本文關(guān)鍵詞：熱打擊致腸上皮細胞損傷機制的中西醫(yī)研究　出處：《廣州中醫(yī)藥大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 中暑 腸粘膜屏障 腸上皮細胞 緊密連接

【摘要】：目的 通過離體細胞和分子水平,研究熱打擊對腸上皮細胞和細胞間連接的影響,為闡明腸機械屏障損傷在中暑腸道病變中的可能作用機制提供實驗依據(jù)。 方法 1.熱打擊對腸上皮細胞形態(tài)與細胞骨架的影響 體外培養(yǎng)正常大鼠腸上皮細胞株(IEC-6)細胞,按不同程度熱打擊進行實驗分組：即將細胞分別置于39℃、41℃、43℃熱打擊1h,對照組為將細胞置于37℃條件同各熱打擊組等時間培養(yǎng),置于倒置顯微鏡下觀察細胞形態(tài)；考馬斯亮藍R250染色,油鏡下觀察細胞形態(tài)的改變及骨架蛋白在不同程度熱打擊下的變化,并照相。 2.熱打擊對腸上皮細胞細胞活力的影響 體外培養(yǎng)正常大鼠腸上皮細胞株(IEC-6)細胞,按不同程度熱打擊進行實驗分組：即將細胞分別置于39℃、41℃、43℃熱打擊0、1、3、5、7h,對照組為將細胞置于37℃條件同各熱打擊組等時間培養(yǎng),于各時間點在相應(yīng)分組的培養(yǎng)細胞上清中加入CCK-8后,并重新置于標(biāo)準37℃、5%C02培養(yǎng)箱中孵育2h,用酶聯(lián)免疫檢測儀在450nm處讀取吸光度值(A)值,計算細胞活性。 3.熱打擊對腸上皮細胞內(nèi)切酶DAO活性的影響 體外培養(yǎng)正常大鼠腸上皮細胞株(IEC-6)細胞,按不同程度熱打擊進行實驗分組：即將細胞分別置于39℃、41℃、43℃熱打擊1h,對照組為將細胞置于37℃條件同各熱打擊組等時間培養(yǎng),待24h后,收集培養(yǎng)上清ELISA法檢測DAO活性。 4.熱打擊對腸上皮細胞細胞旁通透性的影響 體外培養(yǎng)正常大鼠腸上皮細胞株(IEC-6)細胞,按不同程度熱打擊進行實驗分組：即將細胞分別置于39℃、41℃、43℃熱打擊1h,對照組為將細胞置于37℃條件同各熱打擊組等時間培養(yǎng),用Millicell-ERS系統(tǒng)檢測跨上皮電阻(TEER)。 5.熱打擊對腸上皮細胞細胞緊密連接的影響 體外培養(yǎng)正常大鼠腸上皮細胞株(IEC-6)細胞,按不同程度熱打擊進行實驗分組：即將細胞分別置于39℃、41℃、43℃熱打擊1h,對照組為將細胞置于37℃條件同各熱打擊組等時間培養(yǎng),24h后收集細胞蛋白,用Western blotting方法檢測細胞Occludin蛋白表達水平。 結(jié)果 1.熱打擊對腸上皮細胞形態(tài)與細胞骨架的影響 光鏡下可見,熱打擊可使腸上皮細胞(IEC-6)輪廓變圓,偽足變短變粗,細胞間間隙增寬,且隨著熱打擊程度的增加,上述表現(xiàn)越加明顯,至43℃時部分細胞出現(xiàn)壞死�？捡R斯亮藍R250染色法檢測發(fā)現(xiàn),熱打擊可導(dǎo)致IEC-6細胞骨架變粗、變短、紊亂,出現(xiàn)明顯的應(yīng)力纖維,排列呈明顯的非極性單向分布。且溫度越高,變化越明顯。 2.熱打擊對腸上皮細胞細胞活力的影響 CCK-8試驗顯示,熱打擊可造成IEC-6細胞活性下降,且隨熱打擊程度的加深,細胞活性下降越明顯,各熱打擊組均較正常對照組顯著下降(P＜0.01)。 3.熱打擊對腸上皮細胞內(nèi)切酶DAO活性的影響 ELISA檢測結(jié)果顯示,熱打擊后細胞培養(yǎng)上清中DAO活性增高,溫度越高變化越明顯,四組之間比較差異具有統(tǒng)計學(xué)意義(P0.01)；39℃熱打擊組與對照組比較,P㩳0.05；39℃熱打擊組與41℃和43℃熱打擊組比較,P＜0.05。 4.熱打擊對腸上皮細胞細胞旁通透性的影響 TEER檢測結(jié)果顯示,熱打擊可造成TEER水平降低,溫度越高下降越明顯,各熱打擊組與正常對照比較具有統(tǒng)計學(xué)意義(P＜0.05),各熱打擊組之間比較亦有顯著差異(P＜0.01),表明熱打擊可引起腸粘膜通透性增加,并呈溫度依賴性。 5.熱打擊對腸上皮細胞細胞緊密連接的影響 Western blotting結(jié)果顯示熱打擊后緊密連接蛋白Occludin表達增多,且隨溫度升高而增多,各組之間Occludin表達量比較具有統(tǒng)計學(xué)意義P＜0.05) 結(jié)論 一方面,熱打擊對腸上皮細胞造成直接損傷,可引起腸上皮細胞活力下降,甚至引起細胞內(nèi)結(jié)構(gòu)破壞。另一方面,熱打擊還可導(dǎo)致細胞間通透性的增加,主要表現(xiàn)為熱打擊可造成細胞骨架蛋白改變、細胞跨上皮電阻下降。但在我們的研究中還發(fā)現(xiàn),熱打擊可以導(dǎo)致細胞間緊密連接蛋白關(guān)鍵成員——Occludin表達增加,我們推測Occludin在此過程中可能起到了應(yīng)激修復(fù)作用。
[Abstract]:objective
The effects of heat shock on intestinal epithelial cells and intercellular connections were studied at detached cell and molecular level, so as to provide experimental evidence for elucidating the possible mechanism of intestinal mechanical barrier injury in heatstroke intestinal diseases.
Method
The effect of 1. heat shock on the morphology and cytoskeleton of intestinal epithelial cells
In vitro culture of normal rat intestinal epithelial cell line (IEC-6) cells, were grouped according to different degree of heat stroke: the cells were exposed to 39 C, 41 C, 43 C heat against 1H, the control group the cells were placed in the condition of 37 DEG C with the heat stroke group training time, cell morphology was observed under the inverted microscopic under the microscope; Coomassie brilliant blue R250 staining under microscope, oil changes, changes in cell morphology and cytoskeletal proteins in different degrees of heat hit and photography.
The effect of 2. heat shock on the cell viability of intestinal epithelial cells
In vitro culture of normal rat intestinal epithelial cell line (IEC-6) cells, were grouped according to different degree of heat stroke: the cells were exposed to 39 C, 41 C, 43 C heat against 0,1,3,5,7h, the control group the cells were placed in the condition of 37 DEG C with the heat stroke group training time, at each time point in joining CCK-8 the supernatant of cultured cells in the corresponding group, and re placed in standard 37 DEG C, incubation 2H culture 5%C02, using enzyme-linked immunosorbent assay absorbance was read at 450nm (A) values, calculated cell activity.
Effect of 3. heat shock on the activity of endonuclease DAO in intestinal epithelial cells
In vitro culture of normal rat intestinal epithelial cell line (IEC-6) cells, were grouped according to different degree of heat stroke: the cells were exposed to 39 C, 41 C, 43 C heat against 1H, the control group the cells were placed in the condition of 37 DEG C with the heat stroke group training time, to 24h after collecting culture detection of DAO activity in supernatant of ELISA method.
Effect of 4. heat shock on the permeability of intestinal epithelial cells
In vitro culture of normal rat intestinal epithelial cell line (IEC-6) cells, were grouped according to different degree of heat stroke: the cells were exposed to 39 C, 41 C, 43 C heat against 1H, the control group the cells were placed in the condition of 37 DEG C with the heat stroke group culture time Millicell-ERS system for detection of transepithelial electrical resistance (TEER).
The effect of 5. heat shock on the close connection of intestinal epithelial cells
In vitro culture of normal rat intestinal epithelial cell line (IEC-6) cells, were grouped according to different degree of heat stroke: the cells were exposed to 39 C, 41 C, 43 C heat against 1H, the control group the cells were placed in the condition of 37 DEG C with the heat stroke group training time, collected after 24h cell protein. The expression level of Occludin protein was detected in Western cells by blotting method.
Result
The effect of 1. heat shock on the morphology and cytoskeleton of intestinal epithelial cells
Under the light microscope, heat stroke can make the intestinal epithelial cells (IEC-6) of round outline, pseudopodia became shorter and thicker, intercellular space widened, and with the heat hit degree increase, the performance of the more obvious, to 43 degrees C cell necrosis. Coomassie brilliant blue R250 staining method to detect the hot blow can lead to IEC-6 cell skeleton thicker, shorter, disorder, stress fibers were arranged in nonpolar, one-way distribution obviously. The higher the temperature, the more obvious changes.
The effect of 2. heat shock on the cell viability of intestinal epithelial cells
CCK-8 test showed that heat shock could decrease the activity of IEC-6 cells, and with the deepening of heat shock, the cell activity decreased more significantly. The heat shock group was significantly lower than that of the normal control group (P < 0.01).
Effect of 3. heat shock on the activity of endonuclease DAO in intestinal epithelial cells
ELISA test results showed that the heat stress-induced cell culture increased the activity of DAO was, the higher the temperature change was more obvious, with significant differences between the four groups (P0.01); 39 C heat stroke group compared with the control group, P 0.05? 39 DEG C; heat stroke group and 41 degrees and 43 degrees of heat hit group compared to P < 0.05.
Effect of 4. heat shock on the permeability of intestinal epithelial cells
TEER test results showed that the heat stroke can cause the decrease of TEER level, the higher the temperature drop is more obvious, the heat stroke group and the normal control was statistically significant (P < 0.05), there was a significant difference between each group also heat stroke (P < 0.01), indicated that heat stroke can cause an increase in the permeability of the intestinal mucosa, and is temperature dependent.
The effect of 5. heat shock on the close connection of intestinal epithelial cells
Western blotting results showed that the expression of tight junction protein Occludin increased after heat shock, and increased with the increase of temperature. The Occludin expression in each group was statistically significant (P < 0.05).
conclusion
On the one hand, heat shock caused by direct injury of intestinal epithelial cells, intestinal epithelial cell viability decreased, and even cause the cell structure damage. On the other hand, heat stroke can also lead to increased cell permeability, mainly for heat stroke can cause cytoskeletal protein modification, cell transepithelial resistance but also decreased. In our study we found that heat stress can lead to cell tight junctions between members of key protein expression of Occludin, we speculate that Occludin in this process may play a role in repair of stress.

【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

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