血小板源性生长因子受体-β在小鼠脑出血后脑损伤中的作用机制研究
发布时间：2018-10-25 17:06
【摘要】：第一部分小鼠脑出血后脑组织中血小板源性生长因子受体-β及其配体血小板源性生长因子-BB的表达变化目的:应用小鼠脑出血(intracerebral hemorrhage,ICH)模型,探讨小鼠脑出血后不同时间点血小板源性生长因子受体-β(Platelet-derived growth factor receptor-β,PDGFR-β)及其配体血小板源性生长因子-BB(Platelet-derived growth factor-BB,PDGF-BB)的表达变化情况。方法:将42只8周大小、重量约30克的雄性CD1小鼠随机分为六组:对照组和5个时间点组(ICH后3h组、ICH后6h组、ICH后12h组、ICH后24h组、ICH后72h组),每组7只CD1小鼠(n=7)。应用自体动脉血诱导的脑出血模型(blood ICH,b ICH),通过western blot的方法,检测PDGFR-β和PDGF-BB的表达变化情况,通过免疫荧光染色的方法测定小鼠脑出血后PDGFR-β的表达水平和细胞定位情况。结果:Western blot的结果提示小鼠脑出血24小时后同侧大脑半球PDGF-BB的表达水平显著高于对侧大脑半球(p0.05);PDGFR-β的表达从小鼠脑出血后6小时开始升高(p0.05),并且直到脑出血后24小时都维持在升高的水平(p0.05)。免疫荧光染色结果提示在小鼠脑出血后24小时的血肿周围,可以检测到PDGFR-β与血管平滑肌细胞(vascular smooth muscle cell,VSMC)标记蛋白(α-平滑肌肌动蛋白,α-smooth muscle actin,α-SMA)的共表达。结论:小鼠脑出血后PDGFR-β和PDGF-BB的表达增多,并且在血肿周围的血管平滑肌细胞中检测到PDGFR-β的表达。提示PDGFR-β可能参与小鼠脑出血后的脑损伤(brain injury)的过程以及血管平滑肌细胞(VSMC)表型转换(phenotypic transformation)过程。第二部分抑制血小板源性生长因子受体-β改善小鼠脑出血后的脑损伤目的:应用小鼠自体动脉血诱导的脑出血模型,通过小干扰RNA(small interfering RNA,si RNA)技术和PDGFR-β抑制剂(Gleevec)抑制PDGFR-β表达,探讨抑制PDGFR-β是否能够改善小鼠脑出血后的脑损伤情况。方法:将108只8周大小、重量约30克的雄性CD1小鼠随机分为对照组(n=24),ICH组(n=26),ICH+scramble si RNA组(n=26),ICH+PDGFR-βsi RNA组(n=26),ICH+Gleevec组(n=6)。应用小鼠自体动脉血诱导的脑出血模型,在ICH前24小时脑室注射scramble si RNA和PDGFR-βsi RNA,在ICH后1小时腹腔注射PDGFR-β抑制剂(Gleevec)。ICH24和48小时后测定小鼠神经行为学评分情况;ICH24和48小时后处死小鼠,取同侧及对侧大脑半球做标本,测定脑水肿变化情况;ICH24小时后处死小鼠,取脑组织做标本,通过western blot方法和免疫荧光染色的方法测定炎症反应情况和脑血管平滑肌细胞表型转换情况。探讨抑制PDGFR-β是否能够改善小鼠脑出血后的脑损伤情况。结果:与对照组相比,小鼠ICH后24小时和48小时神经行为学评分明显降低(p0.05),而ICH+PDGFR-βsi RNA组相对ICH组行为学评分明显提高(p0.05),ICH+scramble si RNA组则无明显变化。与对照组相比,ICH后24小时和48小时后小鼠同侧基底节脑水肿明显增加(p0.05),而ICH+PDGFR-βsi RNA组相对ICH组脑水肿显著降低(p0.05),ICH+scramble si RNA组则无明显变化。小鼠ICH后24小时,同侧大脑半球非平滑肌肌球蛋白2B(smooth non-muscle myosin IIB,SMemb)、细胞间粘附分子-1(Intercellular adhesion molecule-1,ICAM-1)、髓过氧化物酶(myeloperoxidase,MPO)表达明显增加(p0.05),而ICH+PDGFR-βsi RNA组相对于ICH组明显降低(p0.05),ICH+scramble si RNA组则无明显变化。另外,ICH+Gleevec组小鼠脑组织的SMemb、ICAM-1表达相对于ICH组也显著降低(p0.05)。免疫荧光染色结果表明,在小鼠脑出血后24小时的血肿周围,可以检测到SMemb和ICAM-1与血管平滑肌细胞标记蛋白(α-SMA)的共表达,并且,ICH组和ICH+scramble si RNA组MPO阳性细胞较对照组增多(p0.05),ICH+PDGFR-βsi RNA组MPO阳性细胞则较ICH组和ICH+scramble si RNA组减少。结论:抑制PDGFR-β能够降低ICH引起的小鼠神经行为学评分降低和脑水肿增加,并且能够减轻ICH后的炎症反应和血管平滑肌细胞表型转换过程。提示抑制PDGFR-β能够改善小鼠脑出血后的脑损伤情况。第三部分血小板源性生长因子受体-β调控脑出血后脑血管平滑肌细胞表型转换及炎症反应的分子机制目的:应用小鼠自体动脉血诱导的脑出血模型,通过si RNA技术和PDGFR-β抑制剂(Gleevec)抑制PDGFR-β表达,通过重组PDGF-BB和丝裂原活化蛋白激酶-活化蛋白激酶2(mitogen-activated protein kinase-activated protein kinase 2,MAPKAPK2,MK2)抑制剂(KKKALNRQLGVAA)等干预,检测信号通路相关分子的表达情况,探讨PDGFR-β是否通过p38-MK2信号通路引起血管平滑肌细胞表型转换和加重炎症反应。方法:对照组(n=7),ICH组(n=7),ICH+scramble si RNA组(n=7),ICH+PDGFR-βsi RNA组(n=7)小鼠利用第二部分实验所产生的标本用于分子检测。另将12只8周大小、重量约30克的雄性CD1小鼠随机分为PDGF-BB组(n=6),PDGF-BB+MK2抑制剂组(n=6)。应用小鼠使用立体定向仪向小鼠右侧基底节区注射重组PDGF-BB(200 ng/2ul PBS)和MK2抑制剂KKKALNRQLGVAA(2ug/kg),通过western blot方法和免疫荧光染色的方法检测信号通路相关分子的表达情况,探讨PDGFR-β是否通过p38-MK2信号通路引起血管平滑肌细胞表型转换和加重炎症反应。结果:Western blot检测结果表明,小鼠脑出血后24小时与对照组相比,血肿同侧大脑半球的p-PDGFR-β、p-P38和p-MK2的表达明显增高(P0.05),ICH+scramble si RNA组的p-PDGFR-β、p-P38和p-MK2的表达相比较于对照组也有明显增高(P0.05),ICH+PDGFR-βsi RNA组则相对于ICH组和ICH+scramble si RNA组明显降低(P0.05)。PDGF-BB组小鼠与对照组相比,ICAM-1表达明显增高(P0.05),PDGF-BB+MK2抑制剂组小鼠与PDGF-BB组相比,ICAM-1表达明显降低(P0.05)。免疫荧光染色结果表明,在小鼠脑出血后24小时的血肿周围,可以检测到p-p38和p-MK2与血管平滑肌细胞标记蛋白(α-SMA)的共表达。结论:小鼠ICH后,PDGFR-β通过与PDGF-BB结合,激活p38-MK2信号通路引起血管平滑肌细胞表型转换和加重炎症反应。
[Abstract]:In the first part, the expression of platelet-derived growth factor receptor--1R and its ligand-platelet-derived growth factor-BB in brain tissue after cerebral hemorrhage in the first part of mice was designed to determine the expression of platelet-derived growth factor-BB in brain tissue. To investigate the changes of platelet-derived growth factor receptor (PDGF-BB) and platelet-derived growth factor-BB (PDGF-BB) at different time points after intracerebral hemorrhage in mice. Methods: Forty-eight male CD1 mice weighing about 30 grams were randomly divided into six groups: control group and 5 time-point groups (ICH-3h group, ICH-6h group, ICH-12h group, ICH-24h group, ICH-72h group), and 7 CD1 mice in each group (n = 7). The expression of PDGFR-jun and PDGF-BB were detected by means of western blot, and the expression level and cell localization of PDGFR-BB after intracerebral hemorrhage were determined by immunofluorescent staining. Results: Western blot suggested that the expression level of PDGF-BB on the same side was significantly higher than that of the contralateral hemisphere (P0.05). and remained at an elevated level 24 hours after the cerebral hemorrhage (p0.05). Immunofluorescence staining suggested that PDGFR-actin was co-expressed with vascular smooth muscle cell (VSMC) marker protein (VSMC) marker protein (VSMC) in 24 hours after cerebral hemorrhage in mice. CONCLUSION: The expression of PDGFR-jun and PDGF-BB is increased after cerebral hemorrhage in mice, and the expression of PDGFR-B9 is detected in vascular smooth muscle cells around hematoma. It is suggested that PDGFR-tau may be involved in the process of brain injury (brain injury) after cerebral hemorrhage in mice and the phenotypic transition of vascular smooth muscle cells (VSMC). The second part inhibits platelet-derived growth factor receptor-7721 in improving brain injury after cerebral hemorrhage in mice: The model of cerebral hemorrhage induced by autologous arterial blood of mice is used to inhibit the expression of PDGFR-CoV by small interfering RNA (si RNA) technique and PDGFR-proton pump inhibitor (Gleevec). To investigate whether the inhibition of PDGFR-CoV can improve the brain injury after intracerebral hemorrhage in mice. Methods: 108 male CD1 mice were randomly divided into control group (n = 24), ICH group (n = 26), ICH + scramble si RNA group (n = 26), ICH + PDGFR-Msi RNA group (n = 26), ICH + Gleevec group (n = 6). Intraperitoneal injection of scramble si RNA and PDGFR-Dbsi RNA was performed within 24 hours before ICH using mouse autoarterial blood-induced cerebral hemorrhage model, and PDGFR-proton pump inhibitor (Gleevec) was injected intraperitoneally at 1 hour after ICH. The neurobehavioral score of mice was measured after ICH24 and 48 hours. The changes of cerebral edema were determined by taking samples from the same side and on the hemisphere of the brain, and the mice were sacrificed after ICH24 hours. The brain tissue samples were taken, and the inflammatory response and the phenotype conversion of cerebral vascular smooth muscle cells were determined by western blot and immunofluorescence staining. To investigate whether the inhibition of PDGFR-CoV can improve the brain injury after intracerebral hemorrhage in mice. Results: Compared with the control group, the neurobehavioral score decreased significantly in 24 hours and 48 hours after ICH (P0.05). Compared with the control group, the brain edema at the same side of the mice increased significantly (P0.05) after 24 hours and 48 hours after ICH, while the ICH + PDGFR-Dbsi RNA group was significantly lower than that of the ICH group (P0.05), and the ICH + scramble si RNA group had no obvious change. After ICH for 24 hours, non-smooth muscle Myosin 2B (SMemb), intercellular adhesion molecule-1 (ICAM-1), myelopoxidase (MPO) expression in the same side were significantly increased (P0.05), while the ICH + PDGFR-Msi RNA group was significantly lower than that of ICH group (P0.05). There was no significant change in the ICH + scramble si RNA group. In addition, the expression of SMemb, ICAM-1 in the brain tissue of the ICH + Gleevec group was significantly lower with respect to the ICH group (P0.05). Immunofluorescence staining showed that SMemb and ICAM-1 were co-expressed with vascular smooth muscle cell marker protein (OPG-SMA) around 24 hours after cerebral hemorrhage in mice, and MPO positive cells in ICH group and ICH + scramble si RNA group were higher than those in control group (P0.05). MPO positive cells in the ICH + PDGFR-Msi RNA group were reduced compared to ICH groups and ICH + scramble si RNA groups. Conclusion: It can reduce the decrease of neurobehavioral score and increase of brain edema in mice induced by ICH, and can reduce the inflammatory response after ICH and the phenotype conversion of vascular smooth muscle cells. It is suggested that inhibition of PDGFR-VEP can improve the brain injury after intracerebral hemorrhage in mice. The third part of platelet-derived growth factor receptor-MAA regulates the phenotype transformation of cerebral vascular smooth muscle cells and the molecular mechanism of inflammatory response after cerebral hemorrhage: the model of cerebral hemorrhage induced by autologous arterial blood of mice is used to inhibit the expression of PDGFR-ALK through si RNA technology and PDGFR-proton pump inhibitor (Gleevec). The expression of signal pathway-related molecules was detected by the intervention of recombinant PDGF-BB and p38 pro-activated protein kinase-activated protein kinase 2 (MAPKAPK2, MK2) inhibitor (KKKALNRQLGVAA). To investigate whether PDGFR-IIIa induced vascular smooth muscle cell phenotype conversion and exacerbation of inflammatory response through the p38-MK2 signaling pathway. Methods: The control group (n = 7), ICH group (n = 7), ICH + scramble si RNA group (n = 7), ICH + PDGFR-Alsi RNA group (n = 7) were used for molecular detection by the second partial experiment. Another 12 male CD1 mice were randomly divided into PDGF-BB group (n = 6), PDGF-BB + MK2 inhibitor group (n = 6). The recombinant PDGF-BB (200 ng/ 2ul PBS) and the MK2 inhibitor KKKALNRQLGVAA (2ug/ kg) were injected into the basal ganglia region on the right side of the mouse using a stereotactic instrument, and the expression of the signal pathway-related molecules was detected by western blot and immunofluorescence staining. To investigate whether PDGFR-IIIa induced vascular smooth muscle cell phenotype conversion and exacerbation of inflammatory response through the p38-MK2 signaling pathway. Results: Western blot showed that the expression of p-PDGFR-jun, p-P38 and p-MK2 in the hemisphere of the hematoma was significantly higher than that in the control group (P <0.05). The expression of p-PDGFR-, p-P38 and p-MK2 in the ICH + scramble si RNA group was significantly higher than that in the control group (P0.05). Compared with the control group, the expression of ICAM-1 was significantly increased (P <0.05). The expression of ICAM-1 in the PDGF-BB + MK2 inhibitor group was significantly lower than that in the PDGF-BB group (P <0.05). Immunofluorescence staining showed that p-MK2 and p-MK2 were co-expressed with vascular smooth muscle cell marker protein (OPG-SMA) around the 24-hour hematoma after intracerebral hemorrhage in mice. CONCLUSION: After ICH, PDGFR-B9 is combined with PDGF-BB to activate the signaling pathway of p38-MK2 to induce the phenotype transformation of vascular smooth muscle cells and increase the inflammatory response.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.34
，
本文编号：2294280
[Abstract]:In the first part, the expression of platelet-derived growth factor receptor--1R and its ligand-platelet-derived growth factor-BB in brain tissue after cerebral hemorrhage in the first part of mice was designed to determine the expression of platelet-derived growth factor-BB in brain tissue. To investigate the changes of platelet-derived growth factor receptor (PDGF-BB) and platelet-derived growth factor-BB (PDGF-BB) at different time points after intracerebral hemorrhage in mice. Methods: Forty-eight male CD1 mice weighing about 30 grams were randomly divided into six groups: control group and 5 time-point groups (ICH-3h group, ICH-6h group, ICH-12h group, ICH-24h group, ICH-72h group), and 7 CD1 mice in each group (n = 7). The expression of PDGFR-jun and PDGF-BB were detected by means of western blot, and the expression level and cell localization of PDGFR-BB after intracerebral hemorrhage were determined by immunofluorescent staining. Results: Western blot suggested that the expression level of PDGF-BB on the same side was significantly higher than that of the contralateral hemisphere (P0.05). and remained at an elevated level 24 hours after the cerebral hemorrhage (p0.05). Immunofluorescence staining suggested that PDGFR-actin was co-expressed with vascular smooth muscle cell (VSMC) marker protein (VSMC) marker protein (VSMC) in 24 hours after cerebral hemorrhage in mice. CONCLUSION: The expression of PDGFR-jun and PDGF-BB is increased after cerebral hemorrhage in mice, and the expression of PDGFR-B9 is detected in vascular smooth muscle cells around hematoma. It is suggested that PDGFR-tau may be involved in the process of brain injury (brain injury) after cerebral hemorrhage in mice and the phenotypic transition of vascular smooth muscle cells (VSMC). The second part inhibits platelet-derived growth factor receptor-7721 in improving brain injury after cerebral hemorrhage in mice: The model of cerebral hemorrhage induced by autologous arterial blood of mice is used to inhibit the expression of PDGFR-CoV by small interfering RNA (si RNA) technique and PDGFR-proton pump inhibitor (Gleevec). To investigate whether the inhibition of PDGFR-CoV can improve the brain injury after intracerebral hemorrhage in mice. Methods: 108 male CD1 mice were randomly divided into control group (n = 24), ICH group (n = 26), ICH + scramble si RNA group (n = 26), ICH + PDGFR-Msi RNA group (n = 26), ICH + Gleevec group (n = 6). Intraperitoneal injection of scramble si RNA and PDGFR-Dbsi RNA was performed within 24 hours before ICH using mouse autoarterial blood-induced cerebral hemorrhage model, and PDGFR-proton pump inhibitor (Gleevec) was injected intraperitoneally at 1 hour after ICH. The neurobehavioral score of mice was measured after ICH24 and 48 hours. The changes of cerebral edema were determined by taking samples from the same side and on the hemisphere of the brain, and the mice were sacrificed after ICH24 hours. The brain tissue samples were taken, and the inflammatory response and the phenotype conversion of cerebral vascular smooth muscle cells were determined by western blot and immunofluorescence staining. To investigate whether the inhibition of PDGFR-CoV can improve the brain injury after intracerebral hemorrhage in mice. Results: Compared with the control group, the neurobehavioral score decreased significantly in 24 hours and 48 hours after ICH (P0.05). Compared with the control group, the brain edema at the same side of the mice increased significantly (P0.05) after 24 hours and 48 hours after ICH, while the ICH + PDGFR-Dbsi RNA group was significantly lower than that of the ICH group (P0.05), and the ICH + scramble si RNA group had no obvious change. After ICH for 24 hours, non-smooth muscle Myosin 2B (SMemb), intercellular adhesion molecule-1 (ICAM-1), myelopoxidase (MPO) expression in the same side were significantly increased (P0.05), while the ICH + PDGFR-Msi RNA group was significantly lower than that of ICH group (P0.05). There was no significant change in the ICH + scramble si RNA group. In addition, the expression of SMemb, ICAM-1 in the brain tissue of the ICH + Gleevec group was significantly lower with respect to the ICH group (P0.05). Immunofluorescence staining showed that SMemb and ICAM-1 were co-expressed with vascular smooth muscle cell marker protein (OPG-SMA) around 24 hours after cerebral hemorrhage in mice, and MPO positive cells in ICH group and ICH + scramble si RNA group were higher than those in control group (P0.05). MPO positive cells in the ICH + PDGFR-Msi RNA group were reduced compared to ICH groups and ICH + scramble si RNA groups. Conclusion: It can reduce the decrease of neurobehavioral score and increase of brain edema in mice induced by ICH, and can reduce the inflammatory response after ICH and the phenotype conversion of vascular smooth muscle cells. It is suggested that inhibition of PDGFR-VEP can improve the brain injury after intracerebral hemorrhage in mice. The third part of platelet-derived growth factor receptor-MAA regulates the phenotype transformation of cerebral vascular smooth muscle cells and the molecular mechanism of inflammatory response after cerebral hemorrhage: the model of cerebral hemorrhage induced by autologous arterial blood of mice is used to inhibit the expression of PDGFR-ALK through si RNA technology and PDGFR-proton pump inhibitor (Gleevec). The expression of signal pathway-related molecules was detected by the intervention of recombinant PDGF-BB and p38 pro-activated protein kinase-activated protein kinase 2 (MAPKAPK2, MK2) inhibitor (KKKALNRQLGVAA). To investigate whether PDGFR-IIIa induced vascular smooth muscle cell phenotype conversion and exacerbation of inflammatory response through the p38-MK2 signaling pathway. Methods: The control group (n = 7), ICH group (n = 7), ICH + scramble si RNA group (n = 7), ICH + PDGFR-Alsi RNA group (n = 7) were used for molecular detection by the second partial experiment. Another 12 male CD1 mice were randomly divided into PDGF-BB group (n = 6), PDGF-BB + MK2 inhibitor group (n = 6). The recombinant PDGF-BB (200 ng/ 2ul PBS) and the MK2 inhibitor KKKALNRQLGVAA (2ug/ kg) were injected into the basal ganglia region on the right side of the mouse using a stereotactic instrument, and the expression of the signal pathway-related molecules was detected by western blot and immunofluorescence staining. To investigate whether PDGFR-IIIa induced vascular smooth muscle cell phenotype conversion and exacerbation of inflammatory response through the p38-MK2 signaling pathway. Results: Western blot showed that the expression of p-PDGFR-jun, p-P38 and p-MK2 in the hemisphere of the hematoma was significantly higher than that in the control group (P <0.05). The expression of p-PDGFR-, p-P38 and p-MK2 in the ICH + scramble si RNA group was significantly higher than that in the control group (P0.05). Compared with the control group, the expression of ICAM-1 was significantly increased (P <0.05). The expression of ICAM-1 in the PDGF-BB + MK2 inhibitor group was significantly lower than that in the PDGF-BB group (P <0.05). Immunofluorescence staining showed that p-MK2 and p-MK2 were co-expressed with vascular smooth muscle cell marker protein (OPG-SMA) around the 24-hour hematoma after intracerebral hemorrhage in mice. CONCLUSION: After ICH, PDGFR-B9 is combined with PDGF-BB to activate the signaling pathway of p38-MK2 to induce the phenotype transformation of vascular smooth muscle cells and increase the inflammatory response.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.34
，
本文编号：2294280
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