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
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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
検体肉眼像および組織像
HE x100 HE x200 HE x200
Type II collagen
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
P0軟骨細胞皮下移植 ２週間後
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 HEｘ1.25
初代培養細胞 TBｘ 1.25初代培養細胞 HEｘ 200
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
Human Marrow Stromal Cellsreach 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
Mori, et al., Mol Cell Biol, 2005
Prolongation of the life span of human marrow stromal cellsby 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.
ヒト臍帯血間葉系細胞 s（ UCB）
Po
pu
latio
n D
ou
blin
gs
Cultured Days0 80 180
0
20
40
Methylation assay of p16 promoterMol.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
Terai, et al. Mol Biol Cell, 2005
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
0 0.5 1 2 10
FCS(%)
0
20
40
60
80
100
120
PPL
PLVMSCGM(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。
bPAPBhP
bPAbPBbhP
bPABaEV
PaPEPVabbEbVaEaVEV
-
0 20 40 60 80 100 120
mAbs=450nm
MSC äeëùêBàˆéqÇ…ÇÊÇÈëùêBå¯â
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
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。
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。
MSC bFGF P-AAP-BB
P-AB
+bFGF
P-AA,BB
P-AAP-BB
P-ABPDGF-AA
PDGF-AB
PDGF-BB
A鎖 B鎖
β ﾚｾﾌﾟﾀｰCD140ｂ
α ﾚｾﾌﾟﾀｰCD140ａ
Greb２Nck
Ｒａｓ -GAP
CrkSrc
SHP2PLC-ｒ
PDGF-AA→Competent FactorPDGF-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)‐
MSC
aFG
FbF
GF
EGF
VEGF
PDGF
PDGF+aF
GF
PDGF+bFG
FPDG
F+EGF
PDGF+VEG
F
aFG
F+bFGF
aFG
F+EGF
aFG
F+VEGF
bFGF+EG
FbFG
F+VEGF
EGF+VEG
F
HeL
a
p16p16
βtubulinβtubulin
H4-3Normalized 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, Jackson-Weiss syndrome)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, Jackson-Weiss syndrome)
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 41.2821407 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, Jackson-Weiss syndrome)
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, Jackson-Weiss syndrome)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, Jackson-Weiss syndrome)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, Jackson-Weiss syndrome)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 41.1922568 A 46.9 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome)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, Jackson-Weiss syndrome)
1 A 67.3 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome)1 A 8.2 fgfr2 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome)
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 polypeptide1.0704594 P 4990.3 pdgfra platelet-derived growth factor receptor, alpha polypeptide1.0662563 P 366.9 pdgfrl platelet-derived growth factor receptor-like0.8164289 A 6.2 pdgfra platelet-derived growth factor receptor, alpha polypeptide2.3044996 A 403.1 pdgfra platelet-derived growth factor receptor, alpha polypeptide0.6091959 A 19.5 flt4 fms-related tyrosine kinase 41.7572612 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。
増殖因子 ストレス
Ras
Raf
MEK1/2
ERK1/2
Ets1/2
MLKｓ、 TAKASK1
MKK3/6
ｐ３８ MAPK
A
B
ｐ１６ INK4a
p16ink4a
β-tubulin
X10%FCS
XA B
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
National Center for Child Health and Development
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