察F-actin变化,测定单个细胞荧光强度。结果经形态学观察及免疫组织化学染色鉴定,所培养细胞为颞下颌关节盘细胞。干预24 h时A、C组细胞荧光染色强,保持了细胞正常形态,且分布清晰;B组F-actin排列紊乱;D组F-actin变细,排列混乱。72 h时A、C组F-actin排列整齐;B组F-actin排列紊乱、模糊不清,部分F-actin断裂,形成伪足;D组F-actin变细,排列紊乱、断裂。随时间延长,A、B、D组荧光强度均呈增强趋势,两时间点间比较差异有统计学意义(P<0.05);C组两时间点间比较差异无统计学意义(t=0.284,P=0.781)。干预24 h,C组荧光强度最高,B组最低,与A、D组比较差异均有统计学意义(P<0.05)。72 h时,B、D组荧光强度显著低于A、C组,差异有统计学意义(P<0.05);B、D组间及A、C组间比较差异均无统计学意义(P>0.05)。结论静水压可以引起山羊颞下颌关节盘细胞F-actin发生断裂及重排,IGF-1上调F-actin的表达;静水压联合IGF-1诱导产生的F-actin断裂、重排可能引起细胞生物学行为的改变。
OBJECTIVE: To investigate the effect of hydrostatic pressure combined with IGF-1 on the hydrodynamic status of Fila Actomyosin and F-actin The effect of relationship changes on the change of cell biology behavior. Methods Four temporomandibular joint discs were obtained from 1 month old goats. The temporomandibular joint discs were isolated and cultured by enzymatic digestion, and were identified by type Ⅰ and type Ⅱ collagen immunohistochemistry. The second and third generation of cells were divided into 4 groups according to different intervention methods: group A was cultured in complete medium as a control; group B was treated with hydrostatic pressure of 0.2 MPa and frequency of 1 Hz for 3 h; group C contained 10 ng / mL IGF-1. The rats in group D were incubated with hydrostatic pressure and IGF-1 in the same way as B and C groups. The change of F-actin was observed by immunofluorescence staining 24 and 72 h after the intervention, and the fluorescence intensity of single cells was measured. Results Morphological observation and immunohistochemical staining showed that the cultured cells were temporomandibular joint disc cells. The cells in group A and C were stained fluorescently 24 hours after the intervention, the normal morphology was maintained and the distribution was clear. The F-actin in group B was disordered. The group F was thinner and disordered. At 72 h, the F-actin was arranged neatly in groups A and C; the F-actin in group B was disordered and blurred; some F-actin ruptured to form pseudopodia; group F was thinner and disordered. With time prolonging, fluorescence intensity of A, B and D groups tended to increase, with significant difference between the two time points (P <0.05); there was no significant difference between two groups in time point C (t = 0.284, P = 0.781). After 24 h of intervention, the fluorescence intensity of group C was the highest, the lowest was in group B, and the difference was statistically significant compared with group A and group D (P <0.05). At 72 h, the fluorescence intensity of group B and D was significantly lower than that of group A and C (P <0.05). There was no significant difference between group B and D and group A and C (P> 0.05). Conclusions Hydrostatic pressure can cause the F-actin in the temporomandibular joint of the goat to break and rearrange, and IGF-1 up-regulates the expression of F-actin. F-actin induced by hydrostatic pressure combined with IGF-1 may be disrupted and rearrangement may cause Changes in cellular biological behavior.