1. 1 0 N O V E M B E R 2 0 1 1 | VO L 4 7 9 | N AT U R E | 2 3 3
Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders
Darren J. Baker1,2,3, TobiasWijshake1,4, Tamar Tchkonia3, Nathan K. LeBrasseur3,5, Bennett G. Childs1, Bart van de Sluis4,
James L. Kirkland3 & Jan M. van Deursen1,2,3
清除衰老细胞可以延缓与年龄有关的功能异常
Presented by: Chang chu
Jan.13, 2013

2. 1 http://en.wikipedia.org/wiki/P16_(gene)
Cellular senescence
mouse embryonic fibroblast 
? whether senescent cells are causally implicated in age related
Dysfunction
? whether their removal is beneficial 1
P161: Cyclin-dependent kinase inhibitor 2A
multiple tumor suppressor 1 (MTS-1)
早在五十年前，科学家们就发现，细胞在停止分裂前会经历一个衰老期。这段时间它们既不死亡，也不正常分裂繁殖，而是不断制造出破坏分子，攻击和杀死附近的健康细胞。
学界推测，这种特性是为了防止细胞繁殖失控和癌症的扩散而存在的。
人体内的衰老细胞随着年龄增长越积越多，特别是在皮肤脂肪、骨骼肌肉以及眼部。它们是不是引发人类各种老年病的罪魁祸首呢？科学家们一直心存疑虑，但却很难下定论。因为这种细胞的数量其实很有限，在老年人体内也就占了10%—15%。
Although a universal marker that is solely expressed in senescent cells has not been identified, most senescent cells seem to express p16Ink4a
Additionally, the expression of p16Ink4a is known to increase with ageing in several rodent and human tissues8
p16基因又叫MTS(multiple tumor suppressor 1基因，是1994年美国冷泉实验室Kamb等发现的新抗癌基因，这是一种细胞周期中的基本基因 1)直接参与细胞周期的调控，负调节细胞增殖及分裂，2)稳定抑癌基因P53，在人类50%肿瘤细胞株纯发现有纯合子缺失，突变

3. An earlier mouse model： FAT-ATTAC2 (fat apoptosis through targeted activation of caspase）
Transgenic strategy:
Fabp4 promotor
基于之前一种名为FAT-ATTAC小鼠模型。研究者们用转基因小鼠来进行实验，在它们的衰老细胞中潜伏了一种名为8号蛋白酶的分子。这种蛋白酶一旦遇到给小鼠服用的特殊药物就会被激活，在衰老细胞的外层钻洞，从而让这些细胞自我毁灭，而其他细胞则安然无恙。
2 Pajvani, U. B. et al. Fat apoptosis through targeted activation of caspase 8: a new mouse model of inducible and reversible lipoatrophy. Nature Med. 11, 797–803(2005).

4. BubR1 encodes a key member of the mitotic checkpoint
Pronuclear injection of the INK-ATTAC construct into FVB oocytes
Nine transgenic INK-ATTAC founder lines
BubR1H/H mice
BubR1 encodes a key member
of the mitotic checkpoint
shortened lifespan
age-related phenotypes
纺锤体组装监测点基因 保证在有丝分裂中染色体平均分配到两个子细胞中
随着年龄增长，BubR1显著减少.衰老表现：不育，肌肉减少，白内障，血管硬化等
adipose tissue, skeletal muscle
and eye
inguinal adipose tissue (IAT)

5. 1. Transgenic INK-ATTAC and endogenous p16Ink4a are under the same transcriptional control mechanism
Rosiglitazone
we harvested bone marrow cells from 2-month-old wild-type (WT);INK-ATTAC-3 and -5 mice and cultured them in the absence or presence of rosiglitazone, a drug that can induce cellular senescence and p16 expression.

6. 2. INK-ATTAC is expressed in senescent cells in BubR1 hypomorphic tissue.
SA-b-Gal stained IAT
Expression of senescence markers in tissues
collected from 9-month-old mice of the indicated genotypes.

7. we collected IAT from aged BubR1H/H;INK-ATTAC animals
INK-ATTAC is selectively expressed in p16Ink4a-positive senescent cells

8. 3. INK-ATTAC can eliminate senescent cells
Bone marrow cells of WT;INK-ATTAC transgenic lines 3 and 5
Cultured with rosiglitazone for 5 days
Treated with AP20187 for 48h
野生型转基因小鼠骨髓细胞在罗格列酮中培养5天，用药AP20187，48h后染色
FKBP–Casp8 activation efficiently eliminates p16Ink4a-positive senescent cells in vitro.

9. Sarcopenia, cataracts and loss of adipose tissue
4. Clearance of p16Ink4a-expressing cells from BubR1H/H
mice prevents or delays the onset of age-related phenotypes
BubR1H/H;INK-ATTAC-3 and -5 mice at 3 weeks of age :
AP20187 every third day
Untreated
Sarcopenia, cataracts and loss of adipose tissue
age-associated deficits known to accompany p16Ink4a induction.
Continuous removal of p16Ink4a-expressing cells from BubR1H/H;INK-ATTAC
9-month-old mice

10. prevented loss of adipose tissue
Continuous removal of p16Ink4a-expressing cells from BubR1H/H;INK-ATTAC mice selectively delays age-related phenotypes

11. 5. The delayed onset of age-related pathologies coincided with a reduction in the number of senescent cells in these tissues.
Senescent cells were cleared from tissues and that this delays acquisition of age-related dysfunction in BubR1 hypomorphic mice.

12. 6.Investigating the effect of senescent cell clearance later in life
BubR1H/H at 5 months: age-related phenotypes are apparent
AP20187 treatment
Measured p16Ink4a-dependent age-related phenotypes at 10 months
白内障充分建立，没有好转
muscle fibre diameters and showed improved performance in treadmill exercise tests 脂肪

13. Senescence markers were substantially reduced
Late-life clearance of p16Ink4a positive senescent cells attenuates progression of age-related decline rather than a reversal of ageing in BubR1 hypomorphic mice.

14. A novel transgenic mouse model
Both life-long and late-life clearance of the p16Ink4a expressing senescent cells selectively delayed age-related pathologies in tissues that accumulate these cells.
Therapeutic interventions to clear senescent cells or block their effects may represent an avenue for treating or delaying age-related diseases and improving healthy human lifespan.
a novel transgenic mouse model that allows for the inducible removal ofp16Ink4a-positive senescent cells