Microsoft Word 周龙5.0彩.DOC

Size: px

Start display at page:

Download "Microsoft Word 周龙5.0彩.DOC"

藻田
7 years ago
Views:

1 f zu{ } 19 í } Çf Chinese Journal of Tissue Engineering Research February 5, 2015 Vol.19, No.6 C57L/6 ÙÎÎê ŒÉy» äöýuî ŒqÉä Ú 1 2 Â 2 ˆ ( 1 { œ Þ É { Þ Ô É ) s 1 Íqg p n º t qü ±Îq C57L/6 ÙÎÎÉy ê Œ ~ q«}² Øx ² ùü ÉyÇÎÎê Œ Âµ«Œ Éy ä qñxîî ê Œù Ø ÉäguÎ Œ 2 Í x g«}² y~žùå Ä z ƒ ƒ ÂuqÉyÎÎê Œ q qê Œ gz ùgç uög u{ zu{} ùåz q C57L/6 ÙÎÎê Œ ºÀ Œ» ÎÎê Œ uî Œ ŒÄ Î κ ƒ ä ¹ É ä g«}² gè ê Œ uî Œ ŒÄ Î RNKL ƒ rðîîê ŒqÉy ¼ h ³ Ìn RW264.7 Œ ÉäguÎ Œ rq Éy» ä ¹ C57L/6 ÙÎÎê Œq ÙÎÎê Œƒ màg ºýuÎ ŒÉä Éy C57L/6 Ù Î ŒÎ ÇÎÎ Œ ²zf n Œ ôã Œ ÎÎ Œ²»ý( p 16 h) } 2 ÉyÇ Œ ÿ ŒÄ Î q» f» «q ÙÎÎê Œ ³² ÙÎÎê Œ²Ÿ ä» ²Ÿ m À n Œy ñx ÙÎÎê Œ ŒŸÅ ñ Ø ŒÄ Î κ ƒ ä ÉäguÎ Œ Éyq ÙÎÎê Œ e Ç» 5 d ý Œ e q «ŒÄ Î xéyq ÙÎÎê Œ ±Î Œ É äguî Œ ÌnÂµ«Œ ê Œq«Œ üö ŒÄ Î ²ê Œ«qg Œ Éy z màq ÙÎÎê Œ»q ÙÎÎê Œ gz Ÿ z ²pž² u{ ö 1988 º { x Ôd šì { œ Þ É { Þ Ô É doi: /j.issn [ f É ú:r394.2 h u: ú: (2015) ÈÍ Ú Â ˆ. C57L/6 ÙÎÎê ŒÉy» äöýuî ŒqÉä[J]. f zu{ (6): C57L/6 mouse bone marrow monocytes: isolation, cultivation, purification and differentiation into osteoclasts Zhou Long 1, 2, Chen Xi 2, Luo Zong-ping 1, 2, Yang Hui-lin 1, 2, He Fan 1, 2 ( 1 Department of Orthopaedics, the First ffiliated Hospital of Soochow University, Suzhou , Jiangsu Province, China; 2 Orthopaedic Institute, Soochow University, Suzhou , Jiangsu Province, China) bstract CKGROUND: Isolation and extraction of bone marrow monocytes is mostly realized via osteoclast differentiation induced by RW264.7 cells as reported at home and abroad. OJECTIVE: To explore a new method for the isolation, culture, purification and identification of C57L/6 mouse bone marrow monocytes and to observe the growth features and osteoclast differentiation of mouse bone marrow monocytes in vitro. METHODS: The femurs and tibias of C57L/6 mice were aseptically removed to extract bone marrow cells that were cultured overnight (> 16 hours). During the extraction process, erythrocyte lysate was used to remove red blood cells. t day 2, suspension cells were isolated and cultured with the medium containing macrophage colony-stimulating factor to harvest adhered mouse bone marrow monocytes. The medium was changed to purify and amplify the mouse bone marrow monocytes. Cell morphology was observed, the growth curve was tested and using flow cytometry, the cell surface antigen of primary mouse bone marrow monocytes was detected. Macrophage colony-stimulating factor and receptor activator of nuclear factor-κ ligand were added to differentiate the cells into osteoclasts. Zhou Long, Studying for master s degree, Department of Orthopaedics, the First ffiliated Hospital of Soochow University, Suzhou , Jiangsu Province, China; Orthopaedic Institute, Soochow University, Suzhou , Jiangsu Province, China Corresponding author: He Fan, M.D., ssociate professor, Department of Orthopaedics, the First ffiliated Hospital of Soochow University, Suzhou , Jiangsu Province, China; Orthopaedic Institute, Soochow University, Suzhou , Jiangsu Province, China ccepted: P.O. ox 10002, Shenyang

2 Ú }. C57L/6 ÙÎÎê ŒÉy» äöýuî ŒqÉä RESULTS ND CONCLUSION: The newly separated mouse bone marrow monocytes were circular and feelers were extended from both ends. fter 5 days of culture, the cells were oval-shaped with more obvious feelers, and cell proliferation relied on macrophage colony-stimulating factor. Under the flow cytometry, isolated mouse bone marrow monocytes had high purity and could be induced to differentiate into osteoclasts. It is effective to isolate mouse bone marrow monocytes that grow stably by vigorously promoting the proliferation of monocytes by using different adherent abilities of mesenchymal stem cells and monocytes as well as macrophage colony-stimulating factor. Cultured mouse bone marrow monocytes are phenotypically stable and can be used for further research. Subject headings: Monocytes; Osteoclasts; Macrophage Colony-Stimulating Factor; RNK Ligand Zhou L, Chen X, Luo ZP, Yang HL, He F. C57L/6 mouse bone marrow monocytes: isolation, cultivation, purification and differentiation into osteoclasts. Zhongguo Zuzhi Gongcheng Yanjiu. 2015;19(6): Introduction t º ˆ o Îº ²Ÿ oo ÎÈÎ q ² œƒ Î œƒ Ù t º ˆ ýqg ² öo [1-2] ÈÂ ²Î œƒ Ùqöm f È yvoku{ Å uî Œ Éº Ø uî Œ ² u{ºøœqºà rðuî Œq g mùg Éy ¹Î Î ž/îîê Œ Œ Œ mùgfƒ Œ duî Œ Éy ²zf u ¹ÎÎ quî Œ ±ƒ ÎÎê Œ y dqê Œ ÎÎ Œf ë0.04% Ÿ g uî ŒqÐƒ Œ [3-4] ± žêf Œ quî Œ iî ¹ÎÎ rm quî Œ Î [5] ±Œ Œ Éä i Î [6] C57L/6 Ù º p º t qü ±Î Œ q Íüy t qo o ç uu{f ƒèé [7] Œ y~ q Ÿi qƒ Éy ä ÎÎê Œq ü ³²ê ŒŸÅÉ º ²Ÿ ¹ ùg² ÎÎê Œq u ÎÉäŒ Î«o oofq o nu{} Å qy Œ 1 Materials and methods Œ Í Âö p2014 2Ž11 Îyu{ C57L/6 Ù6ù 812 Ú( 25 g) ÄÄü 3ù «Ž ç y Â¹ú ù ú SCXK( ) Í²zf Ùgq ˆ}ü2009 Ethical issues in animal experimentation rºùg k Âq Í ÙÎÎê Œqƒ Éy» z ÇC57L/6 ÙöŒ Î ŒÎ ÂúfÓ Î n5 ml ž α-mem» ö ± C57L/6 ÙÎÎê ŒqÉy» äö¹ Íg Ïöy Ïöy Ù ÙCD11b ƒ ŒÄ Î (M-csf) κ ƒ ä (RNKL) TRP Ïr FITC-Phalloidin DPI ˆ À Œy ˆ invitrogen¹ú ˆ PEPROTECH¹ú ˆ Sigma¹ú bcam¹ú vp mg ¹ú zeiss ˆ eckman À ÎÎ μm Œ² ² ý1 200 r/miny 5 min Ø 10 Œƒz( 5 ml)q 1X Œ ±± â À 5 min r/min y 5 min xôã Œ n ž α-mem» 2 nÿƒzé 10%qFS 1%PSqα-MEM» 5 ml ÎÎ Œ yp 25 cm 2»mf p37 ƒzé 5%CO 2 ~ Åˆ»² ( 16 h) [8] Ä n ž α-mem»»m r/miny 5 min nÿƒ zé 10%q FS 1%PS 100 μg/l ŒÄ Î qα-mem» 10 ml ê Œý yp 100 mm»rf [9-11]» 3 dý n ž α-mem»»r2 Ø ÿƒzé 10% qfs 1%PS 100 μg/l ŒÄ Î q α-mem» rž Œ Žü² ÙÎÎê ŒmÀ q Í [9] mà qñx ÙÎÎê Œx1 000/ y 96» f É2 Ø100 μg/lq ŒÄ Î ù Ø ŒÄ Î 6 g 100 μl ÉÌ yýq} xmtt Œ ºfØ ŒÄ Î 1 ñ» ý Ø ÿ 10%MTT (5 g/l) q» 100 μl»4 h» ¼ Ø 100 μl DMSO À gµé ý Š o y ü 570 nm ISSN CN /R CODEN: ZLKHH 941

3 942 Ú }. C57L/6 ÙÎÎê ŒÉy» äöýuî ŒqÉä À q 6 q x g ± g ± Í ŒmÀ ÙÎÎê ŒŸÅ ñ¹ màg q ñx ÙÎÎê Œn ŒÍÍ ý ü Ä 2fEP~f y ýôã» 2~ÉÌØ 1%S(n1%NaN 3 )100 μlà Á30 min 1 2 1~gÂ d 2~Ø 0.5 μl Ù ÙCD11b ƒ À Åˆö 30 min y ýø PS 1 ~Ø 600 μlq10 g/l Šn ±zí ~ý ÙÎÎê ŒÉäguÎ Œö u w ä²ýqñx ÙÎÎê Œn ŒÍÍ ýx cm 2 ~xp12» f» ˆp» ~¼(37 ƒzé 5%CO 2 )»5 d d ü 3 ºf d Ø 20 μg/lq ŒÄ Î Ø 20 μg/lq ŒÄ Î 100 μg/lq κ ƒ ä [12-13] 2 d 1 5 dýà Í q üquî Œ Ÿ u w (TRP) Œn40 g/lq Šn 20 min x S-Iw r g gq f37 30 min [14] ôy 1 DPI 3 min ¼ nôy 3 ˆ À d ÙÎÎê ŒÉäguÎ Œö ŒÎ Ù q [15] ü äýqñx ÙÎÎê Œx cm 2 q ~xp12» d ü3» üð»5 dýôã» 40 g/l Šn À 10 min PS Œ2 ý 0.25% Triton (PSz ) 10 min PS 2 Ø FITC-Phalloidin (5 mg/l 1%qSz ) 60 min PS 3 À d g ÙÎÎê Œq ÙÎÎê ŒmÀ q ÙÎÎê ŒŸÅ ñqÿ² ÙÎÎê ŒÉägu Î Œ u w ÙÎÎê ŒÉäguÎ Œ ŒÎ Ù q É Í nx _ ±sÿx nspss 17.0 ±z²ÿt Í P < 0.05Ÿx i 2 Results 2.1 } ¾ Éyq ÙÎÎ ê ŒØ 20 μg/l ŒÄ Î» 1 d ý ± 3 d ý ôã q Œ Œ e Ç 5 d ýe q q Œ± Œ Ž Œ ²Í 90% ù± qnpý Í( 1) (570 nm) d ŒÄ Î » Â(d) 2 ñx ÙÎÎê Œ ŒÄ Î Î q» mà Figure 2 Growth curves of primary monocytes with and without macrophage colony-stimulating factor stimulation ŒÄ Î Œ ² ÙÎÎê Œq mà S Ø ŒÄ Î q ÙÎÎê ŒmÀ ž 2.2 } j g Î«n MTT ÙÎÎê Œ ŒÄ Î z üf Â q i n ù ŒÄ Î Œ q ² ÙÎÎê Œq y e ý² } ÃýŽ ³ ² mà 2 d ý Œ À² ² ú Íq mà S ( 2) Ø ŒÄ Î q ÙÎÎê ŒmÀ ž ù ŒÄ Î ÙÎÎê ŒmÀq 2.3 } Ý { x ñx ÙÎÎê Œ Ÿ² CD11b Ÿ²ig 96.2%( 3) 2.4 } ÝÕ ó Î«ŒÄ Î κ ƒ ä» 5 d ý À Í»gfÇ jƒz (r μm) Ê Œ f TRP xœ f ² 40 f Í È Œ Œ ²n ù q DPI ù dquî Œ 3 f 3 fx Œ ù² qf Œ»qê Œ Ÿ TRP ( 4) 2.5 } Ä ¾ ~ Î«ŒÄ Î κ ƒ ä ý quî Œ q Ù qj Œ ²Š Œ ²n ù q»q d µéê ŒŠÄ ü( 5) P.O. ox 10002, Shenyang

4 周龙等. C57L/6 小鼠骨髓单核细胞分离培养纯化及向破骨细胞的分化 C 第1天第3天第5天图 1 原代单核细胞第天的形态(标尺=100 μm) Figure 1 Morphology of primary monocytes at 1, 3 and 5 days of culture (scale bar=100 μm) 图注第 1 天细胞呈小圆形形态较规整 3 d 后细胞成椭圆形两端伸出触角 5 d 后两端的触角更明显细胞大小一致形态均一细胞密度达到 90% C D 图 4 巨噬细胞集落刺激因子和核因子 κ 受体活化因子配基诱导单核细胞破骨分化的 TRP 及 DPI 染色结果(倒置显微镜 100) Figure 4 TRP and DPI staining of monocytes differentiated into osteoclasts induced by macrophage colony-stimulating factor and receptor activator of nuclear factor-κ ligand (inverted microscope, 100) κ TRP DPI 图注巨噬细胞集落刺激因子和核因子受体活化因子配基诱导培养后逐渐形成细胞核可多达个而未见仍为单核细胞阴性染色阳性的多核破骨细胞实验组图 3 原代小鼠骨髓单核细胞抗原表达阳性率流式细胞仪检测结果 Figure 3 Phenotypes of the primary monocytes shown by flow cytometry CD11b 96.2% 图注结果显示细胞胞表面标志的表达表达阳性率为符合骨髓单核细 3 讨论 Discussion 破骨细胞是一种终末细胞至今仍未有成熟的破骨细胞株获取较困难大多是采用小鼠的单核 /巨噬细胞 RW264.7诱导分化获得但由于RW264 7是细胞系用于具体的实验研究很多机制不是很明确骨髓单核细胞是一种较RW264.7更为前体的破骨前体细胞将其分化为破骨细胞则更有说服力目前单核细胞的分离方法主要有两种密度梯度离心法药物贴壁筛选法密度梯度离心法运用淋巴细胞 [ ] [9 18] [9] ISSN CN /R 染色显示破骨细胞的 CODEN: ZLKHH 图 5 巨噬细胞集落刺激因子和核因子 κ 受体活化因子配基诱导单核细胞破骨分化的荧光染色结果(鬼笔环钛免疫荧光染色荧光显微镜 100) Figure 5 Fluorescence staining of the monocytes differentiated into osteoclasts induced by macrophage colony-stimulating factor and receptor activator of nuclear factor-κ ligand (phalloidin immunofluorescence staining, fluorescence microscope, 100) κ 图注巨噬细胞集落刺激因子和核因子受体活化因子配基诱导形成的多核破骨细胞形成绿色的纤维性肌动蛋白环而可见单核细胞聚集未见融合分离液分离出单个核细胞此法提取的单核细胞纯度较高但是整个分离过程操作步骤多且繁琐影响了获得细胞的增殖活力药物贴壁离心法是利用巨噬细胞集落刺激因子能够促进单核细胞贴壁而间充质干细胞淋巴细胞等不能贴壁从而将单核细胞分离出来本实验对此法加以改进提取过程中先用红细胞裂解 943

5 944 Ú }. C57L/6 ÙÎÎê ŒÉy» äöýuî ŒqÉä ôã Œ ÌnÎÎÂµ«Œ± «qg ÎÎ Œ²»ý( p16 h)»mf«qgâµ«œ [19] Ø»» Œ Äy ýø 100 μg/lq ŒÄ Î 100 mmq»r f» 1 dýž qê Œ«fv q Œ g Œ qâµ«œö «qê Œ 3 dý ôã Œ ùnps À Å 5 dýù ê Œq ²90% ² Žùü É yçîîâµ«œ ê Œ nmà ²ÎÎ ê ŒqmÀ Œ ÎÎê ŒmÀ É }² mà ú öj ŒÄ Î ÎÎê ŒmÀq Ä ñxê ŒmÀz }² e mà e ~x p¹ CD11b ê Œq y ŸÅ ñé [20] ñxê Œq xê Œq g96.2% Ø 20 μg/l q ŒÄ Î 100 μg/lq κ ƒ ä Éä5 dýkù ØÉäg quî Œ qîîê Œùnpý q Íu{ h ³ÎÎê Œ Î q ŒÄ Î Î ù~ px iý ŒŽ Œ«mÀ Çj Ÿ Ãr [11] Íqu{ ñxq ÙÎÎê Œm À³ ± ~x p gz Œ u Â ¼ qê Œ ù Î zf µ y Œ ŽpÎ«o rº ooqk u{ g Îê Œq 1/2 1/3q ŒØ ŒÄ Î» öuîéä ( x) ù ê Œ ŒÄ Î qî Œ «Âµ«Œ Œ} ŒÊ Œ«mÀ Éy ØqºÀ Í nqc57l/6 ÙÎÎ g«}² Éy» äqîîê Œº ê Œq mg g º ~ŽùÅ Ä z ƒ ƒ Âu q Œ Œ ³ mà gz }~ ùgç uög u{ zu{} ùåz qc57l/6 ÙÎÎê Œ Ž { Þ Ô d «h Úe x Ô d d ÌrÀ{ À À í q d k «n t 2009 wethical issues in animal experimentationx üì íd ³ òî s³ Æ À òî òî³ Â p s³ d u k À í À d 4 õ h References [1] ÈÚ,,i,}.t Îº ˆ öoqâ [J].Î º,2003,18(3):210. [2] Ê,Ør,,}.t Î ˆ o ÎÈÎ À Ã [J].f Î,2003,16(9):533. [3] Kartsogiannis V, Ng KW. Cell lines and primary cell cultures in the study of bone cell biology. Mol Cell ndocrinol.2004;228: [4] Scheven, Visser JW, Nijweide PJ. In vitro osteoclast generation from different bone marrow fractions, including a highly enriched haematopoietic stem cell population. Nature. 1986;321: [5] ÀÄ,,,}. 1,25(OH)2 m D3 ÙÎÎê Œ uî Œ[J]. :ç f,2007,38(2): [6] iö,, E Grigoriadis. ÙÎÎ Œ Œ Œ uî Œ Œq ±[J].jxø ç,2006,20: [7] Pittenger MF,Mackay M,eck SC,et al.multilineage potential of adult human mesenchymal stem cells.science.1999; 284(5411): [8] Leung R,Wang Y,Cuddy K,et al.filamin Regulates Monocyte Migration Through Rho Small GTPases During Osteoclastogenesis. J one Miner Res. 2010;25: [9] Takeshita S, Kaji K, Kudo.Identification and Characterization of the New Osteoclast Progenitor with Macrophage Phenotypes eing ble to Differentiate into Mature Osteoclasts.J one Miner Res.2000;15: [10] Kodama H, Nose M, Niida S,et al.essential role of macrophage colony-stimulating factor in the osteoclast differentiation supported by stromal cells. J Exp Med.1991; 173: [11] Tanaka S, Takahashi N, Udagawa N,et al. Macrophage colonystimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors.j Clin Invest.1993; 91: [12] Wang Y, Lebowitz D, Sun C,et al.identifying the relative contributions of Rac1 and Rac2 to osteoclastogenesis.j one Miner Res.2008;23: [13] oyle WJ, Simonet WS, Lacey DL.Osteoclast differentiation and activation. Nature.2003;423: [14] Takahashi N, Yamana H, Yoshiki S, et al. Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures. Endocrinology. 1988;122: [15] Â w,i,â~i,}. «ƒ ä ƒ q duî Œ[J].f zu{,2013,17(24): [16] Kobayashi Y,Mizoguchi T,Take I,et al. Prostaglandin E2 ehances osteoclastic differentiation of precursor cells through protein kinase - dependent phosphorylation of TK1. iol Chem. 2005;280(12): [17] é, pµ,. Ùê ŒRW264.7q Œmg g [J].êé :ç f,2008,36(3): [18] À,i. M-CSF RNKLŠü ÙÎÎ uî Œ q Íu{[J].f Î pîo.2009,8(2): [19],rl,,}. C57L/6 ÙÎÎÂµ«ŒÉy» q ¹ [J].«ç.2012,36(9); [20] Springer T, Galfré G, Secher DS,et al.mac-1:a macrophage differentiation antigen identified by monoclonal antibody. Eur J Immunol.1979;9: P.O. ox 10002, Shenyang

Microsoft Word - 会协_2015_1号.doc

Microsoft Word - 会协_2015_1号.doc k á}» st v Œz Ÿ x v oœ c c g»â» w»â Ä x «g»â» Æm Ã Û»Â Æ m Ä d Ö Ä oø c c g»â» Æm e Ä cà d Ð Àß «Í Èc½ ½ 1 Ä ([FHO ò ([FHO À {Û Ä d d ¼ c Ä œ þ q ì r d Ç } Ä Éxq ì r d ò ~ ~œò d ò ¾ d í àóú å c d ~ ò½