【摘要】：目的 1、尋找一種新的可有效提高神經(jīng)元及肌源性干細胞數(shù)量和質(zhì)量的原代細胞培養(yǎng)方法,為后期實驗提供良好的基礎(chǔ)。 2、將肌源性干細胞與神經(jīng)元共培養(yǎng),觀察肌源性干細胞是否可分化為神經(jīng)元樣細胞。 3、將肌源性干細胞與氧化應(yīng)激神經(jīng)元共培養(yǎng),觀察共培養(yǎng)條件下肌源性干細胞是否具有保護氧化應(yīng)激損傷的神經(jīng)元的作用。 方法 1、采用混合酶消化法分離培養(yǎng)大鼠神經(jīng)元及肌源性干細胞,分別用阿糖胞苷及差速貼壁法純化后,倒置顯微鏡觀察細胞形態(tài),對細胞進行計數(shù),繪制生長曲線,分析肌源性干細胞的增殖能力。并通過結(jié)蛋白(Desmin)及神經(jīng)元特異性烯醇化酶(NSE)免疫組織化學(xué)的方法分別來鑒定肌源性干細胞和神經(jīng)元。 2、將肌源性干細胞與氧化應(yīng)激(損傷組)或未經(jīng)氧化應(yīng)激處理過的神經(jīng)元(未損傷組)共培養(yǎng),同時設(shè)立對照組(干細胞組),將肌源性干細胞單獨培養(yǎng),其間收集三組細胞培養(yǎng)液。培養(yǎng)6天后分別對其進行NSE鑒定,觀察不同組別間肌源性干細胞的分化差異,并對三組培養(yǎng)液進行ELISA檢測,探求其潛在原因。 3、將氧化應(yīng)激神經(jīng)元與肌源性干細胞共培養(yǎng),空白對照組為氧化損傷的神經(jīng)元單獨培養(yǎng)。通過Hoster33258及流式細胞儀檢測不同時間點各組神經(jīng)元存活差異,并用RT-PCR對相關(guān)基因進行分析,探究這種差異的內(nèi)在原因。 結(jié)果 1、經(jīng)過混合酶消化后,成功培養(yǎng)出高純度、高活性的鼠大腦皮層神經(jīng)元(NSE-+95%)和肌源性干細胞Desmin+(陽性率92%)。 2、共培養(yǎng)6天后檢測到損傷組與未損傷組均有部分肌源性干細胞分化為表達NSE的神經(jīng)元樣細胞,前者分化率高于后者,干細胞組未見有細胞分化為神經(jīng)元樣細胞。第2天損傷組BDNF的分泌量高于未損傷組及空白對照組(P0.05),同時,損傷組第2天BDNF的分泌量也高于第4天和第6天BDNF的分泌量(P0.01),也高于其它各組各時間點的分泌量(P0.01)。損傷組與未損傷組在第四天和第六天的分泌量均高于空白對照組。在三個時間點損傷組與未損傷組及空白對照組培養(yǎng)液中BDNF的分泌量均高于MDSCs組(P0.001)。 3、通過Hoster33258及流式細胞儀發(fā)現(xiàn)在氧化應(yīng)激損傷的情況下,損傷組(與肌源性干細胞共培養(yǎng))的神經(jīng)元的存活率要遠遠高于空白對照組(未與肌源性干細胞共培養(yǎng)),RT-PCR發(fā)現(xiàn)這與前者受損神經(jīng)元BCL-2的表達水平升高有關(guān)。 結(jié)論 1、通過NSE和Desmin細胞免疫化學(xué)法證實體外原代培養(yǎng)獲得高純度皮層神經(jīng)元和肌源性干細胞,混合酶消化法是一種簡單有效的體外培養(yǎng)神經(jīng)元和肌源性干細胞的方法。 2、在共培養(yǎng)條件下,大鼠皮層神經(jīng)元可誘導(dǎo)肌源性干細胞為神經(jīng)元樣細胞,這與神經(jīng)元分泌的BDNF有著重要聯(lián)系。肌源性干細胞也可分泌BDNF。 3、在第二天、第四天、第六天三個時間點,損傷組神經(jīng)元的存活率均高于空白對照組,肌源性干細胞可對損傷神經(jīng)元產(chǎn)生保護效應(yīng),Bcl-2基因的上調(diào)以及Bax的下調(diào)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.
【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

【參考文獻】

相關(guān)期刊論文 前1條

1 李進,陳江海,王發(fā)斌,康皓,洪光祥;神經(jīng)干細胞對機械損傷神經(jīng)元保護作用的實驗研究[J];中華手外科雜志;2005年01期

，

本文編號：2486406