【作者】 伦立民；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2008， 博士

【摘要】 机体在受到外伤、炎症和病原感染时，机体发生广泛的生理反应，反应原激活机体的神经内分泌系统，释放儿茶酚胺(catecholamines，CAs)等一系列激素，从而引起机体的急性时相反应(acute phase response，APR)。使机体产生急性时相蛋白(acute phase protein，APPs)等炎症标志物。我们首先利用免疫组化和Western blotting方法，研究了AAT在文昌鱼的组织表达和定位，首次在头索动物文昌鱼中证明了AAT的存在，结果显示，AAT定位于文昌鱼肝盲囊的细胞浆内，从AAT在文昌鱼中的合成部位来看，在功能上文昌鱼的肝盲囊等同于脊椎动物肝脏，肝盲囊是在发育分化过程中消化道组织向右前方突出沿着咽后部向前扩展和延伸形成的一个囊袋状结构。长期以来，文昌鱼的肝盲囊被认为是脊椎动物肝脏的前体，支持了由M(u|¨)ller最早提出的脊椎动物的肝脏是由文昌鱼肝盲囊进化而来的观点。第二，我们用生物化学方法，选取典型的炎性刺激因子-LPS来感染文昌鱼，探讨了文昌鱼体液中的谷丙转氨酶(Alanine aminotransferase，AAT)、谷草转氨酶(Aspartate transaminase，AST)、谷氨酰转肽酶(r-Glutamyltransferase，GGT)、乳酸脱氢酶(Lactate dehydrogenase，LDH)、羟丁酸脱氢酶(α-HydroxybutyrateDehydrogenase，HBDH)、视黄醇结合蛋白(Retinol Binding Protein，RBP)和碱性磷酸酶(Alkaline phosphatase，ALP)七种炎性蛋白的急性时相反应，其中，首次在文昌鱼体液中发现了AAT、AST、GGT、LDH、HBDH和RBP六种炎性蛋白存在，并发现，LPS感染文昌鱼后，在不同时间文昌鱼体液中所含七种蛋白呈现了各自不同的变化趋势，总蛋白浓度在整个实验期内保持了基本恒定的水平，而AAT、AST、GGT、LDH、HBDH和ALP六种蛋白在实验周期内总体呈上升的趋势，其浓度均明显增加，呈正急性时相反应；而RBP实验周期内总体呈下降的趋势，呈负急性时相反应。这都表明：无论正急性时相蛋白还是负急性时相蛋白，在文昌鱼体内都呈现了类似高等哺乳动物急性时相反应特点。因此，我们认为类似哺乳类具有的急性时相反应体系已经存在于头索类动物中了。第三，我们用生物化学方法，选取海洋中广泛存在、众多水产动物的常见致病菌-溶藻弧菌(Vibrio alginolyticus)作为天然反应原感染文昌鱼，从另外一个角度探讨并丰富文昌鱼体液中的八种炎性蛋白的急性时相反应，结果显示，与LPS感染文昌鱼相比，六种正APPs的浓度变化幅度相对较小，曲线相对平缓，但仍然呈正急性时相反应，蛋白电泳也证实了这一点；而二种负APPs：RBP和白蛋白(Albumin，ALB)浓度实验周期内总体呈下降的趋势，呈负急性时相反应，结论与上述LPS相同。第四，我们用高效液相色谱法(High Performance Liquid Chromatography，HPLC)，探讨了LPS感染文昌鱼儿茶酚胺类(CAs)神经递质的变化，优化了同时测定文昌鱼体液中NE、E和DA三种CAs的HPLC方法，并首次发现，文昌鱼体液中同时存在NE、E、和DA三种CAs物质，其中，E在文昌鱼中首次报道，这三种物质在LPS感染文昌鱼后浓度升高非常明显，与同期的APPs相比变化更为迅速，也体现了神经递质的主导作用，文昌鱼的CAs系统也具有了类似脊椎动物CAs系统的功能，可以初步判断CAs系统的进化源头由脊椎动物可推回到头索类。总之，从文昌鱼的CAs类神经递质到文昌鱼机体的APPs，以及参与合成APPs的主要组织器官，这三个方面系统看来，头索类文昌鱼中已经具有了类似哺乳类动物所具有的急性时相反应体系。更多还原

【Abstract】 A prominent systemic physiological reaction of the organism was caused byinfection,tissue injury and trauma,and in turn nervous-endocrine system wasreactivated by reactants and released the catecholamines (CAs).Thus acute phaseresponse was caused in the organism involved in acute phase proteins (APPs).Firstly,the presence and location of AAT in amphioxus was studied by Westernblotting and immunohistochemical methods.The results first revealed that AATpresent in amphioxus,a cephalochordate.Also it was demonstrated that AATdistributed in cellular plasma in hepatic diverticulum,the pouch that protrudesforward as an outpocketing of the digestive tube and extends along the right side ofthe posterior part of the pharynx,which has long been considered to be the precursorof vertebrate liver.In vertebrate,AAT is primary synthesized in liver.In view of thefact that AAT synthesized in hepatic diverticulum of amphioxus,the hepaticdiverticulum of amphioxus is physiological equal to the vertebrate liver.Thissupported the Müller’s argument that vertebrate liver is evolved by the hepaticdiverticulum of amphioxus.Secondly,acute phase response and some APPs were studied in amphioxus.Itwas first revealed that presence of seven kinds of APPs in amphioxus humoral fluid,including Alanine aminotransferase (AAT),Aspartate transaminase (AST),Lactatedehydrogenase (LDH),Alkaline phosphatase (ALP),r-Glutamyltransferase(GGT) andα-Hydroxybutyrate Dehydrogenase (HBDH).Also it was demonstratedthat these proteins content changed respectively after the acute challenge withlipopolysaccharide,while the total protein remains basically unchanged.Among theseproteins,AAT,AST,GGT,LDH,HBDH and ALP increased dramatically after LPStreatment,while the other protein,RBP,decreased significantly.These resultsindicated that the similar acute phase response presented in eutherians has alreadyexisted in amphioxus,a cephalochordate.Thirdly,we studied acute phase response in amphioxus by detection of the change of the above mentioned acute proteins after got amphioxus infected by vibrioalginolyticus,a common disease-baring germ in sea water.The results revealed thatthe changes of these proteins extended the same trends after the disease germ infectedas the changes after LPS challenged.But changes in concentration ranges of six APPswere relatively small and six curves were relatively gentle.These results also provedthe presence of the same acute phase response profile in amphioxus,as observed ineutherians.Thus it pushed the evolutionary existence of the eutherian-like acute phaseresponse back to the protochordate lineage.Fourthly,we determined CAs,including epinephrine(E)、norepinephrin(NE) anddopamine(DA),with HPLC method optimized after LPS challenged amphioxus.Theresults demonstrated that E,DE and DA are present in amphioxus humoral fluid,andE was firstly found in amphioxus.Moreover,it was demonstrated that the threesubstances,E,DE and DA,increased dramatically and quickly after LPS treatment.Also,it was revealed that the changes of three substances were more quickly than thechanges of the above mentioned APPs during the same LPS treatment.Thesesuggested that the three substances existed as neurotransmitter.The presence and thechange of the CAs in amphioxus after treated with LPS indicated the evolutionaryexistence of CAs and acute phase response in the protochordate lineage.In short,from neurotransmitters of CAs to APPs of amphioxus,as well as themain organ involved in the synthesis of APPs,these all indicated that the similar acutephase response system presented in eutherians has already existed in amphioxus,acephalochordate.更多还原