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1、Materials and MethodsMouse ModelsAll animal work was carried out under procedural andethical guidelines of the British Home Office. To determinethe contribution of the BM to hepatic stellate cells and myo-fibroblasts during the development of cirrhosis, we performedsex mismatched BM transplantations
2、 from male donor miceinto female recipients; 6-week-old female Balb/c mice receivedlethal irradiation (8 Gy in a divided dose 4 hours apart) andwhole BM transplants from 6-week-old male donors. Miceimmediately received a tail vein injection of BM; unless stated,this was 1 _ 106 whole BM cells isolat
3、ed from flushing thefemur, tibia, and pelvis of male donor mice with a 29-gaugeneedle containing phosphate-buffered saline (PBS)/2% fetalcalf serum (FCS). Mice were placed on acidified water, and, 4weeks later, mice received intraperitoneal (IP) injections of 1_L per gram body weight of a CCl4/olive
4、 oil mixture (1:7ratio, Sigma-Aldrich, Gillingham, United Kingdom) every 5days. Groups of mice (n _ 4 unless stated) were killed atintervals from 0 to 12 weeks of CCl4, always at 72 hoursfollowing the last injection.To determine whether BM-derived stellate cells and myo-fibroblasts were a stable cel
5、l population after the recovery ofliver injury, BM transplanted mice that had received 8 weeksof CCl4 were allowed to recover for 8 weeks prior to tissueanalysis. A second model of liver damage was also used: femaleBalb/c mice received male BM transplants as before; 4 weekslater, TAA (Sigma, T-8531)
6、 was administered IP at 200mg/kg body weight (diluted in distilled water) 3 times eachweek for 4 weeks. Mice receiving TAA (n _ 8) and controls(no damage, n _ 4) were killed, and tissue was harvested 3days after the final dose of TAA.Cells of BM origin were tracked in liver sections throughthe use o
7、f fluorescent in situ hybridization (FISH) for the Ychromosome. In addition, to confirm the FISH analysis intissue, male and female control mice and a number of mice thathad received BM transplants and 8 weeks of CCl4 had stellatecells isolated from their livers, using collagenase and pronasedigesti
8、on followed by density centrifugation.25 FISH was performedon the isolated stellate cells.To assess whether the BM-derived hepatic myofibroblastswere capable of intrahepatic collagen transcription, 6-week-oldfemale C57/B6 mice (after 10 Gy irradiation in a divided dose4 hours apart) underwent transp
9、lantation with whole BM from6-week-old male Col1a2 mice that express the _-galactosidase(_-gal) reporter gene under control of the _2(I) collagen geneenhancer, giving a direct assay of transcriptional activity forcollagen type I.26 This mouse model activates the transgenefollowing CCl4 injury.27 Con
10、trol mice received BMtransplantsfrom C57/B6 mice; all mice received 12 weeks of CCl4.To analyze whether BM-derived myofibroblasts can determinethe fibrotic phenotype in liver injury, C57/B6 micereceived BM transplants from Col 1a1rr mice (n _ 4). Thesemice have mutated collagen, which is collagenase
11、 resistant,and, when their livers are injured by CCl4, the mice developextensive pericellular fibrosis.28 Control mice received BMtransplants from C57/B6 mice (n _ 4); all mice received 8weeks of CCl4 and were killed 1 week following the finalinjection.To determine whether the hepatic myofibroblasts
12、 were ofMSC or hematopoietic stem cell (HSC) origin, 6-week-oldfemale Balb/c mice were lethally irradiated and received BMfrom donor mice as follows: Group 1 received injections of 1.2_ 106 enriched female MSCs and 2.3 _ 105 enriched maleHSCs (n _ 3). Group 2 received injections of 1.2 _ 106enriched
13、 male MSCs and 2.3 _ 105 enriched female HSCs (n_ 3). All mice received 6 weeks of CCl4. The contribution ofeach BM stem cell fraction to hepatic myofibroblast populationswas assessed by performing immunohistochemistry for_-smooth muscle actin (_-SMA) together with FISH for the Ychromosome.材料与方法小鼠模型
14、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
15、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
