【作者】 章波；

【导师】 栗永萍；

【作者基本信息】 第三军医大学 ， 军事预防医学， 2003， 博士

【摘要】 肠上皮是实现肠道消化、免疫、内分泌等功能的主要组织细胞，也是构筑肠道粘膜屏障的主要成分，同时它对辐射敏感。作为腹腔恶性肿瘤治疗主要手段之一，电离辐射对正常小肠上皮细胞具有毒副作用，限制了其在治疗上的应用。超过一定剂量照射可形成的肠型放射病，导致病死率极高，故对辐射诱导的小肠上皮损伤的研究受到极大的关注。为深入阐明电离辐射致肠上皮损伤的分子机制，寻找促进肠上皮损伤修复和具有辐射保护作用的蛋白质，本研究以离体培养的IEC-6细胞（正常大鼠空肠上皮细胞）和小鼠9Gyγ-射线全身照射所致肠型放射病为模型，采用双向电泳技术分别比较了IEC-6细胞和小鼠小肠上皮细胞照射前后蛋白质表达的差异，通过MALDI-TOF-MS鉴定双向电泳图谱上差异的蛋白质点，采用Western blot和RT-PCR技术进行了部分验证，并集中研究了两个差异蛋白质PrxI和ERP29在电离辐射中的作用。取得了如下的主要结果：一、 损伤模型的确定利用IEC-6细胞作为体外研究模型是合理可行的。首先根据IEC-6细胞在不同照射剂量照射（15-35Gy）和照射后不同时相（1-3d）细胞增殖以及细胞凋亡量来确定合适的照射剂量。结果显示：利用25Gy照射后1天的细胞来研究细胞对电离辐射的反应是较为适合的。体内研究是以肠型放射病为模型。采用不同剂量的γ-射线（7-10Gy）全身照射小鼠，复制肠型放射病模型。结果表明：9Gy照射可导致小鼠出现典型的肠型放射病。9Gy照射3h后，小肠上皮隐窝高度和绒毛高度未见明显缩短，但细胞核分裂相消失，呈严重的损伤性改变；照射后72h，绒毛明显缩短，隐窝明显增高，表明有大量再生上皮细胞，以增殖修复为主。因此，该条件下小鼠能存活一定时间，其早期辐射损伤严重，后期修复明显，是较为适合的研究模型。利用含尿素的细胞裂解液能够较为完全地提取细胞的总蛋白，适合双向电泳制备蛋白样品的要求。二、 IEC-6细胞照射前后蛋白质表达谱的差异采用宽pH范围3-10L的干胶条（IPG、18cm），上样量为500-1000(g，聚焦6-8h，千伏时不低于32000。各选取3张照射前后的蛋白图谱，应用PDQUEST软件进行图像分析，在pH3-10L和Mr 14000-97400Da范围内检测蛋白。电泳图像经过PDQuest处理后，结果表明：在正常IEC-6细胞中检测到608±40个蛋白点，细胞照射后的样<WP=9>品中检测到595±31个蛋白点，其中匹配的蛋白点为396个，电泳图谱的相关系数为0.78。然后，选择了部分差异明显的蛋白点进行肽质量指纹图谱鉴定。在未匹配的蛋白点中选择清晰、较浓的点做好标记，从凝胶上准确切割后进行脱色、酶解、肽片段提取，并测定肽质量指纹图谱。根据质谱图中的肽片段质量进行检索，再结合双向电泳中蛋白点的表观分子量、等电点进行综合分析。本实验共对16个蛋白点进行了肽质量指纹图谱分析，其中11个蛋白点得到相应的蛋白质，这包括一些与代谢、自由基清除、细胞骨架相关的蛋白。其中，电离辐射后降低的蛋白质包括：STRESS-70 PROTEIN，Tubulin beta-5 chain，ATP SYNTHASE BETA CHAIN，Vimentin，PDZ and LIM domain protein 1等；而PEROXIREDOXIN 1，ERP29，Peptidyl-prolyl cis-trans isomerase，RGS4，Aldehyde dehydro- genase（mitochondrial precursor）等在照射后升高。在此基础上，对其中部分蛋白质进行了验证。Western blot结果显示：STRESS-70 PROTEIN在照射后确实被降低了。RT-PCR结果显示：ERP29基因在照射后基因表达增高，这与其蛋白质在照射后升高相吻合。三、 小鼠全身照射前后小肠上皮细胞蛋白质表达谱的差异在9Gy的剂量照射后，分离小肠上皮细胞，并采用Percoil细胞分离液对上皮细胞进行了纯化，得到相对较为单纯的肠上皮细胞。利用含尿素的细胞裂解液提取细胞的总蛋白，对同一样品进行多次电泳。总蛋白的分布模式非常相似：蛋白质斑点相对集中于pH4~7，极端酸性蛋白与碱性蛋白数量较少；蛋白质相对分子质量集中于（15~100）×103，超出此范围的斑点较少。采用PDQuest软件比较各组图谱，在正常对照组、照射后3h组以及照射后72h组分别检测到638±39、566±32和591±29个蛋白质点，总蛋白质点在照射后有所减少。照射后3h组与正常对照组相比有360个点相匹配，电泳图谱的平均相关系数为0.78；照射后72h组与正常对照组相比有312个点相匹配，电泳图谱的平均相关系数为0.82；照后3h组与照后72h组相比有282个相匹配，电泳图谱的平均相关系数为0.76。不匹配的斑点主要为低丰度蛋白质。在差异的蛋白点中选择清晰、较浓的点做好标记，从凝胶上准确切割后进行脱色、酶解、肽片段的提取，并测定肽质量指纹图谱。根据肽质量指纹图中肽片段的质量数进行检索，再结合蛋白质点的表观分子量、进行综合分析。共对28个蛋白点进行了肽质量指纹图谱分析，19个蛋白点得到相应的蛋白质，这包括一些与代谢、过氧化应急、信号转导相关的蛋白。其中，电离辐射后降低的蛋白质包括：Transforming protein RhoB，Antioxidant Protein 2，Q8VC72，O70619等；而PEROXI- REDOXIN 1，ERP29，Rho GDP-dissociation inhibitor 1，Alpha enolase，Prolactin receptor precursor，Glutathione S-transferase P2，Truncated更多还原

【Abstract】 Intestinal epithelial cells (IECs) are the major component of the intestinal mucomembranous barrier, and play important roles in the body owing to its many kinds of functions such as absorption, immunity, and secretion, and so on. However, IECs are sensitive to ionizing radiation (IR), and they are one of the main targets in case of whole-body irradiation. Although much success has been documented with pelvic cancer patients, certain side effects and complications have been encountered which limit its applications in cancer radiotherapy. Intestinal wound induced by large dose of IR can form, thus make the intestine lose its barrier function. This results in gastrointestinal syndrome including intestinal infection of endogenous bacteria, diarrhea and bloody stools. It is the main reason of high death rate of intestinal-type radiation disease. However, its etiology remains unclear. Some studies have showed intestinal epithelial can reconstruct to some extent after IR (non-lethal). This suggested survival IECs can differentiate and re-epithelize intestinal wound. It is possible to study the proteins related to injury and reconstruction of small intestinal epithelium irradiated by γ-Ray. With the accumulating evidence in the literature that new proteins are found to be implicated in radiation response, the molecular mechanism underlying the radiation response of the small intestine remains unknown.Our aim was to identify those proteins in the early-stage of irradiation injury. To achieve this purpose, we have chosen to use comparative proteome approach to identify mouse proteins whose expression is regulated by ionizing irradiation. First, we compare the proteome of normal intestinal epithelial cell line IEC-6 with that of irradiated one at 24h post irradiation. Then, the proteomes of sham irradiated mice and irradiated mice were compared at 3h and 72h post-irradiation. The differential displayed proteins were subject to MALDI-TOF-MS to establish identity. Furthermore, we confirm some differential proteins with the technique such as Western blot and RT-PCR. At last, we focus on the function of 2 proteins, which are up-regulated by ionizing irradiation. New clues are provided to understand the molecular mechanism of the intestinal type of radiation sickness. Main results are listed below.<WP=5>1. Establishment of experimental models It is reasonable and availble to use rat IEC-6 cell line as a vitro model. Dose and time-point was chosen based on our preliminary data of cell death rate and apoptosis rate under different radiation dose and time course. Our results demonstrated that a dose of 25Gy and 24hr post-radiation were suitable to study the proteome of ICE-6 cells. The extent of mouse intestinal injury post-radiation was dose-dependent. Our data showed whole-body radiation at a dose of 9Gy could lead to intestinal type of radiation sickness. Under this condition, the height of intestinal crypts and villi were unchanged 3h post-radiation. However, cell division of intestinal epithelial ceased, and intestine showed severely morphological injury. Further study showed the height of intestinal crypts was obviously increased 72h post-radiation, with villi decreased. This indicated many survival epithelial cells proliferated. 2. Compare the proteome of IEC-6 cell line post-irradiationIsolated total proteins of IEC-6 cells and irradiated cells, and separated them according to the guide-book of 2-dimensional electrophoresis. For isoelectric focusing (IEF), precast IPG strips were used. Samples were applied via rehydration of IPG strips in sample solution more than 12 hours. 500-1000ug of protein was loaded onto an 18cm IPG strip (pH3-10, linear), and isoelectric focusing was run for 32 KVh at a focusing temperature of 20℃. For the second dimensional separation, the concentration of homogeneous SDS-polyacrylamide gels was 13%. We separated the same sample for 3 times. After electrophoresis, the resolved proteins in the 2-DE gels were visualized by Coomassie blue R-250. The gels were scanned, and the ima更多还原