生长分化因子-5诱导人脂肪基质细胞成骨的实验研究

发布时间：2018-04-24 01:21

本文选题：人脂肪基质细胞 + 生长分化因子-5　；参考：《中国人民解放军军医进修学院》2010年博士论文

【摘要】： 外伤、肿瘤切除、先天畸形造成的颌骨缺损在临床较常见,目前常采用自体骨移植、异体骨移植、异种骨移植、金属及人工合成材料替代等治疗方法,尚不能完全满足临床的需要。组织工程技术的发展为颌骨缺损的修复提供了新的途径。种子细胞,生长因子和支架材料是组织工程的三大要素。近年来研究发现脂肪组织中存在一些具有自我更新和多向分化潜能的脂肪基质细胞(adipose derived stromal cells, ADSCs),这些细胞在一定条件下可分化为脂肪细胞、成骨细胞、软骨细胞、肌细胞和神经细胞等多种细胞,具有成骨、成脂、成软骨、成肌肉、成神经等多种潜能。而且脂肪组织还具有取材简便、来源丰富等优势,被认为是组织工程中最有前途的种子细胞。其中,ADSCs用于骨缺损的修复倍受关注。生长因子在ADSCs成骨分化中起关键作用。常用的骨生长因子有骨形态发生蛋白2(bone morphogenetic proteins 2, BMP2),骨形态发生蛋白7(bone morphogenetic proteins 7, BMP7),生长分化因子5(Growth differentiation factor 5, GDF-5),转化生长因子-β(transforming growth factor-β, TGF-β),血小板衍生生长因子(platelet-derived growth factor, PDGF),胰岛素样生长因子(insulin-like growth factor, IGF)等。GDF-5是转化生长因子超家族、骨形态发生蛋白亚家族中的一员,在软骨发生和长骨发育中具有重要作用。目前研究主要集中于其促进软骨分化和形成,对于其促进成骨分化和骨形成的作用,国内外的研究较少。因此本实验应用GDF-5诱导hADSCs向成骨细胞分化,并将诱导后的细胞复合纳米羟基磷灰石／胶原／L-聚乳酸(nHAC/PLA)支架植入裸鼠体内,构建组织工程骨三维培养模型,并通过体内体外研究观察成骨情况,为其将来在颌骨组织工程中的应用奠定实验基础。全文共分为三部分：第一部分：hADSCs分离、培养及生物学特性研究目的：研究hADSCs的生长特性、表面抗原特点,横向分化潜能,在此基础上对其进行鉴定。方法：取健康人吸脂术中取出的新鲜脂肪组织,胶原酶消化法分离其中脂肪基质细胞,倒置相差显微镜观察其形态特征；MTT法测hADSCs的生长增殖状况；流式细胞仪分析其表面抗原；波形丝蛋白、角蛋白免疫荧光染色鉴定其来源；在细胞鉴定基础上矿化液诱导其向成骨细胞分化,通过碱性磷酸酶染色、OPN及Ⅰ型胶原免疫荧光染色、钙结节茜素红染色分析其成骨分化潜能；同时,采用成脂诱导液诱导其向脂肪细胞方向分化,油红0染色进行成脂分化鉴定。结果：原代及传代培养的hADSCs形态均匀,呈类成纤维细胞样形态,生长能力旺盛；P3、P5、P10代hADSCs的增殖曲线大致相同,呈近似“S”形；P2代hADSCs表达CD29、CD44、CD105等间充质细胞表面相对特异性标志蛋白,CD14、CD45阴性表达；P4代hADSCs免疫荧光染色波形丝蛋白阳性,角蛋白为阴性。成骨诱导21d后,碱磷酶染色、OPN、Ⅰ型胶原免疫荧光染色阳性,钙结节茜素红染色表现为红色结节。成脂诱导14d后油红0染色有红色着色的脂滴形成。结论：吸脂术来源的细胞具有未分化hADSCs的特征,在体外诱导条件下,可向成骨、成脂方向分化,具有横向分化潜能。第二部分：GDF-5诱导hADSCs成骨分化作用的体外研究目的：研究GDF-5对hADSCs体外成骨分化能力的影响。方法：将GDF-5分成0,1,10,100,500ng／ml 5个浓度,通过MTT法分析不同浓度GDF-5对hADSCs增殖的影响,通过碱性磷酸酶活性、茜素红染色检测GDF-5对其成骨分化能力的影响,确定GDF-5促进成骨的最适浓度。然后取所确定的GDF-5浓度,分成对照组、矿化组、GDF-5三组,通过测定RT-PCR检测成骨诱导后Runx2、OPN、ColI、VEGF基因的表达；Western Blot检测其成骨诱导后Runx2、OPN、ColI、VEGF蛋白的表达,分析该浓度GDF-5对P4代hADSCs的增殖、分化效应。结果：100ng／ml为GDF-5促进hADSCs成骨的最适浓度；三组培养基均能促进hADSCs增殖,但GDF-5组促增殖作用显著低于另两组(P0.05)；GDF-5组促成骨分化作用最为显著,碱磷酶活性,Runx2、OPN、colI、VEGF基因及蛋白的表达均高于矿化组和细胞组,统计学上有显著性差异(P0.05)。结论：体外研究表明100ng／mlGDF-5有诱导hADSCs分化为成骨细胞的潜能。第三部分：GDF-5诱导hADSCs复合纳米羟基磷灰石／胶原／聚乳酸体内成骨的研究目的：观察GDF-5诱导的hADSCs复合纳米羟基磷灰石／胶原／L-聚乳酸裸鼠皮下成骨的能力方法：取对数生长期hADSCs,接种于纳米羟基磷灰石／胶原／L-聚乳酸支架材料上,构建细胞／支架材料三维培养模型,扫描电镜(SEM)观察细胞在支架上的生长情况。然后,将其分别置于L-DMEM、矿化液、GDF-5体外培养一周后,移植入裸鼠背部皮下,4w、12w后取材,HE染色、Goldner's染色观察支架材料复合体在裸鼠皮下成骨情况。结果：hADSCs能粘附在纳米羟基磷灰石／胶原／L-聚乳酸(nanohydroxyapatite/collagen/L-poly lactic acid, nHAC/PLA)支架材料的表面,生长良好；细胞／支架材料复合体经GDF-5诱导后4w、12w后取材均可见类骨质形成,并逐渐取代支架材料,周围未见明显炎症反应。其中GDF-5组成熟的骨及血管形成数量明显多于其他两组。结论：纳米羟基磷灰石／胶原／L-聚乳酸是有利于ADSCs成骨分化的良好的支架材料；GDF-5可促进hADSCs复合纳米羟基磷灰石／胶原／L-聚乳酸异位成骨效应。
[Abstract]:Bone morphogenetic protein - 2 ( BMP2 ) , bone morphogenetic protein 7 ( BMP7 ) , growth differentiation factor 5 ( GDF - 5 ) , transforming growth factor - 尾 ( transforming growth factor - 尾 ) , platelet - derived growth factor ( PDGF ) , insulin - like growth factor ( insulin - like growth factor ) . GDF - 5 is a member of transforming growth factor superfamily and bone morphogenetic protein subfamily . It plays an important role in the development of cartilage and bone formation .

The full text is divided into three parts :

Part I : Isolation , Culture and Biological Characteristics of hADSCs

Objective : To study the growth characteristics , surface antigen characteristics and lateral differentiation potential of hADSCs .

Methods : The fat stromal cells were isolated from the fresh adipose tissue and collagenase digestion method taken from healthy individuals , and the morphological characteristics were observed by reversed phase contrast microscope .
Growth and proliferation of hADSCs were measured by MTT assay .
The surface antigen was analyzed by flow cytometry .
waveform silk protein , keratin immunofluorescence staining to identify its source ;
On the basis of cell identification , it was induced to differentiate into osteoblast , and the differentiation potential was analyzed by alkaline phosphatase staining , collagen immunofluorescence staining and alizarin red staining of calcium nodules .
At the same time , the fat - forming induction solution was used to induce differentiation of fat cells , and the oil - red - 0 staining was used for the differentiation and identification of fat .

Results : The morphology of hADSCs in primary culture and subculture was uniform , and the growth ability was vigorous .
The proliferation curves of P3 , P5 and P10 hADSCs were approximately the same and approximately " S " ;
The expression of CD29 , CD44 , CD105 and the expression of CD29 , CD44 , CD105 and the expression of CD29 , CD44 and CD105 on the surface of the mesenchymal cells were expressed by the expression of CD29 , CD44 , CD105 and so on .
Immunofluorescence staining of P4 generation hADSCs was positive , keratin was negative . After 21 days of induction of bone formation , alkaline phosphatase staining showed red nodules , and red nodules were stained with alizarin red staining .

Conclusion : The cells from the source of fat - absorbing operation have the characteristics of undifferentiated hADSCs . Under the condition of in vitro induction , they can differentiate into bone - forming and fat - forming directions , and have the potential of lateral differentiation .

Part Two : In Vitro Study on the Osteogenic Differentiation of hADSCs Induced by GDF - 5

Objective : To study the effect of GDF - 5 on the osteogenic differentiation of hADSCs in vitro .

Methods : GDF - 5 was divided into 0 , 1 , 10 , 100 , 500 ng / ml 5 concentrations . The effects of different concentrations of GDF - 5 on proliferation of hADSCs were analyzed by MTT method .
The effects of GDF - 5 on proliferation and differentiation of P4 - generation hADSCs were analyzed by Western Blot .

Results : 100 ng / ml GDF - 5 promoted the optimal concentration of hADSCs into bone ;
The proliferation of hADSCs was promoted by three groups of medium , but the proliferation of GDF - 5 group was significantly lower than that in the other two groups ( P0.05 ) .
The results showed that GDF - 5 group had the most significant effect on bone differentiation , the activity of alkaline phosphatase , Runx2 , osteal , colI , VEGF gene and protein were all higher than those in mineralized group and cell group ( P0.05 ) .

Conclusion : In vitro studies indicate that 100ng / ml GDF - 5 has the potential to induce differentiation of hADSCs into osteoblasts .

The third part : GDF - 5 induced bone formation in hADSCs / collagen / polylactic acid in vivo

Objective : To observe the ability of GDF - 5 - induced hADSCs to synthesize hydroxyapatite / collagen / L - poly ( lactic acid ) nude mice

Methods : The logarithmic growth of hADSCs was carried out on the scaffolds of hydroxyapatite / collagen / L - polylactic acid , and the growth of the cells on the scaffold was observed by scanning electron microscope ( SEM ) . Then , they were cultured in L - DMEM , mineralized fluid and GDF - 5 respectively for one week , then transplanted into the skin of nude mice , and then treated with HE staining and Goldner ' s staining .

Results : The hADSCs were able to adhere to the surface of nano hydroxyapatite / collagen / L - poly lactic acid ( nS / PLA ) scaffold material and grow well .
After GDF - 5 induction , the cells / scaffold composites showed similar bone formation after 4w and 12w , and gradually replaced the scaffold material . There was no obvious inflammatory reaction around them . Among them , GDF - 5 composed of well - cooked bone and blood vessels was significantly larger than the other two groups .

Conclusion : Nano - hydroxyapatite / collagen / L - poly ( lactic acid ) is a good scaffold material for the differentiation of ADSCs into bone .
GDF - 5 can promote the ectopic osteogenesis of hADSCs / collagen / L - polylactic acid .

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

【共引文献】