股骨头坏死组织中Caspase-3调控成骨细胞分化的研究
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摘要
目的:探讨半胱氨酸天冬氨酸蛋白酶(Caspase)-3在无菌性股骨头坏死(Avascular/Aseptic Necrosis of Femoral Head,ANFH)进展过程中的表达及意义。
方法:股骨头坏死患者22例(22髋)取股骨头病灶周围骨松质,其中早期组(6髋),中期组(8髋),晚期组(8髋),另取19例(19髋)新鲜股骨颈骨折的股骨头对应部位骨松质为对照组。采用原位末端标记(TUNEL)法、空骨陷窝计数法检测细胞凋亡水平,比色法检测股骨头组织Caspase-3活性,茜素红染色检测成骨细胞成骨能力。另用不同浓度的Caspase-3特异性抑制剂z-DEVD-FMK处理早期ANFH成骨细胞,RT-PCR检测Caspase-3活性抑制对成骨通路调节因子及成骨标志物mRNA水平的影响。
结果:骨陷窝空虚率、骨细胞凋亡率等凋亡指标均随ANFH临床分期进展而显著升高,Caspase-3活性在ANFH各组均显著高于骨折对照组,但并不随着ANFH病变进展而增强;钙结节染色面积代表的成骨能力在早期ANFH组织高于对照组,同时Caspase-3活性水平升高,成骨能力及Caspase-3活性在中、晚期均进行性下降,组间差异均有统计学显著性(P<0.05);Caspase-3活性与成骨能力指标具有统计学相关性(R2=0.119,P<0.05)。用z-DEVD-FMK高度抑制Caspase-3活性致成骨指标表达显著下调,轻度抑制会提高成骨指标的表达,抑制Caspase-3活性对成骨分化调节因子Dlx5、Msx2及成骨标志物BGP的mRNA表达水平的影响较显著。
结论:1)在ANFH进展过程中,Caspase-3对细胞凋亡有一定的促进作用;2)Caspase-3还能促进ANFH成骨细胞的成骨分化,可能通过影响成骨分化调节因子Dlx5、Msx2的表达,进而调控成骨分化;3)Caspase-3具有执行凋亡和促进成骨的双重作用,ANFH的病变进展是成骨修复先增强后减弱,而凋亡持续增强的过程,适当上调Caspase-3活性的药物或制剂可能成为早期ANFH一种新的治疗手段。
Objective To investigate the expression and role of Caspase-3 during the development of non-traumatic avascular/aseptic necrosis of femoral head(ANFH).
Methods The femoral heads from 22 ANFH patients were devided into groupⅠ(6 cases)、Ⅱ(8 cases)、Ⅲ(8 cases)according to the progress of the disease, those from 19 patients in the acute stage of femoral neck fracture were involved as the control. The level of apoptosis was compared in terms of the percentages of TUNEL positive osteocytes and empty osteocyte lacunae,the activity of Caspase-3 was detected by colorimetric method, the capacity of osteogenesis was established by means of staining cultured osteoblasts with alizarin red S. Furthermore, the osteoblasts from the groupⅠfemoral heads were treated with the specific inhibitor of Caspase-3, and mRNA expression level of both some regulators and an osteogenesis indicating molecule were detected by means of RT-PCR.
Results Both the pecentages of empty osteocyte lacunae and the pecentages of TUNEL positive osteocytes increased significantly with the developing of ANFH. The activity of Caspase-3 in ANFH groups were higher than in the fracture gourp significantly, but did not increase with the progress of the disease. The osteogenetic capacity in terms of the percentage of area stained by aliarin red S was even higher in groupⅠthan in control, so was the expressing level and activity of Caspase-3, both of the two index decreased with further developments of ANFH, with significant statistical differences among groups(P<0.05). Caspase-3 activity was statistically significantly related with the capacity of osteogenesis (P<0.05). Deep suppression of Caspase-3 activity led to significant down-regulation of the index of osteogensis, while modest suppresion was in favor of osteogenesis. It was the expressing level of Dlx5 and Msx2 that changes in Caspase-3 activity could affect more significantly.
Conclusion 1) During the development of ANFH, Caspase-3 is involved in apoptosis in some degree. 2) Furthermore, Caspase-3 could also promote osteogenesis in ANFH, modulating the expression and activity of Dlx5 and Msx2 may be a pathway of regulation on osteogenesis by Caspase-3. 3) Caspase-3 acts as not only the executor of apoptosis, but also a crucial supporter of osteogenesis and repairments, the development of ANFH means the Caspase-3-activity-affected firstly increasing and secondly decreasing of osteogenetic capacity, and progressive rising of apoptosis level.
方法:股骨头坏死患者22例(22髋)取股骨头病灶周围骨松质,其中早期组(6髋),中期组(8髋),晚期组(8髋),另取19例(19髋)新鲜股骨颈骨折的股骨头对应部位骨松质为对照组。采用原位末端标记(TUNEL)法、空骨陷窝计数法检测细胞凋亡水平,比色法检测股骨头组织Caspase-3活性,茜素红染色检测成骨细胞成骨能力。另用不同浓度的Caspase-3特异性抑制剂z-DEVD-FMK处理早期ANFH成骨细胞,RT-PCR检测Caspase-3活性抑制对成骨通路调节因子及成骨标志物mRNA水平的影响。
结果:骨陷窝空虚率、骨细胞凋亡率等凋亡指标均随ANFH临床分期进展而显著升高,Caspase-3活性在ANFH各组均显著高于骨折对照组,但并不随着ANFH病变进展而增强;钙结节染色面积代表的成骨能力在早期ANFH组织高于对照组,同时Caspase-3活性水平升高,成骨能力及Caspase-3活性在中、晚期均进行性下降,组间差异均有统计学显著性(P<0.05);Caspase-3活性与成骨能力指标具有统计学相关性(R2=0.119,P<0.05)。用z-DEVD-FMK高度抑制Caspase-3活性致成骨指标表达显著下调,轻度抑制会提高成骨指标的表达,抑制Caspase-3活性对成骨分化调节因子Dlx5、Msx2及成骨标志物BGP的mRNA表达水平的影响较显著。
结论:1)在ANFH进展过程中,Caspase-3对细胞凋亡有一定的促进作用;2)Caspase-3还能促进ANFH成骨细胞的成骨分化,可能通过影响成骨分化调节因子Dlx5、Msx2的表达,进而调控成骨分化;3)Caspase-3具有执行凋亡和促进成骨的双重作用,ANFH的病变进展是成骨修复先增强后减弱,而凋亡持续增强的过程,适当上调Caspase-3活性的药物或制剂可能成为早期ANFH一种新的治疗手段。
Objective To investigate the expression and role of Caspase-3 during the development of non-traumatic avascular/aseptic necrosis of femoral head(ANFH).
Methods The femoral heads from 22 ANFH patients were devided into groupⅠ(6 cases)、Ⅱ(8 cases)、Ⅲ(8 cases)according to the progress of the disease, those from 19 patients in the acute stage of femoral neck fracture were involved as the control. The level of apoptosis was compared in terms of the percentages of TUNEL positive osteocytes and empty osteocyte lacunae,the activity of Caspase-3 was detected by colorimetric method, the capacity of osteogenesis was established by means of staining cultured osteoblasts with alizarin red S. Furthermore, the osteoblasts from the groupⅠfemoral heads were treated with the specific inhibitor of Caspase-3, and mRNA expression level of both some regulators and an osteogenesis indicating molecule were detected by means of RT-PCR.
Results Both the pecentages of empty osteocyte lacunae and the pecentages of TUNEL positive osteocytes increased significantly with the developing of ANFH. The activity of Caspase-3 in ANFH groups were higher than in the fracture gourp significantly, but did not increase with the progress of the disease. The osteogenetic capacity in terms of the percentage of area stained by aliarin red S was even higher in groupⅠthan in control, so was the expressing level and activity of Caspase-3, both of the two index decreased with further developments of ANFH, with significant statistical differences among groups(P<0.05). Caspase-3 activity was statistically significantly related with the capacity of osteogenesis (P<0.05). Deep suppression of Caspase-3 activity led to significant down-regulation of the index of osteogensis, while modest suppresion was in favor of osteogenesis. It was the expressing level of Dlx5 and Msx2 that changes in Caspase-3 activity could affect more significantly.
Conclusion 1) During the development of ANFH, Caspase-3 is involved in apoptosis in some degree. 2) Furthermore, Caspase-3 could also promote osteogenesis in ANFH, modulating the expression and activity of Dlx5 and Msx2 may be a pathway of regulation on osteogenesis by Caspase-3. 3) Caspase-3 acts as not only the executor of apoptosis, but also a crucial supporter of osteogenesis and repairments, the development of ANFH means the Caspase-3-activity-affected firstly increasing and secondly decreasing of osteogenetic capacity, and progressive rising of apoptosis level.
引文
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