【摘要】：背景和目的：腦缺血后星形膠質(zhì)細胞激活成為反應(yīng)性星形膠質(zhì)細胞,并進一步形成膠質(zhì)疤痕,抑制軸突再生,影響神經(jīng)功能恢復(fù)。目前尚無模擬腦缺血引起的離體膠質(zhì)疤痕模型,本實驗?zāi)康氖墙⑷碧侨毖?Oxygen-Glucose Deprivation, OGD)誘導(dǎo)的離體膠質(zhì)疤痕模型,為更好的研究腦缺血后膠質(zhì)疤痕的形成機制及為尋找促進神經(jīng)功能恢復(fù)的靶點或相關(guān)藥物提供便利。 方法：體外培養(yǎng)的大鼠原代星形膠質(zhì)細胞經(jīng)OGD處理6 h,分別在再灌后0 h、24 h和48 h檢測各類星形膠質(zhì)激活及疤痕形成的指標(biāo),包括利用MTT法和5-溴脫氧尿核苷(5-Bromodeoxyuridine, BrdU)標(biāo)記新生細胞數(shù)量法觀察細胞增殖情況,利用細胞免疫化學(xué)或免疫印記的方法考察膠質(zhì)原纖維酸性蛋白(Glial Fibrillary Acidic Protein, GFAP)的表達和硫酸軟骨素類蛋白聚糖neurocan、phosphacan的表達。并將經(jīng)OGD處理的星形膠質(zhì)細胞與大鼠胚胎皮層神經(jīng)元共培養(yǎng)24 h,觀察對神經(jīng)元突起生長的影響。 結(jié)果：在再灌24h,OGD處理的星形膠質(zhì)細胞增殖明顯,GFAP表達顯著上調(diào),BrdU標(biāo)記的新生細胞數(shù)明顯增加,膠質(zhì)疤痕特異性標(biāo)記蛋白neurocan表達呈顯著上調(diào),而phosphacan的表達在再灌注后呈先下降后恢復(fù)的動態(tài)變化。同時,在共培養(yǎng)模型中,將OGD處理的星形膠質(zhì)細胞在再灌0h時與神經(jīng)元共培養(yǎng),神經(jīng)元突起長度比對照組明顯增長；在再灌24 h時進行共培養(yǎng),兩組間無明顯變化；而在再灌48 h后將兩者共培養(yǎng),則發(fā)現(xiàn)OGD處理組的神經(jīng)元突起生長明顯受到抑制。 結(jié)論：本實驗成功構(gòu)建了一個OGD誘導(dǎo)的離體膠質(zhì)疤痕模型,該模型將可用于缺血后膠質(zhì)疤痕的形成機制研究及神經(jīng)功能保護策略的研究。
[Abstract]:Background & AIM: after cerebral ischemia, astrocytes are activated into reactive astrocytes, and glial scars are formed, axon regeneration is inhibited, and neural function recovery is affected. At present, there is no model of glial scar induced by cerebral ischemia in vitro. The aim of this experiment is to establish an isolated glial scar model induced by glucose deficiency and hypoxia (Oxygen-Glucose Deprivation, OGD). In order to better study the formation mechanism of glial scar after cerebral ischemia and to find the target or related drugs to promote the recovery of neural function. Methods: cultured rat astrocytes were treated with OGD for 6 h. The activation of astrocytes and scar formation were detected at 0 h and 48 h after reperfusion respectively. MTT method and 5-Bromodeoxyuridine (BrdU) labeling method were used to observe cell proliferation and glial fibrillary acidic protein (Glial Fibrillary Acidic Protein,) was detected by immunocytochemistry or immunological imprinting. GFAP and neurocan,phosphacan expression of chondroitin sulfate proteoglycan. The astrocytes treated with OGD were co-cultured with rat embryonic cortical neurons for 24 h to observe the effect on neurite growth. Results: the proliferation of astrocytes, the expression of GFAP, the number of new cells labeled with BrdU and the expression of glial scar specific marker protein (neurocan) were significantly increased after 24 h reperfusion. The expression of phosphacan decreased first and then recovered after reperfusion. At the same time, in co-culture model, the neurite length of astrocytes treated with OGD was significantly longer than that of control group at 0 h after reperfusion, but there was no obvious change between the two groups at 24 h after reperfusion. However, after 48 h of reperfusion, the neurite growth of OGD treated group was obviously inhibited. Conclusion: this experiment successfully constructed an isolated glial scar model induced by OGD, which can be used to study the mechanism of glial scar formation and the neuroprotective strategy after ischemia.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R-332

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