小鼠骨髓移植模型造模方法.docx

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 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 isolated 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 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 cell 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) 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 of 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 pronasedigestion 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 transplantation 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 Control 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 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 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 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