【作者】 龚加庆；

【导师】 田伏洲；

【作者基本信息】 第三军医大学 ， 普通外科， 2011， 博士

【摘要】 一、研究背景急性胰腺炎(acute pancreatitis,AP)是临床常见的急腹症,临床经过复杂、变化迅速、其中约10~20%的患者发展为重症急性胰腺炎(severe acute pancreatitis,SAP)。SAP死亡率高,预后欠佳,死亡率高达15~20%,特别是早期重症胰腺炎(early severe acute pancreatitis,ESAP)合并多脏器功能障碍综合征(MODS)死亡率更高。急性重症胰腺炎被认为本世纪尚待攻克的生命科学的一大难题。如何遏制重症胰腺炎的进行性恶化,促进胰腺损伤的快速修复,近年来,学术界对胰腺炎的发病机制及相应治疗进行了大量的研究和深入探讨,取得了一定成果,但至今还未能够对这个病理过程作出一个完整的、满意的解释。上世纪末、本世纪初,随着干细胞研究逐渐成为热点,胰干细胞参与胰腺损伤修复也备受人们关注。干细胞(stem cell)为一类不分化而长期生存且保持多种分化潜能的细胞群;祖细胞(progenitor cell)为一类分化能力、自我更新、自我维持能力受限制,只有单向或双向分化潜能且生存时间相对较短的细胞群。目前,围绕着干细胞参与胰腺组织的损伤修复主要存在的争论:(1)成体胰腺组织内是否存在胰干细胞?(2)胰干细胞的来源和存在的部位;(3)胰干细胞参与胰腺修复的主要方式和机制。有学者认为成体胰腺组织内原位干细胞可能存在于胰岛、胰腺导管和腺泡上皮。然而也有学者对胰腺组织内存在原位干细胞的观点提出质疑,指出所谓胰腺干细胞实际来自骨髓间充质干细胞(marrow mesenchyme stem cells MSCs)。成体胰腺组织内是否存在原位干细胞?胰腺损伤时,组织内必然伴随有胶原纤维的增生、沉积,干细胞又如何在这种情况下参与组织的损伤修复?我们拟建立大鼠急性胰腺炎动物模型,并对腺泡细胞凋亡进行调控,进一步观察损伤组织胶原的形成和胰干细胞参与组织修复的过程。二、目的本实验通过建立大鼠急性胰腺炎模型,拟行如下内容探讨:(1)大鼠急性胰腺炎的自然病理演变规律;(2)胰腺炎病理过程中腺泡细胞凋亡演变规律及地塞米松对细胞凋亡的影响;(3)通过对胰腺干细胞特异性抗原进行免疫组化染色,探讨胰干细胞在成体胰腺组织内的定位及参与修复时的迁移规律,同时通过realtime-PCR(RT-PCR)技术对不同时段PDX-1mRNA予以实时定量,从基因角度探讨关键因子PDX-1对胰干细胞参与损伤修复时的调控作用;(4)通过对5-溴-2’脱氧脲嘧啶核苷(Brdu)这一特异性细胞增殖抗原进行免疫染色,探讨干细胞参与修复时的增殖规律;(5)探讨Ⅰ型胶原在胰腺损伤时组织内的分布规律,并对其重要调控基因TGF-β1 mRNA进行RT-PCR定量分析,探讨TGF-β1对Ⅰ型胶原合成的调控作用。通过这一课题研究,我们拟初步阐明胰干细胞在胰腺组织中的定位,并进一步阐明胰腺泡细胞凋亡和胰干细胞在胰腺急性损伤修复中的作用,为急性胰腺炎的治疗从细胞学角度开辟一条新的途径。三、方法(一)分组及操作方法清洁级SD大鼠84只,随机分对照组24只、实验组30只和干预组30只,各组再随机分为6个亚组,各亚组分别为4、5、5只。(1)实验组和干预组予以雨蛙素腹腔注射行急性胰腺炎造模,50ug/Kg体重,1次/h,连续4次;(2)对照组于同一时间点腹腔注射生理盐水0.5ml;(3)干预组腹腔注射地塞米松0.5mg/kg,1次/d;(4)三组分别于造模后6h、1d、2d、3 d、5 d、7d各处死一个亚组;(5)处死前6、3 h分别腹腔内注射Brdu标记胰腺增值细胞,100mg/kg体重。(二)手术方法乙醚吸入麻醉,打开腹腔,观察腹腔内各脏器及腹水情况;心尖部穿刺抽血2～4ml,备淀粉酶检测;取胰腺中部组织约200μg,液氮灌内保存,备PCR检测;取胰腺中部组织约500mg,甲醛溶液中固定,备HE染色、凋亡及免疫组化检测。(三)检查项目及方法(1)自动生化系统检测血清淀粉酶;(2)常规HE染色观察胰腺组织的病理变化;(3)按凋亡试剂盒说明行胰腺组织凋亡细胞的检测;(4)免疫组化方法观察胰腺干细胞标志物Nestin、C-kit蛋白和细胞增值蛋白Brdu的表达情况,并观察Ⅰ型胶原在胰腺组织内的分布规律;(5)RT-PCR方法检测胰干细胞重要调节因子胰腺十二指肠同源盒1(PDX-1)基因不同时段的表达量;(6)RT-PCR方法检测Ⅰ型胶原的重要调控因子不同时段TGF-β1的表达量。三、结果(一)病理改变对照组大鼠胰腺呈粉红色,组织柔软,无水肿,光镜下胰腺组织结构清晰。干预组和实验组病理改变享相似,造模6h～1d,腹腔内少量清亮腹水,胰腺水肿明显,镜下可见腺泡细胞水肿明显,腺叶内散在大量空泡样结构,空泡样结构数量于6h～1d达到高峰,1天后,空泡样结构逐渐减少;叶间距明显增宽,叶间区可见少量炎性细胞聚集;造模2~3d,胰腺水肿较前明显减退,可见散在的出血点,镜下可见叶间血管破损,叶间区大量红细胞聚集,部分胰腺小叶呈现片状坏死,大量炎性细胞在坏死区域聚集;造模5~7d,胰腺水肿消退,呈片状暗红色,镜下可见叶间区和腺泡间红细胞聚集明显减少,坏死区域和正常腺泡细胞界限明显,坏死区域内炎细胞大量聚集,腺泡间隙基本恢复正常,空泡样结构少见。(二)凋亡的结果分析正常胰腺组织内可见极少量凋亡腺泡细胞。实验组和干预组大鼠胰腺组织内,凋亡细胞的细胞核和凋亡小体均呈棕黄色;部分凋亡细胞的细胞膜裂解,发生融合,留下一较大空泡区,其内散在数量不等的凋亡小体;实验组和干预组于全程均可发现有凋亡细胞出现;实验组和干预组凋亡趋势基本一致,两组凋亡细胞指数于6h～1d即迅速升高,于第1d达到最大,以后逐渐降低;实验组和干预组凋亡指数均明显高于同期对照组(P<0.05)。(三)血清淀粉酶检测结果正常对照组大鼠血清淀粉酶为1200～1900 U/L,实验组和干预组的淀粉酶走势基本相似。造模6h,实验组和干预组淀粉酶迅速升高,达到病程的最大值,从第1d起,淀粉酶持续下降,第7d,两组淀粉酶接近正常值。从6h～3d,实验组和干预组淀粉酶均明显高于同期对照组(P<0.05),而第7d后,三组淀粉酶值同期无明显差异(P>0.05)。(四)Nestin阳性细胞的分布特征正常胰腺组织内nestin蛋白呈现少量表达;造模6h,nestin阳性细胞主要在叶间血管腔内呈现表达,部分细胞沿血管腔外呈两条平行线分布;造模1～2d,nestin阳性细胞从叶间区向腺小叶和胰岛内扩散迁移;第3d,胰腺小叶内可见大量nestin阳性细胞,且胰岛内阳性细胞也明显增多;第5～7d,nestin阳性细胞在胰腺小叶及胰岛内分布数量持续减少。(五)c-kit阳性细胞的分布特征c-kit阳性染色呈棕黄色,正常组织鲜有c-kit阳性染色细胞。造模6h～7d,c-kit阳性染色始终位于胰岛内,其染色趋势从淡染到浓染,再到淡染,胰岛外未见有阳性染色。(六)Brdu阳性细胞的分布特征正常组织内Brdu阳性细胞非常少见;造模6h~1d,腺泡组织内鲜见有Brdu阳性细胞;第2~3d,叶间区周围可见少量Brdu阳性细胞分布;第5d,胰腺小叶及胰岛内可见大量Brdu阳性细胞分布,炎性细胞的“消散区”,Brdu阳性细胞呈现大量分布;第7d,正常胰腺组织内少有Brdu阳性细胞,炎性细胞消散区,Brdu阳性细胞连接成片。(七)Ⅰ型胶原阳性蛋白的分布特征正常组织内仅于组织间隙见少量阳性染色组织。造模6h,腺泡细胞内即可见阳性染色,主要位于腺叶的周边,颜色淡染;造模1~7d,阳性染色分布位置相对不变,但颜色逐渐浓染,部分呈条带状分布,腺叶中心区淡染,胰岛内未见有阳性染色细胞。(八)PDX-1 mRNA的表达正常胰腺组织内PDX-1 mRNA呈阳性表达,但表达量较低,造模开始后,PDX-1基因的表达量呈逐渐增高趋势,于第3d达到高峰,此后,表达量逐渐降低,但各时间点相对于同时间点的对照组均显著升高(p<0.05)。(九)TGF-β1 mRNA的表达正常胰腺组织内TGF-β1 mRNA呈阳性表达,但表达量较低,相对灰度值于造模6h即开始逐渐升高,到第7d达到高峰,各时间点相对于同时间点的对照组均显著升高(p<0.05)。四、结论(一)腺泡细胞凋亡是胰腺炎发生后的又一瀑布链式反应,是胰腺组织的一种自我防护反应,伴随急性胰腺炎病理全程,以胰腺炎早期最为明显;病理早期的空泡结构很可能是腺泡细胞凋亡后的残留空间;地塞米松对腺泡细胞的凋亡没有明显的促进作用。(二)鉴于腺泡细胞凋亡在胰腺炎病理过程中的重要作用和地位,可将传统胰腺炎的病理分期更改,即水肿凋亡期、出血坏死期、愈合期三期,这样更有助于对胰腺炎病理转归的理解。(三)成体胰腺组织内可能不存在原位干细胞,参与胰腺损伤修复的胰干细胞实际来源可能为MSCs。(四)C-kit阳性细胞可能是一种胰岛祖细胞,仅参与本身胰岛的损伤修复,不具备有迁移特性。(五)PDX-1 mRNA可能在干细胞参与胰腺损伤修复的调控过程中发挥重要作用。(六)Ⅰ型胶原对胰腺损伤修复起到重要作用,TGF-β1 mRNA是参与胰腺损伤修复过程中胶原纤维沉积的重要调控基因。更多还原

【Abstract】 BackgroundAcute pancreatitis is a common clinical acute abdomen.10~20% of the patients develop into severe acute pancreatitis(SAP)。The mortality rate of SAP is about 15~20%,and the prognosis is unfavorable . Especially, The mortality rate of the early severe acute pancreatitis(ESAP) combined with multi-organ dysfunction syndrome (MODS) is more higher. So, SAP is considered one of the unsolved problems of life science in 21st century. How to prevent the progressing aggravation of SAP, and promote quickly reparation of damaged pancreas tissue? In recent years, some authors have made large of penetrating studies for pathogenesis and corresponding therapy methods in pancreatitis, and they have get some outcomes. However, the pathological change can’t be explained perfectly and satisfactorily so far. The research on stem cell has gradually become a hot point in recent ten years. And so, more and more scholars pay close attention to the research on pancreatic stem cell which are involved in the repair of damaged pancreatic tissues..The stem cell is a kind of cell which can keep alive for a long term and has a few of differentiation potencies. While, the progenitor cell is a kind of cell keeping one or double ways of differentiation, and the capability of its differentiation, self-renew and self-maintenance is confined. For the survival time, the progenitor cell is shorter than the stem cell. So far, there are some problems which can not be solved in stem cell involved in pancreas injury reparation. (1)Is really there primary stem cell in adult pancreatic tissue? (2)Where is the stem cell in pancreatic tissue? (3)How does the stem cell involve in pancreas injury reparation? Some authors found that the primary pancreatic stem cell allocates in pancreatic islet, duct epithelium, and gland alveolus epithelium. However, some authors have showed their suspicious that there may be no primary pancreatic stem cell in adult pancreatic tissue, and they were sure that the pancreatic stem cell come from the marrow mesenchymal stem cell actually. Is really there primary pancreatic stem cell in adult pancreatic tissue? The collagen fiber inevitably proliferate and deposit in tissue when pancreas is damaged. In this situation, what a role does the stem cell have in the pathophysiologic process of acute pancreatitis(AP)? In this study, we will establish rat AP animal model, regulate the apoptosis of acinous cell, and observe the pathophysiologic process that the fiber and pancreatic stem cell are involved in pancreatic injury reparation.Objective In this study, we will establish rat AP animal model, and approach the contents as follow: (1) The natural pathophysiologic progress of AP. (2) The apoptosis rule of acinous cell and the influence of dexamethasone for apoptosis in the pathophysiologic process of AP. (3) Approaching the location of primary stem cell and migrating rule of stem cell in damaged pancreatic tissue with immunohistochemistry of specific antigen of pancreatic stem cell. (4) Approaching the generating rule of pancreatic stem cell with immunohistochemistry of Brdu, which is the specific proliferative antigen of cell. (5)Approaching the distribution rule of typeⅠcollagen in pancreatic injury tissue, and approaching the regulatory role of TGF-β1 for typeⅠcollagen with real-time PCR quantitative analysis for TGF-β1 mRNA, which is the important controlling gene of typeⅠcollagen. In this study, we will initially elucidate the location of primary stem cell in the pancreatic tissue, and then, elucidate the role of apoptosis of acinous cell and pancreatic stem cell in pancreatic injury reparation. And further, we could be develop a new way of therapy for AP from cytology.Methods1. Grouping and management methods Eighty four SD rats were randomly divided into experimental (30 rats), interventional (30 rats) and control groups (24 rats). Every group was randomly and averagely divided into 6 sub-groups too, and every sub-group has 5,5,and 4 rats respectively.(1) The experimental and interventional group were given intraperitoneal injections of cerulein to induce acute pancreatitis, 50ug/Kg, once a hour, and continuously 4 times. (2) The control group was given intraperitoneal injections of 0.5 ml normal sodium in the same time. (3) The interventional group were given intraperitoneal injections of dexamethasone, 0.5mg/kg, once a day. (4)At 6 h, 1 d, 2 d, 3 d, 5 d and 7 d following pancreatitis induction, one sub-group rats from every group were sacrificed. (5) Afterwards, 5-bromo-2’-deoxy-uracil nucleotides (Brdu) were intraperitoneally injected 6h and 3h prior to sacrifice in order to label the proliferative pancreatic cells, 100mg/kg once.2. Operational methodsThe rats were anesthetized by aether inhalation. When opened the abdominal, the organs and ascitic fluid were observed; 2~4ml blood was drew from apex of heart, which can be ready for amylase detection; 200μg tissue were obtained from middle of pancreas for RT-PCR, which was stored in liquid nitrogen; 500mg tissue were obtained from middle of pancreas for HE, apoptosis and immunohistochemistry, which was stored with aldehyde fixation.3. Inspection items and methods(1) Serum amylase with automatic biochemistry inspection system; (2) The pathological changes of pancreatic tissue were observed through conventional HE staining.; (3) The cell apoptosis of pancreatic tissue was inspected according to the directions of apoptosis kit; (4) The pancreatic stem cell marker Nestin, C-kit, and the cell proliferation marker Brdu, and then the typeⅠcollagen were detected with immunohistochemistry; (5)The expression level of an important pancreatic stem cell regulatory factor, pancreatic duodenal homeobox 1 (PDX-1), was determined by real-time PCR; (6) The expression level of an important typeⅠcollagen regulatory factor, TGF-β1 was determined by real-time PCR.Results1. The pathophysiological change The pancreatic tissue of control group display pink, soft, and no edema. Under a light microscope, the construction of pancreatic tissue was clear. The pathophysiological progression of experimental and interventional group is similar. Between 6h to 1d, there was small amounts of bright ascites in the abdominal cavity, and the specimens generally display a high degree of edema without obvious hemorrhage. Under a light microscope, acinar cells showed apparent edema with a lot of vacuole-like structures dispersed in the pancreatic acini and islets. The peak amount of vacuole-like structures occurred in 1d. After Day 1, the amount of vacuole-like structures gradually decreased. The interlobular space significantly increased, which was likely caused by inflammatory edema. A small number of inflammatory cells were found to be clustered in the interlobular region and the cells gradually spread between the acini. On 2~3d, the edema of the specimens was significantly diminished compared to the edema observed in the previous phase with dispersed hemorrhagic spots. Interlobular vascular damage was detected with a microscope along with an effusion of red blood cells from the vascular lumen. A large number of red blood cells accumulated in the interlobular region. Some of the pancreatic lobules exhibited patchy necrosis. Acinar-like cells were rarely observed in the necrotic regions, but large amounts of inflammatory cells were found in these areas. On 5 ~7d, pancreatic edema subsided and there were patches of dark red. The reduction in the number of red blood cells accumulated between the acini and in the interlobular region was observed under a microscope. A clear boundary was detected between the necrotic area and the normal acinar cells. Large quantities of inflammatory cells were found in the necrotic region. The interacinar space returned to normal and vacuole-like structures were rarely observed.2. The apoptosis analysisFew apoptosis cells could be seen in normal pancreatic tissue. The apoptosis progression of experimental and interventional group is similar. In damaged pancreatic tissue, the apoptosis cell nucleuses and apoptotic bodies were stained buffy; The cell envelopes of some apoptosis cells had splited, and mixed together, then displayed a big vacuole-like structures, in which some apoptotic bodies could be seen; In any time, the apoptosis cells could be seen in pathophysiological progression; The apoptosis index number (AI) went up quickly after 6h, and the peak amount of AI occurred in 1d. After 1d , AI gradually decrease. The AI of experimental and interventional group was obviously higher than control group in the same time(P<0.05).3. Serum amylase analysisThe quantity of Serum amylase in control group was 1200～1900 U/L; The tendency of Serum amylase in experimental and interventional group was alike; On 6h, the quantity of Serum amylase in experimental and interventional group went up sharply, and got the peak; From 1d, the Serum amylase decreased , and which got to normal in 7d. From 6h~3d, the quantity of Serum amylase in experimental and interventional group was more higher than the control group(P<0.05). However, there was no obviously difference in the 3 groups after Day 7 (P>0.05).4. The distribution characteristics of nestin-positive cellsThe nestin protein was expressed at a low level in normal pancreatic tissues. Six hours after the disease model prepared, the nestin-positive cells were mainly found in the lumen of interlobular vessels in clusters. A small number of positive cells were found around the vascular cavity. One to two day after the cerulein was injected, the nestin-positive cells were still primarily distributed in the interlobular region and in the vascular cavity surrounding the tissues. Some of these cells had an outside distribution along the vascular lumen in two parallel lines. On Day 3, large numbers of nestin-positive cells were found in the pancreatic lobules, and the positive cells in the pancreatic islets increased significantly. On 5~7d, the number of nestin-positive cells were significantly reduced within the pancreatic lobules and the islets. Few nestin-positive cells were observed in the interlobular region and within the lumen of the interlobular vessels.5. The distribution characteristics of C-kit positive cellsThere were few C-kit positive cells in normal pancreatic tissue; In pathophysiological progression(6h~7d), C-kit positive cells could only be found in pancreatic islets all the time, and the color was stained from light to thickness, and then light. The positive cells could not be seen out of islets all along.6. The distribution characteristics of Brdu-positive cellsFew Brdu-positive cells could be seen in normal pancreatic tissue; 6h~1d after the pancreatitis model prepared, there still were few Brdu-positive cells in tissue; On 2~3d, a small amounts of Brdu-positive cells could be found in the interlobular region; On 5d, there were large number of positive cells in glandular lobule, and too, amounts of Brdu-positive cells distributed in the region of few inflammatory cells, and some positive cells connected to sheets. On 7d, there were few positive cells in relatively normal pancreatic tissue, and larges of Brdu- positive cells occupied soundly in the region of few inflammatory cells.7. The distribution characteristics of typeⅠcollagen proteinFew positive protein could be found in normal pancreatic tissue. 6h after the pancreatitis model prepared, the positive stain firstly appeared in acinar cells around the glandular lobules, and which were stained lightly. From 1d to 7d, the position of positive stain kept in the same region. howevere, the color of positive cells gradually become thick, and positive cells gradually distributed to straps. The centre of glandular lobule showed lightly stained, and there were no positive stain in pancreatic islets all the time.8. The expression of PDX-1 mRNAPDX-1 mRNA was positively expressed in the normal pancreatic tissues, but at a low level. After pancreatitis was induced, the expression level of the PDX-1 gene gradually increased and reached its peak on Day 3. The expression gradually decreased afterwards and got close to a normal level on Day 7. The relative quality of expression of experimental group was more higher than which of control group in the same time(p<0.05).9. The expression of TGF-β1 mRNA TGF-β1 mRNA was positively expressed in the normal pancreatic tissues, but at a low level. After pancreatitis was induced, the expression level of the TGF-β1 gene gradually increased and reached its peak on 7d. The relative quality of expression of experimental group was more higher than which of control group in the same time(p<0.05).Concludes1. The apoptosis of acinar cells is a chain reaction like waterfall in pathophysiological progression of AP all the time, and which reaches a peak at the early stage of inflammation due to the self-protective mechanism of pancreatic tissues against self-digestion; The vacuole-like structures in early stage of inflammation are probably the remaining spaces after apoptosis of acinar cells; Dexamethasone can’t obviously promote apoptosis for acinar cells.2. Because of the very important role of apoptisis for acinar cells, The pathological changes of AP can be divided into three phases: the edema and apoptosis phase (6 h– 1 d), the hemorrhagic necrosis phase (2 d– 3 d) and the reconstruction phase (5 d– 7 d). which can offer more help to understand the pathophysiological progression of AP.3. Primary pancreatic stem cells may not exist in the adult pancreatic tissues. The so-called pancreatic stem cells, or nestin-positive cells, may actually originate from bone marrow mesenchyme stem cells.4. The C-kit positive cells are a kind of progenitor cells of pancreatic islets, which only are involved in the repair of damaged pancreatic islets, and have no migrating characteristic.5. PDX-1 mRNA plays an important regulative role in which the stem cells are involved in the repair of damaged pancreatic tissue.6. TypeⅠcollagen plays a important role involving in the repair of damaged pancreatic tissue, and TGF-β1 mRNA is the critical regulative gene of TypeⅠcollagen.更多还原