摘要
目的:观察实验性骨关节炎发生发展过程中软骨组织Akt的表达及卵巢去势后Akt的表达,探讨大鼠骨关节炎进程中雌激素缺乏对软骨组织中Akt的影响,研究其变化规律,为进一步研究雌激素在OA发展过程中所起作用提供依据。
方法:2-3月龄SPF级健康SD雌性大鼠72只,分为三组:16只作为正常对照组,56只为OA建模组,每只鼠行双侧膝关节前交叉韧带切断和部分内侧半月板切除构建OA动物模型。术后第6周处死8只OA建模大鼠及4只正常对照大鼠,取材检测OA建模情况;余下48只建模大鼠再随机分为两组:OA组24只,OA+OVX组24只(行双侧卵巢切除);于第9周、12周、16周再分别处死正常对照组4只, OA组和OA+OVX组各8只大鼠,取材双侧膝关节。HE染色、番红O染色行软骨组织病理学观察;S-P免疫组化法和Western-blotting法分析软骨组织中Akt及P-Akt的表达,SPSS统计软件进行统计学处理。
结果:构建OA动物模型及绝经期动物模型成功。OA造模术后第6周关节软骨出现明确的骨关节炎病理改变,且病变逐渐加重。在各随访时间点上, OA组与OA +OVX组两组组间Mankin’s评分比较未发现两者间有差异(P >0.05),提示组织学上看不出卵巢去势后雌激素缺乏状态下骨关节炎有明显加重趋势。但在各随访时间点上,OA组与OA+OVX组Akt的表达有差异,后者较前者低,差异有显著性(P<0.05)。OA组与OA+OVX组Akt、P-Akt的表达水平均呈现先升高后下降的变化过程,术后第6周到第12周,OA组软骨组织出现轻到中度损伤,此时Akt、P-Akt的表达水平呈现逐渐升高,术后第16周时,关节软骨组织处于中到重度损伤,此时Akt、P-Akt表达水平下降,时间轴上Akt表达量差异有显著性(P<0.01)。
结论:1.通过切断前交叉韧带和切除部分内侧半月板的方法构建早期OA动物模型是成功、可行的。2.在本实验期内,组织学上看不出卵巢去势后骨性关节炎有明显加重趋势。3. Akt、P-Akt的表达水平随OA的发展呈现先升高后下降趋势。4.卵巢切除后,Akt表达水平下降,提示雌激素缺乏可影响软骨组织中Akt的表达。
Objective To research regularity for change of protein Kinase B in the articular cartilage of osteoarthritis(OA) process in OA and OA-ovariectomy rat models.
Methods 72 female Sprague-Dawley rats were used in this study. 16 rats were the control group, 56 rats were performed operation at both knees to induce OA by anterior cruciate ligament transection (ACLT) in combination with resection of part medial menisci (part-MMx). At 6 weeks, we detected the OA model by killing 8 rats(ACLT+ part-MMx), compared with 4 control rats. The other 48 rats were divided into two group: OA group(24 rats) and OVX + OA group(24rats). The OVX + OA group was performed by both side ovariectomy. The articular cartilage in medial femoral condyle and femoral trochlea of control rats(4 ), OA rats(8 ) and OVX + OA rats (8) were acquired at 9, 12, and 16 weeks postoperation, respectively. We observed the morphological change of the articular cartilage in OA rat model by histological technique, including haemotoxylin(H.E.) and safranin O, and detected the expression of Akt and P-Akt by immunohistochemical technique and Western-Blotting from 6 to 16-week post-operation. All data were analyzed in SPSS statistical program.
Results We established the OA and OA-OVX rat model, in which the morphological change of articular cartilage is accordance with the histopathological structure of OA. However, it is no significant morphological change in the articular cartilage following the decrease of estrogen by OVX in OA rat model (P>0.05). Furthermore, we observed that the expression of Akt and P-Akt in articular cartilage in OA-OVX model is significantly lower than that in OA model (P<0.05).But the expression of Akt and P-Akt in articular cartilage gradually increased in a time-dependent manner from 6 to 12-week post-operation (P<0.01), and then the expression of Akt and P-Akt decreased rapidly in 16-week post-operation (P<0.05),whether in OA or in OA-OVX rat model.
Conclusion It is adoptable to establish the OA rat model by ACLT and part-MMX. And it is no more significant effect on the morphological structure of articular cartilage after OVX in OA rat model. It is suggested that the decrease of estrogen by OVX may be associated with the decrease of the expression of Akt and P-Akt.
引文
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