液氮冷冻犬股骨头坏死过程中细胞因子和组织学的动态变化

Changes of cytokines in osteonecrosis of femoral head induced by liquid nitrogen cryopreservation in dogs

目的研究早期犬股骨头缺血坏死(ONFH)发生、发展过程中组织学改变和细胞因子VEGF的动态表达变化,探讨其在股骨头坏死修复中的意义。方法健康比格犬16只,8～12个月龄,体重8～10kg,取其中4只作为正常对照组(A组,共8髋),其余通过液氮冷冻法制成双侧股骨头坏死模型,作为实验组(B组)。分别于术后4、8、12、18周行CT检查后处死,取股骨头标本行HE染色观察骨小梁形态改变,免疫组化及RT-PCR检测VEGF的表达情况。结果 CT扫描发现术后B组股骨头骨密度首先升高,然后逐渐降低,至第18周出现囊性变和钙化灶。HE染色观察实验组术后4、8周可见关节软骨、骨小梁的结构和形态首先出现部分改善,新骨形成,参与修复,之后坏死紊乱吸收。免疫组化示:B组在术后第4周VEGF蛋白呈阳性表达,至第8周表达最高,这两个时期表达强度均高于A组,此后逐渐降低至第18周仅见微弱阳性表达,与A组无明显差异。RT-PCR检测VEGF mRNA的表达情况进一步显示C组在术后第4、8周VEGF mRNA表达率均高于其他各组。通过SPSS13.0分析,有统计学差异(P<0.05)。结论在ONFH发生早期,出现VEGF的高表达,有助于促进坏死股骨头内血管的新生和软骨下骨的增殖,此后骨小梁开始修复重建,这可能有利于坏死股骨头的修复;坏死中后期VEGF表达微弱,骨小梁进一步吸收萎缩、排列紊乱,部分坏死股骨头塌陷,这可能最终导致坏死过程的不可逆性。

Objective To explore the alteration of cytokines in dogs in the process of the osteonecrosis of the femoral head induced by liquid nitrogen cryopreservation.Methods Sixteen dogs aged from 8 to 12 months and weighed from 8- 10Kg were randomly divided into experimental group and control group.Four of them were choosed to be control group （group A,8 hips） and the others were selected to set up the model of ONFH through the method of liquid nitrogen cryopreservation, as the experimental group（group B）.The dogs were executed at the 4^（th）、8^（th）、12^（th） and 18^（th） week after opera- tion.Changes of the femoral head were evaluated by methods of CT,histologic observation,immunohistochemistry staining and quantitative reverse transcription-polymerase chain reaction（RT-PCR）.Results Radiographic examination of CT demonstrated that high density of femoral head in B group at first after operation and then became lower and lower.Cyst formation and calcification can be seen 18 weeks after operation in the necrosis areas.Histologic observation showed that articular cartilage,morphology and structure of trabecular bone in group B was improvement4^（th） and 8^（th） week after operation and new bone could be seen in repaired bone,and then became worse.Immunohistochemistry showed that the VEGF protein positive staining in group B was significantly higher compared with group A、B and C at the 4^（th） and 8^（th） week after operation and the expression was higher at the 6^（th） week after operation.The expression of VEGF mRNA in group B was much more than group A by RT-PCR at the 4^（th） and 8^（th） week after operation.Conclusion At the early stage of the osteonecrosis of the femoral head,there was high levelexpression of VEGF protein and messenger RNA in necrotic femoral head of dogs and high VEGF expressions had a positive correlation with the blood supply of necrotic femoral head,which may accelerate the repairing process of ONFH.At the intermediateandadvanced of the osteonecrosis of the femoral head,there was weekex-pression of VEGF protein and messenger RNA in necrotic femoral head of dogs,which cannot accelerate the repairing process of ONFH.