突破：病人体细胞及肿瘤细胞长期体外培养（附原文）

sunsong7

3 Z# z- c! ]$ o  R7 ?- t2 o! i) h# q
" E4 w# a, G6 Y: d) }正常细胞在体外仅能分裂数次，肿瘤细胞也不例外，许多癌细胞离体后不再生长，乔治敦隆巴迪综合癌症中心科学家采用rho激酶抑制剂（ROCK）+成纤维细胞滋养层的方法，可以让正常细胞和肿瘤细胞转变为干细胞样细胞进行长期体外培养，不久的将来，人们可以建立病人的体细胞库用于疾病的诊疗。' E4 d7 {/ ^; S$ B. d$ ^7 p
New Method Keeps Normal Cells and Tumor Cells Taken from an Individual Cancer Patient Alive
# u3 t. _4 S1 K) m6 A+ ?9 ?# bhttp://www.sciencedaily.com/releases/2011/12/111219102044.htm- w/ z* j* l5 k4 }+ R+ o

) W4 h; e* _7 ^& IScienceDaily (Dec. 19, 2011) — In a major step that could revolutionize biomedical research, scientists have discovered a way to keep normal cells as well as tumor cells taken from an individual cancer patient alive in the laboratory -- which previously had not been possible. Normal cells usually die in the lab after dividing only a few times, and many common cancers will not grow, unaltered, outside of the body.: E9 t0 J% j2 z$ p/ a1 S& R/ v
--------------------------------------------------------------------------------% F5 z# M! k( w! a- Y# g
This new technique, described December 29 online in the American Journal of Pathology, could be the critical advance that ushers in a new era of personalized cancer medicine, and has potential application in regenerative medicine, says the study's senior investigator, Richard Schlegel, M.D., Ph.D., chairman of the department of pathology at Georgetown Lombardi Comprehensive Cancer Center, a part of Georgetown University Medical Center.
) C2 _/ N$ w- C& z& D+ O8 W) s6 j4 ]3 t) W- X0 ?# l7 |
"Because every tumor is unique, this advance will make it possible for an oncologist to find the right therapies that both kills a patient's cancer and spares normal cells from toxicity," he says. "We can test resistance as well chemosensitivity to single or combination therapies directly on the cancer cell itself."( Y6 x8 p: Q, H5 A
$ O6 F. t% c! Z- {
The research team, which also includes several scientists from the National Institutes of Health, found that adding two different substances to cancer and normal cells in a laboratory pushes them to morph into stem-like cells -- adult cells from which other cells are made.
3 a" s0 A! F6 Z3 p5 B6 U& ]
0 p4 A( R: g! p+ M! bThe two substances are a Rho kinase (ROCK) inhibitor and fibroblast feeder cells. ROCK inhibitors help stop cell movement, but it is unclear why this agent turns on stem cell attributes, Schlegel says. His co-investigator Alison McBride, Ph.D., of the National Institute of Allergy and Infectious Diseases, had discovered that a ROCK inhibitor allowed skin cells (keratinocytes) to reproduce in the laboratory while feeder cells kept them alive.
7 q5 ^" W; `2 a+ L& H. z; y
1 n5 ~3 z2 \2 C" D+ ZThe Georgetown researchers -- 13 investigators in the departments of pathology and oncology -- tried ROCK inhibitors and fibroblast feeder cells on the non-keratinocyte epithelial cells that line glands and organs to see if they had any effect. They found that both were needed to produce a dramatic effect in which the cells visibly changed their shape as they reverted to a stem-like state.4 i5 Z8 [; h& D4 d  W

: `- o: ?2 L. ]& s"We tried breast cells and they grew well. We tried prostate cells and their growth was fantastic, which is amazing because it is normally impossible to grow these cells in the lab," Schlegel says. "We found the same thing with lung and colon cells that have always been difficult to grow."
6 n* ?3 e& M' M$ o! z5 _* d8 B( x) `2 y2 p+ {
"In short, we discovered we can grow normal and tumor cells from the same patient forever, and nobody has been able to do that," he says. "Normal cell cultures for most organ systems can't be established in the lab, so it wasn't possible previously to compare normal and tumor cells directly."( U; C% q% C- v
# C4 C6 g5 h# h: Y8 D
The ability to immortalize cancer cells will also make biobanking both viable and relevant, Schlegel says. The researchers further discovered that the stem-like behavior in these cells is reversible. Withdrawing the ROCK inhibitor forces the cells to differentiate into the adult cells that they were initially. This "conditional immortalization" could help advance the field of regenerative medicine, Schlegel says.
' \, s/ N$ c  X! x; k* N( C% S6 X2 s* o. ~- N8 {
However, the most immediate change in medical practice from these findings is the potential they have in "revolutionizing what pathology departments do," Schlegel says.
8 d/ n- V4 N7 m' p9 P
$ z$ ]5 C( D0 H/ M; y"Today, pathologists don't work with living tissue. They make a diagnosis from biopsies that are either frozen or fixed and embedded in wax," he says. "In the future, pathologists will be able to establish live cultures of normal and cancerous cells from patients, and use this to diagnose tumors and screen treatments. That has fantastic potential."
5 v3 T2 y1 v) {' j
9 E4 `: C1 P( Z" D6 gThis research was funded by grants from the National Institutes of Health, Department of Defense fellowship funding, and an internal grant from Georgetown Lombardi's Cancer Center Support Grant from the National Cancer Institute.4 W; U* q6 j% e3 y+ S# g

" R$ S0 g  n2 |& s6 uGeorgetown University and the National Institutes of Health have filed two patent applications on technologies described in this paper. The inventors for the patent application related to immortalization of non-keratinocyte technology described in this paper which is jointly owned by Georgetown and NIH include Schlegel, Xuefeng Liu and Alison McBride. Sandra Chapman and McBride are co-inventors on a separate patent application filed by the National Institutes of Health related to keratinocyte technology described in this paper.
0 Z, `& R1 y9 u. @5 j* i& y
. l( e* y1 R4 F) C
# J) n5 _0 p( q3 s' g15楼原文 感谢subenjinwo 提供

yuhaoze

Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor

yuhaoze

感谢subenjinwo的分享，我从一位朋友那里也得到了这篇文章。ROCK Inhibitor and Feeder Cells Induce the Conditional Reprogramming of Epithelial Cells，讲的是用这两个因子在体外使非角质化上皮细胞永生化的过程，其实感觉这篇文章的前身是2010年发表在J Clin Invest(IF=14.152)的Human keratinocytes are efficiently immotalized by a Rho kinase inhibitor, 可以了解到作者用饲养层细胞和y27632在体外将上皮细胞永生化，代替了导入原癌基因端粒酶基因等有可能使细胞带有致瘤性的方法。得到的永生化上皮细胞体现出了被”部分重编程“的特征，例如端粒酶活性增加等等，并且，在去掉这两个factor其中任何一个的时候，这些细胞都会重新变回原来的上皮细胞，其功能和形态与原代细胞非常相似，并且在传了90多代之后，核型依然正常。2 b1 Z7 d8 i9 K5 H6 k- l/ j
我对这两篇文章非常感兴趣，因为如果真的如作者所发现，这两种因子可以使细胞在体外可以无线增值，并且使其性状保持正常，那么这对医学领域将是一个非常好的消息，会大大facilitate获取细胞的过程。

sunsong7

tpwang 发表于 2012-1-14 15:48
- A+ o% `3 t+ M, J回复 sunsong7 的帖子5 t1 c( J7 x6 i6 q6 T
0 ]+ j! Q# t9 C/ D* I, W9 t8 H
我问的是这个stem cell like细胞除了倒回原始细胞，能否分化为其他细胞。

( t: U3 d- X: u. I呵呵，这么快又进了一步：
" l8 k) g) r1 n# H& [科学家首次将脐带干细胞转化为脑支持细胞！ http://www.stemcell8.cn/thread-51562-1-1.html7 \. z6 g8 q, E9 i/ a

subenjinwo

ROCK Inhibitor and Feeder Cells Induce the Conditional Reprogramming of Epithelial Cells

ly200101042

我也想要全文

yuhaoze

难道就没有原文么！

zhanglianglu

, G! s- d5 s& y) R& `8 V0 }, t. v2 b3 {- s+ B6 A( Q
ROCK Inhibitor and Feeder Cells Induce the Conditional Reprogramming of Epithelial Cells. g8 `6 Q/ S, ?" w$ e5 h& i
We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 106 cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.9 [  u: q0 p7 k0 }6 B  c

' ~  a/ X. B( x5 e

fguw

还有蛮有意思的，不过，细胞是不是真的“正常”？; Z% I. S3 p8 j/ q# d
是不是功能试验需要些，不知道原文有没有

yuhaoze

ROCK Inhibitor and Feeder Cells Induce the Conditional Reprogramming of Epithelial Cells4 z4 a3 l' C8 ^- z6 i! I
http://www.journals.elsevierheal ... /article/S0002-9440(11)01059-5/pdf