大鼠脑缺血再灌注损伤后HIF-1α对胶质亚型细胞修复重塑的调控机制
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摘要
目的:
本实验采用体外重组技术,制备pcDNA3.1-HIF-1α质粒。将HIF-1α质粒经颈部颈外-颈内动脉途径注射入大脑中动脉缺血再灌注损伤的大鼠脑组织中,于6h、12h、24h、72h和7d时,取大鼠脑组织对神经胶质亚型细胞行特异性免疫组织化学染色,观察各组亚型细胞的变化。分别将HIF-1α、VEGF同胶质亚型细胞行荧光双染色,于激光共聚焦显微镜下观察共表达的情况。综合上述实验结果评价重组HIF-1α质粒对于脑梗死的治疗效果,及其对多种细胞修复重塑的调控机制。
方法:
1经体外获得的HIF-1α的cDNA,测序检测后克克隆入真核表达载体pcDNA3.1,酶切鉴定重组子
2大鼠大脑中动脉脑缺血再灌注模型制作参照Zea Longa等报道的方法并加以改进。10%水合氯醛腹腔麻醉,颈部正中切开,分离肌肉组织暴露颈部血管,经颈外动脉向颈内动脉插入线栓,线栓头端距血管分叉处1.8cm左右视为线栓梗死大脑中动脉。2h后取出线栓注入HIF-1α质粒,留取6h、12h、24h、72h和7d观察,对照组注入PBS;
3将相应时间点实验组与对照组大鼠处死后断头取脑,视交叉后做2mm厚连续切片,第一片做TTC染色,其余做石蜡包埋,5μm厚连续石蜡切片,行HE染色并对各胶质亚型细胞行特异的免疫组织化学染色,高倍镜下分别计数不同时间点实验组和对照组各胶质亚型细胞的个数;
4将石蜡切片分别对HIF-1α、VEGF同各胶质亚型细胞做免疫荧光双染色,于激光共聚焦显微镜下观察阳性细胞的重叠情况,分析胶质亚型细胞是否存在与HIF-1α、VEGF的表达;
5统计学分析:使用SPSS15.0统计软件对实验数据进行统计学分析。组间计量资料比较采用t检验,以P<0.05表示差异有统计学意义;
结果:
1 RT-PCR扩增HIF-1αcDNA,经测序结果与Genebank记载完全一致,构建并酶切鉴定后获得pcDNA3.1-HIF-1α质粒;
2大鼠大脑中动脉脑缺血再灌注损伤后,经颈外动脉注射HIF-1α质粒组与PBS组死亡率未见明显差别,但是TTC染色见72h后,治疗组脑梗死面积明显小于对照组,而且存活7d大鼠神经功能缺失评分明显低于PBS对照组;
3 HIF-1α对大鼠脑缺血再灌注后胶质亚型细胞的影响:大鼠脑缺血再灌注6h、12h、24h、72h和7d,经颈外动脉注射HIF-1α质粒组的星形胶质细胞(GFAP阳性细胞)数量均数分别为12.20±2.864、18.80±4.207、30.00±4.301、38.40±2.881和43.20±6.978,注射PBS组的数量均数为15.80±4.494、18.60±1.673、23.00±4.583、31.60±3.361和29.00±6.000,二者相同时间点阳性细胞均数做t检验比较,缺血再灌注损伤24h及以后各时间点,HIF-1α组与对照组阳性细胞数有差异(P<0.05);经颈动脉注射HIF-1α质粒后,小胶质细胞(Isolectin-B4阳性细胞)均数分别为8.400±2.074、14.40±4.393、20.80±2.280、17.00±3.162和11.60±2.408,PBS对照组阳性细胞均数为8.400±2.302、15.40±2.402、25.80±4.147、37.80±6.181和24.80±4.325,相同时间点两组阳性细胞均数做t检验比较,缺血再灌注损伤24h及以后各时间点两组间有差异,有统计学意义(P<0.05)。HIF-1α注射组少突胶质细胞(CNPase阳性细胞)均数分别为17.00±2.916、11.40±2.302、10.00±3.526、16.40±2.302和19.20±2.388,PBS对照组阳性细胞均数分别为16.60±2.408、10.60±2.074、7.200±1.924、4.000±1.581和13.20±2.388,二者相同时间点阳性细胞均数做t检验比较,缺血再灌注损伤72h后,二组间阳性细胞数有统计学差异;
4 HIF-1α、VEGF与胶质亚型细胞共表达:将石蜡切片行免疫荧光双染色,于激光共聚焦显微镜下观察,发现大量星形胶质细胞(GFAP阳性细胞)与HIF-1α、VEGF蛋白存在共表达情况,而未见小胶质细胞(Isolectin-B4阳性细胞)和少突胶质细胞(CNPase阳性细胞)中HIF-1α、VEGF明显表达。
结论:
1经颈外-颈内动脉注射重组pcDNA3.1-HIF-1α质粒可以有效的减少大鼠脑缺血再灌注损伤后脑组织的梗死面积。
2大鼠脑缺血再灌注损伤后,经颈外-颈内动脉注射重组pcDNA3.1-HIF- 1α质粒可以促使星形胶质细胞增加,少突胶质细胞数量增加,并有效抑制小胶质细胞的增殖,起到神经保护作用。
3大鼠脑缺血再灌注损伤后,各神经胶质亚型细胞中,HIF-1α蛋白主要在星形胶质细胞中表达。
Objective:
In the experiment, the vitro recombination technology was used to prepare pcDNA3.1-HIF-1αplasmid. We injected HIF-1αplasmid into the middle cerebral artery (MCA) occlusion reperfusion of rats through external-internal carotid artery. After that, specificity immunohistochemistry was applied to stain for subtypes of glial cells of cerebral tissue in 6h, 12h, 24h, 72h and 7d, respectively. We then observed the changes in each cell group. In this process, double staining immunofluoresence technique was used to stain for HIF-1α,VEGF, and subtypes of glial cells, respectively, and we examined the co-expression of the positive cells under a confocal laser scanning microscope. Based on these experiment results, we finally came to the conclusions and the regulation mechanism of HIF-1αto cell reparation after rats ischemia-reperfusion injury.
Methods:
1 The cDNA of HIF-1αobtained in vitro was cloned into pcDNA3.1 the eukaryotic expression vectors after sequencing and restriction enzyme digestion was employed to identify the recombinants;
2 The modeling for the MCA occlusion reperfusion of rats consulted the reports of Zea Longa, etc and made some improvements. Rats were anaesthetized by intraperitoneal injection of 10% chloral hydrate and were conducted median neck incision. We separated the muscle tissue to have the neck vessels exposed and inserted filament into the internal carotid through the external carotid artery. The filament end which was about 1.8 cm away from the bifurcation of vessels was regarded as the filament infarction MCA. After 2 hours, we removed the filament and injected HIF-1αplasmid. For other time points at 6h、12h、24h、72h and 7d, we carried out the same operations and injected PBS into their control groups;
3 The rats of experimental groups and control groups of corresponding time points were decapitated after brain death. We made 2mm thick serial sections from posterior optic chiasm among which the first one was stained by TTC and the rest ones were embedded in paraffin and made into 5μm thick serial paraffin sections. The paraffin sections were then stained by HE. And specificity immunohistochemistry was applied to stain for subtypes of glial cells. Then, at high magnification, we counted the number of subtypes of glial cells in each group at different time points;
4 HIF-1α,VEGF, and subtypes of glial cells, were stained respectively, by paraffin sections using double staining immunofluoresence technique. We examined the overlapping of the positive cells under a confocal laser scanning microscope and analyzed the problem that whether there was coexpression of the subtypes of glial cells and HIF-1αas well as VEGF;
5 Statistical analysis: Use SPSS15.0 statistical software to conduct statistical analysis of the experimental data. T test was used to compare measurement data between groups. P <0.05 indicated that there were statistically significant differences;
Results:
1 The sequencing results of HIF-1αcDNA amplified by RT-PCR were exactly the same with the Genebank records. We obtained pcDNA3.1-HIF-1αfor experimental use in the following operations;
2 The modeling of MCA occlusion reperfusion of rats was finished. There showed no significant differences in rats death rate between groups whose external carotid artery injected with HIF-1αplasmid and those with PBS. However, the nerve function score of 7-day survival rats were actually obviously lighter than that of the control groups injected with PBS. The staining results by TTC could be observed from 72h groups that the area of cerebral infarction of the treatment groups was smaller than that of the control groups;
3 The effect of HIF-1αto the subtypes of glial cells of rats with ischemia-reperfusion injury: After the carotid artery injection of HIF-1αplasmid, the means of astrocytes (GFAP-positive cells) number were respectively 12.20±2.864, 18.80±4.207, 30.00±4.301, 38.40±2.881 and 43.20±6.978, and the means of GFAP-positive cells number of the PBS injection groups were respectively 15.80±4.494, 18.60±1.673, 23.00±4.583, 31.60±3.361 and 29.00±6.000. Using T test to compare the means of GFAP-positive cell number of the two groups at the same time points, we observed that, after 24h of cerebral infarction treatment, there were significant differences in the numbers of positive cells between the groups injected with HIF-1αand the control groups (P<0.05); After the carotid artery injection of HIF-1αplasmid, the means of microglia (Isolectin-B4 positive cell) number were respectively 8.400±2.074, 14.40±4.393, 20.80±2.280, 17.00±3.162 and 11.60±2.408, and the means of positive cell number of the PBS control groups were respectively 8.400±2.302, 15.40±2.402, 25.80±4.147, 37.80±6.181 and 24.80±4.325. Using T test to compare the means of positive cell number of the two groups at the same time points, we observed that after 24h of cerebral infarction treatment, there were differences between the two groups and they were of statistical significance (P<0.05). The means of oligodendrocyte (CNPase positive cell) number of HIF-1αinjection groups were respectively 17.00±2.916, 11.40±2.302, 10.00±3.526, 16.40±2.302 and 19.20±2.388, and the means of positive cell number of PBS control groups were respectively 16.60±2.408, 10.60±2.074, 7.200±1.924, 4.000±1.581 and 13.20±2.388. Using T test to compare the means of positive cell number of the two groups at the same time points, we observed that after 72h of cerebral infarction treatment, the numbers of positive cells of the two groups were of statistical significance;
4 Coexpression of HIF-1α, VEGF and subtypes of glial cells: We used double staining immunofluoresence technique to stain for the paraffin sections. Under a confocal laser scanning microscope, we oberserved the coexpression phenomenon of a large number GFAP positive cells together with HIF-1αand VEGF protein, whereas we did not find significant overlapping of a large number of Isolectin-B4 positive cells and CNPase positive cells with HIF-1αand VEGF protein.
Conclusions:
1 The pcDNA3.1-HIF-1αplasmid injected through external-internal carotid artery could effectively reduce the infarction size in the rat cerebral tissue.
2 The injection of recombination pcDNA3.1-HIF-1αplasmid after rat ischemia-reperfusion injury.could increase the number of astrocytes and oligodendrocytes, and at the same time, could effectively inhibit microglia cell proliferation to protect the neurons.
3 HIF-1αprotein in subtypes of glial cells was mainly expressed in the form of astrocytes.
本实验采用体外重组技术,制备pcDNA3.1-HIF-1α质粒。将HIF-1α质粒经颈部颈外-颈内动脉途径注射入大脑中动脉缺血再灌注损伤的大鼠脑组织中,于6h、12h、24h、72h和7d时,取大鼠脑组织对神经胶质亚型细胞行特异性免疫组织化学染色,观察各组亚型细胞的变化。分别将HIF-1α、VEGF同胶质亚型细胞行荧光双染色,于激光共聚焦显微镜下观察共表达的情况。综合上述实验结果评价重组HIF-1α质粒对于脑梗死的治疗效果,及其对多种细胞修复重塑的调控机制。
方法:
1经体外获得的HIF-1α的cDNA,测序检测后克克隆入真核表达载体pcDNA3.1,酶切鉴定重组子
2大鼠大脑中动脉脑缺血再灌注模型制作参照Zea Longa等报道的方法并加以改进。10%水合氯醛腹腔麻醉,颈部正中切开,分离肌肉组织暴露颈部血管,经颈外动脉向颈内动脉插入线栓,线栓头端距血管分叉处1.8cm左右视为线栓梗死大脑中动脉。2h后取出线栓注入HIF-1α质粒,留取6h、12h、24h、72h和7d观察,对照组注入PBS;
3将相应时间点实验组与对照组大鼠处死后断头取脑,视交叉后做2mm厚连续切片,第一片做TTC染色,其余做石蜡包埋,5μm厚连续石蜡切片,行HE染色并对各胶质亚型细胞行特异的免疫组织化学染色,高倍镜下分别计数不同时间点实验组和对照组各胶质亚型细胞的个数;
4将石蜡切片分别对HIF-1α、VEGF同各胶质亚型细胞做免疫荧光双染色,于激光共聚焦显微镜下观察阳性细胞的重叠情况,分析胶质亚型细胞是否存在与HIF-1α、VEGF的表达;
5统计学分析:使用SPSS15.0统计软件对实验数据进行统计学分析。组间计量资料比较采用t检验,以P<0.05表示差异有统计学意义;
结果:
1 RT-PCR扩增HIF-1αcDNA,经测序结果与Genebank记载完全一致,构建并酶切鉴定后获得pcDNA3.1-HIF-1α质粒;
2大鼠大脑中动脉脑缺血再灌注损伤后,经颈外动脉注射HIF-1α质粒组与PBS组死亡率未见明显差别,但是TTC染色见72h后,治疗组脑梗死面积明显小于对照组,而且存活7d大鼠神经功能缺失评分明显低于PBS对照组;
3 HIF-1α对大鼠脑缺血再灌注后胶质亚型细胞的影响:大鼠脑缺血再灌注6h、12h、24h、72h和7d,经颈外动脉注射HIF-1α质粒组的星形胶质细胞(GFAP阳性细胞)数量均数分别为12.20±2.864、18.80±4.207、30.00±4.301、38.40±2.881和43.20±6.978,注射PBS组的数量均数为15.80±4.494、18.60±1.673、23.00±4.583、31.60±3.361和29.00±6.000,二者相同时间点阳性细胞均数做t检验比较,缺血再灌注损伤24h及以后各时间点,HIF-1α组与对照组阳性细胞数有差异(P<0.05);经颈动脉注射HIF-1α质粒后,小胶质细胞(Isolectin-B4阳性细胞)均数分别为8.400±2.074、14.40±4.393、20.80±2.280、17.00±3.162和11.60±2.408,PBS对照组阳性细胞均数为8.400±2.302、15.40±2.402、25.80±4.147、37.80±6.181和24.80±4.325,相同时间点两组阳性细胞均数做t检验比较,缺血再灌注损伤24h及以后各时间点两组间有差异,有统计学意义(P<0.05)。HIF-1α注射组少突胶质细胞(CNPase阳性细胞)均数分别为17.00±2.916、11.40±2.302、10.00±3.526、16.40±2.302和19.20±2.388,PBS对照组阳性细胞均数分别为16.60±2.408、10.60±2.074、7.200±1.924、4.000±1.581和13.20±2.388,二者相同时间点阳性细胞均数做t检验比较,缺血再灌注损伤72h后,二组间阳性细胞数有统计学差异;
4 HIF-1α、VEGF与胶质亚型细胞共表达:将石蜡切片行免疫荧光双染色,于激光共聚焦显微镜下观察,发现大量星形胶质细胞(GFAP阳性细胞)与HIF-1α、VEGF蛋白存在共表达情况,而未见小胶质细胞(Isolectin-B4阳性细胞)和少突胶质细胞(CNPase阳性细胞)中HIF-1α、VEGF明显表达。
结论:
1经颈外-颈内动脉注射重组pcDNA3.1-HIF-1α质粒可以有效的减少大鼠脑缺血再灌注损伤后脑组织的梗死面积。
2大鼠脑缺血再灌注损伤后,经颈外-颈内动脉注射重组pcDNA3.1-HIF- 1α质粒可以促使星形胶质细胞增加,少突胶质细胞数量增加,并有效抑制小胶质细胞的增殖,起到神经保护作用。
3大鼠脑缺血再灌注损伤后,各神经胶质亚型细胞中,HIF-1α蛋白主要在星形胶质细胞中表达。
Objective:
In the experiment, the vitro recombination technology was used to prepare pcDNA3.1-HIF-1αplasmid. We injected HIF-1αplasmid into the middle cerebral artery (MCA) occlusion reperfusion of rats through external-internal carotid artery. After that, specificity immunohistochemistry was applied to stain for subtypes of glial cells of cerebral tissue in 6h, 12h, 24h, 72h and 7d, respectively. We then observed the changes in each cell group. In this process, double staining immunofluoresence technique was used to stain for HIF-1α,VEGF, and subtypes of glial cells, respectively, and we examined the co-expression of the positive cells under a confocal laser scanning microscope. Based on these experiment results, we finally came to the conclusions and the regulation mechanism of HIF-1αto cell reparation after rats ischemia-reperfusion injury.
Methods:
1 The cDNA of HIF-1αobtained in vitro was cloned into pcDNA3.1 the eukaryotic expression vectors after sequencing and restriction enzyme digestion was employed to identify the recombinants;
2 The modeling for the MCA occlusion reperfusion of rats consulted the reports of Zea Longa, etc and made some improvements. Rats were anaesthetized by intraperitoneal injection of 10% chloral hydrate and were conducted median neck incision. We separated the muscle tissue to have the neck vessels exposed and inserted filament into the internal carotid through the external carotid artery. The filament end which was about 1.8 cm away from the bifurcation of vessels was regarded as the filament infarction MCA. After 2 hours, we removed the filament and injected HIF-1αplasmid. For other time points at 6h、12h、24h、72h and 7d, we carried out the same operations and injected PBS into their control groups;
3 The rats of experimental groups and control groups of corresponding time points were decapitated after brain death. We made 2mm thick serial sections from posterior optic chiasm among which the first one was stained by TTC and the rest ones were embedded in paraffin and made into 5μm thick serial paraffin sections. The paraffin sections were then stained by HE. And specificity immunohistochemistry was applied to stain for subtypes of glial cells. Then, at high magnification, we counted the number of subtypes of glial cells in each group at different time points;
4 HIF-1α,VEGF, and subtypes of glial cells, were stained respectively, by paraffin sections using double staining immunofluoresence technique. We examined the overlapping of the positive cells under a confocal laser scanning microscope and analyzed the problem that whether there was coexpression of the subtypes of glial cells and HIF-1αas well as VEGF;
5 Statistical analysis: Use SPSS15.0 statistical software to conduct statistical analysis of the experimental data. T test was used to compare measurement data between groups. P <0.05 indicated that there were statistically significant differences;
Results:
1 The sequencing results of HIF-1αcDNA amplified by RT-PCR were exactly the same with the Genebank records. We obtained pcDNA3.1-HIF-1αfor experimental use in the following operations;
2 The modeling of MCA occlusion reperfusion of rats was finished. There showed no significant differences in rats death rate between groups whose external carotid artery injected with HIF-1αplasmid and those with PBS. However, the nerve function score of 7-day survival rats were actually obviously lighter than that of the control groups injected with PBS. The staining results by TTC could be observed from 72h groups that the area of cerebral infarction of the treatment groups was smaller than that of the control groups;
3 The effect of HIF-1αto the subtypes of glial cells of rats with ischemia-reperfusion injury: After the carotid artery injection of HIF-1αplasmid, the means of astrocytes (GFAP-positive cells) number were respectively 12.20±2.864, 18.80±4.207, 30.00±4.301, 38.40±2.881 and 43.20±6.978, and the means of GFAP-positive cells number of the PBS injection groups were respectively 15.80±4.494, 18.60±1.673, 23.00±4.583, 31.60±3.361 and 29.00±6.000. Using T test to compare the means of GFAP-positive cell number of the two groups at the same time points, we observed that, after 24h of cerebral infarction treatment, there were significant differences in the numbers of positive cells between the groups injected with HIF-1αand the control groups (P<0.05); After the carotid artery injection of HIF-1αplasmid, the means of microglia (Isolectin-B4 positive cell) number were respectively 8.400±2.074, 14.40±4.393, 20.80±2.280, 17.00±3.162 and 11.60±2.408, and the means of positive cell number of the PBS control groups were respectively 8.400±2.302, 15.40±2.402, 25.80±4.147, 37.80±6.181 and 24.80±4.325. Using T test to compare the means of positive cell number of the two groups at the same time points, we observed that after 24h of cerebral infarction treatment, there were differences between the two groups and they were of statistical significance (P<0.05). The means of oligodendrocyte (CNPase positive cell) number of HIF-1αinjection groups were respectively 17.00±2.916, 11.40±2.302, 10.00±3.526, 16.40±2.302 and 19.20±2.388, and the means of positive cell number of PBS control groups were respectively 16.60±2.408, 10.60±2.074, 7.200±1.924, 4.000±1.581 and 13.20±2.388. Using T test to compare the means of positive cell number of the two groups at the same time points, we observed that after 72h of cerebral infarction treatment, the numbers of positive cells of the two groups were of statistical significance;
4 Coexpression of HIF-1α, VEGF and subtypes of glial cells: We used double staining immunofluoresence technique to stain for the paraffin sections. Under a confocal laser scanning microscope, we oberserved the coexpression phenomenon of a large number GFAP positive cells together with HIF-1αand VEGF protein, whereas we did not find significant overlapping of a large number of Isolectin-B4 positive cells and CNPase positive cells with HIF-1αand VEGF protein.
Conclusions:
1 The pcDNA3.1-HIF-1αplasmid injected through external-internal carotid artery could effectively reduce the infarction size in the rat cerebral tissue.
2 The injection of recombination pcDNA3.1-HIF-1αplasmid after rat ischemia-reperfusion injury.could increase the number of astrocytes and oligodendrocytes, and at the same time, could effectively inhibit microglia cell proliferation to protect the neurons.
3 HIF-1αprotein in subtypes of glial cells was mainly expressed in the form of astrocytes.
引文
1 Longa EZ.Weinstein PR,Carlson S,eta1.Reversible middle cerebral artery occlusion without crartiectomy in rats[J].Stroke,1989,20:84-91
2王钟秀,杨林花. HIF-1α、VEGF与缺血性脑血管病.血栓与止血学.2007,13:189-192
3 SEMENZA GL,WANG GL.General involvement of hypoxia inducible factor 1 in transcriptional response to hypoxia[J].Proc Natl Acad Sci U S A ,1993,90(9):4304-4308
4 Semenza GL,Shimoda LA,Prabhakar NR.Regulation ofgene expression by HIF-l[J].Novartis Found symp,2006,272:2-8
5 Press K,SchaffA,Ruscher K,et a1.Hypoxia-induced stroke tolerace in the mouseis mediated by erythropoietin [J].Stroke.2003,34(8):1981-1986
6 Bemaudin M ,Nedelec AS,Divoux D,et a1.Normobaric hypoxia induces toleranced to focal permanent cerebral ischemia in association with an increased expression of hypoxia inducible factor-1 and its target genes,erythropoietin and VEGF,in the adult mouse brain[J].J Cereb Blood Flow Metab,2002,22(4):393-403
7 Bergeron M,Yu A Y,Solway K E,et al.Induction of hypoxia-inducible factor-l(HIF-1)and its target genes following focal ischaemia in rat brain[J].Eur J Neumsci,1999,ll(12):4159-4170
8 Sharp F R,Lu A,Tang Y,et al.Multiple molecular penumbras after focal cerebral ischemia[J],J Cereb Blood Flow Metab,2000,20(7):1011-1032
9 Matsuda T,Abe T,wu J L,et al.Hypoxia-inducible factor-1alpha DNA induced angiogenesis in a rat cerebral ischemia model[J].Neurol Res,2005,27(5):503-508
10 Heinl-Green A,Radke PW ,Munkonge FM.et al.The eficacy of a master switch gene HIF-1 alpha in a porcine model of chronic myocardial ischaemi.Eur Heart J,2005.26:1327-1332
11 Vincent KA,ShyN KG,Luo Y,et al.Angiogenesis is induced in ababbit model of hindlimb ischemi a by naked DNA encoding aHIF-1 alpha/VP16 hybrid transeription factor Circulation,2000,102:2255-2261
12赵辉,丁素菊.缺血性脑血管病的基因治疗.国外医学脑血管分册.2004,12:702-705
13谭新杰,胡长林.缺氧诱导因子-1α基因在成年大鼠局灶性脑缺血中的治疗作用研究.中华医学杂志.2006,86(15):1057-1060
14 Memezawa H,Smith ML,Siesjo BK.Penumbral tissue salvaged by reperfusion following middle cetebral artery occlusion in rats.Stroke,1992;23:552
15 Panickar K S and Norenberg M D.Astroeytes in cerebral ischemic injury:morphological and general consid- erations.Glia,2005,50(4):287-298
16 Eng LF.GliaI fibrillary acidic protein(GFAP):the major protein of glial intermediate filaments in differentiated astrocytes,J Neuroimrnunol,1985,8(4-6):203-214
17 Chez Y.Swanson R.A. Astrocytes and brain injury.J Cereb Blood Flow Metab,2003,23(2):137-139
18 Hertz L.Zielke HR.Astrocytic control of glutamatcrgic activity:astrocytes as stars of the show.Trends Neurosci,2004,27(12):735-743
19 Yrjanheikki J,Keinanen R,Pellika M,et al.Tetracyclines inhibit microglial activation and are neuoprotective in global brain ischemia [J].Proc Natl Acad Sci,1998,95,(26):15769-15774
20 Ivacko JA,Sun R,Silverstein FS.Hpoxic-ischemic brain injury induces an acute microglial reaction in perinatal rats [J].Pediatr Res,1996,39(1):39-47
21冯涛,朱克,戚晓昆.大鼠持续性脑缺血后小胶质细胞变化及其与神经元调亡的关系.中风与神经疾病杂志,2001,18(4):201-203
22 Asou H,Hamada K,Miyazaki T,et a1.CNS myelinogenesis in vitro:time course and patten of rat oligodendrocyte development[J].J Neurosci Res,1995,40:519-534
23陈应柱,田野,张志琳.茶多酚对脑缺血大鼠脑内少突胶质细胞的影响.临床神经病学杂志,2007,20(3):211-213
24 Fern R , Moller T. Rapid ischemic cell death in immature oligodendrocytes:a fatal glutamate release feedback loop [J]. J Neurosci, 2000,20(1)34-42
25 FERRARA N,HENZEL WJ.Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells [J].Biochem Biophys Res Commun,1989,161(2):851-858
26 UNEMORI EN ,FERRARA N ,BAUER EA,et a1. VEGF induces interstitial collanenase expression in human endothelial cells[J].Cellphsiol,1992,153:557-558
27 MAEDA K,CLUNG Y,OGAWA Y ,et a1. Progunostic value of vascular endothelial growth factor expression in gastric carcinoma[J].Cancer,1996,77(5):858-860
28 Jin KL,Mao XO,Nagayama T,et a1.Induction of vascular endothelialgrowth factor and hypoxia-inducible factor-1 alpha by global ischemia in rat brain[J].Neuroscience,2000;(99):577-85
1 SEMENZA GL,WANG GL.General involvement of hypoxia inducible factor 1 in transcriptional response to hypoxia[J].Proc Natl Acad Sci U S A ,1993,90(9):4304-4308
2张昱,吉训明,李文斌等.低氧诱导因子在脑缺血中的双重作用.中国病理生理杂志.2009,25(1):197-200
3 Semenza GL,Shimoda LA,Prabhakar NR.Regulation ofgene expression by HIF-l[J].Novartis Found symp,2006,272:2-8
4 SharpF R,Lu A ,Tang Y ,et al .Multiple molecular penumbras after focal cerebral ischemia. J CerebB lood Flow Metab,2000,(20):1011-10 32
5 Bemaudin M ,Nedelec AS,Divoux D ,et al.Normobaric hypoxia induces tolerance to f ocal permanent cerebral ischemia in association With an increased expression of hypoxia– induciblefactor-1 and its targetgenes ,erythropoietin and VEGF,in the adult mouse brain. J Cereb Blood Flow Metab, 2002,(22):393-403
6 Mu D,Jiang X,Sheldon RA,et a1.Regulation of hypoxia-inducible factor 1 alpha and induction of vascular endothelial growth factor in a rat neonatal stroke mode1.Neurobiol Dis,2003,14(3):524-534
7 Makinen K, Manninen H, Hedman M.et a1.Increased vascularity detected by digital subtraction ansiography after VEGF gene transfer to human lower limb artery: A randomized, placebo-controlled, double-blinded phase II study.MMol Ther,2002,6(1):127-133
8 Syu KG,Wang MT,Wang BW,et a1.2002,Intramyocardial injection of naked DNA encoding HIF-l alpha∕ VP16 hybrid to enhance ansiogenesis in an acutemyocardial infarctin model in the rat.Cardiovase Res,2002,54(3):576-583
9李东野,闫艳,朱红等. HIF-1α基因对心肌梗死后心肌细胞凋亡和心功能影响的实验研究.中国病理生理杂志,2008,24(2):275-278
10 Heinl-Green A,Radke PW ,Munkonge FM.et al.The eficacy of a master switch gene HIF-1 alpha in a porcine model of chronic myocardialischaemi.Eur Heart J,2005.26:1327-1332
11王凡,吴海涛,朱玲玲等.低氧诱导因子-1α基因修饰后的成肌细胞移植治疗心肌梗死的研究.中华老年心血管病杂志,2008,10(10):772-774
12 Vincent KA,ShyN KG,Luo Y,et al.Angiogenesis is induced in ababbit model of hindlimb ischemi a by naked DNA encoding aHIF-1 alpha/VP16 hybrid transeription factor Circulation,2000,102:2255-2261
13 Kelly BD,Hackett SF,Hirota K,et al.Cell type-apecific regulation of angiogenic growth factor gene expression and induction of angiogesis in nonischemic tissue by a constitutively active form of hypoxia-inducible 1 [J]. Circ Res,2003, 93:1047
14孙晓峰,谭岩,董春哲等. HIF-1α治疗家兔肢体缺血模型的实验研究.中国实验诊断学,2008,12(12):1504-1506
15陶陶,陈利芬,胡长林等.腺病毒介导的低氧诱导因子-1α基因对大鼠缺血再灌注后脑梗死体积影响的初步观察.复旦学报,2005,32(5):594-596
16谭新杰,胡长林.缺氧诱导因子-1α基因在成年大鼠局灶性脑缺血中的治疗作用研究.中华医学杂志.2006,86(15):1057-1060
1 Rajkowska G, Miguel-Hidalgo J: Gliogenesis and glial pathology in depression. CNS Neurol Disord Drug Targets 2007,6:219-233
2 BROWN AM.Brain glycogen re-awakened[J] . J Neurochem, 2004, 89(3): 537-552
3 SimardM,NedergaardM. The neurobiology of glia in the context of water and ion homeostasis[ J ]. Neuroscience, 2004, 129 (4) : 877-896
4 TakahashiM,Billup sB, RossiD, et al. The role of glutamate transporters in glutamate homeostasis in the brain[ J ]. J Exp B iol, 1997, 200 (2) : 401-409
5 Babot Z, Cristòfol R, Suòol C. Excitotoxic death induced by released glutamate in depolarized p rimary cultures ofmouse cerebellar granule cells is dependent on GABAA recep tors and niflumic acid-sensitive chloride channels[ J ]. Eur J N eurosci, 2005, 21 (1) : 103-12
6 Horio Y. Potassium channels of glial cells:distribution and function[J]. Jpn J Pharmacol, 2001, 87 (1) : 1-6
7 PerezVelazquez J L, Kokarovtseva L, Sarbaziha R, et al. Role of gap junctional coup ling in astrocytic networks in the determination of global ischemia-induced oxidative stress and hippocampal damage[J]. Eur J N eurosci, 2006, 23 (1) : 1-10
8 GABRYELB,TOBOREK T,MALECKI A. immunosuppressive immunophilin ligands attenuate damage in cultured rat astrocytes depleted of glutathione and exposed to simulated ischemia in vitro comparision with N-acetylcysteine[J]. Neuro Toxicology , 2005, 26 (3 ) : 373-384
9 Wang XF, CynaderMS. Astrocytes provide cysteine to neurons by re- leasing glutathione[ J ]. J Neurochem, 2000, 74 (4) : 1434-1442
10 Heales SJ,Lam A A,Duncan A J, et al.Neurodegeneration or neuroprotection: the pivotal role of astrocytes [J]. N eurochem Res,2004, 29 (3) : 513-519
11 Buskila Y, Farkash S, HershfinkelM, et al. Rap id and reactive nitric oxide production by astrocytes in mouse neocortical slices[ J ].Glia, 2005, 52 (3) : 169-76
12 Oderfeld-Nowak B, Orzylowska-SLiwinska O, Soltys Z, et al Concomitant up-regulation of astroglial high and low affinity nervegrowth factor receptors in the CA1 hippocampal area following global transient cerebral ischemia in rat [J]. Neuroscience, 2003, 120:31-40
13 Abiru Y, Katoh-semba R, Nishio C, et al. High potassium enhances secretion of neurotrophic factors from cultured astrocytes [J]. Brain Res,1998, 809:115-126
14刁士元,袁颖,郭海涛,等.神经生长因子对大鼠脑缺血再灌注损伤的保护作用及其对Bax表达的影响[J].临床神经病学杂志, 2005, 18:299-301
15 Dhandapani KM, Hadman M, De Sevilla L, et al. Astrocyte Protection of Neurons:role of transforming growth factor-betasignaling via a c-Jun-AP-1 protective pathway[J]. J BiolChem,2003, 278:43329-43339
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17 Espejo M, Cutillas B, Arenas TE , et al. Increased survival of dopaminergic neurons in striatal grafts of fetal ventral mesencephalic cells exposed to neurotrophin-3 or glial cell linederived neurotrophin factor[J]. Cell Transplant, 2000, 9:45-53
18 Dawson T M . Preconditioning mediated neuroprotection through erythropoietin[J] Lancet,2002;359:96-97
19 Bemadin M.Ned elec AS,Divoux D,et a1.Normobaric hypoxia induces tolerance to focal permanent cerebral ischemia in association with an increased expression of hypoxin—inducible factor-1 and its target genes . Erythropoietin and VEGF . in the adult mouse brain[J].J Cerob Blood Flow Metab,2002;22:393-403
20 Dhandapani KM,Brann DW. Role of astrocytes in estrogen-meditedneuroprotection[ J ]. Exp Gerontol, 2007, 42 (122) : 70-75
21 Areque A, Parpura V, Sanzgiri RP, et al. Tripartite synapses: glia, the unacknowledged partner [ J ]. Trends Neurosci, 1999, 22 (5) : 208-215
22 Araque A, Sanzgiri RP, Parpura V, et al. Astrocyte-induced modulation of synap tic transmission[ J ]. Can J Pharmacol, 1999,77 (9) : 699-706
23 Ullian EM, Sapperstein SK, Christophererson KS, etal . Control of synapse number by glio [ J ] .Science,2001, 291: 657-661
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26 Nicholas RS, WingMG, Compston A. Nonactivated microglia promote oligodendrocyte precursor survival and maturation through thetranscription factorNF-κB. Eur J Neurosci, 2001, 13: 959-967
27 Lin LF, Doherty DH, Lile JD, et al. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science, 1993, 260: 1130-1132
28 Heck S, LezoualchF, Engert S, et al. Insulin-like growth factor-1-mediated neurop rotection against oxidative stress is associated withactivation of nuclear factorκB. Biol Chem, 1999, 274: 9828-9835
29 Azcoitia I, Sierra A, Garcia-Segura LM, et al. Neurop rotective effects of estradiol in the adult rat hippocampus: interaction with insulin-like growth factor-I signalling. J Neurosci Res, 1999, 58:815-822
30 Simakajornboon N, Szerlip NJ, Gozal E, et al. In vivo PDGFβrecep- tor activation in the dorsocaudal brainstem of the rat p revents hypoxia2induced apop tosis via activation of Akt and BAD. Brain Res,2001, 895: 111-118
31 van Landeghem FK, Stover JF, et al. Early expression of glutamate transporter proteins in ramified microglia after controlled cortical impact injury in the rat. Glia, 2001, 35: 167-179
32 Loddick SA, Turnbull AV, RothwellNJ. Cerebral interleukin 6 is neuro-protective during permanent focal cerebral ischemia in the rat e. JCereb Blood FlowMetab, 1998, 18: 176-179
33 Wakita H, Tomimoto H, Akiguchi I, et al. Ibudilast, a phosphodi -esterase inhibitor, protects againstwhite matter damage under chronic cerebral hypoperfusion in the rat. Brain Res, 2003, 992: 53-59
34 Wang Q, Rowan MJ, Anwyl R.β-amyloid-mediated inhibition of NMDA receptor-dependent long-term potentiation induction involves activation of microglia and stimulation of inducible nitric oxide synthase and superoxide. J Neurosci, 2004, 24: 6049-6056
35 Sortwell CE, Daley BF, PitzerMR, et al. Oligodendrocyte-type astrocyte-derived trophic factors increase survival of developing dopamine neurons through the inhibition of apop totic cell death [ J ]. JCom p N eurol, 2000, 426 (1) : 143-153
36 Wilkins A, Majed H, Layfield R, et al. Oligodendrocytes promote Neuronal survival and axonal length by distinct intracellular mechanism: a novel role for oligodendrocyte-derived glial cell line-derived neurotrophic factor[ J ]. J N eurosci, 2003, 23 (12) : 4967-4974
37 Levison SW, Rothstein RP, RomankoMJ, et al. Hypoxia / ischemia depletes the rat perinatal subventricular zone of oligodendrocyte progenitors and neural stem cells[ J ]. Dev N eurosci, 2001, 23 (3): 234-247.38 kerracher L, WintonMJ. Nogo on the go[ J ]. N euron, 2002, 36(3) : 345-348
38 awsonMR, Polito A, Levine JM, et al. NG-2expressing glial progenitor cells:an abundant and widesp read population of cycling cells in the adult rat CNS[ J ]. M ol CellN eurosci, 2003, 24 (2) : 476-488
2王钟秀,杨林花. HIF-1α、VEGF与缺血性脑血管病.血栓与止血学.2007,13:189-192
3 SEMENZA GL,WANG GL.General involvement of hypoxia inducible factor 1 in transcriptional response to hypoxia[J].Proc Natl Acad Sci U S A ,1993,90(9):4304-4308
4 Semenza GL,Shimoda LA,Prabhakar NR.Regulation ofgene expression by HIF-l[J].Novartis Found symp,2006,272:2-8
5 Press K,SchaffA,Ruscher K,et a1.Hypoxia-induced stroke tolerace in the mouseis mediated by erythropoietin [J].Stroke.2003,34(8):1981-1986
6 Bemaudin M ,Nedelec AS,Divoux D,et a1.Normobaric hypoxia induces toleranced to focal permanent cerebral ischemia in association with an increased expression of hypoxia inducible factor-1 and its target genes,erythropoietin and VEGF,in the adult mouse brain[J].J Cereb Blood Flow Metab,2002,22(4):393-403
7 Bergeron M,Yu A Y,Solway K E,et al.Induction of hypoxia-inducible factor-l(HIF-1)and its target genes following focal ischaemia in rat brain[J].Eur J Neumsci,1999,ll(12):4159-4170
8 Sharp F R,Lu A,Tang Y,et al.Multiple molecular penumbras after focal cerebral ischemia[J],J Cereb Blood Flow Metab,2000,20(7):1011-1032
9 Matsuda T,Abe T,wu J L,et al.Hypoxia-inducible factor-1alpha DNA induced angiogenesis in a rat cerebral ischemia model[J].Neurol Res,2005,27(5):503-508
10 Heinl-Green A,Radke PW ,Munkonge FM.et al.The eficacy of a master switch gene HIF-1 alpha in a porcine model of chronic myocardial ischaemi.Eur Heart J,2005.26:1327-1332
11 Vincent KA,ShyN KG,Luo Y,et al.Angiogenesis is induced in ababbit model of hindlimb ischemi a by naked DNA encoding aHIF-1 alpha/VP16 hybrid transeription factor Circulation,2000,102:2255-2261
12赵辉,丁素菊.缺血性脑血管病的基因治疗.国外医学脑血管分册.2004,12:702-705
13谭新杰,胡长林.缺氧诱导因子-1α基因在成年大鼠局灶性脑缺血中的治疗作用研究.中华医学杂志.2006,86(15):1057-1060
14 Memezawa H,Smith ML,Siesjo BK.Penumbral tissue salvaged by reperfusion following middle cetebral artery occlusion in rats.Stroke,1992;23:552
15 Panickar K S and Norenberg M D.Astroeytes in cerebral ischemic injury:morphological and general consid- erations.Glia,2005,50(4):287-298
16 Eng LF.GliaI fibrillary acidic protein(GFAP):the major protein of glial intermediate filaments in differentiated astrocytes,J Neuroimrnunol,1985,8(4-6):203-214
17 Chez Y.Swanson R.A. Astrocytes and brain injury.J Cereb Blood Flow Metab,2003,23(2):137-139
18 Hertz L.Zielke HR.Astrocytic control of glutamatcrgic activity:astrocytes as stars of the show.Trends Neurosci,2004,27(12):735-743
19 Yrjanheikki J,Keinanen R,Pellika M,et al.Tetracyclines inhibit microglial activation and are neuoprotective in global brain ischemia [J].Proc Natl Acad Sci,1998,95,(26):15769-15774
20 Ivacko JA,Sun R,Silverstein FS.Hpoxic-ischemic brain injury induces an acute microglial reaction in perinatal rats [J].Pediatr Res,1996,39(1):39-47
21冯涛,朱克,戚晓昆.大鼠持续性脑缺血后小胶质细胞变化及其与神经元调亡的关系.中风与神经疾病杂志,2001,18(4):201-203
22 Asou H,Hamada K,Miyazaki T,et a1.CNS myelinogenesis in vitro:time course and patten of rat oligodendrocyte development[J].J Neurosci Res,1995,40:519-534
23陈应柱,田野,张志琳.茶多酚对脑缺血大鼠脑内少突胶质细胞的影响.临床神经病学杂志,2007,20(3):211-213
24 Fern R , Moller T. Rapid ischemic cell death in immature oligodendrocytes:a fatal glutamate release feedback loop [J]. J Neurosci, 2000,20(1)34-42
25 FERRARA N,HENZEL WJ.Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells [J].Biochem Biophys Res Commun,1989,161(2):851-858
26 UNEMORI EN ,FERRARA N ,BAUER EA,et a1. VEGF induces interstitial collanenase expression in human endothelial cells[J].Cellphsiol,1992,153:557-558
27 MAEDA K,CLUNG Y,OGAWA Y ,et a1. Progunostic value of vascular endothelial growth factor expression in gastric carcinoma[J].Cancer,1996,77(5):858-860
28 Jin KL,Mao XO,Nagayama T,et a1.Induction of vascular endothelialgrowth factor and hypoxia-inducible factor-1 alpha by global ischemia in rat brain[J].Neuroscience,2000;(99):577-85
1 SEMENZA GL,WANG GL.General involvement of hypoxia inducible factor 1 in transcriptional response to hypoxia[J].Proc Natl Acad Sci U S A ,1993,90(9):4304-4308
2张昱,吉训明,李文斌等.低氧诱导因子在脑缺血中的双重作用.中国病理生理杂志.2009,25(1):197-200
3 Semenza GL,Shimoda LA,Prabhakar NR.Regulation ofgene expression by HIF-l[J].Novartis Found symp,2006,272:2-8
4 SharpF R,Lu A ,Tang Y ,et al .Multiple molecular penumbras after focal cerebral ischemia. J CerebB lood Flow Metab,2000,(20):1011-10 32
5 Bemaudin M ,Nedelec AS,Divoux D ,et al.Normobaric hypoxia induces tolerance to f ocal permanent cerebral ischemia in association With an increased expression of hypoxia– induciblefactor-1 and its targetgenes ,erythropoietin and VEGF,in the adult mouse brain. J Cereb Blood Flow Metab, 2002,(22):393-403
6 Mu D,Jiang X,Sheldon RA,et a1.Regulation of hypoxia-inducible factor 1 alpha and induction of vascular endothelial growth factor in a rat neonatal stroke mode1.Neurobiol Dis,2003,14(3):524-534
7 Makinen K, Manninen H, Hedman M.et a1.Increased vascularity detected by digital subtraction ansiography after VEGF gene transfer to human lower limb artery: A randomized, placebo-controlled, double-blinded phase II study.MMol Ther,2002,6(1):127-133
8 Syu KG,Wang MT,Wang BW,et a1.2002,Intramyocardial injection of naked DNA encoding HIF-l alpha∕ VP16 hybrid to enhance ansiogenesis in an acutemyocardial infarctin model in the rat.Cardiovase Res,2002,54(3):576-583
9李东野,闫艳,朱红等. HIF-1α基因对心肌梗死后心肌细胞凋亡和心功能影响的实验研究.中国病理生理杂志,2008,24(2):275-278
10 Heinl-Green A,Radke PW ,Munkonge FM.et al.The eficacy of a master switch gene HIF-1 alpha in a porcine model of chronic myocardialischaemi.Eur Heart J,2005.26:1327-1332
11王凡,吴海涛,朱玲玲等.低氧诱导因子-1α基因修饰后的成肌细胞移植治疗心肌梗死的研究.中华老年心血管病杂志,2008,10(10):772-774
12 Vincent KA,ShyN KG,Luo Y,et al.Angiogenesis is induced in ababbit model of hindlimb ischemi a by naked DNA encoding aHIF-1 alpha/VP16 hybrid transeription factor Circulation,2000,102:2255-2261
13 Kelly BD,Hackett SF,Hirota K,et al.Cell type-apecific regulation of angiogenic growth factor gene expression and induction of angiogesis in nonischemic tissue by a constitutively active form of hypoxia-inducible 1 [J]. Circ Res,2003, 93:1047
14孙晓峰,谭岩,董春哲等. HIF-1α治疗家兔肢体缺血模型的实验研究.中国实验诊断学,2008,12(12):1504-1506
15陶陶,陈利芬,胡长林等.腺病毒介导的低氧诱导因子-1α基因对大鼠缺血再灌注后脑梗死体积影响的初步观察.复旦学报,2005,32(5):594-596
16谭新杰,胡长林.缺氧诱导因子-1α基因在成年大鼠局灶性脑缺血中的治疗作用研究.中华医学杂志.2006,86(15):1057-1060
1 Rajkowska G, Miguel-Hidalgo J: Gliogenesis and glial pathology in depression. CNS Neurol Disord Drug Targets 2007,6:219-233
2 BROWN AM.Brain glycogen re-awakened[J] . J Neurochem, 2004, 89(3): 537-552
3 SimardM,NedergaardM. The neurobiology of glia in the context of water and ion homeostasis[ J ]. Neuroscience, 2004, 129 (4) : 877-896
4 TakahashiM,Billup sB, RossiD, et al. The role of glutamate transporters in glutamate homeostasis in the brain[ J ]. J Exp B iol, 1997, 200 (2) : 401-409
5 Babot Z, Cristòfol R, Suòol C. Excitotoxic death induced by released glutamate in depolarized p rimary cultures ofmouse cerebellar granule cells is dependent on GABAA recep tors and niflumic acid-sensitive chloride channels[ J ]. Eur J N eurosci, 2005, 21 (1) : 103-12
6 Horio Y. Potassium channels of glial cells:distribution and function[J]. Jpn J Pharmacol, 2001, 87 (1) : 1-6
7 PerezVelazquez J L, Kokarovtseva L, Sarbaziha R, et al. Role of gap junctional coup ling in astrocytic networks in the determination of global ischemia-induced oxidative stress and hippocampal damage[J]. Eur J N eurosci, 2006, 23 (1) : 1-10
8 GABRYELB,TOBOREK T,MALECKI A. immunosuppressive immunophilin ligands attenuate damage in cultured rat astrocytes depleted of glutathione and exposed to simulated ischemia in vitro comparision with N-acetylcysteine[J]. Neuro Toxicology , 2005, 26 (3 ) : 373-384
9 Wang XF, CynaderMS. Astrocytes provide cysteine to neurons by re- leasing glutathione[ J ]. J Neurochem, 2000, 74 (4) : 1434-1442
10 Heales SJ,Lam A A,Duncan A J, et al.Neurodegeneration or neuroprotection: the pivotal role of astrocytes [J]. N eurochem Res,2004, 29 (3) : 513-519
11 Buskila Y, Farkash S, HershfinkelM, et al. Rap id and reactive nitric oxide production by astrocytes in mouse neocortical slices[ J ].Glia, 2005, 52 (3) : 169-76
12 Oderfeld-Nowak B, Orzylowska-SLiwinska O, Soltys Z, et al Concomitant up-regulation of astroglial high and low affinity nervegrowth factor receptors in the CA1 hippocampal area following global transient cerebral ischemia in rat [J]. Neuroscience, 2003, 120:31-40
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