热打击致肠上皮细胞损伤机制的中西医研究

发布时间：2018-01-12 00:35

本文关键词：热打击致肠上皮细胞损伤机制的中西医研究　出处：《广州中医药大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的通过离体细胞和分子水平,研究热打击对肠上皮细胞和细胞间连接的影响,为阐明肠机械屏障损伤在中暑肠道病变中的可能作用机制提供实验依据。方法 1.热打击对肠上皮细胞形态与细胞骨架的影响体外培养正常大鼠肠上皮细胞株(IEC-6)细胞,按不同程度热打击进行实验分组：即将细胞分别置于39℃、41℃、43℃热打击1h,对照组为将细胞置于37℃条件同各热打击组等时间培养,置于倒置显微镜下观察细胞形态；考马斯亮蓝R250染色,油镜下观察细胞形态的改变及骨架蛋白在不同程度热打击下的变化,并照相。 2.热打击对肠上皮细胞细胞活力的影响体外培养正常大鼠肠上皮细胞株(IEC-6)细胞,按不同程度热打击进行实验分组：即将细胞分别置于39℃、41℃、43℃热打击0、1、3、5、7h,对照组为将细胞置于37℃条件同各热打击组等时间培养,于各时间点在相应分组的培养细胞上清中加入CCK-8后,并重新置于标准37℃、5%C02培养箱中孵育2h,用酶联免疫检测仪在450nm处读取吸光度值(A)值,计算细胞活性。 3.热打击对肠上皮细胞内切酶DAO活性的影响体外培养正常大鼠肠上皮细胞株(IEC-6)细胞,按不同程度热打击进行实验分组：即将细胞分别置于39℃、41℃、43℃热打击1h,对照组为将细胞置于37℃条件同各热打击组等时间培养,待24h后,收集培养上清ELISA法检测DAO活性。 4.热打击对肠上皮细胞细胞旁通透性的影响体外培养正常大鼠肠上皮细胞株(IEC-6)细胞,按不同程度热打击进行实验分组：即将细胞分别置于39℃、41℃、43℃热打击1h,对照组为将细胞置于37℃条件同各热打击组等时间培养,用Millicell-ERS系统检测跨上皮电阻(TEER)。 5.热打击对肠上皮细胞细胞紧密连接的影响体外培养正常大鼠肠上皮细胞株(IEC-6)细胞,按不同程度热打击进行实验分组：即将细胞分别置于39℃、41℃、43℃热打击1h,对照组为将细胞置于37℃条件同各热打击组等时间培养,24h后收集细胞蛋白,用Western blotting方法检测细胞Occludin蛋白表达水平。结果 1.热打击对肠上皮细胞形态与细胞骨架的影响光镜下可见,热打击可使肠上皮细胞(IEC-6)轮廓变圆,伪足变短变粗,细胞间间隙增宽,且随着热打击程度的增加,上述表现越加明显,至43℃时部分细胞出现坏死。考马斯亮蓝R250染色法检测发现,热打击可导致IEC-6细胞骨架变粗、变短、紊乱,出现明显的应力纤维,排列呈明显的非极性单向分布。且温度越高,变化越明显。 2.热打击对肠上皮细胞细胞活力的影响 CCK-8试验显示,热打击可造成IEC-6细胞活性下降,且随热打击程度的加深,细胞活性下降越明显,各热打击组均较正常对照组显著下降(P＜0.01)。 3.热打击对肠上皮细胞内切酶DAO活性的影响 ELISA检测结果显示,热打击后细胞培养上清中DAO活性增高,温度越高变化越明显,四组之间比较差异具有统计学意义(P0.01)；39℃热打击组与对照组比较,P㩳0.05；39℃热打击组与41℃和43℃热打击组比较,P＜0.05。 4.热打击对肠上皮细胞细胞旁通透性的影响 TEER检测结果显示,热打击可造成TEER水平降低,温度越高下降越明显,各热打击组与正常对照比较具有统计学意义(P＜0.05),各热打击组之间比较亦有显著差异(P＜0.01),表明热打击可引起肠粘膜通透性增加,并呈温度依赖性。 5.热打击对肠上皮细胞细胞紧密连接的影响 Western blotting结果显示热打击后紧密连接蛋白Occludin表达增多,且随温度升高而增多,各组之间Occludin表达量比较具有统计学意义P＜0.05) 结论一方面,热打击对肠上皮细胞造成直接损伤,可引起肠上皮细胞活力下降,甚至引起细胞内结构破坏。另一方面,热打击还可导致细胞间通透性的增加,主要表现为热打击可造成细胞骨架蛋白改变、细胞跨上皮电阻下降。但在我们的研究中还发现,热打击可以导致细胞间紧密连接蛋白关键成员——Occludin表达增加,我们推测Occludin在此过程中可能起到了应激修复作用。
[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.

【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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