OPN調(diào)控小鼠MSCs向上皮細(xì)胞分化促進(jìn)創(chuàng)面愈合的研究

1、OPN調(diào)控小鼠MSCs向上皮細(xì)胞分化促進(jìn)創(chuàng)面愈合的研究王文平1 李薇2 陳亮 1王珍祥1 李世榮1400038 重慶，第三軍醫(yī)大學(xué)西南醫(yī)院整形外科1；233030 安徽省蚌埠市，蚌埠醫(yī)學(xué)院燒傷整形科2【摘要】目的 實(shí)驗(yàn)以野生型（WT）及OPN-/- (KO)小鼠為對象，探討骨髓間充質(zhì)干細(xì)胞（MSCs）體外、體內(nèi)轉(zhuǎn)分化為上皮細(xì)胞的潛能，并研究OPN在MSCs向上皮細(xì)胞分化過程中的作用。方法 分離培養(yǎng)WT和KO小鼠的MSCs，取其第3代分別在上皮轉(zhuǎn)化培養(yǎng)體系中孵育，通過鏡下觀察、流式細(xì)胞儀、免疫學(xué)、細(xì)胞Western Blot等方法檢測，觀察兩組間的變化與差別。將分離培養(yǎng)的GFP-MSCs注射到兩

2、組小鼠的創(chuàng)面周圍，以注射等量的PBS作為對照組，觀察創(chuàng)面愈合及GFP-MSCs向上皮細(xì)胞分化的情況。結(jié)果 分離培養(yǎng)的MSCs呈梭形或成纖維細(xì)胞樣生長，流式細(xì)胞鑒定MSCs的純度超過91%。對誘導(dǎo)分化第12、17天的兩組細(xì)胞進(jìn)行檢測發(fā)現(xiàn)，WT和KO組的細(xì)胞均表達(dá)CK14，表明MSCs向上皮誘導(dǎo)分化。免疫熒光結(jié)果示W(wǎng)T組CK14的表達(dá)陽性比率為0.936250.0095，而KO組為 0.647450.02313， P0.05，前者明顯高于后者；WB結(jié)果也顯示KO組的CK14表達(dá)量明顯低于WT組。動物實(shí)驗(yàn)示對照組中WT組創(chuàng)面愈合速度快于KO組；注射MSCs的創(chuàng)面愈合速度較注射PBS的快，注射的GFP

3、-MSCs向上皮細(xì)胞分化的能力WT組強(qiáng)于KO組。結(jié)論OPN能夠促進(jìn)創(chuàng)面愈合，MSCs在體內(nèi)、體外可誘導(dǎo)分化為上皮細(xì)胞，敲除OPN能夠減弱MSCs向上皮細(xì)胞分化的能力?！娟P(guān)鍵詞】愈合；間充質(zhì)干細(xì)胞(MSCs)；骨橋蛋白（OPN）；上皮；分化Effects on Osteopontin of Regulating MSCs Differentiated into Epithelial Cells promoting wound healingWang Wenping1,Li Wei2,Chen Liang1,Wang Zhengxiang1,Li Shirong1 (1Department of

4、Medical Center for Cosmetic Plastic Surgery Southwest hospital, the Third Military Medical University,Chongqing,400038；2 Bengbu Medical College, Bengbu City, Anhui Province 233030, China)Abstract Objective Wild type (WT) and OPN-/- (KO) mice were used as experiment objects, to investigate the potent

5、ial of inducing marrow mesenchymal stem cells (MSCs) into epithelial cells in vitro and vivo, and study the role of OPN played in the process. Method WT and KO MSCs were cultured by tissue explants adherent method, took the third-generation respectively to join epithelial transformation culture syst

6、emincubate.Microscopic observation, flow cytometry, cells climbing immunofluorescence staining, western blot methods were used to observe and compare the levels of the two groups. The GFP-MSCs were injected into the wound around of the two groups mice ,equivalent dose of PBS injeted were served as c

7、ontrols , wound healing and GFP-MSCs to differentiate into epithelial cells were observed.Result The isolated and cultured MSCs were seen as a type of fusiform or fibroblast. The purity of separated MSCs, which was identified by flow cytometer, is over 91%. Two groups of induced cells were measured

8、at 12th,17th day showed CK14 was expressed in both WT and KO groups, indicate that MSCs could be induced to differentiate into epithelial cells. Immunofluorescence staining also showed the WT group is obviously higher than KO group (0.936250.0095，vs 0.647450.02313，P0.05) , and western blot also show

9、ed that the expression of CK14 WT group was significantly higher than KO group. Animal experiments also showed wound healing of WT group is significantly faster than the KO group in controls, wound healing of MSCs injected is faster than PBS injected ,the ability of injected GFP-MSCs to differentiat

10、e into epithelial is also stronger than the KO group .Conclusion OPN can promote wound healing ,MSCs can be induced to differentiate into epithelial cells promote wound healing in vitro and vivo, the ability of MSCs differentiate into epithelial cells was weakened by knocking out OPN.Keywords Healin

11、g;Mesenchymal Stem Cells; Osteopontin; Epithelial;Differentiation基金項(xiàng)目：國家自然科學(xué)基金面上項(xiàng)目（81071563，81272102），重慶市應(yīng)用開發(fā)項(xiàng)目（2014yykfA0158）通信作者：李世榮，E-mail: zhengxing 電話:膚的損傷修復(fù)需要上皮細(xì)胞不斷增殖遷移以維持皮膚表面完整性。而間充質(zhì)干細(xì)胞( Mesenchymal Stem Cells，MSCs)具有高度自我更新能力和多向分化潛能，能向多種細(xì)胞及組織分化以修復(fù)缺損，尤其向上皮細(xì)胞的轉(zhuǎn)化已被應(yīng)用于創(chuàng)面組織的修復(fù) ADDIN

12、EN.CITE ADDIN EN.CITE.DATA HYPERLINK l _ENREF_1 o Barry, 2004 #76 1-5。研究證實(shí)，小鼠或人類等不同物種的MSCs均可在一定條件下分化為上皮樣細(xì)胞 ADDIN EN.CITE ADDIN EN.CITE.DATA HYPERLINK l _ENREF_6 o Jiang, 2010 #78 6-9。我們既往研究發(fā)現(xiàn)創(chuàng)面組織內(nèi)MSCs與創(chuàng)面局部Osteopontin（OPN）的高表達(dá)密切相關(guān)，兩者在體內(nèi)體外的定位存在關(guān)聯(lián)性 ADDIN EN.CITE Meng20141101011011017Meng, H.Wang, Z.Wan

13、g, W.Li, W.Wu, Q.Lei, X.Ouyang, X.Liang, Z.Department of Endocrinology, Southwest Hospital of Third Military Medical University, Chongqing, China.Effect of osteopontin in regulating bone marrow mesenchymal stem cell treatment of skin wounds in diabetic miceDiabetes Metab Res RevDiabetes/metabolism r

14、esearch and reviewsDiabetes Metab Res RevDiabetes/metabolism research and reviewsDiabetes Metab Res RevDiabetes/metabolism research and reviews457-663062014/05/162014Sep1520-75522482792810.1002/dmrr.2566Nlmeng HYPERLINK l _ENREF_10 o Meng, 2014 #110 10。OPN是由炎細(xì)胞、上皮細(xì)胞分泌的一種細(xì)胞外基質(zhì)蛋白，具有多種功能，對炎性細(xì)胞、成纖維細(xì)胞、表皮

15、細(xì)胞、內(nèi)皮細(xì)胞等促進(jìn)愈合細(xì)胞的調(diào)控發(fā)揮重要作用 ADDIN EN.CITE Liu20098211828217Liu, Z. J.Zhuge, Y.Velazquez, O. C.The DeWitt Daughtry Family Department of Surgery, Leonard M. Miller School of Medicine, University of MiamiJackson Memorial Medical Center, Miami, FL 33136, USA. ovelazquezTrafficking and differentiation of mes

16、enchymal stem cellsJ Cell BiochemJournal of cellular biochemistryJ Cell BiochemJournal of cellular biochemistryJ Cell BiochemJournal of cellular biochemistry984-9110662009/02/21AnimalsBone Marrow Cells/cytology/physiologyCell Differentiation/*physiologyCell LineageHumansMesenchymal Stem Cell Transpl

17、antationMesenchymal Stromal Cells/cytology/*physiologyMesoderm/cytologySignal Transduction/*physiology2009Apr 150730-23121922987110.1002/jcb.22091Nlmeng HYPERLINK l _ENREF_11 o Liu, 2009 #82 11。OPN調(diào)控創(chuàng)面愈合的多個關(guān)鍵環(huán)節(jié)，但對MSCs向上皮細(xì)胞轉(zhuǎn)化的研究很少，在創(chuàng)面愈合中的作用尚不明確。 本研究分別從小鼠骨髓分離并培養(yǎng)野生型（WT）和OPN-/-（KO）的MSCs，建立MSCs體外上皮細(xì)胞誘導(dǎo)分化

18、系統(tǒng)，誘導(dǎo)其分化為上皮細(xì)胞；以免疫熒光、流式細(xì)胞儀與Western Blot等方法檢測OPN的變化與誘導(dǎo)MSCs分化為上皮細(xì)胞的效率之間的關(guān)系，并通過動物實(shí)驗(yàn)分析研究OPN在MSCs向上皮細(xì)胞轉(zhuǎn)化、促進(jìn)創(chuàng)面愈合過程中的作用，為臨床損傷修復(fù)提供新的理論依據(jù)與治療手段。1.材料與方法1.1主要材料和試劑OPN-/-小鼠(KO)由美國Jackson公司購入, WT-C57BL/6、WT-GFP-C57BL/6小鼠由重慶市大坪醫(yī)院動物實(shí)驗(yàn)中心提供，間充質(zhì)干細(xì)胞完全培養(yǎng)基購買于賽業(yè)（廣州）生物科技有限公司; DMEM/F-12培養(yǎng)基、胎牛血清購自美國GIBC0公司；重組HGF、IGF-I、FGF購買于美

19、國Peprotech公司, CD34-FITC、CD105-PE購買于美國Ebioscience公司; DNA快速提取試劑盒、CK14免疫組化染色試劑盒購自北京中杉金橋生物公司;山羊抗鼠IgG購買于武漢聯(lián)科生物公司等。1.2方法1.2.1骨髓間充質(zhì)干細(xì)胞原代提取培養(yǎng) 取KO及WT新生小鼠（鼠齡1天），75%乙醇溶液浸泡5分鐘放入無菌培養(yǎng)皿內(nèi)，冷無菌PBS溶液沖洗，剪下小鼠四肢，剔出長骨，完全剪碎骨組織，加入BMSCs完全培養(yǎng)液，重懸、混勻后轉(zhuǎn)移至無菌塑料培養(yǎng)瓶內(nèi)培養(yǎng)，于37，5CO2的恒溫培養(yǎng)箱進(jìn)行培養(yǎng)，24h后換液去除組織塊。鏡下見細(xì)胞融合達(dá)80%用0.25%胰酶消化傳代，平均每3-4天傳代

20、一次，實(shí)驗(yàn)采用P3細(xì)胞。1.2.2 BMSCs細(xì)胞純度的流式鑒定收集鏡下達(dá)80%融合的WT及KO兩組P3細(xì)胞，計數(shù)后分裝于EP管內(nèi)，5105cell/管，800rpm離心5min后100ul PBS溶液重懸。設(shè)置分組:空白對照、CD105-PE單染、CD34-FITC單染、CD105-PE及CD34-FITC雙染，按分組分別加入2ul抗體,4孵育1h，加入900ul PBS溶液，800rpm室溫離心5min，棄上清加入PBS 300ul，吹打混勻后轉(zhuǎn)移至流式管內(nèi)，流式細(xì)胞儀檢測CD105及CD34的表達(dá)情況。1.2.3 MSCs體外上皮細(xì)胞誘導(dǎo)分化0.25%胰蛋白酶消化第3代WT和KO MSC

21、s，終止消化，離心，重懸細(xì)胞，調(diào)整細(xì)胞至1106/ml，取1ml加入上皮轉(zhuǎn)化培養(yǎng)體系（F12-DMEM+2%FBS +10ng/ml IGF-I+20ng/ml HGF+10ng/ml b-FGF +100U/ml青霉素+100U/ml鏈霉素），接種于培養(yǎng)瓶，放入37、體積分?jǐn)?shù)5%CO2培養(yǎng)箱中培養(yǎng)，每3d換液1次，每日鏡下觀察細(xì)胞形態(tài)變化至第17天。1.2.4 MSCs體外誘導(dǎo)分化后流式鑒定 收集誘導(dǎo)分化第12天、17天的兩組細(xì)胞，計數(shù)后分裝于EP管，5105cell/管, 4多聚甲醛100ul室溫20min，0.05Triton100ul室溫10min,滴加一抗（CK14）4050l，37

22、60 min，滴加4050l熒光二抗(山羊抗鼠IgG)，3730 min，每兩步間均PBS清洗，800rpm室溫離心5min。最后棄上清加入PBS 300ul，吹打混勻后流式細(xì)胞儀檢測。1.2.5 MSCs體外誘導(dǎo)分化后CK14免疫熒光染色0.25%胰蛋白酶消化第3代WT和KO MSCs，終止消化、離心、重懸、稀釋形成密度為1106/ml 的液體，取100ul置于放有細(xì)胞爬片的12孔板內(nèi)，加入誘導(dǎo)培養(yǎng)體系，17天后取出爬片，溫?zé)酨BS洗5min3次，4%多聚甲醛20min，3%BSA室溫封閉1h，滴加一抗4過夜，滴加熒光二抗37孵育1h,DAPI染核，最后滴加防熒光淬滅封片劑封片，立即在共聚焦

23、顯微鏡下查看。1.2.6 MSCs體外誘導(dǎo)分化后WB檢測CK14表達(dá)情況誘導(dǎo)分化的第17天，根據(jù)蛋白提取試劑盒提取WT組和KO組的細(xì)胞總蛋白, BCA法測蛋白濃度后，8的聚丙烯酰胺凝膠按每孔等量30ug上樣，積層膠80V，分離膠120V電泳，100V轉(zhuǎn)膜90min,轉(zhuǎn)好的PVDF膜5BSA封閉1h，CK14(1:300)、-actin（1:1000）一抗孵育，4振蕩過夜。TBST洗滌后滴加二抗（1:1000），37孵育1h，顯影成像。用Imagej分析軟件掃描特異性條帶凈灰度值后-actin作為內(nèi)參，計算兩者比值分析蛋白相對表達(dá)量。1.2.7 創(chuàng)面模型建立及GFP-MSCs創(chuàng)周注射創(chuàng)面模型建立

24、：將兩組小鼠以1%戊巴比妥150ul腹腔注射麻醉，脫毛膏脫去背部毛發(fā)，勿傷及皮膚，碘伏消毒2次后，打孔器在小鼠背部形成直徑0.5cm深達(dá)筋膜層的創(chuàng)面。GFP-MSCs創(chuàng)周注射：取新生一天的野生型GFP小鼠，MSCs的分離培養(yǎng)方法同前，取培養(yǎng)的第三代GFP-MSCs以PBS重懸形成5105/50ul的細(xì)胞懸液，在距離創(chuàng)緣0.5cm處分別均勻注射于兩組小鼠，同時以等量的PBS注射于兩組小鼠作為對照，觀察創(chuàng)面內(nèi)有無OPN對創(chuàng)面愈合的影響。實(shí)驗(yàn)后1、3、5、7天創(chuàng)面拍照，計算創(chuàng)面愈合百分率，并切取創(chuàng)面組織做組織切片免疫熒光染色檢測MSCs上皮細(xì)胞轉(zhuǎn)化情況。1.2.8 統(tǒng)計學(xué)分析實(shí)驗(yàn)重復(fù)3次以上，數(shù)據(jù)以

25、x表示，使用SPSS13.0統(tǒng)計分析軟件分析，兩組間比較用檢驗(yàn)，以P0.05為差異有統(tǒng)計學(xué)意義。2結(jié)果2.1 原代培養(yǎng)的MSC的形態(tài)學(xué)特征及流式鑒定原代培養(yǎng)的細(xì)胞鏡下見胞體呈多邊形，較小，呈放射狀集落生長；隨著細(xì)胞生長，貼壁細(xì)胞逐漸增大，呈成纖維細(xì)胞樣。觀察發(fā)現(xiàn)WT組的細(xì)胞增殖、生長狀態(tài)優(yōu)于KO組，說明敲除OPN基因?qū)SCs的生長具有一定的影響。MSCs的特征為CD105陽性，而CD34陰性。本實(shí)驗(yàn)兩組MSCs細(xì)胞的CD105陽性而CD34陰性表達(dá)率為：WT組94.04%、KO組91.57% (圖1)，培養(yǎng)的細(xì)胞為高純度MSCs。 A：WT-MSCs；B：KO-MSCs圖1 流式細(xì)胞儀鑒定兩

26、組MSCs2.2 兩組MSCs體外上皮細(xì)胞誘導(dǎo)分化結(jié)果MSCs細(xì)胞貼壁生長。培養(yǎng)第12天部分長梭形細(xì)胞形態(tài)由梭形逐漸變扁變寬（圖2A、C），第17天時細(xì)胞呈現(xiàn)出典型的鋪路石樣改變（圖2B、D），符合上皮分化的特點(diǎn)。A:KO組第12天，細(xì)胞形態(tài)扁寬；B:KO組第17天，細(xì)胞呈鋪路石樣；C:WT組第12天，細(xì)胞形態(tài)扁寬；D:WT組第17天，細(xì)胞呈鋪路石樣； 圖2 MSCs誘導(dǎo)上皮分化后的形態(tài)改變（100）2.3流式鑒定MSCs誘導(dǎo)分化后CK14表達(dá)率細(xì)胞流式鑒定，WT和KO組的細(xì)胞均有上皮細(xì)胞標(biāo)記CK14表達(dá)，表明MSCs向上皮誘導(dǎo)分化成功。第12天時兩組上皮分化率分別為：KO組31.67、WT組

27、37.88；第17天時分別為：KO組63.84、WT組90.40（圖3）。 A:KO-MSCs第12天， B:KO-MSCs第17天 C：WT-MSCs第12天 ，D: WT-MSCs第17天 圖3 MSCs向上皮細(xì)胞分化后流式檢測2.4免疫熒光檢測兩組MSCs誘導(dǎo)分化后CK14表達(dá) 對誘導(dǎo)分化第17天的兩組細(xì)胞進(jìn)行免疫熒光檢測發(fā)現(xiàn)，WT和KO組的細(xì)胞均表達(dá)CK14，表明MSCs向上皮誘導(dǎo)分化。免疫熒光結(jié)果是，WT組CK14的表達(dá)陽性比率為0.936250.0095，而KO組為 0.647450.02313，P0.05，前者明顯高于后者（圖4）,證明了敲除OPN基因后MSCs的上皮分化能力下降

28、。 A:兩組分化細(xì)胞免疫熒光染色；B:免疫熒光染色定量分析；*KO組與WT組比較，P0.05，n=3。圖4 MSCs上皮轉(zhuǎn)化后CK14免疫熒光(400)2.5 WB檢測MSCs體外誘導(dǎo)分化的結(jié)果Western blot結(jié)果顯示，WT組CK14的表達(dá)明顯高于KO組，比值為（0.614550.01534）vs(0.389380.01683)，P0.05，證明WT組的上皮轉(zhuǎn)化明顯高于KO組（圖5）。A:Western blot 檢測（1：KO組；2：WT組）；B:CK14半定量分析；*KO組與WT組比較，P0.05，n=3。 圖5 MSCs上皮轉(zhuǎn)化后WB定量分析 2.6 動物實(shí)驗(yàn)創(chuàng)面愈合率及GFP-

29、MSCs上皮轉(zhuǎn)化檢測成功制作小鼠的圓形創(chuàng)面，實(shí)驗(yàn)當(dāng)日及傷后1、3、5、7天創(chuàng)面拍照，計算創(chuàng)面愈合百分率創(chuàng)面愈合百分率=(初始創(chuàng)面面積觀察日創(chuàng)面面積)初始創(chuàng)面面積100%，結(jié)果以注射PBS的野生型小鼠為對照，注射PBS的敲基因小鼠創(chuàng)面愈合率較前者低，P0.05,表明OPN在創(chuàng)面愈合過程具有重要作用；同樣以注射PBS的野生型小鼠為對照，注射MSCs的野生型小鼠創(chuàng)面愈合率較注射PBS的高，P0.05,表明注射的MSCs能夠加速創(chuàng)面的愈合（圖6、表1）。實(shí)驗(yàn)第3、5、7天，統(tǒng)計完創(chuàng)面大小后將創(chuàng)面組織取下，行組織切片免疫熒光染色檢測GFP-MSCs表達(dá)上皮標(biāo)記CK14的情況，注射入創(chuàng)面內(nèi)的MSCs表達(dá)上

30、皮標(biāo)記物CK14，*KO組與WT組比較，P0.05，說明MSCs在創(chuàng)面內(nèi)分化成為了上皮細(xì)胞參與創(chuàng)面修復(fù)，WT組的分化效率高于KO組（圖7）。圖6 傷后1、3、5、7天創(chuàng)面愈合情況分組 n 1d 3d 5d 7d WT/PBS 5 21.644.90 37.554.97 58.531.59 65.023.11KO/PBS 514.172.45* 26.853.40* 43.663.67* 58.022.56*KO/MSCs 5 20.793.60 44.106.35 58.526.33 69.073.06WT/MSCs 5 39.473.87# 60.172.53# 71.111.87# 80.

31、533.13#表1 四組小鼠創(chuàng)面愈合率（*與WT/PBS組比較，P0.05；#與WT/PBS組比較，P0.05。）A:兩組創(chuàng)面組織第7天免疫熒光染色（200 ）；B:免疫熒光染色定量分析；*KO組與WT組比較，P0.05，n=3。圖7 GFP-MSCs創(chuàng)面注射后免疫熒光染色分析3討論 大面積深度皮膚創(chuàng)傷、自體皮皮源十分缺乏的患者，上皮化不足是創(chuàng)面難以愈合的關(guān)鍵。研究認(rèn)為，以MSCs為種子細(xì)胞，應(yīng)用組織工程皮膚修復(fù)創(chuàng)面可以較好地解決皮源供給以及皮膚結(jié)構(gòu)、功能重建等問題。MSCs經(jīng)過體外擴(kuò)增、純化后仍然保持細(xì)胞的生物學(xué)特性，并能分化為多種類型的細(xì)胞 ADDIN EN.CITE ADDIN EN.C

32、ITE.DATA HYPERLINK l _ENREF_12 o Oswald, 2004 #99 12。在小鼠嵌合體模型中，骨髓MSCs能夠增加創(chuàng)面中表皮與成纖維細(xì)胞數(shù)量；移植到創(chuàng)傷部位的MSCs可以表達(dá)角化細(xì)胞特異性蛋白，形成腺體結(jié)構(gòu)，參與皮膚再生 ADDIN EN.CITE ADDIN EN.CITE.DATA HYPERLINK l _ENREF_13 o Wu, 2007 #84 13。EGF 可誘導(dǎo)MSCs向皮膚組織分化，將兩者復(fù)合移植用于豬創(chuàng)面缺損模型，形成的表皮層厚、角化分層明顯，MSCs細(xì)胞可使經(jīng)久不愈的創(chuàng)面上皮化，達(dá)到愈合重建 ADDIN EN.CITE Badiavas2

33、0039714979717Badiavas, E. V.Falanga, V.Department of Dermatology, Boston University School of Medicine, Boston, MA, USA.Treatment of chronic wounds with bone marrow-derived cellsArch DermatolArchives of dermatologyArch DermatolArchives of dermatologyArch DermatolArchives of dermatology510-613942003/

34、04/23Abdominal WallAdministration, TopicalAgedAged, 80 and overBiopsy*Bone Marrow TransplantationCells, CulturedChronic DiseaseFemaleHumansLegMaleMiddle AgedSkin/pathologySkin Ulcer/pathology/*therapy*Wound Healing2003Apr0003-987X (Print)0003-987x1270709910.1001/archderm.139.4.510Nlmeng HYPERLINK l

35、_ENREF_14 o Badiavas, 2003 #97 14。大量資料說明，MSCs能分化為上皮細(xì)胞。 骨橋蛋白(OPN)是在多種細(xì)胞中廣泛表達(dá)的一種分泌型糖蛋白，參與細(xì)胞的多種生理病理過程。OPN高表達(dá)于創(chuàng)面內(nèi)，趨化MSCs向創(chuàng)面遷移。在創(chuàng)面愈合的關(guān)鍵環(huán)節(jié)中具有多功能調(diào)控的功能，對炎性細(xì)胞、成纖維細(xì)胞、表皮細(xì)胞、內(nèi)皮細(xì)胞等促進(jìn)愈合的功能調(diào)控發(fā)揮重要作用 ADDIN EN.CITE Badiavas20039714979717Badiavas, E. V.Falanga, V.Department of Dermatology, Boston University School of M

36、edicine, Boston, MA, USA.Treatment of chronic wounds with bone marrow-derived cellsArch DermatolArchives of dermatologyArch DermatolArchives of dermatologyArch DermatolArchives of dermatology510-613942003/04/23Abdominal WallAdministration, TopicalAgedAged, 80 and overBiopsy*Bone Marrow Transplantati

37、onCells, CulturedChronic DiseaseFemaleHumansLegMaleMiddle AgedSkin/pathologySkin Ulcer/pathology/*therapy*Wound Healing2003Apr0003-987X (Print)0003-987x1270709910.1001/archderm.139.4.510Nlmeng HYPERLINK l _ENREF_14 o Badiavas, 2003 #97 14。細(xì)胞分泌OPN后，可動員骨髓MSCs增殖分化參與修復(fù)，比如分化為成纖維細(xì)胞。但是目前對此修復(fù)過程MSCs參與過程和機(jī)制的報

38、道較少，難以說明OPN調(diào)控MSCs的重要 ADDIN EN.CITE Badiavas20039714979717Badiavas, E. V.Falanga, V.Department of Dermatology, Boston University School of Medicine, Boston, MA, USA.Treatment of chronic wounds with bone marrow-derived cellsArch DermatolArchives of dermatologyArch DermatolArchives of dermatologyArch D

39、ermatolArchives of dermatology510-613942003/04/23Abdominal WallAdministration, TopicalAgedAged, 80 and overBiopsy*Bone Marrow TransplantationCells, CulturedChronic DiseaseFemaleHumansLegMaleMiddle AgedSkin/pathologySkin Ulcer/pathology/*therapy*Wound Healing2003Apr0003-987X (Print)0003-987x127070991

40、0.1001/archderm.139.4.510Nlmeng HYPERLINK l _ENREF_14 o Badiavas, 2003 #97 14。本研究主要目的在于探究創(chuàng)面修復(fù)過程中OPN與MSCs上皮化之間的關(guān)系。本研究發(fā)現(xiàn)，用MSCs的專用培養(yǎng)基，新生1天的小鼠采用骨組織貼壁法培養(yǎng)的MSCs細(xì)胞活性好、純度高，能夠穩(wěn)定傳代。通過體外對野生型（WT）和OPN-/-(KO)小鼠的MSCs進(jìn)行上皮誘導(dǎo)，經(jīng)多種方法對比分析，WT和KO組的細(xì)胞均表達(dá)CK14，表明MSCs向上皮誘導(dǎo)分化，WT組上皮標(biāo)志物CK14的表達(dá)陽性比率明顯高于KO組，兩組存在明顯差異,證明了OPN基因表達(dá)降低后MSC

41、s的上皮分化能力下降。動物實(shí)驗(yàn)表明敲除OPN基因后創(chuàng)面愈合的速度與正常小鼠相比明顯減慢了。本實(shí)驗(yàn)結(jié)果提示OPN能夠促進(jìn)MSCs向上皮細(xì)胞分化，有利于我們在臨床上能夠更好地應(yīng)用MSCs,提高臨床難愈性創(chuàng)面的治療效果。總之，本研究發(fā)現(xiàn)OPN能夠促進(jìn)MSCs向上皮細(xì)胞分化促進(jìn)創(chuàng)面愈合，但是OPN調(diào)控MSCs向上皮細(xì)胞分化的作用機(jī)制尚不清楚，可能為創(chuàng)面愈合過程中各種因素共同作用的結(jié)果。繼續(xù)研究OPN調(diào)控MSCs的促創(chuàng)面愈合的信號傳導(dǎo)通路，可完善其具體機(jī)制。參考文獻(xiàn) ADDIN EN.REFLIST 1.Barry, F.P. and J.M. Murphy, Mesenchymal stem cell

42、s: clinical applications and biological characterization. Int J Biochem Cell Biol, 2004. 36(4): p. 568-84.2.Hisada, K., et al., Retinoic acid regulates commitment of undifferentiated mesenchymal stem cells into osteoblasts and adipocytes. J Bone Miner Metab, 2013. 31(1): p. 53-63.3.Li, M., et al., C

43、XCR4+ progenitors derived from bone mesenchymal stem cells differentiate into endothelial cells capable of vascular repair after arterial injury. Cell Reprogram, 2010. 12(4): p. 405-15.4.Toma, C., et al., Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine hea

44、rt. Circulation, 2002. 105(1): p. 93-8.5.Zhang, X., et al., Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cell