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In Vitro Differentiation of Mouse Embryonic Stem Cells: Enrichment of Endodermal

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发表于 2009-3-5 10:50 |显示全部帖子
a Department of Surgery, Stem Cell Therapy Center, Soonchunhyang University Hospital, Seoul, Korea;% G& G9 M: X7 U% j5 J7 e8 p
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b Division of Genome and Proteome Research, National Genome Research Institute, NIH Korea, Seoul, Korea;* r+ m8 n# u3 X, T0 G
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c Division of Genetic Disease, Department of Biomedical Science, National Institute of Health, Seoul, Korea;& j2 {1 Y+ f) h- p- V3 m4 W
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d Department of Pathology, Hanyang University Hospital, Seoul, Korea;* r5 @7 B4 x8 S! F/ u2 I
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e Department of Biochemistry, College of Medicine, Ewha Womans University, Seoul, Korea;+ M  O. C/ Z  g9 E
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f Department of Animal Biotechnology, Graduate School of Bio & Information Technology, Hankyong National University, Ansung, Korea;+ L  m& u$ D; e0 [

" K7 c' w+ W( g+ K% R0 zg Department of Surgery, Hanyang University Hospital, Seoul, Korea7 z- L5 Q% u8 S3 o
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Key Words. Embryonic stem cell ? Embryoid body ? Differentiation ? Endoderm, @: \& i5 a# `6 y6 p$ c/ s; C+ H
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Correspondence: Bermseok Oh, Ph.D., Nokbun-Dong 5, Eunpyung-Gu, Seoul, 122-701, Korea. Telephone: 82-2-380-1523; Fax: 82-2-354-1063; e-mail: ohbs@nih.go.kr% ]5 n& B3 D( L' w' x
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ABSTRACT
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" R) a! F4 b8 D7 K+ zEmbryonic stem (ES) cells derived from the inner cell mass of mammalian blastocysts are well known for their potential to maintain the undifferentiated state throughout an extended number of passages . Upon proper stimulation, ES cells differentiate into cells of various lineages, which can be used in cell replacement therapy . Transplantation of neural cells derived from mouse ES cells successfully rescued defective neurons in the central nervous system, proving their potential value in the treatment of neuronal diseases . More recently, ES cells have also been shown to differentiate into insulin-secreting ? cells treating diabetic animal . The development of human ES cell lines has widened the potential usage of ES cells even further, providing an excellent source for cell replacement therapy in various human diseases . Along with the technical progress in the cloning of mammalian cells, it is now quite conceivable to generate patients’ own ES cell lines to develop stem cells of desired lineages .9 Q1 V8 b, z$ [& m  c+ z& N# y
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To prepare ES-derived cells relevant to clinical situations, mouse ES cells have been experimentally differentiated via in vitro suspension culture into the embryoid body (EB), a cell clump comprised of all three germ layers. Various types of differentiated cells, such as neural cells, cardiac and skeletal muscle cells, hematopoietic cells, adipocytes, chondrocytes, and osteoclasts, are found in the EB . Because differentiation of ES cells has been known to recapitulate changes in the embryonic development, factors that partake essential functions during early embryogenesis are also expected to be involved in the formation of EBs. Yet because of the short cultivation time and dearth of information about the cellular characteristics of EBs, little is known about the process guiding the development of three germ layers and specific lineage of cells within the EB.
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* r% z; O! I4 _) n$ q! {/ b( yCell–cell contact mediated by various adhesion molecules and apoptosis play an important role in the early stage of embryonic development. However, the expression or function of catenins and cadherins has not been examined in EB cells. Apoptosis is also expected to be associated with the formation of EBs as well as the formation of three germ layers; however, definite histological location of apoptosis has not been determined within the EB.5 z( z' v% v( I* ~8 Q/ c* m2 q

0 d) F5 g3 K/ e8 Q2 G1 e6 kAmong specific markers of germ layers, GATA-4 and -fetoprotein are considered endodermal markers initially expressed in the primitive endoderm during early postimplantation stages and are maintained in the visceral and parietal endoderm of the yolk sac during gastrulation. Nestin is expressed in the neuroectodermal area. Desmin is expressed in the mesodermal area, especially muscle fibers. These markers are useful for identifying three germ layers in EBs.2 `% Y: p1 Z1 h) F
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In this study, we confirmed the relative location and the expression of marker genes of three germ layers in EBs, especially the cells of endoderm, which thus far have been the least characterized of the three. Furthermore, those endodermal cells were differentiated into hepatocytes by adding hepatotrophic factors. Our findings provide grounds for developing EB-derived cells into various stem cell lineages that can be used in the cell replacement therapy of hard-to-cure diseases.
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/ [/ k) }/ S+ F2 G! sMATERIALS AND METHODS2 F# i/ E; y% d0 J8 e
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Morphological Changes and Apoptosis in the EB& C' |" Q9 h7 Y- p5 @
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To examine the process of cell differentiation during EB formation, undifferentiated ES cell colonies were detached and grown in suspension for 6 weeks. As shown in Figure 1, H&E staining of EBs formed in the suspension culture revealed massive morphological changes accompanied by differentiation of diverse cell types during this period. As early as 1 week, cells within the EB started to show the typical characteristic of large nucleus and scanty cytoplasm outside of it. Chromatin condensation, cytoplasmic vacuolization, disruption of the nuclear membrane, and nuclear fragmentation were also observed in EB cells (Fig. 1A). Various phases of apoptosis were detected with TUNEL assay (Fig. 1G). Starting from the second week, cells with distinct characteristics could be discerned in EBs (Figs. 1B, 1C). At the same time, primitive neural tube–like structure (Fig. 1D), a cylinder-like structure imitating gut tube (Fig. 1E), and squamous epithelium-like structure (Fig. 1F) appeared. Central apoptotic areas were first seen in 1-week-old EBs (Fig. 1G) and then shrunk down to an undetected level by the sixth week (Figs. 1H–1L).2 H0 w' {7 q' g3 J

3 ~. e; ^" i4 y5 q6 x8 yFigure 1. Histological sections (hematoxylin and eosin, x400) and TUNEL assay (x100) in embryoid bodies (EBs). (A, G): 1-week-old EB; (B, H): 2-week-old EB; (C, I): 3-week-old EB; (D, J): 4-week-old EB; (E, K): 5-week-old EB; (F, L): 6-week-old EB. Arrow in (D) indicates rosette formations resembling the early neural tube. Arrow in (F) presents well-differentiated squamous epithelium imitating skin structure. Sections were counterstained with hematoxylin.
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% @- X- D; F, f* x% N; ~% w/ |Expression of Cell Adhesion Molecules During the Development of EBs9 I3 Z) {8 V; z9 H. f- A5 a
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Adhesive characteristics of EB cells were assessed by analyzing the gene expressions of cell adhesion molecules (Fig. 2). E-cadherin, -catenin, -catenin, and desmoglein-2 were all continuously expressed in undifferentiated ES cells as well as in 6-week-old EBs. On the other hand, the message level of N-cadherin and ?-catenin dwindled along with the progression of differentiation. Interestingly, paxillin mRNA, which was abundant in undifferentiated ES cells, showed a transient decrease during the second and fourth weeks but became abundant again by the sixth week.
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Figure 2. mRNA expression analysis of embryoid body (EB) with cell adhesion molecules. Lane 1 shows undifferentiated embryonic stem cells; 2, 1-week-old EB; 3, 2-week-old EB; 4, 3-week-old EB; 5, 4-week-old EB; 6, 5-week-old EB; and 7, 6-week-old EB. ?-Actin and GAPDH were used for the quantitation of RNA.0 ~( f  {" S; a2 p
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We also examined the expression of these adhesion molecules at the protein level by immunohistochemical staining. The expression of -catenin and ?-catenin remained strong throughout the experimental duration (Figs. 3A–3F). -Catenin was scarce in the 1-week-old EB but gradually increased in the following weeks (Figs. 3G–3I). In accordance with the level of their mRNAs, E-cadherin and N-cadherin proteins showed sustained expression throughout the examination period (Figs. 4A–4F). The neural tubes, which were strongly stained with N-cadherin antibody, were not stained with E-cadherin antibody (Figs. 4B, 4E).! Y& J' b. c. G4 T9 F

! e, o% n( D+ H# K, n: ?Figure 3. Immunohistochemical study of cell adhesion molecules in embryoid body (EB) (x400). -Catenin was expressed in (A) 1-week-old, (B) 3-week-old, and (C) 6-week old EBs. ?-Catenin was expressed in (D) 1-week-old, (E) 3-week-old, and (F) 6-week-old EBs. -Catenin was expressed in (G) 1-week old, (H) 3-week-old, and (I) 6-week-old EBs. Sections were counterstained with hematoxylin.
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: S% g6 Q- a' E  kFigure 4. Immunohistochemical study of cell adhesion molecules in embryoid body (EB) (x400). E-cadherin expression was shown in (A) 1-week-old, (B) 3-week-old, and (C) 6-week-old EBs. N-cadherin expression was shown in (D) 1-week-old, (E) 3-week-old, and (F) 6-week-old EBs. Desmoglein-2 was expressed in (G) 1-week-old, (H) 3-week-old, and (I) 6-week-old EBs. Paxillin was expressed in (J) 1-week-old, (K) 3-week-old, and (L) 6-week-old EBs. Arrow in (B) indicates negative staining pattern of E-cadherin to neural tube–like structure. Arrowin (E) indicates specific expression of N-cadherin in neural tube–like structure. Sections were counterstained with hematoxylin.
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Expression of Genes Specific to the Neural Ectoderm and Mesoderm
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The expression of ectoderm and mesoderm markers revealed distinct changes during the process of EB development (Fig. 5). In RT-PCR analysis, mRNAs of nestin, a protein specific to neural stem cells, were abundant in undifferentiated ES cells but slightly decreased from the third week and thereon. The level of Pax-6 transcript, another neural stem cell marker, showed a peak in the 1-week-old EB. Expression of Flk-1 and platelet endothelial cellular adhesion molecule, both known to be mesodermal specific, was differentially regulated during the examination period. The level of collagen IV, a cartilage component, increased during the first week and remained strong throughout the rest of the culture period.& `7 g6 Y9 o* I+ |( _$ s
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Figure 5. mRNA expression analysis of embryoid bodies (EBs) with ectodermal and mesodermal markers. Lane 1 shows undifferentiated embryonic stem cells; 2, 1-week-old EB; 3, 2-week-old EB; 4, 3-week-old EB; 5, 4-week-old EB; 6, 5-week-old EB; 7, 6-week-old EB. ?-Actin and GAPDH were used for the quantitation of RNA. Abbreviation: PECAM, platelet and endothelial cell adhesion molecule.1 J  n. O$ b2 \9 s
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When examined by immunohistochemical staining, nestin protein was abundantly expressed all over the 1-week EB except in the apoptotic areas (Fig. 6A). In later EBs, nestin proteins were detected in focal areas in 2- to 3-week-old EBs (Figs. 6B, 6C) and then in the primitive neural tube–like structures until the fifth week (Figs. 6D, 6E). By 6 weeks, nestin-positive neural tubes were destroyed and lost their cell–cell contact (Fig. 6F).& k7 s+ v( |; U, ~( b) d7 c
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Figure 6. Nestin and desmin expression in embryoid bodies (EBs). Nestin expression in (A) 1-week-old, (B) 2-week-old, (C) 3-week-old, (D) 4-week-old, (E) 5-week-old, and (F) 6-week-old EBs (x400). Desmin expression in 4-week-old EBs (G, x100; J,x 400) and 5-week-old EBs (H and I, x100; K and L, x400). Arrows in (D) and (E) indicate nestin-positive neural tube–like structure. Arrow in (K) indicates intestinal tube–like structure, showing positive staining of desmin. Sections were counterstained with hematoxylin.
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: Y( U. T+ @! RWe also examined the localization of desmin, a mesoderm marker specific to striated and smooth muscle. Desmin proteins were not detectable for the first 3 weeks of culture but appeared in the 4-week-old EB (Figs. 6G, 6H). At 5 weeks, cystic areas in EBs showed internal desmin-positive areas, imitating the typical structure of muscle fibers inside endodermal layers (Figs. 6I, 6J). Desmin proteins were also present in areas outside of the cysts in the 5-week-old EB (Figs. 6K, 6L)., f- @- B. o2 E! a

" r* n- C6 w! v: `9 p5 u4 lExpression of Endoderm-Specific Genes( Q% y1 h: @3 t3 h

, _0 ]* K- I7 C* xNext we compared the expression of endodermal genes in the EB, in 16.5-day embryos, and in the adult liver (Fig. 7). The expression of Oct-4, a specific marker for undifferentiated ES cells, showed a continuous decrease throughout EB development and was absent in the E6.5-day embryo or in the adult liver. GATA-4, which is expressed in visceral endoderm, showed increasing expression in 1- to 2-week-old EBs but decreased thereafter. mRNAs of -fetoprotein and transferrin were not detectable in the ES cells, continuously increased between 1 and 3 weeks, and started to decrease after 5 weeks. Both transcripts were highly expressed in the E16.5-day embryo, but -fetoprotein was absent in the adult liver. Three liver-specific molecules, that is, transthyretin (TTR), aldolase, and albumin, were highly expressed in early weeks and continuously decreased throughout the rest of the culture period." `6 X3 K& R" |/ ^# k# Y9 k
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Figure 7. mRNA expression analysis of embryoid bodies (EBs) with endodermal markers. Lane 1 shows undifferentiated embryonic stem cells; 2,1-week-old EB; 3,2-week-old EB; 4,3-week-old EB; 5,4-week-old EB; 6, 5-week-old EB; 7, 6-week-old EB; 8, 16.5-day-old embryo liver; and 9, adult liver. ?-Actin and GAPDH were used for the quantitation of RNA. Abbreviation: FP, -fetoprotein; TTR, transthyretin.+ }8 r# ?# ^+ v8 R4 ?! M+ Z. [  A
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By immunohistochemical analyses, -fetoprotein protein was localized to the outer layer of the EB (Fig. 8) until the fourth week. After 5 weeks, small areas inside of the EB were positive for -fetoprotein (Figs. 8E, 8F). GATA-4 was present in both outer layers and inner areas of the EB (Figs. 8G–8L) but was avoided in the area around the neural tube–like structures (Fig. 8J).5 |7 G1 w6 m* `& n

0 }+ B5 L; D( m$ T% p/ W7 aFigure 8. -Fetoprotein and GATA-4 expression in embryoid bodies (EBs) (x400). -Fetoprotein expression in (A) 1-week-old, (B) 2-week-old, (C) 3-week-old, (D) 4-week-old, (E) 5-week-old, and (F) 6-week-old EBs. GATA-4 expression in (G) 1-week-old, (H) 2-week-old, (I) 3-week-old, (J) 4-week-old, (K) 5-week-old, and (L) 6-week-old EBs. Sections were counterstained with hematoxylin.! _( ?" N0 C; W/ c( a6 l4 c: L" ^. }7 y
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In Vitro Differentiation of Endoderm-Like EB Cells into Hepatocytes* N, b7 U3 H  v' ]9 E
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To further characterize cells from the outer endodermal layer, 2-week-old EBs were treated with trypsin, and retrieved cells were analyzed by RT-PCR for the expression of endoderm-specific genes such as -fetoprotein, Apo2, and TTR. The expression level of these endodermal genes increased with the duration of trypsin treatment (Figs. 9, 10). Immunohistochemical staining revealed that -fetoprotein is strongly expressed in cells isolated by 3-minute trypsin treatment (Fig. 11).
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Figure 9. mRNA expression of endodermal marker protein in trypsinized 2-week-old embryoid bodies (EBs). Lane 1S shows supernatant of 1-minute trypsin-treated EB; 1R, remnant of 1-minute trypsin-treated EB; 3S, supernatant of 3-minute trypsin-treated EB; 3R; remnant of 3-minute trypsin-treated EB; 6S, supernatant of 6-minute trypsin-treated EB; 6R, remnant of 6-minute trypsin-treated EB; 9S, supernatant of 9-minute trypsin-treated EB; 9R, remnant of 9-minute trypsin-treated EB; EB1, 1-week-old EB; and EB2, 2-week-old EB. ?-Actin was used for the quantitation of RNA. Abbreviation: TTR, transthyretin.
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* l+ E; w) T' m( V; V. @6 ^! T% wFigure 10. -Fetoprotein mRNA expression pattern in trypsinized 2-week-old embryoid bodies (EBs).
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+ P2 z- z0 U$ W+ MFigure 11. -Fetoprotein expression in 3 minute–trypsinized 2-week-old embryoid body (EB) cells. -Fetoprotein staining and nuclear staining with mounting solution of single cells from 3 minute–trypsinized 2-week-old EBs were seen.% j" z( a' p; x6 X4 R

* R" l$ S; G/ h, O: PNext we examined the developmental potential of the dissociated endoderm-like cells by cultivating them with various hepatotropic factors, based on previous report of Hamazaki et al.  on hepatic differentiation. The endodermal cells were cultured in gelatin-coated dishes for 2 days with aFGF and then with HGF for another 2 days. After replating on matrigel matrix–coated dishes, cells were treated with HGF, OSM, and Dex for 4 days, followed by incubation with ITS for 3 days. After this 7-day stimulation regimen of hepatic differentiation, total RNAs were extracted and analyzed for the expression of liver metabolic enzymes. RT-PCR confirmed that the combined stimulation with growth factors, cytokines, and matrigel matrix strongly induced the expression of liver metabolic enzymes, such as G-6-P, PAH, TAT, and cytochrome P450s (P450-2B10 and P450-cb), in these in vitro–maturated endoderm cells (Fig. 12). With this maturation condition, polygonal hepatocyte-like cells were seen after a 7-day maturation period (Figs. 13A, 13B). Binucleated cells suggesting hepatocytes were seen (Fig. 13C). Those cells were stained with albumin, indicating that hepatocyte-like cells were possessing functional hepatic protein (Fig. 13D). Furthermore, these hepatocyte-like cells were stained with periodic acid-Shiff and ICG (Figs. 14A, 14B).
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Figure 12. mRNA expression of hepatic differentiation marker genes in differentiating embryonic stem cells from isolated endodermal cells. Lane 1 shows 2-week-old embryoid body (EB); 2, isolated endodermal cells by trypsin treatment for 3 minutes in 2-week-old EB; 3, acidic fibroblast growth factor (aFGF) alone; 4, aFGF and hepatocyte growth factor (HGF); and 5, aFGF, HGF, oncostatin M (OSM), insulin, transferrin, and selenious acid (ITS), dexamethasone (Dex), and matrigel matrix. Increased hepatic gene expression is seen in the aFGF, HGF, OSM, ITS, Dex, and matrigel matrix maturation condition. Abbreviations: G-6-P, glucose-6-phosphatase; PAH, phenylalanine hydroxylase; TAT, tyrosine aminotransferase.% F+ k( Q& U, ^" N0 w- `5 J! S
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Figure 13. Morphology of hepatocyte-like cells differentiated from isolated endodermal cells. (A, B): Polygonal hepatocyte-like cells are seen in acidic fibroblast growth factor (aFGF), hepatocyte growth factor (HGF), oncostatin M (OSM), insulin, transferrin, and selenious acid (ITS), dexamethasone (Dex), and matrigel matrix maturation condition (A, x100; B, x200). (C): Binuclear cells indicating hepatocyte are seen (x400). (D): Many albumin-positive cells are seen after aFGF, HGF, OSM, ITS, Dex, and matrigel matrix maturation condition (x200).8 _/ Y  V7 k7 P; E0 n+ _! {  F
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Figure 14. Hepatocyte-like cells were stained with periodic acid-Shiff (A, x200) and indocyanine green (B, x200).
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6 d. Z. Q% C8 V" e) S1 X) R) ADISCUSSION1 J. r! L* _+ ~6 A

+ X, u( a; G# R, t; f) e0 H3 _D.C. and H.-J.L. contributed equally to this study. This work was supported by research grants from the Korea National Institutes of Health and from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (00-PJ1-PG1-CH5-0004) and Soonchunhyang University Research Fund (20040161). We thank Dr. Won-Ki Paik for the critical reading and discussion of this manuscript. We are grateful to Dr. Se Jin Jang for technical advice on histological analyses. We also thank Cheol Yong Song, Jae Hyeong Kim, Kuk Hyeon Lee, Tai Ho Im, and Byung-Seok Park for their financial support.
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真是好资料,很有帮助,谢谢了

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希瑞干细胞
任何的限制,都是从自己的内心开始的。  

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干细胞之家微信公众号
一个人最大的破产是绝望,最大的资产是希望。  

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一个人最大的破产是绝望,最大的资产是希望。  

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干细胞治疗糖尿病  

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楼主good  

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顶顶更健康,越顶吃的越香。  

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转基因动物

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加油啊!!!!顶哦!!!!!支持楼主,支持你~  
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