高成脂能力脂肪干细胞系的分离与纯化

发布时间：2018-02-07 14:02

本文关键词： 高成脂脂肪干细胞表面标志 CD54　出处：《南方医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 研究背景: 脂肪组织是修复全身各部位软组织缺损的最佳选择。临床上自体成熟脂肪组织的移植,不论是脂肪块或是脂肪颗粒的注射移植,均可能出现坏死、再吸收等问题,导致术后长期效果不佳。组织工程技术的出现,对组织器官的修复与替代治疗产生了重大意义。组织工程技术的首要条件就是种子细胞。由于胚胎干细胞受到伦理学和来源的限制,故目前使用最多的种子细胞即成体干细胞。作为种子细胞的成体干细胞主要有两种来源,一是骨髓来源的骨髓干细胞(bone marrow-derived stemcells,BMSCs),另一种是脂肪组织来源的脂肪干细胞(Adipose-derived stem cells,ASCs)。相对与BMSCs而言,ASCs取材容易,且细胞量足以满足组织工程的需求,具有很大的优势。但现有的研究表明,通过普通的酶消化法分离出来的ASCs是由极其复杂的各种细胞集合而成,成脂等各项诱导分化的能力及效率都不高。将ASCs进行成脂诱导后两周,其分化比例只为30%～40%,同时ASCs其余的各项分化效率也低于BMSCs。因此,纯化ASCs,提高其诱导分化效率,是优化其成为最佳种子细胞来源之一的有效方法。近年来,有研究发现,成熟脂肪细胞在某种环境中,可去分化回成纤维样细胞状态,这种退回到原始状态的去分化细胞具有多向分化的潜能,且成脂分化能力要高于ASCs。然而这种去分化细胞的高成脂能力是否伴随着某些表面标志的表达,国内外均未见报道。所以,本实验欲比较去分化脂肪细胞与ASCs在表面标志等各方面的不同,寻求出高成脂细胞高表达的表面标志,并希望能通过免疫磁珠分选,提纯出高成脂系ASCs。目的: 探索寻求与高成脂能力相关的干细胞表面分子,并通过分选,纯化得到高成脂系ASCs,从而为提高脂肪组织工程的效率提供有效途径。方法: 1、从人脂肪组织中分离培养ASCs,成脂诱导分化ASCs,得到诱导后脂肪细胞。收集诱导后漂浮于培养基中的脂肪细胞,天花板贴壁培养10d,脂肪细胞贴壁后翻转正常培养得到去分化脂肪细胞。选择同等代数两种细胞,成脂诱导分化并用油红O染色法鉴定其分化效率;细胞计数法绘制细胞生长曲线;流式细胞术测定细胞的表面标志表达,并在上述三方面对ASCs和去分化脂肪细胞进行比较,筛选出可能与高成脂能力相关的表面分子。 2、就筛选出的可能与高成脂能力相关的表面分子,用免疫磁珠分选法分别分选脂肪组织新鲜分离的间质血管碎片(stromal vascular fractions,SVF)及培养后ASCs。流式细胞检测分选前后细胞群体该表面分子阳性表达率;并对两种分选出的细胞与未行分选的ASCs进行成脂、成骨诱导,油红O染色鉴定成脂诱导结果,茜素红染色鉴定成骨诱导结果。结果: 从吸脂术中获得人脂肪组织,培养出ASCs。选择第4代ASCs进行成脂诱导,诱导13d起,收集诱导成熟的脂肪细胞,天花板贴壁培养。培养过程中,多房的脂肪细胞贴壁并逐渐吐出脂泡,最终去分化为成纤维状细胞。去分化细胞与ASCs相比,生长曲线较一致说明两者具有相似的增殖能力;油红O染色鉴定成脂诱导结果表明去分化细胞的成脂分化效率高出ASCs约50%。两种细胞在表面标志的表达上,CD34、CD54的表达阳性率有所差别:去分化细胞的CD34、CD54的表达为阳性,而ASCs的表达均为阴性。用CD54-PE免疫磁珠分选新鲜分离的SVF及经过培养后的ASCs。分选前,流式检测一至三代ASCs,细胞CD54的表达率随着代数的增加而降低。分选后,CD54的表达率在两种细胞群体中均达到90%以上。继续培养数代后,从培养后ASCs分离出的CD54阳性细胞群体CD54表达率维持高水平,新鲜分离SVF中分离出的阳性细胞CD54的表达则降至阴性,未分选的ASCs为阴性表达。分选后的细胞与未分选的ASCs成脂能力比较:从培养后的ASCs分离出的CD54阳性细胞成脂分化率接近100%,未分选的ASCs成脂能力约为30%,而从新鲜分离的SVF中分选出的CD54阳性细胞成脂分化率只为10%左右。成骨诱导结果:ASCs成骨能力为三种细胞中最佳,而从SVF分选出的CD54阳性细胞群的成骨效率最差。结论 1、本实验通过比较去分化细胞与ASCs在增殖能力、成脂分化能力与表面标志表达等方面的差别,认为去分化细胞是一种类似于干细胞但较干细胞具有更高成脂分化能力的细胞。去分化细胞CD54、CD34的表达高于ASCs表明CD54、CD34的表达可能与高成脂能力相关。 2、CD54免疫磁珠分选经过培养的ASCs,能纯化ASCs并分离出高成脂系干细胞。因而,CD54应是高成脂系干细胞特异表达的表面分子之一,从ASCs分离CD54阳性细胞可做为纯化高成脂干细胞系的方法之一。
[Abstract]:Research background:
Adipose tissue is the best choice for soft tissue defect repair in different parts of the body. Clinical autologous mature adipose tissue transplantation, whether it is fat or fat granule injection block transplantation, may appear necrosis, resorption and other issues, leading to long-term results after surgery is poor. Tissue engineering technology, and repair of tissues and organs replacement therapy has great significance.
The first condition is tissue engineering seed cells. Because embryonic stem cells are the source of ethics and restrictions, so that most of the currently used seed cells of adult stem cells as seed cells. Adult stem cells have two main sources, one is bone marrow-derived stem cells (bone marrow-derived stemcells, BMSCs). The other is adipose tissue derived stem cells (Adipose-derived stem fatty cells, ASCs). Compared with BMSCs, ASCs is easy, and the cell amount sufficient to meet the needs of tissue engineering, has a great advantage.
But the existing research indicates that separated by enzyme digestion of the common ASCs is composed of various cell complex assembled into the ability and efficiency of lipid induced differentiation is not high. The ASCs was two weeks after induction of adipogenic differentiation, the proportion is only 30% ~ 40%, and the differentiation of ASCs the rest of the efficiency is lower than BMSCs. so the purification of ASCs, improve the differentiation efficiency, become one of the effective methods is to optimize the best source of seed cells.
In recent years, studies have found that mature fat cells in a certain environment, can go back to the differentiation of fibroblast like cells, the return to the original state of the dedifferentiated cells have multilineage differentiation potential, and adipogenic differentiation ability than ASCs. but to the cell differentiation and adipogenic ability is accompanied by high expression of certain surface mark, have not been reported. Therefore, this experiment to compare different dedifferentiated adipocytes and ASCs in various aspects such as surface marker, seek out high fat high expression of surface markers of the cells, and hope that through the immune magnetic bead separation and purification of high fat line ASCs.
Objective:
We explored the surface molecules of stem cells related to the high adipogenesis ability, and purified high fat ASCs through sorting, which provided an effective way to improve the efficiency of adipose tissue engineering.
Method:
1, ASCs were isolated and cultured from human adipose tissue, adipogenic differentiation of ASCs was induced after collecting fat cells. After induction of floating in the medium fat cells, ceiling adherent culture 10d, fat adherent cells cultured from normal turnover dedifferentiated adipocytes. Two kinds of the same algebra into cells. Differentiation of fat and oil red O staining to identify the differentiation efficiency; cell growth curve was drawn by cell counting; surface markers were determined by flow cytometry and the expression in the above three aspects to adipocyte differentiation of ASCs and comparison, screened with high surface molecules related to the ability of the fat.
2, we screened with high surface molecular ability related lipid, stromal vascular fraction by immunomagnetic separation method were freshly isolated from adipose tissue (stromal vascular, fractions, SVF) and flow cytometry after culture ASCs. cells before and after sorting groups the positive expression rate of surface molecules; and two kinds of sorting the cells without sorting ASCs adipogenic, osteogenic induction, oil red O staining of adipogenic induction, alizarin red staining of bone induction results.
Result:
To obtain human adipose tissue from liposuction, develop ASCs. fourth generation ASCs were induced to differentiate into adipose cells, induced by 13D, collection of induced mature fat cells, ceiling adherent culture. In the process of cultivation, multilocular fat cells adherent and gradually spit out the foam, and eventually to differentiate into fibrous cells. The differentiation of cells compared with ASCs, the growth curve shows that is consistent with a similar proliferation; oil red O staining results show that identification of adipogenic differentiated cells to adipogenic differentiation efficiency of two CD34 ASCs about 50%. cells in the expression of surface markers, and the positive expression rate of CD54 was different: dedifferentiated cells CD34, the expression of CD54 was positive, while ASCs expression was negative.
CD54-PE immunomagnetic separation of freshly isolated SVF and ASCs. after separation after culture, flow cytometry to a three generation of ASCs, the expression of CD54 decreased with the increase of the rate of algebra. After separation, the expression rate of CD54 in the two cell populations were more than 90%. Continue to cultivate generations, CD54 CD54 positive cell populations isolated from cultured ASCs expression rate to maintain a high level, the expression of CD54 positive cells isolated from freshly isolated SVF in the unsorted dropped to negative, the expression of ASCs was negative. After the separation of cells and the unsorted ASCs adipogenic ability: isolated from cultured ASCs CD54 positive cell adipogenic differentiation rate close to 100%, the unsorted ASCs adipogenic capacity is about 30%, while the CD54 positive cells were selected from freshly isolated SVF in adipogenic differentiation rate was only about 10%. Results: ASCs induced osteogenic osteogenic ability of three kinds of cells in the best The most poor osteogenic efficiency of the CD54 positive cell group selected from SVF was the worst.
conclusion
1, this experiment compared to cell differentiation and ASCs in proliferation by adipogenic differentiation and surface marker expression differences, that cell differentiation is similar to stem cells but more stem cells have higher adipogenic differentiation ability of the cells to the differentiation of CD54 cells. The expression of CD34 was higher than that of ASCs. CD54, CD34 expression may be associated with high adipogenic capacity.
2, CD54 immunomagnetic beads sorting and culturing ASCs can purify ASCs and isolate high fat derived stem cells. Therefore, CD54 should be one of the surface molecules specifically expressed on the high fat line stem cells. Separating CD54 positive cells from ASCs can be used as one of the ways to purify the high fat stem cell line.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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