本文關(guān)鍵詞： 高成脂 脂肪干細(xì)胞 表面標(biāo)志 CD54　出處：《南方醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 研究背景: 脂肪組織是修復(fù)全身各部位軟組織缺損的最佳選擇。臨床上自體成熟脂肪組織的移植,不論是脂肪塊或是脂肪顆粒的注射移植,均可能出現(xiàn)壞死、再吸收等問題,導(dǎo)致術(shù)后長期效果不佳。組織工程技術(shù)的出現(xiàn),對組織器官的修復(fù)與替代治療產(chǎn)生了重大意義。 組織工程技術(shù)的首要條件就是種子細(xì)胞。由于胚胎干細(xì)胞受到倫理學(xué)和來源的限制,故目前使用最多的種子細(xì)胞即成體干細(xì)胞。作為種子細(xì)胞的成體干細(xì)胞主要有兩種來源,一是骨髓來源的骨髓干細(xì)胞(bone marrow-derived stemcells,BMSCs),另一種是脂肪組織來源的脂肪干細(xì)胞(Adipose-derived stem cells,ASCs)。相對與BMSCs而言,ASCs取材容易,且細(xì)胞量足以滿足組織工程的需求,具有很大的優(yōu)勢。 但現(xiàn)有的研究表明,通過普通的酶消化法分離出來的ASCs是由極其復(fù)雜的各種細(xì)胞集合而成,成脂等各項(xiàng)誘導(dǎo)分化的能力及效率都不高。將ASCs進(jìn)行成脂誘導(dǎo)后兩周,其分化比例只為30%～40%,同時(shí)ASCs其余的各項(xiàng)分化效率也低于BMSCs。因此,純化ASCs,提高其誘導(dǎo)分化效率,是優(yōu)化其成為最佳種子細(xì)胞來源之一的有效方法。 近年來,有研究發(fā)現(xiàn),成熟脂肪細(xì)胞在某種環(huán)境中,可去分化回成纖維樣細(xì)胞狀態(tài),這種退回到原始狀態(tài)的去分化細(xì)胞具有多向分化的潛能,且成脂分化能力要高于ASCs。然而這種去分化細(xì)胞的高成脂能力是否伴隨著某些表面標(biāo)志的表達(dá),國內(nèi)外均未見報(bào)道。所以,本實(shí)驗(yàn)欲比較去分化脂肪細(xì)胞與ASCs在表面標(biāo)志等各方面的不同,尋求出高成脂細(xì)胞高表達(dá)的表面標(biāo)志,并希望能通過免疫磁珠分選,提純出高成脂系A(chǔ)SCs。 目的: 探索尋求與高成脂能力相關(guān)的干細(xì)胞表面分子,并通過分選,純化得到高成脂系A(chǔ)SCs,從而為提高脂肪組織工程的效率提供有效途徑。 方法: 1、從人脂肪組織中分離培養(yǎng)ASCs,成脂誘導(dǎo)分化ASCs,得到誘導(dǎo)后脂肪細(xì)胞。收集誘導(dǎo)后漂浮于培養(yǎng)基中的脂肪細(xì)胞,天花板貼壁培養(yǎng)10d,脂肪細(xì)胞貼壁后翻轉(zhuǎn)正常培養(yǎng)得到去分化脂肪細(xì)胞。選擇同等代數(shù)兩種細(xì)胞,成脂誘導(dǎo)分化并用油紅O染色法鑒定其分化效率;細(xì)胞計(jì)數(shù)法繪制細(xì)胞生長曲線;流式細(xì)胞術(shù)測定細(xì)胞的表面標(biāo)志表達(dá),并在上述三方面對ASCs和去分化脂肪細(xì)胞進(jìn)行比較,篩選出可能與高成脂能力相關(guān)的表面分子。 2、就篩選出的可能與高成脂能力相關(guān)的表面分子,用免疫磁珠分選法分別分選脂肪組織新鮮分離的間質(zhì)血管碎片(stromal vascular fractions,SVF)及培養(yǎng)后ASCs。流式細(xì)胞檢測分選前后細(xì)胞群體該表面分子陽性表達(dá)率;并對兩種分選出的細(xì)胞與未行分選的ASCs進(jìn)行成脂、成骨誘導(dǎo),油紅O染色鑒定成脂誘導(dǎo)結(jié)果,茜素紅染色鑒定成骨誘導(dǎo)結(jié)果。 結(jié)果: 從吸脂術(shù)中獲得人脂肪組織,培養(yǎng)出ASCs。選擇第4代ASCs進(jìn)行成脂誘導(dǎo),誘導(dǎo)13d起,收集誘導(dǎo)成熟的脂肪細(xì)胞,天花板貼壁培養(yǎng)。培養(yǎng)過程中,多房的脂肪細(xì)胞貼壁并逐漸吐出脂泡,最終去分化為成纖維狀細(xì)胞。去分化細(xì)胞與ASCs相比,生長曲線較一致說明兩者具有相似的增殖能力;油紅O染色鑒定成脂誘導(dǎo)結(jié)果表明去分化細(xì)胞的成脂分化效率高出ASCs約50%。兩種細(xì)胞在表面標(biāo)志的表達(dá)上,CD34、CD54的表達(dá)陽性率有所差別:去分化細(xì)胞的CD34、CD54的表達(dá)為陽性,而ASCs的表達(dá)均為陰性。 用CD54-PE免疫磁珠分選新鮮分離的SVF及經(jīng)過培養(yǎng)后的ASCs。分選前,流式檢測一至三代ASCs,細(xì)胞CD54的表達(dá)率隨著代數(shù)的增加而降低。分選后,CD54的表達(dá)率在兩種細(xì)胞群體中均達(dá)到90%以上。繼續(xù)培養(yǎng)數(shù)代后,從培養(yǎng)后ASCs分離出的CD54陽性細(xì)胞群體CD54表達(dá)率維持高水平,新鮮分離SVF中分離出的陽性細(xì)胞CD54的表達(dá)則降至陰性,未分選的ASCs為陰性表達(dá)。分選后的細(xì)胞與未分選的ASCs成脂能力比較:從培養(yǎng)后的ASCs分離出的CD54陽性細(xì)胞成脂分化率接近100%,未分選的ASCs成脂能力約為30%,而從新鮮分離的SVF中分選出的CD54陽性細(xì)胞成脂分化率只為10%左右。成骨誘導(dǎo)結(jié)果:ASCs成骨能力為三種細(xì)胞中最佳,而從SVF分選出的CD54陽性細(xì)胞群的成骨效率最差。 結(jié)論 1、本實(shí)驗(yàn)通過比較去分化細(xì)胞與ASCs在增殖能力、成脂分化能力與表面標(biāo)志表達(dá)等方面的差別,認(rèn)為去分化細(xì)胞是一種類似于干細(xì)胞但較干細(xì)胞具有更高成脂分化能力的細(xì)胞。去分化細(xì)胞CD54、CD34的表達(dá)高于ASCs表明CD54、CD34的表達(dá)可能與高成脂能力相關(guān)。 2、CD54免疫磁珠分選經(jīng)過培養(yǎng)的ASCs,能純化ASCs并分離出高成脂系干細(xì)胞。因而,CD54應(yīng)是高成脂系干細(xì)胞特異表達(dá)的表面分子之一,從ASCs分離CD54陽性細(xì)胞可做為純化高成脂干細(xì)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.

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

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2 閆迎軍;應(yīng)用脂肪干細(xì)胞與脫細(xì)胞真皮構(gòu)建組織工程皮膚[D];中國協(xié)和醫(yī)科大學(xué);2007年

3 王敏;人脂肪干細(xì)胞在PLGA支架上向肝細(xì)胞誘導(dǎo)分化的實(shí)驗(yàn)研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2009年

4 王福科;臍血來源VECs與ADSCs聯(lián)合培養(yǎng)移植修復(fù)頜骨缺損研究[D];昆明醫(yī)學(xué)院;2009年

5 萬光勇;人脂肪干細(xì)胞與部分脫鈣骨支架構(gòu)建組織工程骨的試驗(yàn)研究[D];山東大學(xué);2009年

6 郝偉;基于兔脂肪干細(xì)胞+I型膠原凝膠+PLGA-β-TCP支架的新型骨組織工程復(fù)合體的構(gòu)建以及修復(fù)自體橈骨缺損的相關(guān)實(shí)驗(yàn)研究[D];第四軍醫(yī)大學(xué);2007年

7 丁小邦;應(yīng)用組織工程技術(shù)和人自體細(xì)胞探索可注射性真皮充填物的實(shí)驗(yàn)研究[D];南方醫(yī)科大學(xué);2007年

8 唐少鋒;脂肪干細(xì)胞分離培養(yǎng)及修復(fù)軟骨缺損的實(shí)驗(yàn)研究[D];昆明醫(yī)學(xué)院;2008年

9 周向陽;脂肪干細(xì)胞移植治療腦冷凍損傷的實(shí)驗(yàn)研究[D];中南大學(xué);2008年

10 張勇;脂肪干細(xì)胞直接修復(fù)兔膝關(guān)節(jié)軟骨損傷的實(shí)驗(yàn)研究[D];第二軍醫(yī)大學(xué);2009年

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1 王宇燕;高成脂能力脂肪干細(xì)胞系的分離與純化[D];南方醫(yī)科大學(xué);2008年

2 楊靚;脂肪干細(xì)胞來源施萬細(xì)胞移植治療大鼠運(yùn)動皮層損傷模型[D];中南大學(xué);2008年

3 王飛;大鼠脂肪干細(xì)胞（ADSCs）的培養(yǎng)、向神經(jīng)細(xì)胞的分化以及BDNF基因和其受體trkB的表達(dá)[D];中南大學(xué);2007年

4 蔣威;體外誘導(dǎo)脂肪干細(xì)胞向血管平滑肌細(xì)胞分化的實(shí)驗(yàn)研究[D];昆明醫(yī)學(xué)院;2008年

5 寧睿明;人吸脂來源的干細(xì)胞與脂肪細(xì)胞共培養(yǎng)的研究[D];大連理工大學(xué);2009年

6 邱婷;利用脂肪源性干細(xì)胞重建角膜上皮的實(shí)驗(yàn)研究[D];復(fù)旦大學(xué);2009年

7 黃俊;不同濃度萬古霉素對大鼠脂肪干細(xì)胞體外定向誘導(dǎo)分化為成骨細(xì)胞的影響[D];中南大學(xué);2009年

8 陳小紅;大鼠脂肪干細(xì)胞（rADSCs）的分離鑒定及向成牙本質(zhì)細(xì)胞分化的實(shí)驗(yàn)研究[D];第三軍醫(yī)大學(xué);2007年

9 李伯休;脂肪來源干細(xì)胞（ADSCs）在周圍神經(jīng)損傷修復(fù)中的研究[D];復(fù)旦大學(xué);2008年

10 李政;經(jīng)靜脈移植脂肪干細(xì)胞在兔激素性股骨頭壞死模型體內(nèi)的示蹤研究[D];福建中醫(yī)學(xué)院;2009年

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