本文選題：冷暴露　切入點：肝組織　出處：《第四軍醫(yī)大學》2012年碩士論文　論文類型：學位論文

【摘要】：【背景】 冷暴露是常見的特殊環(huán)境暴露因素。我們以往的研究發(fā)現(xiàn)，-15℃急性冷暴露0.5h可以引起大鼠肝臟能量水平的改變及相關(guān)細胞通路活性的變化，復(fù)方制劑可以顯著緩解急性冷暴露大鼠體溫下降，但其機制仍不清楚。自噬是細胞正常生理過程，可以清除損傷細胞器，提供能量代謝底物并抑制凋亡發(fā)生；能量應(yīng)激情況下，自噬可以維持細胞ATP水平。我們設(shè)想急性冷暴露能否引起肝臟細胞自噬水平改變及其產(chǎn)生的影響，復(fù)方制劑能否影響急性冷暴露大鼠肝臟能量水平及自噬是否參與復(fù)方制劑提高大鼠耐寒能力的過程值得探討。 【目的】 通過建立大鼠急性冷暴露模型，觀察急性冷暴露對大鼠肝細胞能量水平及細胞凋亡的影響，探討自噬水平變化產(chǎn)生的影響及調(diào)控機制，并進一步探討自噬在復(fù)方制劑調(diào)控急性冷暴露條件下大鼠肝細胞能量水平及細胞凋亡中的作用及機制。為闡明急性冷暴露對機體造成的損傷、急性冷暴露過程中機體能量的調(diào)控及復(fù)方制劑提高耐寒能力的作用機制提供理論依據(jù)，為冷暴露條件下的機體防護措施提供理論基礎(chǔ)。 【方法】 1.急性冷暴露模型的建立：雄性SD大鼠230g±10g體重，獨立暴露于-15℃低溫試驗艙，暴露時間為4h。 2.肝臟糖原含量測定：用肝糖原檢測試劑盒測定大鼠肝組織肝糖原水平。 3.肝臟細胞能量水平測定：用ATP檢測試劑盒，通過多功能酶標儀進行發(fā)光度測定并得到大鼠肝細胞ATP含量值。 4.凋亡水平檢測：用western blot方法測定肝細胞凋亡相關(guān)蛋白caspase-3活化水平及TUNEL染色法觀察TUNEL陽性細胞。 5.自噬水平檢測：用western blot方法測定肝細胞自噬標志物LC3-I和LC3-II的表達變化；用免疫組化方法，通過MDC染色方法進行肝細胞自噬水平的檢測。 6.自噬抑制劑對機體的影響：采用腹腔注射的方法，降低自噬水平，同時觀察其對肝組織冷暴露應(yīng)激狀態(tài)下能量代謝可能產(chǎn)生的影響。 7.復(fù)方制劑對機體的影響：采用連續(xù)灌胃方法，持續(xù)灌胃3天。觀察能量及自噬水平的變化。 【結(jié)果】 1.急性冷暴露對大鼠肝細胞凋亡水平的影響 -15℃急性冷暴露4h引起大鼠肝細胞凋亡增加。急性冷暴露組大鼠肝細胞caspase-3剪切水平顯著增加，TUNEL陽性細胞數(shù)顯著增加（p＜0.05）。 2.急性冷暴露對大鼠肝臟ATP及肝糖原水平的影響 -15℃急性冷暴露4h引起大鼠肝臟細胞ATP、肝糖原水平顯著下降。室溫對照組大鼠肝糖原5.03±0.86mg/g，而冷暴露對照組大鼠肝糖原降至2.24±1.03mg/g；對照組大鼠ATP水平為0.293±0.018μmol/mg，冷暴露4h組大鼠ATP水平降低至0.195±0.044μmol/mg。兩者均具有統(tǒng)計學意義（p＜0.05）。 3.急性冷暴露對肝臟自噬水平的影響 MDC染色檢測發(fā)現(xiàn)，-15℃急性冷暴露4小時引起肝細胞自噬泡數(shù)量增加，與對照組相比具有統(tǒng)計學意義（p＜0.05）；WB檢測發(fā)現(xiàn)LC3-II灰度水平在急性冷暴露后顯著升高（p＜0.05）。提示急性冷暴露引起大鼠肝臟自噬水平顯著增加。 4.自噬抑制劑對急性冷暴露大鼠肝臟細胞凋亡水平、能量水平及自噬水平的影響 腹腔注射自噬抑制劑渥曼霉素可以顯著降低肝細胞自噬水平（p＜0.05），渥曼霉素引起急性冷暴露大鼠肝細胞凋亡水平顯著升高，進一步增加caspase-3蛋白的剪切及TUNEL染色陽性細胞數(shù)量（p＜0.05），并引起肝細胞ATP含量顯著降低，冷暴露抑制劑組大鼠ATP水平為0.146±0.018μmol/mg與冷暴露對照組大鼠ATP水平0.193±0.047μmol/mg相比具有統(tǒng)計學意義（p＜0.05）。而冷暴露抑制劑組大鼠肝糖原水平與冷暴露對照組大鼠相比無明顯差異。 5.復(fù)方制劑對急性冷暴露大鼠肝細胞凋亡水平、肝臟能量水平及肝細胞自噬水平的影響 復(fù)方制劑灌胃3天可以顯著降低急性冷暴露大鼠肝細胞凋亡水平（p＜0.05），并提高急性冷暴露大鼠的肝臟細胞ATP及肝糖原水平，并同時降低肝組織的自噬水平。冷暴露復(fù)方制劑組的大鼠ATP水平為0.236±0.018μmol/mg，與冷暴露對照組大鼠ATP水平0.187±0.030μmol/mg相比具有統(tǒng)計學意義（p＜0.05），冷暴露復(fù)方制劑組大鼠肝糖原2.20±1.24mg/g，而冷暴露對照組大鼠肝糖原降至1.38±0.97mg/g，相比具有統(tǒng)計學意義（p＜0.05）。 【結(jié)論】 1.急性冷暴露可以引起大鼠肝細胞凋亡，，肝細胞能量水平降低，并引起細胞自噬水平增加。 2.寒冷誘導(dǎo)的自噬有助于維持一定的ATP和代謝水平并抑制凋亡的發(fā)生。 3.復(fù)方制劑可以部分逆轉(zhuǎn)急性冷暴露誘導(dǎo)增加的自噬水平，同時減少細胞凋亡水平并提高急性冷暴露大鼠肝細胞ATP水平以及肝糖原水平。提示復(fù)方制劑能夠在一定程度上減輕急性冷暴露對肝臟造成的損傷。
[Abstract]:[background]
Cold exposure is a common specific environmental exposure factors. Our previous study found that -15 C acute cold exposure 0.5h can change caused by the change of rat liver energy level and related cell pathway activity, the compound preparation can significantly relieve the acute cold exposure of rats decreased body temperature, but the mechanism is still unclear. Autophagy is normal process, can the clearance of damaged organelles, energy metabolism substrate and inhibiting apoptosis; energy stress, autophagy can maintain cellular ATP level. We assume that acute cold exposure can cause liver cell autophagy level change and influence of compound preparation can influence the acute cold exposure of rat liver energy level and autophagy is involved in the process improve the cold resistance ability of rat compound preparation is worth exploring.
[Objective]
Through the establishment of acute cold exposure model rats, observe the effect of acute cold exposure on the energy level of liver cells and cell apoptosis in rats, to explore the effect of autophagy level changes and regulation mechanism, and to further explore the regulation of autophagy in compound acute cold exposure effect and mechanism of apoptosis of liver cells and the level of energy under the condition of the cells of rats on the body caused by injury. In order to elucidate the acute cold exposure, acute cold exposure mechanism to improve cold tolerance control and compound body energy in the process to provide a theoretical basis for cold exposure under the condition of body anti theoretical foundation support measures.
[method]
1. the establishment of acute cold exposure model: the male SD rats were 230g + 10g body weight and were exposed to -15 centigrade cryogenic test compartment independently, the exposure time was 4h.
2. determination of liver glycogen content: Determination of liver glycogen level in rat liver tissue with liver glycogen detection kit.
3. determination of liver cell energy level: detection kit by ATP, measurement of luminosity by multi-function enzyme labeling and ATP content of rat liver cells.
4. detection of apoptosis level: the activation level of hepatocyte apoptosis related protein caspase-3 was measured by Western blot method and TUNEL positive cells were observed by TUNEL staining.
5. detection of autophagy level: the expression of LC3-I and LC3-II of hepatocyte autophagy markers were detected by Western blot method. The autophagy level of hepatocytes was detected by immunohistochemical staining and MDC staining.
6., the effect of autophagy inhibitor on the body: the method of intraperitoneal injection can reduce the level of autophagy, and observe its effect on the energy metabolism of liver tissue under cold exposure stress.
7. the effect of compound preparation on the body: continuous gavage method and continuous gavage for 3 days. The changes of energy and autophagy were observed.
[results]
The effect of 1. acute cold exposure on the level of apoptosis in rat liver cells
Acute cold exposure at -15 C increased 4H hepatocyte apoptosis in rats. In acute cold exposure group, the caspase-3 level of liver cells increased significantly, and the number of TUNEL positive cells increased significantly (P < 0.05).
The effect of 2. acute cold exposure on the level of liver ATP and liver glycogen in rats
-15 C acute cold exposure induced by 4H in liver cells of ATP rats, liver glycogen level was significantly decreased at room temperature. The control group of rat liver glycogen was 5.03 + 0.86mg/g, while the control group cold exposure of rat liver glycogen decreased to 2.24 + 1.03mg/g; ATP level of the control group rats was 0.293 + 0.018 mol/mg, ATP level of 4H group exposed to cold rats decreased to 0.195 + 0.044 u mol/mg. both were statistically significant (P < 0.05).
3. effect of acute cold exposure on the level of autophagy in the liver
MDC staining, -15 C acute cold exposure 4 hours caused by global increase in the number of liver cell autophagy, compared with the control group with statistical significance (P < 0.05); WB detected LC3-II gray level significantly increased in acute cold exposure (P < 0.05). Acute cold exposure induced autophagy inrat liver increased significantly.
The effect of 4. autophagy inhibitors on the level of apoptosis, energy level and autophagy in acute cold exposed rats
Intraperitoneal injection of kanamycin fermentation Manchester autophagy inhibitors can significantly reduce the hepatic autophagy level (P < 0.05), Ottawa Manchester kanamycin induced acute cold exposure level of apoptosis of rat liver cells increased significantly, further increase the shear and TUNEL staining of caspase-3 protein positive cells (P < 0.05), and liver cells caused by ATP was significantly decreased that cold exposure level of ATP inhibitor group rats was 0.146 + 0.018 mol/mg and cold exposure control ATP group rats compared to 0.193 + 0.047 mol/mg was statistically significant (P < 0.05). But the cold exposure level of glycogen and cold rats exposed to inhibitors of the rats in the control group had no significant difference.
Effect of 5. compound preparation on the level of hepatocyte apoptosis, liver energy level and the level of autophagy of liver cells in rats with acute cold exposure
The intragastric administration of compound preparation for 3 days can significantly reduce the level of apoptosis of liver cells in rats of acute cold exposure (P < 0.05), and improve the rat liver ATP cells and liver glycogen levels of acute cold exposure, and at the same time reduce the level of autophagy in liver tissue. Cold exposure compound group level of ATP rats was 0.236 + 0.018. Mol/mg, and cold exposure compared to 0.187 + 0.030 mol/mg was statistically significant ATP level of the control group of rats (P < 0.05), cold exposed rats liver glycogen preparation of compound 2.20 + 1.24mg/g, while the control group cold exposure of rat liver glycogen decreased to 1.38 + 0.97mg/g, compared with statistical significance (P < 0.05).
[Conclusion]
1. acute cold exposure can induce apoptosis of hepatocytes in rats, decrease the level of hepatocyte energy, and increase the level of autophagy.
2. cold induced autophagy helps to maintain a certain level of ATP and metabolism and inhibit the occurrence of apoptosis.
3. compound autophagy induced the increase of exposure can partially reverse the acute cold, while reducing the level of apoptosis and improve acute cold exposure level of ATP rats liver cells and liver glycogen level. Suggesting that compound preparation could reduce the liver damage caused by acute cold exposure in a certain extent.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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