中华神经医学杂志
中華神經醫學雜誌
중화신경의학잡지
CHINESE JOURNAL OF NEUROMEDICINE
2012年
4期
325-331
,共7页
蒋士生%贺双腾%韩育明%夏爱民%王红梅%何飞舟
蔣士生%賀雙騰%韓育明%夏愛民%王紅梅%何飛舟
장사생%하쌍등%한육명%하애민%왕홍매%하비주
血红素%原代培养细胞%细胞损伤%神经元%星形胶质细胞%毛细血管内皮细胞
血紅素%原代培養細胞%細胞損傷%神經元%星形膠質細胞%毛細血管內皮細胞
혈홍소%원대배양세포%세포손상%신경원%성형효질세포%모세혈관내피세포
Hemin%Primary cultured cell%Cellular injury%Neuron%Astrocyte%Capillaryendothelial cell
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 (1)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、50 mmol/L)的高铁血红素孵育2h,去血红素后继续培养24或96 h,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.(3)在原代培养细胞上加血红素分别孵育30、60、120 min,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5 mmol/L的血红素处理后24 h,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(5 mmol/L、25 mmol/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(50 mmol/L)在24 h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96 h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.(3)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.(5)神经元接触血红素30 min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)血红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.
目的 研究不同劑量高鐵血紅素對腦內神經元、星形膠質細胞、腦毛細血管內皮細胞(BCEC)的損傷作用. 方法 (1)在原代培養大鼠腦皮質神經元、星形膠質細胞、BCEC上添加不同劑量(0、5、25、50 mmol/L)的高鐵血紅素孵育2h,去血紅素後繼續培養24或96 h,阿拉瑪藍染色檢查細胞存活率,常規生化反應法檢測乳痠脫氫酶(LCD)釋放率,相差光學顯微鏡觀察細胞形態.(2)在原代培養細胞上添加不同劑量血紅素孵育2h,去血紅素後繼續培養4h,以濃甲痠裂解細胞,分光光度計下測定細胞內血紅素含量.(3)在原代培養細胞上加血紅素分彆孵育30、60、120 min,去血紅素後繼續培養4h,以中性福爾馬林液固定細胞,普魯士藍染色檢查細胞內三價鐵離子(Fe3+)染色情況. 結果 (1)神經元經5 mmol/L的血紅素處理後24 h,存活率下降40.2％,LCD釋放率增加22.2％,細胞形態齣現嚴重損傷病變.隨著時間延長和血紅素劑量加大,細胞死亡率和LCD釋放率均增加,細胞病變加重.(2)中、低劑量血紅素(5 mmol/L、25 mmol/L)不引起星形膠質存活率、LCD釋放率及細胞形態改變,高劑量血紅素(50 mmol/L)在24 h後導緻星形膠質細胞存活率下降52.4％,LCD釋放率增加31.8％,併齣現細胞病變；但96 h後損傷細胞得到脩複,細胞重新形成緻密單層,細胞存活率和LCD釋放率均達正常水平.(3)無論低劑量或高劑量血紅素均未引起BCEC存活率下降、LCD釋放率升高或細胞形態學改變.(4)經不同劑量血紅素處理2h後,神經元內血紅素含量均明顯升高,星形膠質細胞內血紅素含量隻有在大劑量血紅素處理後纔升高,BCEC內血紅素含量未見升高.(5)神經元接觸血紅素30 min後細胞內即齣現大量Fe3+染色暘性顆粒,隨著接觸時間延長和劑量加大,Fe3+染色暘性細胞數增多;星形膠質細胞Fe3+染色暘性細胞數明顯少于神經元,BCEC幾乎不齣現Fe3+染色暘性細胞. 結論 (1)血紅素對神經元具有嚴重的直接損傷作用,對星形膠質細胞有可逆的損傷作用,對BCEC無直接損傷作用.(2)血紅素能快速進入神經元併在神經元內積纍,但較少在星形膠質細胞腦積纍,難以在BCEC內積纍.
목적 연구불동제량고철혈홍소대뇌내신경원、성형효질세포、뇌모세혈관내피세포(BCEC)적손상작용. 방법 (1)재원대배양대서뇌피질신경원、성형효질세포、BCEC상첨가불동제량(0、5、25、50 mmol/L)적고철혈홍소부육2h,거혈홍소후계속배양24혹96 h,아랍마람염색검사세포존활솔,상규생화반응법검측유산탈경매(LCD)석방솔,상차광학현미경관찰세포형태.(2)재원대배양세포상첨가불동제량혈홍소부육2h,거혈홍소후계속배양4h,이농갑산렬해세포,분광광도계하측정세포내혈홍소함량.(3)재원대배양세포상가혈홍소분별부육30、60、120 min,거혈홍소후계속배양4h,이중성복이마림액고정세포,보로사람염색검사세포내삼개철리자(Fe3+)염색정황. 결과 (1)신경원경5 mmol/L적혈홍소처리후24 h,존활솔하강40.2％,LCD석방솔증가22.2％,세포형태출현엄중손상병변.수착시간연장화혈홍소제량가대,세포사망솔화LCD석방솔균증가,세포병변가중.(2)중、저제량혈홍소(5 mmol/L、25 mmol/L)불인기성형효질존활솔、LCD석방솔급세포형태개변,고제량혈홍소(50 mmol/L)재24 h후도치성형효질세포존활솔하강52.4％,LCD석방솔증가31.8％,병출현세포병변；단96 h후손상세포득도수복,세포중신형성치밀단층,세포존활솔화LCD석방솔균체정상수평.(3)무론저제량혹고제량혈홍소균미인기BCEC존활솔하강、LCD석방솔승고혹세포형태학개변.(4)경불동제량혈홍소처리2h후,신경원내혈홍소함량균명현승고,성형효질세포내혈홍소함량지유재대제량혈홍소처리후재승고,BCEC내혈홍소함량미견승고.(5)신경원접촉혈홍소30 min후세포내즉출현대량Fe3+염색양성과립,수착접촉시간연장화제량가대,Fe3+염색양성세포수증다;성형효질세포Fe3+염색양성세포수명현소우신경원,BCEC궤호불출현Fe3+염색양성세포. 결론 (1)혈홍소대신경원구유엄중적직접손상작용,대성형효질세포유가역적손상작용,대BCEC무직접손상작용.(2)혈홍소능쾌속진입신경원병재신경원내적루,단교소재성형효질세포뇌적루,난이재BCEC내적루.
Objective To investigate the toxic effect of hemin on primary cultured neurons,astrocytes,and brain capillary endothelial cells (BCECs),and the damage effect of hemin 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 under phase-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 hemin 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 cells was measured with spectrophotometer. (3) Primary cultured cells was exposed to different doses ofhemin 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 ofhemin 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 ofhemin (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 of hemin treatment.(3) Hemin 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 hemin, and Fe3+ stained positive cells in neurons were significantly higher than those in astrocytes and BCECs at any dose ofhemin and any time point ofhemin 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.
