【作者】 戴睿武；

【导师】 田伏洲；

【作者基本信息】 第三军医大学 ， 外科学， 2007， 博士

【摘要】 一、研究背景和研究目的:胰腺创伤发病率约为0.4/10万,仅占腹部外伤的1%～3%。但由于其致伤后病死率高达20%,胰瘘、肠瘘、腹腔感染、出血等严重并发症发生率达40%,因此胰腺创伤在腹部创伤中占重要地位。现有研究主要集中在胰腺创伤后患者的外科学救治、创伤后并发症的防治等方面,对于其伤情变化机制的研究则极为缺乏。研究单器官创伤的伤情变化,有助于排除多器官损伤的影响,更好地了解该器官创伤后的发病机制,掌握其伤情演化特点,提高该病的治疗效果。传统的胰腺损伤动物模型大多存在致伤因素不纯、致伤范围难以控制、重复性差、操作烦琐等问题,故建立单纯的胰腺创伤动物模型,继而对其病理、病理生理学衍变的研究是个急待解决的课题。胰腺创伤后的伤情变化有什么特点呢?这些特点和急性胰腺炎的病情变化是相同的吗?磷脂酶A₂（Phospholipase A₂ ,PLA₂）是一类能够在磷脂甘油部分的Sn - 2位点选择性断裂酯键的酶。急性胰腺炎（AP）时,当PLA₂被激活后,可作用于细胞膜和线粒体膜的甘油磷脂,使其分解为溶血卵磷脂,从而溶解破坏细胞膜和线粒体膜的脂蛋白结构,使细胞死亡。胰腺创伤后,PLA₂是否和AP时一样被激活?胰腺创伤后胰腺细胞PLA₂各基因、蛋白表达水平和组织内定位情况又是怎样的呢?目前没有应用于临床的特异性PLA₂的酶活性抑制剂,最早于1973年在海绵内分离的Variabilin（呋喃二倍半萜烯-4-羟乙酰乙酸内酯）已在体外被证实为PLA₂的有效特异抑制剂,2004年人们用化学合成法得到Variabilin后,其来源问题已成功解决。而Variabilin是否能在生物体内发挥抑制胰酶激活过程中关键步骤―PLA₂激活的目的,发挥创伤后保护作用呢?二、实验方法为了解答上述问题,本研究实验设计如下:首先,利用BIM-Ⅲ型多功能生物撞击机,建立急性胰腺创伤Wistar大鼠模型作为实验组,而对照组为开腹后不激惹胰腺,相同建模开腹时间后关腹。研究在不同压强的外力作用下,模型指定时相点的一般情况和存活率、大体、光镜及电镜下胰腺组织的病理变化和常规酶学变化,得到稳定建模致伤压强;然后,固定致伤压强,研究胰腺创伤后不同时相点的伤情变化（包括一般情况、胰腺组织的病理变化、AMS和LPS活性变化、血常规、血生化、胰腺细胞凋亡以及胰腺组织细胞生长周期变化情况）;再次,采用3H-花生四烯酸标记大肠杆菌膜作为底物的方法检测大鼠胰腺创伤后不同时相点的PLA₂活性,利用荧光定量RT-PCR技术检测其亚型mRNA的表达,利用Western blot和IHC技术检测其蛋白表达量和蛋白组织定位;最后,将Variabilin体内应用于急性胰腺创伤大鼠模型,研究其对模型的保护作用和对PLA₂的抑制效应。三、实验结果及结论1. 400KPa冲击气流作用后24h,大鼠存活率为85%,明显高于600KPa组的40%。因此,稳定建模的冲击气流压力上限为400KPa,可更经济地用于创伤后亚急性期的伤情特点的观察。2.胰腺大体病理变化主要为外力作用后当时,大鼠胰腺局部出血和伤后血性腹水形成,腹水量和胰腺重量与致伤压力大小呈正比。光镜下胰腺细胞空泡形成、水肿、坏死指数以400KPa以上压力组为明显,这与非创伤因素引起的AP时的病理变化相似;但与AP比较,PT炎细胞浸润不多、出血更加明显。电镜下可见内质网扩张、断裂,核糖体脱落,溶酶体增多,血管内皮细胞肿胀、血管淤血等在各压力均可发生,线粒体肿胀变形以400KPa以上压力组为明显。3.不同大小外力作用下,伤后24h血、腹水的AMS、LPS活性均较对照组明显增高,AMS在各实验组间无明显差异,LPS在400KPa以上压力组与其下压力组之间有明显差异, 400KPa以下压力组与对照组比较亦有明显差异。因此可认为在伤后24h内,仅可根据AMS的变化来判断是否存在胰腺创伤,而不能根据AMS值预测创伤的严重程度;而LPS如明显增高可预测存在严重的胰腺创伤。4.固定400KPa压力创伤胰腺后,血性腹水量在6h时达到峰值,24h时减少,而胰腺湿重则在24h时达到峰值,此后逐渐下降。光镜下,出血、空泡形成、水肿、坏死是最显著的变化,而炎性细胞浸润较少。以上病理指数在6-24h时达到高峰,此后逐渐下降,在7d时有明显好转。电镜下,粗面内质网、核糖体、线粒体、溶酶体和毛细血管内皮细胞、细胞间导管早期均有改变,且在72h后开始逐渐恢复正常形态。5.创伤后6h,血液WBC和W-LCR均明显升高,24h最高,此后逐渐下降;RBC、HGB和血钙水平却在72h内逐渐下降,7d时稍有增加;PLT和血钾水平则是先降后升。血AMS在伤后6h开始上升,24h达到高峰,此后逐渐下降。LPS上升亦可在伤后6h开始上升,但在72h才达到高峰,7d时仍维持于较高水平。而腹水AMS、LPS在7d前均较高。这对于病情的判断有较大临床意义:急性胰腺创伤后,血清AMS可作为早期诊断标准,血清LPS可作为特异诊断标准;腹水AMS和LPS不能作为判断预后的标准,只能作为辅助诊断的指标。6.采用TUNEL法和流式细胞技术检测发现,胰腺创伤后细胞凋亡6h就出现且为高峰,此时PI却下降,说明创伤后早期主要为细胞衰亡效应;24h后,AI随着时间的发展逐渐下降,而PI却开始随时间增加而逐渐增加,7d时达到最高值,说明后续细胞修复机制在24h时已开始激活,这种修复机制和破坏机制在创伤后期持续同时存在。7.胰腺创伤后6h大鼠血清和胰腺组织PLA₂活性即开始升高,24h达峰值,此后虽逐渐下降但仍高于对照组。这种变化以胰腺组织的PLA₂活性增高最为显著,而血清PLA₂活性亦有显著增高。因此在胰腺创伤后6h-7d均可检测血PLA₂活性。8.荧光定量RT-PCR显示,胰腺创伤后表达增强的基因主要为sPLA₂,其次为cPLA₂,而iPLA₂则稍有增加。伤后24h前PLA₂各亚型的mRNA表达量均呈随时间而上升的趋势,cPLA₂和sPLA₂在此时出现最高水平,而iPLA₂的表达峰则在72h,这可能和cPLA₂和sPLA₂主要由胰腺组织产生而iPLA₂主要存在于胰腺外组织有关。而Western Blot定量结果也从蛋白水平证实了PLA₂各亚型表达的情况与转录水平一致的特点。9.用IHC技术对酶蛋白亚型组织定位方面:胰腺内的表达以sPLA₂和cPLA₂增加为主,6h时即遍布于胰腺腺泡细胞;24h时由于胰腺细胞的大量破坏,腺泡细胞外均可见sPLA₂和cPLA₂的高表达;72h后细胞内外sPLA₂和cPLA₂逐渐下降。而iPLA₂蛋白组织定位变化并不明显,结合前面的实验结果,初步推断出iPLA₂在胰腺创伤后并不是主要致病因子。10.将海绵提取物Variabilin体内应用于胰腺创伤模型后,我们发现其表现出较强的抑制血清和胰腺细胞内PLA₂活性的作用。使模型存活率提高;可减轻损伤后胰腺坏死和水肿程度;减少血性腹水形成等并发症的发生;促进胰腺损伤修复和机体基本机能恢复,从而发挥良好的保护作用。综上所述,本研究主要结论如下:1.一种有效、稳定、经济的Wistar大鼠急性胰腺创伤动物模型成功建立。2.在胰腺创伤6h-24h是伤情加重的激进期,机体修复机制在创伤后24h时已开始激活,这种修复机制和损伤机制在创伤后期持续同时存在。3.创伤后PLA₂活性增高,主要是sPLA₂和cPLA₂。4. Variabilin在体内可抑制血清和胰腺细胞内PLA₂活性,对胰腺创伤大鼠模型有良好的保护作用。更多还原

【Abstract】 Study background and purpose The incidence of pancreatic trauma （PT） is 0.4/100,000, and it only occupies 1%-3% in abdominal trauma. However, the mortality of PT is highly to 20%,and the incidence of severe complications,such as pancreatic fistula, intestinal fistula, infection of abdominal cavity and bleeding and so on, is 40%. Therefore PT plays an important part in abdominal trauma .At present our study focus on surgical remedy after trauma, prevention and cure to trauma complications, and the study on the mechanism of traumatic conditions variation is severely deficient.The purpose of the study on variation of traumatic conditions in single organ help us to eliminate the influence of other organs’trauma, better understand its pathogenesis after trauma, master the evolutional features of traumatic conditions, and improve its therapeutic efficacy. The traditional animal models of PT mostly have such problems, for instance, complex wound agents, the out of controlled trauma range, mal-reproducibility and complicated performance. Consequently, to found a pure animal model of PT and study its pathologic and physiopathologic development becomes an emergent topic. Meanwhile, what evolutional features will be after PT? And if these features are conformity with those of acute pancreas（AP）?Phospholipase A₂（PLA₂） is a generic name of a group of enzyme which can selectively break in Sn-2 point of the phospholipids’glycerin part. The activated PLA₂ can affect the glycerophospholipide of cell membrane and mitochondria membrane, and decomposing it into lysolecithin at AP. So those construction of lipoprotein are destroyed and cells die.Then is PLA₂ activated at PT likewise at AT? How does the expressing levels of PLA₂ gene and protein, and where does the PLA₂ protein distribute in pancreas at PT?Now there are no specific enzyme inhibiters against PLA₂ for clinical using. Variabilin, which first refined from sponge in 1973, was verified to be a specific active inhibitor against PLA₂.The problem of source was successfully solved after people obtained Variabilin by chemosynthesis method. However, whether Variabilin can inhibit the activity of PLA₂ in vivo and take the protective effect?Methods For answering these formal questions, we designed our experiments as follows: first is founding a model of PT in Wister rats by BIM-Ⅲbiological striking machine, studying its pathologic change and routine enzyme variation at variably powers with different pressures, and get stable trauma pressure of model founded. Second is studying the traumatic condition changes in a choosey pressure at different post-PT phases. Moreover, we detected model PLA₂ activity, express of PLA₂ subtype mRNA by fluorescent quantitative RT-PCR technique, protein express and site by Western blot and IHC at different post-PT phases. At last, we studied the PLA₂ inhibited effects and model protection of Variabilin in vivo.Results and Conclusion1. We found that the survival rate is of 85% at the 24 hours phase after the impact with 400 kPa effect, and it is obviously higher than that of impact with 600 kPa, which is only of 40%.So,the upper limit of model founding pressure is 400 kPa which can be more economically applied to the observation of features of traumatic conditions in subacute stage after trauma.2. The general pathologic changes: pancreas bleeding and blood ascites formation at the strike affecting moment, and the pressure of strike is of direct ratio with blood ascites quantity and pancreas weight. Light microscope: vacuolization, edema, necrosis index of pancreas cells were serious in the groups which pressures were more than 400 kPa, and these were similar with those of AP. But few macrophage infiltration and obviously bleeding were different with those of AP. Electron microscope: endocytoplasmic reticulums expanding and breaking, ribosomes defluxion, lysosomes increasing, vascular endothelial cells swelling and blood vessels congestion appeared in all groups, and the mitochondria swelling is serious in the groups which pressures were more than 400 kPa.3. The AMS, LPS activities in blood and ascitic of experiment groups were obviously high than those of control groups at 24h phase after PT.AMS activities were no obviously difference in any of experiment groups, and LPS activities of hyper-400 kPa groups were higher than those of hypo-400 kPa, and all were different with those of control group. So we can say, AMS is a kind of indication of PT diagnosis other than prediction of PT severity degree. Meanwhile if LPS is obviously high, which can predict the existence of severe pancreatic trauma.4. After PT with 400kPa pressure, the volume of blood ascites was at the top at 6h, and decreased with time gone. Pancreas wet weight was to peak at 24h, after this was falling off. Light microscope: hemorrhage, vacuolization, edema and necrosis were the most notable changes, but inflammatory cells infiltrating was few. These above pathology indexes were to peak at 6-24h, after this fell off and recovered at 7d. Electron microscope: rough endoplasmic reticulum, ribosome, mitochondria, lysosome and capillary endothelium cell, catheter intercells changed early and began to recover after 72h.5. Blood WBC and W-LCR increased after 6h, and were to peak at 24h, then decreased. RBC, HGB and Ca in blood were decreasing since 72h after PT, but increased slightly at 7d. Blood AMS increased at 6h, and hit the peak at 24h, then decreased after this. Blood LPS also increased at 6h, but its peak time was 72h, and was at high levels at 7d.While ascites AMS and LPS were both at high level after 7d.Clinical significance: Blood AMS can be the early diagnosis criteria. Blood LPS can be the specific diagnosis criteria. But AMS and LPS can’t be the criteria of prognostic judgment, only be the indexes of auxiliary diagnosis.6. Apoptosis appeared at 6h and it was the peak time by TUNEL method and FCM technology detection. The condition of PI decreasing at that time illustrated that cells were dying at the beginning after PT. After 24h, AI was decreasing and PI was increasing, and was to peak at 7d.Those demonstrated that the cell repair mechanism was activated at 24h, and it lasted to the later period with destroy mechanism after PT.7. The activity of PLA₂ in blood and pancreas tissue increased at 6h after PT, and its peak time was 24h, then deceased but still to remain higher level than that of control group. This change is more obvious in pancreas than blood. For the activity of PLA₂ in blood also increased, we can detect it from 6h to 7d after PT.8. The level of mRNA and protein expressed were same: the most increased gene was sPLA₂ after PT, then cPLA₂, last one was iPLA₂.All subtypes of PLA₂ were increasing at early after PT. The peak time of cPLA₂ and sPLA₂ was 24h, but iPLA₂ was 72h.The reason maybe is cPLA₂ and sPLA₂ are mostly produced in pancreas, but iPLA₂ is out it.9.The organization fixing of apoenzyme subtypes by IHC: The most increasing expressing were cPLA₂ and sPLA₂ in pancreas, they were found all over in the pancreatic acinar cells in 6h after PT, and spread out from the cells in 24h,then decreased after 72h.But the changes of iPLA₂ organization fixing wasn’t obviously. Associated with the above experimental results, we can say iPLA₂ isn’t the chief causative agent after PT.10. Afer the sponge extractive, Variabilin was applied to the PT model, we found that the activities of PLA₂ in serum and pancreas tissue were inhibited by Variabilin at 24h after trauma, the pancreas tissue swell was lessened under CT, and the morphous and organelle of pancreatic cell were protected by Variabilin under the light and electron microscope. Variabilin can inhibit the activities of PLA₂ in serum and pancreas tissue in vivo and has protective function against acute pancreatic trauma in rat by intra-abdominal administration.In a word, the main results of our study are as follows:1. The pancreatic trauma model in Wistar rat was founded. It is efficient, fine repeatable, economic and ideal for pathologic and therapeutic study further.2. The period from 6h to 24h after PT is the traumatic condition aggressive stage. the cell repair mechanism was activated at 24h,and it lasted to the later period with destroy mechanism after PT.3. The activity of PLA₂ in blood and pancreas tissue increased after PT, mostly for the cPLA₂ and sPLA₂ produced a marked effect.4. Variabilin can inhibit the activities of PLA₂ in serum and pancreas tissue in vivo and has protective function against acute pancreatic trauma in rat by intra-abdominal administration.更多还原