Rhodopsin和recoverin在MNU诱导的光感受器损伤中的表达变化

目的：观察N-甲基-N-亚硝基脲( MNU)诱导的大鼠视网膜光感受器损伤过程中Rhodopsin 和recoverin表达变化与损伤的时效关系。
　　方法：将36只SPF级7周龄大鼠随机分为正常对照组, MNU模型组(6h组,12h组,24h组,3d组,7d组),每组各6只。模型组一次性腹腔注射60mg/kg MNU,正常对照组腹腔注射等量PBS。右眼行HE,TUNEL,透射电镜评估视网膜组织损伤的超微结构变化及细胞凋亡程度,左眼取视网膜组织通过Western blot和免疫荧光观察视网膜组织中Rhodopsin和recoverin的mRNA表达变化。
　　结果：透射电镜观察到MNU注射12 h 后出现凋亡小体,24 h后外核层大部分细胞呈阳性反应；TUNEL 检测发现MNU注射24 h 光感受器细胞凋亡指数最高,达(29.7±2.3)%,与电镜结果吻合。 Western blot 结果表明, MNU注射12 h后表达有极显著性差异( P<0.01),而Recoverin的表达从注射后24h有极显著性差异(P<0.01)。
　　结论：一次性腹腔注射60 mg/kg MNU能特异性诱导SD大鼠视网膜光感受器细胞凋亡, Rhodopsin和recoverin表达下调与MNU诱导光感受器细胞的选择性凋亡有关。

Expression changes of Rhodopsin and recoverin in MNU-induced photoreceptor degeneration in rats

AIM: To investigate the time-effect relationship between the expression of rhodopsin and recoverin and photoreceptor damage induced by N-nethl-N-nitrosourea(MNU).

METHODS: Thirty-six 7-week old Sprague-Dawley(SD)rats were intraperitoneally injected with MNU(60mg/kg)and were put to death by dislocation of cervical vertebra 6, 12, 24h; 3, 7d after injection(6 per group), respectively. As a control, six rats were injected with phosphate buffer saline(PBS)5mL/kg and sacrificed on d3 after injection. The degree of photoreceptor apoptosis was detected by HE staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling(TUNEL)and transmission electron microscope(TEM)in the right eyes. The mRNA expressions of rhodopsin and recoverin were detected different time after injection by Western blot and immunohistochemical method in the left eyes.

RESULTS: The dissolution of photoreceptor nucleus and apoptosis body were first perceived at 12h by TEM; most of cells at outer nuclear layer were presented positive reaction. The apoptotic index reached peak(29.7%±2.3%)at 24h which was coincided with the observation of TEM. The results of immunohistochemistry displayed that rhodopsin and recoverin were on a declining curve with time extension. Furthermore, the results of Western blot indicated that rhodopsin had dramatic decline at 6h after injection(P<0.05), and extremely significant difference comparing to control group after 12h(P<0.01); while recoverin dramatic declined at 12h, and extremely significant difference after 24h(P<0.01).

CONCLUSION: 60mg/kg MNU intraperitoneally injection one-time may specifically induce photoreceptor apoptosis, The mechanism of down-regulation of rhodopsin and recoverin may be related to the selected apoptosis of photoreceptors.