本文選題：脊髓損傷 + 少突膠質(zhì)細(xì)胞��； 參考：《第四軍醫(yī)大學(xué)》2011年博士論文

【摘要】：肌苷是一種在體內(nèi)廣泛存在的小分子物質(zhì),它對多個系統(tǒng)、多種細(xì)胞都存在較好的保護(hù)作用。近年來的有關(guān)肌苷對中樞神經(jīng)系統(tǒng)的保護(hù)作用的研究提示,肌苷不僅能夠調(diào)節(jié)免疫細(xì)胞的作用、促進(jìn)神經(jīng)元軸突廣泛的生長,還對損傷后的神經(jīng)細(xì)胞的有較好的保護(hù)作用,在體外化學(xué)性缺氧環(huán)境中肌苷可以保護(hù)ROC-1細(xì)胞、星形膠質(zhì)細(xì)胞和神經(jīng)元的活性,在體內(nèi)肌苷治療腦缺血損傷大鼠能夠減少神經(jīng)細(xì)胞的凋亡和死亡、抑制血小板的聚積、磷酸肌醇的降解和鈣離子的形成、減輕腦缺血損傷程度,減小腦梗死的體積、抑制谷氨酸的突觸后效應(yīng)、促進(jìn)大鼠神經(jīng)功能恢復(fù)。盡管如此,但是ROC-1是一種大鼠少突膠質(zhì)細(xì)胞和C6膠質(zhì)細(xì)胞融合而成的雜交瘤細(xì)胞,同時具有這兩種細(xì)胞的屬性,無法肯定肌苷對少突膠質(zhì)細(xì)胞缺氧損傷后有保護(hù)作用;另外ROC-1是一種不死的瘤細(xì)胞,且沒有區(qū)分不同的分化階段,無法真正體現(xiàn)少突膠質(zhì)細(xì)胞對病理損傷的敏感性;最后,雖然肌苷治療腦缺血模型大鼠能夠減少神經(jīng)細(xì)胞的凋亡、促進(jìn)神經(jīng)功能的恢復(fù),但肌苷對體內(nèi)缺血缺氧環(huán)境下的少突膠質(zhì)細(xì)胞的保護(hù)作用仍然不清楚。因此,調(diào)查研究肌苷對缺氧環(huán)境下少突膠質(zhì)細(xì)胞的保護(hù)作用有著極其重要的意義。 本研究分為三部分,第一部分調(diào)查研究在化學(xué)性缺氧環(huán)境中肌苷是否對分化后的少突膠質(zhì)細(xì)胞有保護(hù)作用;第二部分調(diào)查研究肌苷在化學(xué)性缺氧環(huán)境中是否對具有增殖能力的少突膠質(zhì)前體細(xì)胞有保護(hù)作用;第三部分調(diào)查研究肌苷治療脊髓挫傷模型7天后對大鼠運(yùn)動功能恢復(fù)和臨近壞死區(qū)的少突膠質(zhì)細(xì)胞存活的影響。 第一部分實(shí)驗(yàn)通過免疫熒光染色等形態(tài)學(xué)觀察,同時結(jié)TUNEL染色、MTT實(shí)驗(yàn)和ATP檢測實(shí)驗(yàn)來評估肌苷對不同分化階段少突膠質(zhì)細(xì)胞在化學(xué)性缺氧損傷環(huán)境下細(xì)胞存活和活性的影響。 結(jié)果表明:采用相同濃度的魚藤酮損傷不同分化階段的少突膠質(zhì)細(xì)胞24 h所造成的損傷不相同。因而,要達(dá)到大致相似的損傷程度所使用的魚藤酮濃度也就不同,未成熟少突膠質(zhì)細(xì)胞的損傷濃度為5μM魚藤酮,成熟少突膠質(zhì)細(xì)胞的損傷濃度為20μM魚藤酮。在未成熟少突膠質(zhì)細(xì)胞和成熟少突膠質(zhì)細(xì)胞大致相似的損傷程度下采用肌苷預(yù)處理受損傷的少突膠質(zhì)細(xì)胞,結(jié)果肌苷能明顯減少未成熟少突膠質(zhì)細(xì)胞的死亡和凋亡,并且肌苷呈劑量依賴性提高受損的未成熟少突膠質(zhì)細(xì)胞的活性,但是肌苷只能減少成熟少突膠質(zhì)細(xì)胞的死亡,不能減少成熟少突膠質(zhì)細(xì)胞的凋亡,并且10 mM肌苷能提高受損成熟少突膠質(zhì)細(xì)胞的活性。肌苷促進(jìn)分化后少突膠質(zhì)細(xì)胞的存活、提高分化后少突膠質(zhì)細(xì)胞的活性和肌苷減少魚藤酮介導(dǎo)細(xì)胞內(nèi)產(chǎn)生的過亞硝酸鹽、提供ATP有一定的相關(guān)性。對未成熟和成熟少突膠質(zhì)細(xì)胞的保護(hù)效果的差異可能與這兩個階段少突膠質(zhì)細(xì)胞上的腺苷受體的表達(dá)差異有關(guān)。 第二部分實(shí)驗(yàn)通過免疫熒光染色等形態(tài)學(xué)觀察,同時結(jié)合MTT實(shí)驗(yàn)來評估肌苷對少突膠質(zhì)前體細(xì)胞在化學(xué)性缺氧損傷環(huán)境下細(xì)胞存活和活性的影響。 結(jié)果表明:20μM魚藤酮能夠明顯地誘導(dǎo)OPCs的死亡、細(xì)胞活性和細(xì)胞總數(shù)下降,阻止NG2抗原的表達(dá),但并沒有誘導(dǎo)OPCs的凋亡,10 mM肌苷預(yù)處理魚藤酮損傷的OPCs并不能夠減少魚藤酮所誘導(dǎo)的OPCs的死亡和活性下降,卻能提高受損細(xì)胞上NG2抗原的表達(dá)。除此之外,值得注意的是10 mM肌苷能夠顯著降低正常培養(yǎng)的OPCs(也就是空白對照組的OPCs)的活性。 第三部分實(shí)驗(yàn)通過行為學(xué)和免疫組織化學(xué)來觀察大鼠腹腔注射肌苷對脊髓挫傷模型大鼠運(yùn)動功能恢復(fù)和臨近壞死區(qū)的少突膠質(zhì)細(xì)胞存活數(shù)目的影響。 結(jié)果表明:給脊髓挫傷大鼠模型腹腔注射肌苷(75mg/kg,每8 h一次)治療對大鼠的運(yùn)動功能和臨近壞死區(qū)的少突膠質(zhì)細(xì)胞存活數(shù)目并沒有改善作用。 在體外實(shí)驗(yàn)中我們確實(shí)觀察到肌苷能明顯促進(jìn)化學(xué)性缺氧損傷的未成熟和成熟少突膠質(zhì)細(xì)胞的存活和活性的提高。對分化后的少突膠質(zhì)細(xì)胞的保護(hù)機(jī)制和肌苷所提供的ATP、肌苷清除過亞硝酸鹽具有一定的相關(guān)性。對化學(xué)性缺氧損傷的少突膠質(zhì)前體細(xì)胞的存活和活性的提高沒有改善作用。同時在用肌苷治療脊髓挫傷大鼠模型時也沒有觀察到損傷區(qū)周邊的少突膠質(zhì)細(xì)胞的數(shù)目增加和大鼠運(yùn)動功能恢復(fù),可能的原因是:脊髓損傷后的給藥劑量、給藥途徑、給藥時間和觀察時間長度可能對肌苷治療脊髓損傷大鼠的療效的存在著不同的影響。
[Abstract]:Inosine is a small molecular substance widely existed in the body. It has a good protective effect on multiple systems and many cells. Recent studies on the protective effect of inosine on the central nervous system suggest that inosine not only regulates the role of immune cells, promotes the extensive growth of neuron axons, but also affects the damaged God. In vitro, inosine can protect ROC-1 cells, astrocytes and neurons in the chemical anoxic environment. In vivo, inosine can reduce the apoptosis and death of neurons, inhibit the accumulation of platelets, degradation of inositol phosphate and the formation of calcium ions, and reduce the formation of calcium ions. The extent of cerebral ischemia, reducing the volume of cerebral infarction, inhibiting the postsynaptic effect of glutamic acid, and promoting the recovery of neural function in rats. However, ROC-1 is a hybridoma cell fused by the oligodendrocytes and C6 glial cells in rats, and has the properties of these two cells, which can not confirm the anoxia of the oligodendrocytes. In addition, ROC-1 is an undead tumor cell, and it does not differentiate between different stages of differentiation and can not truly reflect the sensitivity of oligodendrocytes to pathological damage. Finally, although inosine treatment of cerebral ischemia model rats can reduce the apoptosis of nerve cells and promote the recovery of nerve function, inosine is ischemic in the body. The protective effect of oligodendrocytes in anoxic environment is still not clear. Therefore, it is of great significance to investigate the protective effect of inosine on oligodendrocytes in anoxic environment.
This study is divided into three parts. The first part is to investigate whether inosine has protective effect on the differentiated oligodendrocytes in chemical anoxic environment. The second part is to investigate whether inosine has protective effect on oligodendrocytes with proliferative ability in chemical anoxic environment and the third part of the investigation and study of inosine treatment. The effects of 7 days after spinal cord contusion on the recovery of motor function and the survival of oligodendrocytes in the necrotic area were observed.
In the first part, the effects of inosine on the survival and activity of oligodendrocytes in different stages of hypoxia injury were evaluated by the morphological observation of immunofluorescence staining, TUNEL staining, MTT test and ATP test.
The results showed that the damage caused by 24 h of oligodendrocytes with the same concentration of rotenone at different stages of differentiation was different. Therefore, the concentration of rotenone used to achieve roughly similar damage was also different. The damage concentration of immature oligodendrocytes was 5 UG, and the damage concentration of mature oligodendrocytes was strong. The degree is 20 u M rotenone. Inosine can pretreat damaged oligodendrocytes under the roughly similar damage degree of immature oligodendrocytes and mature oligodendrocytes. Inosine can significantly reduce the death and apoptosis of immature oligodendrocytes, and the inosine dose dependent increase of immature oligodendrocytes Cell activity, but inosine can only reduce the death of mature oligodendrocytes, and can not reduce the apoptosis of mature oligodendrocytes, and 10 mM inosine can improve the activity of damaged mature oligodendrocytes. Inosine promotes the survival of oligodendrocytes after differentiation, improves the activity of oligodendrocytes after differentiation and inosine reduction of the inosine. The difference in the protective effect of immature and mature oligodendrocytes may be related to the difference in the expression of adenosine receptors on oligodendrocytes in these two stages, which is related to the protective effect of the immature and mature oligodendrocytes.
In the second part, the effects of inosine on the survival and activity of oligodendrocyte precursor cells in the environment of chemical anoxic injury were evaluated by immunofluorescence staining and MTT experiments.
The results showed that 20 M rotenone could obviously induce the death of OPCs, the cell activity and the total number of cells decreased, which prevented the expression of NG2 antigen, but did not induce the apoptosis of OPCs. 10 mM inosine pretreated by the pretreatment of rotenone did not reduce the death and activity decline of the rotenone induced OPCs, but could improve the NG2 antigen on the damaged cells. In addition, it is worth noting that 10 mM inosine can significantly reduce the activity of OPCs in normal culture (that is, OPCs in blank control group).
In the third part, the effects of inosine intraperitoneal injection of inosine on the recovery of motor function and the number of oligodendrocytes near the bad dead zone were observed by intraperitoneal injection of inosine in rats.
The results showed that the intraperitoneal injection of inosine (75mg/kg, every 8 h) to the rat model of spinal cord contusion did not improve the motor function of rats and the number of oligodendrocyte survival near the necrotic area.
In vitro experiments we did observe that inosine could significantly promote the survival and activity of immature and mature oligodendrocytes with chemical anoxic damage. The protective mechanism of oligodendrocytes after differentiation and the ATP provided by inosine and the removal of Nitrites by inosine are related to chemical anoxia damage. There is no improvement in the survival and activity of oligodendrocyte precursor cells. At the same time, the number of oligodendrocytes around the injured area and the recovery of motor function are not observed when using inosine to treat the rat model of spinal contusion. The possible reason is the dosage, the way of administration, the time of administration and the time after the injury of the spinal cord. The length of observation time may have different effects on inosine in the treatment of spinal cord injury in rats.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R363

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相關(guān)期刊論文 前2條

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本文編號：1826193