antimiR-138修饰的rBMMSCs膜片的构建及其与种植体复合后的体内外生物学研究
发布时间:2018-03-09 04:13
本文选题:细胞膜片 切入点:骨髓基质干细胞 出处:《第四军医大学》2014年博士论文 论文类型:学位论文
【摘要】:牙科种植技术已成为牙列缺损或缺失的重要修复方法之一,但目前仍存在种植骨愈合时间过长(3-6个月),骨代谢疾病患者(骨质疏松症、糖尿病、放疗术后等)种植骨愈合不良合,失败率较高等问题。本课题拟从促进骨修复再生的角度出发,采用非病毒转染技术构建miRNA修饰的细胞膜片,并对其体内外的成骨活性进行初步研究;在此基础上,拟将miRNA修饰的细胞膜片与微弧氧化钛种植体相复合,构建基因修饰的组织工程化种植体,为种植体的骨愈合的过程提供一个兼有细胞、细胞外基质,以及生物活性分子的微环境,并从基因水平调控骨髓来源的间充质干细胞向成骨细胞转化,从而更好地促进种植体周围的骨形成和骨结合。 第一部分 大鼠骨髓间充质干细胞的分离培养和鉴定 【目的】 体外分离培养并鉴定rBMMSCs的生物学特性 【方法】 采用全骨髓贴壁法分离培养rBMMSCs,并对细胞的形态特征进行观察;采用流式细胞仪进行细胞表面标记的检测;采用MTT及克隆形成检测细胞的自我更新能力;通过体外成骨、成脂诱导分化实验检测细胞的多向分化潜能。 【结果】 全骨髓贴壁法分离培养的rBMMSCs,显微镜下呈梭形或三角形;间充质干细胞的表面标记CD29,CD90,CD105阳性表达,而血细胞标记CD34,CD45,CD31阴性表达;克隆形成能力11.6%;成骨染色可见到钙化结节沉积,成脂染色可见脂滴的形成。 【结论】 (1)全骨髓贴壁法分离培养的rBMMSCs属于间充质来源的细胞,且具有自我更新多和向分化潜能。 (2)该细胞可作为基因治疗的载体细胞,细胞膜片的种子细胞构建miRNA基因修饰的rBMMSCs膜片。 第二部分 细胞膜片基因转染的可行性探索及antimiR-138转染促进rBMMSCs膜片成骨的研究 【目的】 采用非病毒方法转染rBMMSC膜片的可行性进行研究; antimiR-138转染促进rBMMSC膜片成骨的研究。 【方法】 1)antimiR-138转染的rBMMSCs膜片的构建:采用Vc连续诱导法培养rBMMSC膜片,以Lipofectamine2000为载体,通过调整miRNA的用量及转染步骤对rBMMSC膜片进行转染;采用荧光显微观察、流式细胞分选和miRNA-PCR方法比较各组的转染效率的差异;通过大体观察、HE染色和扫描电镜观察rBMMSCs膜片的形态。 2)antimiR-138转染的rBMMSCs膜片的促成骨能力检测:采用ALP、天狼星红、茜素红染色、Real-timePCR和Western blot技术观察转染后细胞膜片成骨分化能力的差异。 3)antimiR-138转染的rBMMSCs膜片的异位成骨能力检测:将细胞膜片/FDB复合物移植到裸鼠皮下,术后4-8周,分别采用Micio-CT、HE和MT染色对膜片的成骨能力进行评估。 【结果】 本实验结果表明,通过恰当的Lipo2000-miRNAs载体体系配比及转染步骤调整,即可将以antimiR-138为代表的miRNA的成功地转染rBMMSCs膜片中,其中150nM组的转染效率可达99%,对内源性miR-138的抑制率可达85%;并且antimiR-138的修饰显著提高了rBMMSCs膜片的体外成骨活性,其中ALP、胶原、茜素红染色,Real-time PCR及Western blot检测成骨相关基因和蛋白的表达均表明实验组膜片的骨向分化能力较两对照组明显增强;体内异位成骨能力检测表明,,实验组移植复合物在术后4周时已经开始有新骨再生,8周时,新生骨不断改建并矿化成熟;而两对照组直到术后8周时才有少量新生骨形成。即antimiR-138转染的rBMMSCs膜片可显著促进并加速骨组织形成。 【结论】 (1)通过对以Lipofectamine2000为载体的转染复合物进行适当的优化,可以构建出具有较高转染效率,良好的细胞相容性以及结构完整的antimiR-138修饰的rBMMSCs膜片。 (2)在体外,antimiR-138可通过激活ERK1/2信号通路有效促进rBMMSCs膜片的成骨分化;在体内,antimiR-138转染的rBMMSCs膜片可显著促进并加速骨组织形成。 第三部分 antimiR-138/rBMMSCs膜片种植体复合物的构建及其骨诱导能力研究 【目的】 构建antimiR-138/rBMMSCs膜片微弧氧化钛种植体;antimiR-138/rBMMSCs膜片微弧氧化钛种植体的成骨活性和骨诱导能力研究 【方法】 1)将antimiR-138转染的rBMMSCs膜片与微弧氧化钛种植体相复合构建基因修饰的组织工程化种植体,并对其表面形貌进行扫描电镜观察。 2)采用ALP、天狼星红、茜素红染色、Real-timePCR和Western blot技术检测antimiR-138/rBMMSCs膜片微弧氧化钛种植体的体外成骨活性。 3)通过异位成骨实验,采用Micio-CT、HE和MT染色,免疫荧光技术对antimiR-138/rBMMSCs膜片微弧氧化钛种植体的骨诱导能力进行评估。 【结果】 antimiR-138转染的rBMMSCs膜片可成功与微弧氧化钛种植体复合,电镜观察亦可见丰富的细胞和胞外基质与种植体多孔结构的表面形成紧密的粘附与嵌合,即构建基因修饰的组织工程化种植体是可行的。体外ALP、胶原、茜素红染色,Real-timePCR及Western blot检测成骨相关基因和蛋白的表达均表明antimiR-138/rBMMSCs膜片与种植体的复合可促进并加速该复合物的体外成骨分化能力。体内的Micro-CT检测、HE和MT染色以及免疫荧光染色均表明antimiR-138/rBMMSCs膜片种植体复合物具有强大的骨诱导能力。 【结论】 (1)将antimiR-138修饰的rBMMSCs膜片与微弧氧化钛种植体复合,构建基因修饰的组织工程化种植体是可行的。 (2)通过体内体外成骨实验证明antimiR-138/rBMMSCs膜片种植体复合物具有优秀的成骨活性和骨诱导能力。 (3)这一技术将为解决临床种植骨愈合时间过长,骨代谢疾病患者种植失败率较高等问题提供新的思路和方法。
[Abstract]:Dental implant technology has become one of the important methods of repairing dentition defect, but there are still growing bone healing time (3-6 months), patients with metabolic bone disease (osteoporosis, diabetes mellitus, radiotherapy after surgery) bone healing, high failure rate and other issues. This paper intends to start from to promote bone repair and regeneration angle, cell membrane to construct miRNA modified by non transfection technique, and to preliminarily study its osteogenic activity in vivo; on this basis, to cell membrane and micro arc titanium oxide modified miRNA composite implant, gene modified tissue engineered implant, a both cell healing process in the implant bone, extracellular matrix, and bioactive molecules micro environment, and the regulation from the gene level of bone marrow derived mesenchymal stem cells to turn into osteoblasts, in order to better Promote bone formation and bone binding around the implant.
Part one
Isolation, culture and identification of rat bone marrow mesenchymal stem cells
[Objective]
Isolation, culture and identification of the biological characteristics of rBMMSCs in vitro
[method]
RBMMSCs were isolated and cultured by whole bone marrow adherent method, and morphological characteristics of cells were observed by detection of cell surface marker; flow cytometry; using MTT and clone detection cell self-renewal ability; in vitro osteogenic differentiation, differentiation into fat test cells.
[results]
The method of whole bone marrow adherent cultured rBMMSCs under the microscope, fusiform or triangle; mesenchymal stem cell markers CD29, CD90, CD105 positive expression, while blood cell markers CD34, CD45, CD31 negative expression; cloning ability 11.6%; bone staining can be seen calcification nodules deposited into lipid staining the formation of visible lipid droplets.
[Conclusion]
(1) all bone marrow adherent methods were isolated and cultured in rBMMSCs, which belonged to the cells of mesenchymal origin, and had the potential of self renewal and differentiation.
(2) the cell can be used as the carrier cell of the gene therapy, and the seed cells of the cell diaphragm construct the miRNA gene modified rBMMSCs film.
The second part
Feasibility of gene transfection of cell membrane and Study on promoting osteogenesis of rBMMSCs membrane by antimiR-138 transfection
[Objective]
The feasibility of using non viral methods to transfect rBMMSC film was studied. AntimiR-138 transfection was used to promote the osteogenesis of rBMMSC film.
[method]
1) construction of rBMMSCs film antimiR-138 transfection: using Vc induced continuously cultured rBMMSC membrane using Lipofectamine2000 as carrier, transfection of rBMMSC diaphragm through adjustment of miRNA and transfection steps; by fluorescence microscopic observation, flow cytometry and miRNA-PCR difference method and compared the transfection efficiency; through general observation, observation rBMMSCs the film morphology of HE staining and scanning electron microscopy.
2) the bone turnover ability of antimiR-138 transfected rBMMSCs membrane was detected by ALP, Sirius red, alizarin red staining, Real-timePCR and Western blot technology, and the difference of osteogenic differentiation ability of cell membrane after transfection was observed.
3) the ectopic osteogenesis ability of antimiR-138 transfected rBMMSCs membrane was detected. The cell membrane /FDB complex was transplanted to the subcutaneous tissue of nude mice. After 4-8 weeks, Micio-CT, HE and MT staining were used to evaluate the osteogenic potential of the membrane.
[results]
The experimental results show that by adjusting the ratio of transfected Lipo2000-miRNAs vector system and appropriate steps, can be successfully represented by antimiR-138 miRNA transfection of rBMMSCs in the membrane of 150nM group transfection efficiency up to 99%, inhibition of endogenous miR-138, the rate reached 85%; and the modification of antimiR-138 significantly enhanced the osteogenic activity of rBMMSCs diaphragm in vitro ALP, collagen, alizarin red staining, the expression of Real-time PCR and Western blot detection of bone related genes and proteins showed that the experimental group of the diaphragm osteogenic differentiation capacity is two in the control group significantly increased; ectopic osteogenesis in experimental group showed that compound transplantation after 4 weeks have begun new bone regeneration, 8 weeks, new bone mineralization and constant alterations to maturity; while the two control groups until after 8 Zhou Shicai a little new bone formation. AntimiR-138 transfected rBMMSCs The diaphragm can promote and accelerate the formation of bone tissue.
[Conclusion]
(1) by optimizing the transfection complex with Lipofectamine2000 as carrier, antimiR-138 modified rBMMSCs membrane with high transfection efficiency, good cytocompatibility and complete structure can be constructed.
(2) in vitro, antimiR-138 can effectively promote the osteogenic differentiation of rBMMSCs membrane by activating ERK1/2 signaling pathway. In vivo, antimiR-138 transfected rBMMSCs membrane can significantly promote and accelerate the formation of bone tissue.
The third part
Study on the construction of antimiR-138/rBMMSCs diaphragm implant complex and its bone induction ability
[Objective]
Construction of antimiR-138/rBMMSCs film microarc titanium oxide implant; osteogenic activity and bone induction ability of antimiR-138/rBMMSCs film microarc titanium oxide implants
[method]
1) we constructed the genetically modified tissue-engineered implants with antimiR-138 transfected rBMMSCs membrane and micro arc titanium implant, and observed the surface morphology of them by scanning electron microscope.
2) ALP, Sirius red, alizarin red staining, Real-timePCR and Western blot technology were used to detect the osteogenic activity of antimiR-138/rBMMSCs diaphragm micro arc titania implant in vitro.
3) by heterotopic bone formation test, Micio-CT, HE and MT staining, immunofluorescence technology were used to evaluate the bone induction ability of antimiR-138/rBMMSCs diaphragm micro arc titanium implant.
[results]
RBMMSCs antimiR-138 can be successfully transfected with diaphragm micro implant titanium oxide composite, adhesion and formation of chimeric tight surface cells and extracellular matrix and implant the porous structure of electron microscope also showed rich, namely the construction of tissue engineered implant gene modification is feasible. In vitro ALP, collagen, alizarin red staining, the expression of Real-timePCR Western and blot detection of bone related genes and proteins showed that antimiR-138/rBMMSCs membrane and implant composite in vitro can promote and accelerate the complex osteogenic differentiation. The in vivo detection of HE and Micro-CT, MT staining and immunofluorescence staining showed that antimiR-138/rBMMSCs membrane implant complexes have strong ability of bone induction.
[Conclusion]
(1) it is feasible to combine the antimiR-138 modified rBMMSCs film with the microarc titanium oxide implants to construct a genetically modified tissue engineered implant.
(2) through the osteogenesis experiment in vitro and in vitro, the antimiR-138/rBMMSCs diaphragm implant complex has excellent osteogenic activity and bone induction ability.
(3) this technique will provide new ideas and methods to solve the problem of long bone healing time and high failure rate for the patients with bone metabolism disease.
【学位授予单位】:第四军医大学
【学位级别】:博士
【学位授予年份】:2014
【分类号】:R783.6
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1 闫钧;antimiR-138修饰的rBMMSCs膜片的构建及其与种植体复合后的体内外生物学研究[D];第四军医大学;2014年
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