Analysis of apoptotic mechanism of cerebellar granular cells

建立了氯化甲基汞(MMC)中毒大鼠小脑颗粒细胞损伤模型。为阐明MMC致脑损伤的分子机制，我们采用HE染色、电子显微镜、TUNEL染色和DNA片段化等方法，初步观察了MMC致脑损伤的病理改变。这些结果表明，有机汞中毒小脑颗粒细胞的死亡是通过一种凋亡过程发生的，且仅限于颗粒细胞，并且凋亡的小脑颗粒细胞在大鼠小脑虫中的分布模式是特有的。其次，用免疫印迹法检测了BDNF、Trk B、Fas配体、RIP、Akt、Bad、Bcl2、Caspase9、Caspase3、CAS、TIAR等凋亡相关分子的表达水平，并用抗Calbindin D免疫组织化学染色检测了浦肯野细胞的功能。此外，我们还观察了MMC中毒后大鼠脑组织基因表达的变化。这些结果表明，…BDNF和Trk B表达上调，RIP、CAS、Fas配体、Bad和Caspase 9表达下调。这一发现表明，在这个阶段，上调的分子起到了抑制细胞凋亡的作用。基因表达分析表明：1)与细胞凋亡相关的基因有蛋白激酶Cγ、白介素13、胰岛素受体底物Z、亲环素、蛋白磷酸酶1和RL/IF-1；2)与激素相关的基因有加压素和催产素；3)与酶相关的基因有血清和糖皮质激素调节的激酶、肥大细胞蛋白酶、丝氨酸蛋白酶、糖蛋白专一性葡萄糖醛酸基转移酶、蛋白酪氨酸磷酸酶；4)7个杂类分子；5)7个未知基因。相反，下调的基因有2个α1珠蛋白基因和2个未知基因。这些结果表明，细胞凋亡抑制基因在小脑细胞凋亡开始时被激活。由此可见，在MMC中毒初期，抗凋亡分子的表达增强，保护细胞死亡的作用强于诱导凋亡分子。较少

We have established a rat model of injury of cerebellar granule cells by intoxication of methylmercury chloride (MMC). To elucidate the molecular mechanisms of brain damage induced by MMC we initial ly examined the pathologic examination by H&E staining, electron microscope, TUNEL staining, and DNA fragmentation. These examination revealed that cerebellar granule cell death by organic mercury intoxication occurs via an apoptotic process and was restricted to the granule cells and that the distributional pattern of apoptotic cerebellar granule cells was specific in rat cerebellar vermis. Secondly, we investigated expression levels of apoptosis related molecules such as; BDNF, Trk B, Fas ligand, RIP, Akt, Bad, Bcl-2, Caspase 9, Caspase 3, CAS, and TIAR using Western blot and also evaluated the function of Purkinje cells by immunostaining with anti-calbindin D. Moreover, we investigated the alteration of gene expression of rat brain after MMC intoxication. These results demonstrated that … More expression of BDNF and Trk B were up-regulated, while RIP, CAS, Fas ligand, Bad, and Caspase 9 were down-regulated. The findings suggest that the up-regulated molecules function to suppress apoptosis at this stage. Gene expression analysis disclosed that among up-regulated genes 1) those related to apoptosis were protein kinase C γ, interleukin 13, insulin receptor substrate Z, cyclophilin, protein phosphatase 1, and RL/IF-1, 2) those related to hormone were vasopressin and oxytocin, 3) those related to enzyme were serum and glucocorticoid-regulated kinase, mast cell protease, serine protease, glycoprotein specific UDP-glucuronyl transferase, protein tyrosine phosphatase, 4) 7 miscellaneous molecules, and 5) 7 unknown genes. In contrast, down-regulated genes were 2α1 globin gene and 2 unknown genes. The results could be summarized that apoptosis suppressive genes were activated at the beginning of cerebellar apoptosis. Thus, it could be concluded that in the initial stage of MMC intoxication the expression of anti-apoptotic molecules were more enhanced to protect cell death than apoptosis-inducing molecules. Less