血小板源性生長因子受體-β在小鼠腦出血后腦損傷中的作用機制研究
發(fā)布時間：2018-10-25 17:06
【摘要】：第一部分小鼠腦出血后腦組織中血小板源性生長因子受體-β及其配體血小板源性生長因子-BB的表達變化目的:應(yīng)用小鼠腦出血(intracerebral hemorrhage,ICH)模型,探討小鼠腦出血后不同時間點血小板源性生長因子受體-β(Platelet-derived growth factor receptor-β,PDGFR-β)及其配體血小板源性生長因子-BB(Platelet-derived growth factor-BB,PDGF-BB)的表達變化情況。方法:將42只8周大小、重量約30克的雄性CD1小鼠隨機分為六組:對照組和5個時間點組(ICH后3h組、ICH后6h組、ICH后12h組、ICH后24h組、ICH后72h組),每組7只CD1小鼠(n=7)。應(yīng)用自體動脈血誘導(dǎo)的腦出血模型(blood ICH,b ICH),通過western blot的方法,檢測PDGFR-β和PDGF-BB的表達變化情況,通過免疫熒光染色的方法測定小鼠腦出血后PDGFR-β的表達水平和細胞定位情況。結(jié)果:Western blot的結(jié)果提示小鼠腦出血24小時后同側(cè)大腦半球PDGF-BB的表達水平顯著高于對側(cè)大腦半球(p0.05);PDGFR-β的表達從小鼠腦出血后6小時開始升高(p0.05),并且直到腦出血后24小時都維持在升高的水平(p0.05)。免疫熒光染色結(jié)果提示在小鼠腦出血后24小時的血腫周圍,可以檢測到PDGFR-β與血管平滑肌細胞(vascular smooth muscle cell,VSMC)標記蛋白(α-平滑肌肌動蛋白,α-smooth muscle actin,α-SMA)的共表達。結(jié)論:小鼠腦出血后PDGFR-β和PDGF-BB的表達增多,并且在血腫周圍的血管平滑肌細胞中檢測到PDGFR-β的表達。提示PDGFR-β可能參與小鼠腦出血后的腦損傷(brain injury)的過程以及血管平滑肌細胞(VSMC)表型轉(zhuǎn)換(phenotypic transformation)過程。第二部分抑制血小板源性生長因子受體-β改善小鼠腦出血后的腦損傷目的:應(yīng)用小鼠自體動脈血誘導(dǎo)的腦出血模型,通過小干擾RNA(small interfering RNA,si RNA)技術(shù)和PDGFR-β抑制劑(Gleevec)抑制PDGFR-β表達,探討抑制PDGFR-β是否能夠改善小鼠腦出血后的腦損傷情況。方法:將108只8周大小、重量約30克的雄性CD1小鼠隨機分為對照組(n=24),ICH組(n=26),ICH+scramble si RNA組(n=26),ICH+PDGFR-βsi RNA組(n=26),ICH+Gleevec組(n=6)。應(yīng)用小鼠自體動脈血誘導(dǎo)的腦出血模型,在ICH前24小時腦室注射scramble si RNA和PDGFR-βsi RNA,在ICH后1小時腹腔注射PDGFR-β抑制劑(Gleevec)。ICH24和48小時后測定小鼠神經(jīng)行為學(xué)評分情況;ICH24和48小時后處死小鼠,取同側(cè)及對側(cè)大腦半球做標本,測定腦水腫變化情況;ICH24小時后處死小鼠,取腦組織做標本,通過western blot方法和免疫熒光染色的方法測定炎癥反應(yīng)情況和腦血管平滑肌細胞表型轉(zhuǎn)換情況。探討抑制PDGFR-β是否能夠改善小鼠腦出血后的腦損傷情況。結(jié)果:與對照組相比,小鼠ICH后24小時和48小時神經(jīng)行為學(xué)評分明顯降低(p0.05),而ICH+PDGFR-βsi RNA組相對ICH組行為學(xué)評分明顯提高(p0.05),ICH+scramble si RNA組則無明顯變化。與對照組相比,ICH后24小時和48小時后小鼠同側(cè)基底節(jié)腦水腫明顯增加(p0.05),而ICH+PDGFR-βsi RNA組相對ICH組腦水腫顯著降低(p0.05),ICH+scramble si RNA組則無明顯變化。小鼠ICH后24小時,同側(cè)大腦半球非平滑肌肌球蛋白2B(smooth non-muscle myosin IIB,SMemb)、細胞間粘附分子-1(Intercellular adhesion molecule-1,ICAM-1)、髓過氧化物酶(myeloperoxidase,MPO)表達明顯增加(p0.05),而ICH+PDGFR-βsi RNA組相對于ICH組明顯降低(p0.05),ICH+scramble si RNA組則無明顯變化。另外,ICH+Gleevec組小鼠腦組織的SMemb、ICAM-1表達相對于ICH組也顯著降低(p0.05)。免疫熒光染色結(jié)果表明,在小鼠腦出血后24小時的血腫周圍,可以檢測到SMemb和ICAM-1與血管平滑肌細胞標記蛋白(α-SMA)的共表達,并且,ICH組和ICH+scramble si RNA組MPO陽性細胞較對照組增多(p0.05),ICH+PDGFR-βsi RNA組MPO陽性細胞則較ICH組和ICH+scramble si RNA組減少。結(jié)論:抑制PDGFR-β能夠降低ICH引起的小鼠神經(jīng)行為學(xué)評分降低和腦水腫增加,并且能夠減輕ICH后的炎癥反應(yīng)和血管平滑肌細胞表型轉(zhuǎn)換過程。提示抑制PDGFR-β能夠改善小鼠腦出血后的腦損傷情況。第三部分血小板源性生長因子受體-β調(diào)控腦出血后腦血管平滑肌細胞表型轉(zhuǎn)換及炎癥反應(yīng)的分子機制目的:應(yīng)用小鼠自體動脈血誘導(dǎo)的腦出血模型,通過si RNA技術(shù)和PDGFR-β抑制劑(Gleevec)抑制PDGFR-β表達,通過重組PDGF-BB和絲裂原活化蛋白激酶-活化蛋白激酶2(mitogen-activated protein kinase-activated protein kinase 2,MAPKAPK2,MK2)抑制劑(KKKALNRQLGVAA)等干預(yù),檢測信號通路相關(guān)分子的表達情況,探討PDGFR-β是否通過p38-MK2信號通路引起血管平滑肌細胞表型轉(zhuǎn)換和加重炎癥反應(yīng)。方法:對照組(n=7),ICH組(n=7),ICH+scramble si RNA組(n=7),ICH+PDGFR-βsi RNA組(n=7)小鼠利用第二部分實驗所產(chǎn)生的標本用于分子檢測。另將12只8周大小、重量約30克的雄性CD1小鼠隨機分為PDGF-BB組(n=6),PDGF-BB+MK2抑制劑組(n=6)。應(yīng)用小鼠使用立體定向儀向小鼠右側(cè)基底節(jié)區(qū)注射重組PDGF-BB(200 ng/2ul PBS)和MK2抑制劑KKKALNRQLGVAA(2ug/kg),通過western blot方法和免疫熒光染色的方法檢測信號通路相關(guān)分子的表達情況,探討PDGFR-β是否通過p38-MK2信號通路引起血管平滑肌細胞表型轉(zhuǎn)換和加重炎癥反應(yīng)。結(jié)果:Western blot檢測結(jié)果表明,小鼠腦出血后24小時與對照組相比,血腫同側(cè)大腦半球的p-PDGFR-β、p-P38和p-MK2的表達明顯增高(P0.05),ICH+scramble si RNA組的p-PDGFR-β、p-P38和p-MK2的表達相比較于對照組也有明顯增高(P0.05),ICH+PDGFR-βsi RNA組則相對于ICH組和ICH+scramble si RNA組明顯降低(P0.05)。PDGF-BB組小鼠與對照組相比,ICAM-1表達明顯增高(P0.05),PDGF-BB+MK2抑制劑組小鼠與PDGF-BB組相比,ICAM-1表達明顯降低(P0.05)。免疫熒光染色結(jié)果表明,在小鼠腦出血后24小時的血腫周圍,可以檢測到p-p38和p-MK2與血管平滑肌細胞標記蛋白(α-SMA)的共表達。結(jié)論:小鼠ICH后,PDGFR-β通過與PDGF-BB結(jié)合,激活p38-MK2信號通路引起血管平滑肌細胞表型轉(zhuǎn)換和加重炎癥反應(yīng)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R743.34
，
本文編號：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R743.34
，
本文編號：2294280
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