本文選題：鉛 + 錳　； 參考：《細(xì)胞與分子免疫學(xué)雜志》2016年03期

【摘要】：目的以小膠質(zhì)細(xì)胞與星形膠質(zhì)細(xì)胞建立共培養(yǎng)模型,研究鉛、錳單獨(dú)及聯(lián)合暴露下不同類型膠質(zhì)細(xì)胞的反應(yīng)及小膠質(zhì)細(xì)胞活化對星形膠質(zhì)細(xì)胞功能的影響。方法先通過MTT法篩選對C6星形膠質(zhì)細(xì)胞生長無影響的鉛和錳的暴露劑量和作用時間,再選取BV2小膠質(zhì)細(xì)胞和C6細(xì)胞以鉛、錳單獨(dú)及聯(lián)合染毒建立細(xì)胞模型。分為直接刺激法、條件培養(yǎng)基法和共培養(yǎng)法3種細(xì)胞模型。直接刺激法采用完全培養(yǎng)基或含醋酸鉛、氯化錳培養(yǎng)基直接作用于C6細(xì)胞24 h;條件培養(yǎng)基法采用完全培養(yǎng)基或含鉛、錳培養(yǎng)基作用于BV2細(xì)胞24 h,取上清經(jīng)離心后作用于C6細(xì)胞24 h;共培養(yǎng)法將BV2細(xì)胞接種于TranswellTM共培養(yǎng)模型上層小室內(nèi),再將上層小室置入空白12孔板內(nèi),將C6細(xì)胞接種于另一12孔板內(nèi),以完全培養(yǎng)基或含鉛、錳培養(yǎng)基作用接種于TranswellTM共培養(yǎng)模型上層小室內(nèi)的BV2細(xì)胞,24 h后棄去培養(yǎng)基,以完全培養(yǎng)基輕柔洗滌后,將含BV2細(xì)胞的上層小室置入接種C6細(xì)胞的12孔板內(nèi)繼續(xù)培養(yǎng)24 h。采用Western blot法檢測補(bǔ)體受體3(CR3/CD11b/OX42)、膠質(zhì)纖維酸性蛋白(GFAP)水平,確定鉛、錳單獨(dú)及聯(lián)合暴露下對不同類型膠質(zhì)細(xì)胞的影響;檢測谷氨酰胺合成酶(GS)水平確定小膠質(zhì)細(xì)胞的活化對星形膠質(zhì)細(xì)胞谷氨酸-谷氨酰胺循環(huán)環(huán)路的影響。結(jié)果直接刺激模型中,10μmol/L鉛、100μmol/L錳作用星形膠質(zhì)細(xì)胞24 h,鉛、錳單獨(dú)及聯(lián)合暴露不能引起星形膠質(zhì)細(xì)胞顯著活化及其GS表達(dá)的改變;條件培養(yǎng)基模型中,10μmol/L鉛、100μmol/L錳作用于BV2小膠質(zhì)細(xì)胞24 h,BV2小膠質(zhì)細(xì)胞OX42表達(dá)水平顯著升高,將其培養(yǎng)基作用于C6星形膠質(zhì)細(xì)胞24 h后,C6細(xì)胞GFAP表達(dá)顯著性升高,GS表達(dá)顯著性降低;共培養(yǎng)模型中,10μmol/L鉛、100μmol/L錳作用于BV2小膠質(zhì)細(xì)胞24 h,BV2小膠質(zhì)細(xì)胞OX42表達(dá)水平顯著升高,將其洗滌后與C6星形膠質(zhì)細(xì)胞共培養(yǎng)24 h后,星形膠質(zhì)細(xì)胞GFAP表達(dá)顯著性升高,GS表達(dá)顯著性降低。結(jié)論低劑量鉛、錳單獨(dú)及聯(lián)合暴露能夠引起體外培養(yǎng)的小膠質(zhì)細(xì)胞活化,而活化的小膠質(zhì)細(xì)胞能夠誘導(dǎo)星形膠質(zhì)細(xì)胞活性增強(qiáng)及GS表達(dá)水平降低,且鉛錳聯(lián)合暴露比單獨(dú)暴露效應(yīng)更為顯著。
[Abstract]:Objective to establish a co-culture model of microglia and astrocytes to study the effects of different types of glial cells exposed to lead and manganese alone or in combination and the effects of microglial activation on the function of astrocytes. Methods the exposure dose and time of exposure to lead and manganese which had no effect on the growth of C6 astrocytes were screened by MTT assay. Then BV2 microglial cells and C6 cells were selected to be exposed to lead and manganese alone or in combination to establish the cell model. It was divided into three cell models: direct stimulation method, conditioned medium method and co-culture method. Direct stimulation was carried out on C6 cells in full medium or lead acetate, manganese chloride medium was directly applied to C6 cells for 24 h, and conditional medium method was used in complete medium or lead. BV2 cells were treated with manganese medium for 24 h, the supernatants were centrifuged for 24 h, BV2 cells were co-cultured in the upper chamber of Transwell TM co-culture model, and then the upper chamber was placed into a blank 12-well plate. C6 cells were inoculated in another 12-well plate. BV2 cells in the upper chamber of Transwell TM co-culture model were inoculated in full medium or lead and manganese medium for 24 hours. The upper chamber containing BV2 cells was cultured in a 12-well plate inoculated with C6 cells for 24 h. The levels of complement receptor 3 (CR3 / CD11b / OX42) and glial fibrillary acidic protein (GFAP) were determined by Western blot assay to determine the effects of lead and manganese exposure alone or in combination on different types of glial cells. The level of glutamine synthase (GS) was measured to determine the effect of microglia activation on the glutamate-glutamine loop in astrocytes. Results in the direct stimulation model, 10 渭 mol / L lead and 100 渭 mol / L manganese could not induce the activation of astrocytes and the changes of GS expression in astrocytes after exposure to lead and manganese alone or in combination for 24 h. In the conditioned medium model, the expression of OX42 in BV2 microglia cells was significantly increased by 100 渭 mol / L mn (10 渭 mol / L) on BV2 microglia cells, and the expression of GFAP in C6 astrocytes was significantly increased after 24 hours of treatment with the medium and the expression of GS in BV2 microglial cells was significantly decreased. The expression of GFAP in BV2 microglial cells was significantly decreased after treated with 10 渭 mol / L lead 100 渭 mol / L manganese for 24 h. In the co-culture model, 10 渭 mol 路L ~ (-1) lead and 100 渭 mol 路L ~ (-1) manganese increased the expression of OX42 in BV2 microglia for 24 h, and then co-cultured with C6 astrocytes for 24 h after washing. The expression of GFAP in astrocytes increased significantly and the expression of GS decreased significantly. Conclusion low dose lead and manganese exposure alone and in combination can induce activation of microglia in vitro, while activated microglia can induce increased activity of astrocytes and decrease of GS expression. The combined exposure of lead and manganese was more significant than that of single exposure.
【作者單位】： 第四軍醫(yī)大學(xué)軍事預(yù)防醫(yī)學(xué)系軍隊勞動與環(huán)境衛(wèi)生學(xué)教研室;
【基金】：國家自然科學(xué)基金(81230063,81472942)
【分類號】：R114

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