本文選題：氯胺酮 + PC12細(xì)胞��； 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：氯胺酮（ketamine）作為一種非競(jìng)爭(zhēng)性NMDA（N-methyl-D-aspartate）受體阻滯劑，可產(chǎn)生劑量相關(guān)的意識(shí)和感覺分離的麻醉狀態(tài)，廣泛用于成人和小兒麻醉。然而越來越多的研究結(jié)果顯示，全身麻醉藥氯胺酮可導(dǎo)致神經(jīng)損傷，引起神經(jīng)退行性疾病和長(zhǎng)期的神經(jīng)認(rèn)知缺陷。因此對(duì)氯胺酮神經(jīng)毒性的研究引起人們的關(guān)注。 已有研究報(bào)道氯胺酮可導(dǎo)致嚙齒類、斑馬魚、非人類靈長(zhǎng)類神經(jīng)細(xì)胞凋亡。細(xì)胞凋亡是細(xì)胞死亡的主要形式之一，有關(guān)細(xì)胞凋亡的發(fā)生機(jī)制尚未充分闡明。現(xiàn)在認(rèn)為有三條細(xì)胞信號(hào)通路參與凋亡的發(fā)生：即線粒體通路、死亡受體通路和內(nèi)質(zhì)網(wǎng)通路。有研究報(bào)道氯胺酮可通過線粒體途徑導(dǎo)致體外培養(yǎng)的人胚胎干細(xì)胞發(fā)生凋亡，但氯胺酮是否可以通過內(nèi)質(zhì)網(wǎng)應(yīng)激（Endoplasmic reticulum stress, ERS）途徑誘導(dǎo)細(xì)胞凋亡還未明確。 已有研究表明，氧自由基、應(yīng)激激素和細(xì)胞因子等都可以影響內(nèi)質(zhì)網(wǎng)功能，導(dǎo)致未/錯(cuò)誤折疊蛋白堆積，激活內(nèi)質(zhì)網(wǎng)應(yīng)激。適度的內(nèi)質(zhì)網(wǎng)應(yīng)激可提高內(nèi)質(zhì)網(wǎng)處理未折疊或錯(cuò)誤折疊蛋白的能力，維持細(xì)胞的生存；如果應(yīng)激程度過強(qiáng)或時(shí)間過長(zhǎng)，細(xì)胞內(nèi)的穩(wěn)態(tài)不能恢復(fù)，內(nèi)質(zhì)網(wǎng)應(yīng)激最終將導(dǎo)致細(xì)胞發(fā)生凋亡。在一些神經(jīng)退行性疾病如帕金森病，阿爾茨海默病和亨廷頓病等的發(fā)病機(jī)制研究中已證實(shí)內(nèi)質(zhì)網(wǎng)應(yīng)激可以引起神經(jīng)細(xì)胞的凋亡。葡萄糖調(diào)節(jié)蛋白78（78-kDa glucose-regulated protein，GRP78），也被稱為免疫球蛋白重鏈結(jié)合蛋白（Immunoglobulin heavy chain binding protein，Bip），是未折疊蛋白反應(yīng)的主調(diào)控者。當(dāng)鈣超載、氧化應(yīng)激等因素導(dǎo)致內(nèi)質(zhì)網(wǎng)腔中未折疊蛋白聚集時(shí)，GRP78/Bip釋放跨膜蛋白，引起ERS激活。因此，GRP78/Bip被視為ERS的標(biāo)志性蛋白。Caspase12是caspase家族成員，它僅產(chǎn)生于內(nèi)質(zhì)網(wǎng)。Caspase12僅在內(nèi)質(zhì)網(wǎng)應(yīng)激時(shí)被活化，是內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)凋亡途徑的關(guān)鍵蛋白酶。 基于以上研究背景，本實(shí)驗(yàn)中我們研究氯胺酮對(duì)PC12細(xì)胞的毒性作用，觀察內(nèi)質(zhì)網(wǎng)應(yīng)激在氯胺酮誘導(dǎo)PC12細(xì)胞損傷的過程中是否發(fā)揮了作用，為氯胺酮神經(jīng)毒性機(jī)制研究提供新的思路。 方法： 1MTT實(shí)驗(yàn)觀察0.5、1、1.5、2、2.5mM氯胺酮分別作用6、12、24h對(duì)PC12細(xì)胞存活率的影響；20、30、40M Salubrinal（內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑）單獨(dú)或與氯胺酮共同作用對(duì)PC12細(xì)胞存活率的影響。 2Western blot方法檢測(cè)內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志分子GRP78/Bip、內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)凋亡分子Caspase12蛋白的表達(dá):1、1.5、2mM氯胺酮作用24h對(duì)PC12細(xì)胞GRP78/Bip、Caspase12蛋白的影響；1.5mM氯胺酮作用6、12、24h對(duì)PC12細(xì)胞GRP78/Bip、Caspase12蛋白的影響；加用內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑Salubrinal共同作用于PC12細(xì)胞后對(duì)兩種蛋白的影響。 3為了明確氯胺酮是否導(dǎo)致了PC12細(xì)胞的凋亡，采用流式細(xì)胞術(shù)Annexin V-PI雙標(biāo)染色法檢測(cè)PC12細(xì)胞凋亡率，并觀察內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑Salubrinal對(duì)氯胺酮所致PC12細(xì)胞凋亡的影響。 4數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(Mean±SD)表示，用SPSS17.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析，各組均數(shù)的比較行單因素方差分析（ANOVA），以p＜0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1PC12細(xì)胞存活率的變化：與正常對(duì)照組相比，0.5mM氯胺酮作用于PC12細(xì)胞6h后細(xì)胞存活率沒有明顯變化（p0.05），作用12、24h后，PC12細(xì)胞存活率明顯降低（p0.05）；1、1.5、2、2.5mM氯胺酮作用于PC12細(xì)胞6、12、24h后，PC12細(xì)胞存活率明顯降低（p0.05），且呈濃度依賴性；20、30、40M Salubrinal單獨(dú)作用于PC12細(xì)胞24h對(duì)其存活率無明顯影響；30或40M Salubrinal可抑制1.5mM氯胺酮所致PC12細(xì)胞存活率的下降（p0.05）。 2內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志分子GRP78/Bip蛋白表達(dá)的變化：與正常對(duì)照組相比，1.5、2mM氯胺酮作用于PC12細(xì)胞24h后，，GRP78/Bip蛋白表達(dá)明顯增高（p0.05or p0.01），呈濃度依賴性；1.5mM氯胺酮作用于PC12細(xì)胞12、24h后，GRP78/Bip蛋白表達(dá)明顯增高（p0.05or p0.01），呈時(shí)間依賴性；30M Salubrinal可抑制1.5mM氯胺酮所致PC12細(xì)胞GRP78/Bip蛋白表達(dá)的增高。 內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)凋亡分子Caspase12蛋白表達(dá)的變化：與正常對(duì)照組相比，1.5、2mM氯胺酮作用于PC12細(xì)胞24h后，Caspase12蛋白表達(dá)明顯增高（p0.05），呈濃度依賴性；1.5mM氯胺酮作用于PC12細(xì)胞6、12、24h后，Caspase12蛋白表達(dá)明顯增高（p0.05），呈時(shí)間依賴性；30M Salubrinal可抑制1.5mM氯胺酮所致PC12細(xì)胞Caspase12蛋白表達(dá)的增高。 3PC12細(xì)胞凋亡率的變化：與對(duì)照組相比，1.5mM氯胺酮作用24h后PC12細(xì)胞總凋亡率明顯升高（p0.05）；30M Salubrinal可抑制1.5mM氯胺酮所致PC12細(xì)胞凋亡率的增高（p0.05）；30M Salubrinal單獨(dú)作用后細(xì)胞凋亡率較對(duì)照組相比差異無統(tǒng)計(jì)學(xué)意義（p0.05）。 結(jié)論： 氯胺酮可誘導(dǎo)PC12細(xì)胞發(fā)生凋亡，且呈濃度依賴和時(shí)間依賴性；內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)了氯胺酮誘導(dǎo)的PC12細(xì)胞凋亡。
[Abstract]:Objective : ketamine ( ketamine ) , as a non - competitive NMDA ( N - methyl - D - aspartate ) receptor blocker , can produce dose - related conscious and sensory isolated anesthesia , widely used in adult and pediatric anesthesia .

It has been reported that ketamine can induce apoptosis in rodents , zebrafish and non - human primates . Apoptosis is one of the main forms of cell death . There are three cell signaling pathways involved in apoptosis : mitochondrial pathway , death receptor pathway and endoplasmic reticulum pathway .

Studies have shown that oxygen free radicals , stress hormones , cytokines and the like can affect the function of endoplasmic reticulum , lead to accumulation of unfolded protein and activate endoplasmic reticulum stress . Appropriate endoplasmic reticulum stress can improve the ability of endoplasmic reticulum to treat unfolded or misfolded protein , and maintain cell survival ;
In some neurodegenerative diseases such as Parkinson ' s disease , Alzheimer ' s disease and Huntington ' s disease , GRP78 / Bip release the transmembrane protein .

Based on the above research background , we studied the toxicity of ketamine on PC12 cells , and observed whether the endoplasmic reticulum stress played a role in the induction of PC12 cell injury induced by ketamine , and provided a new idea for the study of ketamine neurotoxicity .

Method :

The effects of 0.5 , 1 , 1.5 , 2 , 2.5 mM ketamine on the survival rate of PC12 cells were observed by MTT assay .
Effects of 20 , 30 , 40 M ulbrinal ( endoplasmic reticulum stress inhibitors ) on the survival of PC12 cells alone or in combination with ketamine .

2Western blot was used to detect the expression of GRP78 / Bip , the endoplasmic reticulum stress - mediated apoptosis molecule Caspase12 protein : 1 , 1.5 , 2 mM ketamine on the expression of GRP78 / Bip , Caspase12 protein in PC12 cells .
The effects of 1.5mM ketamine on GRP78 / Bip , Caspase12 protein in PC12 cells were studied .
The effect of the stress inhibitor of endoplasmic reticulum stress on the two proteins was studied in PC12 cells .

3 In order to clarify whether ketamine led to apoptosis of PC12 cells , the apoptosis rate of PC12 cells was detected by flow cytometry with V - PI double standard staining , and the effects of the endoplasmic reticulum stress inhibitor on apoptosis of PC12 cells induced by ketamine were observed .

The mean 鹵 SD of 4 data was expressed by mean 鹵 SD . Statistical analysis was performed with SPSS 17.0 software . The comparison of the mean number of each group was statistically significant ( p < 0.05 ) .

Results :

The survival rate of PC12 cells was significantly lower than that in the normal control group ( p < 0.05 ) . The survival rate of PC12 cells decreased significantly after 12 h and 24 h ( p < 0.05 ) .
1 , 1.5 , 2 , 2.5 mM ketamine , the survival rate of PC12 cells decreased significantly after 6 , 12 and 24 h of PC12 cells ( p < 0.05 ) , and the PC12 cells were dose - dependent ;
The survival rate of PC12 cells was not significantly affected by 20 , 30 , and 40 M brinal alone .
30 or 40 M . brinal inhibited the decrease in the survival rate of PC12 cells induced by 1.5 mM ketamine ( p . 05 ) .

2 . The expression of GRP78 / Bip protein in the endoplasmic reticulum stress marker molecule was significantly higher than that of the normal control group ( p0.01 or p0.01 ) , and the expression of GRP78 / Bip protein was significantly higher than that of the normal control group ( p0.05 or p0.01 ) .
After 12 h and 24 h , the expression of GRP78 / Bip protein was significantly increased ( p0.05 or p0.01 ) .
The expression of GRP78 / Bip protein in PC12 cells induced by 1 . 5mM ketamine was inhibited by 30 mg of ketamine .

Compared with the control group , the expression of Caspase - 12 protein in PC12 cells increased significantly ( p < 0.05 ) , and the expression of Caspase - 12 protein was significantly increased ( p < 0.05 ) .
After 6 , 12 and 24 h of PC12 cells , the expression of caspase12 increased significantly ( p < 0.05 ) .
The expression of Caspase12 protein in PC12 cells induced by 1.5 mM ketamine was inhibited by 30 mg of ketamine .

Compared with the control group , the apoptosis rate of PC12 cells increased significantly after 24 h ( p < 0.05 ) .
The apoptosis rate of PC12 cells induced by 1 . 5mM ketamine was inhibited by 30 mg of ketamine ( p < 0.05 ) .
Compared with the control group , the apoptosis rate was not significantly higher than that of the control group ( p < 0.05 ) .

Conclusion :

The apoptosis of PC12 cells can be induced by ketamine , and concentration - dependent and time - dependent ;
The endoplasmic reticulum stress mediated the apoptosis of PC12 cells induced by ketamine .
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R614

【引證文獻(xiàn)】

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本文編號(hào)：1987238