本帖最后由 qianqianlaile 于 2011-3-20 08:26 编辑 , H' m' b/ u8 d) i! H( ~( ?" {5 h 7 e' d( p' B8 \6 sOriginal Article* c: g9 _8 b2 k! g- a4 t
Development of a Serum-Free System to Expand Dental-Derived Stem Cells: PDLSCs and SHEDs ; X1 S1 @7 S, [- y% aTarle S.A.,1Shi S.,2 *Kaigler D.,1,3,4 . x( N) X/ I, S8 I1 |1 y( _3 b1 Department of Periodontics and Oral Medicine, University of Michigan; Ann Arbor, MI USA; Y2 t( u3 F# ]& t6 F9 Z
2 Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern ! Z5 ^5 `2 V$ P- N: ZCalifornia, Los Angeles, CA USA; * ?2 v+ Y, U& Z$ A: q, U. M3 Michigan Center for Oral Health Research; Ann Arbor, MI USA; 9 h$ k! X! J; a1 R+ a3 w% Z j4 Department of Biomedical Engineering, University of Michigan; Ann Arbor, MI USA ' z U. P. V: A0 A% }% T: LKey Words: PDLSCs SHEDs serum-free media regenerative medicine dental stem cells , E& M3 D0 b! F8 b; M* Z/ D* kFigures: 5 9 A8 Z7 ^% t, b8 M: |' V5 kTables: 4 ' F) @3 R5 m, X( ^( N; J6 C9 s
*Corresponding Author: Darnell Kaigler, DDS, MS, PhD 8 K) v( e' E. |* O; S7 w! w# K
Department of Periodontics and Oral Medicine $ u# @( o5 |4 C, V6 _University of Michigan ( K3 j! q- [2 y5 I) D1011 N. University % V" _6 g+ q5 ^6 P' {' iAnn Arbor MI, 48109, USA 6 M' i9 q4 |2 v$ T
Email: dkaigler@umich.edu% u& A% x3 o9 H' {7 N G0 J
Tel: 734-615-4023 - p4 M- e* \7 B6 p+ s( S7 L
Fax: 734-763-5503 : J+ j3 v9 R) m$ u5 AContract grant sponsor: Burroughs Wellcome Fund; Contract grant number: CAMS-1006918.- h# r0 o+ s! @7 v
Received 13 May 2010; Revised 21 June 2010; Accepted 23 June 2010 y4 C, q% a$ L
ABSTRACT 3 \2 e3 i" e& b$ f1 \Recently, extracted teeth have been identified as a viable source of stem cells for tissue ! I$ Q6 l8 u% wregenerative approaches. Current expansion of these cells requires incorporation of animal sera; 2 e0 } U6 \. o/ F) r
yet, a fundamental issue underlying cell cultivation methods for cell therapy regards concerns in ; m: R& ^# h7 \4 `5 q3 L
using animal sera. In this study, we investigated the development of a chemically-defined, 1 L% `% Q$ `! I. l; X5 Userum-free media (K-M) for the expansion of human periodontal ligament stem cells (PDLSCs) 4 i: M, A8 O+ n, d7 P9 l
and human stem cells from exfoliated deciduous teeth (SHEDs). Proliferation assays were 8 J" y; p1 a8 Q8 q
performed comparing cells in serum-containing media (FBS-M) with cells cultured in four* _5 p1 t' z: x, K# S5 H3 b& N. z
different serum-free medium and these demonstrated that in these medium, the cell proliferation ( P; s# f' `% i' Q: {! ^3 C' hof both cell types was significantly less than the proliferation of cells in FBS-M. Additional $ K) v% [4 `, m- F; _proliferation assays were performed using pre-coated fibronectin (FN) tissue culture plates and 6 t" _) A6 |5 Q! G
of the four serum-free medium, only K-M enabled PDLSCs and SHEDs to proliferate at higher % n* ^' u; D4 y+ c' }/ @
rates than cells cultured in FBS-M. Next, alkaline phosphatase activity showed that PDLSCs and SHEDs 5 U& B# z8 O. t3 Nexhibited similar osteogenic potential whether cultured in K-M or FBS-M, and, + X0 W3 o' u( i; Eadditionally, cells retained their multipotency in K-M as seen by expression of chondrogenic and . }2 Q& S( m2 Padipogenic genes, and positive Von Kossa, Alcian blue, and Oil Red O staining. Finally, ( e H, a" ^* q4 e/ ~5 Mdifferential expression of 84 stem cell associated genes revealed that for most genes, PDLSCs ! U A- G0 {* l
and SHEDs did not differ in their expression regardless of whether cultured in K-M or FBS-M. 2 l# K7 P' r: |0 m: j8 F FTaken together, the data suggest that K-M can support the expansion of PDLSCs and SHEDs and 5 [, Q* ~2 u8 Z# B* X+ B$ e
maintainence of their multipotency.) _* k j) s5 l/ w: e 作者: qianqianlaile 时间: 2011-3-20 08:22
原文 q3 T' l5 a; G/ K& c无血清系统培养扩增牙源性干细胞：牙周膜干细胞和脱落乳牙干细胞的进展7 P" s, u$ k+ c" J) i0 S
Tarle S.A.,1 Shi S.,2 *Kaigler D.,134 6 t/ X1 u. M4 P! n) x) @4 `
1 MI美国安娜堡密西根大学，牙周病学和口腔医学系；( C5 L: L8 ?$ U- T
2 CA美国洛杉矶南加利福尼亚大学牙科学院颅面分子生物中心& m6 i, i3 {& y/ x% y
3 MI美国安娜堡密西根口腔健康研究中心; {- e0 g% D# ~3 j
4 MI美国安娜堡密西根大学生物医学工程部/ M# [, @4 k' l" g3 U
! O" o% y) w0 H8 } U2 E9 P/ M
关键词：牙周膜干细胞 脱落乳牙来源干细胞 无血清 培养基 再生医学 牙干细胞 3 E6 d' d# u; A8 v标注：SHEDs stem cell from exfoliated deciduous teeth脱落乳牙来源干细胞 2 L, r4 ]" l( W$ S 3 Q$ [ F+ S# }; d4 P) h! l图片：53 {( E' _3 K- C; a, f$ q) _& R* o
表格：4 4 H. q4 w" K$ x通信作者：Darnell Kaigler, DDS, MS, PhD5 i! t! q: M ?5 P
美国48109MI安娜堡大学密西根大学1011N.牙周病学和口腔医学院! [1 r7 o0 h; y# D2 Y
电邮地址：dkaigler@umich.Edu 7 y: q' o" a* ^7 t) R9 T7 z电话：734-615-4023 2 p0 Y* R2 f- ^# `- x$ Q传真：734-763-5503: }0 {. Q, T+ K& P* T- ], I5 P, r
合同授予赞助商：伯勒斯威康基金；' k; Y/ F- @: _
合同授予号码：CAMS-1006918 " X2 r; v3 h0 M7 d, T f, j2010年5月13日接收；2010年6月21日修改；2010年6月23日发表 6 W. R9 Q7 f$ t* s: M, T" w摘要% y7 |+ l5 a: S$ A9 ^
近来，研究者认为拔除的牙可为组织再生的方法提供干细胞。这些细胞目前的扩增需要加入动物血清，然而，细胞疗法中细胞培养方法的根本问题与用动物血清密切相关。本研究中，我们研究了一种化学合成的培养人牙周膜干细胞（PDLSCs）和人脱落乳牙来源干细胞（SHEDs）的无血清培养基的研制。分析比较含胎牛血清培养基培养的细胞和四种不同无血清培养基培养的细胞的增殖状况，结果表明，无血清培养基培养的两种细胞增殖均明显少于胎牛血清培养基培养的细胞。用预包裹的纤连蛋白组织培养板后分析细胞增殖，结果表明，四中无血清培养基中仅K-M中培养的人牙周膜干细胞和人脱落乳牙来源干细胞增长率比胎牛血清培养基培养的高。其次，无论用K-M还是胎牛血清培养基培养人牙周膜干细胞和人脱落乳牙来源干细胞，碱性磷酸酶活性检测表明它们都拥有相似的成骨潜力；另外，K-M培养的细胞表达成软骨和成脂肪基因，钙结节染色、阿尔新蓝染色、油红O染色均阳性，说明细胞保持了它们的多向分化潜能。最后，84种干细胞相关基因的不同表达，说明无论用K-M还是胎牛血清培养基培养，人牙周膜干细胞和人脱落乳牙来源干细胞中大多数基因表达没有差异。总而言之，研究数据表明, K-M有利于人牙周膜干细胞和人脱落乳牙来源干细胞的扩增，并能保持其多向分化潜能。 : i _, }2 y( l1 I/ v作者: tpwang 时间: 2011-3-20 19:14
本帖最后由 tpwang 于 2011-3-20 19:16 编辑 0 C$ j- L# j* ~+ X 0 h0 P) J! e" O I g; g回复 qianqianlaile 的帖子- w; I: B0 e4 E$ x
! Z" K7 N, W- m$ D; P) u都是自己翻译的吗？细节锻炼也很重要。两个小地方： 8 r3 W$ l4 t5 D) l4 LOriginal article在这里是指期刊上的原发研究文章，以区别于评述、社论等其他文章，原文是指被翻译的原作。$ J' C0 i% p' q8 U0 R/ G
University of Michigan; Ann Arbor, MI USA，Ann Arbor是密大所在的城市，一般不把它放在大学名称前面。; }' {1 M+ M% \) L R0 \3 E/ X
, O! i, G: u E5 m8 W( B
无血清系统培养扩增牙源性干细胞：牙周膜干细胞和脱落乳牙干细胞的进展 . e7 A: q8 G+ P: @; A9 ZDevelopment of a Serum-Free System to Expand Dental-Derived Stem Cells: PDLSCs and SHEDs7 o8 Q3 d& i3 P
“一种用于扩增牙源性干细胞即牙周膜干细胞和脱落乳牙干细胞的无血清培养系统的建立”。Development这里指的是这个系统的建立过程。0 x. o. d. Y& g( X
2 M4 S# U8 B* g9 z近来，研究者认为拔除的牙可为组织再生的方法提供干细胞。Recently, extracted teeth have been identified as a viable source of stem cells for tissue regenerative approaches. + W: ]: ^5 R/ _* L' B
“近来，拔除的牙已被确认为一种组织再生干细胞的可靠来源。”/ z8 C) [ F& E/ z' D
: p5 }7 m; j) [( f
这些细胞目前的扩增需要加入动物血清，然而，细胞疗法中细胞培养方法的根本问题与用动物血清密切相关。 9 o. y* g( C8 j1 E- ~$ e" `Current expansion of these cells requires incorporation of animal sera; yet, a fundamental issue underlying cell cultivation methods for cell therapy regards concerns in using animal sera. & i2 K4 j- Q% P3 Z0 V# {- O“目前扩增这些细胞需要添加动物血清，而动物血清正是细胞治疗所采用的细胞培养方法中需要克服的一个基本问题。”concerned issue带有负面的意思，即担忧的问题。 $ z0 J' Z2 j. p6 w5 {7 u; y/ |1 V7 z% U% ?
本研究中，我们研究了一种化学合成的培养人牙周膜干细胞（PDLSCs）和人脱落乳牙来源干细胞（SHEDs）的无血清培养基的研制。 $ e6 X0 {1 ?: \一句话中重复了三个“研究”“研制”。investigate=探索、探究；development=发展、建立（见标题）。 6 y! r& m3 }$ \8 B2 [/ T% x9 i9 \6 x; \. t7 E! ~* w/ }- `: Y4 Q
最后，84种干细胞相关基因的不同表达，说明无论用K-M还是胎牛血清培养基培养，人牙周膜干细胞和人脱落乳牙来源干细胞中大多数基因表达没有差异。+ k- U. h6 H1 M& V1 d
Finally, differential expression of 84 stem cell associated genes revealed that for most genes, PDLSCs and SHEDs did not differ in their expression regardless of whether cultured in K-M or FBS-M. + W$ n* v- e; P* g M$ B, p1 e这句翻译的不准确而且拗口。differential expression不是指“不同的表达”。 4 `5 v# E$ U* a 0 u8 \0 e& ?- G' i9 c总而言之，研究数据表明, K-M有利于人牙周膜干细胞和人脱落乳牙来源干细胞的扩增，并能保持其多向分化潜能。' r, M6 y, P3 [! j
Taken together, the data suggest that K-M can support the expansion of PDLSCs and SHEDs and maintainence of their multipotency.+ ~8 k0 o, A6 v4 S
“上述结果提示，K-M支持人牙周膜干细胞和人脱落乳牙来源干细胞的扩增并维持其分化潜能。”5 u7 H* N/ J; q4 v
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常见不少朋友翻译文章结论时随心所欲，其实结论的用词非常讲究，不能随意改变，细微的用词区别恰恰是研究的价值的精确评定的需要。科技文献的翻译不能一方面过于直译和死板，同时又随意发挥变动。作者: qianqianlaile 时间: 2011-3-20 22:36
都是我翻译的，不过这篇好像难些，翻译后自己也没完全理顺 7 E; n7 b, A- @5 h, N( W% j初次修正译文如下： ( m/ E0 I. e8 N d原发研究文章 ; z% J5 r2 a6 Z a1 l$ o7 X1 O一种用于扩增牙源性干细胞即牙周膜干细胞和脱落乳牙干细胞的无血清培养系统的建立( F% R0 G' ]# ~
Tarle S.A.,1 Shi S.,2 *Kaigler D.,134 ( Q" E4 j5 a+ e& F L
1 MI美国，安娜堡，密西根大学，牙周病学和口腔医学系； ' H$ }# r i; r* I& E2 CA美国，洛杉矶，南加利福尼亚大学牙科学院颅面分子生物中心8 M1 y \8 |' K
3 MI美国，安娜堡，密西根口腔健康研究中心 6 O: a. l8 Y0 w" L, U0 f6 y5 E2 W4 MI美国，安娜堡，密西根大学生物医学工程部: [& r# v: {' _
本帖最后由 qianqianlaile 于 2011-3-21 11:29 编辑 $ {2 W& B M8 l3 O, {9 k) w) n; `9 {7 e0 ^! p4 \, }# c Z t
INTRODUCTION ' G D4 {" l# [3 ECell therapy has tremendous potential in regenerative medicine, yet, there are concerns in ( D _; T' F( |! J
the utility of cell therapy due to questions regarding different cell harvest and cultivation - R7 b: g- K: m, R' I) \( fmethods (Haack-Sorensen et al., 2008; Mannello and Tonti, 2007). Bone marrow derived , F% X v/ e' Z4 [# }- o) H9 Ustem cells have been identified for a number of years, and there are a number of " v! ^3 q0 w7 K% h d# O
ongoing clinical trials exploring the safety and efficacy of their use for a number of 2 a! a, D' m# ?5 i5 s
clinical applications (Battiwalla and Hematti, 2009; Sadan et al., 2009; Satija et al., ; W- ]$ O* `5 z8 K2 @/ b2 S2 K
2009). There has been an increased interest in recent years in the potential of oral-' {0 Y( E: H6 J; G) |; s' Q
derived stem cells for cell therapy, primarily because they can be derived from a readily 3 R+ Y1 g2 r4 Q" `/ [( a2 }available source, extracted teeth (Gronthos et al., 2000; Miura et al., 2003; Seo et al., ' |, G9 V) r% ?8 k8 E6 D2004). These cells exhibit multipotency and regenerative capacities characteristic of # ]+ c# X2 s- I! Z$ Y) \& b" \8 Mmesenchymal stem cells (Batouli et al., 2003; Shi et al., 2005) and have the capacity to 0 W. `! }- X8 ~+ R: x8 @
repair and regenerate tooth structures in vivo (Krebsbach and Robey, 2002; Mao et al., 2006). 7 d1 c% y5 L% W7 t+ _ Because oral-derived stem cells have been more recently identified, clinical 8 T2 c( T2 {8 }7 k% Y0 E9 L
protocols are still being developed for their use. Regardless of the specific protocol % P) O/ E+ [) P8 X$ c
used, most current cell therapy approaches rely upon ex vivo cell expansion in order to / Y2 B3 W- E. o2 G4 qproduce sufficient cell numbers for transplantation. Though a wide variety of protocols e/ Y" g" `# N$ z, \
and culturing methods exist, one common aspect to most of them is the inclusion of ; j' E9 t8 }5 O3 l7 `, d8 U* f4 p6 sanimal sera for cell expansion, in that it contains a rich source of nutrients and growth 5 P* m& E; E, pfactors (Mannello and Tonti, 2007). Despite the widespread standard use of animal % e: d7 m- x" K' t; S8 O5 E: I: {
sera for in vitro cell culture (Freshney, 2000), there are several problems which exist ) K* I1 L8 N+ q+ t0 ?" frelative to its use for clinical application. 0 g+ S/ ]; H, }$ B: y# p! Y
One of the central issues regarding limitations in using animal sera for clinical cell # p5 V( ]/ }: @5 w/ {2 xtherapy protocols is that its components are highly variable and, in many cases, ( M& ~+ f$ i% `9 l! q- [: b+ ^unknown. Though components of sera have identified, it has also been demonstrated 0 ^ i6 P( z: e3 G# `4 ]
that consistency between different lots cannot be assured (Price and Gregory, 1982). In " Z4 m* O6 Z8 r" ?9 t% @
the context of multipotent stem cells, serum components and concentrations have / J4 M3 u- `) \significant impact on cell survival and proliferative capacity, phenotype, and multipotent 6 V5 O y4 p, ~0 g2 N6 lpotential (Agata et al., 2009; Sotiropoulou et al., 2006). Additionally, for clinical use, the k( h0 t9 g/ R! Y" d) o+ U, linclusion of xenogeneic serum for cell expansion carries immunological risks associated ; X6 c, Z( [ d- \6 F6 s9 Twith the immunogenicity of serum proteins and the potential of transmission of prion ( H$ X) M: ?0 ], Q/ [; |6 V0 c0 Cdiseases and zoonoses (Shahdadfar et al., 2005). These concerns have led to efforts 6 E4 Z# c3 z+ @aimed at incorporating FBS alternatives in cell expansion protocols, including the use of 9 L& T" j4 P4 l3 W1 d4 W \7 P' Qautologous and allogeneic sera, and the proprietary manufacturing of serum-free media " g I! s w7 `% t* w9 l
formulations by different companies (Nakamura et al., 2008). Even with these approaches,6 ?3 U3 ?- w9 E, d& q0 C
there are limitations in the availability of both autologous and allogeneic , w0 K. c- v2 t ^+ \: esera and companies do not freely disclose their proprietary “serum-free” media 4 R3 S' Y: C' e1 d% `3 C
components. These factors not only prohibit clinical translation, but also limit K9 ?3 f, `, M% l, t0 L
widespread use and study of more basic fundamental questions regarding specific 7 ?' z% h, A _' e) ?mechanisms involved in the modulation of these media on cell function. As such, there : }0 _# d3 C1 F: b# d( {, Qexists a need for the development of chemically-defined media which can propagate the 7 W0 L# l- r: m; C. r5 `cultivation of stem cells without adversely affecting cell function and phenotype 1 h, h+ t3 E) z# c4 j(Mannello and Tonti, 2007). 9 e# F+ h' ?3 v$ W4 HIn this study, we aimed to develop a serum-free media (K-M) for the expansion o7 \7 h8 w& i5 a6 ^$ R4 M8 G' U' ~
dental-derived stem cells, including stem cells-derived from exfoliated deciduous (baby) 9 p, M8 L% D6 l( a* F0 Hteeth (SHEDs) and periodontal ligament stems cells (PDLSCs). Cell expansion in this # Y, ?* U5 V' a6 y
media was compared to standard FBS containing media used to culture these cells, 5 |7 Z6 H( q* V" Jas well as three other serum-free media formulations (two of which are commercially available) 3 k/ L* b2 K, Yused for culture of mesenchymal stem cells. Additionally, through ; @7 h; v% }2 A3 x+ Q. a5 K
differentiation assays and microarray analyses, multipotency and differential gene 6 F& r5 z1 w2 x, i) ]expression of 84 stem cell associated genes was examined between cells cultured in K- + d, i7 D% l" Z7 X+ kM vs. those cultured in FBS- containing media.作者: qianqianlaile 时间: 2011-3-21 12:38
本帖最后由 tpwang 于 2011-3-21 21:24 编辑 ! Q* ?. N* R8 ?7 N. ^
( o. ?* m! a/ F 回复 qianqianlaile 的帖子 + D8 A0 }2 _1 ^1 E1 o& ?3 p" Z" o8 B+ r( F0 C3 Y% S8 j$ }% L
这篇翻译的中间一段问题比较多，感觉把握的不够细。' D6 R9 V# ?2 Q; d$ L% d' R
' J5 C5 l( z- `3 C$ |限制动物血清用于临床细胞疗法的核心问题之一是动物血清组成成分高度变化，并且多数情况下变化是不可知的。 * Q; o, \- |: D6 Q) I, S# OOne of the central issues regarding limitations in using animal sera for clinical cell-therapy protocols is that its components are highly variable and, in many cases,unknown.5 D% e: ?+ D" P& P
: O. {( H) C( W3 a- u+ m5 a“动物血清产品用于临床细胞治疗的核心问题之一是动物血清成分非常不稳定，而且很多情况下不明确。”unknow也指的是components，而不是指variable。 . x; {7 U2 P8 C+ L$ H( H' N 8 I. j4 s# @4 a* D% B1 Q尽管血清的组织成分已经确定，但Price 和Gregory的研究证实不同地域的血清浓度不同。 . Z/ R7 U- ?6 _Though components of sera have identified, it has also been demonstratedthat consistency between different lots cannot be assured (Price and Gregory, 1982). # B3 P9 B* k1 `* M: Z3 t) ]
3 {! r% l9 z" g; l/ X2 x
“即使确定了血清成分，研究表明不同批次产品的成分的一致性难以保证。”lots这里指产品的“批次”，并非“地域”。生物制品的一个质量控制问题就是不同批次产品的一致性。其次这里不是指浓度而是指成分。 0 `2 W% T7 J$ n! P Z; t4 X2 Y* `. y5 j6 @# s" V7 p; E
另外，将外源血清扩增的细胞用于临床…… |' A+ L! n1 ^3 l0 y; r" TAdditionally, for clinical use, the inclusion of xenogeneic serum for cell expansion... , a2 y+ x% a" v- w+ x; i: e/ e8 X( X
exnogeneic指异基因的，这里应该是指异种（即动物血清），而外源可以是同种的。8 j4 L h- F1 x7 Z- k- I) @
; C6 V' `& q' a' |& X" B* l
……包括用自体血清和同种异体血清以及不同公司专有制造的无血清培养基配方。. ]$ k7 l# W, F2 U6 Q/ m
including the use of autologous and allogeneic sera, and the proprietary manufacturing of serum-free media formulations by different companies 2 d' m1 B m" f: {7 c- m3 @/ U& y3 b1 {
这里的proprietary manufacturing可以翻译为“独家生产的”，意思是这些产品的信息不是公开的，是生产者自己保有的，这就为研究这些无血清培养基的细胞功能调控作用设置了障碍。! r8 h9 g8 k- w5 H
, h+ t2 [& |( D5 U
……而且商家无法详细说明他们专有的“无血清培养基”的成分。# f) ~, q) |" H" f
...and companies do not freely disclose their proprietary “serum-free” media components.! H+ j7 D) n8 w" ]
4 t4 G- Z: L% W/ ]0 }# b
“而且厂家不愿免费公开他们的专有无血清培养基的成分。”do not freely disclose不是无办法，而是有办法而无意愿。这一句是上一句proprietary manufacturing的进一步注解。+ e% v! Z3 a5 N- B+ ?4 G
. G# I: H& n3 K5 m( ]1 L( p
因此，需要研制一种能使培养的干细胞增殖又不影响细胞功能及表型的化学合成培养基。 , f: k2 Y7 r$ |% a2 {As such, there exists a need for the development of chemically-defined media which can propagate the cultivation of stem cells without adversely affecting cell function and phenotype. D: C6 p) |4 |; r4 b) k8 [( }
+ `# O) |9 N s3 q2 S# {0 o# p( e“有鉴于此，需要研制具有明确化学成分的培养基……以便……。”有鉴于上面提到的动物血清不稳定不安全、商业公司自产的成分保密替代产品的局限等，所以要研制一种具有明确（公开）化学构成的培养基。: J0 X' G) b$ P0 z3 b4 l
3 B7 W, s, ~' o! F此外，与其粗制十书，不如精抠一篇。非此，不能提升境界，只能原地重复。 9 C" y$ K4 x) ~; n ' j: Y. z. |! w; L" a( U. u话说得重点，只是好意。另外，与各位翻译者讨论翻译内容，是一个非常好的学习机会，可以了解很多不在自己知识范围内的东西。 9 }! K1 p2 S/ A6 @4 R- L作者: qianqianlaile 时间: 2011-3-22 08:46
听君一席话，胜读十年书。1 s h" s4 n$ A
真正会在我面前说我不足之处的人是朋友和亲人，我父母也常说我。在我身边只说好话是于我有害的。前辈能跟我说这么多是把我当朋友。 * C4 F5 x, `8 g& ]4 S. y前辈提出我的问题的同时还教了我解决的方法，感激不尽。 5 N: f! U% R8 ~作者: qianqianlaile 时间: 2011-3-22 22:19
本帖最后由 qianqianlaile 于 2011-3-22 22:26 编辑 ; A, H3 N; E8 Z0 n3 N9 j
, D: E2 N6 Y4 W) z* Z; \ MATERIALS AND METHODS % x' [9 H0 l' \ x+ i5 n5 zIsolation of Dental-Derived Stem Cells (PDLSCs, SHEDs) 7 n* h( Y$ F8 W& R& q5 m) _
PDLSCs and SHEDs were harvested as previously described (Miura et. al, 2003; Seo et. + `2 u' r n8 l( s. }al, 2004). Briefly, PDLSCs were scraped from the root surface of a tooth into a p60 dish o( u0 G9 E& N: t
containing minimum essential alpha medium (DMEM, Gibco) and SHEDs were harvested by ' o9 ?( d9 r( ^ o- C0 R+ ascraping out the dental pulp tissue from a deciduous tooth into a p60 dish containing DMEM. & v5 P0 j/ H, H7 |4 t% s3 yAfter collection, the cells were centrifuged at 1600 rpm for 5 minutes at room temperature. The + V {/ ?% ^/ \$ ~: P- @9 xsupernatant was aspirated and the cells were resuspended in a phosphate buffered saline (PBS; 2 k9 z% X5 h; @+ P" @2 A1 Y9 N
Gibco #14190) solution with 4 mg/ml Dispase II (Roche #04 942 078 001) and 2 mg/ml' ?2 L& T+ e7 @0 x1 O
Collagenase Type II (Worthington # LS004196) and incubated at 37°C for 60 minutes. The : s/ @# p* q- s" i$ E- `enzyme solution was inactivated with 5 ml of DMEM- 15% FBS- 100µM ascorbic acid 2 ) ~! {, m! _7 Y; {8 ^) ^" G9 ]; fphosphate (ASAP, Sigma A-8960) and centrifuged at 1600 rpm for 5 minutes at room : o5 p1 s: F7 M5 ~# R* `* mtemperature. Cells were resuspeneded in 5 ml DMEM- 15% FBS- 0.1mM ASAP and transferred * ]3 _& p3 M8 L! J6 W9 _
to T-25 flasks. Media was changed the next day and then every 2-3 days. 2 h @# l: O6 _+ y7 k6 ^, v) H Cell Culture * N# G* a8 v8 k, o: p3 m7 t Cells were expanded in culture in DMEM, Iscove’s modified Dulbecco’s media (IMDM, [9 b. B) G, I- U$ w7 p& K
Gibco-Invitrogen #12571), Gibco Stem Pro Mesenchymal Stem Cell Serum-Free Media 4 e. e9 q c+ l+ ^0 g1 z7 c! U
(MSCSFM; Invitrogen# A1033401) or Lonza Therapeak Mesenchymal Stem Cell Growth 9 x h; Q/ X1 v5 m; s7 \: `
Media- Chemically Defined (MSCGM-CD; Lonza #00190632) and grown in a 37°C humidified ! D" l& ~* d R! R0 O9 o( wtissue culture incubator at 5% CO2. Media formulations are as follows: DMem (Gibco-% j" u3 X4 ^& G' J5 l
Invitrogen #12571) with 15% FBS (Gibco-Invitrogen-16000), 100µM ASAP and 5 µg/ml( {/ a2 H# F* ]0 g
Gentamicin (Invitrogen # 15750060) (FBS-M); DMem with 2% bovine serum albumin (BSA; , _( V3 Q: J* \4 o8 [( ZSigma A7888), 10ug/ml human insulin (Sigma), 4ug/ml low density lipoprotein, 200ug/ml 7 S) G6 h+ O+ s* J |# i) o7 mtransferrin, 10 nM dexamethasone, 100 uM ASAP, 50 uM ȕ-mercaptoethanol, 5 ug/ml $ E; F) c. }1 s& ~+ T$ h# zgentamicin, 10ng/ml platelet-derived growth factor (PDGF; Sigma), 10ng/ml epidermal growth ) H3 [* G; Y3 @4 V7 V: a
factor (EGF; R&D Systems), 10ng/ml basic fibroblast growth factor (b-FGF, Sigma) (SDM);* |4 r6 q! {4 }( t
IMDM with 0.2% BSA, SITE 3 (Sigma #S5295), 384µM ASAP, 10 ng/ml PDGF, 10ng/ml, O: V i! R h% }
hydrocortisone 5ng/ml b-FGF, 1 ng/ml EGF, 10-7 - a j3 L) S) z2 Y3 k mgm/ml parathyroid hormone (PTH) and 5 % B' ^! H+ N: Q. p6 }
µg/ml gentamicin (K-M). Media on the cells were changed every 2 or 3 days. Cells were grown & g, a4 Z8 M& C" E+ U2 o4 p$ Rin T-150 flasks to about 80% confluency then media was aspirated from the flasks, cells were ) Q# H! I2 `) Mwashed with PBS and trypsinized with TrypLE Express (Gibco#12605) before being split into 12! D7 r% F! ^* b: }0 X9 {' M! s+ z, r `
well plates for the assays. 8 L' r- Z( }4 V& o6 ?$ u- NFibronectin Coating of Tissue Culture Plates; H1 ]- Q8 \% [ P: J7 X% B
Fibronectin (FN) was coated on the plates and flasks to provide growth and attachment# l& r, w2 O4 A4 Q8 r9 a
support for cells grown in the serum-free, IMDM media. For the 12 well plates, 0.1% FN ; e) F7 R/ e5 nsolution (Sigma F-1141) was diluted in PBS so that each well received 3.8 micrograms per well % K0 [ |* Z6 m- @$ Y(1µg FN/cm2). The T-150 flasks were coated so that each received 150 micrograms of FN (1µg $ F6 n0 ^$ W% }FN /cm2). The plates and flasks were tilted back and forth to ensure complete coverage of the 1 G: \: a3 b: h# o# g
FN solution. The FN coating was allowed to stand at room temperature for 90 minutes. The FN 3 H. \+ Y& p0 ^6 S4 G% l
solution was then aspirated before the resuspended cells were transferred to the flasks and plates. 1 u0 v( ], j% g( ~1 {Proliferation Assays 8 v, k4 C" d4 }+ g% q/ [After trypsinization cells were resuspended in an equal amount of the appropriate media # X' A" k% B5 k: z5 \( Zbefore an aliquot was removed for counting on a hemocytometer to determine the concentration. ( L4 B+ G5 N" d6 UThe cells were then centrifuged at ~1600 rpm for 5 minutes at room temperature. Cells were * K, c+ [% a) V: S
resuspended in the appropriate media at a concentration of 3800 cells per ml. One milliliter of % u u& w7 G3 Z% m7 o
cells was dispensed into each well of a 12 well plate. K-M plates were precoated with FN & ~1 A2 D0 n7 j4 A
solution (as outlined above). Four plates for each cell type and media condition were plated and : U7 B8 e. X& k k z' f0 D
counted on a hemocytometer at days 1, 3, 5 and 7 to determine the cell numbers within each % E; M$ b! r; e: x& J1 U
well. All experiments were performed in triplicate. - ?" g0 A/ b) {; F/ ?% G0 W+ n9 t& lRNA Isolation and Purification for MicroArray 7 s# N, u, ]" l! @1 W# I V: Y
PDLSCs and SHEDs were grown in T-75 flasks to 80% confluency before the cells were 6 O0 H' p ]; d2 o) U/ sharvested for RNA. The Trizol method (Invitrogen) was used for RNA isolation. This involved : ]( o. w& o& z) q, @& o0 e0 Kwashing the cell layer with PBS, adding Trizol directly to the cells and transferring this cell ; V% X9 c# F& ^+ q& W% Dsuspension to polypropylene tubes. RNA was isolated from the cells by a Trizol-choloroform8 \2 H! d0 ]. t$ w2 z7 C
extraction, isopropanol precipitation, an ethanol rinse and resuspension of theRNA pellet in ; p O6 |8 s- C% s+ xDiethylpyrocarbonate (DEPC) water. The RNA was further purified by column$ S u! a: M+ e* K, V: {- }
chromatography, following manufacturer’s instructions (Qiagen RNeasy Kit # 74104), and 9 |" j4 E! R% ~5 _: ^2 }/ P
resuspended in DEPC water. RNA concentration was determined by the 260/280 absorbance 7 x4 B# z* [8 {) F E2 y+ Emeasurement using a Beckman DU540 spectrophotometer. ! N2 D2 R3 r; M3 J: ^8 H Q9 bIn Vitro Multilineage Differentiation 9 M! g0 O. l/ {
Multipotency of PDLSCs and SHEDs was determined through lineage specific / S; N8 v, f+ n! V
osteogenic, chondrogenic, and adipogenic induction, according to previously described methods ( x+ S7 z# \) k0 \* i5 s(Pittenger et. al, 1999). Briefly, cells were plated at a density of 30,000 cells per well in 12 well 1 J1 a# A/ n6 A' k: Nplates. At 80% confluency cells were induced with osteogenic [Growth media plus 5mM E-7 L& w+ s- v% r0 b
glycerophosphate, 100nM dexamethasone, 50µM ascorbic acid 2-phosphate] or chondrogenic . ]9 v5 U/ M! L, F
[growth media plus 50µM ascorbic acid 2-phosphate, 100nM dexamethasone, 5 µg/ml human" Q+ k: F9 S/ u& D
insulin (Sigma I-9278), 1 ng/ml TGFE, 400µM proline, 1X Non essential amino acids] or ( V) @- F+ z$ E5 @3 W
adipogenic [growth media plus 0.5mM IBMX, 1 µM dexamethasone, 10 µg/ml human insulin, ! M+ }3 @' |. ?! p4 _
200µM indomethacin] induction media. Cells were grown at 37°C in a humidified 5% CO2 ) Y4 f; x3 ^" u( H) gincubator. The media was changed every 2-3 days. At three weeks the cells were fixed and 8 ]. v; Y) @! o8 q( |2 ~8 X8 fstained as outlined below. ' x! h" @, Z' x4 ?0 b# eMultipotent Staining of PDLSCs and SHEDs 2 Y" P% O( b1 O" N, CTo identify the mineralized nodules, induced PDLSC, SHED and DPSC were fixed in 4% ; y7 o1 x: W. J4 ^' d
paraformaldehyde for 30 minutes, immersed in fresh 5% silver nitrate and incubated in the dark . X S! b' E" V6 y1 cfor 30 minutes. After washing in water the PDLSC, SHED and DPSC were exposed to / h% l6 c: E# C* d8 N! l' a8 ?
ultraviolet light for 30 minutes followed by a four minute incubation in 1% sodium thiosulfate to 0 j7 p( W) r: }# j" K. o! N& Q8 A( y* yneutralize the silver nitrate. Cells were washed twice with water before 1 ml of PBS was added 6 ^5 t6 w2 l; I: b' ^, ~& B0 V" ]
to each well and viewed. Plates were stored at 4°C. ; Q+ Q: z, K( f% U. V& L, V9 D! E* ~0 x8 wTo detect chondrogenic differentiation induced PDLSC, SHED and DPSC were fixed in 0 U# i, i0 X2 E p* h0 t" Pcold 100% methanol for 30 minutes and then exposed to 1% alcian blue in 0.1N HCl for 30 7 w% C. b' k3 Q* e' u( Z8 zminutes. Cells were washed twice with 0.1N HCl before 1 ml of PBS was added to each well / d; O; {9 \) ~! Z! [; O d
and viewed. Plates were stored at 4°C.0 O' {9 d) [: n7 v7 N( L
To detect adipogenic differentiation by identifying lipid vesicles, induced PDLSC, SHED & Z c, ^9 r7 y/ T) i( Wand DPSC were fixed in 4% paraformaldehyde for 30 minutes, and then immersed in 0.3% oil 5 J/ O8 W. h+ ^' t) e$ Ured O solution for 30 minutes. Cells were washed twice with water before 1 ml of PBS was / F5 P* l# a7 f! A: Dadded to each well and viewed. Plates were stored at 4°C. % B, n$ N; ~' L+ z8 L. s$ _/ E Alkaline Phosphatase Activity and Detection 9 Y# G* h$ R3 c( ?2 p% d* bEarly osteogenic differentiation was detected and quantified by the alkaline phosphatase 7 @8 h: Y5 ~3 q6 m7 i# `' f- v
(ALP) enzyme assay. Cells were plated at a density of 30,000 cells per well in 12 well plates.2 q, O, E( q5 G# ]; p" d5 [
At 80% confluence, cells were induced with osteogenic media as described above. The media % @0 {2 g- d5 B1 P- k$ e7 Zwas changed every 2-3 days and after one week, ALP activity was measured.& X) f/ I+ N( u$ g3 E0 A9 J% ]
To detect phosphatase activity, PDLSCs and SHEDs were fixed in 70% ethanol for 30 ) Q9 [: Q* ~4 W9 C5 v( S3 Xminutes. They were then incubated with freshly made substrate containing naphthol AS-TR * Z* N3 h0 `) F6 ]# Q
phosphate (Sigma) and Fast blue (Sigma) for 30 minutes. Cells were washed twice with PBS then " R9 `! d9 v" P2 ~3 t4 }. [
viewed or stored at 4’C. $ x! E( R; O. Z$ g6 JTo quantify the ALP activity and normalize the results, cells were lysed in Passive Lysis ) w( z7 I+ R6 }9 g2 }3 X/ C
Buffer (Promega) according to manufacturer’s instructions. Cell lysates were then sonicated, $ L% f( V* A$ i5 {6 mand centrifuged (10,000 rpm for 10 minutes at 4°C). The supernatant was recovered for the & x. y! ]5 m3 P
quantitative colormetric ALP assay (Manolagas et al., 1981) and the cell pellet was used for 4 q' [# J3 z6 ^. SDNA isolation and the determination of the DNA concentration using the Quant-iT™ dsDNA # m1 s% L. `. x, [ k. Y( JBR Assay (Invitrogen) per the manufacturer’s instructions. 5 R. }: l$ J) r$ e0 O& U Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) j0 l& v3 \3 D4 n7 aTo confirm chondrogenic and adipogenic differentiation, total PDLSC and SHED cellular - O- q3 Q6 O5 ~' JRNA was extracted, reverse transcribed, and amplified using osteoblast specific gene primers.1 O& r) @3 O/ E& J/ |
Media from the wells of induced and uninduced PDLSCs and SHEDs were aspirated. Cells k! `% b) T, w$ U3 r Z& Z3 Kwere immediately resuspended in 1 ml of Trizol (Invitrogen) and RNA was isolated according to , y& [+ V+ i; W( i C# I V. j7 U
the manufacturer’s instructions. Synthesis of cDNA was performed using Invitrogen’s 8 e, G5 Q5 n+ [3 ]' H- @SuperScriptII kit and oligo dT. PCR reaction components and concentrations were as described ( r) X+ [8 b% ~4 T, m K( p6 T% n! |in the Invitrogen Platinum Taq polymerase instructions using the primer sets below. An MJ % p, j% c' d* o0 bthemorcycler was used for the following two PCR reaction conditions: 4 W& i4 L4 U/ |( ?! R*94°C 2 minutes [94°C 45” 56°C 45” 72°C 1’] X 35 cycles 72°C 15’ 1 r7 W- m9 N: d- D1 g- M8 \8 Tor 9 X9 N9 U& U- v0 C) {- m**94°C 2 minutes [94°C 45” 67°C 45” 72°C 1’] X 35 cycles 72°C 15’ 1 e, b/ a" R1 o* l5 X5 N% J' nPCR Primer Pairs ' I" r" m7 D& a5 L9 y8 `! u0 D( |4 ^' zPrimer Name Primer Sequence Product 9 P5 M; ^! v1 P' U4 R. b, ^1 nSize( Z' y$ F1 l0 j2 c
Accession ' l" V/ w5 v- I( TNumber, _) s; k# L. z. X ]
*GAPDH FWD AGCCGCATCTTCTTTTGCGTC 815 bp NM_002046 % G3 F! T' d& v1 B*GAPDH REV TCATATTTGGCAGGTTTTTCT+ h/ r3 [. k; J( Y$ _' X4 p0 R2 h
PPARJ2 FWD GCTGTGCAGGAGATCACAGA 226 bp NM_005037 # _* S5 b3 i8 s QPPARJ2 REV GGGCTCCATAAAGTCACCAA- ~. F$ I4 u9 e9 D! f9 ^" g
Lipoprotein lipase FWD GTCCGTGGCTACCTGTCATT 212 bp NM_000237 ! H$ A: O) u8 k9 _8 ~5 SLipoprotein lipase REV TGTCCCACCAGTTTGGTGTA `4 h! V, P' L
Sox 9 FWD TTGAGCCTTAAAACGGTGCT 224 bp NM000346 5 j" z5 ^' _1 J" @; F, DSox 9 REV CTGGTGTTCTGAGAGGCACA + M. r$ U7 N/ C" ]* `* iType X collagen FWD TGAGCAGCAACGTAAAAACG 471 bp NM_00049% s2 R$ y u& v
Type X collagen REV AGGAAATGCCGAGTTTCTCA * _: ]* {7 n8 IStatistical Analysis 0 U$ K: v6 Z3 q5 _
Statistical analysis was performed with the use of Instat software (GraphPad Software, San " n a0 v3 _6 {* Y: E
Diego, CA, USA). All data were plotted as mean ± standard error of the mean (SEM), unless 7 d6 ~8 a$ B: F# o4 X
otherwise noted. Statistically significant differences were determined by two-tailed Student t % {2 q0 N4 k6 E4 L5 Mtests, and statistical significance was defined as p < 0.05. 作者: qianqianlaile 时间: 2011-3-23 12:10
本帖最后由 qianqianlaile 于 2011-3-23 12:14 编辑 ; u. q# t5 V. U2 Q
5 C% y+ ], M/ w0 Z% A$ [ 材料和方法3 u( x! P Q8 E3 l; J) C" |7 \
牙源性干细胞的分离（PDLSc，SHEDs）# D: u% D3 v3 o& n" w
如前所述方法获得牙周膜干细胞和脱落乳牙来源的干细胞。简而言之，将从牙根表面刮取的牙周膜干细胞浸入p60培养皿内最少量的基本α培养基（αMEM，Gibco）中，并将从乳牙牙髓组织中分离出的干细胞浸入p60培养皿的α培养基中。室温下每分钟1600转离心收集到的细胞5分钟。去除上清并用溶解了4mg/ml II型中性蛋白酶和2mg/ml II型胶原蛋白酶的PBS液重悬细胞，37OC培养60分钟。用5ml含15%胎牛血清和100μmol/L的抗坏血酸-2-磷酸（ASAP）的αMEM终止酶液消化，随后室温下每分钟1600转离心5分钟。将5ml含15%胎牛血清和0.1mmol/L的ASAP的αMEM重悬了的细胞悬液移入T-25烧瓶中。第二天更换培养基，之后每2到3天更换培养基。2 ^2 s/ O, W* }$ l 细胞培养 6 l# e/ Q1 _! t ~0 y, T将四种培养基即αMEM，IMDM，Gibco Stem Pro间充质干细胞无血清培养基或Lonza Therapeak 化学合成间充质干细胞生长培养基分别培养扩增的细胞置于37OC，5%CO2湿培养箱中培养。培养基具体配方如下：含15%胎牛血清、100μmol/L的ASAP和5μg/ml庆大霉素的αMEM（FBS-M）；含2%牛血清白蛋白、10μg/ml人胰岛素、4μg/ml低密度脂蛋白、200μg/ml转铁蛋白、10nmol/L地塞米松、100μmol/LASAP、50μmol/L β-巯基乙醇、5μg/ml庆大霉素、10ng/ml 血小板来源生长因子、10ng/ml 表皮生长因子、10ng/ml碱性成纤维生长因子的αMEM（SDM）；含0.2%牛血清白蛋白、SITE3、384μmol/LASAP、10 ng/ml 血小板来源生长因子、10 ng/ml肾上腺皮质素、5ng/ml碱性成纤维细胞生长因子、1 ng/ml表皮生长因子、10-7mgm/ml（mgm是否是印刷错误？）甲状旁腺激素（PTH）和5μg/ml庆大霉素的IMDM（K-M）。每2到3天更换培养基。T-150烧瓶内的细胞生长至80%融合时，去除烧瓶内培养基并用PBS液冲洗细胞，之后TrypLE Express胰酶消化细胞并将其移至12孔板内待检测。 7 R$ o+ |4 p- T: X5 y7 k( \( @7 Q纤连蛋白包裹组织培养板9 I2 C% ?, n% ~, Z8 U
培养板和烧瓶上的纤连蛋白涂层为无血清培养基即IMDM培养基中生长的细胞提供生长和粘附支持。将PBS液稀释的0.1%纤连蛋白（FN）溶液加入12孔培养板中，使每孔获得3.8μg纤连蛋白（1μg FN/cm2）。同理使每个T-150烧瓶获得150μg的纤连蛋白（1μg FN/cm2）。来回震荡培养板和烧瓶以确保纤连蛋白溶液完全覆盖(器皿表面)。室温下保留纤连蛋白液90分钟。之后去除纤连蛋白溶液并将重悬的细胞移入培养板和烧瓶。. ~* |" E+ V/ x 增殖分析" o& r0 I7 @% n( l* \. _
用等量适当培养基重悬胰酶消化了的细胞后，用血球计计算传代前的细胞以检测细胞浓度。然后室温下每分钟1600转离心细胞5分钟。适量培养基重悬细胞使其浓度为每毫升3800个，纤连蛋白溶液（如上所述）预涂布K-M培养板后向12孔板的各孔中分别加入1ml细胞悬液。将不同培养基培养的不同细胞分别接种到四个培养板内，血球计分别计算第1,3,5,7天的细胞数以检测每孔的细胞数量。所有标本重复3个。 $ T/ K- }# h! g' m& k% M$ _/ c. URNA分离和纯化获得微矩阵 ' s1 ^+ ?7 {7 c. t: G( p+ e采用Trizol法分离T-75烧瓶中80%汇合的人牙周膜干细胞和脱落乳牙来源干细胞的RNA。步骤如下：直接将Trizol加至PBS冲洗后的细胞层上，再将细胞悬液移入聚丙烯管中。用Trizol-choloroform提取法从细胞中提取RNA后异丙醇沉淀，再用乙醇冲洗，焦磷酸二乙酯（DEPC）水重悬RNA小球。根据使用说明柱层析进一步纯化RNA后再用焦磷酸二乙酯水重悬RNA。用贝克曼DU540分光光度计测量吸光度获得A260/A280的值以检测RNA浓度。, U q u4 [* G5 ~6 `: _ 体外多向分化 8 r0 H0 u' t1 }* K0 ~- [根据先前研究描述的方法，通过具体谱系即成骨、成软骨和成脂诱导确定人牙周膜干细胞和脱落乳牙来源干细胞多向潜能性。简而言之，细胞以每孔30000个的密度接种到12孔板中。待细胞生长至80%融合时，用成骨（生长培养基中加入5mmol/Lβ-甘油磷酸，100nmol/L地塞米松，50μmol/L抗坏血酸-2-磷酸）或成软骨（生长培养基加入50μmol/L抗坏血酸-2-磷酸，100nmol/L地塞米松，5μg/ml人胰岛素，1ng/ml转化生长因子β，400μmol/L脯氨酸，1X非必须氨基酸）或成脂（生长培养基加入0.5mmol/L3-异丁基-1-甲基-黄嘌呤，1μmol/L地塞米松，10μg/ml人胰岛素，200μmol/L消炎痛）诱导培养基培养细胞，并将其置于37OC，5%CO2湿培养箱中培养。每2到3天更换培养基。第三周固定细胞并按如下概述染色。+ |9 i$ R% U( _# V1 j2 f 人牙周膜干细胞和脱落乳牙来源干细胞多能染色 4 R, |% A# _+ K+ Z; J: Z为检测矿化结节，用4%多聚甲醛固定成骨诱导培养基培养的人牙周膜干细胞、脱落乳牙来源干细胞和牙髓干细胞30分钟，再将其浸入现配的5%硝酸银中并于黑暗中静置30分钟。用水冲洗细胞后紫外灯照射30分钟，之后加入1%硫代硫酸钠中孵育4分钟中和硝酸银。接下来用水冲洗细胞两次，向每孔加入1mlPBS并观察。培养板置于4OC环境中保存。 6 V/ r0 H T3 s为检测成软骨分化，用100%冷甲醇固定成软骨诱导培养基培养的人牙周膜干细胞、脱落乳牙来源干细胞和牙髓干细胞30分钟，再用溶于0.1N HCl的1%阿辛蓝浸泡30分钟。之后用0.1N HCl冲洗细胞两次后每孔加入1mlPBS并观察。培养板置于4OC环境中保存。 ( M! }& S4 j" q$ C, N# j% a为检测成脂分化，用4%多聚甲醛固定成脂诱导培养基培养的人牙周膜干细胞、脱落乳牙来源干细胞和牙髓干细胞30分钟，再用0.3%油红O溶液浸泡细胞30分钟。之后再用水冲洗细胞两次后每孔加入1mlPBS并观察。培养板置于4OC环境中保存。& b4 i* g1 q/ i7 X' p 碱性磷酸酶活性及其检测 9 Q |( U- K# l1 t3 v' Z碱性磷酸酶分析检测并定量早期成骨向分化。向12孔板的各孔中接种30000个细胞。待细胞长至80%融合时，用上述成骨诱导培养基培养细胞。每2到3天更换一次培养基并于一周后定量检测碱性磷酸酶活性。# ^. B, h0 M3 \7 `9 O
为检测磷酸酶活性，用70%乙醇固定人牙周膜干细胞和脱落乳牙来源干细胞30分钟。之后用新鲜的含奈酚AS-TR磷酸盐和快蓝的基底培养30分钟，PBS冲洗细胞两次后观察或将细胞储存在4OC环境中。2 Z* I1 h3 Q7 \- d( s
为定量碱性磷酸酶活性并使结果正常化，根据说明书用Passive裂解液溶解细胞。之后声波处理（sonicated）并离心细胞裂解物（4OC每分钟10000转离心10分钟），收集上清液并用比色法定量检测碱性磷酸酶，从细胞沉淀中提取DNA后再根据说明书用Quant-iT™ 双链DNA BR分析法检测DNA浓度。! w. b- n- r0 \& t1 s' c* H- S- y 逆转录聚合酶链反应6 h. D* U0 x( F* Y4 T9 ?4 d. z' b e+ s
为证实成软骨和成脂分化，提取人牙周膜干细胞和脱落乳牙干细胞的总RNA后逆转录并用成骨特定基因引物放大。具体操作如下：吸出已诱导和未诱导的人牙周膜干细胞和脱落乳牙干细胞培养孔中的培养基后立即用1ml曲拉通（Trizol）重悬细胞并根据说明书提取RNA。用Invitrogen公司的SuperScriptII kit 和oligo dT合成cDNA。PCR反应所需试剂及其浓度如Invitrogen公司Platinum Taq 聚合酶使用说明中所述，使用如下引物。一个MJ themorcycler被用于以下两种PCR反应条件中：: n9 M* b+ Y* c X ?1 p+ f
94°C 2分钟→(94°C 45” → 56°C 45” → 72°C 1’) X 35 循环→72°C 15’或者 ' c$ i& C8 B0 z" ?9 l# [ \94°C 2分钟→(94°C 45” → 67°C 45” → 72°C 1’) X 35 循环→72°C 15’4 @, ^. E& l, V% A3 {( G
PCR引物组8 p% p: z+ m# r" y9 U' \' L' d
引物名称 引物序列 产品大小 Accession号 ! O8 ?, E* e* l8 O1 m6 T*GAPDH FWD AGCCGCATCTTCTTTTGCGTC 815 bp NM_002046 4 o" r# _) ]- E6 ?" l9 f7 @; H*GAPDH REV TCATATTTGGCAGGTTTTTCT ( o& L6 h+ `0 uPPARJ2 FWD GCTGTGCAGGAGATCACAGA 226 bp NM_0050377 v% T/ Y6 P$ b4 O, J7 z+ j
PPARJ2 REV GGGCTCCATAAAGTCACCAA / m$ W. S( U" j4 s" X; X# l9 ?Lipoprotein lipase FWD GTCCGTGGCTACCTGTCATT 212 bp NM_000237 $ V/ P. Q0 W: o$ f5 N2 T$ o! TLipoprotein lipase REV TGTCCCACCAGTTTGGTGTA # G( m* ~' \, pSox 9 FWD TTGAGCCTTAAAACGGTGCT 224 bp NM000346, O% f- E# B. i1 O Q2 R9 F
Sox 9 REV CTGGTGTTCTGAGAGGCACA / z( C+ F) U" A: ]( x& J9 ~
Type X collagen FWD TGAGCAGCAACGTAAAAACG 471 bp NM_00049 2 M/ D+ c- P: ?4 h' oType X collagen REV AGGAAATGCCGAGTTTCTCA & Z/ R8 ~* D5 h j
统计分析) T6 P, h. |) f! ?5 U
用instat软件进行统计分析。除特殊说明外所有数据均用均数±标准误表示。双侧t检验结果有显著性差异，p<0.05时有统计学意义。 & G2 }- J# V' l ]作者: tpwang 时间: 2011-3-24 14:51
，脑子里也没有独家生产这个概念
，所以没理解。