【作者】 张海波；

【导师】 夏求明；

【作者基本信息】 哈尔滨医科大学 ， 外科学， 2002， 博士

【摘要】 背景:在当前医学界,细菌感染性疾病仍然占有全世界的第一位的发病率,极大地威胁着人们的身体健康安全。目前细菌感染的表现也呈现着多样化的形式,不仅仅局限于经典意义上的感染和炎症,而是出现包括器官移值后感染、慢性感染、感染后细胞恶性改变、感染后冠状动脉粥样硬化、HIV等后天性免疫系统抑制性感染、细菌耐药性感染、机会致病菌的感染等等许多表现形式。尤其应该指出的是,在当前医学治疗中普遍存在的抗生素泛滥应用的现象,使得尽管抗生素不断推陈出新,仍然解决不了细菌的耐药问题,反而加大了抗菌素的毒副作用。往往是一种新型抗生素临床试用了一段较短的时间后,就发现有耐药菌株的出现,给临床治疗感染性疾病带来了令人头痛的难题。尽管全世界的很多科学家们都致力于这个问题的研究,但目前对这种问题还没有一个较理想的解决办法。自然抗菌多肽在地球上多种动植物体内都广泛性的存在,从植物的种子到低等无脊椎动物,一直到人类的体内都存在着这类结构上具有高度保守性结构的抗菌多肽。人防御素是其中非常重要的一类天然抗菌性多肽。它们是生物体在长期的进化过程中演化出来的一类与以往的抗生素不同的高效广谱抗菌性多肽,而且有成分天然,取之于人可用之于人,不易引起毒副作用和细菌耐药等问题的特点。因此是一种新型具有临床应用前景的抗菌性药物。另外,这类天然防御素的基因表达调控也与传统的抗生素不同,使由单基因进行表达的,因而使得转基因的治疗方法就成为可能,目的:1997年发现的人β防御素2（HBD-2）是首次发现的可诱生性的人防御素,对细菌、真菌、病毒和支原体都有具有强效广谱的杀灭作用。本研究目的是尝试构建HBD-2逆转录病毒载体对哺乳动物细胞进行转染,并从分子、蛋白、免疫、组织工程模型和动物模型等方面对转基因的细胞的生物学功能进行检测。方法:利用限制性酶切、低溶点琼脂糖电泳、核酸分子去磷酸化等分子克隆技术构建并扩增逆转录病毒载体pBabeNeo-HBD-2,结合Lipofectin法对多种哺乳动物细胞系进行转基因。用ELISA法检测HBD-2生成情况;提取细胞的mRNA,并制备p³²标记探针进行Northem Blot杂交,检测HBD-2的mRNA表达:利用CM Extraction和ZipTip技术由细胞培养液提取HBD-2,利用AU胶和Geloverlay技术检测生物学活性;利用Western Blot技术检测其免疫学活性;建立离体组织工程模型和SCID小鼠在体模型检测其抗菌生物学活性;并利用ELISA和RT-PCR技术对转染细胞生物学功能进行检测。结果:成功构建pBabeNeo-HBD-2载体,并从分子、细胞、蛋白质和免疫学等多个水平角度证明了HBD-2基因在多种哺乳动物细胞中转染成功,并可转录为mRNA,并表达合成HBD-2分子。而且无论在分子大小,电泳能力,抗菌活性,还是与抗体的特异性结合方面,都与标准的HBD-2是一致的。在离体和载体模型也显示其具有抗菌活性。结论:本研究在世界上首次对哺乳动物细胞进行HBD-2转基因的尝试,结果证明利用新构建的逆转录病毒载体成功地对多种细胞进行了转染,并且合成的HBD-2分子与标准的提取自人体组织的HDB-2是一致的,给今后对HBD-2的研究利用,以及转基因技术在器官移植后抗感染增强免疫能力等领域的应用开辟了一条新的途径,具有非常重要的研究和应用意义。更多还原

【Abstract】 Research on the construction of retrovirus vector pBabeNeo-HBD-2 and the gene transfer of HBD-2 into mammary cellsBackground: Infectious disease is still the most prevalent disease in the world, which consists variety of new challenges, including chronic infection, arthrosclerosis induced by infection, infection after transplantation, infection due to the resistance of bacteria, et al. Moreover, the overdose of antibiotics brings another problem that the resistant bacteria appears quickly after the administration of new kind of antibiotics. Defensin is a natural peptide, which were found among many kinds of creatures on the earth, and has a wide functional spectrum, such as bacteria, fungus, virus, PPLO, et al. The most interesting feature is that it is hard for the bacteria to generate a resistant type to the denfensins although they have met with each other for billions of years on the earth. Considering these properties the defensin really is a new kind of effective antibiotic.Objective: Human β defensin 2(HBD-2) is the first inducible human defensin which was discovered in 1997.This research is going to design a retrovirus vector carrying the gene of HBD-2 and infect different kinds of mammary cells. Then the functional properties of the cells and HBD-2 extracted were examined. And also the antibacterial biological function of transduced cells was examined in in vitro and in vivo model.Methods: The retrovirus vector pBabeNeo-HBD-2 was constructed and used to infect some kinds of mammary cells. The the genetic, protein, immune and biological functions were examined with Northern Blot, Western Blot, ELISA, CM Extraction, ZipTip and Geloverlay methods. And the in vitro tissue-engineering and SCID mice model were used to check the anti-bacteria function.Results: The retrovirus vector pBabeNeo-HBD-2 was constructed successfully and used to infect the cells. The HBD-2 extracted from the culture supernatant was proven to have the similar biological function as the original one from the human skin. And the anti-bacteria function were shown in both in vitro and in vivo model.Conclusions: The research is the first time to try to transfer the HBD-2 gene into mammary cell and examine its biological properties. It showed that many kinds of transduced mammary cell can express the functional HBD-2, which was similar as the original one from the human skin. This successful gene transfer of HBD-2 is the first step towards the clinical therapy use of defensins.更多还原