【摘要】：研究背景和目的隨著國(guó)內(nèi)人口年齡結(jié)構(gòu)逐漸步入老齡化,許多老齡化相關(guān)的疾病日漸引起醫(yī)學(xué)界的廣泛重視。骨質(zhì)疏松作為一種老年人群高發(fā)的疾病,尤其重度骨質(zhì)疏松具有較高的骨折風(fēng)險(xiǎn),同時(shí)此類骨折發(fā)生后容易遷延不愈,是嚴(yán)重影響老年人健康和生活質(zhì)量的一類骨科門急診常見病。世界衛(wèi)生組織已將骨質(zhì)疏松、糖尿病與心血管病一起列為影響中老年人健康的“三大殺手”。此外,臨床上因嚴(yán)重暴力所致的大段骨缺損、骨折后不愈合以及骨不連一直以來都是創(chuàng)傷骨科中的治療難點(diǎn)。以上疾病的發(fā)生和發(fā)展均與機(jī)體內(nèi)骨代謝失衡,成骨能力受損密切相關(guān)。因此,對(duì)成骨機(jī)制的研究和探索,將可能為這些臨床問題的解決提供支持。人骨髓間充質(zhì)干細(xì)胞(human bone marrow mesenchymal stem cells,hBMSCs)作為成骨細(xì)胞重要的一種前體細(xì)胞,在骨的形成和成骨分化上都起著重要的作用。隨著對(duì)間充質(zhì)干細(xì)胞成骨機(jī)理的研究不斷深入,將有助于我們更好地理解多種骨病的病理過程,為此類疾病的治療尋找新的思路。此外,hBMSCs具有較強(qiáng)的增殖能力和多向分化潛能,是骨組織工程領(lǐng)域最重要的種子細(xì)胞來源,同時(shí)在干細(xì)胞移植領(lǐng)域也具有廣泛的應(yīng)用前景。microRNA(miRNAs)是一類廣泛存在于真核生物細(xì)胞內(nèi)的非編碼單鏈小分子RNA,它能夠與靶基因的3’非翻譯區(qū)部分堿基互補(bǔ)結(jié)合,繼而調(diào)控基因的表達(dá),在生物體內(nèi)眾多基本的生理和病理過程中都扮演著重要的角色。近年來,越來越多的研究表明miRNAs在成骨細(xì)胞和破骨細(xì)胞的動(dòng)態(tài)平衡中起著重要的調(diào)控作用,并且直接參與了諸多骨病如骨質(zhì)疏松、關(guān)節(jié)炎等疾病的發(fā)生和發(fā)展過程。有研究發(fā)現(xiàn),miR-125b在成骨過程中下調(diào),但具體的調(diào)控機(jī)制尚不完全明了。本研究選定miR-125b作為研究對(duì)象,希望通過對(duì)miR-125b調(diào)控人骨髓間充質(zhì)干細(xì)胞成骨分化的機(jī)制研究。進(jìn)一步豐富干細(xì)胞成骨定向分化的理論體系,同時(shí)可加深對(duì)骨骼發(fā)生、發(fā)育的分子機(jī)制認(rèn)識(shí),為骨質(zhì)疏松防治,骨折治療,骨缺損修復(fù)尋找新的治療思路并提供理論支持。研究方法1.人骨髓間充質(zhì)干細(xì)胞的分離、培養(yǎng)和鑒定使用密度梯度離心的方法,從健康成年志愿者捐獻(xiàn)的骨髓中分離獲取hBMSCs,通過傳代、培養(yǎng)、純化,獲取穩(wěn)定的干細(xì)胞來源。培養(yǎng)過程中注意觀察貼壁細(xì)胞形態(tài)變化,使用MTT法檢測(cè)其增殖活力。經(jīng)流式細(xì)胞術(shù)檢測(cè)細(xì)胞表面抗原對(duì)細(xì)胞屬性進(jìn)行鑒定,通過成骨、成脂、成軟骨誘導(dǎo)分化,對(duì)hBMSCs的多向分化能力即干性進(jìn)行鑒定。2.miR-125b對(duì)hBMSCs成骨分化的影響用慢病毒轉(zhuǎn)染的方式對(duì)細(xì)胞進(jìn)行干預(yù)。首先將人工合成的miR-125-pre和miR-125b-inhibitor核酸序列構(gòu)建到慢病毒載體并轉(zhuǎn)染hBMSCs。對(duì)轉(zhuǎn)染后的細(xì)胞轉(zhuǎn)染效率和增殖能力進(jìn)行觀察檢測(cè)。然后,采用Real Time RT-PCR技術(shù)檢測(cè)轉(zhuǎn)染后細(xì)胞內(nèi)miR-125b表達(dá)量的變化。最后,使用Real Time RT-PCR和Western Blot對(duì)間充質(zhì)干細(xì)胞成骨分化過程中重要的成骨因子包括ALP,Runx2,OSX以及OCN的表達(dá)情況進(jìn)行核酸和蛋白水平上的檢測(cè),觀察其表達(dá)變化情況。采用堿性磷酸酶染色和茜素紅染色的方法對(duì)轉(zhuǎn)染后的細(xì)胞進(jìn)行染色觀察,通過細(xì)胞顯色情況鑒定其成骨分化能力。3.miR-125b調(diào)控hBMSCs的作用靶點(diǎn)預(yù)測(cè)及驗(yàn)證首先,選用生物信息學(xué)的方法,使用Target Scan,miRanda及Pic Tar作為靶基因預(yù)測(cè)工具,對(duì)可能與miR-125b結(jié)合的靶基因序列進(jìn)行篩選預(yù)測(cè),選擇可能與其結(jié)合的潛在靶點(diǎn)BMPR1b作為研究對(duì)象。隨后,構(gòu)建BMPR1b的熒光素酶報(bào)告載體,使用雙熒光素酶報(bào)告的方法對(duì)所預(yù)測(cè)的靶基因進(jìn)行初步驗(yàn)證。最后,將hBMSCs分別轉(zhuǎn)染miR-125b的上調(diào)和下調(diào)慢病毒載體,使用Real Time RT-PCR和Western Blot檢測(cè)當(dāng)細(xì)胞內(nèi)miR-125b表達(dá)水平變化時(shí),BMPR1b的mRNA和蛋白表達(dá)變化。4.miR-125b抑制hBMSCs成骨分化的機(jī)制首先使用Real Time RT-PCR和Western Blot檢測(cè)hBMSCs成骨過程中BMPR1b的表達(dá)變化。隨后,采用RNAi技術(shù),將BMPR1b的siRNA載體轉(zhuǎn)染hBMSCs,使用Real Time RT-PCR和Western Blot檢測(cè)Runx2,OSX以及OCN的mRNA和蛋白表達(dá)變化。采用堿性磷酸酶染色和茜素紅染色的方法對(duì)轉(zhuǎn)染后的細(xì)胞進(jìn)行染色觀察,鑒定成骨分化能力。最后,將miR-125b-inhibitor與si-BMPR1b共轉(zhuǎn)染hBMSCs,使用Real Time RT-PCR和Western Blot檢測(cè)Runx2,OSX以及OCN的mRNA和蛋白表達(dá)變化。采用堿性磷酸酶染色和茜素紅染色的方法對(duì)轉(zhuǎn)染后的細(xì)胞進(jìn)行染色觀察,鑒定成骨分化能力。5.miR-125b調(diào)控hBMSCs成骨分化的體內(nèi)研究首先,建立裸鼠股骨原位骨缺損模型。隨后,將miR-125b-inhibitor轉(zhuǎn)染hBMSCs,將細(xì)胞復(fù)合至DBM后進(jìn)行成骨誘導(dǎo),再通過手術(shù)的方式將DBM移植至裸鼠體內(nèi),利用micro CT觀測(cè)新骨生成狀況,采用組織切片,HE染色和Masson染色,從細(xì)胞的微觀角度評(píng)估成骨能力。研究結(jié)果1.本實(shí)驗(yàn)所采用的密度梯度離心法分離獲取hBMSCs,培養(yǎng)過程中細(xì)胞各項(xiàng)指標(biāo)表明hBMSCs細(xì)胞形態(tài)正常,具有較強(qiáng)的增殖活力。hBMSCs的陽(yáng)性表面抗原CD73,CD90,和CD105陽(yáng)性率均在95%以上,而陰性抗原CD34,CD45,CD14,CD19,HLA-DR陽(yáng)性率在5%以下。符合hBMSCs鑒定標(biāo)準(zhǔn)。此外,分離培養(yǎng)的hBMSCs具備成骨、成軟骨和成脂的多向分化能力,具備良好的干性。2.在hBMSCs向成骨分化的過程中,miR-125b處于低水平表達(dá)的狀態(tài)。慢病毒載體對(duì)hBMSCs轉(zhuǎn)染效果較好,且對(duì)細(xì)胞的增殖不產(chǎn)生影響。在hBMSCs成骨分化過程中,上調(diào)miR-125b表達(dá)能夠抑制成骨。相反地,下調(diào)miR-125b則能夠促進(jìn)hBMSCs的成骨分化。3.生物信息學(xué)預(yù)測(cè)出BMPR1b基因可能是miR-125的作用靶點(diǎn)。雙熒光素酶報(bào)告實(shí)驗(yàn)證實(shí)miR-125b在細(xì)胞內(nèi)能與BMPR1b的3’UTR部分堿基序列存在結(jié)合位點(diǎn)。在hBMSCs中,上調(diào)miR-125b能夠抑制BMPR1b的mRNA和蛋白表達(dá)。相反,下調(diào)miR-125b則能夠增加BMPR1b的mRNA和蛋白表達(dá)量。4.hBMSCs成骨分化過程中,BMPR1b呈高表達(dá)狀態(tài)。沉默BMPR1b的基因后,hBMSCs的成骨能力下降。miR-125b-inhibitor與si-BMPR1b共轉(zhuǎn)染hBMSCs后,hBMSCs的成骨分化能力同樣受到抑制。5.對(duì)miR-125b調(diào)節(jié)成骨分化的體內(nèi)研究發(fā)現(xiàn),轉(zhuǎn)染miR-125b-inhibitor后的hBMSCs,micro CT和組織切片染色其體內(nèi)成骨能力強(qiáng)于陰性對(duì)照組。結(jié)論1.本實(shí)驗(yàn)所分離培養(yǎng)的hBMSCs增殖活躍,符合間充質(zhì)干細(xì)胞的一般形態(tài)學(xué)特點(diǎn)。干細(xì)胞特性明顯,經(jīng)不同條件誘導(dǎo),能向成骨、成脂以及成軟骨細(xì)胞分化。純度較高,干細(xì)胞來源穩(wěn)定可靠。2.miR-125b能夠負(fù)向調(diào)控hBMSCs成骨分化。3.miR-125b通過與靶基因BMPR1b的3’UTR部分堿基互補(bǔ)結(jié)合,從而抑制BMPR1b的表達(dá)。4.miR-125b通過抑制其靶基因BMPR1b的表達(dá),進(jìn)而抑制hBMSCs的成骨分化。5.復(fù)合hBMSCs的DBM骨移植材料,能夠在體內(nèi)較好地修復(fù)骨缺損。下調(diào)miR-125b的表達(dá)后,能夠進(jìn)一步提高h(yuǎn) BMSC在體內(nèi)的成骨能力。
[Abstract]:The background and purpose of the study, as the age structure of the population of the country has gradually entered the age of aging, many of the aging-related diseases are increasing the attention of the medical community. Osteoporosis is a high-incidence disease of the old people, especially the severe osteoporosis has a higher risk of fracture, and at the same time, it is easy to move after the fracture, which is a kind of common diseases in the department of orthopedics that seriously affects the health and quality of life of the old people. The World Health Organization has included osteoporosis, diabetes and cardiovascular disease as a "the three killers" that affects the health of the middle-aged and the elderly. In addition, the clinical experience of the large-segment bone defect due to the severe violence, the non-union after the fracture, and the non-union of the bone all the time are the treatment difficulties in the trauma orthopedics. The occurrence and development of the above diseases are closely related to the imbalance of bone metabolism and the damage of osteogenesis in the body. Therefore, the research and exploration of the osteogenic mechanism will provide support for the solution of these clinical problems. Human bone marrow mesenchymal stem cells (hBMSCs) play an important role in the formation of bone and the differentiation of osteogenesis. With the development of the bone formation mechanism of the mesenchymal stem cells, it will help us to better understand the pathological process of various bone diseases, and find a new way for the treatment of such diseases. In addition, hBMSCs have a strong proliferation ability and multi-directional differentiation potential, which are the most important seed cell sources in the field of bone tissue engineering, and have a wide application prospect in the field of stem cell transplantation. The microRNA (miRNAs) is a kind of non-coding single-chain small-molecule RNA which is widely present in eukaryotes, which can be combined with the base of the 3 'non-translation region of the target gene, and then regulate the expression of the gene, and plays an important role in many basic physiological and pathological processes in the organism. In recent years, more and more studies have shown that miRNAs play an important role in the dynamic balance of osteoblasts and osteoclasts, and are directly involved in the occurrence and development of many bone diseases such as osteoporosis and arthritis. It has been found that miR-125b is down-regulated in the process of osteogenesis, but the specific regulatory mechanism is not entirely clear. In this study, miR-125b was selected as the subject of study, and it was hoped to study the mechanism of human bone marrow-derived mesenchymal stem cells in human bone marrow by the mechanism of miR-125b. The theory system of stem cell osteogenesis and orientation differentiation is further enriched, and the molecular mechanism of bone formation and development can be enhanced, and a new treatment idea and theoretical support are provided for the prevention and treatment of osteoporosis, fracture treatment and bone defect repair. Study Method 1. The method for separating, culturing and identifying human bone marrow mesenchymal stem cells using density gradient centrifugation is to separate and obtain hBMSCs from the bone marrow donated by healthy adult volunteers, and to obtain stable stem cell sources by passage, culture and purification. In the process of culture, the morphological changes of the adherent cells were observed, and the proliferative activity of the adherent cells was detected by the MTT method. The cell surface antigen was identified by flow cytometry. The differentiation of hBMSCs was induced by osteogenic, adipogenic and chondrogenic differentiation, and the multi-directional differentiation ability of hBMSCs was identified. The synthetic miR-125-pre and miR-125b-inhitor nucleic acid sequences are first constructed to a lentiviral vector and transfected with hBMSCs. And the transfection efficiency and the proliferation ability of the transfected cells are observed and detected. Then, the expression of miR-125b in the transfected cells was detected by Real Time RT-PCR. Finally, the expression of ALP, Runx2, OSX and OCN was detected by using Real Time RT-PCR and Western Blot to detect the expression of the nucleic acid and protein. The method of using alkaline phosphatase staining and fluorescein red staining to dye and observe the transfected cells, and the bone differentiation capacity of the transfected cells is identified by the color development of the cells.3. The method for predicting and verifying the action target of the hBMSCs by using the miR-125b is first, the method of bioinformatics is selected, the target Scan is used, MiRANDa and Pic Tar are used as target gene prediction tools to screen and predict target gene sequences that may be combined with miR-125b to select potential targets BMPR1b that may be combined with them as the study target. Subsequently, the luciferase reporter vector of BMPR1b was constructed, and the predicted target gene was initially validated using a double luciferase reporter method. Finally, hBMSCs were transfected with the up-regulated and down-regulated lentiviral vector of miR-125b, respectively, and the expression level of miR-125b in the cell was detected by Real Time RT-PCR and Western Blot. 4. The expression of BMPR1b was detected by using Real Time RT-PCR and Western Blot, and the expression of BMPR1b was detected by using Real Time RT-PCR and Western Blot. Subsequently, the siRNA vector of BMPR1b was transfected into hBMSCs by using the RNAi technology, and the mRNA and protein expression of the Runx2, OSX and OCN were detected by Real Time RT-PCR and Western Blot. The cells of the transfected cells were stained with alkaline phosphatase and fluorescein red staining, and the osteogenic differentiation ability was identified. Finally, hBMSCs were cotransfected with miR-125b-inhitor and si-BMPR1b, and the mRNA and protein expression of Runx2, OSX and OCN were detected by Real Time RT-PCR and Western Blot. 5. The in vivo study of the osteogenic differentiation of hBMSCs by miR-125b was first established, and the in-situ bone defect model of the femur in nude mice was established. Subsequently, the hBMSCs were transfected with miR-125b-inhitor, the cells were combined to DBM, and the bone formation was induced, and the DBM was transplanted into the nude mice by operation. The new bone formation was observed by microCT, and the osteogenic ability was assessed from the micro-angle of the cells by using tissue sections, HE staining and Masson staining. Study Results 1. In this experiment, hBMSCs were obtained by density gradient centrifugation, and the indexes of the cells in the culture process showed that the morphology of hBMSCs was normal and the proliferation of hBMSCs was strong. The positive rate of CD73, CD90 and CD105 of hBMSCs was more than 95%, while the positive rate of negative antigen CD34, CD45, CD14, CD19 and HLA-DR was below 5%. The criteria for the identification of hBMSCs were met. In addition, the cultured hBMSCs have the ability of osteogenesis, chondrogenesis and adipocyte differentiation, and have a good dry.2. MiR-125b is at a low level of expression during the differentiation of hBMSCs into osteogenesis. The effect of lentiviral vector on hBMSCs was better, and the effect of lentiviral vector on the proliferation of cells was not affected. The up-regulation of the expression of miR-125b in the osteogenesis of hBMSCs can inhibit the formation of bone. In contrast, down-regulation of miR-125b can promote the osteogenic differentiation of hBMSCs. Bioinformatics predicts that the BMPR1b gene may be the target of miR-125. The double luciferase reporter assay demonstrated that miR-125b can bind to the 3 'UTR partial base sequence of BMPR1b in the cell. In hBMSCs, up-regulation of miR-125b can inhibit the mRNA and protein expression of BMPR1b. In contrast, down-regulation of miR-125b can increase the mRNA and protein expression of BMPR1b. After the gene of BMPR1b was silent, the osteogenic ability of hBMSCs decreased. The osteogenic differentiation of hBMSCs was also inhibited by the co-transfection of hBMSCs with miR-125b-inhitor and si-BMPR1b. In vivo study of the regulation of the osteogenic differentiation of miR-125b, the ability of hBMSCs, micro-CT and tissue sections transfected with miR-125b-inhitor was stronger than that of the negative control group. Conclusion 1. The proliferation of hBMSCs isolated and cultured in this experiment is in accordance with the general morphological characteristics of the mesenchymal stem cells. The characteristics of stem cells are distinct and can be induced by different conditions, and can be differentiated into osteogenesis, adipogenesis and chondrocyte differentiation. 3. miR-125b can inhibit the expression of BMPR1b by combining with the base of the 3 'UTR part of the target gene BMPR1b, thereby inhibiting the expression of the BMPR1b. The DBM bone graft material of the composite hBMSCs can be used for repairing the bone defect in vivo. After the expression of miR-125b is down-regulated, the osteogenic ability of the h BMSC in the body can be further improved.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R580

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