| 摘要: | 當細胞處於壓力下，如：熱休克、癲癇、腦部缺血和機械性的腦部外傷等會誘發熱休克蛋白質70 (Heat shock protein70；HSP70)產生。過去研究顯示在顳葉癲癇病人（temporal lobe epilepsy；簡稱TLE）及Kainic acid (KA)誘發癲癇的動物腦內會有HSP70存在，而這些HSP70會延遲神經細胞的凋亡1。但HSP70在腦部的機制仍不是很清楚，故本實驗主要目的探討HSP70在癲癇中的主要機制。
本實驗共分為三種研究方式，第一人體組織：共收集6例癲癇與3例非癲癇病人的顳葉海馬迴檢體，第二動物模式：經注射KA誘發大白鼠（Male Sprague–Dawley；SD）產生癲癇發作之動物模組，第三細胞模式：培養SD胚胎鼠之初代海馬迴神經細胞、顳葉皮質星細胞及經轉殖HSP70-siRNA之初代海馬迴神經細胞後，添加KA誘發產生HSP70；利用HE（Hematoxylin & Eosin）染色、免疫化學染色、免疫螢光染色及凋亡分析法觀察細胞變化。
由實驗觀察到TLE病人海馬迴CA3區域與腦內海馬迴注射KA的大白鼠在CA3區域會有神經細胞減少及膠細胞增生聚集的情形。雙重螢光染色結果中，顯示HSP70是與神經細胞(MAP2)共存且在注射KA 2天達到最高的表現，而PCNA（Proliferating Cell Nuclear Antigen）和GFAP在注射KA 5天後具有顯著增加。由TUNEL和FJB（FLUORO-JADE® B；FJB）結果得知凋亡神經細胞在注射KA 5天最多（TUNEL：控制組2.625±1.06%、KA 2天：39.37±2.97%、KA 5天：62.25±12.4%；p<0.001）（FJB：控制組4.245±2.2%、KA 2天73.5±13.84%、KA 5天98.75±9.6%；p<0.001）且凋亡的神經細胞不會有HSP70表現。同樣在初代細胞中經KA誘發的HSP70表現位置也是在神經細胞。經轉殖HSP70-siRNA添加KA刺激後可觀察到神經凋亡與HSP70的存在與否並無顯著關係。
結果顯示，KA會誘發神經細胞產生HSP70，但神經細胞轉殖HSP70-siRNA抑制HSP70後，再添加KA，發現細胞凋亡與HSP70的存在沒有絕對相關性，所以我們推論HSP70可能是一種壓力標誌(stress marker)，與細胞凋亡保護機制無明顯關係。
Heat shock protein 70 (HSP70) is well induced during press. Previous reports indicated induction of inducible HSP70 response to stressful conditions, such as heat shock, epilepsy, ischemia, and trauma. HSP70 was found in hippocampus of the temporal lobe epilepsy (TLE), and the treatment with Kainic acid (KA) in vivo had shown, however, these mechanisms in the brain are still poorly elucidated. In this aim of research is to explore the mechanism of HSP70 in epilepsy.
This experiment is totally divided three kinds of research ways. First, human sample: collect 6 TLE patients’ and 3 non- epilepsy patients’.Second, animal model: Male Sprague–Dawley（SD） rats, KA was microinjected into the CA1 area of the hippocampus to induce seizure.Third, cell model: primary neuron culture of embryonic day 18 SD rat pups (E18), primary cortex astrocytes cultures were prepared from newborn rat brains (1–2-day-old SD rat), and neuron culture was transfected with HSP70-siRNA（small interfering RNA）.Treat them KA to induce HSP70.We use Hematoxylin & Eosin（HE） stain, immunohistochemical stain, double immunofluorescence assay and analysis of apoptosis to study cell morphology.
Our observations suggest that HE stain shows that neuronal degeneration and gliosis are found in the CA3 area of TLE patients and treated KA animal model. By using double-immunofluorescence of HSP70 with MAP2 show that HSP70 is found mainly within neuron.Proliferating Cell Nuclear Antigen (PCNA）and GFAP double immunostaining show that KA induced epilepsy is evoked and increase of PCNA, whereas glia cells proliferation are detected 5 days after epileptic seizures. According to TUNEL and FJB（FLUORO-JADE® B）, we found denatured neuron obvious after treating KA 5 days （TUNEL：control 2.625±1.06%、KA 2days 39.37±2.97%、KA 5days 62.25±12.4%；p<0.001）（FJB：control 4.245±2.2%、KA 2 days 73.5±13.84%、KA 5 days 98.75±9.6%；p<0.001）.By using double-immunofluorescence of FJB and HSP70, we found that HSP70 is not expression in degenerate neuron. In KA treated neuron culture transfected with HSP70-siRNA found that the neuron apoptosis are unrelated with existence of the HSP70. So we suggest that HSP70 is just a stress marker. |
