血红素对脑神经元、星形胶质细胞、毛细血管内皮细胞的不同损伤作用

Different toxic effect of hemin on primary cultured neurons, astrocytes and brain capillary endothelial cells

目的研究不同剂量高铁血红素对脑内神经元、星形胶质细胞、脑毛细血管内皮细胞（BCEC）的损伤作用。方法（11在原代培养大鼠脑皮质神经元、星形胶质细胞、BCEC上添加不同剂量（0、5、25、50mmol／L）的高铁血红素孵育2h，去血红素后继续培养24或96h，阿拉玛蓝染色检查细胞存活率，常规生化反应法检测乳酸脱氢酶（LCD）释放率，相差光学显微镜观察细胞形态。（2）在原代培养细胞上添加不同剂量血红素孵育2h，去血红素后继续培养4h，以浓甲酸裂解细胞．分光光度计下测定细胞内血红素含量。（31在原代培养细胞上加血红素分别孵育30、60、120min，去血红素后继续培养4h，以中性福尔马林液固定细胞，普鲁士蓝染色检查细胞内三价铁离子（Fe3＋）染色情况。结果（1）神经元经5mmol／L的血红素处理后24h，存活率下降40_2％，LCD释放率增加22．2％，细胞形态出现严重损伤病变。随着时间延长和血红素剂量加大，细胞死亡率和LCD释放率均增加，细胞病变加重。（2）中、低剂量血红素（5mmol／L、25mmol／L）不引起星形胶质存活率、LCD释放率及细胞形态改变．高剂量血红素（50mmol／L）在24h后导致星形胶质细胞存活率下降52．4％，LCD释放率增加31．8％，并出现细胞病变；但96h后损伤细胞得到修复，细胞重新形成致密单层，细胞存活率和LCD释放率均达正常水平。f31无论低剂量或高剂量血红素均未引起BCEC存活率下降、LCD释放率升高或细胞形态学改变。（4）经不同剂量血红素处理2h后，神经元内血红素含量均明显升高，星形胶质细胞内血红素含量只有在大剂量血红素处理后才升高．BCEC内血红素含量未见升高。（51神经元接触血红素30min后细胞内即出现大量Fe3＋染色阳性颗粒，随着接触时间延长和剂量加大，Fe3＋染色阳性细胞数增多：星形胶质细胞Fe3＋染色阳性细胞数明显少于神经元，BCEC几乎不出现Fe3＋染色阳性细胞。结论（1）Ng_素对神经元具有严重的直接损伤作用，对星形胶质细胞有可逆的损伤作用，对BCEC无直接损伤作用。（2）血红素能快速进入神经元并在神经元内积累，但较少在星形胶质细胞脑积累，难以在BCEC内积累。

Objective To investigate the toxic effect of hemin on primary cultured neurons, astrocytes, and brain capillary endothelial cells （BCECs）, and the damage effect of heroin with different concentrations on the above cells. Methods （1） Primary cultured neurons, astrocytes and BCECs from the cortex of rats were exposed to different doses of hemin for 2 h, and continue culture of these cells for 24 to 96 h after withdrawing hemin was performed; the cellular morphology was examined underphase-contrast microscope; cellular survival rate was measured with Alama blue staining; and the releasing rate of lactate dehydrogenasing （LDH） was detected with regular biochemical method. （2） Primary cultured cells were exposed to different doses of heroin for 2 h, and continue culture of the cells for 4 h was performed after washing out the hemin; and then, concentrated formic acid was employed to dissociate the cells, and heme content in dissociated ceils was measured with spectrophotometer. （3） Primary cultured cells was exposed to different doses of hemin for 30, 60 and 120 min, respectively, and continue culture of the cells for 4 h was performed after washing out hemin; and then, intracellular Fe3~ was examined with Prussian blue staining. Results （1） Cultured neurons were injured by a low dose ofhemin （5 mmol/L） with a decreased survival rate by 40.2% and an increased LDH releasing rate by 22.2%; and the pathological changes of cellular morphology were severe after 24 h of exposure to hemin. Following the increased doses of heroin and time of post-exposure, the cellular death and LDH releasing were increased, and the morphological changes of cells were much severe. （2） The low and medium doses of hemin （5 mmol/L and 25 mmol/L） did not induce cellular death, LDH releasing and morphological changes in astrocytes; and a high dose of hemin （50 mmol/L） could induce a death rate of astrocytes decreasing by 52.4%, a LDH releasing rate increasing by 31% and obvious morphological changes of astrocytes; however, the injured astrocytes could regenerate fluent cellular monolayer 96 h after exposing to high dose ofhemin treatment. （3） Heroin with either low or high dose did not induce any changes in cellular survival, LDH releasing and cellular morphology of BCECs. （4） The heme content in cultured neurons was significantly higher than that in astrocytes and BCECs after hemin treatment for 2 h. （5） The blue Fe3~ stained granules appeared in neurons as early as 30 min after neurons being exposed to heroin, and Fe3＋ stained positive cells in neurons were significantly higher than those in astrocytes and BCECs at any dose of hemin and any time point of hemin treatment. Conclusion Hemin is highly toxic to neurons, but it can only injure astrocytes at a high dose and it can not induce direct damage in BCECs; free hemin could rapidly enter and accumulate in neurons, but less accumulate in astrocytes and not accumulate in BCECs.