【作者】 高广周；

【导师】 孙涛； 金博； 浦江；

【作者基本信息】 第二军医大学 ， 内科学， 2010， 硕士

【摘要】 肠功能障碍可使整个机体处于较为严重的营养不良状态。而在目前的治疗方案中,肠外营养支持可改善患者的营养状况,维护肠屏障功能,但长期肠外营养可出现肝功能损害和骨质疏松症等并发症。肠移植可从根本上治愈肠功能障碍,但其的较高的总体的失败率和高费用又限制了其在临床中的广泛开展。骨髓间充质干细胞(bone marrow mesenchymal stem cells BMSCs)是来源于中胚层的多能祖细胞,具有自我更新和向中胚层其它细胞分化的能力。在机体组织受损伤时,经骨髓动员到达损伤部位,可在局部微环境诱导下分化为特异的组织细胞,参与自身修复。加之骨髓间充质干细胞具有的低免疫原性、取材方便,易培养,增殖快的特点,已使之成为组织工程中理想的种子细胞。骨髓间充质干细胞作为细胞治疗和基因治疗的种子细胞,已经广泛应用于心血管、神经系统、呼吸和创伤等方面的基础研究,部分成果已用于临床。而在骨髓间充质干细胞治疗肠道方面,国内外相关研究较少,本课题将骨髓间充质干细胞治疗应用于肠功能障碍的大鼠模型,并观察其在肠道内的定植和疗效。研究目的1、完成雄性大鼠的骨髓间充质干细胞分离、培养、鉴定、染色工作。2、制备腹部开放伤合并海水浸泡的大鼠模型,并完善相关指标的检测,证实其存在肠功能障碍,为后续的骨髓间充质干细胞回输治疗和检测奠定基础。3、采用荧光标记和基因标记双示踪的方法来观察回输的雄性大鼠的骨髓间充质干细胞在雌性大鼠致伤肠道内的定植,并检测空肠组织中的丙二醛(malondialdehydeMDA)、超氧化物歧化酶(superoxide dismutase SOD)含量。研究方法1、分离、培养、鉴定、染色雄性大鼠的骨髓间充质干细胞。2、制备腹部开放伤合并海水浸泡致肠功能障碍的雌性大鼠模型,检测不同时间段空肠组织中的MDA和SOD的含量,制作HE染色病理切片。3、致伤模型建立后即通过鼠尾静脉将以CM-DiI染色标记的雄性大鼠的骨髓间充质干细胞输入治疗组的雌性大鼠体内,对照组仅输入生理盐水。4、治疗结束后分别于3天、7天、14天、28天、56天分批取空肠组织,制备冰冻切片在荧光显微镜观察供体细胞在肠道的定植,组织匀浆后行RT-PCR检测雄性大鼠的性别决定基因(sex determination region Y gene SRY)和MDA和SOD含量。结果1、与正常对照组比较,腹部开放伤合并海水浸泡结束后1小时至第3天的肠组织中MDA含量呈进行性上升,而SOD含量则出现进行性下降,随后二者与对照组的差异呈逐渐减小的趋势,14天后与对照组比较已无明显差异。2、雌性大鼠肠组织中SRY的RT-PCR检测结果为：3天阳性率为50%,7天阳性率66.6%,14天阳性率33.3%,28天和56天阳性率为16.6%。3、骨髓间充质干细胞治疗组第3天和第7天的空肠组织中的MDA、SOD含量较之对照组有统计学差异。结论1、腹部开放伤合并海水浸泡可导致大鼠肠功能障碍。MDA和SOD是反映肠组织氧自由基损伤和抗氧化修复能力较好的指标.2、移植的雄性大鼠的骨髓间充质干细胞可在雌性大鼠致伤肠组织内定植,但随着时间延长,存活细胞数量逐渐下降。3、骨髓间充质干细胞移植治疗可加速肠道损伤的恢复,其发挥疗效主要是通旁分泌的形式促进机体内源性修复,而不是直接分化受损组织细胞。更多还原

【Abstract】 BackgroundIntestinal dysfunction can bring the whole body in a relatively severe state of malnutrition. Current available therapy includes:parenteral nutrition capable of improving the nutritional status of patients and maintaining the intestinal barrier function. However long-term parenteral nutrition can lead to liver injury and other serious complications. the intestinal transplantation is limited by high failure rate and cost.Bone marrow mesenchymal stem cells(BMSCs) are considered as multipotent progenitor cells of mesodermal origin due to their potential of self-renewal and differentiation.When body was injuried, the mobilized BMSCs would move to the injury site, and differentiate into specific tissue cells so as to being involved in self-repair under the local micro-environment. BMSCs are characterized by of low immunogenicity, drawing convenience, ease of culture, and rapid proliferation. Based on these properties, BMSCs are regarded as promising cell therapy in regenerative medicine. BMSCs can be employed in cell therapy and gene therapy, they have been widely used in cardiovascular, nervous, respiratory and trauma disorders. Clinical application has conducted under certain condition. However, we known little about how BMSCs was involved the repair of intestinal injury and fulfill their functions. The study was designed to use BMSCs in the rat model of intestinal dysfunction in vivo to observe their colonization in the intestine and their therapeutic effect.Objective1. To explore the methods of rat BMSCs isolation, culture, characterization and staining.2. Base on our previous studies, the open abdominal injury and seawater immersion rat model was employed, and pathophyiologiacal indices was characterized to conform the existence of intestinal dysfunction.3. By using fluorescent labeling and genetic markers, the colonization of male rat BMSCs in female rats’injuried intestinal tract was detected and the content of malondialdehyde (MDA) and superoxide dismutase (SOD) were investigated. Methods1. BMSCs were isolated, cultured, characterized, stained.2. The intestinal contents of MDA and SOD were investigated in the female rats model with open abdominal injury and seawater immersion. The intestinal sample obtained by biopsy were stained by HE.3. BMSCs from male rats labeled by CM-DiI were transfusion into abdominal injuried female rats through the tail vein after injury. The control group accepted saline transfusion.4. After treatment, sample from jejunum was obtained in 3,7,14,28,56 days respectively, and sectioned to detect the colonization of BMSCs in intestine by fluorescence microscopy. On the condition of lavage of circulation system,RT-PCR was applied to detect the positivity of sex determination region Y gene (SRY). Also, the content of MDA and SOD in the intestinal tissue was also detected.Results1. Compared with normal controls, the content of MDA in intestinal tract of the open abdominal injury and seawater immersion rat increased progressively from 1 hour to 3days, but the content of SOD decreased dramatically. The difference was reduced gradually and completely diminished after 14 days.2. The results of RT-PCR revealed that, the positivity of the SRY in the intestinal tissue of female rats was 50%,66.6%,33.3%,16.6%,16.6% in 3,7,14,28,56 days respectively.3. The contents of MDA and SOD in BMSCs treated rats had statistical significance in 3 and 7 days in comparison with controls.Conclusion1. The open abdominal injury with seawater immersion can lead to intestinal dysfunction in rats. MDA, SOD are better indicators of oxidative damage and antioxidative repair, and are able to reflect the intestinal damage of oxygen free radicals and antioxidant repair.2. BMSCs from male rats can colonize in the intestine injured female rat models. But the survival of cells is decreased in the long run.3.The BMSCs transplantation can promote the recovery of intestinal function. The beneficial effects of MSC are primarily mediated via paracrine actions and not by their differentiation into target cells.更多还原