【摘要】：背景:細(xì)胞共培養(yǎng)技術(shù)是在體外條件下將細(xì)胞與另一種細(xì)胞共同培養(yǎng)。對(duì)于成骨細(xì)胞和脂肪細(xì)胞橫向分化研究多采用體外條件下成脂誘導(dǎo)液誘導(dǎo)成骨細(xì)胞發(fā)生橫向分化,而忽略了體內(nèi)環(huán)境中細(xì)胞互相作用的因素。目的:探討在Transwell間接共培養(yǎng)條件下成熟脂肪細(xì)胞誘導(dǎo)成骨細(xì)胞成脂橫向分化的能力。方法:(1)選取小鼠成纖維樣3t3-l1前體脂肪細(xì)胞進(jìn)行成脂誘導(dǎo)為成熟脂肪細(xì)胞;(2)實(shí)驗(yàn)分組:A組接種成熟脂肪細(xì)胞(Transwell間接共培養(yǎng)體系下室),B組接種小鼠成骨樣細(xì)胞mc3t3-e1(以1∶4比例接種到Transwell間接共培養(yǎng)體系上室),C組單獨(dú)培養(yǎng)的小鼠成骨樣細(xì)胞mc3t3-e1;(3)檢測(cè)指標(biāo):分別在7,14,21 d倒置相差顯微鏡下觀察細(xì)胞形態(tài)變化,檢測(cè)各組細(xì)胞三酰甘油相對(duì)含量、成脂目的蛋白PPARγ2表達(dá),并進(jìn)行油紅O染色以及茜素紅染色觀察;以0,24,36 h為截點(diǎn)檢測(cè)B組與C組細(xì)胞增殖抑制率的變化。結(jié)果與結(jié)論:(1)培養(yǎng)2周時(shí),3t3-l1細(xì)胞由長(zhǎng)梭形變?yōu)閳A形,胞質(zhì)內(nèi)脂滴增多呈亮圓形,油紅O染色鑒定后備用;(2)細(xì)胞形態(tài):B組共培養(yǎng)7 d時(shí)呈長(zhǎng)梭形,未見(jiàn)透明脂滴;14 d時(shí)胞質(zhì)內(nèi)出現(xiàn)黑色顆粒及小圓形脂滴;21 d細(xì)胞形態(tài)由長(zhǎng)梭形變?yōu)闄E圓形或圓形,脂滴變大;(3)茜素紅染色:7,14,21 d時(shí)B組細(xì)胞呈染色區(qū)減少的趨勢(shì),C組細(xì)胞無(wú)明顯變化均呈大片著色區(qū);(4)油紅O染色:7,14,21 d時(shí),B組細(xì)胞染色區(qū)呈遞增趨勢(shì)且與時(shí)間成正比,C組細(xì)胞染色陰性且無(wú)明顯變化;(5)三酰甘油:B組細(xì)胞三酰甘油聚集量呈遞增趨勢(shì)且與時(shí)間成正比,B組組間及與A組相比差異有顯著性意義(P0.05),C組無(wú)明顯變化(P0.05);(6)MTT抑制率:B組細(xì)胞增殖抑制率與時(shí)間成正比,與C組差異有顯著性意義(P0.05);(7)Western blot實(shí)驗(yàn):B組細(xì)胞PPARAγ2蛋白表達(dá)量呈遞增趨勢(shì)且與時(shí)間成正比,與A、C組相比,差異均有顯著性意義(P0.05);(8)結(jié)果提示,在Transwell間接共培養(yǎng)體系中,成骨細(xì)胞在成熟脂肪細(xì)胞誘導(dǎo)下呈現(xiàn)出成脂橫向分化趨勢(shì),其中脂肪細(xì)胞代謝產(chǎn)物及細(xì)胞因子可明顯抑制成骨細(xì)胞的增殖能力,其是否可以完全橫向分化為脂肪細(xì)胞有待進(jìn)一步研究。
[Abstract]:Background: co-culture of cells is to co-culture cells with another cell in vitro. For the lateral differentiation of osteoblasts and adipocytes, osteoblasts were induced by adipogenic medium in vitro, but the factors of cell interaction in vivo were neglected. Aim: to investigate the ability of adipocytes to induce adipogenic transversal differentiation of osteoblasts in Transwell indirect co culture. Methods: (1) Mouse fibroblast-like 3t3-l1 precursor adipocytes were selected for adipogenic induction to mature adipocytes. (2) experiment group: group A was inoculated with mature adipocytes (), B group was inoculated with mc3t3-e1 of mouse osteoblast in Transwell indirect co-culture system) (mc3t3-e1 was inoculated into the upper chamber of Transwell indirect co-culture system at 1:4). Mc3t3-e1; of osteoblast-like cells isolated in group C (3) Indexes: the morphological changes of the cells were observed under the inverted phase contrast microscope at day 714 and 21, respectively. The relative content of triglyceride and the expression of PPAR 緯 2 in each group were detected, and the oil red O staining and alizarin red staining were carried out. The cell proliferation inhibition rate of group B and group C was measured at 24 h. Results and conclusion: (1) after 2 weeks of culture, 3t3-l1 cells changed from long fusiform to round, and the increase of lipid droplets in the cytoplasm was bright and round. (2) Cell morphology: in group B, the cells were long fusiform at 7 d, no hyaline lipid droplets, black particles and small circular lipid droplets in the cytoplasm at 14 d, and changed from long fusiform to elliptic or round at 21 d, and the lipid droplets became larger. (3) alizarin red staining: group B cells showed a decreasing trend of staining area at day 21, while group C cells showed no obvious changes in large areas of staining; (4) Oil red O staining: the staining area of cells in group B showed an increasing trend and was proportional to the time at day 21, but the staining of cells in group C was negative and had no obvious change. (5) triacylglycerol: the accumulation of triglyceride in group B showed an increasing trend and was in direct proportion to the time, and there was a significant difference between group B and group A (P0.05) in), C group (P0.05); (6) inhibition rate of MTT: the inhibition rate of cell proliferation in group B was in direct proportion to time, and there was significant difference between group B and group C (P0.05). (7) Western blot test: the expression of PPARA 緯 2 protein in group B showed an increasing trend and was in direct proportion to time, and there was significant difference compared with group C (P0.05). (8) the results suggested that in Transwell indirect co-culture system, osteoblasts showed a tendency of adipogenic transversal differentiation induced by mature adipocytes, and adipocyte metabolites and cytokines could significantly inhibit the proliferation of osteoblasts. Whether it can be completely transversely differentiated into adipocytes remains to be further studied.
【作者單位】： 遵義醫(yī)學(xué)院第五附屬(珠海)醫(yī)院骨科二病區(qū);
【分類(lèi)號(hào)】：R318.08;R68

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