【作者】 陆阳；

【导师】 刘伟；

【作者基本信息】 上海交通大学 ， 外科学（整复外科）， 2013， 博士

【摘要】 干细胞体外扩增和干性维持是本领域的研究热点和难点,已知体内干细胞特性维持与自我更新依靠其所处的微环境,其中干细胞之间,以及与周围细胞的接触与信息交换起着重要的作用,而传统体外干细胞培养方法破坏了细胞间的接触与信息交换,降低了扩增效率。我们提出了局部高密度细胞接种的设想,通过高密度接种骨髓细胞的方法来增加细胞之间的相互接触,达到扩增骨髓中成血管类祖细胞的目的。目的本研究旨在通过局部高密度培养增加细胞-细胞间接触,进而达到扩增骨髓中成血管类祖细胞的目的,并通过基因芯片分析,定量PCR等手段,发现并验证与高密度培养相关的特异性分子,阐明高密度培养扩增并维持成血管类祖细胞特性的可能机制。该研究将解决骨髓成血管类祖细胞体外扩增难题,并为研究细胞-细胞接触建立重要的体外研究模型。方法我们从wistar大鼠的股骨与胫骨中取出骨髓,进行原代培养。原代培养的细胞消化后,以2×10~5/cm~2为密度点状接种直径10cm培养皿,均匀分布6点接种,每皿细胞总量9×10~5,以相同数量细胞均匀接种于相同大小培养皿作为普通密度培养对照组。在培养15天后,我们通过流式细胞术对传统密度已经局部高密度培养出的骨髓细胞进行表型的鉴定,并在体外与体内验证了局部高密度培养得到的细胞与传统密度培养得到的细胞在血管再生能力上的差异。结果流式细胞分析结果显示与传统密度相比,局部高密度培养的骨髓细胞与原代细胞的表型更为接近,高表达CD14((18)(20)(13)(22)(4)),CD133((19)(22)(13)(20)(4)),CD45((19)(24)(13)(24)(4)),KDR((18)(17)(13)(24)(4)),CD144((18)(19)(13)(20)(4)),CD31((18)(21)(4))与CD34((22)(13)(20)(4))这些与造血和成血管类祖细胞密切相关的表面标志,显示了局部高密度培养的骨髓细胞可能具有很强的血管再生能力。我们通过在matrigel上的成管实验证明了局部高密度培养的骨髓细胞在体外具有更加良好的血管形成能力,结果显示传统密度形成的分支节点数为7.2个,而局部高密度形成的分支节点数为29.8个。我们通过对下肢缺血裸鼠的细胞治疗来证明了局部高密度扩增细胞具有更好的潜在治疗价值。结果显示下肢缺血裸鼠治疗三周后,局部高密度治疗组能够更好的改善缺血裸鼠的预后:出现下肢坏死的比例为30%,作为对照PBS组坏死率为100%,传统密度组坏死率为90%。下肢血流改变反映了同样的趋势:高密度治疗组3周后患/健血流比为0.82,而PBS组与传统密度组患/健血流比分别为0.4与0.53。我们检测了缺血下肢肌肉内的血管密度,结果显示高密度治疗组的血管密度为776.9/mm~2,而PBS组与传统密度组血管密度分别为400/mm~2与423.4/mm~2。最后我们通过基因芯片以及定量PCR的方法初步探讨了局部高密度扩增骨髓内成血管类祖细胞的可能机制。实验结果显示局部高密度扩增的细胞高表达整合素类相关表面分子,整合素类分子与细胞接触密切相关,同时也可以影响细胞的成血管能力,因此可能整合素类分子的上调在局部高密度扩增成血管类祖细胞中起到了重要的作用。另外我们发现了在局部高密度培养体系,一些与血管再生密切相关的生长因子无论在基因还是在蛋白上的表达均明显高于传统密度培养体系,这提示生长因子的差异也可能是局部高密度扩增成血管干细胞的可能机制之一。结论局部高密度培养大鼠骨髓细胞可以扩增出骨髓中的成血管类祖细胞,这些细胞具有很强的体内外形成血管的能力并具有很好的治疗缺血性疾病的价值。这些成血管类祖细胞在局部高密度体系内的扩增可能与整合素相关分子的上调,细胞外基质相关分子的上调以及成血管类生长因子的高表达有着密切的联系。更多还原

【Abstract】 Background—Adult stem cells are distributed in various tissues and their stemness is maintained by the stem cell―niche‖.Mimicking the niche in vitro remains a great challenge.Growing evidence suggests that cell-cell interactions play an important role in the niche.We hypothesized that seeding cells at high density to physically maintain cell-cell interactions in culture would be beneficial for stem cell expansion in vitro.Methods—Rat bone marrow cells were extracted from the femurs and tibias of4-week-old male Wistar rats.Primary culture of bone marrow cells was carried out by seeding the cells at 1.6×10~4 cells/cm~2 in DMEM.For regular density culture,9×10~5primary cultured cells(P0)were seeded evenly at a density of 1.6×10~4 cells/cm~2 in a10-cm diameter tissue culture dish,for high density dot culture,9×10~5 primary cells were suspended in 300μl of culture medium,and then six drops(50μl each)of cell suspension were dot-seeded separately onto a 10-cm diameter culture dish.The average diameter of each dot was about 1 cm,resulting in a final local cell density of2×10~5 cells/cm~2.After 15 days,flow cytometric analyses were used to test the angiogenic markers,tube formation assay and hindlimb ischemia model were used to test the angiogenic ability of those cells in regular density culture or high density dot culture.At last,we carried out microarray analysis to compare global gene expression patterns between high density and regular density cultured cells.Results—After 15 days,flow cytometric analyses showed that cells in high density culture expressed higher levels of angiogenic markers,like CD14,CD133,CD45,KDR,CD144,CD31与CD34,than those of cells in regular density culture.In tube formation assays,the number of branch points per field,which were formed by cells from the high density culture,was significantly higher than those formed by cells from the regular density culture.In addition a significant difference in the physiological outcomes of ischemic limbs was observed.Injection of high density cultured cells achieved limb salvage in seven out of 10 mice(70%),whereas only one out of 10(10%)mice were fully protected by injection of regular density cultured cells,and limb salvage was not achieved in the PBS-treated group.Laser Doppler image analysis revealed that blood flow was restored to 80%in the high density group,whereas blood flow was restored to 55%and 40%in regular density and PBS groups,and histological analysis confirmed that the number of microvessels in ischemic muscles was significantly higher in the group treated with high density cultured cells compared with that in the group treated with regular density cultured cells or PBS.Global gene expression analyses revealed that high density cultured cells represented a population that highly expressed focal adhesion,ECM-receptor interaction and proangiogenic genes.Conclusions—This study establishes a novel and simple cell culture method that can mimic the bone marrow niche to maintain and expand endothelial progenitor cells without additional growth factors.Cells from this type of culture can serve as a new therapeutic cell source for treating ischemic diseases.更多还原