本文關(guān)鍵詞： 骨科組織工程 脂肪干細(xì)胞 去分化脂肪細(xì)胞 成骨能力 成軟骨能力　出處：《遵義醫(yī)學(xué)院》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：比較兔去分化脂肪細(xì)胞(Dedifferentiated adipocytes, DA)和脂肪干細(xì)胞(Adipose-derived stem cells, ADSCs)在體外成骨及成軟骨的能力,為骨科組織工程中種子細(xì)胞的選擇提供依據(jù)。 方法：①取4月齡新西蘭大白兔腹股溝脂肪組織,機(jī)械分離和酶消化法提取成熟脂肪細(xì)胞(Mature adipocytes, MA)和ADSCs。②天花板貼壁培養(yǎng)法誘導(dǎo)MA去分化,獲得DA。③將ADSCs和DA以2×104個(gè)/ml接種到24孔板中,每日倒置顯微鏡下觀察細(xì)胞生長情況,并細(xì)胞計(jì)數(shù)法檢測(cè)細(xì)胞增殖數(shù)量,取平均值通過Excel2007軟件繪制原代至第三代的生長曲線圖,計(jì)算兩種細(xì)胞增殖倍增時(shí)間。④選取第三代細(xì)胞進(jìn)行實(shí)驗(yàn)分組：ADSCs加入成骨誘導(dǎo)液(β-甘油磷酸鈉、VitC、地塞米松和含10%FBS的基礎(chǔ)培養(yǎng)基),設(shè)為Ao組,DA加入成骨誘導(dǎo)液,設(shè)為Do組；ADSCs加入成軟骨誘導(dǎo)液(胰島素、TGF-β1、VitC、轉(zhuǎn)鐵蛋白、地塞米松和含10%FBS的基礎(chǔ)培養(yǎng)基),設(shè)為Ac組,DA加入成軟骨誘導(dǎo)液,設(shè)為Dc組,各組都設(shè)立空白對(duì)照組。各組細(xì)胞誘導(dǎo)第14天時(shí),Ao組和Do組行成骨細(xì)胞茜素紅、Ⅰ型膠原免疫組化染色；Ac組和Dc組行軟骨細(xì)胞甲苯胺藍(lán)、Ⅱ型膠原免疫組化染色。⑤取各組培養(yǎng)第14天及第21的細(xì)胞,RT-PCR半定量檢測(cè)Ao組和Do組中Ⅰ型膠原mRNA表達(dá)量,所得數(shù)據(jù)采用t檢驗(yàn)行組間和組內(nèi)比較；同法檢測(cè)Ac組和Dc組中Ⅱ型膠原mRNA表達(dá)量及統(tǒng)計(jì)學(xué)分析。比較ADSCs和DA成骨及成軟骨的能力。 結(jié)果：①原代MA呈小圓形指環(huán)狀、形態(tài)規(guī)則,聚集分布。天花板貼壁培養(yǎng)后,MA中脂質(zhì)部分逐漸由細(xì)胞胞質(zhì)內(nèi)脫出,由小圓形變?yōu)闄E圓形,最終變?yōu)殚L梭形成纖維細(xì)胞狀；原代ADSCs呈長梭形或小多角形,大小均勻并散在分布。②傳代培養(yǎng)后,兩種細(xì)胞亦呈長梭形、密集分布,“漩渦樣”生長。③原代培養(yǎng)時(shí),細(xì)胞計(jì)數(shù)法檢測(cè)ADSCs對(duì)數(shù)生長期終末細(xì)胞數(shù)為5.9±0.41,DA為6.3±0.36,t檢驗(yàn)兩者無統(tǒng)計(jì)學(xué)差異(p0.05)；傳代培養(yǎng)后,第一代、第二代和第三代的ADSCs及DA對(duì)數(shù)生長期終末細(xì)胞數(shù)差異不大(p0.05)。兩種細(xì)胞倍增時(shí)間在58-61小時(shí)之間(p0.05),說明兩種細(xì)胞增殖能力相似。④Ao組和Do組茜素紅和Ⅰ型膠原免疫組化染色陽性,對(duì)照組陰性；Ac組和Dc組甲苯胺藍(lán)及Ⅱ型膠原免疫組化染色陽性,對(duì)照組陰性。⑤RT-PCR檢測(cè)第14天和第21天Ao組中Ⅰ型膠原mRNA表達(dá)量為0.375±0.018、0.908±0.017,Do組Ⅰ型膠原表達(dá)量為0.246±0.014、0.821±0.012,相同時(shí)間點(diǎn)Ao組表達(dá)量明顯高于Do組(p0.05),且Ⅰ型膠原表達(dá)量與培養(yǎng)時(shí)間成正比(p0.05)；Ac組和Dc組中Ⅱ型膠原mRNA表達(dá)量分別為0.316±0.027、0.458±0.020和0.204±0.024、0.377±0.021,Ac組中Ⅱ型膠原表達(dá)量也均明顯較Dc組高(p0.05),隨著培養(yǎng)時(shí)間的延長Ⅱ型膠原表達(dá)量增加(p《0.05)。兩種細(xì)胞空白對(duì)照組中均無Ⅰ型膠原和Ⅱ型膠原mRNA的表達(dá),與實(shí)驗(yàn)組相比具有統(tǒng)計(jì)學(xué)差異(p0.05)。 結(jié)論：天花板貼壁培養(yǎng)法可誘導(dǎo)MA去分化,獲得DA；體外培養(yǎng)時(shí),DA與ADSCs形態(tài)上均呈長梭形成纖維細(xì)胞狀,沒有明顯的形態(tài)學(xué)差異；DA和ADSCs在體外增殖能力相似；兩種細(xì)胞都可通過體外定向誘導(dǎo)向成骨細(xì)胞及軟骨細(xì)胞分化,且ADSCs成骨和成軟骨分化能力均高于DA; ADSCs和DA都可以考慮作為骨科組織工程的種子細(xì)胞。
[Abstract]:Objective: To compare the ability of Dedifferentiated adipocytes (DA) and Adipose-derived stem cells (ADSCs) to induce osteogenesis and chondrogenesis in vitro, and to provide basis for selection of seed cells in tissue engineering of Department of orthopedics.
Methods: a total of 4 month old New Zealand rabbits inguinal adipose tissue from mature fat cells mechanical separation and enzymatic digestion method (Mature adipocytes, MA ADSCs.) and the ceiling adherent culture method to induce MA differentiation, DA. 3 and DA ADSCs with 2 * 104 /ml were inoculated into 24 well plate, to observe the growth of the cells under inverted microscope daily, and cell proliferation was detected by cell counting number, the average growth curve by Excel2007 software to draw the original to the third generation, calculation of two proliferating cell doubling time. The third generation cells were selected experimental groups: ADSCs with osteogenic medium (beta glycerophosphate, VitC the foundation, dexamethasone and 10%FBS containing medium), group Ao, DA with the osteogenic induction medium, group Do; ADSCs into chondrogenic media (insulin, TGF- beta 1, VitC, transferrin, dexamethasone and 10%FBS containing basic training Yang Ji), group Ac, DA joined the chondrogenic media, as group Dc, each group a blank control group. The cells were cultured for fourteenth days, Ao group and Do group for osteoblast alizarin red, type I collagen immunohistochemical staining; Ac group and Dc group for chondrocyte toluidine blue. Type II collagen immunohistochemical staining. 5 were taken and cultured for fourteenth days and 21 cells, the expression of RT-PCR semi quantitative detection of type Ao collagen in the mRNA group and Do group, the data obtained using the t test for comparison between and within group; analysis and statistics with type II was detected in Ac group and Dc group expression of collagen mRNA. Comparison of ADSCs and DA into the bone and cartilage.
Results: the primary MA were small round ring, regular shape, aggregation distribution. The ceiling after the adherent, MA lipid fraction gradually from the cytoplasm of prolapse, composed of small round into oval, eventually become spindle shaped fibroblast like; primary ADSCs were fusiform or small angle shape, uniform size and scattered. The passage, two cells were fusiform, densely distributed, the vortex like growth. The primary culture, detection of ADSCs cell count at the end of the logarithmic growth cells was 5.9 + 0.41, DA = 6.3 + 0.36, t test two statistical difference (P0.05); after subculture, the first generation, second generation and third generation of ADSCs and DA in the final at the end of the logarithmic growth cells had no difference (P0.05). Two kinds of cell doubling time in 58-61 hours (P0.05), shows two kinds of cell proliferation are similar. The Ao group and Do group alizarin red and collagen type I immune Positive staining, negative control group; Ac group and Dc group, toluidine blue staining and type II collagen immunohistochemical staining was positive and negative in the control group. The RT-PCR expression of type fourteenth and twenty-first days in group Ao, collagen mRNA was 0.375 + 0.018,0.908 + 0.017, Do group of type I collagen expression was 0.246 + 0.014,0.821 0.012, at the same time in Ao group was significantly higher than that in group Do (P0.05), and the expression of type I collagen and the amount of training time is proportional to the type II (P0.05); Ac group and Dc group in the expression of collagen mRNA were 0.316 + 0.027,0.458 + 0.020 and 0.204 + 0.024,0.377 + 0.021, type II in Ac group. The expression of collagen were significantly higher than group Dc (P0.05), with the extension of time the expression of type II collagen (p<0.05). The amount of training to increase the expression of two kinds of cells in the control group had no collagen type I and type II collagen mRNA, compared with the experimental group with significant difference (P0.05).
Conclusion: the ceiling adherent culture method to induce MA differentiation in vitro, DA; DA and ADSCs morphology were long spindle shaped fibroblast like morphology, no obvious difference; DA and ADSCs are similar in vitro proliferation ability; two kinds of cells can induce osteogenic and chondrogenic differentiation by in vitro, ADSCs and osteogenic and chondrogenic differentiation ability were higher than that of DA; ADSCs and DA can be considered as the seed cells for tissue engineering of the Department of orthopedics.

【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 廖云君;高建華;姜平;魯峰;;缺氧對(duì)成熟脂肪細(xì)胞去分化影響的實(shí)驗(yàn)研究[J];南方醫(yī)科大學(xué)學(xué)報(bào);2008年03期

2 楊震;簡月奎;安榮澤;敖俊;王兆杰;;SD大鼠脂肪干細(xì)胞的體外分離培養(yǎng)及實(shí)驗(yàn)研究[J];貴州醫(yī)藥;2008年05期

3 Jie-fei Shen;Atsunori Sugawara;Joe Yamashita;Hideo Ogura;Soh Sato;;Dedifferentiated fat cells: an alternative source of adult multipotent cells from the adipose tissues[J];International Journal of Oral Science;2011年03期

4 孫煒;王吉興;金大地;劉曉霞;劉安慶;;iNOS抑制劑對(duì)兔關(guān)節(jié)軟骨修復(fù)組織膠原表達(dá)的影響[J];實(shí)用骨科雜志;2009年02期

5 安榮澤;王兆杰;張強(qiáng);楊震;于立志;齊新文;楊晉;;改良誘導(dǎo)方案誘導(dǎo)SD大鼠脂肪源性干細(xì)胞定向分化為軟骨細(xì)胞[J];中國組織工程研究與臨床康復(fù);2009年36期

6 安榮澤;王兆杰;劉凡凡;齊新文;袁小洪;陳軍平;趙俊延;胡小軍;楊晉;趙豪;;重組pcDNA3.1-hBMP-2轉(zhuǎn)染兔脂肪干細(xì)胞體外誘導(dǎo)的成骨分化[J];中國組織工程研究與臨床康復(fù);2011年41期

7 楊國慶;王兆杰;安榮澤;趙俊延;齊新文;;兔脂肪干細(xì)胞在含有轉(zhuǎn)化因子和轉(zhuǎn)鐵蛋白培養(yǎng)基中向軟骨細(xì)胞的分化[J];中國組織工程研究與臨床康復(fù);2011年45期

8 史永紅;任韞卓;趙松;郝軍;姚芳;劉巍;吳海江;段惠軍;;細(xì)胞因子信號(hào)傳導(dǎo)抑制蛋白1對(duì)糖基化終末產(chǎn)物誘導(dǎo)的腎小管細(xì)胞轉(zhuǎn)分化的影響[J];中國藥理學(xué)通報(bào);2010年02期

9 金長禮;李德華;李洪秀;樸仁京;;GDF-5誘導(dǎo)hADSCs分化為軟骨樣細(xì)胞[J];中國實(shí)用醫(yī)藥;2012年01期

相關(guān)碩士學(xué)位論文 前1條

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

，

本文編號(hào)：1495170