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1 htgf raav

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2 2

3 TGF 2 TGF 2 PLGA 1. O PPARγ PPARγ 2. TGF 2 htgf 2 RT-PCR II 3

4 RT-PCR II II TGF 2 3. htgf 2 pcdna3.1(+)/htgf 2 G RT-PCR Western-blot htgf 2 mrna htgf 2 II Aggrecan pcdna3.1(+)/htgf 2 htgf 2 htgf 2 PLGA II 12 PLGA 4. ALP ALP Von Kossa Western Blot BMP-2 OPN ALP P<0.01 BMP-2 OPN PLGA TGF 2 4

5 Research of adipose mesenchymal stem cells in cartilage and bone tissue engineering Abstract Articular cartilage has little or no potential for intrinsic repair, injury or disease of the joints related to cartilage damage is the primary complaint of orthopedic patients. Numerous strategies currently in clinical treatment, such as lavage and debridement, subcondral drilling, transplantation of chondrocytes and osteochondral autografts or allografts, have no fine effects that can restore a damaged cartilage surface and prevent the outcome of degenerative joint disease except joint replacement. In recent years, cartilage tissue engineering displays a favourable prospect on cartilage repair, however the source of seeding cells is the main barrier for clinical application. The research of stem cells has given rise to the new field of tissue engineering. In this study, we investigated the feasibility of chondrogenic differentiation of adipose mesenchymal stem cells (AMSCs) as seeding cells in cartilage tissue engineering. In addition, whereas adipose mesenchymal stem cells have the multi-lineage potential, we also directed them to differentiated into osteblasts. The whole study consisted of four parts: 1. The biological characteristics of adipose mesenchymal stem cells Adipose mesenchymal stem cells were obtained from the fat tissue of 3-week-old Lewis rat by digested with 0.1% collagenase type and then cell growth curve was ploted. After treated with insulin and dexamethasone, AMSCs were observed using Oil red O staining and PPARγ immunocytohistochemistry. AMSCs assumed a spindle-shaped and fibroblastic morphology and showed strong capacity of proliferation, whose population doubling time was approximately 55h. After being induced with insulin and dexamethasone, 60% of AMSCs differentiated into mature adipocytes and expression of PPARγ significantly increased. 2. TGF 2 induced adipose mesenchymal stem cells into chondrocytes in vitro. To initiate chondrogenic induction, medium were changed to chondrogenic induction 5

Numerous strategies currently in clinical treatment, such as lavage and debridement, subcondral drilling, transplantation of chondrocytes and osteochondral autografts or allografts, have no fine

6 medium consisting of 10 ng/ml TGF 2, 6.25 ug/ml insulin, 50nM ascorbate-2-phosphate in micromass culture and monoplayer culture. At different time, the cells were were assessed by histological staining. Immunohistochemical analyses and RT-PCR were also performed to evaluate collagen type II synthesis. After 21 days, the cell pellet in micromass culture appeared entirely cartilaginous and histological staining displayed chondroid structure, such as cartilage lacuna. The treated cells expressed and produced collagen type II and aggrecan. But the cells in monoplayer culture did not express collagen type II and cartilage matrix. These results demonstrate that TGF 2 can induce AMSCs into chondrocytes in vitro, and three-dimensional construction environment is necessary in this process. 3. Gene-enhanced cartilage tissue engineering The effect of htgf 2 on AMSCs chondrogenesis in monolayer culture was studied using gene transfection technology. A recombinant pcdna3.1(+)/htgf 2 construct containing a full-length htgf 2 was lipofected into AMSCs. Immunohistochemistry, RT-PCR and Western blot analyses were performed to identify the expression of htgf 2 and cartilage associated genes and proteins. The results showed that htgf 2 was expressed throughout the culture period. The transfected cells expressed and produced collagen type II and aggrecan. This shows that transfection of pcdna3.1(+)/htgf 2 into AMSCs is able to provide transient and persistent expression of cartilage specific genes and proteins, and suggests that the differentiation of AMSC into chondrocyte may be induced by TGF 2 gene transfection. The transfected cells were seeded onto sponge PLGA scaffolds and then the complex of cell-scaffolds were transplanted to subcutaneous of nude mouse. The complex were assessed by histological staining of H-E, Alcian blue and Immunohistochemical analyses of collagen type II. After cultured in vivo for 12 weeks, the complex grew into hyaline cartilage-like tissues. This may suggest that transfected AMSCs could differentiate into chongdrocytes in vivo and serve as seeding cell of cartilage tissue engineering. 4. Osteoblast differentiation of adipose mesenchymal stem cells After treated with dexamethasone, ascorbic acid and beta-sodium glycerophosphate, cellular alkaline phosphatase activities and Von Kossa staining of bone nodules were 6

After 21 days, the cell pellet in micromass culture appeared entirely cartilaginous and histological staining displayed chondroid structure, such as cartilage lacuna.

7 tested, and changes of BMP 2, OPN expression were detected by immunocytohistochemistry or Western Blot. Results showed that cellular ALP activities significantly increased (P<0.01), bone nodules became more and larger than the control, and expression of BMP 2 and OPN increased. These results suggest that adipose mesenchymal stem cell can differentiated into osteoblast when cultured in vitro, and adipose mesenchymal stem cells can likely be served as optimal autogenous cell source for bone tissue engineering. Key Words: Articular Cartilage; Adipose tissue; Mesenchymal stem cell; Chondrocye; Tissue Engineering; PLGA; TGF 2 ; Transplant; Gene transfection; Differentiation 7

01), bone nodules became more and larger than the control, and expression of BMP 2 and OPN increased.

8 [1] 3mm [2] 4mm [3,4] [5] 80, (cell-based therapy) [6] 1987 (NSF) LakeTahoe Tissue Engineering is the application of principles and methods of engineering and life sciences toward the fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and development of biological substitutes to restore, maintain, or improve tissue function 8

fundamental understanding of structure-function relationships in normal and pathological

9 extrocellular matrix, ECM ECM ECM [7] mesenchymal stem cell, MSC [7,8] 2. II IV IX X XI II IX XI II 9

10 IX XI IX IX X [9,10] 95 5 glycosaminoglycan, GAG GAGs GAGs [7,9,11] anchorin CII [7] 80 ECM GAG ECM [7,9] 3. 4 ECM [7,9] ECM [7,9] [7,9] 10

11 10 [12] [7,9] :(1) (2) (3) [13,14,15] 1. [7,16,17], 6 [18,19,20,21,22] (dedifferentiation) ( ) [23] immortalitying chondrocyte [24] 40 T SV40LTAg 11

12 / p53 [25,26,27,28] Takazawa [28] SV40LTAg 6 Steimberg [29] SV40LTAg Co12a1 pco12sv 80 9 Goldering [30] [30, 31] SV40LTAg [32] 2. (mesenchymal stem cells, MSCs) MSCs MSCs MSCs 2.1 (hematopoietic 12

13 stem cell, HSC) [33] 130 Cohnheim analine [34] [34,35] BMSC MSC MSC BMSC BMSC [36,37] BMSC [38,39] BMSC G0/G1 [37] BMSC 15 4 MSC MSC BMSC BMSC BMSC BMSC BMSC SH2 SH3 SH4 SB210 CD44 CD45 CD34 CD14 [40,41,42] MSC BMSC BMSC 13

[37] BMSC 15 4 MSC 1 10 6 2 5 MSC BMSC BMSC BMSC BMSC BMSC

14 BMSC BMSC HSC BMSC MSC BMSC CD44 CD44 BMSC Wakitani [43] 1994 BMSC Miriam [44] BMSC 2 3 BMSC BMSC TGF Johnstone [45] (1998 ) BMSC TGF 1 TGF 1 BMSC 20 BMSC BMSC Worster [46] BMSC Yoo [22] BMSC BMSC 14

15 , BMSC Martin [47] BMSC 4 BMSC TGF 1 TGF 3 [48,49] BMSC : BMSC BMSC BMSC [43,50] 2.2 MSC Pate [51] Williams [52] [53] MSC [40,54] MSC [55] MSC 15

16 [56] [57] MSC MSC [58] MSC Adachi [59] MSC Lacz MSC 2 24 MSC MSC 2.3 MSC MSC, ml ( 1 MSC/10 5 ) [40], Zuk PA [60] FBS DMEM processed lipoaspirate, PLA adipose-derived adult stem (ADAS) cells ADAS ADAS 16

3 MSC MSC, 10 20 ml ( 1 MSC/10 5 ) [40], Zuk PA [60] 0.

17 3. embronic stem cell, ES 200 ES (embronic body EB) 1981 Evans Martin ES 1995 Thomson [61] ES 1998 ES [62] 2000 Reubinoff [63] ESCs ESCs [64] [65] [66] 2 (BMP-2 ) BMP-4 ESCs [67] ESCs ESCs ESCs 17

18 : 90% [68] (Fibrin, FN) (PolyglycolicAcid, PGA) (Poly-L-lacticAcid, PLLA) (CarbonFiber) [69,70,71,72,73] PGA PLLA PGA 1. PGA PGA PGA [74] PGA PGA PGA ph PGA PGA PGA PGA PGA PGA PGA PLLA (PGA-PLLA) 18

19 PGA PGA PLLA [75] [76] PGA-PLLA PLGA PLGA [75] PLGA [77] ph CaCO 3 Na 2 CO 3 ph [77] PLGA PGA [76] PLA PLGA PLGA [75] 2. PLGA PLGA 1984 KimuraT [78] 1989 Wkitanis [79] QuiW [80] 19

20 [81] 90% UV Nehrer [82] UV / RGD ( - - ) X3B1 X4B1 XVB3 - - (RGD) PGA-PLA PLGA PLGA 3. PLGA Haisch [83] 20

21 Haisch Homminga [84] [85] 4. 8 GAG Aggrecan [86,87,88] 12 [89,90] [poly(ethylene oxide) PEO] [P(EO-co-PO)] Sims [91] PEO 12 P(EO-co-PO) PGA [92] Bonaventure [93] 21

22 : (simulated microgravity in rotating bioreactors) (solid body rotation in rotating bioreactors) (turbulent mixing in spinner flasks) (orbital mixing in petridishes) ph [94] [95] Gooch [96] GAG CO 2 O 2 Freed [74] GAG ECM Sittinger [97] ph - (growth factor, GF) 22

23 Langer Vacanti (transforming growth factor TGF ) TGF DNA [99] matrix metalloproteinases, MMPs [18,99,100,101] TGF BMSC Johnstone BMSC C TGF 1 2 TGF 1 2. (bone morphogenetic protein BMP) BMP TGF [102] BMP-3 BMP-3 BMP BMP Cook [103] BMP-7 12 BMP-7 BMP-3 BMP-4 BMP-7 II I BMP GAG [104,105,106] 3. (insulin-like growth factor IGF) 23

24 IGF IGF-1 IGF-2 IGF-1 [107] IGF-1 IGF-1 IGF-1 [108] IGF-1 IGF-1 TGF DNA [109] Fortier [110] IGF-1 8 IGF-1 4. (basic fabroblast growth factor bfgf) basic fibroblast growth factor bfgf 146 Sox9 Sry-type high-mobility-group box gene [111] bfgf [112] bfgf 1 7 [113] 5. -I(chondromodulin-I ChM-I) ChM-I ChM-I 24

医学科研方法

医学科研方法医学科研方法主讲苏秀兰细胞生物学教授第一讲绪论一概念 : 在医学科学研究活动中, 运用科学的实践和理论思维的技巧, 充分发挥自己的智慧, 去寻找观念世界与现实世界之间的联系或规律, 从而发现新现象, 提出新理论的手段二医