本文選題：脂肪細胞　切入點：脂肪組織來源干細胞　出處：《南方醫(yī)科大學》2008年碩士論文

【摘要】： 背景 組織工程技術(shù)的出現(xiàn)為臨床上徹底解決組織缺損等難題提供了一條革命性的途徑。然而如何獲得來源廣泛、數(shù)量充足、功能良好的細胞,是解決種子細胞問題的關(guān)鍵。 近年來,吸脂來源的人脂肪組織來源的干細胞(Adipose derived stemcells,ASCs)成為當今干細胞研究領域的一大熱點。但ASCs是位于人脂肪組織內(nèi)的一個混雜細胞群體。只有大約40%的細胞能向脂肪細胞分化。因此,ASCs并不能滿足脂肪組織工程的發(fā)展需要。尋找一種分離容易、增殖能力強、成脂分化率高的更優(yōu)秀的種子細胞仍然是研究熱點。 脂肪組織內(nèi)含有多種不同的細胞,主要包含有成熟脂肪細胞、血管內(nèi)皮細胞、成纖維細胞、組織細胞和干細胞等。大量的脂質(zhì)聚集使脂肪細胞具有的漂浮性,導致細胞難于體外培養(yǎng),因此對該細胞的特性研究很少。脂肪細胞由此被認為是處于分化終末期,缺乏增殖能力的一種細胞。國外有研究表明,成熟脂肪細胞在體外培養(yǎng)的過程中,能發(fā)生脂質(zhì)分解變化,重新啟動增殖機制,開始分裂增生。我們把這種生理轉(zhuǎn)變稱之為脂肪細胞去分化。 脂肪細胞去分化將開創(chuàng)細胞生理學一個令人驚喜的領域,改變?nèi)藗儗τ诮M織再生能力的許多觀點。然而,關(guān)于去分化脂肪細胞的細胞生物學特性研究還很少。本實驗中我們嘗試從脂肪抽吸物的脂質(zhì)部分分離純的成熟脂肪細胞,誘導成熟脂肪細胞去分化,獲得去分化脂肪細胞(Dedifferentiated adipocytes,DA)。同時對DA和脂質(zhì)部分來源的ASCs進行生長動力學、形態(tài)學、表面標志物、分化能力和成脂分化率五個方面的鑒定比較。為獲得、研究DA提供新的途徑,并首次為DA作為脂肪組織工程種子細胞的應用提供實驗依據(jù)。 目的: 1、探索從人脂肪抽吸物中脂質(zhì)部分分離、提純成熟脂肪細胞,體外培養(yǎng),以及誘導其去分化,獲得去分化脂肪細胞的方法。 2、通過與ASCs的比較,于形態(tài)學、生長動力學、表面標記物、分化能力及成脂分化率五個方面對DA進行研究,為其廣泛應用于組織工程提供實驗依據(jù)。 方法: 自成人吸脂術(shù)后抽吸物提取成熟脂肪細胞及ASCs,天花板貼壁培養(yǎng)法誘導成熟脂肪細胞去分化,觀察細胞形態(tài)變化,獲得DA。相同的條件下,MTT比色法比較DA、ASCs活性并繪制細胞生長曲線;流式細胞儀鑒定DA、ASCs表面分子的表達;油紅O染色、茜素紅染色、阿爾辛藍染色分別鑒定DA、ASCs成脂分化、成骨分化、成軟骨分化能力:大體鏡下觀察、油紅O染色分光光度法、油紅O染色細胞計數(shù)法比較DA、ASCs成脂分化能力。 結(jié)果: 人成熟脂肪細胞在體外培養(yǎng)環(huán)境下能去分化為成纖維細胞狀DA;MTT比色法測細胞活性:DA、ASCs都有很強的增殖能力,兩者無顯著性差異;流式細胞儀測定:DA、ASCs中HLA-ABC、CD29、CD44都為陽性,CD45、CD34、CD106都為陰性;成骨分化兩周,茜素紅染色可見DA、ASCs內(nèi)出現(xiàn)紅色鈣鹽沉積;成軟骨分化兩周,阿爾辛藍染色可見DA、ASCs內(nèi)軟骨基質(zhì)沉積;成脂分化兩周,油紅O染色可見DA、ASCs內(nèi)出現(xiàn)紅色脂滴;大體鏡下觀察顯示DA脂滴聚集能力顯著大于ASCs;油紅O染色分光光度法顯示DA在誘導第4天后出現(xiàn)顯著性脂滴聚集,ASCs在誘導第10天后才出現(xiàn)顯著性脂滴聚集,誘導第12天后DA的吸光值大于ASCs的吸光值,出現(xiàn)顯著性差異;油紅O染色細胞計數(shù)法示DA的平均成脂分化率約為65%,ASCs的約為35%,兩者有顯著性差異。 結(jié)論: 從成人脂肪抽吸物脂質(zhì)部分中可以分離、提純得到大量的成熟脂肪細胞。成熟的脂肪細胞在體外培養(yǎng)條件下可成為DA,DA與ASCs在細胞的生長動力學、形態(tài)學、表面標志物和分化能力等方面具有相似的特征,具有很強的增殖活性,表達部分干細胞特征性表面蛋白,有成骨、成軟骨分化能力及強大的成脂分化能力,有望成為脂肪組織工程優(yōu)秀的種子細胞。
[Abstract]:background
The emergence of tissue engineering technology provides a revolutionary way for solving the problems of tissue defects in clinical practice. However, how to get a wide range of cells with sufficient quantity and good function is the key to solve the problem of seed cells.
In recent years, liposuction from human adipose tissue derived stem cells (Adipose derived stemcells, ASCs) has become a hot research field of stem cells. But ASCs is a mixed cell population in human adipose tissues. Only about 40% of the cells to adipose cell differentiation. Therefore, ASCs does not meet the the need of the development of adipose tissue engineering. Looking for an easy separation, strong proliferation ability, a more excellent seed cell fat high differentiation rate is still the focus of research.
Contains a variety of different cells in adipose tissue, are mainly composed of mature fat cells, vascular endothelial cells, fibroblasts, cells and tissue stem cells. A large number of lipid accumulation in fat cells with the floating cells in vitro, resulting in difficult, so little research on the characteristics of the cell. The fat cells is thus considered is in the terminal stage of differentiation, the lack of a cell proliferation. Foreign studies have shown that mature adipocytes in vitro, lipolysis changes can occur, restart the proliferation mechanism, begin to proliferate. The physiological changes known to differentiate into fat cells.
Fat cells to differentiation of cell physiology will create a surprise for many people, change view regeneration ability of organization. However, the research on the biological characteristics of cells to adipocyte differentiation are rare. In this experiment we attempt to separate pure mature fat cells from the lipid portion of liposuction, induction of mature fat cells to get to the differentiation of adipocyte differentiation (Dedifferentiated, adipocytes, DA). At the same time, DA and ASCs are the source of the lipid portion of the growth kinetics, morphology, surface markers, differentiation and adipogenic differentiation rate of identification in five aspects. In order to obtain and provide a new way of DA, and for the first time for the application of DA as adipose tissue engineering seed cells and provide experimental basis.
Objective:
1, we need to explore the way to isolate and purify mature adipocytes from human liposuction, to induce dedifferentiation, and to get dedifferentiated adipocytes.
2, by comparing with ASCs, we studied DA in five aspects: morphology, growth kinetics, surface markers, differentiation ability and adipogenic differentiation rate, providing experimental evidence for its wide application in tissue engineering.
Method:
Since adults after liposuction aspirates from mature fat cells and ASCs ceiling adherent induced dedifferentiation of mature adipocytes were cultured. The morphological changes were observed for DA. under the same conditions, MTT colorimetric method DA, ASCs activity and cell growth curve; flow cytometry identification DA surface expression of ASCs molecules; oil red O staining, alizarin red staining, alcian blue staining were identified as DA ASCs, adipogenic differentiation, osteogenic differentiation and chondrogenic differentiation ability: observed under microscope oil red O staining and spectrophotometry, oil red O staining cell count is compared with DA, ASCs and adipogenic differentiation ability.
Result:
Human mature adipocytes in vitro environment can differentiate into fibroblast like DA; MTT cell activity assay: DA and ASCs had strong proliferation ability, no significant difference; flow cytometry: DA, ASCs HLA-ABC, CD29, CD44 were positive for CD45. CD34, CD106 were negative; osteogenic differentiation for two weeks, alizarin red staining showed red DA, calcium salt deposition within ASCs; chondrogenic differentiation for two weeks, alcian blue staining, DA, ASCs in cartilage matrix deposition; adipogenic differentiation for two weeks, oil red O staining showed DA, ASCs appeared in red lipid droplets; general microscopy showed that DA lipid droplet aggregation ability was significantly greater than ASCs; oil red O staining spectrophotometric method showed that DA in fourth days after the induction of significant lipid droplet aggregation, ASCs appeared significant lipid droplet aggregation in tenth days after induction, Twelfth days after the induction of DA light absorption value is greater than ASCs value, there was significant difference The oil red O staining cell count method showed that the average fat differentiation rate of DA was about 65%, and that of ASCs was about 35%, and there was a significant difference between them.
Conclusion:
Can be isolated from adult adipose aspirates lipid fraction, purified mature fat cells. Mature adipocytes into DA in vitro, DA and ASCs on the growth kinetics, cell morphology, surface markers and differentiation capacity were similar characteristics, with strong proliferation, expression some characteristics of stem cell surface protein, osteogenic, chondrogenic differentiation ability and strong adipogenic differentiation ability, is expected to become fat seed cells for tissue engineering of excellent.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329

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