本帖最后由 qianqianlaile 于 2011-3-20 08:26 编辑 0 i- t& O, w0 K& n" k
4 D% C4 J% f, l& `( j, bOriginal Article - ^# P* l) u& c! j6 W% NDevelopment of a Serum-Free System to Expand Dental-Derived Stem Cells: PDLSCs and SHEDs# P' R$ {- a" I' r' s- }
Tarle S.A.,1Shi S.,2 *Kaigler D.,1,3,4 " o; o0 j" P; Y7 K' |; t
1 Department of Periodontics and Oral Medicine, University of Michigan; Ann Arbor, MI USA; $ y4 z# m) Y+ N8 d, k( P+ C* Y- ~7 p
2 Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern 8 n5 x2 A0 v" s6 l1 j/ @$ n( rCalifornia, Los Angeles, CA USA;5 z- v5 y+ t. w+ j
3 Michigan Center for Oral Health Research; Ann Arbor, MI USA;. g1 o9 T+ c, ^
4 Department of Biomedical Engineering, University of Michigan; Ann Arbor, MI USA " C0 ?' Q2 X0 G! |+ {
Key Words: PDLSCs SHEDs serum-free media regenerative medicine dental stem cells $ V; I9 {. j/ P+ CFigures: 5 # j$ }) ?8 _" aTables: 4 0 I$ P U8 `* G4 E6 w*Corresponding Author: Darnell Kaigler, DDS, MS, PhD 1 `6 V9 l: ? P: V( U, k- E' F
Department of Periodontics and Oral Medicine 9 Q s, H3 a1 m( C
University of Michigan * S# |( @) \* b4 g! q+ F+ Y
1011 N. University 6 D/ I) c7 V7 J: }- uAnn Arbor MI, 48109, USA : c- z U7 ~0 m; F( I' z' D- OEmail: dkaigler@umich.edu 6 w7 L# y( h O0 oTel: 734-615-4023 , h" [7 A* U4 {% L4 V% c
Fax: 734-763-5503 . b% V" Y c$ m3 CContract grant sponsor: Burroughs Wellcome Fund; Contract grant number: CAMS-1006918. q; p9 _% K; g( E; \
Received 13 May 2010; Revised 21 June 2010; Accepted 23 June 2010 ' N$ w4 U" a4 c' J) L
ABSTRACT; ]. l: Y/ c! @' ^) S4 e2 G* P
Recently, extracted teeth have been identified as a viable source of stem cells for tissue u$ U, z, g: k4 nregenerative approaches. Current expansion of these cells requires incorporation of animal sera; ( ^+ k/ t6 g# h: ~6 m0 A6 M
yet, a fundamental issue underlying cell cultivation methods for cell therapy regards concerns in , t$ G y1 E" q6 t
using animal sera. In this study, we investigated the development of a chemically-defined, + e5 `; q0 n8 Q$ s4 f. A* Oserum-free media (K-M) for the expansion of human periodontal ligament stem cells (PDLSCs) ( ]9 \8 H4 I, n8 V( C; mand human stem cells from exfoliated deciduous teeth (SHEDs). Proliferation assays were 2 h2 L2 b. t0 V( @ y$ Fperformed comparing cells in serum-containing media (FBS-M) with cells cultured in four+ \6 Y! U0 l) Z/ ~
different serum-free medium and these demonstrated that in these medium, the cell proliferation 8 u" b9 r7 j2 H1 i) r5 M; Dof both cell types was significantly less than the proliferation of cells in FBS-M. Additional ; `) R7 O: J3 M m* g& lproliferation assays were performed using pre-coated fibronectin (FN) tissue culture plates and 0 C4 e& n1 r! l# y5 x7 f [/ ?0 zof the four serum-free medium, only K-M enabled PDLSCs and SHEDs to proliferate at higher 6 `$ a+ y# ~4 ^ ]1 } N2 z' arates than cells cultured in FBS-M. Next, alkaline phosphatase activity showed that PDLSCs and SHEDs , F6 ]5 @; L0 g8 ^# Wexhibited similar osteogenic potential whether cultured in K-M or FBS-M, and, 7 S" a6 E9 w* ~
additionally, cells retained their multipotency in K-M as seen by expression of chondrogenic and / p+ B5 B. |9 R: {: W, badipogenic genes, and positive Von Kossa, Alcian blue, and Oil Red O staining. Finally, ( c7 u; z$ L4 {0 |6 v! A0 K
differential expression of 84 stem cell associated genes revealed that for most genes, PDLSCs 1 z5 [) v+ a+ B* E: o7 m( R/ p- L X: Y
and SHEDs did not differ in their expression regardless of whether cultured in K-M or FBS-M. 9 P9 a* b0 t+ m# t* r' E/ ]Taken together, the data suggest that K-M can support the expansion of PDLSCs and SHEDs and 0 B* ?7 @7 r! b c2 |" b8 Imaintainence of their multipotency.+ z7 F+ f/ H7 S 作者: qianqianlaile 时间: 2011-3-20 08:22
原文0 \$ x6 b! O- h7 W4 V# d
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 & p# i4 {6 L% ~, jTarle S.A.,1 Shi S.,2 *Kaigler D.,134 $ K( q& K q' D i# t5 `1 H# n" k
1 MI美国安娜堡密西根大学，牙周病学和口腔医学系； + T1 C6 A& c% a& G. k2 G r- _" s2 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 F: h8 o @8 k, z3 MI美国安娜堡密西根口腔健康研究中心8 y# }6 q3 v" ]% f B) H$ K
4 MI美国安娜堡密西根大学生物医学工程部1 M( z4 ?" t* W* `4 K* M1 F$ S% M0 U
9 q7 a6 |8 z4 `
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 f: e; \: b* p# y2 E* p标注：SHEDs stem cell from exfoliated deciduous teeth脱落乳牙来源干细胞 ) q: c$ X7 Z1 r " h& z+ U/ X, v* t! `图片：5( o/ I! q0 W" b+ N, _6 i- Y
表格：4, p6 b% R( ^7 N3 Y) V# J
通信作者：Darnell Kaigler, DDS, MS, PhD1 U( ]6 J$ ~- N1 W4 y% {
美国48109MI安娜堡大学密西根大学1011N.牙周病学和口腔医学院) T7 _/ s) o- I& F" p/ }! H
电邮地址：dkaigler@umich.Edu% v. o: w9 |1 t1 l
电话：734-615-4023- D6 ^ R/ q- k) c2 e# r$ Y
传真：734-763-5503# c4 O( o8 ^; p" R
合同授予赞助商：伯勒斯威康基金；. M3 ~% h' w" D4 g" G
合同授予号码：CAMS-1006918) Z, d! z! ~! x+ c
2010年5月13日接收；2010年6月21日修改；2010年6月23日发表4 L2 y0 {5 L2 y8 [8 ^" I
摘要 6 X+ R5 g( N$ ~& P0 T& Q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。 8 P# y" U4 d9 f, b; D- Y8 h, a/ U3 X7 w作者: tpwang 时间: 2011-3-20 19:14
本帖最后由 tpwang 于 2011-3-20 19:16 编辑 ( S: Y% W, z- l/ ^0 M
0 A4 R$ r. F! Q% ]* P 回复 qianqianlaile 的帖子' _4 k: L5 g9 N; ~; p4 K2 y
% h5 @7 G- [8 v ]- ]
都是自己翻译的吗？细节锻炼也很重要。两个小地方：9 c; g& p' c2 e, h) o$ K3 f
Original 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。' ? a" l* O6 n
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。! D; a$ r' ?' z8 Q
: i: j7 @1 m% U. Z9 a
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 W' | x6 I+ y& e1 ?. f# iDevelopment of a Serum-Free System to Expand Dental-Derived Stem Cells: PDLSCs and SHEDs. v9 O, ?( r% s3 C1 L" U _7 Q
“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。# Y* i' j' R" k3 L" q+ O
# i2 _! H9 e! ?$ L; T% g+ ~ s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. 6 H3 V. \+ j: Q7 C3 a
“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。” 8 F4 O; q8 U0 ~% @2 U/ y! @4 y! w! s& k# j# A7 O$ z
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。( h q, w$ z3 C, o$ R3 z& j
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. : r" x) s# O/ L
“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。 3 y6 i6 f; k# Y- h : V: m( ` y0 J* X# P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。 ) d/ N& R# l& J: t% r, M' P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）。 , p, X1 Y2 V8 d/ w2 m" y( N! A- ?, B' s! {9 B
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。 4 v1 J" w; ]; F: ]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.2 H7 o. ]: ]! l+ D! N! ]2 `* Q Q
这句翻译的不准确而且拗口。differential expression不是指“不同的表达”。, ?- k8 R4 ]" B) o& @) C
+ Q. {# j6 T. k% \
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。3 | M+ S% T. z! r
Taken together, the data suggest that K-M can support the expansion of PDLSCs and SHEDs and maintainence of their multipotency. * ^( _' N4 p" g* z3 U“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。”; P- E4 v# p! `
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: Q0 t; J. R) C* \7 ~+ d. ^
初次修正译文如下： 0 Y* m, R) u0 A" R2 C( U原发研究文章. c; y- z$ c$ p b
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 ' `/ Q1 z5 M8 GTarle S.A.,1 Shi S.,2 *Kaigler D.,134 , l" B6 R6 f6 I) Q$ c$ u$ |
1 MI美国，安娜堡，密西根大学，牙周病学和口腔医学系； 8 E$ z4 K3 }6 P" o$ R5 p/ U( F2 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; z" }" ]* v7 h5 n3 f0 e3 o, t/ w
3 MI美国，安娜堡，密西根口腔健康研究中心 / z* S& \. L* l2 F) }3 M4 MI美国，安娜堡，密西根大学生物医学工程部 ) e7 i, P4 O8 Y; `; l2 Y# U' b 5 L+ o% G2 _6 n# \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 % y$ q( E, ~, ]* @7 j( k# s/ Z标注：SHEDs stem cell from exfoliated deciduous teeth脱落乳牙来源干细胞 1 g8 {6 W. \. j' ]# ^( H& K* p1 W r * e! }7 u, b6 O# S# u图片：5 " w8 ^; Z8 G3 W/ ~9 I O表格：4 / J* T8 ~; l$ }) a通信作者：Darnell Kaigler, DDS, MS, PhD/ H! O2 l1 A; ~* H4 b4 K: l
美国48109MI，安娜堡，密西根大学1011N.牙周病学和口腔医学院 4 R. G+ j9 m0 t3 \# y8 J% ?电邮地址：dkaigler@umich.Edu& E- m3 b) G6 |/ x0 w
电话：734-615-4023 2 u( r/ \: g! a* N o: z传真：734-763-5503 * A# ~! g* K4 X( y3 ?8 _2 m6 z合同授予赞助商：伯勒斯威康基金； g6 X% s; g; {/ B; u0 s- R
合同授予号码：CAMS-1006918 " Y/ r; k/ P. T2010年5月13日接收；2010年6月21日修改；2010年6月23日发表 5 P2 {) _2 t4 i摘要 5 s& m, U5 L! t( `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。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疗法所采用的细胞培养方法中需要克服的一个基本问题。本研究中，我们探索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）的无血清培养基的建立。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，四种无血清培养基中仅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。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。7 E; J7 Q. Q- K- s! I& B1 z: ~& L 作者: tpwang 时间: 2011-3-20 23:03
回复 qianqianlaile 的帖子 : A9 |. J: W2 K% N9 p3 Z# {2 a ! F( |+ P5 C3 m3 g8 Pdifferential gene expression是一种方法，翻译为“差异化基因表达”或“差别基因表达”。因此，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.这句的翻译应该为：+ p C$ B$ ?8 v$ A e5 _3 }
) z4 Z& B3 D. t7 Y( {4 e n8 b( t
最后，84个干细胞相关基因的差异表达分析表明，PDESCs和SHEDs的大多数基因的表达在K-M或FBS-M这两种培养条件下均没有差别。9 g5 g4 O* R1 z5 E. V; E6 A
8 r o8 L1 `( H1 r! n8 Q一是说两种细胞没有差别，二是说两种细胞在两种条件下没有差别。 - r l% S3 a. i7 }% X* y2 l r+ V3 s9 q0 k* b" @% s
这一系列文章有原文吗？: v- |9 V+ A; j. t8 N5 O0 ] 作者: qianqianlaile 时间: 2011-3-21 09:29
本帖最后由 qianqianlaile 于 2011-3-21 11:29 编辑 % X& v4 [4 b! x* R& ? w/ D1 ]& {; s. {4 `2 K; p5 i! N
INTRODUCTION: C* K8 {" B6 y1 g4 b6 u
Cell therapy has tremendous potential in regenerative medicine, yet, there are concerns in % v! G a7 R8 K J
the utility of cell therapy due to questions regarding different cell harvest and cultivation & _0 S# @+ n2 n1 L5 Y4 @$ C6 f o8 D
methods (Haack-Sorensen et al., 2008; Mannello and Tonti, 2007). Bone marrow derived , {% Y# h3 h2 R: |& x# Astem cells have been identified for a number of years, and there are a number of . l# }9 Z/ ~6 j, F# O' Uongoing clinical trials exploring the safety and efficacy of their use for a number of : N" d6 D; V) q( U
clinical applications (Battiwalla and Hematti, 2009; Sadan et al., 2009; Satija et al., 9 P0 M+ O# L0 E( a+ Z9 D t& L( U2009). There has been an increased interest in recent years in the potential of oral- . K/ j# i: p! G7 ?; T3 Q& ^derived stem cells for cell therapy, primarily because they can be derived from a readily $ i- ]8 i( h6 g% {* ?% g2 Gavailable source, extracted teeth (Gronthos et al., 2000; Miura et al., 2003; Seo et al., " c$ e& q; O0 X
2004). These cells exhibit multipotency and regenerative capacities characteristic of ' @% Q* u9 p' ]1 N, P. K: U: Z) p
mesenchymal stem cells (Batouli et al., 2003; Shi et al., 2005) and have the capacity to - J" G8 F3 {+ M+ D8 P7 hrepair and regenerate tooth structures in vivo (Krebsbach and Robey, 2002; Mao et al., 2006). N4 e' c) r5 a6 { Because oral-derived stem cells have been more recently identified, clinical 1 o e; V+ i8 \" J* n2 o
protocols are still being developed for their use. Regardless of the specific protocol , ]6 Z. |' i7 Vused, most current cell therapy approaches rely upon ex vivo cell expansion in order to 4 l0 S4 G' B. x% r( R! N1 g* ~
produce sufficient cell numbers for transplantation. Though a wide variety of protocols ! R) Q( ]4 E1 F- v
and culturing methods exist, one common aspect to most of them is the inclusion of # U# X0 I# r* T& r) m0 B3 ]animal sera for cell expansion, in that it contains a rich source of nutrients and growth " {4 x* ^7 k/ n ?factors (Mannello and Tonti, 2007). Despite the widespread standard use of animal * [1 J7 _" ?0 n. e0 G. D; [5 ysera for in vitro cell culture (Freshney, 2000), there are several problems which exist / u$ J" r- u+ q: s% s
relative to its use for clinical application. 7 A* e8 f$ Z2 k. G: [7 @
One of the central issues regarding limitations in using animal sera for clinical cell / T: C* K2 l9 O% ntherapy protocols is that its components are highly variable and, in many cases, 2 x4 j% b& P0 @% B/ d# t& _. junknown. Though components of sera have identified, it has also been demonstrated 8 ]7 F* O0 s; B6 }! I
that consistency between different lots cannot be assured (Price and Gregory, 1982). In : ~8 P/ j( O1 C1 \the context of multipotent stem cells, serum components and concentrations have % {9 K9 H2 ^ b+ a+ c7 e) u5 Hsignificant impact on cell survival and proliferative capacity, phenotype, and multipotent / n' y$ `0 `1 b9 A" ]2 Zpotential (Agata et al., 2009; Sotiropoulou et al., 2006). Additionally, for clinical use, the ' J* }) {4 e7 s4 t
inclusion of xenogeneic serum for cell expansion carries immunological risks associated ( s: C2 J. Z( [7 m. T
with the immunogenicity of serum proteins and the potential of transmission of prion 0 D5 x) D, s0 D6 f* e$ q
diseases and zoonoses (Shahdadfar et al., 2005). These concerns have led to efforts 9 g9 F! x! j/ j) Y7 R( l7 X' m ?0 e
aimed at incorporating FBS alternatives in cell expansion protocols, including the use of $ m# u6 \; B' `4 c- W g
autologous and allogeneic sera, and the proprietary manufacturing of serum-free media $ O0 o! |5 {% A# F9 c- @
formulations by different companies (Nakamura et al., 2008). Even with these approaches, 0 r) r5 E7 H4 T there are limitations in the availability of both autologous and allogeneic " t: M e3 n5 s" I; s
sera and companies do not freely disclose their proprietary “serum-free” media 5 B: p$ x7 @5 e: L" h& d3 ^components. These factors not only prohibit clinical translation, but also limit " d: E4 i6 Y$ ~( swidespread use and study of more basic fundamental questions regarding specific d6 q, h' s" q0 h/ t* ]
mechanisms involved in the modulation of these media on cell function. As such, there & y9 J- O: O% d; \+ u9 F% H- o
exists a need for the development of chemically-defined media which can propagate the ) t* }5 X- I+ z" hcultivation of stem cells without adversely affecting cell function and phenotype ! \7 O4 Q, _8 @4 b8 e* J(Mannello and Tonti, 2007). # |' r i' S0 O3 BIn this study, we aimed to develop a serum-free media (K-M) for the expansion o. J' `( q- r" l) R1 e
dental-derived stem cells, including stem cells-derived from exfoliated deciduous (baby) 9 G$ K4 \/ N, H0 t7 ]5 Lteeth (SHEDs) and periodontal ligament stems cells (PDLSCs). Cell expansion in this 0 K4 K$ E! o) y3 _) l
media was compared to standard FBS containing media used to culture these cells, & m: p6 ]" {" b" o/ K: \0 d5 Aas well as three other serum-free media formulations (two of which are commercially available) : u8 q$ [4 P5 i. O
used for culture of mesenchymal stem cells. Additionally, through 5 |8 P1 s7 V4 w0 T2 D! Bdifferentiation assays and microarray analyses, multipotency and differential gene 8 d8 J7 @3 C' ~2 j. T1 d
expression of 84 stem cell associated genes was examined between cells cultured in K-1 X2 p; z/ X4 q( I( K7 y4 t5 U# N
M vs. those cultured in FBS- containing media.作者: qianqianlaile 时间: 2011-3-21 12:38
引言; D. F M6 a% F Z1 S
细胞疗法在再生医学中有着巨大的潜力，然而不同细胞收获和培养方法带来的问题使得研究者们担忧细胞疗法的效用。通过数年的努力骨髓源性干细胞被鉴定，而且大量正在进行的临床试验探索着该细胞用于临床的安全性和效能。近年来研究者们越来越关心口腔源性干细胞用于细胞治疗的潜力，主要是因为该细胞能从拔除的牙中分离获得。这些细胞拥有间充质干细胞的多向分化潜能和再生特性，并能在体内修复和再生牙齿结构。因为口腔源性干细胞最近被鉴定，所以其用于临床的操作仍在建立中。无论使用何种特殊方法，最新的细胞疗法仍需依赖于体外细胞扩增为移植提供有效的细胞数量。虽然有多种程序和方法培养细胞，但大部分都用含有丰富的营养和生长因子的动物血清培养扩增细胞。尽管用动物血清培养体外细胞是普遍的使用标准，然而动物血清的临床应用还存在很多问题。 4 C0 W7 @. o, H: m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。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。用血清培养多能干细胞，血清组成成分和浓度显著影响细胞的生存和增殖能力、表型及其多能潜力。另外，将外源血清扩增的细胞用于临床会带来免疫风险，如血清蛋白的免疫源性和朊病毒疾病的潜在感染、人畜共患疾病的传播。这些担忧使研究者们致力于用胎牛血清的替代物来扩增细胞，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。即使用这些方法，自体血清和同种异体血清的效用仍有限，而且商家无法详细说明他们专有的“无血清培养基”的成分。这些因素不仅妨碍了临床应用，而且限制了其广泛使用以及对这些培养基调节细胞功能的具体机制相关基础问题的研究。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。1 }& _& o, L/ W) Y
本研究中我们研制了一种扩增牙源性干细胞即脱落乳牙来源的干细胞和牙周膜干细胞的无血清培养基（K-M）。比较了K-M培养基、常用标准胎牛血清培养基和其他三种常用于培养间充质干细胞的无血清培养基中细胞的扩增情况。另外分化实验和微矩阵列分析检测并比较了K-M培养基和含胎牛血清培养基培养的细胞的多潜能性和84种干细胞相关基因的基因差异。 - |/ j% Q5 }8 x W+ r. ?2 y" R作者: tpwang 时间: 2011-3-21 21:22
; b7 Z$ b! g# T# E* d* w" \8 m回复 qianqianlaile 的帖子$ f1 `6 a) |6 v8 Z% ~ B* p
9 W# P% g! E2 F) s: b" `$ H这篇翻译的中间一段问题比较多，感觉把握的不够细。 , n) B' B4 G" N0 N : \, a9 }6 q0 f- `% J. w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。9 Y8 V+ J6 R8 ]0 N
One 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. 9 d. \* [8 k$ }, Q # F4 F7 f x2 ~: r# 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。 * |1 h( R& F4 }5 i2 [* @, q6 K5 A0 u: `% Q/ r3 R' M# s0 p! ~7 _
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。6 J J3 E# G- s
Though components of sera have identified, it has also been demonstratedthat consistency between different lots cannot be assured (Price and Gregory, 1982). - y/ ^% {8 A% |* _. M' { 5 S3 S! `- z8 k" B5 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。/ }* i9 U) U5 z# [7 q8 E# I
0 b/ {& N% B( x/ F' }
另外，将外源血清扩增的细胞用于临床……# n3 F A' _; Z
Additionally, for clinical use, the inclusion of xenogeneic serum for cell expansion...4 [- d$ V( _- X; [! \* c* a7 ~, q2 D
& J. ]/ R3 B2 x* K" ~ a4 ?+ z0 K
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。2 L/ Y* ~0 r! M# R/ A1 y$ H
, i' h# A: C- r8 f, h; \
……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。 . V5 F3 B ^2 C. M6 p/ W6 @, I! U0 rincluding the use of autologous and allogeneic sera, and the proprietary manufacturing of serum-free media formulations by different companies 4 L4 ]6 F4 r/ o! w5 B% V( N+ a - R U- _2 d8 I$ T8 X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。 ' Z, M# v) Q6 K) c& r$ ]1 l. e0 Y' R* B; v9 Z( |5 l: a4 M! S4 t
……而且商家无法详细说明他们专有的“无血清培养基”的成分。 w& N5 `! c' q# C1 Q5 P...and companies do not freely disclose their proprietary “serum-free” media components., q2 O1 u% [( |; u S- H
. C7 \3 ~6 [- p4 P“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。 ; L3 c8 \! D3 s: g9 ~+ } % @, d5 _" J9 C% r+ p7 K$ o' b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。# _, V0 Y/ l5 G# q% O3 B
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./ u, b) k% x8 V
/ i) P, x8 i' P回复 qianqianlaile 的帖子 ( ?4 v5 D( L2 {7 [. l, t4 {" Q w+ \7 m$ \( e9 v p! l
没有充分通读原文就看一句翻译一句，其次翻译完后没有耐心地修订，这是通病。不养成好习惯的话，还不如不练，因为反而会把不好的习惯固化下来，以后就积习难改了。 * [6 p# d7 G# c& V; s( }4 e' P3 D9 x
大部分的文字都是“自圆其说”，意思是通过上下文都可以读通。尤其是科技文献，逻辑性非常强。本来读文章是有规律的，即一定要一段一段读，不要一句一句读，尤其不要半句半句读。遇到一下子不通的地方，先顺下去，通过后面的内容来理解和验证前面的疑问。比如这一篇里面的proprietary，即使一下子不知道该如何理解，但往下读到下一句，就应该猜个差不多，然后再查字典等确定准确的意思。不少人阅读的毛病，尤其是读英文，只会从头一字一字往下读，不懂得从后往前“读”。作者表达一个意思往往不是一句话就能说明白的，需要一段甚至在文章结尾才能说清楚。古代有倒背如流的典故，其实说的就是这个道理。英文泛读练习规则里很重要的一条，就是要限定时间，逼着读者“跳过”暂时不懂得地方往下读，然后把握总体意思的基础上再来理解不懂得局部。翻译这种精读不存在时间限制，反而不容易训练整体把握和局部理解的技能和习惯。; {* E8 P$ Q8 n; l7 s6 Z
7 R, H9 E( i! h" q8 F1 _从另一个角度来说，即使自己水平有限，还是有很多主观能动性可以调动的，比如把自己没有把握的地方标注出来，把自己的想法和疑问提出来。一来方便征求别人意见，二来自己思考过了才有真正的进步积累，三来表示自己的诚意。要是将来投文章，这些基本的非内容错误编辑是不会给你改的，直接就退了。 * l, p0 ~9 F# A, P1 J; t. I. W. k3 i! d1 |* W
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。5 f; A* m. j! G* E: I8 G
$ ?1 n, n; J9 z2 }9 D. B. d+ Y$ tMATERIALS AND METHODS # W+ x. F, y- a% M, mIsolation of Dental-Derived Stem Cells (PDLSCs, SHEDs) . \- }3 U; _/ \9 X( u/ _( mPDLSCs and SHEDs were harvested as previously described (Miura et. al, 2003; Seo et. 9 {! y) C; {1 S( f
al, 2004). Briefly, PDLSCs were scraped from the root surface of a tooth into a p60 dish + _4 E1 T2 v9 g xcontaining minimum essential alpha medium (DMEM, Gibco) and SHEDs were harvested by 9 C& Z' q3 M" z: T' U/ u! L' o
scraping out the dental pulp tissue from a deciduous tooth into a p60 dish containing DMEM., m; A4 Q8 |# E, I; Q) ]; n0 O
After collection, the cells were centrifuged at 1600 rpm for 5 minutes at room temperature. The ( N8 s. b/ `8 e# o/ L- Q" k1 c
supernatant was aspirated and the cells were resuspended in a phosphate buffered saline (PBS; 7 ?) Y+ `, G% `' m+ M5 ^. s
Gibco #14190) solution with 4 mg/ml Dispase II (Roche #04 942 078 001) and 2 mg/ml: r) d' w' W1 y! @, _# Z. V5 j
Collagenase Type II (Worthington # LS004196) and incubated at 37°C for 60 minutes. The S) l6 n8 H5 y9 x; ]. y- N3 }( d
enzyme solution was inactivated with 5 ml of DMEM- 15% FBS- 100µM ascorbic acid 2 8 s h) @- n( `- \6 e1 ~
phosphate (ASAP, Sigma A-8960) and centrifuged at 1600 rpm for 5 minutes at room6 B1 H4 v6 q/ K; f; V
temperature. Cells were resuspeneded in 5 ml DMEM- 15% FBS- 0.1mM ASAP and transferred 3 F6 ~4 g4 ~/ k: \6 |! \0 |) q) }
to T-25 flasks. Media was changed the next day and then every 2-3 days. 8 D0 x1 D2 p+ e' k' n( g& w% H Cell Culture 6 k( b1 w, A* }$ N Cells were expanded in culture in DMEM, Iscove’s modified Dulbecco’s media (IMDM, 9 O4 a" l! c U
Gibco-Invitrogen #12571), Gibco Stem Pro Mesenchymal Stem Cell Serum-Free Media 7 y7 y; u4 X& A! O. |2 ^$ }
(MSCSFM; Invitrogen# A1033401) or Lonza Therapeak Mesenchymal Stem Cell Growth # W3 Z# F/ k- T d% x% k1 eMedia- Chemically Defined (MSCGM-CD; Lonza #00190632) and grown in a 37°C humidified - u- R' q9 X, B3 p m! Z. t( Ntissue culture incubator at 5% CO2. Media formulations are as follows: DMem (Gibco- 7 o, j( e: K7 U3 w; L- ^Invitrogen #12571) with 15% FBS (Gibco-Invitrogen-16000), 100µM ASAP and 5 µg/ml & }8 X+ B$ y: o! r% Y: [3 W0 tGentamicin (Invitrogen # 15750060) (FBS-M); DMem with 2% bovine serum albumin (BSA; 9 D# A4 T9 e* A' S* R/ d9 j% GSigma A7888), 10ug/ml human insulin (Sigma), 4ug/ml low density lipoprotein, 200ug/ml ( l* w( j# {6 Q* q! itransferrin, 10 nM dexamethasone, 100 uM ASAP, 50 uM ȕ-mercaptoethanol, 5 ug/ml 4 |2 H) u* J8 Wgentamicin, 10ng/ml platelet-derived growth factor (PDGF; Sigma), 10ng/ml epidermal growth 8 v5 V9 X+ H1 X9 |0 ^0 x% wfactor (EGF; R&D Systems), 10ng/ml basic fibroblast growth factor (b-FGF, Sigma) (SDM); ! s- s% x% w+ }, t' R3 AIMDM with 0.2% BSA, SITE 3 (Sigma #S5295), 384µM ASAP, 10 ng/ml PDGF, 10ng/ml , F+ H* X4 a* E) r* {' Hhydrocortisone 5ng/ml b-FGF, 1 ng/ml EGF, 10-7( Q7 u0 x8 j( r1 b3 L, l C3 c; w
mgm/ml parathyroid hormone (PTH) and 5 - S/ b: K0 x. Q# m3 _; }1 W% b
µg/ml gentamicin (K-M). Media on the cells were changed every 2 or 3 days. Cells were grown t8 t3 s& z: ^7 ?0 Oin T-150 flasks to about 80% confluency then media was aspirated from the flasks, cells were 1 r" L5 F! N# U# {) ]- l4 dwashed with PBS and trypsinized with TrypLE Express (Gibco#12605) before being split into 12& G& ?! p6 T3 j+ O8 P7 c
well plates for the assays. / Z. w" a: \. H' F( r Fibronectin Coating of Tissue Culture Plates0 i$ m0 [; ^, X# \/ Y
Fibronectin (FN) was coated on the plates and flasks to provide growth and attachment ( m2 `( H+ r8 H. s1 l8 T1 dsupport for cells grown in the serum-free, IMDM media. For the 12 well plates, 0.1% FN . V4 w* T4 L+ Y. L5 ksolution (Sigma F-1141) was diluted in PBS so that each well received 3.8 micrograms per well ( r; ^4 ^2 K: V8 z. H" b* s5 K- a( \; K(1µg FN/cm2). The T-150 flasks were coated so that each received 150 micrograms of FN (1µg 1 g) t5 \" S# H6 }6 b7 NFN /cm2). The plates and flasks were tilted back and forth to ensure complete coverage of the ) }8 M% Q5 J8 `; I; c
FN solution. The FN coating was allowed to stand at room temperature for 90 minutes. The FN 1 C8 f! Q7 m$ v- w% `solution was then aspirated before the resuspended cells were transferred to the flasks and plates. + q6 X$ D, u0 ~+ ~) b* t# a6 j4 ?Proliferation Assays 6 P0 Q; g# z9 F7 V4 ~After trypsinization cells were resuspended in an equal amount of the appropriate media% V. I" z! ~$ a. {$ j3 h# e2 y
before an aliquot was removed for counting on a hemocytometer to determine the concentration. + a2 G, J+ e3 z5 Q1 C: E! DThe cells were then centrifuged at ~1600 rpm for 5 minutes at room temperature. Cells were # m) ^& F' n, W% M2 O4 f4 Cresuspended in the appropriate media at a concentration of 3800 cells per ml. One milliliter of ! `$ b/ X' v2 Q! ?! C
cells was dispensed into each well of a 12 well plate. K-M plates were precoated with FN # K6 Q" L C! i. Y/ Isolution (as outlined above). Four plates for each cell type and media condition were plated and ; F5 W- \3 r8 a5 y* v* Scounted on a hemocytometer at days 1, 3, 5 and 7 to determine the cell numbers within each , t4 t5 p0 Q; q' }1 \- `( z5 X/ F
well. All experiments were performed in triplicate. 5 q* J- ?/ j3 K% {' k$ m RNA Isolation and Purification for MicroArray 7 `# T% u: w4 f. Z
PDLSCs and SHEDs were grown in T-75 flasks to 80% confluency before the cells were + F$ A. j$ u& O: ]1 u% H
harvested for RNA. The Trizol method (Invitrogen) was used for RNA isolation. This involved 0 I. r2 M; t3 L$ L; M& M
washing the cell layer with PBS, adding Trizol directly to the cells and transferring this cell " d2 n/ T7 p; [suspension to polypropylene tubes. RNA was isolated from the cells by a Trizol-choloroform3 T2 ^, p+ C5 D
extraction, isopropanol precipitation, an ethanol rinse and resuspension of theRNA pellet in 5 Z$ A0 ^% \( X) n
Diethylpyrocarbonate (DEPC) water. The RNA was further purified by column2 a1 f Y; ^( P$ r t- g# \* z5 i
chromatography, following manufacturer’s instructions (Qiagen RNeasy Kit # 74104), and # G8 B& x* l' _% O+ V$ K
resuspended in DEPC water. RNA concentration was determined by the 260/280 absorbance 2 Z/ O* y3 m0 U1 q/ j, qmeasurement using a Beckman DU540 spectrophotometer. 4 @( d- V& q* H7 v$ v3 WIn Vitro Multilineage Differentiation 0 a+ |6 _' p6 c# [
Multipotency of PDLSCs and SHEDs was determined through lineage specific 3 a2 R: Q1 J1 s& k* Aosteogenic, chondrogenic, and adipogenic induction, according to previously described methods5 h+ L; A7 }. J# F. |7 u" b" I
(Pittenger et. al, 1999). Briefly, cells were plated at a density of 30,000 cells per well in 12 well + O5 X' f! L M/ z0 l" `3 p! K
plates. At 80% confluency cells were induced with osteogenic [Growth media plus 5mM E-1 z; g8 \1 @: v% X- z+ {
glycerophosphate, 100nM dexamethasone, 50µM ascorbic acid 2-phosphate] or chondrogenic ! _, x% ?: X' U7 M6 {; b
[growth media plus 50µM ascorbic acid 2-phosphate, 100nM dexamethasone, 5 µg/ml human2 A2 J; p* A' I: ^% e' D; n
insulin (Sigma I-9278), 1 ng/ml TGFE, 400µM proline, 1X Non essential amino acids] or 1 R) i0 M; ?0 l% Cadipogenic [growth media plus 0.5mM IBMX, 1 µM dexamethasone, 10 µg/ml human insulin, % Q" ~, a6 V W4 k. @8 G
200µM indomethacin] induction media. Cells were grown at 37°C in a humidified 5% CO27 ^, J/ g5 V6 V' w' w' K$ o* {
incubator. The media was changed every 2-3 days. At three weeks the cells were fixed and : V5 d+ _- }( s& E+ q
stained as outlined below. ' j) @; {0 e9 K: w0 v Multipotent Staining of PDLSCs and SHEDs ) O4 F$ B" i5 l9 A! O$ v. HTo identify the mineralized nodules, induced PDLSC, SHED and DPSC were fixed in 4% c8 G! S1 N4 C5 I% ]' [+ X, Zparaformaldehyde for 30 minutes, immersed in fresh 5% silver nitrate and incubated in the dark . V' _% b2 y/ Y' s
for 30 minutes. After washing in water the PDLSC, SHED and DPSC were exposed to " m0 D- t9 l* [ H% w$ _5 Z! U
ultraviolet light for 30 minutes followed by a four minute incubation in 1% sodium thiosulfate to ; x) ?5 ^6 |1 t3 z- s' d3 [7 f: t
neutralize the silver nitrate. Cells were washed twice with water before 1 ml of PBS was added 4 @/ Y. b# O( d
to each well and viewed. Plates were stored at 4°C. 8 }/ S9 M. S! P! M
To detect chondrogenic differentiation induced PDLSC, SHED and DPSC were fixed in 4 c6 |! B1 \9 Q) p6 q# k
cold 100% methanol for 30 minutes and then exposed to 1% alcian blue in 0.1N HCl for 30 0 u9 h0 a4 d# L3 D* H$ `
minutes. Cells were washed twice with 0.1N HCl before 1 ml of PBS was added to each well + m& Q, @( F# c: [/ Z
and viewed. Plates were stored at 4°C.* [ G7 R% v' q" v: D5 v
To detect adipogenic differentiation by identifying lipid vesicles, induced PDLSC, SHED " K- c# c3 l1 F3 x9 M& [
and DPSC were fixed in 4% paraformaldehyde for 30 minutes, and then immersed in 0.3% oil 5 u. _( |, h; \% m/ G. [- w8 @/ M
red O solution for 30 minutes. Cells were washed twice with water before 1 ml of PBS was # _; }1 v9 c' s. g1 g0 nadded to each well and viewed. Plates were stored at 4°C. - d: T; f4 ~' p! IAlkaline Phosphatase Activity and Detection , T) H6 _$ h0 JEarly osteogenic differentiation was detected and quantified by the alkaline phosphatase 2 e) z" ?' j3 O! z- L, W(ALP) enzyme assay. Cells were plated at a density of 30,000 cells per well in 12 well plates.) K. o4 L/ |6 J$ v/ x
At 80% confluence, cells were induced with osteogenic media as described above. The media& {6 W) C+ Y+ w7 z h: W5 D
was changed every 2-3 days and after one week, ALP activity was measured. . q0 q' d& e S3 ^ o+ x; J$ V2 ]8 aTo detect phosphatase activity, PDLSCs and SHEDs were fixed in 70% ethanol for 30 4 l# D) \0 n) Q& @6 }! ^
minutes. They were then incubated with freshly made substrate containing naphthol AS-TR & O1 q4 D" p8 L" wphosphate (Sigma) and Fast blue (Sigma) for 30 minutes. Cells were washed twice with PBS then * h% s' ?1 p4 T0 k' @6 [0 V/ g$ ~
viewed or stored at 4’C. , H$ x& k) A% Q8 _2 @2 MTo quantify the ALP activity and normalize the results, cells were lysed in Passive Lysis ; F) }. n! d0 S8 F* s
Buffer (Promega) according to manufacturer’s instructions. Cell lysates were then sonicated, * p! v6 g H4 u# y' w3 ^and centrifuged (10,000 rpm for 10 minutes at 4°C). The supernatant was recovered for the , d8 O' p: z4 H& y
quantitative colormetric ALP assay (Manolagas et al., 1981) and the cell pellet was used for % Z+ f+ g( w: B" Z
DNA isolation and the determination of the DNA concentration using the Quant-iT™ dsDNA & c+ V% ? j% |! w3 }; v# _$ d
BR Assay (Invitrogen) per the manufacturer’s instructions. . C% o' {4 }0 v5 M$ G$ a. d7 x Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) 5 d9 C; Y- @# _+ W9 {To confirm chondrogenic and adipogenic differentiation, total PDLSC and SHED cellular - i' y' C6 W2 m8 w% oRNA was extracted, reverse transcribed, and amplified using osteoblast specific gene primers. 9 P3 A7 ?5 `" }3 `/ zMedia from the wells of induced and uninduced PDLSCs and SHEDs were aspirated. Cells % c& N1 O; ^4 C! H, L' u
were immediately resuspended in 1 ml of Trizol (Invitrogen) and RNA was isolated according to 5 g r5 x+ x- k+ r, P
the manufacturer’s instructions. Synthesis of cDNA was performed using Invitrogen’s ; G' U; O, \2 DSuperScriptII kit and oligo dT. PCR reaction components and concentrations were as described 7 x7 s, n# ^& ?3 y k4 B7 ~$ F
in the Invitrogen Platinum Taq polymerase instructions using the primer sets below. An MJ % g1 c: x8 Y1 j6 cthemorcycler was used for the following two PCR reaction conditions: ( o) g- v6 {. z3 u
*94°C 2 minutes [94°C 45” 56°C 45” 72°C 1’] X 35 cycles 72°C 15’ $ s" Z! }" n) M' n
or' ]; I, }' E" C
**94°C 2 minutes [94°C 45” 67°C 45” 72°C 1’] X 35 cycles 72°C 15’ ! @. z! {9 x: t% ^5 YPCR Primer Pairs . [- C" |0 \: H% p9 X- }4 gPrimer Name Primer Sequence Product) i% N) H/ g4 r2 g6 J6 P
Size 7 `9 N( j4 [2 r3 u X7 V( t* a' kAccession+ ^; t3 z* ^# m0 L8 k& S
Number 1 B+ \3 R3 c& C% m6 _*GAPDH FWD AGCCGCATCTTCTTTTGCGTC 815 bp NM_002046" |- {! ^, D! |6 X! X
*GAPDH REV TCATATTTGGCAGGTTTTTCT ! B7 D$ r. A( |3 l( NPPARJ2 FWD GCTGTGCAGGAGATCACAGA 226 bp NM_005037 ( f: |1 C7 C' }5 u$ w) q8 ]9 F _PPARJ2 REV GGGCTCCATAAAGTCACCAA 3 U: s; b2 q1 [9 S) W5 T0 G+ rLipoprotein lipase FWD GTCCGTGGCTACCTGTCATT 212 bp NM_000237 z0 a; I- i, g0 K2 C5 x; E* X
Lipoprotein lipase REV TGTCCCACCAGTTTGGTGTA9 X* I( y }& o- _
Sox 9 FWD TTGAGCCTTAAAACGGTGCT 224 bp NM000346 . i5 p; F3 M; I/ f8 L% NSox 9 REV CTGGTGTTCTGAGAGGCACA9 k1 K; g0 o/ w- r2 g
Type X collagen FWD TGAGCAGCAACGTAAAAACG 471 bp NM_000494 l( i5 {& t( T4 i5 W+ V6 b
Type X collagen REV AGGAAATGCCGAGTTTCTCA 1 A+ `1 n4 _, x1 @Statistical Analysis ) @9 g8 C( V$ I" R' U. TStatistical analysis was performed with the use of Instat software (GraphPad Software, San " J( K: w& g* {+ f: Z5 J S* Y5 jDiego, CA, USA). All data were plotted as mean ± standard error of the mean (SEM), unless i( C) d8 ~3 Q. }
otherwise noted. Statistically significant differences were determined by two-tailed Student t$ z; {1 Y& M0 W
tests, and statistical significance was defined as p < 0.05. 作者: qianqianlaile 时间: 2011-3-23 12:10
: Q0 t; J. R) C* \7 ~+ d. ^
，脑子里也没有独家生产这个概念
，所以没理解。, Y" @0 g! x( l+ c! V