Gann 112.006

1. 間葉系細胞間葉系細胞
間葉系細胞の供給源間葉系細胞の供給源
骨髄骨髄
多指症多指症
子宮内膜子宮内膜
内膜幹細胞内膜幹細胞
月経血月経血
臍帯血臍帯血
胎盤胎盤
分化能分化能
成育バイオリソース成育バイオリソース
支持能支持能

2. 検体肉眼像および組織
像
HE x100 HE x200 HE x200
Type II collagen

3. 軟骨培養細胞の形態像 (Papanicolau 染色、各倍率 200 倍 )
P0 P1
P10
p16 免疫染色

4. P0 軟骨細胞皮下移植 ２週間後

5. 軟骨培養細胞皮下移植 ２週間後
初代培養細胞移植 初代培養細胞移植、浮遊組織を再移植 一回継代細胞移植
初代培養細胞 HE ｘ
1.25
初代培養細胞 TB ｘ 1.25初代培養細胞 HE ｘ 200

6. HE X1.5 HE X100
Type II collagen X100 S-100 X200
HE X200
T.Blue X100
P0 軟骨細胞移植部 組織形態
像

7. Human Marrow Stromal Cells
reach senescence after a limited number of cell division
H4-1H4-1
H4-1H4-1
慶應義塾大学医学部慶應義塾大学医学部
血液内科血液内科
木崎 昌弘 木崎 昌弘
福地 由美 福地 由美
H4-1, senescenceH4-1, senescence

8. Mori, et al., Mol Cell Biol, 2005

10. Prolongation of the life span of human marrow stromal cell
by transferring Bmi-1, E6, E7, and TERT
The cells with the extended life span retained the multipotency
into osteocytes, chondrocytes, adipocytes, and myocytes in vitro.

11. ヒト臍帯血間葉系細胞 s
（ UCB ）
PopulationDoublings
Cultured Days
0 80 180
0
20
40
Methylation assay of p16 promoter
Mol.Cell.Biol 1999, 5642-51 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
HMEC6/40:LXSN-16E6E7(@p4) p=16 1 0
HMEC6/40:LXSN-16E6E7(@p4) p=16 2 0
HMEC6/8:LXSN-16E6SD(@p4) p=31 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14
HMEC6/8:LXSN-16E6SD(@p4) p=31 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 23
UCB (primary) p=5 1 1 1 2
UCB (primary) p=5 2 1 1 2
UCB (primary) p=5 3 0
UCB (primary) p=5 4 0
UCB (primary) p=5 5 1 1 2
UCB (primary) p=5 6 1 1 2
UCB p=9 1 0
UCB p=9 2 0
UCB p=9 3 0
3 4 5 7 8 9 16 HeLa
p16
Actin

12. Terai, et al. Mol Biol Cell,

13. 0
20
40
60
80
100
120
140
0 50 100 150 200 250
培養日数
E6E7
Mock
Infection
hTERT
Mock
hTERT
E6E7
細胞増殖曲線
細
胞
分
裂
回
数

14. 0 0.5 1 2 10
FCS(%)
0
20
40
60
80
100
120
P
PL
PLV
MSCGM(10%FCS)
UEET-12 無血清培養
改良培地は無血清下でも血清含有培地と同等の増殖性を有する
b
PA
PB
hP
bPA
bPB
bhP
bPAB
a
E
V
Pa
PE
PV
ab
bE
bV
aE
aV
EV
-
0 20 40 60 80 100 120
mAbs=450nm
MSC äeëùêBàˆéqÇ…ÇÊÇÈëùêBå¯â

15. 時間
分裂回数
血清培地
増殖因子
ＭＳＣＧＭ X
ｐ１６ＩＮＫ４ａ
β tubulin‐
蘇生後の継代 １ １ ２ ２ ３ １ １ ２ ２ ３

16. ｐ１６ＩＮＫ４ａ
β tubulin‐
X ＋ FCS -IT
+FCS
-IT -PDGF
- ｂ FGF
-EOP -Sel -P,b
+FCS
-P,b MSC
p16 誘導の主要要因は PDGF, ｂ FGF である。

17. ｐ１６ＩＮＫ４ａ
β tubulin‐
X-P,b X-P,b/10%FCS MSCGM
PDGF+bFGF(ng/ml) 0 1 10 0 1 10 0 1 10
ｂＦＧＦ，ＰＤＧＦが p16 発現を誘導する。

18. MSC bFGF P-AA
P-BB
P-AB
+bFGF
P-AA,BB
P-AA
P-BB
P-AB
PDGF
-AA
PDGF
-AB
PDGF
-BB
A 鎖 B 鎖
β ﾚｾﾌﾟﾀｰ
CD140 ｂ
α ﾚｾﾌﾟﾀｰ
CD140 ａ
Greb ２
Nck
Ｒａｓ -
GAP
Crk
Src
SHP2
PLC- ｒ
PDGF-AA→Competent Factor
PDGF-BB→Competent and Prouression Factor
IgG CD140a CD140b
p16
βtubulin
― ＋
MSC の p16 発現は PDGF-B 鎖による。
( 細胞 /H10 8)‐

19. M
SC
aFG
F
bFG
F
EG
F
VEG
FPDG
FPDG
F+aFG
F
PDG
F+bFG
F
PDG
F+EG
F
PDG
F+VEG
F
aFG
F+bFG
F
aFG
F+EG
F
aFG
F+VEG
F
bFG
F+EG
F
bFG
F+VEG
F
EG
F+VEG
F
H
eLa
p16p16
βtubulinβtubulin
H4-3
Normalized Flags Raw Common Description
1.0818335 P 2172.2 egfr epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)
1.1522822 P 657.3 egfr epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)
0.6146532 A 337.2 egfr epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)
0.8937527 A 17.8 egfr epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)
1.0583736 A 167.9 egfr epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)
0.8768445 A 358.1 egfr ErbB1-S; Human epidermal growth factor receptor precursor (EGFR) mRNA, complete cds.
1.0504522 A 10.4 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jac
0.7824624 A 21.5 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jac
0.417403 A 9.8 fgfr3 fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism)
0.54518497 A 27.2 fgfr3 fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism)
1 A 38.3 fgfr4 fibroblast growth factor receptor 4
1.2821407 A 125.7 fgfr1 fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome)
1 P 652.2 fgfr1 fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome)
1.4052641 A 14.3 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jac
0.75333506 A 59.2 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jac
1.3886433 P 124.5 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jac
1.1120172 A 13.6 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jac
1.0663294 P 2619.3 fgfr1 fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome)
1 A 11.1 fgfr4 fibroblast growth factor receptor 4
1.1922568 A 46.9 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jac
3.1074321 A 73.5 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jac
1 A 67.3 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jac
1 A 8.2 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jac
1.3065747 P 3125.7 fgfr1 fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome)
3.3271298 P 4049.1 pdgfrb platelet-derived growth factor receptor, beta polypeptide
1.0704594 P 4990.3 pdgfra platelet-derived growth factor receptor, alpha polypeptide
1.0662563 P 366.9 pdgfrl platelet-derived growth factor receptor-like
0.8164289 A 6.2 pdgfra platelet-derived growth factor receptor, alpha polypeptide
2.3044996 A 403.1 pdgfra platelet-derived growth factor receptor, alpha polypeptide
0.6091959 A 19.5 flt4 fms-related tyrosine kinase 4
1.7572612 A 61.6 kdr kinase insert domain receptor (a type III receptor tyrosine kinase)
aFGF-R ○
bFGF-R ○
EGF-R ◎
VEGF-R ×
PDGF-R ◎
受容体の有無
p16 発現は増殖因子の
種類による。

20. 増殖因子 ストレス
Ras
Raf
MEK1/2
ERK1/2
Ets1/2
MLK ｓ、 TAK
ASK1
MKK3/6
ｐ３８ MAPK
A
B
ｐ１６ INK4a
p16ink4a
β-tubulin
X
10%FCS
X
A B

21. 慶應義塾大学
病理学教室
森 泰昌
生理学教室
岡野栄之
島崎琢也
埼玉医科大学
五條理志
許 俊英
整形外科学教室
戸山芳昭
今林英明
越智健介
微生物学教室
瀬川 薫
NIH, USA
洪 実
National Cancer Center, Jpn
清野 透
Helix 研究所
磯貝 隆夫
杉山 友康
入江 亮太郎
中外製薬
東佐由美
大川広行
Mori

22. National Center for Child Health and Development