本文選題：脂肪間充質(zhì)干細胞 + 富血小板血漿��； 參考：《大連醫(yī)科大學》2011年碩士論文

【摘要】：背景:自體脂肪移植已有上百年的歷史,因其來源豐富、取材容易、操作簡單、充盈外形好、無排異反應等優(yōu)點,倍受整形美容外科醫(yī)生的重視,但同時也存在不足之處,其中最主要是移植后脂肪細胞的成活率較低(60%)。如何提高脂肪細胞的增殖及分化能力,是提高移植成活率的重要環(huán)節(jié)。隨著脂肪組織工程的發(fā)展及研究,尋求來源廣、易獲取、損傷小、穩(wěn)定擴增的種子細胞和無免疫反應、高效、廉價、配比協(xié)同作用好的生長因子是關鍵問題。 目的:分離培養(yǎng)兔脂肪來源干細胞(adipose-derived stem cells,ADSCs),并觀察ADSCs體外生長的生物學特性及增殖分化潛能,探討自體富血小板血漿(platelet-rich plasma,PRP)對ADSCs增殖的影響。 方法:1.實驗采用新西蘭大白兔,取兔頸背部區(qū)脂肪墊,貼壁法及Ⅰ型膠原酶消化法體外培養(yǎng)兔ADSCs,觀察其形態(tài)特征。 2.取第3代ADSCs進行成脂、成骨誘導分化,兩周后油紅O成脂染色及三周后茜素紅成骨染色鑒定。 3.抽取兔心臟血,采用二次離心法制備PRP,并行全血及PRP血小板計數(shù)。 4.將第3代細胞分為對照組(細胞180ul +單純DMEM培養(yǎng)基20ul)、全血組(細胞180ul +全血血漿20ul)和PRP組(細胞180ul+ PRP 20ul),采用CCK-8法(Cell Counting Kit-8)檢測不同作用時間(24h、48h、72h)各組對ADSCs增殖活動的影響。 結(jié)果:1.原代培養(yǎng)的細胞24h后見少量橢圓形細胞貼壁,大部分未貼壁細胞為圓形的紅細胞及筋膜雜質(zhì)。48h首次換液,除去未貼壁的紅細胞及筋膜雜質(zhì),見小部分貼壁細胞呈短梭形,其間也可見三角形、多角形細胞。3d后見部分細胞已貼壁伸展,呈長梭形,可見核分裂相,梭形細胞逐漸增多。7d后見細胞80%-90%融合,細胞呈漩渦樣生長,大小不一,細胞多為長梭形成纖維細胞樣。傳代細胞4-6h即可貼壁,細胞形態(tài)與原代相似,細胞生長速度較原代細胞明顯增快。 2.細胞成脂誘導兩周后油紅O染色呈陽性,對照組為陰性;細胞成骨誘導三周后茜素紅染色呈陽性,對照組為陰性。 3.全血血小板計數(shù)為(313.0±137.5)×10~9/L, PRP血小板計數(shù)為(1267.0±760.2)×10~9/L ,PRP血小板計數(shù)是全血的4.08倍。 4.CCK-8法顯示PRP組在24h、48h、72h時較對照組增殖明顯(P0.01)。且在第24h、48h、72h時,與全血組有統(tǒng)計學差異(P0.05)。 結(jié)論:1.通過膠原酶消化法從兔頸背部區(qū)提取的脂肪組織分離得到的細胞,經(jīng)鑒定證實為ADSCs。采用這種方法獲得的細胞增殖迅速,可長期傳代并維持穩(wěn)定的增殖能力。 2.ADSCs具有多向分化潛能,在特定條件下能夠向成骨細胞、脂肪細胞方向誘導分化,可作為組織工程的種子細胞。 3.通過二次離心成功提取兔PRP,血小板計數(shù)后,PRP的血小板計數(shù)控制在全血的4倍以上,二次離心法是制備PRP的有效方法。 4.自體PRP對體外培養(yǎng)的兔ADSCs增殖有明顯的促進作用。
[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 鹵137.5 脳 10 ~ (9) / L, the platelet count of PRP was 1267.0 鹵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.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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