小鼠骨髓移植模型造模方法.docx

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，
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
针含有磷酸盐缓冲液（ PBS ） / 2 ％胎儿
小牛血清（ FCS ）的。小鼠放在酸化水，并在四日
两周后，小鼠腹腔（ IP ）的针剂1
_l每克体重一ccl4/olive油混合物（ 1时07分
比，西格玛-爱秩序， Gillingham ，英国） ，每5
天。组小鼠（ n _四日除非另有说明）被打死在
间隔从0到12个星期的四氯化碳，始终处于72小时
继去年注资。
，以确定是否骨髓源星状细胞和肌-
成纤维细胞是一种稳定的细胞群体之后的复苏
肝损伤，骨髓移植小鼠已收到8周
四氯化碳被允许恢复为8周之前组织
分析。第二个模型的肝损伤还用于：女
BALB / C小鼠收到男性骨髓移植手术，因为之前;四周
后来，权限与问责表（西格玛，的T - 8531年）是经管的IP在200
毫克/公斤体重（摊薄在蒸馏水）的3倍，每
本周4周。小鼠接受权限与问责表（ _ 8 ）和管制
（没有损坏， n _ 4 ）被杀害，并组织收割三日
几天后，最后剂量的权限与问责表。
细胞的骨髓来源地进行了追踪肝路段通过
利用荧光原位杂交技术（ FISH ）的Y
染色体。此外，以确定鱼分析
组织中，男性和女性对照组小鼠和一些基因小鼠
收到骨髓移植和8周的四氯化碳了星状
细胞中分离出自己的肝脏，用胶原酶和pronase
消化其次密度centrifugation.25鱼类演出
对离体星形细胞。
评估其是否骨髓源性肝肌纤维母细胞
有能力肝内胶原转录， 6周岁
女性c57/b6小鼠（经过10 Gy的照射在一个分裂的剂量
4个小时之遥） ，经历了移植骨髓的整体，从
6周岁男col1a2小鼠表达_ -半乳糖苷酶
（ _ -半乳糖）记者基因控制的_2 （一）胶原基因
增强器，使直接测定的转录活性，为
Ⅰ型胶原i.26这个小鼠模型激活基因
以下四氯化碳injury.27对照组小鼠收到bmtransplants
从c57/b6小鼠;所有小鼠收到12个星期的四氯化碳。
分析是否骨髓源肌纤维母细胞可确定
纤维化表型的肝损伤， c57/b6小鼠
收到骨髓移植，由中校1a1rr小鼠（ n _ 4 ） 。这些
老鼠突变胶原蛋白，这是胶原酶，耐
，并在其肝脏有损伤大鼠，小鼠的发展
广泛pericellular fibrosis.28对照组小鼠所收到的BM
移植手术从c57/b6小鼠（ n _ 4 ） ;所有小鼠获得8
周四氯化碳和死亡一周后，最后
注射。
，以确定是否肝肌纤维母细胞的人
海安或造血干细胞（ HSC ）的原产地， 6周岁
雌性BALB / C小鼠致死量照射，并收到的BM
从供鼠如下：第一组接受注射1.2
_ 106丰富了女性的MSCs和2.3 _ 105丰富男
造血干细胞（ _ 3 ） 。第二组接受注射1.2 _ 106
丰富的男性充和2.3 _ 105丰富女性造血干细胞（
_ 3 ） 。所有小鼠收到6周的四氯化碳。贡献
每一个骨髓干细胞组分，以肝细胞群体
被评估的表演免疫组化
_ -平滑肌肌动蛋白（ _ -平滑肌肌动蛋白） ，连同鱼的Y