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  本文關(guān)鍵詞：miR-30a-5p在人骨髓間充質(zhì)干細胞體外向成骨細胞分化中的生物學功能驗證　出處：《第四軍醫(yī)大學》2011年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細胞 miRNA 成骨細胞 生物學功能研究

【摘要】：背景 組織工程干細胞(SC)的研究作為生物醫(yī)學領(lǐng)域的研究熱點,為組織器官缺損的修復重建提供了一條全新而極具臨床應用前景的途徑。成體干細胞(ASC)作為早期未分化細胞具有強大的增殖更新及定向分化為成骨細胞、軟骨細胞、神經(jīng)細胞、脂肪細胞、骨骼肌細胞、真皮層細胞、肝細胞等的潛能。因其還具有來源廣泛、易于體外擴增且可行個體化治療從而避免排斥反應等眾多優(yōu)點而被密切關(guān)注并于多層面展開進行深入研究。多項研究證實骨髓間充質(zhì)干細胞(BMSCs)的增殖、分化受到各種細胞因子、信號通路、細胞內(nèi)外微環(huán)境等多方面因素的聯(lián)合作用影響。同時大量研究還證實microRNA(miRNA)也參與了干細胞的各種生物學行為的調(diào)控。 miRNA是一類平均含有22nt、主要于基因轉(zhuǎn)錄后水平行使調(diào)控功能的小分子非蛋白編碼RNA。近年來大量研究表明在多個物種中存在著一個異常龐大的miRNA家族,不同物種中其類型、數(shù)量及表達時序性均具有差異性。另外,在生物個體的生長發(fā)育、信號轉(zhuǎn)導、組織器官分化、疾病的發(fā)生發(fā)展等過程中miRNA也參與了重要調(diào)控,并決定了該生物體的生理、病理和行為等的變化。 本課題組前期實驗通過高通量基因芯片檢測技術(shù)檢測BMSCs及其誘導分化的成骨細胞中的miRNA基因表達譜,分析篩選出一系列成骨誘導分化前后差異性表達miRNA,本實驗從該結(jié)果中選取成骨性表達量顯著增高的miRNA-30a-5p,來驗證其在成骨中的生物學作用,并對其產(chǎn)生的該生物學作用的靶基因進行預測。 目的 探討并驗證重組miRNA-30a-5p體外轉(zhuǎn)染對人骨髓間充質(zhì)干細胞向成骨細胞分化的生物學作用。并通過生物信息學研究方法來預測其可能的作用靶基因。 方法 人工重組合成從人骨髓MSCs定向成骨分化差異性表達miRNA基因芯片結(jié)果中篩選成骨性表達量顯著增高的miRNA-30a-5p(3組樣品結(jié)果篩選結(jié)果參見附錄2)。從人體骨髓中分離培養(yǎng)BMSCs,并于體外轉(zhuǎn)染重組miRNA-30a-5p后繼續(xù)行成骨誘導培養(yǎng),第14、21天時分別取細胞爬片通過茜素紅S法染色檢測鈣鹽沉積、堿性磷酸酶(ALP)鈣鈷法染色及ALP比色法定量檢測成骨細胞ALP活性,對比觀察研究重組miRNA-30a-5p在人骨髓MSCs定向成骨誘導分化過程中的生物學功能。 結(jié)果 成功分離培養(yǎng)人骨髓MSCs并誘導其成骨定向分化。經(jīng)體外向人BMSCs轉(zhuǎn)染miRNA-30a-5p并誘導其成骨定向分化,轉(zhuǎn)染效率為37.32%±2.43%。分別于誘導培養(yǎng)第14天、21天行ALP染色觀察、ALP活性測定、茜素紅鈣鹽結(jié)節(jié)染色檢測各組細胞成骨活性,結(jié)果顯示miRNA-30a-5p mimics轉(zhuǎn)染組MSCs成骨分化特性顯著性增強(P0.05)。 結(jié)論 (1)用percoll密度梯度離心法結(jié)合全骨髓貼壁培養(yǎng)法可以從新鮮骨髓液中分離得到數(shù)量充足、純度較高的人BMSCS,并可在體外誘導培養(yǎng)促使其向成骨細胞分化。(2)miRNA-30a-5p在MSCs定向成骨分化的過程中具有一定的促進增強作用,被轉(zhuǎn)染的MSCs向成骨細胞定向分化表達有所增加,為進一步闡明人骨髓MSCs定向成骨分化的分子生化機制,細胞移植修復治療骨缺損奠定了理論基礎(chǔ)。
[Abstract]:background
Tissue engineering stem cells (SC) as a research focus in the field of biomedical research, provides a way to a new and very prospect of clinical application for repair and reconstruction of tissue and organ defects. Adult stem cells (ASC) as early undifferentiated cells have strong self-renewal and proliferation and differentiated into osteoblasts, cartilage cells. Nerve cells, fat cells, skeletal muscle cells, dermal cells, liver cells and other potential. Because it also has a wide range of sources, easy to amplify in vitro and individual treatment to avoid possible rejection by many advantages closely and conduct research on many levels. Studies have shown that bone marrow mesenchymal stem cells (BMSCs) proliferation and differentiation by various cytokines, signaling pathway, the combined effects of extracellular micro environment and other factors. At the same time, the study confirmed that a large number of microRNA (miRNA) are also involved in The regulation of various biological behavior of stem cells.
MiRNA is a small molecule containing an average of 22nt, mainly in the post transcriptional gene regulates the non protein encoding RNA. in recent years a large number of studies show that in many species there is an unusually large miRNA family in different species of the type, quantity and time expressions are different. In addition, signal transduction in individuals'growth,, tissue and organ differentiation, the occurrence and development process of disease in miRNA also play an important role in regulation, and determines the organism's physiological, pathological changes and behavior.
Our previous studies by high-throughput gene chip detection technology BMSCs and its differentiation into bone cells in miRNA gene expression profiles analysis screened a series of osteogenic differentiation and expression of miRNA, the experimental results from the selected osteogenic significantly increased expression of miRNA-30a-5p, to verify the in the biological effects of bone in the target gene of the biological function and the forecast.
objective
Objective to investigate and verify the biological function of recombinant miRNA-30a-5p transfection on human bone marrow mesenchymal stem cells to differentiate into osteoblasts, and to predict the possible target genes by bioinformatics.
Method
The artificial synthesis of recombinant human bone marrow MSCs from osteoblast specific expression of miRNA gene chip results in screening of osteogenic significantly increased expression of miRNA-30a-5p (3 samples the screening results see Appendix 2). BMSCs was isolated from human bone marrow, and continue to osteoblast induced in vitro after transfected with recombinant miRNA-30a-5p, 14,21 when the days were taken cell sheets by alizarin red S staining method to detect calcium deposition, alkaline phosphatase (ALP) calcium cobalt staining and ALP colorimetric method for quantitative detection of ALP activity in osteoblasts, a comparative study of biological function of recombinant miRNA-30a-5p in human bone marrow MSCs osteogenic differentiation process.
Result
Culture of human bone marrow MSCs and induce osteogenic differentiation. The separation successfully transfected to human BMSCs in vitro by miRNA-30a-5p and induce osteogenic differentiation, the transfection efficiency was 37.32% + 2.43%. were induced for fourteenth days, 21 days were observed by ALP staining, ALP activity, alizarin red calcium nodules staining cells were detected with osteogenic activity the results showed that miRNA-30a-5p mimics transfection group, MSCs osteogenic differentiation increased significantly (P0.05).
conclusion
(1) by Percoll density gradient centrifugation combined with whole bone marrow adherent culture method can obtain a sufficient number of isolated from fresh bone marrow, high purity of BMSCS, and can be cultured to promote its osteogenic differentiation in vitro. (2) miRNA-30a-5p in the MSCs process oriented differentiation has certain promotion enhanced expression of MSCs transfected into osteoblasts differentiation increased, in order to further clarify the orientation of human bone marrow MSCs into molecular and biochemical mechanisms of osteogenic differentiation, cell transplantation of bone defect repair treatment has laid a theoretical foundation.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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