蘇華斌 胡波涌 葉俊杰

摘要：目的 ?探究miR-449是否具有調(diào)控骨髓間充質(zhì)干細(xì)胞（MSCs）成骨分化的作用。方法 ?分離、培養(yǎng)骨髓MSCs，采用流式細(xì)胞儀檢測(cè)CD44、CD29、CD34、CD45等表面相關(guān)標(biāo)志抗原的表達(dá)鑒定骨髓MSCs;將MSCs分為對(duì)照組、miR-449a組、miR-449b組及anti-miR-449組，對(duì)照組用成骨誘導(dǎo)培養(yǎng)基培養(yǎng)，其余各組分別經(jīng)miR-449a/b或inhibitors轉(zhuǎn)染后在成骨誘導(dǎo)培養(yǎng)基培養(yǎng)，分別于培養(yǎng)第3、6、9、12天檢測(cè)各組堿性磷酸酶活性，qRT-PCR檢測(cè)miR-449a/b對(duì)成骨特異性基因Runx2、Osterix表達(dá)的影響。結(jié)果 ?①第4代間充質(zhì)干細(xì)胞CD44、CD29表達(dá)陽性，而CD34 和CD45表達(dá)陰性，符合骨髓間充質(zhì)干細(xì)胞的特征。②成骨誘導(dǎo)培養(yǎng)3、6、9、12 d后，MSCs細(xì)胞堿性磷酸酶活性較上一時(shí)間點(diǎn)升高，且呈時(shí)間依賴性，差異有統(tǒng)計(jì)學(xué)意義（P<0.05）;MSCs成骨誘導(dǎo)培養(yǎng)3、6、9、12 d后，Runx2、Osterix表達(dá)水平逐漸升高。③miR-449a組和miR-449b組AKP活性低于對(duì)照組，而anti-miR-449組AKP活性升高，差異有統(tǒng)計(jì)學(xué)意義（P<0.05）;miR-449a組和miR-449b組 Runx2、Osterix表達(dá)較對(duì)照組下調(diào)，差異有統(tǒng)計(jì)學(xué)意義（P<0.05）。結(jié)論 ?本次實(shí)驗(yàn)成功分離、培養(yǎng)了骨髓MSCs，其具有體外成骨分化能力，而miR-449能抑制骨髓MSCs向成骨分化。

關(guān)鍵詞：miR-449 a/b;間充質(zhì)干細(xì)胞;成骨分化

中圖分類號(hào)：R329 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 文獻(xiàn)標(biāo)識(shí)碼：A ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? DOI：10.3969/j.issn.1006-1959.2020.04.021

文章編號(hào)：1006-1959（2020）04-0069-04

Abstract：Objective ?To investigate whether miR-449 can regulate the osteogenic differentiation of bone marrow mesenchymal stem cells （MSCs）. Methods ?Bone marrow MSCs were isolated and cultured. Flow cytometry was used to detect the expression of surface-associated marker antigens such as CD44， CD29， CD34， and CD45. Bone marrow MSCs were identified. The MSCs were divided into control group， miR-449a group， miR-449b group，and anti-miR-449 group， the control group was cultured with osteogenic induction medium， and the remaining groups were cultured in osteogenic induction medium after transfection with miR-449a / b or inhibitors， respectively in culture 3， 6， 9， and12 d， the alkaline phosphatase activity was detected in each group， and the effect of miR-449a / b on the expression of osteogenic specific genes Runx2 and Osterix was detected by qRT-PCR. Results ?①The fourth generation of mesenchymal stem cells was positive for CD44 and CD29， while the expression of CD34 and CD45 was negative， which is in line with the characteristics of bone marrow mesenchymal stem cells. ②After 3， 6， 9， 12 d of osteogenic induction culture， the alkaline phosphatase activity of MSCs cells increased compared with the previous time point， and it was time-dependent，the difference was statistically significant （P<0.05）. After 3，6，9，12 d of osteogenic induction of MSCs， the expression levels of Runx2 and Osterix gradually increased. ③The AKP activity of miR-449a group and miR-449b group was lower than the control group， while the anti-miR-449 group increased AKP activity， the difference was statistically significant （P<0.05）; miR-449a group and miR-449b group Runx2， Osterix expression was down-regulated compared with the control group，the difference was statistically significant （P<0.05）. Conclusion ?This experiment successfully isolated and cultured bone marrow MSCs， which has the ability to differentiate into bone in vitro， and miR-449 can inhibit the differentiation of bone marrow MSCs into osteogenesis.

3討論

BMP/ Smad信號(hào)通路調(diào)控成骨化和成軟骨化，是骨形成中最重要的一條信號(hào)通路。其中Smad4（mothers against decapentaplegic homolog 4）是BMP/Smad骨形成信號(hào)通路的中心介導(dǎo)者。Smadl/5/8 磷酸化后與Smad4形成復(fù)合物后進(jìn)入到細(xì)胞核內(nèi)調(diào)控成骨分化關(guān)鍵基因Runx2、Osterix等的轉(zhuǎn)錄，從而實(shí)現(xiàn)對(duì)成骨分化和骨成熟的調(diào)控[4，5]。本研究通過Targetscan等在線軟件預(yù)測(cè)miR-449靶基因，發(fā)現(xiàn)Smad4是miR-449a/b潛在的靶基因。因此推測(cè)miR-449可能通過靶向調(diào)節(jié)Smad4表達(dá)來調(diào)控成骨分化關(guān)鍵基因Runx2、Osterix等的轉(zhuǎn)錄，從而影響MSCs成骨分化。

miRNAs作為一種新型的基因轉(zhuǎn)錄后調(diào)控方式，與組織器官發(fā)育、細(xì)胞生長、分化、凋亡、細(xì)胞運(yùn)動(dòng)、新陳代謝和疾病發(fā)生等生命活動(dòng)密切相關(guān)[6]。越來越多的研究表明miRNA 在干細(xì)胞的自我更新和多向分化過程中發(fā)揮重要的調(diào)控作用[7]。已有研究[9]發(fā)現(xiàn)miRNAs在調(diào)控MSCs或前體細(xì)胞成骨、成軟骨及成脂分化過程中也發(fā)揮重要生物學(xué)功能。miR-449能抑制MSCs向成骨分化，但具體的分子機(jī)制或靶基因調(diào)控作用尚不明確。

本研究通過分離、培養(yǎng)骨髓MSCs，鑒定其成骨分化能力，在骨髓MSCs轉(zhuǎn)染經(jīng)特殊化學(xué)修飾的miR-449 a/bmimics或inhibitors，通過qRT-PCR檢測(cè)過表達(dá)miR-449a/b對(duì)成骨特異性基因Runx2、Osterix表達(dá)的影響。結(jié)果顯示過表達(dá)miR-449a/b后Runx2及Osterix表達(dá)較對(duì)照細(xì)胞下調(diào)，表明miR-449能通過下調(diào)Runx2及Osterix表達(dá)抑制骨髓MSCs細(xì)胞向成骨分化。從而可能在骨質(zhì)疏松、骨折愈合與骨不連的發(fā)生發(fā)展中發(fā)揮重要作用，因此，以miR-449為靶點(diǎn)開發(fā)的藥物有望用于骨質(zhì)疏松等多種骨代謝疾病的治療或者逆轉(zhuǎn)骨質(zhì)疏松患者自體分離的間充質(zhì)干細(xì)胞的成骨能力表現(xiàn)出明顯的缺陷，從而將自體干細(xì)胞治療應(yīng)用于骨質(zhì)疏松條件下的骨修復(fù)成為現(xiàn)實(shí)。

綜上所述，miR-449能抑制骨髓間充質(zhì)干細(xì)胞向成骨分化，未來可以miR-449為靶點(diǎn)開發(fā)相關(guān)藥物治療多種骨代謝疾病。

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收稿日期：2019-12-04;修回日期：2019-12-23

編輯/成森