钙敏感受体调控神经干细胞增殖、凋亡、迁移和分化
发布时间：2018-09-10 19:45
【摘要】：研究背景:钙敏感受体(CaSR)属于细胞膜表面鸟嘌呤核苷酸调节蛋白(G蛋白)偶联受体(GPCRs),广泛表达在调节钙平衡的组织器官中,维持机体的钙稳态。CaSR也在中枢神经系统大部分脑区表达,并具有调节神经元突起生长和少突胶质细胞分化等功能。本实验室前期研究证实CaSR基因敲除(CaSR~(-/-))小鼠存在高钙、高甲状旁腺素血症,并在出生后的发育过程中出现脑内神经元、星形胶质细胞和少突胶质细胞分化、成熟障碍等表型。为了排除了体内高钙、高甲状旁腺素血症的干扰,进一步探讨CaSR基因缺失所导致的小鼠脑发育障碍的确切机制,我们分离CaSR~(+/+)和CaSR~(-/-)新生小鼠室管膜下区(SVZ)的神经干细胞(NSCs)进行体外培养,研究CaSR基因缺失对NSCs增殖、凋亡、迁移和分化的影响及机制。 研究方法和内容:(1)应用神经球检测法测定CaSR~(+/+)和CaSR~(-/-)NSCs自我更新能力;(2)采用BrdU掺入法测定不同的细胞外钙浓度( [Ca~(2+)]0 )下,两种基因型的NSCs增殖能力;(3)通过Hoechst染色和TUNEL法观察NSCs在该过程中的凋亡情况;(4)检测神经干细胞球贴壁后放射状迁移的距离,从而比较两种基因型的NSCs迁移能力;(5)1%胎牛血清诱导NSCs分化6天、10天后,应用免疫细胞化学方法测定两种基因型的神经元、星形胶质细胞和少突胶质细胞形态和数量的变化;(6)Western blot检测CaSR缺失对NSCs增殖和分化过程中ERK1/2、JNK磷酸化水平的影响。 结果:(1)在基础状态1mM [Ca~(2+)]0下,CaSR~(+/+)组与CaSR~(-/-)组NSCs自我更新及增殖能力无差异。(2)在生理范围[Ca~(2+)]0 (1-3 mM)内,随着[Ca~(2+)]0升高,CaSR促进NSCs增殖,抑制凋亡,促进NSCs存活;当[Ca~(2+)]0超过生理范围时( 5 mM ),CaSR促进NSCs增殖、抑制NSCs凋亡的作用减弱。在NSCs增殖过程中,CaSR~(+/+)组ERK1/2的磷酸化水平高于CaSR~(-/-)组。(3)CaSR基因缺失明显抑制NSCs迁移。(4)CaSR基因缺失延迟NSCs向神经元、星型胶质细胞和少突胶质细胞分化,并抑制神经元和少突胶质细胞突起的生长。(5)NSCs分化2、6天时CaSR~(+/+)组ERK1/2磷酸化水平比CaSR~(-/-)组高,但第10天CaSR~(-/-)组ERK1/2磷酸化水平较CaSR~(+/+)组高。在上述时间点,两种基因型NSCs的JNK磷酸化水平无明显差异。 结论:上述结果提示了CaSR参与调控离体NSCs增殖、凋亡、迁移和分化等基本生物学特性,并通过ERK1/2信号通路调节NSCs增殖和分化过程。
[Abstract]:Background: calcium sensitive receptor (CaSR) is a guanine nucleotide-regulated protein (G protein) coupled receptor (GPCRs), on the surface of cell membrane, which is widely expressed in tissues and organs that regulate calcium balance. Calcium homeostasis. CaSR is also expressed in most brain regions of the central nervous system and has the functions of regulating neurite growth and oligodendrocyte differentiation. Our previous study confirmed that CaSR knockout (CaSR~ (-r -) mice had high calcium and hyperparathyroidism. During postnatal development, the phenotypes of neurons, astrocytes and oligodendrocytes, and dysmaturation were found in the brain. In order to eliminate the interference of hypercalcemia and hyperparathyroiemia in vivo, the exact mechanism of brain development disorder caused by CaSR gene deletion in mice was further explored. We isolated neural stem cells (NSCs) from subependymal (SVZ) of CaSR~ (/) and CaSR~ (-r -) newborn mice to investigate the effect of CaSR gene deletion on NSCs proliferation, apoptosis, migration and differentiation. Methods and contents of the study: (1) the self-renewal ability of CaSR~ (/) and CaSR~ (-r -) NSCs was measured by the method of neurosphere detection; (2) the extracellular calcium concentration ([Ca~ (2)] 0) was measured by BrdU incorporation method. The proliferative ability of NSCs of two genotypes; (3) observing the apoptosis of NSCs in the process by Hoechst staining and TUNEL method; (4) detecting the distance of radial migration of neural stem cell ball after adherent to the wall, and (3) observing the apoptosis of NSCs in the process by means of Hoechst staining and TUNEL method. The NSCs migration ability of the two genotypes was compared. (5) after 1% fetal bovine serum induced NSCs differentiation for 6 days or 10 days, the neurons of the two genotypes were detected by immunocytochemistry. Changes of astrocytes and oligodendrocytes in morphology and quantity. (6) Western blot was used to detect the effect of CaSR deletion on the level of ERK1/2,JNK phosphorylation in the proliferation and differentiation of NSCs. Results: (1) there was no difference in self-renewal and proliferative ability of NSCs between 1mM [Ca~ (2)] 0 and CaSR~ (-r -) groups at basal state [Ca~ (2)] 0. (2) in the physiological range [Ca~ (2)] 0 (1-3 mM), CaSR promoted NSCs proliferation, inhibited apoptosis and promoted NSCs survival with increasing [Ca~ (2)] 0; When [Ca~ (2)] 0 exceeded the physiological range (5 mM) CaSR promoted the proliferation of NSCs and inhibited the apoptosis of NSCs. During the proliferation of NSCs, the phosphorylation level of ERK1/2 in ERK1/2 group was higher than that in CaSR~ group. (3) CaSR gene deletion significantly inhibited NSCs migration. (4) CaSR gene deletion delayed the differentiation of NSCs into neurons, astrocytes and oligodendrocytes. (5) the ERK1/2 phosphorylation level of CaSR~ (/) group was higher than that of CaSR~ (-r-) group at 2d after NSCs differentiation, but the ERK1/2 phosphorylation level of CaSR~ (-r-) group was higher than that of CaSR~ (/) group on the 10th day. There was no significant difference in JNK phosphorylation between the two genotypes of NSCs at the above time points. Conclusion: these results suggest that CaSR plays an important role in regulating the proliferation, apoptosis, migration and differentiation of NSCs in vitro, and regulates the proliferation and differentiation of NSCs through ERK1/2 signaling pathway.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
[Abstract]:Background: calcium sensitive receptor (CaSR) is a guanine nucleotide-regulated protein (G protein) coupled receptor (GPCRs), on the surface of cell membrane, which is widely expressed in tissues and organs that regulate calcium balance. Calcium homeostasis. CaSR is also expressed in most brain regions of the central nervous system and has the functions of regulating neurite growth and oligodendrocyte differentiation. Our previous study confirmed that CaSR knockout (CaSR~ (-r -) mice had high calcium and hyperparathyroidism. During postnatal development, the phenotypes of neurons, astrocytes and oligodendrocytes, and dysmaturation were found in the brain. In order to eliminate the interference of hypercalcemia and hyperparathyroiemia in vivo, the exact mechanism of brain development disorder caused by CaSR gene deletion in mice was further explored. We isolated neural stem cells (NSCs) from subependymal (SVZ) of CaSR~ (/) and CaSR~ (-r -) newborn mice to investigate the effect of CaSR gene deletion on NSCs proliferation, apoptosis, migration and differentiation. Methods and contents of the study: (1) the self-renewal ability of CaSR~ (/) and CaSR~ (-r -) NSCs was measured by the method of neurosphere detection; (2) the extracellular calcium concentration ([Ca~ (2)] 0) was measured by BrdU incorporation method. The proliferative ability of NSCs of two genotypes; (3) observing the apoptosis of NSCs in the process by Hoechst staining and TUNEL method; (4) detecting the distance of radial migration of neural stem cell ball after adherent to the wall, and (3) observing the apoptosis of NSCs in the process by means of Hoechst staining and TUNEL method. The NSCs migration ability of the two genotypes was compared. (5) after 1% fetal bovine serum induced NSCs differentiation for 6 days or 10 days, the neurons of the two genotypes were detected by immunocytochemistry. Changes of astrocytes and oligodendrocytes in morphology and quantity. (6) Western blot was used to detect the effect of CaSR deletion on the level of ERK1/2,JNK phosphorylation in the proliferation and differentiation of NSCs. Results: (1) there was no difference in self-renewal and proliferative ability of NSCs between 1mM [Ca~ (2)] 0 and CaSR~ (-r -) groups at basal state [Ca~ (2)] 0. (2) in the physiological range [Ca~ (2)] 0 (1-3 mM), CaSR promoted NSCs proliferation, inhibited apoptosis and promoted NSCs survival with increasing [Ca~ (2)] 0; When [Ca~ (2)] 0 exceeded the physiological range (5 mM) CaSR promoted the proliferation of NSCs and inhibited the apoptosis of NSCs. During the proliferation of NSCs, the phosphorylation level of ERK1/2 in ERK1/2 group was higher than that in CaSR~ group. (3) CaSR gene deletion significantly inhibited NSCs migration. (4) CaSR gene deletion delayed the differentiation of NSCs into neurons, astrocytes and oligodendrocytes. (5) the ERK1/2 phosphorylation level of CaSR~ (/) group was higher than that of CaSR~ (-r-) group at 2d after NSCs differentiation, but the ERK1/2 phosphorylation level of CaSR~ (-r-) group was higher than that of CaSR~ (/) group on the 10th day. There was no significant difference in JNK phosphorylation between the two genotypes of NSCs at the above time points. Conclusion: these results suggest that CaSR plays an important role in regulating the proliferation, apoptosis, migration and differentiation of NSCs in vitro, and regulates the proliferation and differentiation of NSCs through ERK1/2 signaling pathway.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
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