董 晶 聶李平 周 宇
人血小板裂解液促進(jìn)人羊膜來源間充質(zhì)干細(xì)胞的成骨分化
董 晶 聶李平 周 宇
目的:研究人血小板裂解液(HPL)對(duì)羊膜來源間充質(zhì)干細(xì)胞(AMMSCs)成骨分化的作用。方法:分別以含7% HPL(HPL組)和10%胎牛血清(FBS,F(xiàn)BS組)的LG-DMEM培養(yǎng)介質(zhì)擴(kuò)增AMMSCs,比較兩組擴(kuò)增AMMSCs效率及其免疫表型SSEA-3和SSEA-4的表達(dá)差異。使用MSCs成骨分化培養(yǎng)液誘導(dǎo)AMMSCs成骨,對(duì)比觀察兩組鈣鹽沉積量、鈣化結(jié)節(jié)、堿性磷酸酶(ALP)活性;提取兩組成骨誘導(dǎo)后AMMSCs總RNA,采用RT-PCR檢測成骨分化調(diào)節(jié)因子RUNX-2和ALP mRNA相對(duì)表達(dá)量。結(jié)果:HPL組擴(kuò)增速度快于FBS組;AMMSCs的 SSEA-3和SSEA-4表達(dá)較FBS組明顯下調(diào)(P<0.05)。HPL組鈣鹽沉積量、鈣化結(jié)節(jié)數(shù)量、ALP活性以及RUNX-2和ALP mRNA表達(dá)量均明顯高于FBS組(P均<0.05)。結(jié)論:含HPL培養(yǎng)介質(zhì)可促進(jìn)AMMSCs成骨分化。
人血小板裂解液;間充質(zhì)干細(xì)胞;羊膜來源;成骨分化
研究表明,羊膜來源間充質(zhì)干細(xì)胞(Amniotic Membrane-derived Mesenchymal Stem Cells, AMMSCs)的免疫表型與骨髓來源間充質(zhì)干細(xì)胞(Bone Marrow-derived Mesenchymal Stem Cells, BMMSCs)相似,具有多向分化能力,并且比BMMSCs有更強(qiáng)的擴(kuò)增能力[1,2]。羊膜來源廣泛,受社會(huì)倫理學(xué)限制少,是MSCs來源的重要研究目標(biāo)。血小板富含的各種生長因子能促進(jìn)MSCs增殖,加速種植體周圍骨再生和修補(bǔ)骨缺損[3]。本實(shí)驗(yàn)觀察含人血小板裂解液(Human Platelet Lysate, HPL)培養(yǎng)液擴(kuò)增AMMSCs并誘導(dǎo)其成骨分化,探討HPL對(duì)AMMSCs成骨分化的影響,分析該方式的優(yōu)勢,為利用AMMSCs進(jìn)行骨缺損治療提供依據(jù)。
1.1 主要試劑和儀器
流式細(xì)胞儀(XL-4,Beckman,美國),全自動(dòng)生化儀(AU-5400,Olympus,日本),基因擴(kuò)增儀(ABI-7500,Gene,美國),胎牛血清(FBS,Gibco,美國,635742),LG-DMEM (Hyclone,美國,NXH0681),0.25% Trypsin (Hyclone,美國,NWD0400)。堿性磷酸酶(ALP)活性檢測試劑盒(Beckman,美國,AUZ1780),Ca2+濃度檢測試劑盒(Beckman,美國,AUX2129),細(xì)胞裂解液Triton X-100 (Roche,瑞士) ,胚胎干細(xì)胞標(biāo)志物鼠抗人單克隆抗體SSEA-4-PE、 SSEA-3-Alexa Fluor 488及同型對(duì)照均為美國BioLegend公司產(chǎn)品,MSCs成骨分化誘導(dǎo)培養(yǎng)液、茜素紅S均購自美國Cyagen公司。
1.2 AMMSCs的分離、擴(kuò)增和成骨誘導(dǎo)分化
1.2.1 細(xì)胞培養(yǎng)液的制備及AMMSCs分離與擴(kuò)增: 參照作者前期方法[2],制備含F(xiàn)BS或HPL的細(xì)胞培養(yǎng)液,根據(jù)文獻(xiàn)[1]通過兩步酶消化法分離人羊膜組織收獲AMMSCs,將AMMSCs分別加入含F(xiàn)BS(FBS組)或HPL(HPL組)細(xì)胞培養(yǎng)液中并接種于培養(yǎng)皿,在37℃、5% CO2培養(yǎng)箱中培養(yǎng),每3天換液1次。當(dāng)細(xì)胞生長至80%-90%融合時(shí),以0.25% Trypsin消化傳代。
1.2.2 AMMSCs的成骨誘導(dǎo)分化:當(dāng)?shù)?代AMMSCs生長至80%-90%融合時(shí),以0.25% Trypsin消化,中和胰酶后150g離心10min,按3 × 103/cm2接種于預(yù)先以明膠包被的6孔板,分別加入含F(xiàn)BS或HPL的培養(yǎng)液至2ml。置于37℃,5% CO2培養(yǎng)箱中培養(yǎng)24h后,吸出培養(yǎng)液。再各加入2ml MSCs成骨分化培養(yǎng)液培養(yǎng)。每3天換液一次,共14天。
1.3 AMMSCs的擴(kuò)增速度和免疫表型鑒定
從原代培養(yǎng)48h開始觀察,比較兩組培養(yǎng)達(dá)到80%-90%融合時(shí)所需的時(shí)間。待第3代AMMSCs達(dá)80%-90%融合時(shí),制成1.0×109/ L的細(xì)胞懸液,分別加入熒光標(biāo)記抗體SSEA-4-PE、SSEA-3-Alexa Fluor 488及其陰性對(duì)照單抗,室溫反應(yīng)30min,再用PBS洗滌并重懸細(xì)胞。用流式細(xì)胞儀檢測。
1.4 成骨指標(biāo)的檢測
1.4.1 鈣鹽沉積量測定:成骨誘導(dǎo)7天、14天后PBS洗滌培養(yǎng)板內(nèi)細(xì)胞層,加入1mol/L HCl 2ml,充分振蕩后密封置于4℃冰箱保存過夜,使沉積在胞外的鈣基質(zhì)充分溶解,24h后以1 500g離心10min,用Ca2+試劑盒在自動(dòng)生化儀上測定上清液中的Ca2+濃度。
1.4.2 鈣化結(jié)節(jié)染色:成骨誘導(dǎo)培養(yǎng)14天后PBS洗滌培養(yǎng)板內(nèi)細(xì)胞層,4%甲醛固定30min,再次使用PBS洗滌細(xì)胞2次后,0.1%茜素紅S染色3-5min,去離子水沖洗,鈣化結(jié)節(jié)被染成紅色。HPL組和FBS組各取3個(gè)培養(yǎng)孔,顯微鏡下每孔隨機(jī)選3個(gè)視野(×100),計(jì)數(shù)鈣結(jié)節(jié)數(shù)量,取9個(gè)視野均數(shù)進(jìn)行統(tǒng)計(jì)學(xué)分析。
1.4.3 ALP活性測定:成骨誘導(dǎo)7天、14天PBS洗滌培養(yǎng)板內(nèi)細(xì)胞層后,每孔加入3ml 細(xì)胞裂解液(1%Triton X-100)置于4℃冰箱內(nèi)保存,12h后吹打使細(xì)胞充分破碎,1 500g離心5min,采用全自動(dòng)生化儀測定上清液中ALP活性。
1.4.4 成骨相關(guān)基因RUNX-2及ALP mRNA檢測:成骨誘導(dǎo)第7天、14天按常規(guī)方法提取AMMSCs總RNA,以1.0%瓊脂糖凝膠電泳檢測無降解后,取1μg總RNA反轉(zhuǎn)錄為cDNA作為模板,采用反轉(zhuǎn)錄聚合酶鏈反應(yīng)檢測RUNX-2 mRNA和ALP mRNA表達(dá),擴(kuò)增體系及反應(yīng)條件均參照說明書設(shè)定。β-actin為內(nèi)參,所用引物序列如表1。以目的基因與β-actin mRNA比值表示目的基因表達(dá)量。
表1 RT-PCR的引物序列
1.5 統(tǒng)計(jì)學(xué)處理
2.1 AMMSCs的生長速度和形態(tài)
AMMSCs原代培養(yǎng)48h后,可觀察到貼壁生長的長梭形成纖維細(xì)胞樣細(xì)胞小克隆形成,HPL組7-10天即可達(dá)到80%-90%融合,F(xiàn)BS組則需10-14天。傳代后細(xì)胞呈渦旋狀或平行生長(圖1)。
2.2 兩組AMMSCs免疫表型陽性率
流式細(xì)胞儀檢測結(jié)果顯示,部分AMMSCs表達(dá)胚胎干細(xì)胞標(biāo)志SSEA-4和SSEA-3,HPL組AMMSCs的SSEA-4和SSEA-3陽性率顯著低于FBS組(t值分別為3.843和4.608,P均<0.05),見圖2和表2。
注:A,F(xiàn)BS組;B,HPL組圖2 FBS組和HPL組第3代AMMSCs的SSEA-3和SSEA-4表達(dá)流式圖
表2 兩組AMMSCs的SSEA-3和SSEA-4陽性率均=5)
注:與FBS組比較,1)P<0.05
2.3 兩組鈣鹽沉積量
AMMSCs成骨分化后第7天和第14天,HPL組的Ca2+總量均高于FBS組(表3)。差異有統(tǒng)計(jì)學(xué)意義(t值分別為9.439和17.750,P均<0.05)。
表3 兩組Ca2+總量均=5)
注:與FBS組比較,1)P<0.05
2.4 兩組鈣化結(jié)節(jié)形成及其數(shù)量
HPL組AMMSCs成骨誘導(dǎo)培養(yǎng)2天后,細(xì)胞開始變短,外形由長梭形向多邊形轉(zhuǎn)變,培養(yǎng)7 天后細(xì)胞基質(zhì)中即有鈣沉積,培養(yǎng)14天時(shí)形成鈣化結(jié)節(jié)。茜素紅S染色顯示HPL組鈣化結(jié)節(jié)數(shù)量多于FBS組,且大而密集(圖3)。計(jì)數(shù)分析結(jié)果表明兩組差異有統(tǒng)計(jì)學(xué)意義( 22.90±6.20 vs 10.80±3.30,t=12.389,P<0.05)。
2.5 兩組ALP活性
AMMSCs成骨分化后第7天和第14天,HPL組ALP活性均明顯高于FBS組(表4),差異有統(tǒng)計(jì)學(xué)意義(t值分別為13.035和11.231,P均<0.05)。
表4 兩組ALP活性均=5)
注:與FBS組比較,1)P<0.05
2.6 兩組成骨相關(guān)基因表達(dá)
PCR結(jié)果顯示,成骨誘導(dǎo)第7天和第14天,HPL組的成骨分化特異性基因RUNX-2 mRNA和ALP mRNA的相對(duì)表達(dá)量均高于FBS組,差異有統(tǒng)計(jì)學(xué)意義(圖4)。
[本文圖1、圖3見封3]
MSCs具有多向分化潛能、低免疫原性和修復(fù)作用,這些特點(diǎn)使其在再生醫(yī)學(xué)中有良好利用價(jià)值[4,5]。胎盤組織富含干細(xì)胞[6],足月羊膜是MSCs的高通量來源[1],對(duì)其研究不涉及倫理道德問題。HPL富含促進(jìn)MSCs增殖的血小板衍生生長因子(Platelet-derived Growth Factor,PDGF)、成纖維細(xì)胞生長因子(Fibroblast Growth Factor,bFGF)、轉(zhuǎn)化生長因子-β(Transforming Growth Factor-β,TGF-β)等生長因子[7],成為擴(kuò)增臨床應(yīng)用規(guī)模MSCs最合適的人源化FBS替代品[8], 是目前骨組織工程學(xué)領(lǐng)域有關(guān)種子細(xì)胞的成骨分化能力和體外擴(kuò)增能力研究的有益嘗試。本文研究HPL對(duì)AMMSCs的擴(kuò)增和成骨分化能力,為AMMSCs在臨床骨組織工程學(xué)上的應(yīng)用提供實(shí)驗(yàn)室依據(jù)。
SSEA-3和SSEA-4為胚胎干細(xì)胞的標(biāo)志,在分化后的干細(xì)胞中不能被檢測到[9]。本文結(jié)果中,HPL介質(zhì)擴(kuò)增的AMMSCs的SSEA-3和SSEA-4表達(dá)陽性率顯著低于FBS介質(zhì)擴(kuò)增的AMMSCs,說明HPL更能促進(jìn)AMMSCs的分化,而FBS介質(zhì)擴(kuò)增的AMMSCs處于更原始狀態(tài)。與有關(guān)文獻(xiàn)[10]認(rèn)為HPL富含的TGF-β、PDGF能促進(jìn)MSCs生長,同時(shí)也能誘導(dǎo)MSCs分化的結(jié)論一致。而且HPL中所含bFGF這一能支持未分化人胚胎干細(xì)胞生長的物質(zhì)含量相對(duì)很低[11],故而不會(huì)影響HPL對(duì)MSCs的分化[6-8]。
注:與FBS組第7天比較,*P<0.05;與FBS組第14天比較,#P<0.05圖4 兩組成骨相關(guān)基因RUNX-2 mRNA和ALP mRNA相對(duì)表達(dá)量(n均=6)
ALP能使磷酸根離子濃度增加而啟動(dòng)細(xì)胞鈣化,尤其在鈣鹽沉積中起關(guān)鍵作用,其活性升高是成骨分化的特異性標(biāo)志之一。本實(shí)驗(yàn)成骨分化第7天和第14天,HPL組ALP活性和Ca2+總量較FBS組均明顯增高,PCR結(jié)果提示HPL組ALP mRNA明顯增多,表明HPL組的AMMSCs成骨分化程度較高。鈣鹽是骨骼的重要組成部分,茜素紅S染色顯示HPL組的鈣化結(jié)節(jié)較FBS組數(shù)量多且大而密集,進(jìn)一步說明HPL能促進(jìn)成骨過程中鈣鹽的沉積。
成骨分化過程經(jīng)多能干細(xì)胞定向分化為前成骨細(xì)胞、幼稚的成骨細(xì)胞直至成熟的成骨細(xì)胞。RUNX-2是成骨分化過程的重要監(jiān)控因子,從成骨分化的啟動(dòng)階段即開始參與成骨分化的調(diào)控,通過上調(diào)I型膠原蛋白、骨橋蛋白、骨分泌蛋白和骨鈣蛋白等多種骨基質(zhì)蛋白基因,引導(dǎo)多能干細(xì)胞向非成熟成骨細(xì)胞分化,對(duì)骨組織的形成起著重要作用[12]。本文結(jié)果表明,HPL組AMMSCs的RUNX-2 mRNA表達(dá)水平明顯高于FBS組,亦即HPL培養(yǎng)介質(zhì)能更好促進(jìn)AMMSCs的成骨分化。與HPL能增強(qiáng)BMMSCs成骨分化基因、ALP表達(dá)、礦物質(zhì)沉積、增強(qiáng)體外成骨分化和體內(nèi)異位骨形成的研究報(bào)道相符合[13,14],也與醫(yī)學(xué)上應(yīng)用富血小板血漿促進(jìn)骨折愈合的臨床實(shí)踐相符合。
綜上所述,含HPL的培養(yǎng)介質(zhì)能快速擴(kuò)增AMMSCs,并能促進(jìn)AMMSCs成骨分化,為骨組織工程研究及臨床上促進(jìn)骨折愈合提供了一種新的AMMSCs擴(kuò)增體系。
?
本文第一作者簡介:
董 晶(1981-),男,漢族,碩士,主管技師
1 Alviano F, Fossati V, Marchionni C, et al. Term amniotic membrane is a high throughput source for multipotent mesenchymal stem cells with the ability to differentiate into endothelial cells in vitro[J]. BMC Dev Biol, 2007, 7(11): 1-14.
2 董 晶,聶李平,周 宇,等.血小板裂解液快速擴(kuò)增人羊膜來源間充質(zhì)干細(xì)胞[J].微循環(huán)學(xué)雜志,2012, 22 (4): 13-16.
3 Crespo-Diaz R, Behfar A, Butler GW, et al. Platelet lysate consisting of a natural repair proteome supports human mesenchymal stem cell proliferation and chromosomal stability[J]. Cell Transplant,2011,20(6):797-811.
4 Sensebé L, Krampera M, Schrezenmeier H, et al. Mesenchymal stem cells for clinical application[J]. Vox Sang, 2010, 98(2): 93-107.
5 English K, French A, Wood KJ. Mesenchymal stromal cells: facilitators of successful transplantation[J]. Cell Stem Cell, 2010, 7(4): 431-442.
6 Parolini O, Alviano F, Bagnara GP, et al. Concise review: isolation and characterization of cells from human term placenta: outcome of the first international workshop on placenta derived stem cells[J]. Stem Cells, 2008, 26(2): 300-311.
7 Bernardo ME, Avanzini MA, Perotti C, et al. Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute[J]. J Cell Physiol, 2007, 211(1): 121-130.
8 Schallmoser K, Bartmann C, Rohde E, et al. Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells[J]. Transfusion, 2007, 47(8): 1 436-1 446.
9 Takashi M, Hisako M. Glycoconjugate antigens expressed on stem cells and early embryonic cells[J]. Glycoconjugate,2004, 24(1): 41-45.
10 Ng F, Boucher S, Koh S, et al. PDGF, TGF-β, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages[J]. Blood, 2008, 112(2): 295-307.
11 Xu C, Rosler E, Jiang J, et al. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium[J]. Stem Cells, 2005, 23(3): 315-323.
12 Igarashi M,Kamiya N,Hasegawa M,et a1.Inductive effects of dexamethasone on the gene expression of Cbfa1,Osterix and bone matrix proteins during differentiation of cultured primary rat osteoblasts[J]. J Mol Histol, 2004, 35(1): 3-10.
13 Chen B, Sun HH, Wang HG, et al. The effects of human platelet lysate on dental pulp stem cells derived from impacted human third molars[J]. Biomaterials, 2012,33(20): 5 023-5 035.
14 Xia W, Li H, Wang Z, et al. Human platelet lysate supports ex vivo expansion and enhances osteogenic differentiation of human bone marrow-derived mesenchymal stem cells[J]. Cell Biol Int, 2011, 35(6): 639-643.
Human Platelet Lysate Enhances Human Amnion-derived Mesenchymal Stem Cells (AMMSCs) Osteogenic Differentiation
DONG Jing,NIE Li-ping,ZHOU Yu
Peking University Shenzhen Hospital,Shenzhen 518036,China
Objective: To investigate the effect of human platelet lysate (HPL) on amnion-derived mesenchymal stem cells (AMMSCs) osteogenic differentiation.Method: AMMSCs were amplified by LG-DMEM medium supplemented with 7% HPL(HPL group) or 10% fetal bovine serum (FBS,FBS group). The surface molecular markers potential SSEA-3 and SSEA-4 were compared. When AMMSCs were used for osteogenic differentiation, ALP activity and calcium deposition were comparatively observed. Total RNA was isolated and the gene expression of RUNX-2 and ALP were investigated by RT-PCR.Results: HPL-containing medium significantly accelerated MSCs proliferation as compared with FBS containing medium. Expressions of embryonic stem cell markers SSEA-3 and SSEA-4 on AMMSCs were promoted down-regulation by HPL-containing medium than that of FBS-containing medium. HPL-containing medium significantly improved ALP activity and calcium deposition, which also enhanced the mRNA level of RUNX-2 and ALP (P<0.05).Conclusion: HPL-containing medium promotes osteogenic differentiation of AMMSCs.
Human platelet lysate; Mesenchymal stem cells; Amnion; Osteogenic differentiation
北京大學(xué)深圳醫(yī)院,深圳 518036
本文2015-01-10收到,2015-03-18修回
Q343.6
A
1005-1740(2015)02-0022-05