缺氧条件下鼠胚软骨c-Fos和巢蛋白表达及当归的保护作用

Effect of Chinese angelica on the expression of c-Fos and nidogen in embryonic cartilage of rats during hypoxia stage

目的:探讨宫内缺氧对胚胎大鼠软骨组织内c-Fos和巢蛋白表达的影响,以及中药当归注射液对缺氧状态下其表达的调节。方法:于2004-06/12在四川省泸州医学院动物科将成年发情期雌性Wis-tar大鼠(220～250g)12只,分别与1只雄鼠合笼饲养,于次日上午8:00发现阴栓记孕鼠怀孕0d,饲养雌鼠至孕15d。将12只孕鼠随机分为对照组、缺氧组和当归组各4只(每只孕鼠产胎鼠5～8只)。当归组孕鼠经尾静脉注入当归注射液,然后用低张性缺氧模型致鼠胚宫内缺氧;缺氧组用生理盐水代替当归注射液,其余同当归组;对照组不缺氧,其余同缺氧组。然后麻醉状态下处死孕鼠,快速剖腹取胎,每组中随机选取20个鼠胚,将各组胎鼠头部软骨组织作c-Fos和巢蛋白免疫组化双标染色后,进行观察分析。结果分析与判断:每张切片用DP12数码相机在400倍下照相,并随机选择1个软骨组织视野观察计数。c-Fos免疫组化反应阳性细胞为胞核着蓝色,巢蛋白免疫组化反应阳性细胞胞质染红色。将凡是胞质染红色的细胞根据红色深浅分为3个等级:深红色计为、红色计为、淡红色计为+,分别对其记数,然后将同一组内20个鼠胚3个等级的阳性细胞数分别相加,得出该组的巢蛋白免疫组化结果。既有胞核染蓝色,又有胞质染红色的为c-Fos/巢蛋白免疫组化双染阳性细胞,计数每例鼠胚软骨组织双染阳性细胞的数量。结果:60个鼠胚头部软骨组织进入结果分析。①对照组软骨组织c-Fos和巢蛋白有弱表达;与对照组相比,缺氧组软骨组织c-Fos和巢蛋白表达增强[c-Fos:(5.35±1.42),(14.55±3.37)个/视野,q=16.48,P<0.05;巢蛋白:193,427个/20个鼠胚,RA-RB=66.78,P<0.05]。②与缺氧组相比,当归组软骨组织c-Fos和巢蛋白表达减弱[c-Fos:(8.00±2.31)个/视野,q=11.73,P<0.05;巢蛋白:250个/20个鼠胚,RA-RB=51.83,P<0.05]。结论:①缺氧可刺激鼠胚软骨组织巢蛋白的表达,c-Fos可能是缺氧刺激巢蛋白表达的机制之一。②当归注射液对缺氧鼠胚软骨组织可能有保护作用。

AIM：To study the effect of hypoxia in uterus on the expression of c-Fos and nidogen in embryonic cartilage tissue of rats and the regulative role of angelica parenteral solution in the expression of them during hypoxia stage. METHODS： The experiment was completed at Animal Department of Luzhou Medical College from June to December 2004. Totally 12 female Wistar rats in rut weighting 220-250 g were fed with a male rat respectively. At 8 o＇clock of next morning, rats recorded with negative lock were conceived for 0 day, but female rats were conceived for 15 days. All 12 conceived rats were divided randomly into the control group （CG）, the hypoxia group （HG） and the angelica group （AG） with 4 in each group （with 5-8 fetal rats in each mother rat）. Rats in angelica group were injected with angelica parenteral solution through caudal veins before their embryos were in hypotonic hypexia in uterus. Rats in hypoxia group were injected with saline and other processes were as the same as those in angelica group. Rats in control group were not treated with hypoxia and others were as the same as those in hypoxia group, Then conceived rats were sacrificed under anesthesia, and fetuses were obtained after cutting the belly open. Twenty embryos were selected randomly from each group, and the expression of c-Fos and nidogen in cartilage tissue of embryos was studied with immunohistochmistry double staining and the results were observed and analyzed under light microscope. Analysis and judgement： Each section was photographied with DP12 digital camera （×400）, and sight of 1 cartilage tissue was selected randomly to observe the account. Nucleus of positive cell was blue with c-Fos immunohistochmistry, and cytoplasm of positive cell was red with nidogen immunohistochmistry. Red cytoplasm was classified into three grades according to color level： deep red was ＋＋＋, red was ＋＋, and light red was ＋. The account was recorded respectively. Posi-tive cells in the three grades of embryo in the same group were added to work out the results of nidogen immunohistochmistry. Cells showed both blue nucleus and red cytoplasm were regarded as double staining positive cells with c-Fos/nidogen immunohistochmistry, and the numbers double staining positive cells were accounted in cartilage tissue in each group. RESULTS： Totally cartilage tissue in brain of 60 rats entered the final analysis. ① c-Fos and nidogen in cartilage tissue had mild expression in control group. Expression of c-Fos and nidogen in cartilage tissue was increased in hypoxia group as compared with that in control group [c-Fos： （5.35±1.42）, （14.55±3.37）/sight, q=16.48, P 〈 0.05; nidogen： 193, 427/20 embryo, ｜R^-A-R^-B｜=66.78, P 〈 0.05]. ②Expression of c-Fos and nidogen in cartilage tissue was decreased in angelica group as compared with that in hypoxia group [c-Fos： （8.00±2.31） /sight, q=11.73, P 〈 0.05; nidogen： 250/20 embryo, {R^-A-R^-B}=51.83, P 〈 0.05]. CONCLUSION： ① Hypoxia can stimulate the expression of nidogen in embryonic cartilage tissue of rats, and c-Fos may be one of the mechanism of expression of nidogen stimulated by hypoxia. ②Angelica parenteral solution can protect embryonic cartilage tissue of rats during hypoxia stage.