【作者】 张丽新；

【导师】 安铁洙；

【作者基本信息】 东北林业大学 ， 生理学， 2008， 硕士

【摘要】 糖尿病是一类严重的代谢疾病,正危害着世界上越来越多人口的健康。胰岛移植是一种治疗糖尿病的有效方法,但因供体缺乏和移植后免疫排斥问题制约了其广泛应用。干细胞为具有强增殖能力和多向分化潜能的细胞,是利用细胞替代疗法治疗重大疾病的细胞来源,其中成体干细胞因不存在胚胎干细胞的致瘤性及伦理道德问题而被人们寄予厚望。但目前国际上对成体胰腺干细胞的研究进展缓慢,其主要障碍在于缺少公认的特异性标记物和可靠的检测系统,而无法准确指认胰腺干细胞。因此,目前的主要任务是准确定位、指认成体胰腺干细胞。本研究通过对新生1d小鼠皮下注射和E13.5d孕鼠腹腔注射Brdu不同剂量,摸索BrdU高效率标记小鼠胰腺细胞的最佳方法;然后对E13.5d孕鼠腹腔注射5针Brdu处理的小鼠进行长期追踪,确定LRCs在胰腺中的活体定位;通过STZ处理追踪小鼠诱导的糖尿病模型,研究小鼠胰腺活体损伤后LRCs增殖和分化能力,尤其是向β细胞分化的能力;通过G418处理、彻底消化所得胰腺单细胞分阶段接种及分阶段消化所得胰腺单细胞分阶段接种三种离体培养方法,研究LRCs的自我更新能力。结果表明:对E13.5d孕鼠腹腔注射5针Brdu、标记胎鼠胰腺细胞的方法是相对最好的;通过这种方法标记小鼠胰腺细胞后,经过10周追踪,这些LRCs在胰腺活体中大多数位于胰岛区,在腺泡中间也有少量分布,导管中极少见;小鼠胰腺中LRCs在受到STZ诱导的胰腺损伤刺激7天后能够发生增殖,10天后能够向胰岛素分泌细胞分化;在离体培养方法中,用50μg/mL G418处理48h,能达到去除成纤维细胞、纯化上皮细胞效果,但是得到的胰腺上皮细胞很难形成单细胞集落;彻底消化所得胰腺单细胞分阶段接种法,达到了去除成纤维细胞,富集胰腺上皮细胞目的,但是没有达到筛选出具有强增殖能力、能形成大的单细胞集落的成体胰腺干/祖细胞的目的;分阶段消化所得胰腺单细胞分阶段接种法中,通过将成体小鼠胰腺组织进行头20min消化,然后将所收集到的胰腺单细胞悬液分别进行头40min、过夜及再过夜三阶段接种、2%血清培养基继续培养这种方法,从过夜及再过夜接种培养的胰腺上皮细胞中筛选出了一群具有强增殖能力、能形成大的单细胞集落的细胞,经检测,这些具有集落形成能力的细胞来源于LRCs。活体和离体实验结果表明:成体小鼠胰腺LRCs中确实存在一群成体胰腺干/祖细胞。这为今后进一步研究胰腺干细胞增殖与定向分化调控机制以及它们特异性标志分子等问题奠定基础,也为今后成体干细胞真正应用于人类临床上糖尿病等胰腺相关疾病的治疗准备了条件。更多还原

【Abstract】 Diabetes mellitus is a group of metabolic diseases,which is threatening many people’s health.Transplantation of islets of Langerhans offers an attractive strategy for diabetes therapies,but its wide application has been limited by donor shortage and immunological rejection after transplantation.Adult pancreatic stem cells with strong proliferatve capacity and multipotential may be potential cell source since they have no ethical issues and teratoma formation.But researches on adult pancreatic stem cell progress slowly because of lacking recognized specific markers and credible detecting systems.So,the major task is localizing and identifying adult pancreatic stem cells.In this research,different dosages of BrdU were injected subcutaneously to neonatal mice for 6 times or interperitoneally to pregnant 14th day mice for 5 to 6 times,by which we tried to find out the best method of labeling mouse pancreatic cells with high efficiency;then,the neonatal mice born by pregnant mice injected interperitoneally five times of BrdU were long time traced to make certain the localization of label-retaining cells(LRCs) in pancreas in vivo; The diabetic BrdU-chasing mice induced by streptozotocin were used to explored the LRCs′proliferative capacity and multipotential,especially their potential of differentiation intoβcells;LRCs′proliferative capacity in vitro was explored by three kinds of culturing methods, including in vitro-G418 treatment,classifying inoculation of pancreatic single cells got from complete digestion or multistep digestion of pancreas.The results showed that injecting interperitoneally pregnant 14th day mice for 5 times is the best method of labeling mouse pancreas high efficiently;By this method,after the neonatal mice were chasing for 10 weeks,the LRCs were found in the islets and around acini,but not in big ducts;the LRCs in mouse pancreas can proliferate after STZ administration for 7 days and differentiate into insulin-secreting cells after STZ administration for 10 days;in the in vitro culture tests,50μg/mL G418 added in culture medium for 48h could wipe off fibroblasts and purify epithelial cells,but the left epithelial cells were hard to proliferate into single cell clusters;the method of classifying inoculating of pancreatic single cells got from complete digestion of pancreas could reach the aim of removing fibroblasts and purifying epithelial cells, but couldn’t achieve the aim of screening out adult pancreatic stem/progenitor cells;In the third strategy,the pancreatic single cell suspensions after digestion for 20min were inoculated and culture for the first 40min,the unattached cells were moved to another plate and culture for 24 hours,then the unattached cells were moved to a new plate and culture for another 24 hours, at last the upper unattached cells were discarded.All the attached cells in the foregoing steps were cultured with medium containing 2%FBS for 7 to 10 days.By this method,we screened out a group of cells with strong proliferative capacity and cluster formation capability from the first and second 24 hours attached cells,these ceils were demonstrated to be candidate pancreatic stem/progenitor cells by RT-PCR and immuno-stainning analysis.And also some cells were proved deriving from LRCs with immuno-stainning detection.The in vivo and in vitro experiment results suggested that LRCs were a group of adult pancreatic stem/progenitor cells in adult mouse pancreas.These results may help for further exploring the specific markers of pancreatic stem cells,mechanism of their proliferation and directional differentiation,,and also preparing the conditions for applications of adult stem cells in human clinic therapy of pancreatic diseases in future.更多还原