小鼠骨髓移植模型造模方法

Materials and Methods

Mouse Models

All animal work was carried out under procedural and

ethical guidelines of the British Home Office. To determine

the contribution of the BM to hepatic stellate cells and myo-

fibroblasts during the development of cirrhosis, we performed

sex mismatched BM transplantations from male donor mice

into female recipients; 6-week-old female Balb/c mice received

lethal irradiation (8 Gy in a divided dose 4 hours apart) and

whole BM transplants from 6-week-old male donors. Mice

immediately received a tail vein injection of BM; unless stated,

this was 1 _ 106 whole BM cells isolated from flushing the

femur, tibia, and pelvis of male donor mice with a 29-gauge

needle containing phosphate-buffered saline (PBS)/2% fetal

calf serum (FCS). Mice were placed on acidified water, and, 4

weeks later, mice received intraperitoneal (IP) injections of 1

_L per gram body weight of a CCl4/olive oil mixture (1:7

ratio, Sigma-Aldrich, Gillingham, United Kingdom) every 5

days. Groups of mice (n _ 4 unless stated) were killed at

intervals from 0 to 12 weeks of CCl4, always at 72 hours

following the last injection.

To determine whether BM-derived stellate cells and myo-

fibroblasts were a stable cell population after the recovery of

liver injury, BM transplanted mice that had received 8 weeks

of CCl4 were allowed to recover for 8 weeks prior to tissue

analysis. A second model of liver damage was also used: female

Balb/c mice received male BM transplants as before; 4 weeks

later, TAA (Sigma, T-8531) was administered IP at 200

mg/kg body weight (diluted in distilled water) 3 times each

week for 4 weeks. Mice receiving TAA (n _ 8) and controls

(no damage, n _ 4) were killed, and tissue was harvested 3

days after the final dose of TAA.

Cells of BM origin were tracked in liver sections through

the use of fluorescent in situ hybridization (FISH) for the Y

chromosome. In addition, to confirm the FISH analysis in

tissue, male and female control mice and a number of mice that

had received BM transplants and 8 weeks of CCl4 had stellate

cells isolated from their livers, using collagenase and pronase

digestion followed by density centrifugation.

25

FISH was performed

on the isolated stellate cells.

To assess whether the BM-derived hepatic myofibroblasts

were capable of intrahepatic collagen transcription, 6-week-old

female C57/B6 mice (after 10 Gy irradiation in a divided dose

4 hours apart) underwent transplantation with whole BM from

6-week-old male Col1a2 mice that express the _-galactosidase

(_-gal) reporter gene under control of the _2(I) collagen gene

enhancer, giving a direct assay of transcriptional activity for

collagen type I.

26

This mouse model activates the transgene

following CCl4 injury.

27

Control mice received BMtransplants

from C57/B6 mice; all mice received 12 weeks of CCl4.

To analyze whether BM-derived myofibroblasts can determine

the fibrotic phenotype in liver injury, C57/B6 mice

received BM transplants from Col 1a1rr mice (n _ 4). These

mice have mutated collagen, which is collagenase resistant,

and, when their livers are injured by CCl4, the mice develop

extensive pericellular fibrosis.

28

Control mice received BM

transplants from C57/B6 mice (n _ 4); all mice received 8

weeks of CCl4 and were killed 1 week following the final

injection.

To determine whether the hepatic myofibroblasts were of

MSC or hematopoietic stem cell (HSC) origin, 6-week-old

female Balb/c mice were lethally irradiated and received BM

from donor mice as follows: Group 1 received injections of 1.2

_ 106 enriched female MSCs and 2.3 _ 105 enriched male

HSCs (n _ 3). Group 2 received injections of 1.2 _ 106

enriched male MSCs and 2.3 _ 105 enriched female HSCs (n

_ 3). All mice received 6 weeks of CCl4. The contribution of

each BM stem cell fraction to hepatic myofibroblast populations

was assessed by performing immunohistochemistry for

_-smooth muscle actin (_-SMA) together with FISH for the Y

chromosome.

材料与方法

小鼠模型

所有动物进行训练工作，根据程序和

道德准则的英国家庭办公。确定

贡献的骨髓，以肝星状细胞和肌

-

成纤维细胞发育过程中的肝硬化，我们演出

性别错配骨髓移植手术，由男供鼠

到女受助人

; 6

周岁的女

BALB / C

小鼠收到

致命的辐射（

8

照射在一个分裂的剂 量

4

小时之遥）

，并

整个骨髓移植，从

6

周龄雄性捐助者。小鼠

立即收到了尾静脉注射骨髓

;

除非另有说明，

< br>

这是一

_ 106

整个骨髓细胞分离冲水

股骨， 胫骨，骨盆的男性供鼠与一个

29

轨距