【作者】 杨焕纳；

【导师】 崔正军；

【作者基本信息】 郑州大学 ， 外科学， 2011， 硕士

【摘要】 背景与目的皮下软组织充填一直是整形外科常见的治疗手段,对于体表的皮下软组织欠丰满、凹陷、萎缩畸形目前常采用自体游离脂肪或各种人工材料进行充填。人工材料因存在排异、创伤大及安全性等问题难以满足临床需要,而自体游离脂肪的移植成活率仅为40%～50%,这使得其临床应用受到很大限制。脂肪移植物被吸收的原因之一是成熟脂肪细胞对缺氧、损伤的耐受力低,且不能增殖更新。而脂肪源性干细胞(Adipose derived stem cells, ADSCs)则具有强大的增殖分化潜力,对缺氧耐受力更强,且自体干细胞用于移植町避免排异反应。更加安全脂肪源性干细胞不仅具有跟骨髓间充质干细胞相似的多向分化潜能,而且具有取材方法简便、来源广泛、患者创伤小等优势,这都使其成为近年来干细胞研究领域中的热点。干细胞应用的一个重要方向就是脂肪组织工程,由于脂肪干细胞具有上述优点,这无疑令脂肪干细胞成为构建工程脂肪组织的种子细胞的最佳选择。如何提高脂肪源性干细胞的增殖分化能力是构建工程脂肪组织重要的研究课题。前列腺素El(prostaglandinEl, PGE1)是血管内皮细胞产生的一种保护因子,是一种有效的血管扩张剂,长期以来主要用于肺动脉高压的治疗,随着临床应用、研究的进展,人们逐渐认识到PGE1具有抑制血小板聚集和血栓形成、松弛支气管平滑肌、保护缺血性心肌、缩小梗塞面积、抑制巨噬细胞和白细胞脱颗粒、改善红细胞变形及正性肌力等作用,已广泛应用于冠心病心绞痛、心力衰竭、高脂血症、糖尿病神经病变、慢性肾功能不全等。已有研究发现米索前列醇(PGE1类似物)对人前脂肪细胞的增殖和分化均有较强的刺激作用,而PGEl对ADSCs增殖的作用影响尚未见报道,研究前列腺素对脂肪源性干细胞的增殖及分化作用,对探求提高脂肪源性干细胞移植的成活率具有临床实用意义,因此设计实验,初步探索PGE1对ADSCs细胞增殖的作用效果。方法用酶消化法提取4例临床患者腹部皮下脂肪组织中的干细胞,体外进行细胞培养。对分离得到的脂肪细胞在显微镜下进行细胞形态观察,流式细胞术检测细胞表面标志物。当脂肪源性干细胞一次性培养的数量过多,短期内又不会完全使用时,可以将部分冻存起来,以备日后实验时再复苏使用。用五种不同浓度的PGE1处理培养的细胞,其中3μmol/L,6μmol/L,9μmol/L和12μmol/L作为实验组,0μmol/L作为对照组,观察经不同浓度的PGE1处理后细胞的增殖情况。结果ADSCs培养种植入培养皿48h后有大量梭形细胞贴壁,呈成纤维细胞样生长,胞浆和核仁丰富,细胞形态不一,有短突起,呈短梭形或小多角形,细胞核居中。5d后,细胞达到对数生长期,细胞成长梭形融合聚集,紧密排列有一定方向性,呈漩涡状或平行状排列。9d后细胞长满培养瓶,集落彼此融合呈束状或漩涡状排列。13d后为典型成纤维细胞样生长。流式细胞术检测结果显示ADSCs表达相对特异分子CD44,CD105,CD34,CD45,CDHLA-DR。通过实验观察和检测,证明实验收获的细胞确实为形态完整,功能健全的脂肪源性干细胞。各实验组的细胞数量均有显著升高,其差异有统计学意义(与对照组相比p=0.004),各实验组内相比,部分差异有统计学意义(实验组组内相比p<0.05)。结论1.实验中用酶消化法分离培养的细胞,经组织学及表面标志物鉴定确实为脂肪源性干细胞；2.脂肪源性干细胞经不同浓度的PGE1处理后,细胞数目呈现递增趋势,再根据统计结果,组间差异有统计学意义,初步说明了PGE1能够促进脂肪源性干细胞的增值作用。3.应用统计软件进行组内比较发现部分实验组之间差异有统计学意义,说明PGE1的浓度达到一定高度时,细胞数量增值的程度与PGE1具有一定的剂量依赖性。更多还原

【Abstract】 Background and ObjectiveSubcutaneous soft tissue filling plastic surgery has been a common treatment, the surface of the skin due to soft tissue fullness, depression, abnormal contraction present, the common or autologous free fat filling all kinds of artificial materials. Rejection due to the existence of artificial materials, such as trauma and safety problems which are difficult to meet clinical needs, and free autogenous fat graft survival rate of only 40% to 50%, which makes its clinical application is greatly limited. Absorption of fat grafts is one of the reasons is the mature fat cells to hypoxia, damage tolerance is low, and not the proliferation of updates. The adipose derived stem cells (ADSCs) is a strong potential for proliferation and differentiation, greater tolerance to hypoxia, and autologous stem cell transplantation for the town to avoid rejection. More secure.Just like bone marrow mesenchymal stem cells(BM-MSCs), aidpose derived stemcells(ADSCs)have the capacity of multi-differentiation. However,ADSCs can be isolatedeasily with abundant rcsoBrces and little injury to patients. Thus more and more researchersbecame interested in the study of ADSCs. One of the important applications of ADSCs is Tissue Engineering. It’s very clear that ADSCs are the most suitable seed cells for the construction of engineering adipose tissues. Thus, it is meaningful to study the proliferation and differentiation of ADSCs.prostaglandinEl (PGE1) as a peotect factor derived from vascular endothelial cells, It is an effective vasodilating agent. For a long time, PGE1 is apply to the treatmem of pulmonary artery hypenension. With me development of clinical application and research, it is gradually realized that PGE1 can inhibit platelet aggregation and mrombogenesis, relax the smooth muscle of bronchus, prevent thrombogenesis, improve ischemic myocardium, inhibit the degranulation of macrophage and blood corpuscle, improvered blood cell defomation and has positive inotropic effect. Which is generally used to coronary heart disease, heart failure, hyperlipemia, diabetic neuropathy and chronic renal insuffciency.Studies have found that misoprostol (PGE1 analogue) on human preadipocyte proliferation and differentiation has a strong stimulating effect, while the effect of PGE1 on the proliferation of ADSCs has not been reported, of prostaglandins on the proliferation of stem cells and Differentiation effect on the quest to improve survival rate of transplanted adipose derived stem cells clinically practical significance. Therefore the design of the experiment, preliminary exploration PGE1 on cell proliferation effect of ADSCs.MethodsThe mesenchymal stem cells in adipose tissue were extracted from abdomen wall of 4 patients, donors undergoing the lipesuction in clinic, then these cells were induced to differentiate to the adipogenic cells in vitro. Cell morphology was observed under the microscope, Flow cytometric analysis of the cell surface antigens. When the adipose derived stem cells in an excessive number of one-time training, short term and will not be fully used, it can be part of the cryopreserved for future use of the experiment and then recovery. The cultured ADSCs were treated with five different concentration of PGE1, which were 3μmol/L,6μmol/L,9μmol/L,12μmol/L, and 0μmol/L (control group). After 3 days, observe the cell morphology and proliferation. ResultsADSCs into the culture dish 48 hours after planting a large number of adherent spindle cells, showing fibroblast-like growth, rich in cytoplasm and nucleolus, cell morphology varies with short processes, were small polygonal or short spindle nuclei Center.5 days, cells reached the logarithmic growth phase, growth of spindle cells fused together, closely packed a certain direction, or parallel-like arrangement was swirling.9 days after cell confluence culture bottles, each colony was fused bundle or whirlpool order.13 days later, a typical fibroblast-like growth. ADSCs flow cytometry showed that the relative expression of specific molecules CD44-, CD105, CD34, CD45, CDHLA-DR. By experimental observation and testing to show that the cells harvested in this experiment indeed form a complete, fully functioning stem cells, There was no obervious different among the experimental groups. And the cell number of each experimental group was obervious elevated, comparing with control group, The difference was statistically significant (p<0.05), Compared to the experimental group, some differences were statistically significant (the experimental group compared with group p=0.004).Conclusions1. The enzyme digestion experiments using cultured cells isolated by histology and identification of surface markers indeed adipose-derived stem cells;2. Adipose-derived stem cells were treated with different concentrations of PGE1, the number of cells showed an increasing trend, according to statistics, there was significant difference between the groups, the initial description of the PGE1 can proliferation of adipose-derived stem cells.3. Application of statistical software to compare the group discovered that some of the differences between the experimental group was statistically significant, indicating that PGE1 concentrations reach a certain height, cell number and degree of value-added PGE1 in a dose dependent manner.更多还原