兔脂肪来源干细胞分离培养及富血小板血浆对其增殖的影响

发布时间：2018-04-21 03:23

本文选题：脂肪间充质干细胞 + 富血小板血浆　；参考：《大连医科大学》2011年硕士论文

【摘要】：背景:自体脂肪移植已有上百年的历史,因其来源丰富、取材容易、操作简单、充盈外形好、无排异反应等优点,倍受整形美容外科医生的重视,但同时也存在不足之处,其中最主要是移植后脂肪细胞的成活率较低(60%)。如何提高脂肪细胞的增殖及分化能力,是提高移植成活率的重要环节。随着脂肪组织工程的发展及研究,寻求来源广、易获取、损伤小、稳定扩增的种子细胞和无免疫反应、高效、廉价、配比协同作用好的生长因子是关键问题。目的:分离培养兔脂肪来源干细胞(adipose-derived stem cells,ADSCs),并观察ADSCs体外生长的生物学特性及增殖分化潜能,探讨自体富血小板血浆(platelet-rich plasma,PRP)对ADSCs增殖的影响。方法:1.实验采用新西兰大白兔,取兔颈背部区脂肪垫,贴壁法及Ⅰ型胶原酶消化法体外培养兔ADSCs,观察其形态特征。 2.取第3代ADSCs进行成脂、成骨诱导分化,两周后油红O成脂染色及三周后茜素红成骨染色鉴定。 3.抽取兔心脏血,采用二次离心法制备PRP,并行全血及PRP血小板计数。 4.将第3代细胞分为对照组(细胞180ul +单纯DMEM培养基20ul)、全血组(细胞180ul +全血血浆20ul)和PRP组(细胞180ul+ PRP 20ul),采用CCK-8法(Cell Counting Kit-8)检测不同作用时间(24h、48h、72h)各组对ADSCs增殖活动的影响。结果:1.原代培养的细胞24h后见少量椭圆形细胞贴壁,大部分未贴壁细胞为圆形的红细胞及筋膜杂质。48h首次换液,除去未贴壁的红细胞及筋膜杂质,见小部分贴壁细胞呈短梭形,其间也可见三角形、多角形细胞。3d后见部分细胞已贴壁伸展,呈长梭形,可见核分裂相,梭形细胞逐渐增多。7d后见细胞80%-90%融合,细胞呈漩涡样生长,大小不一,细胞多为长梭形成纤维细胞样。传代细胞4-6h即可贴壁,细胞形态与原代相似,细胞生长速度较原代细胞明显增快。 2.细胞成脂诱导两周后油红O染色呈阳性,对照组为阴性;细胞成骨诱导三周后茜素红染色呈阳性,对照组为阴性。 3.全血血小板计数为(313.0±137.5)×10~9/L, PRP血小板计数为(1267.0±760.2)×10~9/L ,PRP血小板计数是全血的4.08倍。 4.CCK-8法显示PRP组在24h、48h、72h时较对照组增殖明显(P0.01)。且在第24h、48h、72h时,与全血组有统计学差异(P0.05)。结论:1.通过胶原酶消化法从兔颈背部区提取的脂肪组织分离得到的细胞,经鉴定证实为ADSCs。采用这种方法获得的细胞增殖迅速,可长期传代并维持稳定的增殖能力。 2.ADSCs具有多向分化潜能,在特定条件下能够向成骨细胞、脂肪细胞方向诱导分化,可作为组织工程的种子细胞。 3.通过二次离心成功提取兔PRP,血小板计数后,PRP的血小板计数控制在全血的4倍以上,二次离心法是制备PRP的有效方法。 4.自体PRP对体外培养的兔ADSCs增殖有明显的促进作用。
[Abstract]:Background: autogenous fat transplantation has a history of more than one hundred years. Because of its rich sources, easy materials, simple operation, good filling appearance, no rejection reaction and so on, autologous fat transplantation has attracted the attention of plastic and cosmetic surgeons, but it also has some shortcomings. The most important is the survival rate of fat cells after transplantation is lower than 60%. How to improve the proliferation and differentiation of adipocytes is an important link to improve the survival rate of transplantation. With the development and research of adipose tissue engineering, it is a key problem to seek for the growth factors with wide source, easy to obtain, small damage, stable expansion of seed cells and no immune response, high efficiency, low cost and good synergistic effect. Aim: to isolate and culture adipose-derived stem cells from rabbit adipose-derived stem cells, observe the biological characteristics and proliferation and differentiation potential of ADSCs in vitro, and investigate the effect of platelet-rich plasma-rich plasma from autologous platelet-rich plasma on the proliferation of ADSCs. Method 1: 1. In this experiment, New Zealand white rabbits were used to culture ADSCsin vitro, and the morphological characteristics of ADSCs were observed by using fat pad in the cervical and dorsal region of rabbits, adherent method and type I collagenase digestion method in vitro. 2. The third generation of ADSCs was taken for adipogenesis and osteogenic differentiation. Oil red O fat-forming staining was performed two weeks later and alizarin red osteogenic staining was performed three weeks later. 3. Rabbit heart blood was extracted and prepared by secondary centrifugation. The whole blood and PRP platelets were counted. 4. The third passage cells were divided into three groups: control group (cell 180ul alone DMEM medium 20ulus, whole blood group (cell 180ul whole blood plasma 20ull) and PRP group (180ul PRP 20uln). The proliferation of ADSCs was detected by using CCK-8 method for 24 h, 48 h and 72 h). The result is 1: 1. After 24 hours of primary culture, a small number of oval cells adhered to the wall, and most of the unadherent cells were round red blood cells and fascia impurities for the first time. The removal of non-adherent red blood cells and fascia impurities showed that a small number of adherent cells were short fusiform. There were also triangles. After 3 days, some of the polygonal cells were adherent to the wall and appeared to be fusiform, showing mitotic phase. The fusiform cells gradually increased .7d later, 80-90% of the cells were fused, and the cells grew like whirlpool and varied in size. The cells were mostly fusiform fibroblasts. The passage cells could adhere to the wall for 4-6 hours. The morphology of the cells was similar to that of the primary cells, and the growth rate of the cells was much faster than that of the primary cells. 2. After two weeks of adipogenic induction, the oil red O staining was positive, the control group was negative, and the alizarin red staining was positive after three weeks of osteogenesis, but the control group was negative. 3. The platelet count of whole blood was 313.0 卤137.5 脳 10 ~ (9) / L, the platelet count of PRP was 1267.0 卤760.2) 脳 10 ~ (9) / L, the platelet count of PRP was 4.08 times of that of the whole blood. 4.CCK-8 assay showed that the proliferation of PRP group was significantly higher than that of control group at 24 h and 48 h / 72 h compared with that of control group (P 0.01). There was a significant difference between the 24 h group and the whole blood group at 48 h and 72 h (P 0. 05). Conclusion 1. The cells isolated from adipose tissue of rabbit neck and back area by collagenase digestion were identified as ADSCs. The cells obtained by this method proliferate rapidly, can be subcultured for a long time and maintain stable proliferative ability. 2.ADSCs has the potential to differentiate into osteoblasts and adipocytes and can be used as seed cells for tissue engineering. 3. Rabbit PRP was successfully extracted by secondary centrifugation. After platelet count, the platelet count of rabbit PRP was controlled more than 4 times of that of the whole blood. Secondary centrifugation was an effective method for the preparation of PRP. 4. Autologous PRP can promote the proliferation of rabbit ADSCs in vitro.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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