本文選題：稀土離子Ce~(3+) + 骨髓間充質(zhì)干細(xì)胞�。� 參考：《山東大學(xué)》2014年博士論文

【摘要】：目的： 稀土包括化學(xué)元素周期表中的鑭系元素以及與鑭系元素化學(xué)性質(zhì)相似的其他兩種元素—鈧(Sc)和釔(Y),統(tǒng)稱(chēng)為稀土元素。我國(guó)是稀土資源最豐富的國(guó)家,產(chǎn)量和儲(chǔ)量占世界首位。鈰(Ce)是鑭系元素的一種,具有抗菌、止吐以及抗癌等多種功能。由于稀土離子Ce3+與鈣離子離子半徑相似,電荷相較鈣離子大,對(duì)細(xì)胞內(nèi)鈣離子位點(diǎn)更具有親和性,從而可能影響骨細(xì)胞的功能和干預(yù)骨重建過(guò)程。關(guān)于稀土離子Ce3+對(duì)成骨細(xì)胞的影響早在20世紀(jì)就有學(xué)者研究報(bào)道,研究發(fā)現(xiàn)稀土離子Ce3+可以促進(jìn)成骨細(xì)胞增殖、分化和成骨。稀土離子對(duì)成骨細(xì)胞的作用依賴(lài)其金屬離子濃度的雙向性,并且與稀土離子的種類(lèi)以及細(xì)胞屬性相關(guān)。 頜骨是口腔頜面部主要支架骨,行使咀嚼、吞咽以及語(yǔ)言等主要功能。炎癥、外傷、腫瘤術(shù)后等原因都能造成頜骨缺損,降低生活質(zhì)量。骨缺損修復(fù)是眾多學(xué)者研究熱點(diǎn)和重點(diǎn),近年來(lái)組織工程骨的發(fā)展為骨缺損修復(fù)提供了一條新的途徑。骨髓間充質(zhì)干細(xì)胞(BMSCs)是組織工程骨的一類(lèi)種子細(xì)胞,具有強(qiáng)大的增殖能力和多向分化潛能、體外培養(yǎng)技術(shù)簡(jiǎn)單成熟能建立穩(wěn)定的細(xì)胞系、取材方便等多項(xiàng)優(yōu)點(diǎn)。BMSCs在體內(nèi)外誘導(dǎo)環(huán)境下可以分化為成骨細(xì)胞,在BMSCs定向轉(zhuǎn)化為成骨細(xì)胞的過(guò)程中,成骨細(xì)胞表達(dá)特異性成骨標(biāo)志物,如ALP、Runx2、OCN、 Satb2、BSP等。在成骨細(xì)胞分化晚期,細(xì)胞開(kāi)始礦化,細(xì)胞內(nèi)ALP減少,骨鈣素等非膠原蛋白分泌到細(xì)胞外基質(zhì)中,形成羥基磷灰石結(jié)晶,最終鈣鹽沉積形成“鈣結(jié)節(jié)”。ALP、Runx2、Satb2是成骨細(xì)胞早期標(biāo)志基因,而OCN是分化晚期的標(biāo)志基因。 骨缺損修復(fù)是一個(gè)復(fù)雜的過(guò)程,多種因素能夠影響骨修復(fù)過(guò)程中細(xì)胞增殖、分化及骨基質(zhì)形成,其中研究新骨形成較深入的為骨形態(tài)發(fā)生蛋白(BMPs)。 BMPs家族中最具代表性的為BMP2,它是一類(lèi)骨誘導(dǎo)因子,可在骨受損部位促進(jìn)細(xì)胞增殖,提高ALP活性,并且可促進(jìn)BMSCs向成骨細(xì)胞分化。細(xì)胞外BMP2與細(xì)胞膜上的受體(BMPR Ⅱ)結(jié)合,使BMPR Ⅱ磷酸化從而激活BMPRI,活化的BMPR Ⅰ使細(xì)胞內(nèi)的Smadl/5/8磷酸化形成復(fù)合物,該復(fù)合物進(jìn)入到細(xì)胞核內(nèi),結(jié)合到DNA序列上,從而調(diào)節(jié)BMP靶基因的轉(zhuǎn)錄。 綜上所述,稀土離子Ce3+可以促進(jìn)成骨細(xì)胞增殖和成骨,但是稀土離子Ce3+對(duì)BMSCs成骨作用的影響國(guó)內(nèi)外鮮有報(bào)道。BMSCs具有多向分化潛能,可以被誘導(dǎo)成骨,但是由于研究手段的限制,信號(hào)通路的復(fù)雜性以及細(xì)胞因子網(wǎng)絡(luò)性調(diào)控的特點(diǎn),BMSCs誘導(dǎo)成骨的機(jī)制尚不明確。本研究通過(guò)體內(nèi)外實(shí)驗(yàn)研究稀土離子Ce3+對(duì)BMSCs成骨作用的影響,初步探討稀土離子Ce3+誘導(dǎo)BMSCs成骨作用的分子機(jī)制,從而為BMSCs成骨功能提供新的理論依據(jù),為臨床治療骨缺損提供新思路。 方法： 1.小鼠BMSCs培養(yǎng)、鑒定和Ce3+對(duì)BMSCs增殖、成骨及相關(guān)基因表達(dá)的影響 1)小鼠BMSCs分離、培養(yǎng)及鑒定 采用全骨髓貼壁培養(yǎng)法分離、培養(yǎng)小鼠來(lái)源的BMSCs;采用換液、有限稀釋法純化BMSCs; MTT法繪制細(xì)胞生長(zhǎng)曲線(xiàn)；流式細(xì)胞儀檢測(cè)BMSCs表面抗原；BMSCs成骨、成脂誘導(dǎo)分化； 2)稀土離子Ce3+對(duì)BMSCs增殖、成骨及相關(guān)基因表達(dá)的影響 首先取對(duì)數(shù)生長(zhǎng)期的BMSCs,利用CCK-8法檢測(cè)稀土離子Ce3+對(duì)BMSCs細(xì)胞增殖影響；然后通過(guò)檢測(cè)細(xì)胞ALP活性、“鈣結(jié)節(jié)”茜素紅S染色測(cè)定稀土離子Ce3+對(duì)BMSCs早晚期成骨作用的影響。最后通過(guò)qRT-PCR技術(shù)和Western blot技術(shù)檢測(cè)Ce3+離子干預(yù)培養(yǎng)7天后的細(xì)胞成骨基因Runx2、Satb2和OCNmRNA和相關(guān)蛋白的表達(dá)情況。 2.稀土離子Ce3+促進(jìn)BMSCs遷移以及成骨分化的分子機(jī)制 1)稀土離子Ce3+對(duì)BMSCs遷移的影響 利用Transwell小室檢測(cè)稀土離子Ce3+對(duì)BMSCs遷移的影響；SDF-1是干細(xì)胞遷移的主要趨化因子,CXCR4是SDF-1的天然受體,通過(guò)qRT-PCR技術(shù)檢測(cè)Ce3+離子干預(yù)培養(yǎng)7天后的細(xì)胞SDF-1和CXCR4基因表達(dá)情況。 2)稀土離子Ce3+促進(jìn)BMSCs遷移和成骨的分子機(jī)制 首先利用qRT-PCR技術(shù)檢測(cè)Ce3+離子干預(yù)培養(yǎng)7天后的細(xì)胞BMP2mRNA表達(dá)；然后Western blot技術(shù)檢測(cè)Ce3+離子干預(yù)細(xì)胞培養(yǎng)0、10、20、30min后p-Smad1/5/8蛋白表達(dá)情況以及細(xì)胞免疫熒光技術(shù)檢測(cè)Ce3+離子干預(yù)細(xì)胞培養(yǎng)30min后p-Smadl/5/8蛋白在細(xì)胞內(nèi)的位置變化；最后BMSCs加入LDN-193189(BMP抑制劑)和Ce3+離子一起干預(yù)培養(yǎng)后,通過(guò)Western blot技術(shù)檢測(cè)p-Smad1/5/8蛋白表達(dá)情況；然后通過(guò)qRT-PCR技術(shù)檢測(cè)BMP2.SDF-1及Runx2基因表達(dá)情況。 3.稀土離子Ce3+干預(yù)培養(yǎng)BMSCs細(xì)胞膜片修復(fù)小鼠顱骨缺損的實(shí)驗(yàn)研究 選取7周齡雄性C57BL/6小鼠,在顱骨頂部雙側(cè)各制備出直徑4mm的骨缺損,不要損傷硬腦膜,隨機(jī)分為三組,其中6只為實(shí)驗(yàn)組,植入BMSCs+Ce細(xì)胞膜片復(fù)合CBB；6只為BMSCs對(duì)照組,植入BMSCs細(xì)胞膜片復(fù)合CBB。6只為空白對(duì)照組無(wú)任何植入。術(shù)后4周處死動(dòng)物,留取標(biāo)本制成組織切片,HE染色觀察骨缺損成骨情況；計(jì)算新骨形成率,觀察骨缺損愈合情況,從而研究稀土離子Ce3+對(duì)顱骨缺損成骨情況的影響；通過(guò)對(duì)標(biāo)本進(jìn)行qRT-PCR險(xiǎn)測(cè)稀土離子Ce3+對(duì)成骨基因BSP、OCN表達(dá)情況的影響。 結(jié)果： 1.小鼠BMSCs培養(yǎng)、鑒定和Ce3+對(duì)BMSCs增殖、成骨及相關(guān)基因表達(dá)的影響 1)小鼠BMSCs分離培養(yǎng)、鑒定 通過(guò)全骨髓貼壁法獲得BMSCs,經(jīng)過(guò)換液、有限稀釋法使細(xì)胞純化,細(xì)胞大小、形態(tài)穩(wěn)定；MTT法繪制BMSCs生長(zhǎng)曲線(xiàn)為“S”,前2天生長(zhǎng)緩慢,第3天開(kāi)始進(jìn)入對(duì)數(shù)生長(zhǎng)期,7-8天生長(zhǎng)又開(kāi)始緩慢；流式細(xì)胞儀檢測(cè)BMSCs細(xì)胞表面表達(dá)CD29、CD44,而不表達(dá)CD3、CD45; BMSCs成骨誘導(dǎo)經(jīng)茜素紅染色發(fā)現(xiàn)細(xì)胞外有大量鈣基質(zhì)沉積；成脂誘導(dǎo)經(jīng)油紅O染色發(fā)現(xiàn)細(xì)胞內(nèi)有大量脂滴形成。通過(guò)以上結(jié)果我們確定分離培養(yǎng)的細(xì)胞為BMSCs,并且BMSCs具有多向分化潛能。 2)稀土離子Ce3+對(duì)BMSCs增殖、成骨及相關(guān)基因表達(dá)的影響 通過(guò)CCK8檢測(cè)細(xì)胞增殖、ALP活性測(cè)定,發(fā)現(xiàn)稀土離子Ce3+在濃度為0.001μM明顯促進(jìn)細(xì)胞增殖和增加ALP活性；茜素紅染色測(cè)定細(xì)胞晚期成骨,結(jié)果發(fā)現(xiàn)稀土離子Ce3+明顯促進(jìn)鈣結(jié)節(jié)生成；qRT-PCR和Western blot技術(shù)測(cè)定結(jié)果發(fā)現(xiàn)稀土離子Ce3+能顯著促進(jìn)成骨基因Runx2、satb2和OCN以及相關(guān)蛋白的表達(dá)。 2.稀土離子Ce3+促進(jìn)BMSCs遷移以及成骨分化的分子機(jī)制 1)稀土離子Ce3+對(duì)BMSCs遷移的影響 稀土離子Ce3+通過(guò)上調(diào)遷移趨化因子SDF-1基因表達(dá)從而促進(jìn)BMSCs遷移,但是對(duì)CXCR4基因表達(dá)無(wú)明顯影響。 2)稀土離子Ce3+促進(jìn)BMSCs遷移和成骨的分子機(jī)制 稀土離子Ce3+能夠促進(jìn)BMP2基因和p-Smad1/5/8蛋白表達(dá),并且p-Smad1/5/8蛋白表達(dá)出現(xiàn)時(shí)間依賴(lài)性；然后發(fā)現(xiàn)稀土離子Ce3+處理過(guò)的BMSCs細(xì)胞中p-Smad1/5/8蛋白主要位于細(xì)胞核內(nèi),而沒(méi)經(jīng)過(guò)稀土離子Ce3+處理過(guò)的BMSCs細(xì)胞中p-Smad1/5/8蛋白主要位于細(xì)胞漿內(nèi)；最后發(fā)現(xiàn)經(jīng)過(guò)LDN-193189處理過(guò)的BMSCs細(xì)胞中p-Smad1/5/8蛋白、SDF-1和Runx2基因表達(dá)都降低,但對(duì)BMP2基因表達(dá)無(wú)影響。 3.稀土離子鈰干預(yù)培養(yǎng)BMSCs細(xì)胞膜片修復(fù)小鼠顱骨缺損的實(shí)驗(yàn)研究 通過(guò)動(dòng)物模型試驗(yàn),稀土離子Ce3+可以促進(jìn)顱骨骨缺損區(qū)新骨形成,并對(duì)顱骨缺損區(qū)成骨基因BSP、OCN呈現(xiàn)正調(diào)節(jié)作用。 結(jié)論： 1.全骨髓貼壁法分離培養(yǎng)骨髓間充質(zhì)干細(xì)胞操作簡(jiǎn)單,易于掌握；BMSCs具有多向分化潛能,是骨組織工程理想的種子細(xì)胞。 2.稀土離子Ce3+具有促進(jìn)BMSCs增殖、成骨及募集BMSCs的作用。 3.稀土離子Ce3+通過(guò)BMP-Smad信號(hào)通路促進(jìn)BMSCs遷移和成骨。 4.稀土離子Ce3+可以促進(jìn)體內(nèi)成骨。
[Abstract]:Objective:
Rare earth elements, including lanthanide elements in the periodic table of chemical elements and other two other elements similar to lanthanide elements - scandium (Sc) and yttrium (Y), are collectively known as rare earth elements. China is the richest country with rare earth resources, and the yield and reserves are the first in the world. Cerium (Ce) is one of the lanthanide elements, which has many kinds of work such as antibacterial, antiemetic and anticancer. As the rare earth ion Ce3+ is similar to the radius of the calcium ion, the charge phase is more affinity to the intracellular calcium ion site than the calcium ion, which may affect the function of bone cells and interfere with the process of bone reconstruction. The effect of rare earth ion Ce3+ on osteoblasts has been reported in twentieth Century, and the rare earth ion C has been discovered. E3+ can promote osteoblast proliferation, differentiation and osteogenesis. The effect of rare earth ions on osteoblasts depends on the bi-directional concentration of metal ions, and is related to the species of rare earth ions and cell properties.
Maxillofacial bone is the main stenting bone in the oral and maxillofacial region, which exercises the main functions of chewing, swallowing and language. Inflammation, trauma, and postoperative tumor can cause the defect of the jaw and reduce the quality of life. The repair of bone defect is a hot spot and focus of many scholars. In recent years, the development of tissue engineering bone has provided a new way for the repair of bone defect. Bone marrow mesenchymal stem cell (BMSCs) is a kind of seed cells of tissue engineering bone. It has strong proliferation ability and multidirectional differentiation potential. In vitro culture technology is simple and mature to establish stable cell lines, and the advantages of.BMSCs can be differentiated into osteoblasts under the induced environment in vitro and in the BMSCs direction into osteogenic fine. In the process of cell, osteoblasts express specific osteogenic markers, such as ALP, Runx2, OCN, Satb2, BSP and so on. In the late differentiation of osteoblasts, the cells begin to mineralized, the intracellular ALP decreases, and the osteocalcin and other non collagen secretes into the extracellular matrix to form hydroxyapatite nodules, and eventually calcium salts are deposited to form a "calcium nodule".ALP, Runx2, Satb2 is The early marker genes of osteoblasts, while OCN is a marker of late differentiation.
Bone defect repair is a complex process. A variety of factors can affect cell proliferation, differentiation and bone matrix formation during the process of bone repair, in which new bone formation is BMPs. The most representative of the BMPs family is BMP2. It is a kind of bone inducer, which can promote cell proliferation at the damaged part of bone. The high ALP activity can promote the differentiation of BMSCs into the osteoblast. The extracellular BMP2 is combined with the receptor on the cell membrane (BMPR II), which makes BMPR II phosphorylation and activates the BMPRI. The activated BMPR I makes the Smadl/5/8 phosphorylation in the cell to form a complex. The complex enters the nucleus and combines to the DNA sequence, thereby regulating the transfer of the BMP target gene. Record.
To sum up, rare earth ion Ce3+ can promote osteoblast proliferation and osteogenesis, but the effect of rare earth ion Ce3+ on BMSCs osteogenesis is rarely reported that.BMSCs has multidirectional differentiation potential and can be induced to osteogenesis, but the complexity of signal pathway and the characteristics of cytokine network regulation, BMSC The mechanism of s induction of osteogenesis is not clear. In this study, the effect of rare earth ion Ce3+ on the osteogenesis of BMSCs was studied in vitro and in vitro, and the molecular mechanism of BMSCs osteogenesis induced by rare earth ion Ce3+ was preliminarily discussed, which provided a new theoretical basis for the osteogenesis of the bone and provided a new idea for the clinical treatment of bone defect.
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