培養(yǎng)體系對(duì)維持人臍帶間充質(zhì)干細(xì)胞特性及其體外擴(kuò)增效率的影響

1、培養(yǎng)體系對(duì)維持人臍帶間充質(zhì)干細(xì)胞特性及其體外擴(kuò)增效率的影響孫亞如，張炳強(qiáng)，王福斌，許 評(píng)，王二樸，李翠翠(青島市立醫(yī)院衛(wèi)生部細(xì)胞移植重點(diǎn)實(shí)驗(yàn)室臨床中心，山東省青島市 266000)引用本文：孫亞如，張炳強(qiáng)，王福斌，許評(píng)，王二樸，李翠翠. 培養(yǎng)體系對(duì)維持人臍帶間充質(zhì)干細(xì)胞特性及其體外擴(kuò)增效率的影響J.中國(guó)組織工程研究，2017，21(13):2023-2028.DOI:7.13.010 ORCID: 0000-0002-7756-9487(孫亞如)文章快速閱讀：不同培養(yǎng)體系中人臍帶間充質(zhì)干細(xì)胞的特性及體外擴(kuò)增效率消化傳代培養(yǎng)14 d后：比較各培養(yǎng)體系對(duì)人臍帶間充質(zhì)干細(xì)胞形態(tài)、表面標(biāo)記物、分化及增

2、殖能力的影響。6組培養(yǎng)體系：MesenCult無血清完全培養(yǎng)基中添加2%人血小板裂解液；StemPro無血清完全培養(yǎng)基中添加2%人血小板裂解液；MesenCult無血清完全培養(yǎng)基；StemPro無血清完全培養(yǎng)基；低糖DMEM中添加體積分?jǐn)?shù)10%胎牛血清；低糖DMEM中添加10%人血小板裂解液。結(jié)果表明：無血清和人血小板裂解液相結(jié)合的培養(yǎng)基能較好地維持間充質(zhì)干細(xì)胞的特性及擴(kuò)增效率。原代培養(yǎng)：臍帶間充質(zhì)干細(xì)胞文題釋義：臍帶間充質(zhì)干細(xì)胞的特性：臍帶來源間充質(zhì)干細(xì)胞的原始豐度高于骨髓、骨膜、胎盤來源間充質(zhì)干細(xì)胞，其數(shù)量依然需要經(jīng)過體外數(shù)代擴(kuò)增才能達(dá)到臨床應(yīng)用的需求。然而，臍帶間充質(zhì)干細(xì)胞并非永生化的干

3、細(xì)胞，在多次傳代后，會(huì)隨擴(kuò)增的數(shù)量增多而逐漸分化，最終喪失干細(xì)胞特性，進(jìn)而失去治療功能。因此選擇能最大限度維持干細(xì)胞特性及高擴(kuò)增效率的培養(yǎng)體系至關(guān)重要。無血清培養(yǎng)體系：是一種無任何血清成分，通過像雞尾酒式的添加營(yíng)養(yǎng)成分和生長(zhǎng)因子就可以維持間充質(zhì)干細(xì)胞在體外生長(zhǎng)繁殖的培養(yǎng)體系。它不僅能夠避免異種血清可能對(duì)細(xì)胞帶來的異源性污染，而且較胎牛血清和人血小板裂解液培養(yǎng)體系在細(xì)胞增殖效率方面有較大提升。盡管如此，實(shí)驗(yàn)數(shù)據(jù)顯示單獨(dú)應(yīng)用無血清培養(yǎng)體系作為臨床級(jí)臍帶間充質(zhì)干細(xì)胞的培養(yǎng)體系尚有其不足之處：細(xì)胞在無血清培養(yǎng)體系中隨傳代次數(shù)遞增容易出現(xiàn)分化，難以維持干細(xì)胞的特性；隨傳代數(shù)次遞增擴(kuò)增效率顯著下降。摘要背

4、景：前期數(shù)據(jù)顯示，應(yīng)用人血小板裂解液培養(yǎng)體系對(duì)人臍帶間充質(zhì)干細(xì)胞進(jìn)行體外培養(yǎng)時(shí)，較無血清培養(yǎng)體系能更好地維持干細(xì)胞特性，而無血清培養(yǎng)體系相較于人血小板裂解液培養(yǎng)體系能一定程度上提高間充質(zhì)干細(xì)胞的體外增殖能力。目的：篩選能最大限度維持人臍帶間充質(zhì)干細(xì)胞特性及擴(kuò)增效率高的培養(yǎng)體系。方法：將6份人臍帶標(biāo)本分別接種于6種培養(yǎng)體系中，進(jìn)行臍帶間充質(zhì)干細(xì)胞原代培養(yǎng)，6種培養(yǎng)體系分別為MesenCult無血清完全培養(yǎng)基中添加2%人血小板裂解液(A組)、StemPro無血清完全培養(yǎng)基中添加2%人血小板裂解液(B組)、MesenCult無血清完全培養(yǎng)基(C組)、StemPro無血清完全培養(yǎng)基(D組)、低糖DME

5、M中添加體積分?jǐn)?shù)10%胎牛血清(E組)、低糖DMEM中添加10%人血小板裂解液(F組)，14 d后進(jìn)行消化傳代培養(yǎng)，比較各培養(yǎng)體系對(duì)人臍帶間充質(zhì)干細(xì)胞形態(tài)、表面標(biāo)記物、分化及增殖能力的影響。孫亞如，男，1983年生，山東省青島市人，漢族，2007年英國(guó)謝菲爾德大學(xué)畢業(yè)，碩士，主要從事干細(xì)胞相關(guān)技術(shù)的研究。并列第一作者：張炳強(qiáng)，男，1979年生，山東省青島市人，漢族，碩士，主要從事干細(xì)胞研究。 通訊作者：孫亞如，青島市立醫(yī)院衛(wèi)生部細(xì)胞移植重點(diǎn)實(shí)驗(yàn)室臨床中心，山東省青島市 266000中圖分類號(hào):R394.2文獻(xiàn)標(biāo)識(shí)碼:A文章編號(hào):2095-4344(2017)13-02023-06稿件接受：20

6、16-12-16Sun Ya-ru, Master, Ministry of Health Cell Transplantation Laboratory, Qingdao Municipal Hospital, Qingdao 266000, Shandong Province, China Zhang Bing-qiang, Master, Cell Transplantation Laboratory of Ministry of Health, Qingdao Municipal Hospital, Qingdao 266000, Shandong Province, ChinaSun

7、 Ya-ru and Zhang Bing-qiang contributed equally to this work. Correspondence author: Sun Ya-ru, Cell Transplantation Laboratory of Ministry of Health, Qingdao Municipal Hospital, Qingdao 266000, Shandong Province, China結(jié)果與結(jié)論：D組第3代細(xì)胞細(xì)長(zhǎng)，大小不均；E、F組第3代細(xì)胞扁平，其余組第3代細(xì)胞為長(zhǎng)梭形且大小較均一。A、B組第5代細(xì)胞形態(tài)及大小較第3代時(shí)無明顯變化，C組第5

8、代細(xì)胞趨于扁平且大小不均一，D組第5代細(xì)胞形態(tài)較第3代時(shí)細(xì)長(zhǎng)，E組第5代細(xì)胞形態(tài)較第3代時(shí)扁平，F(xiàn)組第5代細(xì)胞形態(tài)較第3代時(shí)無明顯變化；各組細(xì)胞表面標(biāo)志物檢測(cè)結(jié)果無明顯區(qū)別；A、B組成脂、成骨誘導(dǎo)率較高，E、F組次之，C、D組最低；A組各代次細(xì)胞擴(kuò)增數(shù)量顯著高于C組，B組各代次細(xì)胞擴(kuò)增數(shù)量顯著高于D組，E、F組擴(kuò)增數(shù)量顯著低于培養(yǎng)組A、B組；結(jié)果表明，無血清和人血小板裂解液相結(jié)合的培養(yǎng)基能較好地維持間充質(zhì)干細(xì)胞的特性及擴(kuò)增效率。關(guān)鍵詞：干細(xì)胞；臍帶臍血干細(xì)胞；臍帶；間充質(zhì)干細(xì)胞；培養(yǎng)體系；培養(yǎng)基；篩選；形態(tài)；表型；分化；擴(kuò)增主題詞：干細(xì)胞；臍帶；間質(zhì)干細(xì)胞；組織工程Effect of cult

9、ivation systems on the maintenance of human umbilical cord mesenchymal stem cell characteristics and their proliferation rate in vitro Sun Ya-ru, Zhang Bing-qiang, Wang Fu-bin, Xu Ping, Wang Er-pu, Li Cui-cui (Cell Transplantation Laboratory of Ministry of Health, Qingdao Municipal Hospital, Qingdao

10、 266000, Shandong Province, China)AbstractBACKGROUND: Preliminary data showed that the application of human platelet lysate to human umbilical cord mesenchymal stem cell culture can better maintain the characteristics of stem cells than the application of serum-free medium. However, the serum-free m

11、edium can better improve the proliferation of mesenchymal stem cells in vitro than the human platelet lysate.OBJECTIVE: To screen out a better mesenchymal stem cell cultivation system that can greatly maintain the characteristics and proliferation rate of human umbilical cord mesenchymal stem cells.

12、METHODS: Six human umbilical cord specimens were inoculated in six culture systems, and the primary culture of umbilical cord mesenchymal stem cell was performed. These six culture systems were respectively MesenCult serum-free medium with 2% human platelet lysate (group A), StemPro serum-free mediu

13、m with 2% human platelet lysate (group B), MesenCult serum-free medium (group C), StemPro serum-free medium (group D), low glucose-DMEM with 10% fetal bovine serum (group E), low glucose-DMEM with 10% human platelet lysate (group F). The cells were subcultured at 14 days after inoculation to compare

14、 the effects of different culture systems on the morphology, surface markers, differentiation and proliferation of human umbilical cord mesenchymal stem cells.RESULTS AND CONCLUSION: (1) The morphology of passage 3 cells in group D was elongated and uneven in size. The morphology of passage 3 cells

15、was flattened in groups E and F, but the cells in the other groups were spindle-shaped and uniform. There were no significant changes in morphology and size between passage 3 and 5 cells in A and B. In group C, the morphology of passage 5 cells was more flattened and uneven in size compared with pas

16、sage 3 cells. In group D, the morphology of passage 5 cells was more elongated than that of passage 3 cell. In group E, the morphology of passage 5 cells was more flattened than that of passage 3 cells. There was no significant difference in morphology between passage 3 and 5 cells in group F. (2) T

17、he expression rate of cell surface markers had no significant difference at different passages in each group. (3) The adipoinduction and osteoinduction rates were relatively higher in groups A and B compared with groups E and F, and lowest in groups C and D. (4) The cell proliferation rate for each

18、passages in group A was significantly higher than that in group C. The cell proliferation rate for each passage in group B was significantly higher than that in group D. The cell proliferation rate for each passage in groups E and F was significantly lower than that in groups A and B. To conclude, t

19、hese results suggest that the combination of serum-free medium with human platelet lysate could better maintain the characteristics and the proliferation efficiency of mesenchymal stem cells.Subject headings: Stem Cells; Umbilical Cord; Mesenchymal Stem Cells; Tissue EngineeringCite this article: Su

20、n YR, Zhang BQ, Wang FB, Xu P, Wang EP, Li CC. Effect of cultivation systems on the maintenance of human umbilical cord mesenchymal stem cell characteristics and their proliferation rate in vitro. Zhongguo Zuzhi Gongcheng Yanjiu. 2017;21(13):2023-2028.0 引言 Introduction間充質(zhì)干細(xì)胞是一群具有自我更新及多向分化潛能的成體干細(xì)胞，因其

21、具有免疫調(diào)控、細(xì)胞因子分泌、組織器官定植等特點(diǎn)而成為細(xì)胞治療的理想種子細(xì)胞1。目前，間充質(zhì)干細(xì)胞已被廣泛用于臨床試驗(yàn)研究，在移植治療移植物抗宿主病、急性心肌梗死、糖尿病、脊髓損傷、軟骨和骨損傷、克羅恩病等方面均顯示出良好的效果2-11。臍帶是連于胎兒臍部與胎盤間的條索狀結(jié)構(gòu)。從臍帶組織華通氏膠中分離出的臍帶間充質(zhì)干細(xì)胞，拓展了間充質(zhì)干細(xì)胞的來源途徑。與其他來源的間充質(zhì)干細(xì)胞相比，臍帶間充質(zhì)干細(xì)胞具有取材方便、免疫原性低、原始豐度高且增殖能力強(qiáng)等優(yōu)點(diǎn)，成為更為理想的間充質(zhì)干細(xì)胞來源12-15。盡管臍帶來源間充質(zhì)干細(xì)胞的原始豐度高于骨髓、骨膜、胎盤來源間充質(zhì)干細(xì)胞，其數(shù)量依然需要經(jīng)過體外數(shù)代擴(kuò)增才

22、能達(dá)到臨床應(yīng)用的需求。然而，臍帶間充質(zhì)干細(xì)胞并非永生化的干細(xì)胞，在多次傳代后，會(huì)隨擴(kuò)增的數(shù)量增多而逐漸分化，最終喪失干細(xì)胞特性，進(jìn)而失去治療功能16。因此選擇能最大限度維持干細(xì)胞特性及高擴(kuò)增效率的培養(yǎng)體系至關(guān)重要。實(shí)驗(yàn)前期數(shù)據(jù)顯示，應(yīng)用人血小板裂解液培養(yǎng)體系對(duì)人臍帶間充質(zhì)干細(xì)胞進(jìn)行體外培養(yǎng)時(shí)，較無血清培養(yǎng)體系能更好地維持干細(xì)胞特性，而無血清培養(yǎng)體系相較于人血小板裂解液培養(yǎng)體系能一定程度上提高間充質(zhì)干細(xì)胞的體外增殖能力。結(jié)合人血小板裂解液培養(yǎng)體系和無血清培養(yǎng)體系的優(yōu)勢(shì)，實(shí)驗(yàn)研究在胎牛血清培養(yǎng)體系、人血小板裂解液培養(yǎng)體系和無血清培養(yǎng)體系的基礎(chǔ)上，加入無血清和人血小板裂解液相結(jié)合的培養(yǎng)體系，對(duì)人臍帶

23、間充質(zhì)干細(xì)胞進(jìn)行體外連續(xù)傳代培養(yǎng)，比較各培養(yǎng)體系對(duì)臍帶間充質(zhì)干細(xì)胞的細(xì)胞形態(tài)、細(xì)胞表面標(biāo)記物、細(xì)胞分化及增殖能力的影響，為兼具干細(xì)胞特性維持和高擴(kuò)增效率的臨床級(jí)臍帶間充質(zhì)干細(xì)胞培養(yǎng)體系的篩選提供數(shù)據(jù)支持。1 材料和方法 Materials and methods 1.1 設(shè)計(jì) 體外細(xì)胞學(xué)觀察實(shí)驗(yàn)。1.2 時(shí)間及地點(diǎn) 實(shí)驗(yàn)于2016年3至5月在青島市立醫(yī)院衛(wèi)生部細(xì)胞移植重點(diǎn)實(shí)驗(yàn)室臨床中心完成。1.3 材料 選用血液微生物檢測(cè)合格的6份臍帶樣本，新生兒男女各半，采自青島市立醫(yī)院產(chǎn)科，產(chǎn)婦均簽署實(shí)驗(yàn)知情同意書。主要試劑與儀器：MesenCult無血清完全培養(yǎng)基(Stem cell Technolog

24、ies)；StemPro無血清完全培養(yǎng)基、間充質(zhì)干細(xì)胞成脂誘導(dǎo)試劑盒、間充質(zhì)干細(xì)胞成骨誘導(dǎo)試劑盒(Life Technologies)；胎牛血清(Biochrom)；低糖DMEM B A F E D C B A F E A C B a E B E D F A C b D F圖1 各培養(yǎng)體系中人臍帶間充質(zhì)干細(xì)胞的形態(tài)對(duì)比(相差顯微鏡，×100)Figure 1 Comparison of human umbilical cord mesenchymal stem cell morphology in different cultivation systems (phase contra

25、st microscope, ×100)圖注：圖中a為第3代臍帶間充質(zhì)干細(xì)胞，b為第5代臍帶間充質(zhì)干細(xì)胞。A為MesenCult無血清完全培養(yǎng)基中添加2%人血小板裂解液培養(yǎng)體系，B為StemPro無血清完全培養(yǎng)基中添加2%人血小板裂解液培養(yǎng)體系，C為MesenCult無血清完全培養(yǎng)基培養(yǎng)體系，D為StemPro無血清完全培養(yǎng)基培養(yǎng)體系，E為低糖DMEM中添加體積分?jǐn)?shù)10%胎牛血清培養(yǎng)體系，F(xiàn)為低糖DMEM中添加10%人血小板裂解液培養(yǎng)體系。成脂誘導(dǎo)分化，油紅O染色 a D C成骨誘導(dǎo)分化，茜素紅染色 b圖3 各培養(yǎng)體系中第5代人臍帶間充質(zhì)干細(xì)胞分化能力的比較(×400)Fi

26、gure 3 Comparison of passage 5 human umbilical cord mesenchymal stem cell differentiation potential in different cultivation systems (×400)圖注：圖中a為成脂誘導(dǎo)，b為成骨誘導(dǎo)。A為MesenCult無血清完全培養(yǎng)基中添加2%人血小板裂解液培養(yǎng)體系，B為StemPro無血清完全培養(yǎng)基中添加2%人血小板裂解液培養(yǎng)體系，C為MesenCult無血清完全培養(yǎng)基培養(yǎng)體系，D為StemPro無血清完全培養(yǎng)基培養(yǎng)體系，E為低糖DMEM中添加體積分?jǐn)?shù)10%胎牛血

27、清培養(yǎng)體系，F(xiàn)為低糖DMEM中添加10%人血小板裂解液培養(yǎng)體系。圖2 第5代人臍帶間充質(zhì)干細(xì)胞表面標(biāo)記物的表達(dá)率Figure 2 Comparison of passage 5 human umbilical cord mesenchymal stem cell surface marker expression rate in different cultivation systems151050A組 B組 C組 D組 E組 F組原代 第1代 第2代 第3代 第4代 第5代細(xì)胞數(shù)(×106)圖4 各培養(yǎng)體系中人臍帶間充質(zhì)干細(xì)胞擴(kuò)增能力的比較Figure 4 Comparison o

28、f human umbilical cord mesenchymal stem cell proliferation potential in different cultivation systems 圖注： A組各細(xì)胞代次細(xì)胞擴(kuò)增數(shù)量顯著高于C相組對(duì)應(yīng)的細(xì)胞代次(P < 0.001)，B組各細(xì)胞代次細(xì)胞擴(kuò)增數(shù)量顯著高于D組相對(duì)應(yīng)細(xì)胞代次(P < 0.001)；E、F組擴(kuò)增數(shù)量顯著低于A、B組(P < 0.001)。A為MesenCult無血清完全培養(yǎng)基中添加2%人血小板裂解液培養(yǎng)體系，B為StemPro無血清完全培養(yǎng)基中添加2%人血小板裂解液培養(yǎng)體系，C為MesenCul

29、t無血清完全培養(yǎng)基培養(yǎng)體系，D為StemPro無血清完全培養(yǎng)基培養(yǎng)體系，E為低糖DMEM中添加體積分?jǐn)?shù)10%胎牛血清培養(yǎng)體系，F(xiàn)為低糖DMEM中添加10%人血小板裂解液培養(yǎng)體系。(Hyclone)；人血小板裂解物(捐獻(xiàn)者自體血漿)；CD34-PE、CD45-FITC、CD105-PE、CD44-FIT、CD14-PE、CD31-FITC、HLA-DR-PE、CD90-FITC、流式細(xì)胞儀(Beckman)。1.4 實(shí)驗(yàn)方法人臍帶間充質(zhì)干細(xì)胞的原代分離及擴(kuò)增培養(yǎng)：6份臍帶樣本均于采集后的24 h內(nèi)送到青島市立醫(yī)院衛(wèi)生部細(xì)胞移植重點(diǎn)實(shí)驗(yàn)室臨床中心，進(jìn)行臍帶的血管剝離及臍帶組織處理。將每份處理后的臍

30、帶組織塊分別接種于6種培養(yǎng)體系進(jìn)行細(xì)胞原代培養(yǎng)，分別為MesenCult無血清完全培養(yǎng)基中添加2%人血小板裂解液(A組)、StemPro無血清完全培養(yǎng)基中添加2%人血小板裂解液(B組)、MesenCult無血清完全培養(yǎng)基(C組)、StemPro無血清完全培養(yǎng)基(D組)、低糖DMEM中添加體積分?jǐn)?shù)10%胎牛血清(E組)、低糖DMEM中添加10%人血小板裂解液(F組)。14 d后進(jìn)行消化傳代培養(yǎng)。將消化獲得的臍帶間充質(zhì)干細(xì)胞以1×104/cm2的密度接種于直徑100 mm的無菌培養(yǎng)皿中，行體外連續(xù)傳代培養(yǎng)。1.5 主要觀察指標(biāo)細(xì)胞形態(tài)學(xué)觀察：應(yīng)用倒置相差顯微鏡觀察臍帶間充質(zhì)干細(xì)胞的形態(tài)，

31、比較各培養(yǎng)體系對(duì)臍帶間充質(zhì)干細(xì)胞形態(tài)學(xué)的影響。細(xì)胞表面標(biāo)志物檢測(cè)：將各組臍帶間充質(zhì)干細(xì)胞傳代至第5代，流式檢測(cè)表面標(biāo)記物。方法如下，取5×106細(xì)胞，經(jīng)洗滌、過濾、固定、熒光抗體標(biāo)記、再洗滌，流式細(xì)胞儀上樣，檢測(cè)表面標(biāo)記物CD105、CD90、CD44、CD34、CD45、CD14、CD31和HLA-DR的表達(dá)率，比較各培養(yǎng)組間細(xì)胞表面標(biāo)志物表達(dá)的差異，分析各培養(yǎng)體系對(duì)細(xì)胞表面標(biāo)志物表達(dá)的影響。細(xì)胞誘導(dǎo)分化能力檢測(cè)：將各組臍帶間充質(zhì)干細(xì)胞傳代至第3代，以1×104/cm2的密度接種24孔板，于細(xì)胞融合度達(dá)到100%時(shí)全量更換成脂誘導(dǎo)試劑，每三四天半量更換新鮮誘導(dǎo)劑，14 d

32、后行油紅O染色，比較各培養(yǎng)體系對(duì)臍帶間充質(zhì)干細(xì)胞成脂誘導(dǎo)分化能力的影響；將各組臍帶間充質(zhì)干細(xì)胞傳代至第5代，以1×104/cm2的密度接種24孔板，于細(xì)胞融合度達(dá)到80%左右時(shí)全量更換成骨誘導(dǎo)試劑，每三四天更換新鮮誘導(dǎo)劑，28 d后行茜素紅染色，比較各培養(yǎng)體系對(duì)臍帶間充質(zhì)干細(xì)胞成骨誘導(dǎo)分化能力的影響。細(xì)胞增殖能力檢測(cè)：應(yīng)用6種培養(yǎng)體系將原代獲得的臍帶間充質(zhì)干細(xì)胞以1×104/cm2的密度接種于100 mm無菌培養(yǎng)皿，進(jìn)行傳代培養(yǎng)，每4 d傳代，每代均以1×104/cm2的密度接種，傳至第5代。將每次傳代過程中消化獲得的細(xì)胞進(jìn)行計(jì)數(shù)，比較各組臍帶間充質(zhì)干細(xì)胞的擴(kuò)增效

33、率。1.6 統(tǒng)計(jì)學(xué)分析 采用SPSS 18.0統(tǒng)計(jì)學(xué)軟件進(jìn)行t 檢驗(yàn)。2 結(jié)果 Results 2.1 培養(yǎng)體系對(duì)臍帶間充質(zhì)干細(xì)胞形態(tài)學(xué)的影響 D組第3代細(xì)胞形態(tài)細(xì)長(zhǎng)、大小不均，E、F組第3代細(xì)胞形態(tài)扁平，其余組第3代細(xì)胞均為長(zhǎng)梭形且大小較均一(圖1a)。將各組細(xì)胞傳代至第5代，結(jié)果顯示A、B組細(xì)胞形態(tài)及大小較第3代時(shí)無明顯變化，C組細(xì)胞形態(tài)較第3代時(shí)趨于扁平且大小不均一，D組細(xì)胞形態(tài)較第3代時(shí)細(xì)長(zhǎng)，E組細(xì)胞形態(tài)較第3代時(shí)扁平，F(xiàn)組細(xì)胞形態(tài)較第3代時(shí)形態(tài)無明顯變化(圖1b)。2.2 培養(yǎng)體系對(duì)臍帶間充質(zhì)干細(xì)胞表面標(biāo)志物表達(dá)率的影響 表面標(biāo)志物流式檢測(cè)結(jié)果顯示，各組CD105、CD90、CD44

34、陽(yáng)性表達(dá)率均大于95%，且組間無明顯表達(dá)差異；CD34、CD45、CD14、CD31和HLA-DR陽(yáng)性表達(dá)率均小于2%，各組間無明顯差異(圖2，表1)。表1 各組培養(yǎng)體系中第5代人臍帶間充質(zhì)干細(xì)胞表面標(biāo)記物的表達(dá)率 (%)Table 1 Surface marker expression rate of passage 5 human umbilical cord mesenchymal stem cells in different cultivation systems組別CD105CD90CD44CD34CD45CD14CD31HLA-DRA組98.4399.7898.610.280.3

35、40.220.340.65B組99.8899.9299.730.260.260.670.320.38C組98.9699.9899.690.180.460.040.380.39D組99.6899.7399.840.200.790.270.450.30E組99.0099.1350.140.060.50F組99.7399.9499.810.370.090.800.140.52表注：A組為MesenCult無血清完全培養(yǎng)基中添加2%人血小板裂解液培養(yǎng)體系，B組為StemPro無血清完全培養(yǎng)基中添加2%人血小板裂解液培養(yǎng)體系，C組為MesenCult無血清完全培養(yǎng)基培養(yǎng)體系，D組

36、為StemPro無血清完全培養(yǎng)基培養(yǎng)體系，E組為低糖DMEM中添加體積分?jǐn)?shù)10%胎牛血清培養(yǎng)體系，F(xiàn)組為低糖DMEM中添加10%人血小板裂解液培養(yǎng)體系。2.3 培養(yǎng)體系對(duì)臍帶間充質(zhì)干細(xì)胞誘導(dǎo)分化能力的影響 第5代細(xì)胞成脂誘導(dǎo)結(jié)果顯示，A、B組成脂誘導(dǎo)率較高，脂滴分布較培養(yǎng)E、F組密集，C、D組誘導(dǎo)率較低(圖3a)。第5代細(xì)胞成骨誘導(dǎo)結(jié)果顯示，A、B組鈣鹽沉淀著色較E、F組深，C、D組鈣鹽沉淀較少(圖3b)。2.4 培養(yǎng)體系對(duì)臍帶間充質(zhì)干細(xì)胞擴(kuò)增效率的影響 A組各細(xì)胞代次細(xì)胞擴(kuò)增數(shù)量顯著高于C相組對(duì)應(yīng)的細(xì)胞代次(P < 0.001)，B組各細(xì)胞代次細(xì)胞擴(kuò)增數(shù)量顯著高于D組相對(duì)應(yīng)細(xì)胞代次(P

37、 < 0.001)；E、F組擴(kuò)增數(shù)量顯著低于A、B組(P < 0.001)，見圖4。3 討論 Discussion目前，干細(xì)胞行業(yè)采用的主流間充質(zhì)干細(xì)胞培養(yǎng)體系分為以下幾種：以胎牛血清為添加物的培養(yǎng)基；以人血小板裂解物為添加物的培養(yǎng)基；成分確定的無血清培養(yǎng)基17-26。其中，胎牛血清作為培養(yǎng)基添加成分容易帶來動(dòng)物源病毒及人畜共患病交叉感染的風(fēng)險(xiǎn)，同時(shí)因胎牛血清含有多種熱源成分且難以去除，因此臨床上一直在探尋胎牛血清的替代品。人血小板裂解液是將人自體血漿的血小板進(jìn)行反復(fù)凍融裂解的產(chǎn)物，其含有來自血小板中的數(shù)量豐富的血小板衍生生長(zhǎng)因子，能促進(jìn)間充質(zhì)干細(xì)胞的增殖并較好地維持其干細(xì)胞特性1

38、7-19。研究顯示人血小板裂解液含量為10%時(shí)，能最大化提升間充質(zhì)干細(xì)胞的增殖效率18，并且在安全性上優(yōu)于胎牛血清20-24，然而此次實(shí)驗(yàn)數(shù)據(jù)顯示，10%人血小板裂解液培養(yǎng)體系在連續(xù)傳代后雖然能一定程度上維持干細(xì)胞特性，但擴(kuò)增效率不及無血清培養(yǎng)體系。再者，由于人血小板裂解液來源于供者的自體血漿，其有限的獲取量也使得它難以滿足間充質(zhì)干細(xì)胞的大規(guī)模傳代擴(kuò)增需求25。無血清培養(yǎng)體系是一種無任何血清成分，通過像雞尾酒式的添加營(yíng)養(yǎng)成分和生長(zhǎng)因子就可以維持間充質(zhì)干細(xì)胞在體外生長(zhǎng)繁殖的培養(yǎng)體系。它不僅能夠避免異種血清可能對(duì)細(xì)胞帶來的異源性污染，而且較胎牛血清和人血小板裂解液培養(yǎng)體系在細(xì)胞增殖效率方面有較大提

39、升26-40。盡管如此，實(shí)驗(yàn)數(shù)據(jù)顯示單獨(dú)應(yīng)用無血清培養(yǎng)體系作為臨床級(jí)臍帶間充質(zhì)干細(xì)胞的培養(yǎng)體系尚有其不足之處：細(xì)胞在無血清培養(yǎng)體系中隨傳代次數(shù)遞增容易出現(xiàn)分化，難以維持干細(xì)胞的特性；隨傳代數(shù)次遞增擴(kuò)增效率顯著下降。由于人血小板裂解液培養(yǎng)體系維持干細(xì)胞特性的能力優(yōu)于無血清培養(yǎng)體系，而無血清培養(yǎng)體系的細(xì)胞增殖能力優(yōu)于人血小板裂解液。因此實(shí)驗(yàn)結(jié)合兩者優(yōu)勢(shì)，加入人血小板裂解液與無血清相結(jié)合的培養(yǎng)體系進(jìn)行對(duì)比，結(jié)果顯示，雖然人血小板裂解液與無血清相結(jié)合的培養(yǎng)體系同樣像無血清培養(yǎng)體系一樣，隨細(xì)胞傳代次數(shù)遞增擴(kuò)增效率有所下降，但增殖能力顯著高于無血清培養(yǎng)體系，且在維持干細(xì)胞特性方面優(yōu)于人血小板裂解液培養(yǎng)體系

40、和無血清培養(yǎng)體系。更重要的，采用2%人血小板裂解液與無血清相結(jié)合的培養(yǎng)體系較10%人血小板裂解液培養(yǎng)體系能夠很好地解決自體人血小板裂解液獲取量有限的問題。后續(xù)實(shí)驗(yàn)研究將采用不同濃度人血小板裂解液和無血清相結(jié)合的方式對(duì)人臍帶間充質(zhì)干細(xì)胞進(jìn)行培養(yǎng)，對(duì)其干細(xì)胞特性的維持和擴(kuò)增效率進(jìn)行進(jìn)一步評(píng)估，從而優(yōu)選最佳的人血小板裂解液添加濃度，并對(duì)這種培養(yǎng)體系傳代后細(xì)胞的染色體及基因穩(wěn)定性進(jìn)行測(cè)定，篩選出既安全又適合大規(guī)模生產(chǎn)的臨床級(jí)臍帶間充質(zhì)干細(xì)胞培養(yǎng)體系。作者貢獻(xiàn)：孫亞如、張炳強(qiáng)進(jìn)行實(shí)驗(yàn)設(shè)計(jì)，實(shí)驗(yàn)實(shí)施為孫亞如、王二樸、李翠翠，實(shí)驗(yàn)評(píng)估為孫亞如、張炳強(qiáng)，資料收集為孫亞如、張炳強(qiáng)、王福斌、許評(píng)，成文為孫亞如、張

41、炳強(qiáng)，審校為孫亞如。利益沖突：所有作者共同認(rèn)可文章無相關(guān)利益沖突。倫理問題：臍帶供者對(duì)實(shí)驗(yàn)知情同意。研究用人體組織的實(shí)驗(yàn)方案符合相關(guān)倫理學(xué)要求，文章的撰寫與編輯修改后文章遵守了國(guó)際醫(yī)學(xué)期刊編輯委員會(huì)學(xué)術(shù)研究實(shí)驗(yàn)與報(bào)告和醫(yī)學(xué)期刊編輯與發(fā)表的推薦規(guī)范。文章查重：文章出版前已經(jīng)過CNKI反剽竊文獻(xiàn)檢測(cè)系統(tǒng)進(jìn)行3次查重。文章外審：文章經(jīng)國(guó)內(nèi)小同行外審專家雙盲外審，符合本刊發(fā)稿宗旨。作者聲明：通訊作者對(duì)研究和撰寫的論文中出現(xiàn)的不端行為承擔(dān)責(zé)任。論文中涉及的原始圖片、數(shù)據(jù)(包括計(jì)算機(jī)數(shù)據(jù)庫(kù))記錄及樣本已按照有關(guān)規(guī)定保存、分享和銷毀，可接受核查。文章版權(quán)：文章出版前雜志已與全體作者授權(quán)人簽署了版權(quán)相關(guān)協(xié)議。

42、開放獲取聲明：這是一篇開放獲取文章，文章出版前雜志已與全體作者授權(quán)人簽署了版權(quán)相關(guān)協(xié)議。根據(jù)知識(shí)共享許可協(xié)議“署名-商業(yè)性使用-相同方式共享3.0”條款，在合理引用的情況下，允許他人以非商業(yè)性目的基于原文內(nèi)容編輯、調(diào)整和擴(kuò)展，同時(shí)允許任何用戶閱讀、下載、拷貝、傳遞、打印、檢索、超級(jí)鏈接該文獻(xiàn)，并為之建立索引，用作軟件的輸入數(shù)據(jù)或其它任何合法用途。4 參考文獻(xiàn) References1 Parekkadan B,Milwid JM.Mesenchymal stem cells as therapeutics.Annu Rev Biomed Eng.2010;12:87-117.2 Servais

43、S,Beguin Y,Delens L,et al.Novel approaches for preventing acute graft-versus-host disease after allogeneic hematopoieticstem cell transplantation.Expert Opin Investig Drugs.2016;25(8):957-972. 3 Wystrychowski W,Patlolla B,Zhuge Y,et al.Multipotency and cardiomyogenic potential of human adipose-deriv

44、ed stem cells from epicardium, pericardium, and omentum.Stem Cell Res Ther.2016;7(1):84-95. 4 Jiang R,Han Z,Zhuo G,et al.Transplantation of placenta-derived mesenchymal stem cells in type 2 diabetes: a pilot study.Front Med.2011;5(1):94-100.5 Deng P,Torrest A,Pollock K,et al.Clinical trial perspecti

45、ve for adult and juvenile Huntington's disease using genetically-engineered mesenchymal stem cells.Neural Regen Res.2016;11(5):702-705. 6 Labrador S,Alonso ML,Alvarez S,et al.Mesenchymal stem cell therapy in retinal and optic nerve diseases: An update of clinical trials.World J Stem Cells.2016;8

46、(11):376-383.7 Moussa L,Pattappa G,Doix B, et al.A biomaterial-assisted mesenchymal stromal cell therapy alleviates colonic radiation-induced damage.Biomaterials.2017;115:40-52.8 Mao X,Liu Y,Chen C,et al.Mesenchymal Stem Cells and Their Role in Dental Medicine. Dent Clin North Am.2017;61(1): 161-172

47、. 9 Cui GH,Wang YY,Li CJ,et al.Efficacy of mesenchymal stem cells in treating patients with osteoarthritis of the knee: A meta-analysis.Exp Ther Med.2016;12(5):3390-3400. 10 Oner A,Gonen ZB,Sinim N,et al.Subretinal adipose tissue-derived mesenchymal stem cell implantation in advanced stage retinitis

48、 pigmentosa: a phase I clinical safety study.Stem Cell Res Ther.2016;7(1):178.11 Dothel G,Raschi E,Rimondini R,et al.Mesenchymal stromal cell-based therapy: Regulatory and translational aspects in gastroenterology.World J Gastroenterol.2016; 22(41):9057- 9068.12 Can A,Balci D.Isolation,culture,and c

49、haracterization of human umbilical cord stroma-derived mesenchymal stem cells. Methods Mol Biol.2011;698:51-62.13 Gong W, Han Z, Zhao H,et al.Banking human umbilical cord-derived mesenchymal stromal cells for clinical use.Cell Transplant.2012;21(1):207-216.14 Zhou HX,Liu ZG,Liu XJ,et al.Umbilical co

50、rd-derived mesenchymal stem cell transplantation combined with hyperbaric oxygen treatment for repair of traumatic brain injury.Neural Regen Res.2016;11(1): 107-113.15 Guo ZY,Sun X,Xu XL,et al.Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms. Neura

51、l Regen Res. 2015;10(4): 651-658. 16 Zhao Q,Wang XY,Yu XX,et al.Expression of human telomerase reverse transcriptase mediates the senescence of mesenchymal stem cells through the PI3K/AKT signaling pathway.Int J Mol Med.2015;36(3):857-864.17 Mizuno M,Katano H,Otabe K,et al.Platelet-derived growth fa

52、ctor (PDGF)-AA/AB in human serum are potential indicators of the proliferative capacity of human synovial mesenchymal stem cells.Stem Cell Res Ther.2015;6:243-253.18 Esmaeli A,Moshrefi M,Shamsara A,et al.Xeno-free culture condition for human bone marrow and umbilical cord matrix-derived mesenchymals

53、tem/stromal cells using human umbilical cord blood serum.Int J Reprod Biomed (Yazd). 2016; 14(9):567-576.19 Fazzina R,Iudicone P,Fioravanti D,et al.Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues. Stem Cell Res Ther.2016; 7(1):122.20

54、Smith JR,Pfeifer K,Petry F,et al.Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method.Stem Cells Int.2016;2016:6810980.21 Kocaoemer A,Kern S,Klüter H,et al.Human AB serum and thrombin-activate

55、d platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue.Stem Cells. 2007;25(5):1270-1278. 22 Tateishi K,Ando W,Higuchi C,et al.Comparison of human serum with fetal bovine serum for expansion and differentiation of human syn

56、ovial MSC: Potential feasibility for clinical applications.Cell Transplant.2008;17(5):549-557.23 Yilmaz M,Ovali E,Akdogan E,et al.Autologous serum is more effective than fetal bovine serum on proliferation of bone marrow derived human mesenchymal stem cells.Saudi Med J.2008;29(2):306-309.24 Juhl M,Tratwal J,Follin B,et al.Comparison of clinical grade human platelet lysates for cultivation of mesenchymal stromal cells from bone marrow and adipose tissue.Scand J Clin Lab Invest.2016;76(2):93-104. 25 Stute