体外冲击波对成骨细胞与破骨细胞共培养系中破骨细胞形成及活性影响的研究

发布时间：2018-04-05 17:05

本文选题：体外冲击波　切入点：破骨细胞　出处：《河北医科大学》2011年硕士论文

【摘要】：目的:健康骨骼处于成骨细胞(osteoblast, OB)骨形成与破骨细胞(osteoclast, OC)骨吸收动态平衡的状态,这种平衡一旦被打破可导致骨质疏松(osteoporosis)、骨硬化病(osteopetrosis)等多种骨代谢性疾病。力学刺激在调节骨骼重塑和维持骨量方面具有重要的作用,以往研究已证明有效力学刺激能促进成骨。破骨细胞是唯一具有骨吸收功能的细胞,它由来源于单核/巨噬细胞谱系(monocyte/macrophage lineage),通过多个单核的祖细胞融合形成多核巨细胞。力学刺激对破骨细胞形成和活性的影响研究少见。体外冲击波(Extracorporeal Shock Wave,ESW)是由压力瞬间急剧变化产生的一种机械波,它产生的能量可经相应仪器二次聚焦形成高能量冲击波,产生治疗作用。体外冲击波具有可定量作用于治疗部位、无创、并发症少、治疗周期短、风险低等许多优势。实验研究发现适宜能量的冲击波可以增加成骨细胞的成骨活性,进而促进骨折愈合。但冲击波作为一种能量刺激形式,对破骨细胞的形成及骨吸收活性的影响,目前未见国内外有相关研究。本实验通过将体外冲击波作用于成骨细胞与破骨前体细胞共培养体系,模拟破骨细胞在体内与成骨细胞相互作用的环境,通过观察破骨细胞形成过程中的形态变化、RT-PCR分析形成过程中调控基因的表达水平、ELISA检测破骨细胞特异性蛋白分泌水平及电镜下定量分析其骨吸收能力,探讨冲击波对破骨细胞形成及骨吸收作用的影响。方法: 1骨髓血分离hMSCs及成骨诱导:选择10名志愿者(排除代谢性疾病),各抽取骨髓20ml,梯度离心法获得hMSCs,常规培养1周后获得大量hMSCs,采用经典成骨培养基(10nM地塞米松、50uM抗坏血酸、10mMβ-甘油磷酸钠)经4周诱导,获得成骨细胞,ALP染色、茜素红染色验证成骨细胞活性。 2成骨细胞和破骨细胞共培养体系建立:10名志愿者再次骨髓穿刺获得骨髓血20ml,密度梯度离心法获得骨髓单个核细胞(bone marrow mononuclear cells, BMMCs)。将BMMCs加入预先放置骨片的悬挂式培养皿,培养液中加入0.1μM的1,25(OH)2VD3为破骨诱导剂。由此建立底层为成骨细胞,上层为破骨细胞,大分子能相互影响的共培养系统。 3体外冲击波对共培养系中破骨细胞的作用:BMMCs贴壁后12小时,使用500频次0.12mJ/mm2的体外冲击波对培养系进行干预,在光镜下连续观察细胞形态变化,培养一周后ELISA法分析检测培养液中组织蛋白酶K、抗酒石酸酸性磷酸酶5b、白细胞介素1β,肿瘤坏死因子α的含量,RT-PCR分析破骨细胞RANK,NFATc1和c-Fos表达水平,行TRAP染色,定量分析ESW对破骨细胞形态及融合的影响,骨片电镜扫描分析骨陷窝数量及面积。以单个核细胞单独培养ESW干预组,单独培养组及共培养非干预组组为对照。对所有数据x±s表示统计学分析,方差分析比较其差异,设定P0.05为有统计学意义。结果: 1骨髓间充质干细胞经成骨诱导28天后,经ALP和茜素红染色,可见钙结节,证明已获得高活性成骨细胞。加入单个核细胞建立共培养体系后,各组单个核细胞在1-3天都可见聚集趋势,聚集密度2-10个不等,但此时仍可见细胞膜完整,未发生融合。4-6天可见细胞逐渐融合,形成2-6个核不等的多个核巨细胞。经ESW干预组与未干预组相比,细胞融合发生较晚,融合后形成的细胞核数量较少。 2 ELISA法检测破骨细胞特异性蛋白分泌水平,共培养干预组组织蛋白酶K 2.23±0.35ng/ml,TRAP 5b 1.53±0.29ng/ml,IL1β53.28±7.45 ng/ml,TNFα5.87±0.80ng/ml,经统计学分析发现明显低于非ESW干预组,但高于BMMC单独培养ESW干预组(P0.05),显示共培养中成骨细胞分泌的相关因子促进了破骨细胞特异性蛋白的分泌,但ESW对这些蛋白的分泌有抑制作用。RT-PCR检测RANK,NFATc1和c-Fos基因表达水平,冲击波干预组荧光亮度低于未干预组,证明冲击波抑制了破骨细胞形成过程中关键基因的表达。 3培养后的单个核细胞经TRAP染色,各组细胞均被染为酒红色,证明各组均表达特异性抗酒石酸酸性磷酸酶,所产生的细胞为破骨细胞。破骨细胞融合指数ESW干预共培养组为0.46±0.10,未干预组为0.73±0.14,两者间有显著性差异,证明ESW抑制了共培养系中BMMC的融合(P0.05)。扫描电镜分析骨陷窝面积,ESW干预共培养组为12963±952μm2,未干预组为16257±1423μm2,两者之间的差别具有统计学意义(P0.05)。证明ESW干预后破骨细胞骨吸收能力降低。结论: 1采用成骨细胞及BMMCs诱导培养可建立人成骨细胞与破骨细胞共培养体系,可获得足够破骨细胞用于体外研究。 2在共培养系中,成骨细胞具有促进BMMCs融合生成破骨细胞的作用。 3 ESW可有效抑制共培养系中破骨细胞形成相关基因表达、特异性蛋白分泌、抑制其骨吸收能力,ESW对破骨细胞的形成及活性具有明显的抑制作用。
[Abstract]:Objective : To study the dynamic balance of bone resorption in osteoblasts ( OB ) and osteopetrosis , which can lead to osteoporosis , osteopetrosis and other bone metabolic diseases .

The extracorporeal shock wave is a kind of mechanical wave produced by the rapid change of pressure . The energy generated by it can form high energy shock wave through the secondary focusing of the corresponding instrument . The extracorporeal shock wave has many advantages , such as treatment site , non - invasive , few complications , short treatment period and low risk . The experimental study finds that the shock wave of suitable energy can increase the osteogenic activity of osteoblasts and promote the healing of fracture .

In this experiment , the effects of shock wave on the formation and bone resorption of osteoblasts were investigated by observing the morphological changes in the formation of osteoblasts and the expression level of the regulatory genes in the formation of osteoblasts by observing the morphological changes in the process of formation and the quantitative analysis of the bone resorption ability by ELISA .

Method :

hMSCs were isolated from bone marrow and induced by bone formation : 10 volunteers ( excluding metabolic diseases ) were selected , 20 ml of bone marrow were extracted , and hMSCs were obtained by gradient centrifugation . After 1 week of routine culture , a large amount of hMSCs were obtained . After 1 week of routine culture , a large amount of hMSCs were obtained . Osteoblasts , ALP staining and alizarin red staining were used to verify the activity of osteoblasts .

Bone marrow mononuclear cells ( BMMCs ) were obtained from bone marrow mononuclear cells ( BMMCs ) by density gradient centrifugation . BMMCs were added to a suspension culture dish in which bone fragments were pre - placed . 0.1 . m u.M of 1 , 25 ( OH ) 2VD3 was added to the culture solution to break the bone inducing agent .

3 In vitro shock wave was used to observe the effect of culture system in the co - culture system : 12 hours after the BMMCs were applied , the culture system was observed with 500 frequency of 0.12 mJ / mm2 . After a week of culture , the levels of tissue proteinase K , anti - tartrate acid phosphatase 5b , interleukin 1 尾 and tumor necrosis factor 伪 were observed under light microscope .

Results :

After bone marrow mesenchymal stem cells were cultured for 28 days , ALP and alizarin red staining , calcium nodules were observed to show that high - activity osteoblasts were obtained . After addition of mononuclear cells to establish the co - culture system , the cells of each group showed a tendency of aggregation in 1 - 3 days .

2 . The level of specific protein secretion was detected by ELISA . The tissue proteinase K 2 . 23 卤 0 . 35ng / ml , TRAP 5b 1 . 53 卤 0 . 29ng / ml , IL - 1尾 53.28 卤 7.45 ng / ml , IL - 1尾 53.28 卤 7.45 ng / ml , TNF - 伪 were 5.87 卤 0.80 ng / ml .

All the cells were stained red with TRAP staining , and the cells were stained red to prove that the specific anti - tartrate acid phosphatase was expressed in each group . There was significant difference between the two groups ( P 0.05 ) . The difference between the two groups was 12963 卤 95渭m 2 , and 16257 卤 1423渭m in the untreated group . The difference between the two groups was statistically significant ( P0.05 ) .

Conclusion :

1 . Osteoblasts were cultured in vitro and cultured with BMMCs to establish the co - culture system of human osteoblasts and Osteoclasts , which can be used for in vitro study .

2 In the co - culture system , the osteoblast has the function of promoting the fusion of BMMCs to form the broken cell .

3 . The expression of related genes , specific protein secretion and inhibition of bone resorption in the co - culture system can be effectively inhibited in the co - culture system .

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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