【摘要】 目的:探讨电针对D-半乳糖处理后衰老大鼠神经细胞凋亡的抑制作用。方法:实验于2000-10/2003-01在上海中医药大学生理学教研室完成。选取雄性Wistar大鼠42只,随机分为正常组、模型组、电针组,14只/组。正常组每天皮下注射生理盐水1mL,连续6周;模型组每天皮下注射D-半乳糖800mg/kg,连续6周;电针组每天皮下注射同剂量D-半乳糖,第18天开始电针,电针穴位根据动物“针灸穴位图谱”选择百会、大椎,采用电流强度5mA、频率10Hz的断续波,15min/d,至第6周末结束。凋亡细胞以原位末端标记法检测,凋亡相关因子蛋白行免疫组织化学法检测,线粒体形态的改变用体视学方法分析。结果:42只大鼠全部进入结果分析。①各组脑海马锥体细胞的超微结构比较:正常组核周边内质网、线粒体、高尔基体等细胞器丰富,核大而圆,染色质密度均一,核仁明显。模型组粗面内质网大量增生,线粒体嵴变短、不规则或消失,溶酶体数量增加,细胞内出现自嗜体,核不规则固缩,染色质向核膜凝集成块,有核仁裂解现象,核膜、细胞器膜、细胞膜完整,提示模型组神经细胞有凋亡样形态学改变。电针组与正常组接近,主要表现为线粒体的肿胀不明显,溶酶体数量有所下降,核结构接近正常。②各组大鼠脑海马CA1区线粒体比表面情况:与正常组比较,模型组变小,电针组基本无变化(25.02±14.66,21.03±10.75,23.87±14.00,P<0.05,P>0.05)。③各组大鼠脑海马CA1区线粒体平均轴比情况:与正常组比较,模型组变大,电针组基本无变化(1.57±0.72,2.04±0.83,1.63±0.69,P<0.05,P>0.05);电针组明显高于模型组(P<0.05)。④凋亡阳性细胞率:与正常组比较,模型组显著增加,电针组略高(0.25±0.11,1.07±0.31,0.50±0.34,P<0.05),与模型组比较,电针组明显降低(P<0.05)。⑤Bax与Bcl-2阳性细胞数检测结果:与正常组Bax比较,模型组呈过表达,电针组略高(66.4±12.8,140.4±41.0,77.1±18.1,P<0.05),电针组明显低于模型组(P<0.05);与正常组Bcl-2比较,模型组表达明显降低,电针组略低(157.0±59.7,56.9±15.3,108.5±25.8,P<0.05),电针组明显高于模型组(P<0.05)。⑥Bax与Bcl-2积分吸光度检测结果:与正常组Bax比较,模型组呈过表达,电针组略高(4.2±2.1,10.6±5.9,7.5±5.7,P<0.05),电针组明显低于模型组(P<0.05);与正常组Bcl-2比较,模型组表达明显降低,电针组升高(8.0±1.7,1.2±0.8,13.9±4.1,P<0.05),电针组明显高于模型组(P<0.05)。结论:D-半乳糖致衰老时,大鼠脑海马组织中存在凋亡阳性神经细胞增加的现象。电针干预后,凋亡抑制基因Bcl-2蛋白的表达量显著增强,线粒体形态得到改善,凋亡阳性细胞率也有所下降,但对促凋亡基因Bax蛋白的调节不很明显。因而推断电针是通过调节Bcl-2蛋白的表达来抑制D-半乳糖所引起的神经细胞凋亡的。更多还原

【Abstract】 AIM To observe the inhibition of electro-acupuncture on neural cell apoptosis induced by D-galactose in senility rats. METHODS The experiment was done in Staff Room of Physiology of Shanghai University of Traditional Chinese Medicine from October 2000 to January 2003. Forty-two male Wistar rats were selected and divided randomly into normal group model group and electro-acupuncture group with 14 in each group. The rats in normal group were injected with 1 mL saline subcutaneous injection every day for 6 weeks The rats in model group were injected with 800 mg/kg D-galactose subcutaneous injection every day for 6 weeks The rats in electro-acupuncture group were injected with the same D-galactose subcutaneous injection every day and treated with electric needle at the 18th day. According to animal acupoint pattern Paihui Du20 and Tachui DU 14 were selected as acupuncture point with 5 mA current strength CS 10 Hz frequency 15 minutes per day lasting to the end of the 6th week. Apoptosis of neurons was determined with In situ end labeling ISEL. The protein expression of relative factors was observed by using immunohistochemistry. The changes of morphology of mitochondria was analyzed with stereology method. RESULTS Totally 42 rats were involved in the result analysis. ① The comparison of ultrastructure of brain hippocampus pyramidal cell in every group Endoplasmic reticulum mitochondrion and golgi apparatusetc. were rich around nucleolus which was big and round with even chromatin density and obvious nucleolus. The rough endoplasmic reticulum RER in model group was proliferated in a large number and the mitochondrial cristae became short irregular or disappeared. The number of lysosomes increased and autophagosome was appeared in cells with irregular pycnosis of accessory nucleus of oculomotor nerve. The agglutination of chromatin formed block toward karyotheca. There were the phenomena of nucleolus burst but karyotheca organell lemma and cell membrane were intact which indicated that there were apoptosis morphological changes of nerve cell in model group. It was similar between that in electro-acupuncture group and in normal group which expressed that the mitochondrial swelling was insignificant. The number of lysosome reduced and the structure of nucleus stone was closed to normal situation. ② The facial condition of mitochondrion in brain hippocampi CA1 area in every group Compared with that in normal group it became shorter in model group and no change in electro-acupuncture group basically (25.02±14.66,21.03±10.75,23.87±14.00,P < 0.05,P > 0.05). ③ The average axial wall condition in brain hippocampi CA1 area in every group Compared with that in normal group it became bigger in model group and no change in electro-acupuncture group basically (1.57±0.72,2.04±0.83,1.63±0.69,P < 0.05,P > 0.05). It was significantly higher in electro-acupuncture group than in model group (P < 0.05). ④ The apoptosis positive cell rate Compared with that in normal group it increased significantly in model group and became a little higher in electro-acupuncture group(0.25±0.11,1.07±0.31,0.50±0.34,P < 0.05). Compared with that in model group it decreased significantly in electro-acupuncture group (P < 0.05). ⑤ The detection result of the number of Bax and Bcl-2 positive cell Compared with Bax in normal group it passed in model group and it was a little higher in electro-acupuncture group(66.4±12.8,140.4±41.0,77.1±18.1,P < 0.05). It was significantly lower in electro-acupuncture group than that in model group (P < 0.05);Compared with Bcl-2 in normal group it decreased significantly in model group and it was a little lower in electro-acupuncture group (157.0±59.7,56.9±15.3,108.5±25.8,P < 0.05). It was significantly higher in electro-acupuncture group than that in model group (P < 0.05). ⑥ The absorbance detection result of Bax and Bcl-2 integra Compared with Bax in normal group it passed in model group and it was a litter higher in electro-acupuncture group (4.2±2.1,10.6±5.9,7.5±5.7,P < 0.05). It was significantly lower in electro-acupuncture group than that in model group (P < 0.05). Compared with Bcl-2 in normal group it decreased significantly in model group and it decreased in electro-acupuncture group (8.0±1.7,1.2±0.8,13.9±4.1,P < 0.05). It was significantly higher in electro-acupuncture group than that in model group (P < 0.05=.CONCLUSION When D-galactose induces senility the phenomena of the increase of positive apoptotic cells in brain hippocampus tissue in rats is found. After the intervention of electro-acupucture the protein expression of apoptosis inhibiting gene Bcl-2 increases significantly and the morphology of mitochondria has been ameliorated and the rats of positive apoptotic cells decreases also a little. However it is insignificant of the adjustment of Bax protein which leads to apoptosis. Thus it concludes that electro-acupuncture inhibits nerve apoptosis due to D-galactose through the adjustment of Bcl-2 protein expression.更多还原