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  本文選題：細胞原代培養(yǎng)　切入點：海馬神經(jīng)元　出處：《貴州醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:原代培養(yǎng)SD新生鼠海馬神經(jīng)元,倒置顯微鏡觀察海馬神經(jīng)元的形態(tài),透射電鏡觀察其超微結(jié)構(gòu);Western blotting檢測MLCK、p-MLC表達水平;免疫熒光染色,激光共聚焦顯微鏡下觀察F-actin的變化;探討糖濃度增加海馬神經(jīng)元中MLCK升高對神經(jīng)元微絲骨架的影響,推測其改變在糖尿病認知功能障礙中的機制。方法:1.原代培養(yǎng)SD新生鼠海馬神經(jīng)元及其純度鑒定:無菌條件下分離SD新生鼠海馬并進行原代培養(yǎng),倒置相差顯微鏡觀察細胞形態(tài);每2天采用CCK-8檢測海馬神經(jīng)元活性,細胞培養(yǎng)至第7天,NSE免疫組化染色鑒定其純度;2.實驗分組:對照組、高糖組、ML-7組。其中,對照組用葡萄糖濃度為25mmol/L的培養(yǎng)基培養(yǎng);高糖組細胞培養(yǎng)至第5天換用糖濃度為45mmol/L培養(yǎng)基培養(yǎng)48小時;ML-7組在高糖作用的同時加入MLCK抑制劑ML-7(20μmol/L)作用8小時。3.Western blot檢測各組細胞內(nèi)MLCK、p-MLC表達水平。4.透射電鏡觀察各組海馬神經(jīng)元超微結(jié)構(gòu)改變。5.進行免疫熒光染色,激光共聚焦顯微鏡下觀察各組微絲成分F-actin的變化。結(jié)果:1.成功培養(yǎng)海馬神經(jīng)元,倒置相差顯微鏡下觀察培養(yǎng)7d的神經(jīng)元形態(tài)成熟,體積較大,聚集存活,光暈明顯;突起發(fā)達并向外延伸,互相連接形成密集交錯的網(wǎng)絡(luò)。且培養(yǎng)7天的海馬神經(jīng)元細胞活性達到最高,之后產(chǎn)生短暫的平臺期,11d后活性逐漸下降,經(jīng)鑒定細胞純度達90%以上;2.與對照組相比,高糖組細胞內(nèi)p-MLC、MLCK均上調(diào)(P0.05),ML-7組p-MLC、MLCK均較高糖組表達下調(diào)(P0.05);3.與對照組相比,高糖組海馬神經(jīng)元突觸縮短,超微結(jié)構(gòu)顯示海馬神經(jīng)元細胞核膜皺縮,細胞核形態(tài)不規(guī)則,核內(nèi)染色質(zhì)分布不均、顆粒化、凝集成塊并邊集,細胞內(nèi)質(zhì)網(wǎng),線粒體有囊泡化擴張,伴隨有自噬現(xiàn)象的產(chǎn)生,且微絲成分減少;與高糖組相比,ML-7組在細胞及線粒體方面無明顯變化,細胞中出現(xiàn)極少自噬現(xiàn)象,內(nèi)質(zhì)網(wǎng)等細胞器改變不大,微絲更豐富。4.免疫熒光顯示高糖組細胞形態(tài)改變,F-actin發(fā)生解聚、重排,且軸突、樹突退縮;與高糖組比較ML-7組細胞形態(tài)完整,F-actin帶清晰。結(jié)論:1.糖濃度增加使海馬神經(jīng)元內(nèi)MLCK、p-MLC表達增加;2.糖濃度增加導(dǎo)致海馬神經(jīng)元形態(tài)及超微結(jié)構(gòu)發(fā)生改變;3.MLCK和p-MLC表達升高,引起F-actin解聚,神經(jīng)元細胞骨架重排;MLCK抑制劑ML-7可阻斷F-actin的解聚。
[Abstract]:Objective: to observe the morphology of hippocampal neurons in primary cultured neonatal SD rats, observe the morphology of hippocampal neurons by inverted microscope, detect the expression of MLCK-p-MLC by transmission electron microscopy (TEM), detect the expression of MLCK-p-MLC by Western blotting, and observe the changes of F-actin by immunofluorescence staining and confocal laser microscopy. To investigate the effect of increased glucose concentration on the microfilament skeleton in hippocampal neurons. Methods 1. Primary culture of neonatal SD rat hippocampal neurons and their purity identification: isolation and primary culture of SD newborn rat hippocampus under aseptic condition. The morphology of the cells was observed by inverted phase contrast microscope, the activity of hippocampal neurons was detected by CCK-8 every 2 days, and the purity of neurons was identified by immunohistochemical staining on the 7th day of culture. The experiment was divided into two groups: control group, high glucose group and ML-7 group. The control group was cultured on 25mmol/L medium with glucose concentration. Cells in high glucose group were cultured on 45mmol/L medium for 48 hours. ML-7 group was treated with MLCK inhibitor ML-7(20 渭 mol / L for 8 hours. 3. Western blot was used to detect the expression level of MLCKp-MLC. 4. Transmission electron microscope was used to observe the expression of MLCKp-MLC in cells of each group. The ultrastructural changes of hippocampal neurons in group A. 5. immunofluorescence staining, Results 1. Hippocampal neurons were successfully cultured. The neurons cultured under inverted phase contrast microscope were observed to be mature in shape, large in size, aggregating and surviving, and obvious halo. The neurites developed and extended outward, connected with each other to form a dense interlaced network. The activity of hippocampal neurons reached its highest level after 7 days of culture, and then decreased gradually after 11 days of transient plateau phase. The purity of the cells was over 90%. Compared with the control group, the expression of p-MLCnMLCK in the high glucose group was significantly up-regulated than that in the high glucose group. Compared with the control group, the synapses of hippocampal neurons in the high glucose group were shorter than those in the high glucose group, and the expression of p-MLCU MLCK in the ML-7 group was significantly lower than that in the high glucose group. The ultrastructure showed that the nuclear membrane of hippocampal neurons shrank, the shape of nucleus was irregular, the distribution of chromatin in nucleus was uneven, granulation, agglutination, endoplasmic reticulum, cysts and mitochondria, accompanied by autophagy. Compared with high glucose group, ML-7 group had no obvious changes in cell and mitochondria, few autophagy in cells, and little change in endoplasmic reticulum and other organelles. Immunofluorescence showed that F-actin was depolymerized, rearranged, axon and dendritic retreated in high glucose group. Conclusion 1. The increase of glucose concentration can increase the expression of MLCKP-MLC in hippocampal neurons. 2. The increase of glucose concentration leads to changes in morphology and ultrastructure of hippocampal neurons. 3. The expression of MLCK and p-MLC increases and F-actin deaggregates. Neuronal cytoskeleton rearrangement (MLCK) inhibitor ML-7 can block the depolymerization of F-actin.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R587.2;R749.1

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