發(fā)布時間：2018-03-20 19:47

  本文選題：創(chuàng)傷性顱腦損傷　切入點：腸道　出處：《蘇州大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：創(chuàng)傷性顱腦損傷（traumatic brain injury, TBI）是神經(jīng)外科最常見的疾病，隨著工業(yè)及交通運輸業(yè)的發(fā)展，其發(fā)病率逐年升高。因為其高致死率和致殘率，TBI一直以來都被臨床醫(yī)生和眾多學(xué)者重視。而事實上，相當(dāng)一部分TBI患者并非原發(fā)病致死，而是多器官功能障礙（multiple organ dysfunction syndrome, MODS）致死。許多研究表明，，腸道不僅是MODS的靶器官，更是MODS的啟動者。鑒于此，人們越來越重視TBI后腸功能紊亂的情況，對TBI后腸道屏障功能的研究也成為當(dāng)今醫(yī)學(xué)領(lǐng)域的一個重要課題和熱點。 TBI后常發(fā)生腸功能障礙。腸功能障礙主要表現(xiàn)為胃腸動力下降，腸黏膜營養(yǎng)吸收障礙，黏膜破壞等。已有的研究主要集中在腸黏膜的顯微結(jié)構(gòu)改變，黏膜屏障破壞，黏膜血流變化以及炎癥反應(yīng)等，確切的機制仍不清楚。為了保證重要器官如心腦的血供，TBI后腸黏膜上皮細(xì)胞常處于缺血或低灌注狀態(tài)。不幸地是，腸黏膜上皮細(xì)胞能量儲備有限，不足以應(yīng)對缺血或低灌注的狀態(tài)。所以，腸黏膜上皮細(xì)胞對缺血缺氧非常敏感。一旦TBI發(fā)生，腸黏膜上皮細(xì)胞將面對強烈的氧化應(yīng)激。 線粒體作為機體氧化磷酸化和能量供應(yīng)的"重要工廠"，在氧化應(yīng)激中扮演了重要的角色。一方面，線粒體呼吸作用可以產(chǎn)生機體所需的ATP，同時，呼吸作用也會產(chǎn)生大量反應(yīng)性氧族（reactive oxygen species, ROS），從而進(jìn)一步發(fā)生氧化損傷。雖然線粒體與能量代謝密切相關(guān)，但尚未發(fā)現(xiàn)有關(guān)TBI后腸道黏膜上皮細(xì)胞線粒體功能變化的研究。 第一部分兩步法制備高質(zhì)量大鼠小腸上皮細(xì)胞線粒體 目的：探討一種兩步制備高質(zhì)量大鼠小腸上皮細(xì)胞線粒體的方法。方法：首先，用膠原酶和透明質(zhì)酸酶消化法制備高質(zhì)量的大鼠小腸上皮細(xì)胞；第二步再參考從培養(yǎng)的組織細(xì)胞中提取線粒體的方法從第一步制備的大鼠小腸上皮細(xì)胞中提取線粒體。BCA蛋白定量試劑盒測定制備的線粒體蛋白濃度；Western-blotting檢測樣品及對照品中Pax-5、β-actin、Cox IV的濃度來反映制備的線粒體純度及污染情況；測定制備的線粒體完整性、穩(wěn)定性以及生物活性。結(jié)果：檢測制備的20例大鼠小腸上皮細(xì)胞線粒體樣本，最低濃度為2.2108μg/μl，最高濃度為4.4516μg/μl，平均濃度為3.1932μg/μl；制備的線粒體樣品中Pax-5、β-actin表達(dá)量比較低，Cox IV表達(dá)量較高；以細(xì)胞色素C氧化酶為標(biāo)志酶在0~4℃保溫2h，檢測線粒體完整性仍在97%以上；制備的線粒體中細(xì)胞色素C氧化酶能很好的氧化NADH和蘋果酸，且其活性可以被1mmol/L KCN抑制；制備的線粒體ATP酶含量與對照組（從培養(yǎng)的IEC-6細(xì)胞株制備的線粒體）無明顯差別。結(jié)論：兩步法制備大鼠腸道上皮細(xì)胞線粒體方法簡便，制備的線粒體蛋白濃度和純度較高，胞質(zhì)和細(xì)胞核污染少，線粒體完整性和穩(wěn)定性較好，線粒體活性也保持地較好，適于線粒體功能實驗研究。 第二部分創(chuàng)傷性顱腦損傷（TBI）后大鼠腸道上皮細(xì)胞線粒體呼吸功能及相關(guān)酶活性的變化 目的：觀察創(chuàng)傷性顱腦損傷（TBI）后大鼠腸道上皮細(xì)胞線粒體呼吸功能及相關(guān)酶活性的變化情況。方法：雄性SD大鼠56只，隨機分為7組（每組8只）,包括對照組、腦損傷后6、12、24h和2、3、7d各1組。采用Feeney自由落體撞擊法制作TBI模型。使用Clark氧電極測定線粒體呼吸功能（呼吸控制率和ADP/O比）；線粒體呼吸鏈復(fù)合物I-IV以及相關(guān)酶（丙酮酸脫氫酶、α-酮戊二酸脫氫酶、蘋果酸脫氫酶）活性用分光光度計測定。結(jié)果：與對照組相比，創(chuàng)傷性顱腦損傷后大鼠腸道上皮細(xì)胞線粒體呼吸控制率在傷后6h開始下降，并且一直持續(xù)到傷后7d仍低于對照組(control,5.42±0.46;6h,5.20±0.18;12h,4.55±0.35;24h,3.75±0.22;2d,4.12±0.53;3d,3.45±0.41;7d,5.23±0.24; P＜0.01)。磷氧比在傷后12h開始顯著下降，一直持續(xù)到術(shù)后3d(12h,3.30±0.10;24h,2.61±0.21;2d,2.95±0.18;3d,2.76±0.09; P＜0.01)。傷后24h和3d各可以觀察到1個峰值。線粒體呼吸鏈復(fù)合物I (6h:110±10,12h:115±12,24h:85±9,2d:80±15,3d:65±16; P＜0.01)和復(fù)合物II (6h:105±8,12h:110±92,24h:80±10,2d:76±8,3d:68±12; P＜0.01)的活性在傷后6h和12h先增高，然后從傷后24h開始下降，并持續(xù)到傷后3d。與對照組相比，線粒體呼吸鏈復(fù)合物III和復(fù)合物IV傷后變化無明顯差別。此外，與對照組相比，丙酮酸脫氫酶活性在傷后6h開始下降，并且一直持續(xù)到傷后7d仍低于對照組(6h:90±8,12h:85±10,24h:65±12,2d:60±9,3d:55±6,7d:88±11; P＜0.01)。α-酮戊二酸脫氫酶活性變化規(guī)律與丙酮酸脫氫酶類似，但其活性下降是從傷后12h開始的(12h:90±12,24h:80±9,2d:76±15,3d:68±7,7d:90±13; P＜0.01)。蘋果酸脫氫酶活性傷后無明顯變化。結(jié)論：創(chuàng)傷性顱腦損傷后線粒體呼吸功能和部分相關(guān)酶活性收到抑制，線粒體功能不良可能在創(chuàng)傷性顱腦損傷后腸道功能障礙的發(fā)生中扮演重要角色。 第三部分創(chuàng)傷性顱腦損傷后大鼠腸道上皮細(xì)胞線粒體膜電位和通透性轉(zhuǎn)換的改變 目的：觀察創(chuàng)傷性顱腦損傷（TBI）后大鼠腸道上皮細(xì)胞線粒體膜電位和通透性轉(zhuǎn)換的改變情況。方法：雄性SD大鼠96只，體重220～260g，按隨機數(shù)字法分均為假手術(shù)對照組和TBI組，每組按照術(shù)后3、6、12h和1、3、7d分為6個亞組（n=8）：采用Feeney自由落體撞擊法制作TBI模型，假手術(shù)組對照組僅行右側(cè)頂部開窗而無TBI。分別于TBI后3、6、12h和1、3、7d處死各組8只大鼠，分離腸黏膜上皮細(xì)胞，用流式細(xì)胞檢測細(xì)胞凋亡指數(shù)，成功提取腸道上皮細(xì)胞線粒體后分別測定線粒體膜電位（MMP）和通透性轉(zhuǎn)換孔（MPTP）活性。結(jié)果：與假手術(shù)對照組比較，TBI組凋亡指數(shù)從術(shù)后6h起明顯升高，一直持續(xù)到術(shù)后7d(P＜0.05），從術(shù)后3h起腸道上皮細(xì)胞線粒體MMP即明顯降低(P＜0.05）而MPTP活性明顯增高(P＜0.05）。結(jié)論：大鼠TBI后腸道上皮細(xì)胞凋亡率增加，腸道上皮細(xì)胞線粒體MMP及MPTP活性的改變可能在這一病理生理過程中發(fā)揮重要作用。
[Abstract]:Traumatic brain injury (traumatic brain, injury, TBI) is the most common disease in Department of Neurosurgery, along with the development of industry and transportation industry, its incidence increased year by year. Because of its high mortality rate and disability rate, TBI has always been clinicians and many scholars attention. In fact, quite a part of TBI patients is not the primary disease but death, multiple organ dysfunction (MODS multiple organ dysfunction syndrome) to death. Many studies show that intestinal tract is not only the target organ of MODS, MODS is a start. In view of this, people pay more and more attention to TBI functional bowel disorder situation, research on the intestinal barrier function after TBI has become an important issue and a hot field in modern medicine.
Intestinal dysfunction often occurs after TBI. Intestinal dysfunction mainly decreased gastrointestinal motility, intestinal malabsorption, mucosal damage. The change of microstructure research has been mainly concentrated in the intestinal mucosa, mucosal barrier damage, mucosal blood flow changes and inflammatory reaction, the exact mechanism is still not clear. In order to ensure the important organs such as heart the blood supply of TBI intestinal epithelial cells in ischemia or hypoperfusion. Unfortunately, intestinal epithelial cells and energy reserves is limited, not enough to cope with ischemia or low perfusion state. Therefore, the intestinal mucosal epithelial cells are very sensitive to ischemia and hypoxia. Once TBI occurs, the intestinal epithelial cells will face oxidative stress strong.
As the body of mitochondrial oxidative phosphorylation and energy supply of the "factory", plays an important role in oxidative stress. On the one hand, mitochondrial respiration can produce the body's ATP, at the same time, the respiration will produce large amounts of reactive oxygen species (reactive oxygen, species, ROS), so as to further oxidative damage although closely related to mitochondrial energy metabolism, but have not yet found the changes of mitochondrial function of intestinal mucosal epithelial cells of the TBI.
The first part of the two step method to prepare the mitochondria of small intestinal epithelial cells in high quality rats
Objective: To investigate the two step method for preparing high quality mitochondria of intestinal epithelial cells in rats. Methods: first, the production of high quality with collagenase and hyaluronidase digestion method of intestinal epithelial cells in rats; the second step according to the determination of the concentration of mitochondrial protein extract preparation of mitochondrial.BCA protein assaykit intestinal epithelial cells the extraction method of mitochondria from cultured cells from the first step of preparation in rats; Western-blotting samples and the control sample in Pax-5, beta -actin, IV concentration of Cox to reflect the purity of mitochondria and the pollution of preparation; Determination of mitochondrial integrity of preparation, stability and biological activity. Results: detection 20 cases of rat intestinal epithelial cell mitochondrial sample preparation, the lowest concentration of 2.2108 mu g/ Mu L, the highest concentration of 4.4516 mu g/ Mu L, the average concentration of 3.1932 mu g/ Mu L; mitochondrial sample preparation In Pax-5, beta -actin expression is relatively low, Cox IV high expression; the cytochrome C oxidase enzyme marked 2H insulation at 0~4 DEG C, detection of mitochondrial integrity is still more than 97%; the preparation of mitochondrial cytochrome C oxidase in good oxidation of NADH and malic acid, and its activity can be 1mmol/L KCN inhibition; preparation of mitochondrial ATP enzyme and the content of the control group (IEC-6 cells cultured from the mitochondrial preparation) had no significant difference. Conclusion: the two step preparation of mitochondria in intestinal epithelial cells in rats, simple preparation, high purity and concentration of mitochondrial protein, cytoplasmic and nuclear pollution, mitochondrial integrity and good stability, mitochondrial activity also maintained good, suitable for experimental study of mitochondrial function.
The changes of mitochondrial respiratory function and related enzyme activity in the intestinal epithelial cells of the second part of traumatic brain injury (TBI)
Objective: To observe the effect of traumatic brain injury (TBI) changes of mitochondrial respiratory function of intestinal epithelial cells of rats and related enzyme activity. Methods: 56 male SD rats were randomly divided into 7 groups (n = 8), including control group, 6,12,24h after brain injury and 1 2,3,7d group. The TBI model Feeney free fall impact method. Determination of mitochondrial respiratory function using Clark oxygen electrode (respiratory control ratio and ADP/O ratio); mitochondrial respiratory chain complex I-IV and related enzymes (pyruvate dehydrogenase, - ketoglutarate dehydrogenase, malate dehydrogenase) activity was measured by a spectrophotometer. Results: compared with control group, traumatic brain after the injury of mitochondria in rat intestinal epithelial cells of respiratory control rate at 6h after injury began to decline, and continued until 7d after injury is still lower than that of the control group (control, 5.42 + 0.46 + 0.18; 6h, 5.20; 12h, 4.55 + 0.35 + 0.22; 24h, 3.75; 2D, 4.12 + 0.5 3;3d,3.45鹵0.41;7d,5.23鹵0.24; P錛

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