本文選題：細胞膜片　切入點：骨髓基質(zhì)干細胞　出處：《第四軍醫(yī)大學》2014年博士論文　論文類型：學位論文

【摘要】：牙科種植技術已成為牙列缺損或缺失的重要修復方法之一，但目前仍存在種植骨愈合時間過長（3-6個月），骨代謝疾病患者（骨質(zhì)疏松癥、糖尿病、放療術后等）種植骨愈合不良合，失敗率較高等問題。本課題擬從促進骨修復再生的角度出發(fā)，采用非病毒轉染技術構建miRNA修飾的細胞膜片，并對其體內(nèi)外的成骨活性進行初步研究；在此基礎上，擬將miRNA修飾的細胞膜片與微弧氧化鈦種植體相復合，構建基因修飾的組織工程化種植體，為種植體的骨愈合的過程提供一個兼有細胞、細胞外基質(zhì)，以及生物活性分子的微環(huán)境，并從基因水平調(diào)控骨髓來源的間充質(zhì)干細胞向成骨細胞轉化，從而更好地促進種植體周圍的骨形成和骨結合。 第一部分 大鼠骨髓間充質(zhì)干細胞的分離培養(yǎng)和鑒定 【目的】 體外分離培養(yǎng)并鑒定rBMMSCs的生物學特性 【方法】 采用全骨髓貼壁法分離培養(yǎng)rBMMSCs，并對細胞的形態(tài)特征進行觀察；采用流式細胞儀進行細胞表面標記的檢測；采用MTT及克隆形成檢測細胞的自我更新能力；通過體外成骨、成脂誘導分化實驗檢測細胞的多向分化潛能。 【結果】 全骨髓貼壁法分離培養(yǎng)的rBMMSCs，顯微鏡下呈梭形或三角形；間充質(zhì)干細胞的表面標記CD29，CD90，CD105陽性表達，而血細胞標記CD34，CD45，CD31陰性表達；克隆形成能力11.6%；成骨染色可見到鈣化結節(jié)沉積，成脂染色可見脂滴的形成。 【結論】 (1)全骨髓貼壁法分離培養(yǎng)的rBMMSCs屬于間充質(zhì)來源的細胞，且具有自我更新多和向分化潛能。 (2)該細胞可作為基因治療的載體細胞，細胞膜片的種子細胞構建miRNA基因修飾的rBMMSCs膜片。 第二部分 細胞膜片基因轉染的可行性探索及antimiR-138轉染促進rBMMSCs膜片成骨的研究 【目的】 采用非病毒方法轉染rBMMSC膜片的可行性進行研究; antimiR-138轉染促進rBMMSC膜片成骨的研究。 【方法】 1）antimiR-138轉染的rBMMSCs膜片的構建：采用Vc連續(xù)誘導法培養(yǎng)rBMMSC膜片，以Lipofectamine2000為載體，通過調(diào)整miRNA的用量及轉染步驟對rBMMSC膜片進行轉染；采用熒光顯微觀察、流式細胞分選和miRNA-PCR方法比較各組的轉染效率的差異；通過大體觀察、HE染色和掃描電鏡觀察rBMMSCs膜片的形態(tài)。 2）antimiR-138轉染的rBMMSCs膜片的促成骨能力檢測：采用ALP、天狼星紅、茜素紅染色、Real-timePCR和Western blot技術觀察轉染后細胞膜片成骨分化能力的差異。 3）antimiR-138轉染的rBMMSCs膜片的異位成骨能力檢測：將細胞膜片/FDB復合物移植到裸鼠皮下，術后4-8周，分別采用Micio-CT、HE和MT染色對膜片的成骨能力進行評估。 【結果】 本實驗結果表明，通過恰當?shù)腖ipo2000-miRNAs載體體系配比及轉染步驟調(diào)整，即可將以antimiR-138為代表的miRNA的成功地轉染rBMMSCs膜片中，其中150nM組的轉染效率可達99%，對內(nèi)源性miR-138的抑制率可達85%；并且antimiR-138的修飾顯著提高了rBMMSCs膜片的體外成骨活性，其中ALP、膠原、茜素紅染色，Real-time PCR及Western blot檢測成骨相關基因和蛋白的表達均表明實驗組膜片的骨向分化能力較兩對照組明顯增強；體內(nèi)異位成骨能力檢測表明，，實驗組移植復合物在術后4周時已經(jīng)開始有新骨再生，8周時，新生骨不斷改建并礦化成熟；而兩對照組直到術后8周時才有少量新生骨形成。即antimiR-138轉染的rBMMSCs膜片可顯著促進并加速骨組織形成。 【結論】 （1）通過對以Lipofectamine2000為載體的轉染復合物進行適當?shù)膬?yōu)化，可以構建出具有較高轉染效率，良好的細胞相容性以及結構完整的antimiR-138修飾的rBMMSCs膜片。 （2）在體外，antimiR-138可通過激活ERK1/2信號通路有效促進rBMMSCs膜片的成骨分化；在體內(nèi)，antimiR-138轉染的rBMMSCs膜片可顯著促進并加速骨組織形成。 第三部分 antimiR-138/rBMMSCs膜片種植體復合物的構建及其骨誘導能力研究 【目的】 構建antimiR-138/rBMMSCs膜片微弧氧化鈦種植體；antimiR-138/rBMMSCs膜片微弧氧化鈦種植體的成骨活性和骨誘導能力研究 【方法】 1）將antimiR-138轉染的rBMMSCs膜片與微弧氧化鈦種植體相復合構建基因修飾的組織工程化種植體，并對其表面形貌進行掃描電鏡觀察。 2）采用ALP、天狼星紅、茜素紅染色、Real-timePCR和Western blot技術檢測antimiR-138/rBMMSCs膜片微弧氧化鈦種植體的體外成骨活性。 3）通過異位成骨實驗，采用Micio-CT、HE和MT染色，免疫熒光技術對antimiR-138/rBMMSCs膜片微弧氧化鈦種植體的骨誘導能力進行評估。 【結果】 antimiR-138轉染的rBMMSCs膜片可成功與微弧氧化鈦種植體復合，電鏡觀察亦可見豐富的細胞和胞外基質(zhì)與種植體多孔結構的表面形成緊密的粘附與嵌合，即構建基因修飾的組織工程化種植體是可行的。體外ALP、膠原、茜素紅染色，Real-timePCR及Western blot檢測成骨相關基因和蛋白的表達均表明antimiR-138/rBMMSCs膜片與種植體的復合可促進并加速該復合物的體外成骨分化能力。體內(nèi)的Micro-CT檢測、HE和MT染色以及免疫熒光染色均表明antimiR-138/rBMMSCs膜片種植體復合物具有強大的骨誘導能力。 【結論】 (1)將antimiR-138修飾的rBMMSCs膜片與微弧氧化鈦種植體復合，構建基因修飾的組織工程化種植體是可行的。 (2)通過體內(nèi)體外成骨實驗證明antimiR-138/rBMMSCs膜片種植體復合物具有優(yōu)秀的成骨活性和骨誘導能力。 (3)這一技術將為解決臨床種植骨愈合時間過長，骨代謝疾病患者種植失敗率較高等問題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.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R783.6

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