劉真 張浩然
【摘要】 目的:研究生長分化因子-5(GDF-5)對間充質(zhì)干細(xì)胞向肌腱細(xì)胞分化及遷移潛能的影響。方法:用GDF-5對骨髓間充質(zhì)干細(xì)胞(MSCs)進(jìn)行14 d誘導(dǎo)分化,取樣對細(xì)胞外基質(zhì)合成及向肌腱細(xì)胞分化相關(guān)基因表達(dá)進(jìn)行測定分析;通過體外三維細(xì)胞遷移模型,評價(jià)GDF-5對間充質(zhì)干細(xì)胞遷移能力的作用及影響。結(jié)果:20、100、500 ng/mL GDF-5誘導(dǎo)4 d后的細(xì)胞數(shù)均高于0 ng/mL GDF-5誘導(dǎo)(P<0.05),且隨著GDF-5濃度增高增殖細(xì)胞數(shù)呈現(xiàn)增加的趨勢。100 ng/mL GDF-5誘導(dǎo)4、12 d的細(xì)胞數(shù)均高于0 ng/mL GDF-5誘導(dǎo)(P<0.05),隨著培養(yǎng)時(shí)間的延長,差異更加明顯。100 ng/mL GDF-5誘導(dǎo)分化骨髓干細(xì)胞12 d后,Tenascin-C與Ⅰ型膠原蛋白的基因表達(dá)量均高于0 ng/mL GDF-5誘導(dǎo)(P<0.05)。100 ng/mL GDF-5誘導(dǎo)的細(xì)胞遷移能力大于0 ng/mL GDF-5誘導(dǎo)(P<0.05)。結(jié)論:GDF-5可以促進(jìn)間充質(zhì)干細(xì)胞向肌腱細(xì)胞分化,并誘導(dǎo)活化間充質(zhì)干細(xì)胞的遷移,該機(jī)制可能在肌腱損傷的修復(fù)重建中具有重要意義。將GDF-5與間充質(zhì)干細(xì)胞進(jìn)行聯(lián)合治療可為肌腱損傷的修復(fù)提供新的治療方法。
【關(guān)鍵詞】 骨髓間充質(zhì)干細(xì)胞 細(xì)胞遷移 生長分化因子-5
Effects of GDF-5 on Differentiation into Tendon Cells and Migration of Bone Marrow Derived Mesenchymal Stem Cells/LIU Zhen, ZHANG Haoran. //Medical Innovation of China, 2021, 18(20): 0-032
[Abstract] Objective: To investigate the effects of the growth and differentiation factor-5 (GDF-5) on differentiation into tendon cells and migration of bone marrow derived mesenchymal stem cells (BMSCs). Method: BMSCs were treated with GDF-5 for 14 d, and the samples were collected. The extracellular matrix synthesis and the expression of genes related to differentiation into tendon cells were analyzed. The effects of GDF-5 on the migration ability of mesenchymal stem cells were evaluated by three-dimensional cell migration model in vitro. Result: The number of cells induced by 20, 100 and 500 ng/mL GDF-5 for 4 d were higher than that induced by 0 ng/mL GDF-5 (P<0.05), and the number of proliferating cells increased with the increase of GDF-5
concentration. The number of cells induced by 100 ng/mL GDF-5 on day 4 and 12 were higher than that induced by 0 ng/mL GDF-5 (P<0.05), and the difference was more obvious with the extension of culture time. After inducing bone marrow stem cells with 100 ng/mL GDF-5 for 12 d, the gene expression levels of Tensascin-C and Ⅰ type collagen were both higher than those of 0 ng/mL GDF-5 induction (P<0.05). The cell migration ability induced by 100 ng/mL GDF-5 was higher than that induced by 0 ng/mL GDF-5 (P<0.05). Conclusion: GDF-5 can promote differentiation into tendon cells of mesenchymal stem cells and induce migration of activated mesenchymal stem cells, which may play an important role in the repair and reconstruction of tendon injury. Combined treatment of GDF-5 with mesenchymal stem cells may provide a new therapeutic method for tendon injury repair.
[Key words] BMSCs Cell migration GDF-5
First-author’s address: Shanghai Xuhui Central Hospital, Shanghai 200031, China
doi:10.3969/j.issn.1674-4985.2021.20.007
肌腱的損傷斷裂是骨科領(lǐng)域的重要疾病,尤其在運(yùn)動損傷中更為常見[1-2]。由于肌腱組織難于修復(fù),且修復(fù)后組織結(jié)構(gòu)較弱,極易發(fā)生再次斷裂和粘連等并發(fā)癥[3]。因此,利用組織工程及再生醫(yī)療技術(shù)提高肌腱的愈合近年來成為骨科領(lǐng)域關(guān)注的焦點(diǎn)[4-5],尤其是相關(guān)生長因子結(jié)合干細(xì)胞治療更是有望成為修復(fù)肌腱損傷的新手段[6-7]。
骨髓干細(xì)胞具有肌肉骨組織多向分化潛能,如在體外適當(dāng)誘導(dǎo)下可以分化為成骨細(xì)胞、軟骨細(xì)胞、脂肪細(xì)胞、肌細(xì)胞等[8-11]。間充質(zhì)干細(xì)胞已被應(yīng)用于軟組織損傷的修復(fù)。然而,骨髓間充質(zhì)干細(xì)胞向肌腱細(xì)胞分化的相關(guān)研究報(bào)道極少。
生長分化因子-5(growth differentiation factor-5,GDF-5)又被稱為軟骨來源成形蛋白-1或BMP-14,在骨關(guān)節(jié)形成、軟骨內(nèi)成骨和肌腱韌帶損傷修復(fù)等生理過程中其重要作用[12-15]。有動物實(shí)驗(yàn)結(jié)果表明利用GDF-5基因治療可以增強(qiáng)跟腱修復(fù)強(qiáng)度[16]。本研究將通過體外實(shí)驗(yàn)考察GDF-5對骨髓干細(xì)胞向肌腱細(xì)胞分化的誘導(dǎo)作用及遷移能力的影響,為闡明GDF-5促進(jìn)肌腱修復(fù)的機(jī)制提供理論基礎(chǔ),為肌腱修復(fù)提供新的治療手段,現(xiàn)報(bào)道如下。
1 材料與方法
1.1 實(shí)驗(yàn)動物和試劑 選用6周齡SD雄性大鼠,由哈爾濱醫(yī)科大學(xué)實(shí)驗(yàn)動物中心提供。DMEM培養(yǎng)液、胎牛血清、Ⅰ型膠原酶和胰蛋白酶購自Gibico公司。GDF-5購自Peprotech公司。MTT購自 Sigma Chemical公司。QuantiTect SYBR Green PCR試劑盒購自Qiagen公司。
1.2 方法
1.2.1 骨髓干細(xì)胞的分離與原代培養(yǎng) 取SD大鼠雙側(cè)股骨、脛骨,剪去兩側(cè)骺端,用10 mL注射器針頭抽取適量含10% FBS的完全培養(yǎng)基沖洗骨髓腔,制成單細(xì)胞懸液。細(xì)胞懸液在1 500 r/min離心5 min后,去上清,用含10% FBS的DMEM培養(yǎng)液重懸后轉(zhuǎn)移至培養(yǎng)瓶中常規(guī)傳代培養(yǎng)。
1.2.2 細(xì)胞增殖測定 將骨髓干細(xì)胞以5.0×104個(gè)/孔的密度種植于6孔板中,分別用含有0、20、100、500 ng/mL GDF-5的培養(yǎng)基培養(yǎng)4 d后,每孔加入30 μL MTT,孵箱內(nèi)作用3 h后加棄去上清。加入二甲基亞砜。20 min后用酶標(biāo)儀測定490 nm波長吸光度,與細(xì)胞數(shù)標(biāo)準(zhǔn)曲線的吸光度值相對比計(jì)算出細(xì)胞數(shù)。
1.2.3 向肌腱細(xì)胞分化基因表達(dá)測定 將骨髓干細(xì)胞以5.0×104個(gè)/孔的密度種植于6孔板中,分別用含有100 ng/mL GDF-5和不含有GDF-5(0 ng/mL)的培養(yǎng)基培養(yǎng)12 d后,提取細(xì)胞總RNA,通過逆轉(zhuǎn)錄反應(yīng)(退火,70 ℃,10 min;cDNA合成,42 ℃,60 min;熱滅活,95 ℃,5 min)制備cDNA,于-70 ℃保存。根據(jù)QuantiTect SYBR Green PCR試劑盒操作手冊進(jìn)行定量PCR測定,應(yīng)用25 μL SYBR Green反應(yīng)體系,分別加入300 nmol上游和下游引物,具體如下:Tenomodulin上游引物 5’-GGA CTT TGA GGA GGA TGG-3’,下游引物5’-CGC TTG CTT GTC TGG TGC-3’;Tenascin-C上游引物5’-GCT ACT CCA GAC GGT TTC-3’,下游引物5’-TTC CAC GGC TTA TTC CAT-3’;ColⅠ (α1) typeⅠ上游引物5’-AGG CTT TGA TGG ACG CAA TG-3’,下游引物5’-GCG GCT CCA GGA AGA CC-3’。加入3 μL cDNA,充分混合后離心,用PCR儀進(jìn)行測定分析(共40循環(huán):變性溫度為94 ℃,30 s;退火溫度為55 ℃,30 s;擴(kuò)增溫度為72 ℃,30 s)。
1.2.4 三維細(xì)胞誘導(dǎo)遷移實(shí)驗(yàn) 用類組織缺損體外細(xì)胞遷移評價(jià)模型對干細(xì)胞在GDF-5的誘導(dǎo)下遷移能力進(jìn)行評價(jià)。將含有100 ng/mL GDF-5和不含有GDF-5(0 ng/mL)的改性天然水凝膠羥苯基丙酸/明膠(Gtn-HPA)水凝膠灌注于模具中固化得到直徑8.66 mm、厚約4 mm的核,周圍灌注Ⅰ型膠原和骨髓干細(xì)胞,這樣就構(gòu)建了一個(gè)類似于內(nèi)部為水凝膠填充的缺損、周圍有類組織包繞的缺損模型,分別在第4、8、12天通過倒置顯微鏡觀察干細(xì)胞向水凝膠內(nèi)遷移的數(shù)量和距離,并作定量分析。
1.3 統(tǒng)計(jì)學(xué)處理 采用SPSS 19.0軟件對所得數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析,計(jì)量資料用(x±s)表示,比較采用單因素方差分析,以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
2 結(jié)果
2.1 GDF-5可促進(jìn)骨髓干細(xì)胞增殖 20、100、500 ng/mL GDF-5誘導(dǎo)4 d后的細(xì)胞數(shù)分別為(13.1±0.5)、(13.6±0.2)、(13.8±0.1)×104個(gè)/孔,均顯高于0 ng/mL GDF-5誘導(dǎo)的(10.2±0.3)×104個(gè)/孔(P<0.05),且隨著GDF-5濃度增高增殖細(xì)胞數(shù)呈現(xiàn)增加的趨勢。100 ng/mL GDF-5誘導(dǎo)4、12 d的細(xì)胞數(shù)分別為(13.6±0.2)、(33.4±2.3)×104個(gè)/孔,均高于0 ng/mL GDF-5誘導(dǎo)的(10.2±0.3)、(22.2±1.5)×104個(gè)/孔(P<0.05),隨著培養(yǎng)時(shí)間的延長,差異更加明顯。見圖1。
2.2 骨髓干細(xì)胞特異性向肌腱細(xì)胞分化基因的表
達(dá) 100 ng/mL GDF-5誘導(dǎo)分化骨髓干細(xì)胞12 d后,測得骨髓干細(xì)胞的Tenomodulin基因表達(dá)未見顯著性增加(P>0.05),而Tenascin-C的表達(dá)量的差異倍數(shù)為(2.2±0.2)高于0 ng/mL GDF-5誘導(dǎo)的(1.30±0.05)(P<0.05),Ⅰ型膠原蛋白的基因表達(dá)量的差異倍數(shù)為(4.3±0.4),高于0 ng/mL GDF-5誘導(dǎo)的(0.90±0.04)(P<0.05)。見圖2。
2.3 骨髓干細(xì)胞的細(xì)胞遷移 類組織缺損體外細(xì)胞遷移評價(jià)模型對干細(xì)胞在GDF-5的誘導(dǎo)下遷移能力進(jìn)行了評價(jià),見圖3A。100 ng/mL GDF-5誘導(dǎo)4、8、12 d后每厘米遷移界面細(xì)胞遷移數(shù)分別為14.6(3.6,58.6)、256.6(168.4,291.3)、344.8(256.1,383.4)個(gè)/cm,而0 ng/mL GDF-5誘導(dǎo)下分別為0(0,0)、7.4(0,36.7)、124.9(88.4,161.7)個(gè)/cm,100 ng/mL GDF-5誘導(dǎo)8、12 d的每厘米遷移界面細(xì)胞遷移數(shù)均大于0 ng/mL GDF-5(P<0.05)。100 ng/mL GDF-5誘導(dǎo)12 d,骨髓干細(xì)胞遷移能力大于0 ng/mL GDF-5。見圖3B。
3 討論
肌腱缺損的愈合過程包括含有肌腱細(xì)胞在內(nèi)的細(xì)胞成分在損傷局部分裂、增殖、分化、細(xì)胞外基質(zhì)的沉積與重建等過程,相關(guān)細(xì)胞因子在這一修復(fù)過程中也起著重要的調(diào)控作用[17]。由于成熟肌腱細(xì)胞進(jìn)一步分裂、增殖潛力有限,因此,本研究通過考察GDF-5對骨髓干細(xì)胞向肌腱細(xì)胞分化的誘導(dǎo)作用及遷移能力的影響,為二者聯(lián)合應(yīng)用促進(jìn)肌腱愈合的可能性提供理論依據(jù)。
骨髓干細(xì)胞具有多向分化潛能,易于提取和分離,不僅在硬組織領(lǐng)域已經(jīng)得到一定的應(yīng)用,在軟組織修復(fù)方面,也被越來越多的證實(shí)有著良好的應(yīng)用前景。骨髓干細(xì)胞在體外適當(dāng)誘導(dǎo)條件下可以分化為成骨細(xì)胞,軟骨細(xì)胞,脂肪細(xì)胞,肌細(xì)胞等,然而,骨髓干細(xì)胞向肌腱細(xì)胞誘導(dǎo)分化的相關(guān)報(bào)道極少。
GDF-5又被稱為軟骨來源成形蛋白-1或BMP-14,越來越多的證據(jù)表明其在肌腱韌帶的發(fā)育和損傷修復(fù)過程中起重要的調(diào)節(jié)作用。GDF-5基因缺陷會導(dǎo)致小鼠肌腱愈合障礙[18];還有動物實(shí)驗(yàn)結(jié)果表明GDF-5修飾的縫線可以增加修復(fù)后肌腱的厚度[19]。此外,用負(fù)載GDF-5的膠原海綿橋接修復(fù)跟腱缺損,可以顯著增加修復(fù)后跟腱的強(qiáng)度[20]。上述結(jié)果都表明了GDF-5可以促進(jìn)肌腱的重建與修復(fù),但是目前為止,其在體外對干細(xì)胞的作用的相關(guān)研究尚少見報(bào)道。
本研究考察了不同濃度GDF-5對骨髓干細(xì)胞增殖的影響,結(jié)果顯示,各濃度GDF-5作用下細(xì)胞增殖數(shù)均明顯高于0 ng/mL GDF-5(P<0.05),且隨著GDF-5濃度增高增殖細(xì)胞數(shù)呈現(xiàn)增加的趨勢,100 ng/mL GDF-5誘導(dǎo)4、12 d的細(xì)胞數(shù)均高于0 ng/mL GDF-5誘導(dǎo)(P<0.05),隨著培養(yǎng)時(shí)間的延長,差異更加明顯。因此,GDF-5可以促進(jìn)骨髓干細(xì)胞的增殖,其作用在500 ng/mL范圍內(nèi)隨著濃度的增高而增強(qiáng)。結(jié)果顯示,100 ng/mL GDF-5誘導(dǎo)12 d后特異性向肌腱細(xì)胞分化標(biāo)志物Tenascin-C、肌腱細(xì)胞外主要基質(zhì)Ⅰ型膠原蛋白的基因表達(dá)均顯著提高(P<0.05)。說明GDF-5可以促進(jìn)骨髓干細(xì)胞向肌腱細(xì)胞分化和細(xì)胞外基質(zhì)的合成,從而增加肌腱修復(fù)缺損過程中肌腱細(xì)胞表型的維持、干細(xì)胞向肌腱細(xì)胞分化和基質(zhì)沉積,進(jìn)而提高肌腱修復(fù)強(qiáng)度,這與文獻(xiàn)[21-22]在脂肪干細(xì)胞上取得的研究結(jié)果相似。本研究用類組織缺損體外細(xì)胞遷移評價(jià)模型對干細(xì)胞在GDF-5的誘導(dǎo)下遷移能力進(jìn)行了評價(jià),結(jié)果顯示,100 ng/mL GDF-5誘導(dǎo)的細(xì)胞遷移能力大于0 ng/mL GDF-5誘導(dǎo),所以,GDF-5誘導(dǎo)可促進(jìn)骨髓干細(xì)胞的細(xì)胞遷移,該機(jī)制在肌腱修復(fù)與重建中可能起著重要作用。Date等[23]的研究也表明,GDF-5可以促進(jìn)肌腱纖維母細(xì)胞的遷移,且這種遷移為整合素-α2介導(dǎo)。因此,GDF-5可能通過細(xì)胞誘導(dǎo)遷移作用[24],使肌腱損傷周邊的內(nèi)源性干細(xì)胞遷移至缺損區(qū),參與修復(fù)重建過程。
本實(shí)驗(yàn)結(jié)果提示,如果將GDF-5與骨髓干細(xì)胞聯(lián)合應(yīng)用修復(fù)肌腱損傷,一方面GDF-5可以促進(jìn)骨髓干細(xì)胞的增殖和向肌腱細(xì)胞分化,還可以誘導(dǎo)內(nèi)源性干細(xì)胞遷移入肌腱損傷局部參與修復(fù)重建過程,這為肌腱的損傷修復(fù)細(xì)胞治療提供了新的治療思路。
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(收稿日期:2021-06-01) (本文編輯:田婧)