人參皂苷Rg1對腦損傷后神經干細胞增殖、分化的作用及其表觀遺傳調控機制
發(fā)布時間：2018-08-12 18:37
【摘要】：神經干細胞(neural stem cells,NSCs)是可以自我增生和復制,并能分化為構成中樞神經系統的包括神經元、星形膠質細胞、少突膠質細胞的細胞類型。神經前體細胞(neural progenitor cells,NPCs)是神經干細胞在分化過程中初步分化形成的具備分化為單一成熟神經細胞的單能神經干細胞。在胚胎早期,腦室區(qū)NSCs是處于對稱分裂狀態(tài)中,從而達到增加數量的目的。接下來,神經干細胞再通過不對稱分裂,分裂為神經干細胞和神經元來繼續(xù)神經發(fā)生的過程。胚胎發(fā)展過程中,一部分神經干細胞是具備分裂成一個神經干細胞和一個NPCs的能力。神經發(fā)生后期,NSCs在分化成神經元的基礎上,還可以分化產生為星型膠質細胞和少突膠質細胞。上述過程是受很多因素調節(jié)才得以發(fā)生的,其中表觀遺傳學是重要的調節(jié)因素。表觀遺傳學(epigenetics)是一種指不涉及DNA序列改變的基因或者蛋白表達的變化,并可以在發(fā)育和細胞增殖過程中穩(wěn)定傳遞的遺傳學,即可以從一個細胞被傳遞到其子代的基因調節(jié)類型。表觀遺傳性狀的變化具有可繼承性,又有一定程度的可逆性。表觀遺傳學在環(huán)境因素和轉錄因子等因素的調控下可以調節(jié)神經干細胞的增殖和分化。表觀遺傳學包括很多方式,對神經干細胞的增殖分化都存在著影響。因此揭示表觀遺傳學如何調節(jié)神經干細胞的增殖分化的機制,對中樞神經退行性病變的防治有著重要的意義。1、人參皂苷Rg1對離體缺血再灌注神經干細胞增殖分化的作用采用氧糖剝奪/再灌注(OGD/R)細胞模型模擬腦缺血再灌注造成的腦組織損傷,將體外分離培養(yǎng)的神經干細胞分為OGD/R后復氧2h、4h、6h共3個時間點,每個時間點分為對照組、缺血再灌注組和人參皂苷Rg1組,對照組細胞正常培養(yǎng),缺血再灌注組和人參皂苷Rg1組缺氧培養(yǎng)4h后,再分別正常培養(yǎng)2h、4h、6h,各時間點結束后,各組神經干細胞增殖標記物(Brdu)、神經干細胞特異性標志物巢蛋白(Nestin)、神經元祖細胞特異性標記物(Tuj-1)及星形膠質細胞祖細胞標記物(Vimentin)的表達都應用免疫熒光雙標染色方法進行檢測,統計各組陽性細胞的個數、光密度、面密度。結果發(fā)現正常情況下,Nestin、Tuj-1和Vimentin均能在神經干細胞中有所表達。缺血再灌注組與對照組相比,免疫熒光觀察發(fā)現,神經干細胞的陽性細胞數、光密度、面密度明顯減少和降低,差異具有統計學意義(P0.05);人參皂苷Rg1組與缺血再灌注組相比,神經干細胞的陽性細胞數、光密度、面密度明顯增加,差異具有統計學意義(P0.05)。而且隨著復氧時間的延長,神經干細胞陽性細胞數、光密度、面密度逐漸增加。從形態(tài)學上說明人參皂苷Rg1可以在缺血缺氧的條件下通過促進神經干細胞的增殖分化,從而促進中樞神經損傷的修復。2、人參皂苷Rg1對缺血再灌注神經干細胞microRNA-21/210表達的影響實時熒光定量PCR的方法檢測microRNA-21/210的表達量與神經干細胞增殖分化的關系,與對照組相比,缺血再灌注組miR-21在復氧4h和6h的表達呈現上調表達,miR-210在三個時間點均呈現上調表達,差異具有統計學意義(P0.05),這說明在缺氧可以促進miR-21和miR-210的表達,從而促進神經干細胞的增殖。人參皂苷Rg1干預后,與缺血再灌注組相比,人參皂苷Rg1組miR-21在在復氧4h和6h的表達呈現上調表達,miR-210在三個時間點均呈現上調表達差異具有統計學意義(P0.05),這說明人參皂苷Rg1可以促進缺氧狀態(tài)下的神經干細胞中miR-21和miR-210的表達增加,促進神經干細胞在缺氧狀態(tài)下的修復。這說明人參皂苷Rg1可能通過miR-21和miR-210參與腦損傷的神經干細胞修復。3、人參皂苷Rg1對缺血再灌注神經干細胞DNMT1/DNMT3a表達的影響實時熒光定量PCR發(fā)現,與對照組相比,三個時間點的缺血再灌注組中的DNMT1和DNMT3a的表達均有所增加,且差異有統計學意義(p0.05)。但人參皂苷Rg1藥物干預后,與缺血再灌注組相比,三個時間點下人參皂苷Rg1組中的DNMT1表達是沒有顯著變化的,而DNMT3a的表達呈現顯著上調,且差異有統計學意義(p0.05)。Western blot實驗發(fā)現,神經干細胞中的DNMT3a在缺血再灌注組中,即缺血缺氧的條件下,與對照組相比,三個時間點的表達均是增加的,差異具有統計學意義(P0.05)。這說明DNMT3a可能促進神經干細胞在缺血缺氧條件下的自我修復和增殖。在人參皂苷Rg1的干預下,與缺血再灌注組相比,三個時間點的DNMT3a的表達也是增加的。說明人參皂苷Rg1可能通過DNMT3a促進神經干細胞的自我更新和增殖。綜上所述,人參皂苷Rg1可以在缺血缺氧的條件下促進神經干細胞的增殖分化,并可以促進缺氧狀態(tài)下的神經干細胞中miR-21和miR-210以及DNMT3a的表達增加。這證明人參皂苷Rg1可能通過增強miR-21/miR-210以及DNMT3a表達,從而能促進了缺血再灌注神經干細胞的存活、自我復制和分化,參與腦組織缺血、損傷的神經修復。
[Abstract]:Neural stem cells (NSCs) can self-proliferate and replicate and differentiate into cell types that comprise neurons, astrocytes and oligodendrocytes in the central nervous system. In the early embryonic stage, NSCs in the ventricular region are in a state of symmetrical division, thereby increasing the number of neural stem cells. Next, neural stem cells are divided asymmetrically into neural stem cells and neurons to continue the process of neurogenesis. During embryonic development, a part of the nerve is involved. Stem cells have the ability to divide into a neural stem cell and a NPCs. At the late stage of neurogenesis, NSCs can also differentiate into astrocytes and oligodendrocytes on the basis of differentiation into neurons. Epigenetics is a type of genetics that does not involve changes in DNA sequence or protein expression and can be transmitted steadily during development and cell proliferation. Reversibility. Epigenetics regulates the proliferation and differentiation of neural stem cells by environmental factors and transcription factors. Epigenetics involves many ways that affect the proliferation and differentiation of neural stem cells. Therefore, it is necessary to reveal how epigenetics regulates the proliferation and differentiation of neural stem cells and the central nervous system. The effect of ginsenoside Rg1 on the proliferation and differentiation of neural stem cells after ischemia-reperfusion in vitro was studied. The brain tissue injury induced by cerebral ischemia-reperfusion was simulated by oxygen-glucose deprivation/reperfusion (OGD/R) cell model. The neural stem cells isolated and cultured in vitro were divided into OGD/R 2 h, 4 h and 6 h after reoxygenation. At each time point, the cells were divided into control group, ischemia-reperfusion group and ginsenoside Rg1 group, control group were cultured normally, ischemia-reperfusion group and ginsenoside Rg1 group were cultured under hypoxia for 4 hours, then normal culture for 2 hours, 4 hours, 6 hours, after the end of each time point, neural stem cell proliferation markers (Brdu), neural stem cell specific markers nested eggs. The expression of Nestin, Tuj-1 and Vimentin were detected by immunofluorescence double labeling. The number, optical density and surface density of positive cells were counted. The results showed that Nestin, Tuj-1 and Vimentin could be found in neural stem cells under normal conditions. Compared with the control group, the number of positive cells, optical density and area density of neural stem cells in the ischemia-reperfusion group were significantly decreased (P 0.05), and the number of positive cells, optical density and area density of neural stem cells in the ginsenoside Rg1 group were significantly higher than those in the ischemia-reperfusion group. Morphologically, ginsenoside Rg1 can promote the proliferation and differentiation of neural stem cells under hypoxic and ischemic conditions, thereby promoting the repair of central nervous system injury. 2, ginsenoside R Effect of G1 on the expression of microRNA-21/210 in ischemia-reperfusion neural stem cells The difference was statistically significant (P 0.05), indicating that hypoxia can promote the expression of microRNAs-21 and microRNAs-210, thus promoting the proliferation of neural stem cells. The difference was statistically significant (P 0.05), indicating that ginsenoside Rg1 can promote the expression of microRNAs-21 and microRNAs-210 in neural stem cells under hypoxia, and promote the repair of neural stem cells under hypoxia. This suggests that ginsenoside Rg1 may participate in the repair of neural stem cells after brain injury through microRNAs-21 and microRNAs-210. The expression of DNMT1 and DNMT3a in the ischemia-reperfusion group at three time points was higher than that in the control group, and the difference was statistically significant (p0.05). The expression of DNMT1 in the Rg1 group was not significantly changed, but the expression of DNMT3a was significantly up-regulated (p0.05). Western blot showed that the expression of DNMT3a in neural stem cells was increased in the ischemia-reperfusion group, i.e. in the condition of ischemia-hypoxia, compared with the control group at three time points. The difference was statistically significant (P 0.05). This suggests that DNMT3a may promote the self-repair and proliferation of neural stem cells under hypoxic and ischemic conditions. In conclusion, ginsenoside Rg1 can promote the proliferation and differentiation of neural stem cells under hypoxic and ischemic conditions, and increase the expression of microRNAs-21, microRNAs-210 and DNMT3a in neural stem cells under hypoxic and ischemic conditions. It can promote the survival, self-replication and differentiation of neural stem cells after ischemia-reperfusion, and participate in the nerve repair of cerebral ischemia and injury.
【學位授予單位】：北京中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285
本文編號：2179942
[Abstract]:Neural stem cells (NSCs) can self-proliferate and replicate and differentiate into cell types that comprise neurons, astrocytes and oligodendrocytes in the central nervous system. In the early embryonic stage, NSCs in the ventricular region are in a state of symmetrical division, thereby increasing the number of neural stem cells. Next, neural stem cells are divided asymmetrically into neural stem cells and neurons to continue the process of neurogenesis. During embryonic development, a part of the nerve is involved. Stem cells have the ability to divide into a neural stem cell and a NPCs. At the late stage of neurogenesis, NSCs can also differentiate into astrocytes and oligodendrocytes on the basis of differentiation into neurons. Epigenetics is a type of genetics that does not involve changes in DNA sequence or protein expression and can be transmitted steadily during development and cell proliferation. Reversibility. Epigenetics regulates the proliferation and differentiation of neural stem cells by environmental factors and transcription factors. Epigenetics involves many ways that affect the proliferation and differentiation of neural stem cells. Therefore, it is necessary to reveal how epigenetics regulates the proliferation and differentiation of neural stem cells and the central nervous system. The effect of ginsenoside Rg1 on the proliferation and differentiation of neural stem cells after ischemia-reperfusion in vitro was studied. The brain tissue injury induced by cerebral ischemia-reperfusion was simulated by oxygen-glucose deprivation/reperfusion (OGD/R) cell model. The neural stem cells isolated and cultured in vitro were divided into OGD/R 2 h, 4 h and 6 h after reoxygenation. At each time point, the cells were divided into control group, ischemia-reperfusion group and ginsenoside Rg1 group, control group were cultured normally, ischemia-reperfusion group and ginsenoside Rg1 group were cultured under hypoxia for 4 hours, then normal culture for 2 hours, 4 hours, 6 hours, after the end of each time point, neural stem cell proliferation markers (Brdu), neural stem cell specific markers nested eggs. The expression of Nestin, Tuj-1 and Vimentin were detected by immunofluorescence double labeling. The number, optical density and surface density of positive cells were counted. The results showed that Nestin, Tuj-1 and Vimentin could be found in neural stem cells under normal conditions. Compared with the control group, the number of positive cells, optical density and area density of neural stem cells in the ischemia-reperfusion group were significantly decreased (P 0.05), and the number of positive cells, optical density and area density of neural stem cells in the ginsenoside Rg1 group were significantly higher than those in the ischemia-reperfusion group. Morphologically, ginsenoside Rg1 can promote the proliferation and differentiation of neural stem cells under hypoxic and ischemic conditions, thereby promoting the repair of central nervous system injury. 2, ginsenoside R Effect of G1 on the expression of microRNA-21/210 in ischemia-reperfusion neural stem cells The difference was statistically significant (P 0.05), indicating that hypoxia can promote the expression of microRNAs-21 and microRNAs-210, thus promoting the proliferation of neural stem cells. The difference was statistically significant (P 0.05), indicating that ginsenoside Rg1 can promote the expression of microRNAs-21 and microRNAs-210 in neural stem cells under hypoxia, and promote the repair of neural stem cells under hypoxia. This suggests that ginsenoside Rg1 may participate in the repair of neural stem cells after brain injury through microRNAs-21 and microRNAs-210. The expression of DNMT1 and DNMT3a in the ischemia-reperfusion group at three time points was higher than that in the control group, and the difference was statistically significant (p0.05). The expression of DNMT1 in the Rg1 group was not significantly changed, but the expression of DNMT3a was significantly up-regulated (p0.05). Western blot showed that the expression of DNMT3a in neural stem cells was increased in the ischemia-reperfusion group, i.e. in the condition of ischemia-hypoxia, compared with the control group at three time points. The difference was statistically significant (P 0.05). This suggests that DNMT3a may promote the self-repair and proliferation of neural stem cells under hypoxic and ischemic conditions. In conclusion, ginsenoside Rg1 can promote the proliferation and differentiation of neural stem cells under hypoxic and ischemic conditions, and increase the expression of microRNAs-21, microRNAs-210 and DNMT3a in neural stem cells under hypoxic and ischemic conditions. It can promote the survival, self-replication and differentiation of neural stem cells after ischemia-reperfusion, and participate in the nerve repair of cerebral ischemia and injury.
【學位授予單位】：北京中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285
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