细胞共培养条件下肌源性干细胞对氧化应激损伤神经元的保护作用
发布时间：2019-05-27 19:03
【摘要】：目的 1、寻找一种新的可有效提高神经元及肌源性干细胞数量和质量的原代细胞培养方法,为后期实验提供良好的基础。 2、将肌源性干细胞与神经元共培养,观察肌源性干细胞是否可分化为神经元样细胞。 3、将肌源性干细胞与氧化应激神经元共培养,观察共培养条件下肌源性干细胞是否具有保护氧化应激损伤的神经元的作用。 方法 1、采用混合酶消化法分离培养大鼠神经元及肌源性干细胞,分别用阿糖胞苷及差速贴壁法纯化后,倒置显微镜观察细胞形态,对细胞进行计数,绘制生长曲线,分析肌源性干细胞的增殖能力。并通过结蛋白(Desmin)及神经元特异性烯醇化酶(NSE)免疫组织化学的方法分别来鉴定肌源性干细胞和神经元。 2、将肌源性干细胞与氧化应激(损伤组)或未经氧化应激处理过的神经元(未损伤组)共培养,同时设立对照组(干细胞组),将肌源性干细胞单独培养,其间收集三组细胞培养液。培养6天后分别对其进行NSE鉴定,观察不同组别间肌源性干细胞的分化差异,并对三组培养液进行ELISA检测,探求其潜在原因。 3、将氧化应激神经元与肌源性干细胞共培养,空白对照组为氧化损伤的神经元单独培养。通过Hoster33258及流式细胞仪检测不同时间点各组神经元存活差异,并用RT-PCR对相关基因进行分析,探究这种差异的内在原因。 结果 1、经过混合酶消化后,成功培养出高纯度、高活性的鼠大脑皮层神经元(NSE-+95%)和肌源性干细胞Desmin+(阳性率92%)。 2、共培养6天后检测到损伤组与未损伤组均有部分肌源性干细胞分化为表达NSE的神经元样细胞,前者分化率高于后者,干细胞组未见有细胞分化为神经元样细胞。第2天损伤组BDNF的分泌量高于未损伤组及空白对照组(P0.05),同时,损伤组第2天BDNF的分泌量也高于第4天和第6天BDNF的分泌量(P0.01),也高于其它各组各时间点的分泌量(P0.01)。损伤组与未损伤组在第四天和第六天的分泌量均高于空白对照组。在三个时间点损伤组与未损伤组及空白对照组培养液中BDNF的分泌量均高于MDSCs组(P0.001)。 3、通过Hoster33258及流式细胞仪发现在氧化应激损伤的情况下,损伤组(与肌源性干细胞共培养)的神经元的存活率要远远高于空白对照组(未与肌源性干细胞共培养),RT-PCR发现这与前者受损神经元BCL-2的表达水平升高有关。 结论 1、通过NSE和Desmin细胞免疫化学法证实体外原代培养获得高纯度皮层神经元和肌源性干细胞,混合酶消化法是一种简单有效的体外培养神经元和肌源性干细胞的方法。 2、在共培养条件下,大鼠皮层神经元可诱导肌源性干细胞为神经元样细胞,这与神经元分泌的BDNF有着重要联系。肌源性干细胞也可分泌BDNF。 3、在第二天、第四天、第六天三个时间点,损伤组神经元的存活率均高于空白对照组,肌源性干细胞可对损伤神经元产生保护效应,Bcl-2基因的上调以及Bax的下调是其原因之一。
[Abstract]:Purpose 1. Find a new primary cell culture method which can effectively improve the quantity and quality of the neuron and the myogenic stem cells, and provide good for later experiments. 2. co-culture the myogenic stem cells with the neurons to observe whether the myogenic stem cells can be differentiated into the gods through the co-culture of the myogenic stem cells and the oxidative stress neurons, the cell-like cells are co-cultured with the oxidative stress neurons to observe whether the myogenic stem cells have the protection of oxidative stress loss under the co-culture conditions, bruised Methods 1. The rat's neurons and myogenic stem cells were isolated and cultured by using a mixed-enzyme digestion method. The cell morphology was observed by an inverted microscope, and the cells were counted and the growth curve was drawn. The proliferative ability of the myogenic stem cells was analyzed, and the method of immunohistochemistry of the neuron-specific enolase (NSE) was carried out by the binding protein (Desmin) and the neuron-specific enolase (NSE). The myogenic stem cells and the neurons were identified.2. The myogenic stem cells were co-cultured with the oxidative stress (injury group) or the untreated neurons without oxidative stress (non-damaged group), and the control group (stem cell group) was established to dry the myogenic stem cells. The cells were cultured separately, and three groups of cell culture medium were collected. After 6 days of culture, the NSE was identified and the differentiation of the myogenic stem cells in different groups was observed, and the three groups of culture medium were observed. An enzyme-linked immunosorbent assay (ELISA) was used to investigate the potential causes.3. The co-culture of oxidative stress and myogenic stem cells The survival difference of the neurons in different time points was detected by the Hoster33258 and the flow cytometry, and the neurons were detected by RT-PCR. related Results 1. After the digestion of the mixed enzyme, the high-purity and high-activity rat cerebral cortex neurons (NSE-+) were successfully cultured. (95%) and Desmin + (positive rate of 92%) of myogenic stem cells. After 6 days of co-culture, some of the myogenic stem cells were differentiated into neuron-like cells expressing NSE. In the second day, the amount of BDNF in the injured group was higher than that of the non-injured group and the blank control group (P0.05), and the secretion of BDNF on the second day of the injury group was higher than that of the BDNF in the 4th and 6th days (P0.05). (P0.01), and also higher than that of other groups (P0.01). The secretion of the injury group and the non-damaged group in the fourth day and the sixth day was higher than that of the blank control group. The injury group and the non-damaged group and the blank control group were damaged in three time points The amount of BDNF in the culture medium was higher than that of the MDSCs (P 0.001).3. In the case of oxidative stress injury, the injured group (co-cultured with the myogenic stem cells) was found by the Hoster33258 and flow cytometry. The survival rate of neurons in the control group was much higher than that of the blank control group (not co-cultured with the myogenic stem cells). ,R T-PCR was found to be related to the level of expression of BCL-2 in damaged neurons of the former. A simple and effective method for the in-vitro culture of neurons and myogenic stem cells, which is a simple and effective method for the in vitro culture of neurons and myogenic stem cells. rat cortical neuron can induce myogenic stem cell For neuron-like cells, this is related to the BDNF secreted by the neurons. The myogenic stem cells can also secrete the BDNF.3. The survival rate of the injured group neurons is higher than that of the blank control group on the next day, the fourth day, the sixth day and the third time point.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2486406
[Abstract]:Purpose 1. Find a new primary cell culture method which can effectively improve the quantity and quality of the neuron and the myogenic stem cells, and provide good for later experiments. 2. co-culture the myogenic stem cells with the neurons to observe whether the myogenic stem cells can be differentiated into the gods through the co-culture of the myogenic stem cells and the oxidative stress neurons, the cell-like cells are co-cultured with the oxidative stress neurons to observe whether the myogenic stem cells have the protection of oxidative stress loss under the co-culture conditions, bruised Methods 1. The rat's neurons and myogenic stem cells were isolated and cultured by using a mixed-enzyme digestion method. The cell morphology was observed by an inverted microscope, and the cells were counted and the growth curve was drawn. The proliferative ability of the myogenic stem cells was analyzed, and the method of immunohistochemistry of the neuron-specific enolase (NSE) was carried out by the binding protein (Desmin) and the neuron-specific enolase (NSE). The myogenic stem cells and the neurons were identified.2. The myogenic stem cells were co-cultured with the oxidative stress (injury group) or the untreated neurons without oxidative stress (non-damaged group), and the control group (stem cell group) was established to dry the myogenic stem cells. The cells were cultured separately, and three groups of cell culture medium were collected. After 6 days of culture, the NSE was identified and the differentiation of the myogenic stem cells in different groups was observed, and the three groups of culture medium were observed. An enzyme-linked immunosorbent assay (ELISA) was used to investigate the potential causes.3. The co-culture of oxidative stress and myogenic stem cells The survival difference of the neurons in different time points was detected by the Hoster33258 and the flow cytometry, and the neurons were detected by RT-PCR. related Results 1. After the digestion of the mixed enzyme, the high-purity and high-activity rat cerebral cortex neurons (NSE-+) were successfully cultured. (95%) and Desmin + (positive rate of 92%) of myogenic stem cells. After 6 days of co-culture, some of the myogenic stem cells were differentiated into neuron-like cells expressing NSE. In the second day, the amount of BDNF in the injured group was higher than that of the non-injured group and the blank control group (P0.05), and the secretion of BDNF on the second day of the injury group was higher than that of the BDNF in the 4th and 6th days (P0.05). (P0.01), and also higher than that of other groups (P0.01). The secretion of the injury group and the non-damaged group in the fourth day and the sixth day was higher than that of the blank control group. The injury group and the non-damaged group and the blank control group were damaged in three time points The amount of BDNF in the culture medium was higher than that of the MDSCs (P 0.001).3. In the case of oxidative stress injury, the injured group (co-cultured with the myogenic stem cells) was found by the Hoster33258 and flow cytometry. The survival rate of neurons in the control group was much higher than that of the blank control group (not co-cultured with the myogenic stem cells). ,R T-PCR was found to be related to the level of expression of BCL-2 in damaged neurons of the former. A simple and effective method for the in-vitro culture of neurons and myogenic stem cells, which is a simple and effective method for the in vitro culture of neurons and myogenic stem cells. rat cortical neuron can induce myogenic stem cell For neuron-like cells, this is related to the BDNF secreted by the neurons. The myogenic stem cells can also secrete the BDNF.3. The survival rate of the injured group neurons is higher than that of the blank control group on the next day, the fourth day, the sixth day and the third time point.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
【参考文献】
相关期刊论文 前1条
1 李进,陈江海,王发斌,康皓,洪光祥;神经干细胞对机械损伤神经元保护作用的实验研究[J];中华手外科杂志;2005年01期
，本文编号：2486406
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