神經(jīng)生長(zhǎng)因子對(duì)山羊骨髓間充質(zhì)干細(xì)胞增殖及向成骨分化的調(diào)節(jié)作用
發(fā)布時(shí)間：2018-11-15 11:30
【摘要】： 目的體外培養(yǎng)山羊骨髓間充質(zhì)干細(xì)胞(Bone marrow Mesenchymal stem cells,BMSCs)并向成骨方向進(jìn)行誘導(dǎo),通過加入神經(jīng)生長(zhǎng)因子(Nerve growth factor,NGF),研究NGF在成骨培養(yǎng)基中對(duì)山羊BMSCs增殖及分化的影響。為闡明NGF促進(jìn)牽張成骨術(shù)(Distraction osteogenesis, DO)中骨形成的機(jī)理提供實(shí)驗(yàn)依據(jù),希望所建立的實(shí)驗(yàn)方法能為將來(lái)進(jìn)一步的臨床和科研工作建立穩(wěn)定的細(xì)胞模型,從而使?fàn)繌埑晒切g(shù)中促進(jìn)牽張區(qū)新骨生成,解決臨床問題提供一種可能。 方法抽取山羊骨髓,利用Percoll分離液進(jìn)行密度梯度離心結(jié)合貼壁法分離山羊BMSCs,體外培養(yǎng)擴(kuò)增,觀察其形態(tài)學(xué)變化。體外培養(yǎng)至第三代后,首先用流式細(xì)胞儀檢測(cè)細(xì)胞表面標(biāo)志物,以鑒定細(xì)胞純度,排除了雜質(zhì)細(xì)胞的干擾;再將第三代BMSCs分為A、B、C、D四組,A組(空白對(duì)照組):用含10%胎牛血清的低糖DMEM常規(guī)培養(yǎng)液培養(yǎng)。B組(成骨誘導(dǎo)對(duì)照組):成骨誘導(dǎo)液為常規(guī)培養(yǎng)液中加入10-8mol/L地塞米松、50mg/L維生素C、10mmol/Lβ-甘油磷酸鈉。C組(NGF組):常規(guī)培養(yǎng)液中加入終濃度為100ng/ml的NGF。D組(實(shí)驗(yàn)組):成骨誘導(dǎo)液中加入終濃度為100ng/ml的NGF。于第3,5,7天利用MTT法測(cè)定各培養(yǎng)組細(xì)胞增殖活性,第14天檢測(cè)各組細(xì)胞堿性磷酸酶(ALP)活性和骨鈣素(OC)水平、第21天應(yīng)用Von Kossa染色的方法檢測(cè)鈣結(jié)節(jié)的形成,比較各組細(xì)胞成骨性能,觀察NGF對(duì)山羊BMSCs增殖及向成骨分化的影響。 結(jié)果1.利用Percoll分離液進(jìn)行密度梯度離心結(jié)合貼壁法分離、純化山羊BMSCs,是一種簡(jiǎn)便可行的方法,可獲得較高的純度,流式細(xì)胞術(shù)測(cè)得CD90、CD105為強(qiáng)陽(yáng)性表達(dá),CD34、CD45為陰性。 2.MTT法檢測(cè)細(xì)胞增殖實(shí)驗(yàn)中,A組和C組細(xì)胞增殖率相似,B組和D組細(xì)胞增殖率相似,但B、D兩組明顯低于A、C兩組,差異有顯著性意義(p0.05)。 3.各組細(xì)胞成骨性能檢測(cè)結(jié)果:A組和C組ALP和OC表達(dá)均無(wú)明顯差異(p0.05),B組和D組ALP和OC表達(dá)明顯高于A組和C組,且D組較B組表達(dá)水平增高,差異有顯著性意義(p0.05)。A、C兩組細(xì)胞Von Kossa染色陰性,未見明顯的鈣化結(jié)節(jié)出現(xiàn);B組細(xì)胞Von Kossa染色陽(yáng)性,可見黑色鈣化結(jié)節(jié),D組較成骨誘導(dǎo)對(duì)照組鈣化結(jié)節(jié)數(shù)目明顯增多,面積增大。 結(jié)論密度梯度離心結(jié)合貼壁法是分離、純化山羊BMSCs的簡(jiǎn)便實(shí)用的方法,傳至第三代即可獲得較高的純度。單純使用NGF對(duì)山羊BMSCs并無(wú)明顯促進(jìn)增殖及分化的作用,但NGF在山羊BMSCs向成骨細(xì)胞分化過程中具有明顯的促進(jìn)作用。這為加快骨形成,進(jìn)一步進(jìn)行相關(guān)的臨床研究提供了實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Objective to culture goat bone marrow mesenchymal stem cells (Bone marrow Mesenchymal stem cells,BMSCs) in vitro and induce them to osteogenesis. By adding nerve growth factor (Nerve growth factor,NGF), the effect of NGF on the proliferation and differentiation of goat BMSCs in osteoblasts was studied. In order to elucidate the mechanism of NGF in promoting bone formation in distraction osteogenesis (Distraction osteogenesis, DO), it is hoped that the established experimental method can establish a stable cell model for further clinical and scientific research in the future. So that distraction osteogenesis can promote the formation of new bone in distraction zone and provide a possibility to solve clinical problems. Methods Goat bone marrow was extracted and BMSCs, was isolated by density gradient centrifugation and adherent method. The morphological changes of goat BMSCs, were observed. After the third passage in vitro, the cell surface markers were detected by flow cytometry in order to identify the cell purity and eliminate the interference of impurity cells. The third generation of BMSCs was divided into four groups, Group A (blank control group) was cultured with low glucose DMEM medium containing 10% fetal bovine serum. Group B (osteogenic control group): 10-8mol/L dexamethasone and 50mg/L vitamin C were added to osteogenic medium. 10mmol/L 尾 -glycerophosphate. C group (NGF group): NGF.D group with 100ng/ml final concentration in conventional culture medium (experimental group): NGF. with 100ng/ml final concentration in osteoblast inducer The proliferative activity of the cultured cells was measured by MTT method on the 7th day, the alkaline phosphatase (ALP) activity and osteocalcin (OC) level were measured on the 14th day, and the formation of calcium nodules was detected by Von Kossa staining on the 21st day. The effects of NGF on the proliferation and osteogenic differentiation of goat BMSCs were observed. Result 1. It is a simple and feasible method to purify goat BMSCs, by density gradient centrifugation combined with adherent method. The purity of goat BMSCs, can be obtained by flow cytometry. The strong positive expression of CD90,CD105 and negative expression of CD34,CD45 were detected by flow cytometry. The cell proliferation rate of group A and group C was similar to that of group B and group D by 2.MTT assay, but the cell proliferation rate of group B and D was significantly lower than that of group A and C (p0.05). 3. There was no significant difference in the expression of ALP and OC between group A and group C (p0. 05), B and group D were significantly higher than those in group A and C, and the expression of ALP and OC in group D was higher than that in group B. The difference was significant (p0.05). Von Kossa staining was negative and no calcified nodules were found in the two groups. In group B, the number and area of calcified nodules were significantly increased and the number of calcified nodules in group D was significantly higher than that in the control group. Conclusion density gradient centrifugation combined with adherent method is a simple and practical method for the separation and purification of goat BMSCs. NGF alone did not promote the proliferation and differentiation of goat BMSCs, but NGF could promote the differentiation of goat BMSCs into osteoblasts. This provides experimental basis for accelerating bone formation and further related clinical research.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329
本文編號(hào)：2333189
[Abstract]:Objective to culture goat bone marrow mesenchymal stem cells (Bone marrow Mesenchymal stem cells,BMSCs) in vitro and induce them to osteogenesis. By adding nerve growth factor (Nerve growth factor,NGF), the effect of NGF on the proliferation and differentiation of goat BMSCs in osteoblasts was studied. In order to elucidate the mechanism of NGF in promoting bone formation in distraction osteogenesis (Distraction osteogenesis, DO), it is hoped that the established experimental method can establish a stable cell model for further clinical and scientific research in the future. So that distraction osteogenesis can promote the formation of new bone in distraction zone and provide a possibility to solve clinical problems. Methods Goat bone marrow was extracted and BMSCs, was isolated by density gradient centrifugation and adherent method. The morphological changes of goat BMSCs, were observed. After the third passage in vitro, the cell surface markers were detected by flow cytometry in order to identify the cell purity and eliminate the interference of impurity cells. The third generation of BMSCs was divided into four groups, Group A (blank control group) was cultured with low glucose DMEM medium containing 10% fetal bovine serum. Group B (osteogenic control group): 10-8mol/L dexamethasone and 50mg/L vitamin C were added to osteogenic medium. 10mmol/L 尾 -glycerophosphate. C group (NGF group): NGF.D group with 100ng/ml final concentration in conventional culture medium (experimental group): NGF. with 100ng/ml final concentration in osteoblast inducer The proliferative activity of the cultured cells was measured by MTT method on the 7th day, the alkaline phosphatase (ALP) activity and osteocalcin (OC) level were measured on the 14th day, and the formation of calcium nodules was detected by Von Kossa staining on the 21st day. The effects of NGF on the proliferation and osteogenic differentiation of goat BMSCs were observed. Result 1. It is a simple and feasible method to purify goat BMSCs, by density gradient centrifugation combined with adherent method. The purity of goat BMSCs, can be obtained by flow cytometry. The strong positive expression of CD90,CD105 and negative expression of CD34,CD45 were detected by flow cytometry. The cell proliferation rate of group A and group C was similar to that of group B and group D by 2.MTT assay, but the cell proliferation rate of group B and D was significantly lower than that of group A and C (p0.05). 3. There was no significant difference in the expression of ALP and OC between group A and group C (p0. 05), B and group D were significantly higher than those in group A and C, and the expression of ALP and OC in group D was higher than that in group B. The difference was significant (p0.05). Von Kossa staining was negative and no calcified nodules were found in the two groups. In group B, the number and area of calcified nodules were significantly increased and the number of calcified nodules in group D was significantly higher than that in the control group. Conclusion density gradient centrifugation combined with adherent method is a simple and practical method for the separation and purification of goat BMSCs. NGF alone did not promote the proliferation and differentiation of goat BMSCs, but NGF could promote the differentiation of goat BMSCs into osteoblasts. This provides experimental basis for accelerating bone formation and further related clinical research.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329
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