本文選題：BMSCs + 脂肪細(xì)胞�。� 參考：《石河子大學(xué)》2016年碩士論文

【摘要】：目的觀察不同時(shí)期骨髓間充質(zhì)干細(xì)胞來(lái)源的脂肪細(xì)胞內(nèi)分泌功能的差異;采用細(xì)胞共培養(yǎng)觀察不同大小脂肪細(xì)胞對(duì)成骨和破骨細(xì)胞誘導(dǎo)分化的影響;觀察不同濃度脂肪酸對(duì)成骨細(xì)胞誘導(dǎo)分化的影響。方法1.各型細(xì)胞誘導(dǎo)分化及鑒定:選取4周齡Wistar雄性大鼠,分離骨髓間充質(zhì)干細(xì)胞(Bone marrow mesenchymal stem cells,BMSCs),分別向脂肪細(xì)胞和成骨細(xì)胞方向誘導(dǎo)分化;油紅O染色鑒定成熟脂肪細(xì)胞,茜素紅染色鑒定成骨細(xì)胞;收集Wistar大鼠全骨髓液,誘導(dǎo)分化為破骨細(xì)胞,抗酒石酸酸性磷酸酶染色鑒定。2.BMSCs來(lái)源的脂肪細(xì)胞與成骨細(xì)胞、破骨細(xì)胞共培養(yǎng):1)BMSCs來(lái)源的脂肪細(xì)胞成熟后繼續(xù)誘導(dǎo)分化,每隔2 d收集一次脂肪細(xì)胞培養(yǎng)液上清,-20℃凍存(采樣時(shí)間點(diǎn):21 d、24 d、27 d、30 d、33 d、36 d、39 d、42 d、45d、48 d、50d),所有樣本收集完成后監(jiān)測(cè)炎癥細(xì)胞因子;同時(shí)觀察脂肪細(xì)胞體積和數(shù)量,細(xì)胞體積用標(biāo)尺測(cè)量,細(xì)胞數(shù)量在400×顯微鏡下計(jì)數(shù)6個(gè)視野,取平均值。綜合以上監(jiān)測(cè)數(shù)據(jù),確定共培養(yǎng)的時(shí)間點(diǎn)。2)脂肪細(xì)胞誘導(dǎo)劑誘導(dǎo)P3代BMSCs向脂肪細(xì)胞方向分化,分別誘導(dǎo)到第21 d和40 d進(jìn)行共培養(yǎng)。3)非直接接觸共培養(yǎng):更換脂肪培養(yǎng)基為脂肪維持培養(yǎng)基,每隔一天分別將脂肪細(xì)胞培養(yǎng)皿中的培養(yǎng)基吸出并在其中添加成骨細(xì)胞誘導(dǎo)劑/破骨誘導(dǎo)劑,進(jìn)行成骨細(xì)胞/破骨細(xì)胞的誘導(dǎo)。4)直接接觸共培養(yǎng):分別在培養(yǎng)皿中刮去約10 cm2的脂肪細(xì)胞,在其中接種新鮮的BMSCs,培養(yǎng)基更換為成骨細(xì)胞誘導(dǎo)劑加脂肪細(xì)胞維持培養(yǎng)液,向成骨細(xì)胞方誘導(dǎo)分化;將加有完全培養(yǎng)基的骨髓細(xì)胞于細(xì)胞培養(yǎng)箱孵育24小時(shí),收集非貼壁細(xì)胞,分別在培養(yǎng)皿中刮去約10 cm2的脂肪細(xì)胞,在其中接種非貼壁細(xì)胞,培養(yǎng)基更換為破骨細(xì)胞誘導(dǎo)劑加脂肪細(xì)胞維持培養(yǎng)液,向破骨細(xì)胞方向誘導(dǎo)分化。5)培養(yǎng)基中炎癥細(xì)胞因子及骨代謝因子表達(dá)水平的檢測(cè)。運(yùn)用ELISA方法檢測(cè)炎癥因子運(yùn)用RT-PCR方法檢測(cè)骨代謝因子。3.不同濃度脂肪酸對(duì)成骨細(xì)胞分化的影響:Wistar大鼠P3代BMSCs向成骨細(xì)胞誘導(dǎo)分化,在成骨誘導(dǎo)培養(yǎng)基中加入不同濃度(25m M、50 m M、100 m M和200 m M)油酸和棕櫚酸,觀察兩種脂肪酸對(duì)成骨細(xì)胞分化的影響。結(jié)果1.Wistar大鼠BMSCs 15 d左右培養(yǎng)成功,傳三代后其表面分子標(biāo)志物CD29和CD44呈陽(yáng)性。P3代BMSCs在脂肪誘導(dǎo)培養(yǎng)基作用下21 d分化為脂肪細(xì)胞,油紅O染色陽(yáng)性;在成骨誘導(dǎo)培養(yǎng)基作用下21 d分化為成骨細(xì)胞,茜素紅染色陽(yáng)性。Wistar大鼠全骨髓液在破骨細(xì)胞培養(yǎng)基作用下28 d分化為破骨細(xì)胞,抗酒石酸酸性磷酸酶染色陽(yáng)性。2.脂肪細(xì)胞成熟后,繼續(xù)誘導(dǎo)分化,第21 d脂肪細(xì)胞數(shù)量顯著高于第40 d(P0.001),但體積顯著低于第40 d脂肪細(xì)胞(P0.05)。隨著誘導(dǎo)時(shí)間增加,脂肪細(xì)胞分泌TG、TNF-α及IL-6表達(dá)量呈上升趨勢(shì),誘導(dǎo)至40 d表達(dá)量均顯著高于第21 d(P0.01),在40 d-50 d基本保持水平不變。3.在非直接接觸和直接接觸共培養(yǎng)過程中,誘導(dǎo)至21 d的脂肪細(xì)胞與成骨細(xì)胞共培養(yǎng)組鈣結(jié)節(jié)數(shù)量比陽(yáng)性對(duì)照組顯著增加(P0.001),誘導(dǎo)至40 d的脂肪細(xì)胞與成骨細(xì)胞共培養(yǎng)組鈣結(jié)節(jié)數(shù)量比陽(yáng)性對(duì)照組顯著減少(P0.05)。4.誘導(dǎo)至21 d脂肪細(xì)胞共培養(yǎng)組比誘導(dǎo)至40 d脂肪細(xì)胞共培養(yǎng)組成骨細(xì)胞的OPG表達(dá)量顯著增高(P0.001),RANKL的表達(dá)量顯著降低(P0.001)。5.在成骨誘導(dǎo)培養(yǎng)基中加入不同濃度油酸(25m M、50m M、100m M、200m M),BMSCs多數(shù)分化為脂肪細(xì)胞,且隨著濃度的增加,脂肪細(xì)胞的數(shù)量和體積都增加,但未觀察到有成骨細(xì)胞分化。不同濃度棕櫚酸均對(duì)細(xì)胞有毒性作用,細(xì)胞死亡。200m M油酸可作為誘導(dǎo)劑直接誘導(dǎo)骨髓間充質(zhì)干細(xì)胞分化為脂肪細(xì)胞,此方法誘導(dǎo)的脂肪細(xì)胞數(shù)量比傳統(tǒng)方法顯著增多(P0.01),并且體積顯著增大(P0.01)。結(jié)論1.骨髓腔內(nèi)小脂肪細(xì)胞與成骨細(xì)胞共培養(yǎng),可上調(diào)OPG的表達(dá),促進(jìn)成骨細(xì)胞形成。2.骨髓腔內(nèi)大脂肪細(xì)胞與成骨細(xì)胞共培養(yǎng),可下調(diào)OPG的表達(dá),抑制成骨細(xì)胞形成。
[Abstract]:Objective To observe the differences in the endocrine function of adipocyte derived from bone marrow mesenchymal stem cells in different periods; to observe the effect of different adipocytes on the induction of differentiation of osteoblasts and osteoclasts by cell co culture, and to observe the effect of different concentrations of fatty acids on the induced differentiation of osteoblasts. Method 1. the differentiation and identification of different types of cells were induced. 4 weeks old Wistar male rats were selected to separate bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs) and differentiate into adipocytes and osteoblasts respectively. Oil red O staining was used to identify mature adipocytes and alizarin red staining was used to identify osteoblasts, and the whole bone marrow fluid of Wistar rats was collected to differentiate into osteoclast and anti alcohol. Stone acid acid phosphatase staining identified.2.BMSCs derived adipocytes and osteoblasts, osteoclasts and osteoclasts co culture: 1) BMSCs derived adipocytes continued to induce differentiation after maturation, and collected a fat cell culture supernatant every 2 D intervals, and -20 C (sampling time points: 21 d, 24 D, 27 D, 30 d, 33 D, 36 D, 39 D, 42 d, 48, 42 d, 48), all samples The volume and quantity of adipocyte were monitored at the same time, and the volume and quantity of adipocyte were observed. The cell volume was measured by a scale. The number of cells was counted in 6 fields under 400 x microscope. The average value was taken. The time point.2 of the co culture was determined by the comprehensive monitoring data. The direction of the adipocyte inducer was induced to differentiate into the adipocytes in the P3 generation BMSCs, respectively. Induction of twenty-first D and 40 d for co culture.3) non direct contact co culture: replacement of fat medium as fat maintenance medium, sucking out culture medium in adipocyte culture dish every other day and adding osteoblast inducer / osteoclast inducer and inducing osteoblast / osteoclast induced.4) direct contact co culture. Do not scrape about 10 cm2 of adipocytes in a Petri dish, inoculate the fresh BMSCs, replace the medium with the osteoblast inducer and maintain the adipocyte culture medium, induce the differentiation to the osteoblast side, and incubate the bone marrow cells with the complete medium in the cell culture box for 24 hours, collect the non adherent cells, and scrape them in the culture dish. About 10 cm2 of adipocytes, in which non adherent cells were inoculated, the culture medium was replaced by osteoclast inducer and adipocyte maintenance medium, and the expression level of inflammatory cytokines and bone metabolic factors in the medium of osteoclast induced differentiation.5) was detected. ELISA method was used to detect the inflammatory factors by using RT-PCR method to detect bone The effects of different concentrations of metabolic factor.3. on osteoblast differentiation: Wistar rats were induced to differentiate into osteoblasts by P3 generation BMSCs, adding different concentrations (25m M, 50 m M, 100 m M and 200 m M) with oleic acid and palmitic acid in the osteogenic induction medium. The effect of two fatty acids on osteoblast differentiation was observed. After three generations, the surface molecular markers CD29 and CD44 were positive.P3 generation BMSCs differentiated into adipocytes with 21 d under the action of fat induced medium, and the oil red O staining was positive. 21 d differentiated into osteoblasts under the action of osteogenic induction medium, and the whole bone marrow fluid of alizarin red staining positive.Wistar rats was used in osteoclast culture medium. The lower 28 d was differentiated into osteoclast, and the.2. adipocyte resistant to tartaric acid acid phosphatase stained positive adipocytes continued to induce differentiation. The number of twenty-first D adipocytes was significantly higher than fortieth D (P0.001), but the volume was significantly lower than fortieth D adipocytes (P0.05). As the induction time increased, the expression of TG, TNF- A and IL-6 increased in the adipocytes. The expression of the induced 40 d was significantly higher than that of twenty-first D (P0.01). In the course of non direct contact and direct contact co culture at 40 d-50 D, the number of calcium nodules in the co culture group of adipocytes and osteoblasts induced to 21 d increased significantly (P0.001), and the induction to 40 d of adipocytes and osteoblasts was in common. The number of calcium nodules in the culture group was significantly lower than that in the positive control group (P0.05).4. induced to 21 d adipocyte co culture group, the expression of OPG expression in the co culture of 40 d adipocytes was significantly higher (P0.001), and the expression of RANKL was significantly reduced (P0.001) in the osteogenic inducement medium with different concentrations of oleic acid (25m M, 50m) 200m M), the majority of BMSCs differentiated into adipocytes, and with the increase of concentration, the number and volume of adipocytes increased, but the osteoblast differentiation was not observed. The different concentrations of palmitic acid were toxic to the cells, and the cell death.200m M oleic acid could be used as an inducer to induce bone marrow mesenchymal stem cells to differentiate into adipocytes. The number of adipocytes induced by the method increased significantly (P0.01) and increased significantly (P0.01). Conclusion 1. the co culture of small fat cells in bone marrow cavity and osteoblast can increase the expression of OPG and promote the formation of.2. bone marrow cells and osteoblasts to co culture the osteoblasts, which can reduce the expression of OPG and inhibit the osteogenesis. Cell formation.

【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R589.2;R580

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