【摘要】：研究背景:鈣敏感受體(CaSR)屬于細(xì)胞膜表面鳥嘌呤核苷酸調(diào)節(jié)蛋白(G蛋白)偶聯(lián)受體(GPCRs),廣泛表達(dá)在調(diào)節(jié)鈣平衡的組織器官中,維持機(jī)體的鈣穩(wěn)態(tài)。CaSR也在中樞神經(jīng)系統(tǒng)大部分腦區(qū)表達(dá),并具有調(diào)節(jié)神經(jīng)元突起生長和少突膠質(zhì)細(xì)胞分化等功能。本實驗室前期研究證實CaSR基因敲除(CaSR~(-/-))小鼠存在高鈣、高甲狀旁腺素血癥,并在出生后的發(fā)育過程中出現(xiàn)腦內(nèi)神經(jīng)元、星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞分化、成熟障礙等表型。為了排除了體內(nèi)高鈣、高甲狀旁腺素血癥的干擾,進(jìn)一步探討CaSR基因缺失所導(dǎo)致的小鼠腦發(fā)育障礙的確切機(jī)制,我們分離CaSR~(+/+)和CaSR~(-/-)新生小鼠室管膜下區(qū)(SVZ)的神經(jīng)干細(xì)胞(NSCs)進(jìn)行體外培養(yǎng),研究CaSR基因缺失對NSCs增殖、凋亡、遷移和分化的影響及機(jī)制。 研究方法和內(nèi)容:(1)應(yīng)用神經(jīng)球檢測法測定CaSR~(+/+)和CaSR~(-/-)NSCs自我更新能力;(2)采用BrdU摻入法測定不同的細(xì)胞外鈣濃度( [Ca~(2+)]0 )下,兩種基因型的NSCs增殖能力;(3)通過Hoechst染色和TUNEL法觀察NSCs在該過程中的凋亡情況;(4)檢測神經(jīng)干細(xì)胞球貼壁后放射狀遷移的距離,從而比較兩種基因型的NSCs遷移能力;(5)1%胎牛血清誘導(dǎo)NSCs分化6天、10天后,應(yīng)用免疫細(xì)胞化學(xué)方法測定兩種基因型的神經(jīng)元、星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞形態(tài)和數(shù)量的變化;(6)Western blot檢測CaSR缺失對NSCs增殖和分化過程中ERK1/2、JNK磷酸化水平的影響。 結(jié)果:(1)在基礎(chǔ)狀態(tài)1mM [Ca~(2+)]0下,CaSR~(+/+)組與CaSR~(-/-)組NSCs自我更新及增殖能力無差異。(2)在生理范圍[Ca~(2+)]0 (1-3 mM)內(nèi),隨著[Ca~(2+)]0升高,CaSR促進(jìn)NSCs增殖,抑制凋亡,促進(jìn)NSCs存活;當(dāng)[Ca~(2+)]0超過生理范圍時( 5 mM ),CaSR促進(jìn)NSCs增殖、抑制NSCs凋亡的作用減弱。在NSCs增殖過程中,CaSR~(+/+)組ERK1/2的磷酸化水平高于CaSR~(-/-)組。(3)CaSR基因缺失明顯抑制NSCs遷移。(4)CaSR基因缺失延遲NSCs向神經(jīng)元、星型膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞分化,并抑制神經(jīng)元和少突膠質(zhì)細(xì)胞突起的生長。(5)NSCs分化2、6天時CaSR~(+/+)組ERK1/2磷酸化水平比CaSR~(-/-)組高,但第10天CaSR~(-/-)組ERK1/2磷酸化水平較CaSR~(+/+)組高。在上述時間點,兩種基因型NSCs的JNK磷酸化水平無明顯差異。 結(jié)論:上述結(jié)果提示了CaSR參與調(diào)控離體NSCs增殖、凋亡、遷移和分化等基本生物學(xué)特性,并通過ERK1/2信號通路調(diào)節(jié)NSCs增殖和分化過程。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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