前列腺素E1对人脂肪源性干细胞体外增殖影响的初步研究

发布时间：2018-02-25 07:13

本文关键词： 前列腺素E1 脂肪源性干细胞细胞分离细胞增殖　出处：《郑州大学》2011年硕士论文　论文类型：学位论文

【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p0.05)。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。
[Abstract]:Background and purpose
The subcutaneous soft tissue filling plastic surgery has been a common treatment for subcutaneous soft tissue skin less fullness, depression, atrophy deformity is often used in autologous free fat or filling all kinds of artificial materials. Due to the existence of artificial materials rejection, trauma and safety problems such as difficult to meet clinical needs, and free autogenous fat graft survival rate of only 40% to 50%, which makes its clinical application is limited. One of the reasons for absorption of fat grafts is mature fat cells to hypoxia, damage tolerance is low, and cannot be updated. The proliferation of adipose derived stem cells (Adipose derived stem cells, ADSCs) has the potential for proliferation and differentiation strong, stronger tolerance to hypoxia, and autologous stem cell transplantation for the town to avoid rejection more secure.
Adipose derived stem cells not only have bone marrow mesenchymal stem cell differentiation of similar, and has drawn a simple method, wide source, small trauma and other advantages, which make it become hot in recent years in the field of stem cell research. The use of stem cells is an important direction for adipose tissue engineering. Because fat stem cells have the advantages, which undoubtedly become the best choice of adipose derived stem cells in adipose tissue engineering seed cells. How to improve the ability of proliferation and differentiation of adipose derived stem cells is an important research topic in the engineering construction of adipose tissue.
Prostaglandin El (prostaglandinEl, PGE1) is a protective factor produced by vascular endothelial cells, is a potent vasodilator, mainly for a long time for the treatment of pulmonary arterial hypertension, with the clinical application, the progress of the research, people gradually realize that PGE1 can inhibit platelet aggregation and thrombosis, relaxation of bronchial smooth muscle, protection ischemic myocardium, reduce infarct size, macrophage and leukocyte degranulation, improve erythrocyte deformability and positive inotropic effect, has been widely used in angina pectoris of coronary heart disease, heart failure, hyperlipidemia, diabetic neuropathy, chronic renal insufficiency.
Studies have found that misoprostol (PGE1 analogue) on human preadipocyte proliferation and differentiation has a strong stimulating effect, while the effect of PGEl on the proliferation of ADSCs has not been reported of prostaglandins on proliferation and differentiation of adipose derived stem cells, to explore the improvement of adipose derived stem cell survival rate has clinical and practical significance transplantation, therefore the design of the experiment, explore the effect of PGE1 on proliferation of ADSCs cells.
Method
The extraction of 4 cases of abdominal subcutaneous adipose tissue in patients with stem cells by enzyme digestion, cells were cultured in vitro. The observation of the isolated adipose cell morphology under the microscope, flow cytometry to detect the surface markers. When adipose derived stem cell number of one-time training too much, but in the short term will not be fully used, can be stored, and then recovery in preparation for days after the experiment. With five different concentrations of PGE1 treatment of cultured cells, including 3 mol/L, 6 mol/L, 9 mol/L and 12 mol/L as experimental group, 0 mol/L as control group, to observe the proliferation of were treated with different concentrations of PGE1 cells.
Result
ADSCs cultivated into a Petri dish of 48h after a large number of fusiform adherent cells were fibroblast like growth, cytoplasm and nucleolus rich cell morphology, short processes, showed short fusiform shape or polygonal small, centrally located nuclei after.5d cells reached the logarithmic growth phase, spindle cell growth fusion aggregation closely arranged in a certain direction, whirlpool or parallel arranged.9d cells covered the culture bottle, the colony of each other were bundle or whorls after.13d for typical fibroblast growth. Flow cytometry showed that the expression of ADSCs specific molecules CD44, CD105, CD34, CD45, CDHLA-DR. by experimental observation and testing, the experimental results proved the harvested cells indeed form a complete, perfect function of adipose derived stem cells. The number of cells in each experimental group were significantly higher, the difference was statistically significant (p=0.004 compared with the control group, the experimental group) The differences were statistically significant (compared with P0.05 in the experimental group).
conclusion
1. in the experiment, the cells were isolated by enzyme digestion, and they were identified as adipose derived stem cells by histology and surface markers.
2., after treated with different concentrations of PGE1, the number of adipose derived stem cells showed an increasing trend. According to the statistical results, there was a statistically significant difference between the groups. It preliminarily indicated that PGE1 could promote the increment of adipose derived stem cells.
3., compared with the statistical software, it was found that the difference between the experimental groups was statistically significant, indicating that the degree of cell number increment has a dose-dependent relationship with PGE1 when the concentration of PGE1 reaches a certain height.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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