RNA干扰敲减视网膜小胶质细胞中组织型纤溶酶原激活物的表达对其活化反应的影响
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摘要
第一部分:原代培养的大鼠视网膜小胶质细胞中组织型纤溶酶原激活物的表达
应用原代培养分离的S-D大鼠视网膜小胶质细胞(MG),并通过不同浓度(0,3,10,30,100,300ng/ml)的脂多糖(LPS)刺激来建立小胶质细胞体外活化的模型,行细胞免疫荧光双标观察MG中OX42和t-PA的表达,同时应用Westernblot检测MG中t-PA的表达。
实验结果显示:分离的小胶质细胞贴壁24h后呈较小细胞体,大多数为圆形,少数呈短杆状。随着培养时间的延长,细胞逐渐伸出细长的分支,7d时易见多极或双极分枝状细胞。荧光显微镜下观察静息的小胶质细胞,细胞形态小,细胞突起少,OX42染色浅;而活化的小胶质细胞,细胞体增大,突起增多,OX42染色加深。未受刺激的小胶质细胞中可以观察到较微弱的t-PA的表达,呈淡红色弱荧光,经LPS作用后MG中的t-PA表达明显增强,并且随LPS的浓度增加而增强。Western blot的结果显示,各组MG中均有t-PA蛋白条带的表达,随着LPS激活的浓度的增加,条带的灰度逐渐增强。
本部分研究建立了视网膜小胶质细胞的体外培养和分离的成熟的方法,研究发现小胶质细胞可以被LPS作用后活化,活化的MG可以表达产生t-PA,并随着LPS刺激的浓度增大,表达增强。
第二部分:应用RNA干扰的方法敲减小胶质细胞中组织型纤溶酶原激活物的表达
我们根据文献报道和RNAi设计软件(oligoengine RNAi)模拟验证,筛选高效的RNAi序列,将化学合成的siDNA片断与慢病毒载体进行定向连接和重组,其产物转化细菌感受态细胞,对长出的克隆先进行酶切鉴定,再对酶切鉴定阳性的克隆进行测序和分析比对以确定载体的构建成功。将构建好的载体进行超纯去内毒素抽提,脂质体法共转染293T细胞,36-48h后通过荧光显微镜观察共表达的EGFP,测量获得的病毒载体的病毒滴度。然后将携带t-PA siRNA的慢病毒(PTM-sit-PA)或空白病毒转染小胶质细胞,流式细胞仪测量转染的效率,通过Real Time-PCR检测方法检测目的基因t-PA的表达抑制情况。
研究结果发现:酶切后的阳性克隆经过测序和分析比对发现与目的基因完全一致,说明载体构建成功。慢病毒质粒能够很好的转染293T细胞,转染阳性率85%,这样就确保了病毒包装的成功。最终获得慢病毒的滴度大约在5×10~8TU/ml。包装好的慢病毒(PTM-sit-PA)转染染小胶质细胞(MOI=10),两天后荧光显微镜下观察发现细胞高效表达EGFP,利用流式细胞仪检测感染小胶质细胞的荧光表达效率约88%。Real Time PCR检测发现PTM-sit-PA转染小胶质细胞的t-PA mRNA表达受到明显抑制,抑制效率约80%。
本部分实验成功的合成了大鼠t-PA基因的siRNA的慢病毒表达系统,研究结果提示该病毒能够高效率的转染小胶质细胞,高效率的下调t-PA的mRNA的表达水平。为进一步研究t-PA在小胶质细胞中活化中的作用奠定了基础。
第三部分:RNA干扰敲减小胶质细胞t-PA表达后对其活化反应的影响
应用携带t-PA siRNA的慢病毒(RNA干扰组)或空白对照病毒(对照组)分别转染小胶质细胞(MOI=10),转染2天后更换不含血清的含30ng/mlLPS的培养液活化MG,加入LPS后1,3,6,12,24小时各取培养液100μl,应用Elisa法检测培养液IL-1β和TNF-α的浓度。24小时后取小胶质细胞行OX42和Iba-1免疫荧光。
实验结果发现OX42和Iba-1均可以在两组细胞表达,OX42的表达两组之间无明显差异,而Iba-1的表达RNA干扰组比对照组明显减弱。Elisa结果发现的IL-1β浓度除1小时两组之间无明显差别外,其余各时间点RNA干扰组均显著低于对照组,TNF-α浓度在各时间点RNA干扰组均低于对照组。
本部分研究表明在RNA干扰下调了小胶质细胞中t-PA的表达后,Iba-1的表达下调,小胶质细胞对IL-1β和TNF-α分泌减少。提示t-PA表达被敲减后,小胶质细胞的活化受到一定程度的抑制。
Part one:The expression of tissue plasminogen activator(t-PA) in primary cultured rat retinal microglia
The primary cultured microglia cells were isolated from retina of S-D rats and activated with different concentration of LPS(0,3,10,30,100,300ng/ml).Double immunofluorescence(OX42 and t-PA) of microglia were performed and western blot were used to detect t-PA expression.
The results showed that the isolated microglia could adhere 24 hours later,and mostly had small round cell body.As time went by,the microglia cells gradully grew short thin processes and became multi or bipolar ramified cells on the 7th day.The silent microglia had small cell body,little processes,and weak OX42 stain;the activated microglia had larger cell body,more processes,and stronger OX42 stain. Unstimulated microglia only had weak t-PA expression,with carmoisine t-PA stain; Under the stimulation of LPS,the t-PA expression became strong,and the t-PA stain appeared crimson in colour.The result of Western blot showed that all groups of microglia had a band of t-PA expression,and the gray scale increased gradually with increasing concentrations of LPS.
In this part of experiment,we demonstrated a successful method of primary culture and isolation of retinal microglia.The conclusions are that microglia can be activated by LPS,and the activated microglia can express t-PA in a dose related pattern.
Part two:Knockdown of the expression of t-PA of microglia by RNA interference
According to literature and RNAi design software(oligoengine RNAi),we selected a highly effective RNAi sequence,and got the siDNA with chemical synthesis method. Then the siDNA was inserted into the lentivirus RNAi vector,the production was used to transfect the bacterium competent cells,then the positive clone was identified by sequencing to confirm that the preparation of the vetor was successful.The successfully prepared vector was ultrapure extracted and cotransfected the 293T cells with liposome method.The expression of EGFP was observed using a fluorescent microscope,then the virus titer of lentivirus vector was analyzed.Microglia was transfected with t-PA siRNA lentivirus or blank control lentivirus.The efficiency of transfection was determined by flow cytometry,and the efficiency of the objective gene t-PA supression was identified by Real Time PCR.
The results showed that the sequence of positive clone which was analyzed by seguencing was extremely same to the objective gene and it proved that the preparation of vetor was successful.The efficiency of transfection of lentivirus to 293T cells was as high as 85%,and this ensured the success of lentivirus package. The lentivirus titer was about 5×10~8 TU/ml.The efficiency of transfection of lentivirus to microglia was 88%which was determined by flow cytometry.The results of Real Time PCR showed that the inhibitory efficiency of t-PA mRNA expression was 80%.
In this part of investigation,the system of t-PA siRNA lentivirus expressive vector was successfully synthesized.The lentivirus system could transfect microglia and downregulate the expression of t-PA with a high efficacy.All of these results built a good foundation of further investigation of the role of t-PA during process of the activation of microglia.
Part three:The inhibitory effect on microglia activation when t-PA expression was downregulated by RNA interference
Microglia cells were transfected with t-PA siRNA lentivirus(RNA interference group) or blank control lentivirus(control group)(MOI=10),two days later stimulated by 30ng/ml LPS.Then 100μl culture media was acquired every 1,3,6,12,24 hours later.The concentration of IL-1βand TNF-αin the media was assessed by Elisa.The microglia cells were harvested 24 hours later for OX42 and Iba-1 immuno-fluorescence.
The results showed that OX42 and Iba-1 stain of both groups were positive,there was no difference of OX42 expression between two groups,but the Iba-1 expression of RNA interference group was downregulated markedly.The concentration of TNF-αin RNA interference group was significant lower than control group at all times,and the IL-1βconcentration was significant lower than control group at all times except the collection one hour after LPS treatment.
After downregulation of t-PA expression by RNA interference,the Iba-1 expression was weakened and the secretion of IL-1βand TNF-αdecreased significantly.It hint that the activation of microglia induced by LPS is inhibited when t-PA expression is knocked down by RNA interference.
应用原代培养分离的S-D大鼠视网膜小胶质细胞(MG),并通过不同浓度(0,3,10,30,100,300ng/ml)的脂多糖(LPS)刺激来建立小胶质细胞体外活化的模型,行细胞免疫荧光双标观察MG中OX42和t-PA的表达,同时应用Westernblot检测MG中t-PA的表达。
实验结果显示:分离的小胶质细胞贴壁24h后呈较小细胞体,大多数为圆形,少数呈短杆状。随着培养时间的延长,细胞逐渐伸出细长的分支,7d时易见多极或双极分枝状细胞。荧光显微镜下观察静息的小胶质细胞,细胞形态小,细胞突起少,OX42染色浅;而活化的小胶质细胞,细胞体增大,突起增多,OX42染色加深。未受刺激的小胶质细胞中可以观察到较微弱的t-PA的表达,呈淡红色弱荧光,经LPS作用后MG中的t-PA表达明显增强,并且随LPS的浓度增加而增强。Western blot的结果显示,各组MG中均有t-PA蛋白条带的表达,随着LPS激活的浓度的增加,条带的灰度逐渐增强。
本部分研究建立了视网膜小胶质细胞的体外培养和分离的成熟的方法,研究发现小胶质细胞可以被LPS作用后活化,活化的MG可以表达产生t-PA,并随着LPS刺激的浓度增大,表达增强。
第二部分:应用RNA干扰的方法敲减小胶质细胞中组织型纤溶酶原激活物的表达
我们根据文献报道和RNAi设计软件(oligoengine RNAi)模拟验证,筛选高效的RNAi序列,将化学合成的siDNA片断与慢病毒载体进行定向连接和重组,其产物转化细菌感受态细胞,对长出的克隆先进行酶切鉴定,再对酶切鉴定阳性的克隆进行测序和分析比对以确定载体的构建成功。将构建好的载体进行超纯去内毒素抽提,脂质体法共转染293T细胞,36-48h后通过荧光显微镜观察共表达的EGFP,测量获得的病毒载体的病毒滴度。然后将携带t-PA siRNA的慢病毒(PTM-sit-PA)或空白病毒转染小胶质细胞,流式细胞仪测量转染的效率,通过Real Time-PCR检测方法检测目的基因t-PA的表达抑制情况。
研究结果发现:酶切后的阳性克隆经过测序和分析比对发现与目的基因完全一致,说明载体构建成功。慢病毒质粒能够很好的转染293T细胞,转染阳性率85%,这样就确保了病毒包装的成功。最终获得慢病毒的滴度大约在5×10~8TU/ml。包装好的慢病毒(PTM-sit-PA)转染染小胶质细胞(MOI=10),两天后荧光显微镜下观察发现细胞高效表达EGFP,利用流式细胞仪检测感染小胶质细胞的荧光表达效率约88%。Real Time PCR检测发现PTM-sit-PA转染小胶质细胞的t-PA mRNA表达受到明显抑制,抑制效率约80%。
本部分实验成功的合成了大鼠t-PA基因的siRNA的慢病毒表达系统,研究结果提示该病毒能够高效率的转染小胶质细胞,高效率的下调t-PA的mRNA的表达水平。为进一步研究t-PA在小胶质细胞中活化中的作用奠定了基础。
第三部分:RNA干扰敲减小胶质细胞t-PA表达后对其活化反应的影响
应用携带t-PA siRNA的慢病毒(RNA干扰组)或空白对照病毒(对照组)分别转染小胶质细胞(MOI=10),转染2天后更换不含血清的含30ng/mlLPS的培养液活化MG,加入LPS后1,3,6,12,24小时各取培养液100μl,应用Elisa法检测培养液IL-1β和TNF-α的浓度。24小时后取小胶质细胞行OX42和Iba-1免疫荧光。
实验结果发现OX42和Iba-1均可以在两组细胞表达,OX42的表达两组之间无明显差异,而Iba-1的表达RNA干扰组比对照组明显减弱。Elisa结果发现的IL-1β浓度除1小时两组之间无明显差别外,其余各时间点RNA干扰组均显著低于对照组,TNF-α浓度在各时间点RNA干扰组均低于对照组。
本部分研究表明在RNA干扰下调了小胶质细胞中t-PA的表达后,Iba-1的表达下调,小胶质细胞对IL-1β和TNF-α分泌减少。提示t-PA表达被敲减后,小胶质细胞的活化受到一定程度的抑制。
Part one:The expression of tissue plasminogen activator(t-PA) in primary cultured rat retinal microglia
The primary cultured microglia cells were isolated from retina of S-D rats and activated with different concentration of LPS(0,3,10,30,100,300ng/ml).Double immunofluorescence(OX42 and t-PA) of microglia were performed and western blot were used to detect t-PA expression.
The results showed that the isolated microglia could adhere 24 hours later,and mostly had small round cell body.As time went by,the microglia cells gradully grew short thin processes and became multi or bipolar ramified cells on the 7th day.The silent microglia had small cell body,little processes,and weak OX42 stain;the activated microglia had larger cell body,more processes,and stronger OX42 stain. Unstimulated microglia only had weak t-PA expression,with carmoisine t-PA stain; Under the stimulation of LPS,the t-PA expression became strong,and the t-PA stain appeared crimson in colour.The result of Western blot showed that all groups of microglia had a band of t-PA expression,and the gray scale increased gradually with increasing concentrations of LPS.
In this part of experiment,we demonstrated a successful method of primary culture and isolation of retinal microglia.The conclusions are that microglia can be activated by LPS,and the activated microglia can express t-PA in a dose related pattern.
Part two:Knockdown of the expression of t-PA of microglia by RNA interference
According to literature and RNAi design software(oligoengine RNAi),we selected a highly effective RNAi sequence,and got the siDNA with chemical synthesis method. Then the siDNA was inserted into the lentivirus RNAi vector,the production was used to transfect the bacterium competent cells,then the positive clone was identified by sequencing to confirm that the preparation of the vetor was successful.The successfully prepared vector was ultrapure extracted and cotransfected the 293T cells with liposome method.The expression of EGFP was observed using a fluorescent microscope,then the virus titer of lentivirus vector was analyzed.Microglia was transfected with t-PA siRNA lentivirus or blank control lentivirus.The efficiency of transfection was determined by flow cytometry,and the efficiency of the objective gene t-PA supression was identified by Real Time PCR.
The results showed that the sequence of positive clone which was analyzed by seguencing was extremely same to the objective gene and it proved that the preparation of vetor was successful.The efficiency of transfection of lentivirus to 293T cells was as high as 85%,and this ensured the success of lentivirus package. The lentivirus titer was about 5×10~8 TU/ml.The efficiency of transfection of lentivirus to microglia was 88%which was determined by flow cytometry.The results of Real Time PCR showed that the inhibitory efficiency of t-PA mRNA expression was 80%.
In this part of investigation,the system of t-PA siRNA lentivirus expressive vector was successfully synthesized.The lentivirus system could transfect microglia and downregulate the expression of t-PA with a high efficacy.All of these results built a good foundation of further investigation of the role of t-PA during process of the activation of microglia.
Part three:The inhibitory effect on microglia activation when t-PA expression was downregulated by RNA interference
Microglia cells were transfected with t-PA siRNA lentivirus(RNA interference group) or blank control lentivirus(control group)(MOI=10),two days later stimulated by 30ng/ml LPS.Then 100μl culture media was acquired every 1,3,6,12,24 hours later.The concentration of IL-1βand TNF-αin the media was assessed by Elisa.The microglia cells were harvested 24 hours later for OX42 and Iba-1 immuno-fluorescence.
The results showed that OX42 and Iba-1 stain of both groups were positive,there was no difference of OX42 expression between two groups,but the Iba-1 expression of RNA interference group was downregulated markedly.The concentration of TNF-αin RNA interference group was significant lower than control group at all times,and the IL-1βconcentration was significant lower than control group at all times except the collection one hour after LPS treatment.
After downregulation of t-PA expression by RNA interference,the Iba-1 expression was weakened and the secretion of IL-1βand TNF-αdecreased significantly.It hint that the activation of microglia induced by LPS is inhibited when t-PA expression is knocked down by RNA interference.
引文
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[34]Perletti G, Osti D , Marras E , et al . Generation of VSVG pseudotyped lentiviral particles in 293 T cells [J]. J Cel 1 Mol Med, 2004, 8 (1): 142-143.
[35]Hannon GJ. RNA interference [J ]. Nature, 2002,418 (6894): 244-51.
[36]Czauderna F, Fechtner M, Dames S, et al . Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells[J]. Nucleic Acids Res, 2003, 31 (11): 2705-2716.
[37]Seeds NW, Friedman G, Hayden S, Thewke D, Haffke S, McGuire P,Krystosek A. Plasminogen activators and their interaction with the extracellular matrix in neural development, plasticity and regeneration. Semin Neurosci, 1996, 8:405-412.
[38] Tsirka SE, Gualandris A, Amaral DG, Strickland S. Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature 1995.377:340-344.
[39]Stolzing A, Wengner A, Grune T. Degradation of oxidized extracellular proteins by microglia. Arch Biochem Biophys[J]. 2002;400(2):171-179.
[40]Collen D. The plasminogen (fibrinolytic) system. Thromb Haemost. 1999; 82:259-270.
[41]Tsirka S, Rogove A, Bugge T, Degen J, Strickland S. An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus. J Neurosci. 1997; 17:543-552.
[42]Chen Z-L, Strickland S. Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell. 1997; 91:917-925.
[43]Chao, C. C., Hu, S. and Peterson, P. K. Glia, cytokines, and neurotoxicity. Crit. Rev. Neurobiol. 1995; 9:189-205.
[44]Gehrmann J, Matsumoto Y, and Kreutzberg G W. Microglia: intrinsic immuneffector cell of the brain. Brain Res Brain Res Rev .1995; 20:269-87.
[45] Zeng, H. Y., Zhu, X. A., Zhang, C, Yang, L. P., Wu, L. M., Tso, M. O. Identification of sequential events and factors associated with microglial activation, migration, and cytotoxicity in retinal degeneration in rd mice. Invest. Ophthalmol. Vis. Sci. 2005; 46:2992-2999.
[46]Ying-qin Ni, Ge-zhi Xu, Wen-zheng Hu, Le Shi, Yao-wu Qin, and Cui-di Da. Neuroprotective effects of naloxone against light-induced photoreceptor degeneration through inhibiting retinal microglial activation. Invest. Ophthalmol. Vis. Sci.2008; 49: 2589-2598.
[47]Siao CJ, Tsirka SE. Tissue plasminogen activator mediates microglial activation via its finger domain through annexin Ⅱ. J Neurosci. 2002; 22:3352-3358.
[48]Liu B, Du L, Hong JS. Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation. J Pharmacol Exp Ther[J]. 2000;293(2):607-617.
[49]Ryu JK, Franciosi S, Sattayaprasert P, et al.Minocycline inhibits neuronal death and glial activation induced by beta-amyloid peptide in rat hippocampus. Glia[J]. 2004; 48(1): 85-90.
[50]Imai Y and Kohsaka S. Intracellular signaling in M-CSF-induced microglia activation: role of Ibal. Glia. 2002;40:164-174.
[51]Sindhu S, GueyShuang Wu, and Narsing A. Rao. Retinal Microglial Activation and Chemotaxis by Docosahexaenoic Acid Hydroperoxide. Invest Ophthalmol Vis Sci. 2006; 47:3656-3663.
[52]Gualandris A, Jones T, Strickland S, Tsirka S. Membrane depolarization induces the Ca 2+-dependent release of tissue plasminogen activator. J Neurosci. 1996; 16:2220-2225.
[53]Akiyama H, Ikeda K, Kondo H, McGeer P, McGeer E. Immunohistochemical study of type-1 plasminogen activator inhibitor(PAI-1) in brain. Soc Neurosci Abstr. 1995; 21:741.
[54]Krueger SR, Ghisu GP, Cinelli P, Gschwend TP, Osterwalder T, Wolfer DP, Sonderegger P. Expression of neuroserpin, an inhibitor of tissue plasminogen activator, in the developing and adult nervous system of the mouse. J Neurosci. 1997; 17:8984-8996.
[55]Seeds N, Basham M, Haffke S. Neuronal migration is retarded in mice lacking the tissue plasminogen activator gene. Proc Natl Acad Sci USA. 1999; 96:14118-14123.
[56]Wu Y-P, Siao C-J, Lu W, Sung T-C, Frohman M, Milev P, Bugge T, Degen J, Levine J, Margolis R, Tsirka S. The t-PA/plasmin extracellular proteolytic system regulates seizure-induced hippocampal mossy fiber outgrowth through a proteoglycan substrate. J Cell Biol. 2000; 148:1295-1304.
1.Tsirka SE, Rogove AD, Strickland S. 1996. Neuronal cell death and t-PA. Nature 384:123-124.
2.Seeds NW, Friedman G, Hayden S, Thewke D, Haffke S, McGuire P,Krystosek A. 1996. Plasminogen activators and their interaction with the extracellular matrix in neural development, plasticity and regeneration. Semin Neurosci 8:405-412.
3.Huang YY, Bach ME, Lipp HP,et al. 1996. Mice lacking the gene encoding tissue-type plasminogen activator show a selective interference with late-phase longterm potentiation in both Schaffer collateral and mossy fiber pathways. Proc Natl Acad Sci USA 93:8699-8704.
4.Melchor JP, Pawlak R, Strickland S. 2003. The tissue plasminogen activator-plasminogen proteolytic cascade accelerates amyloid-beta (Abeta) degradation and inhibits Abeta-induced neurodegeneration. J. Neurosci., 2003, 23(26):8867- 8871
5.Kim YH, Park JH, Hong SH, Koh JY. 1999. Nonproteolytic neuroprotection by human recombinant tissue plasminogen activator. Science 284:647-650.
6.Siddiq M, Tsirka SE. 2004. Modulation of zinc toxicity by tissue plasminogen activator. Mol Cell Neurosci 25:162-171.
7.Stolzing A, Wengner A, Grune T. Degradation of oxidized extracellular proteins by microglia [J]. Arch Biochem Biophys, 2002 ,400 (2): 171 - 1791.
8.Wang YF, Tsirka SE, Strickland S, Stieg PE, Soriano SG, Lipton SA. Tissue plasminogen activator (t-PA) increases neuronal damage after focal cerebral ischemia in wild-type and t-PA-deficient mice. Nat Med 1998,4:228-231.
9.Nagai N, De Mol M, Lijnen HR, Carmeliet P, Collen D. 1999. Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice. Circulation 99:2440-2444.
10.Tsirka SE, Gualandris A, Amaral DG, Strickland S. 1995. Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature 377:340-344.
11.Tsirka SE, Rogove AD, Bugge TH, Degen JL, Strickland S. 1997. An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus. J Neurosci 17:543-552.
12. Nicole O, Docagne F, Ali C, et al. 2001. The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat Med 7:59-64.
13. M(?)nica Fern(?)ndez-Monreal,Dulin, Fabienne Mackenzie. Arginine 260 of the amino-terminal domain of NR1 subunit is critical for tissue-type plasminogen activator-mediated enhancement of N-methyl-D-aspartate receptor signaling. J Biol Chem. Volume(2004) 279: 50850-6.
14. Indyk JA, Chen ZL, Tsirka SE, et al. Laminin chain expression suggests that laminin-10 is a major isoform in the mouse hippocampus and is degeraded by the tissue plasminogen activator/plasm in protease cascade during excitotoxic injury. J. Neuroscience, 2003 ,116(2):359-371.
15. Xiaoying Wang , Sun-Ryung Leel, Ken Arail, et al. Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator. Nat Med 2003(9): 1313-1317.
16. Manel G, Medina et al. Tissue plasminogen activator mediates amyloid induced neurotoxicity via Erkl/2 activation. The EMBO Journal (2005) 24, 1706-1716.
17. M.P. Flavin, G. Zhao, and L.T. Ho. Microglial Tissue Plasminogen Activator (t-PA) Triggers Neuronal Apoptosis In Vitro. GLIA 29:347-354, 2000.
18. Dong Liu, Tong Cheng, Huang Guo et al. Tissue plasminogen activator neurovascular toxicity is controlled by activated protein C. Nat. Med (2004)10 :1379-1383.
19. Ge'raldine Liot, Benoit D. Roussel, Nathalie Lebeurrier et al. Tissue-type plasminogen activator rescues neurones from serum deprivation-induced apoptosis through a mechanism independent of its proteolytic activity. J. Neurochem. (2006) 98: 1458-1464.
20. Rogove AD, Tsirka SE. Neurotoxic responses by microglia elicited by excitotoxic injury in the mouse hippocampus. Curr Biol.1997.8:19-25.
21. Rogove AD, Lu W, Tsirka SE. Microglial activation and recruitment, but not proliferation, suffice to mediate neurodegeneration. Cell Death and Differentiation(2002)9:801 -806.
22. Siao CJ, Tsirka SE. Tissue plasminogen activator mediates microglial activation via its finger domain through annexin Ⅱ. J Neurosci(2002)22:3352-3358.
23. Ling Q, Jacovina AT, Deora A, et al. Annexin II regulates fibrin homeostasis and neoangiogenesis in vivo. J Clin Invest (2004)113:38-48.
24. Chia-Jen Siao, Susana R. Fernandez and Stella E. Tsirkal. Cell Type-Specific Roles for Tissue Plasminogen Activator Released by Neurons or Microglia after Excitotoxic Injury. J. Neurosci. 2003 · 23(8):3234 -3242
25. John J. Sheehan, Chun Zhou, Iordanis Gravanis, et al. Proteolytic Activation of Monocyte Chemoattractant Protein-1 by Plasmin Underlies Excitotoxic Neurodegeneration in Mice. J. Neurosci., 2007, 27(7):1738 - 1745
26. Chunzhang Cao, Daniel A Lawrence, Yang Li, et al. Endocytic receptor LRP together with t-PA and PAI-1 coordinates Mac-1-dependent macrophage migration. The EMBO Journal (2006) 25: 1860-1870.
27. Ghazi, N.G., Noureddine, B., Haddad, R.S., Jurdi, F.A., Bashshur, Z.F.,2003. Intravitreal tissue plasminogen activator in the management of central retinal vein occlusion. Retina 23, 780-784.
28. Schulze, S.D., Hesse, L., 2002. Tissue plasminogen activator plus gas injection in patients with subretinal hemorrhage caused by age-related macular degeneration: predictive variables for visual outcome. Graefes Arch. Clin. Exp. Ophthalmol. 240,717-720.
29. Zhang, X., Chaudhry, A., Chintala, S.K., 2003. Inhibition of plasminogen activation protects against ganglion cell loss in a mouse model of retinal damage. Mol. Vis. 9, 238-248.
30. J.E. Collinge, V.N. Simirskii, M.K. Duncan. Expression of tissue plasminogen activator during eye development. Experimental Eye Research, 2005,81:90-96.
31. San-Ni Chen, Te-Cheng Yang, Cheng-Lien ho,et al. Retinal toxicity of intravitreal tissue plasminogen activator. Ophthalmology, 2003,110(4):704-708.
32. M. Kumada, M. Niwa, Xiaodan Wang, et al. Endogenous tissue type plasminogen activator facilitates NMDA-induced retinal damage. Toxicology and Applied Pharmacology 2004,200: 48-53.
33. M. Kumada, M. Niwa, A. Hara, Tissue Type Plasminogen Activator Facilitates NMDA-Receptor-Mediated Retinal Apoptosis through an Independent Fibrinolytic Cascade. Invest Ophthalmol Vis Sci. 2005;46:1504-1507.
34. Raghuveer S. Mali, Mei Cheng, and Shravan K. Chintalal. Plasminogen activators promote excitotoxicity-induced retinal damage. FASEB J. 2005,19:1280-1289.
35. Harvey and Chintala. Inhibition of plasminogen activators attenuates the death of differentiated retinal ganglion cells and stabilizes their neurite network in vitro. Invest. Ophthalmol. Vis. Sci..2007; 48: 1884-1891.
36. Rakic, J.M., Lambert, V., Munaut, C.,et al. Mice without uPA, t-PA, or plasminogen genes are resistant to experimental choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 2003, 44: 1732-1739.
37. Wolfgang Schacke, Karl-Friedrich Beck, Josef Pfeilschifter et al. Modulation of tissue plasminogen activator and plasminogen activator inhibitor-1 by transforming growth factor-P in human retinal glial cells. Invest Ophthalmol Vis Sci. 2002,43:2799-2805.
38. Rogove AD, Siao C, Keyt B, Strickland S, Tsirka SE. Activation of microglia reveals a non-proteolytic cytokine function for tissue plasminogen activator in the central nervous system. J Cell Sci,1999, 112:4007-4016
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[13]Nagai N,De Mol M,Lijnen HR,Carmeliet P,Collen D.1999.Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice. Circulation 99:2440-2444.
[14]Rogove AD, Tsirka SE. Neurotoxic responses by microglia elicited by excitotoxic injury in the mouse hippocampus. Curr Biol.1997.8:19-25.
[15]Nicole O, Docagne F, Ali C, et al. 2001. The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat Med 7:59-64.
[16]M(?)nica Fern(?)ndez-Monreal,Dulin, Fabienne Mackenzie. Arginine 260 of the amino-terminal domain of NR1 subunit is critical for tissue-type plasminogen activator-mediated enhancement of N-methyl-D-aspartate receptor signaling. J Biol Chem. Volume(2004) 279: 50850-6.
[17]Indyk JA, Chen ZL, Tsirka SE, et al. Laminin chain expression suggests that laminin-10 is a major isoform in the mouse hippocampus and is degeraded by the tissue plasminogen activator/plasm in protease cascade during excitotoxic injury. J. Neuroscience, 2003 ,116(2):359-371.
[18]M(?)nica Fern(?)ndez-Monreal,Dulin, Fabienne Mackenzie. Arginine 260 of the amino-terminal domain of NR1 subunit is critical for tissue-type plasminogen activator-mediated enhancement of N-methyl-D-aspartate receptor signaling. J Biol Chem. Volume(2004) 279: 50850-6.
[19]Manel G, Medina et al. Tissue plasminogen activator mediates amyloid induced neurotoxicity via Erkl/2 activation. The EMBO Journal (2005) 24,1706-1716.
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[33]Uchida H, Tanaka T, Sasaki K, et ,al . Adenovirus-mediated transfer of siRNA against survivin induced apoptosis and attenuated tumor cell growth in vitro and in vivo [ J ] . Mol Ther, 2004, 10 (1): 162-171.
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[35]Hannon GJ. RNA interference [J ]. Nature, 2002,418 (6894): 244-51.
[36]Czauderna F, Fechtner M, Dames S, et al . Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells[J]. Nucleic Acids Res, 2003, 31 (11): 2705-2716.
[37]Seeds NW, Friedman G, Hayden S, Thewke D, Haffke S, McGuire P,Krystosek A. Plasminogen activators and their interaction with the extracellular matrix in neural development, plasticity and regeneration. Semin Neurosci, 1996, 8:405-412.
[38] Tsirka SE, Gualandris A, Amaral DG, Strickland S. Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature 1995.377:340-344.
[39]Stolzing A, Wengner A, Grune T. Degradation of oxidized extracellular proteins by microglia. Arch Biochem Biophys[J]. 2002;400(2):171-179.
[40]Collen D. The plasminogen (fibrinolytic) system. Thromb Haemost. 1999; 82:259-270.
[41]Tsirka S, Rogove A, Bugge T, Degen J, Strickland S. An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus. J Neurosci. 1997; 17:543-552.
[42]Chen Z-L, Strickland S. Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell. 1997; 91:917-925.
[43]Chao, C. C., Hu, S. and Peterson, P. K. Glia, cytokines, and neurotoxicity. Crit. Rev. Neurobiol. 1995; 9:189-205.
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[46]Ying-qin Ni, Ge-zhi Xu, Wen-zheng Hu, Le Shi, Yao-wu Qin, and Cui-di Da. Neuroprotective effects of naloxone against light-induced photoreceptor degeneration through inhibiting retinal microglial activation. Invest. Ophthalmol. Vis. Sci.2008; 49: 2589-2598.
[47]Siao CJ, Tsirka SE. Tissue plasminogen activator mediates microglial activation via its finger domain through annexin Ⅱ. J Neurosci. 2002; 22:3352-3358.
[48]Liu B, Du L, Hong JS. Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation. J Pharmacol Exp Ther[J]. 2000;293(2):607-617.
[49]Ryu JK, Franciosi S, Sattayaprasert P, et al.Minocycline inhibits neuronal death and glial activation induced by beta-amyloid peptide in rat hippocampus. Glia[J]. 2004; 48(1): 85-90.
[50]Imai Y and Kohsaka S. Intracellular signaling in M-CSF-induced microglia activation: role of Ibal. Glia. 2002;40:164-174.
[51]Sindhu S, GueyShuang Wu, and Narsing A. Rao. Retinal Microglial Activation and Chemotaxis by Docosahexaenoic Acid Hydroperoxide. Invest Ophthalmol Vis Sci. 2006; 47:3656-3663.
[52]Gualandris A, Jones T, Strickland S, Tsirka S. Membrane depolarization induces the Ca 2+-dependent release of tissue plasminogen activator. J Neurosci. 1996; 16:2220-2225.
[53]Akiyama H, Ikeda K, Kondo H, McGeer P, McGeer E. Immunohistochemical study of type-1 plasminogen activator inhibitor(PAI-1) in brain. Soc Neurosci Abstr. 1995; 21:741.
[54]Krueger SR, Ghisu GP, Cinelli P, Gschwend TP, Osterwalder T, Wolfer DP, Sonderegger P. Expression of neuroserpin, an inhibitor of tissue plasminogen activator, in the developing and adult nervous system of the mouse. J Neurosci. 1997; 17:8984-8996.
[55]Seeds N, Basham M, Haffke S. Neuronal migration is retarded in mice lacking the tissue plasminogen activator gene. Proc Natl Acad Sci USA. 1999; 96:14118-14123.
[56]Wu Y-P, Siao C-J, Lu W, Sung T-C, Frohman M, Milev P, Bugge T, Degen J, Levine J, Margolis R, Tsirka S. The t-PA/plasmin extracellular proteolytic system regulates seizure-induced hippocampal mossy fiber outgrowth through a proteoglycan substrate. J Cell Biol. 2000; 148:1295-1304.
1.Tsirka SE, Rogove AD, Strickland S. 1996. Neuronal cell death and t-PA. Nature 384:123-124.
2.Seeds NW, Friedman G, Hayden S, Thewke D, Haffke S, McGuire P,Krystosek A. 1996. Plasminogen activators and their interaction with the extracellular matrix in neural development, plasticity and regeneration. Semin Neurosci 8:405-412.
3.Huang YY, Bach ME, Lipp HP,et al. 1996. Mice lacking the gene encoding tissue-type plasminogen activator show a selective interference with late-phase longterm potentiation in both Schaffer collateral and mossy fiber pathways. Proc Natl Acad Sci USA 93:8699-8704.
4.Melchor JP, Pawlak R, Strickland S. 2003. The tissue plasminogen activator-plasminogen proteolytic cascade accelerates amyloid-beta (Abeta) degradation and inhibits Abeta-induced neurodegeneration. J. Neurosci., 2003, 23(26):8867- 8871
5.Kim YH, Park JH, Hong SH, Koh JY. 1999. Nonproteolytic neuroprotection by human recombinant tissue plasminogen activator. Science 284:647-650.
6.Siddiq M, Tsirka SE. 2004. Modulation of zinc toxicity by tissue plasminogen activator. Mol Cell Neurosci 25:162-171.
7.Stolzing A, Wengner A, Grune T. Degradation of oxidized extracellular proteins by microglia [J]. Arch Biochem Biophys, 2002 ,400 (2): 171 - 1791.
8.Wang YF, Tsirka SE, Strickland S, Stieg PE, Soriano SG, Lipton SA. Tissue plasminogen activator (t-PA) increases neuronal damage after focal cerebral ischemia in wild-type and t-PA-deficient mice. Nat Med 1998,4:228-231.
9.Nagai N, De Mol M, Lijnen HR, Carmeliet P, Collen D. 1999. Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice. Circulation 99:2440-2444.
10.Tsirka SE, Gualandris A, Amaral DG, Strickland S. 1995. Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature 377:340-344.
11.Tsirka SE, Rogove AD, Bugge TH, Degen JL, Strickland S. 1997. An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus. J Neurosci 17:543-552.
12. Nicole O, Docagne F, Ali C, et al. 2001. The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat Med 7:59-64.
13. M(?)nica Fern(?)ndez-Monreal,Dulin, Fabienne Mackenzie. Arginine 260 of the amino-terminal domain of NR1 subunit is critical for tissue-type plasminogen activator-mediated enhancement of N-methyl-D-aspartate receptor signaling. J Biol Chem. Volume(2004) 279: 50850-6.
14. Indyk JA, Chen ZL, Tsirka SE, et al. Laminin chain expression suggests that laminin-10 is a major isoform in the mouse hippocampus and is degeraded by the tissue plasminogen activator/plasm in protease cascade during excitotoxic injury. J. Neuroscience, 2003 ,116(2):359-371.
15. Xiaoying Wang , Sun-Ryung Leel, Ken Arail, et al. Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator. Nat Med 2003(9): 1313-1317.
16. Manel G, Medina et al. Tissue plasminogen activator mediates amyloid induced neurotoxicity via Erkl/2 activation. The EMBO Journal (2005) 24, 1706-1716.
17. M.P. Flavin, G. Zhao, and L.T. Ho. Microglial Tissue Plasminogen Activator (t-PA) Triggers Neuronal Apoptosis In Vitro. GLIA 29:347-354, 2000.
18. Dong Liu, Tong Cheng, Huang Guo et al. Tissue plasminogen activator neurovascular toxicity is controlled by activated protein C. Nat. Med (2004)10 :1379-1383.
19. Ge'raldine Liot, Benoit D. Roussel, Nathalie Lebeurrier et al. Tissue-type plasminogen activator rescues neurones from serum deprivation-induced apoptosis through a mechanism independent of its proteolytic activity. J. Neurochem. (2006) 98: 1458-1464.
20. Rogove AD, Tsirka SE. Neurotoxic responses by microglia elicited by excitotoxic injury in the mouse hippocampus. Curr Biol.1997.8:19-25.
21. Rogove AD, Lu W, Tsirka SE. Microglial activation and recruitment, but not proliferation, suffice to mediate neurodegeneration. Cell Death and Differentiation(2002)9:801 -806.
22. Siao CJ, Tsirka SE. Tissue plasminogen activator mediates microglial activation via its finger domain through annexin Ⅱ. J Neurosci(2002)22:3352-3358.
23. Ling Q, Jacovina AT, Deora A, et al. Annexin II regulates fibrin homeostasis and neoangiogenesis in vivo. J Clin Invest (2004)113:38-48.
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