Rescue of Hemostasis-deficiency States

止血不足状态的救援

• 批准号: 6853271
• 负责人: ELLIOT David ROSEN
• 金额: $ 43.19万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6853271
• 关键词: biotechnology; blood coagulation; blood coagulation disorders; cell line; cell population study; cell transplantation; coagulation factor X; disease /disorder model; embryo /fetus therapy; gene expression; gene mutation; gene targeting; genetic disorder; genetic manipulation; genetically modified animals; hemostasis; in utero transplantation; laboratory mouse; liver cells; model design /development; molecular pathology; nonhuman therapy evaluation; pathologic process; therapy design /development

Factor X (FX)-deficient embryos suffer partial embryonic lethality with approximately 30% of the embryos arresting at midgestation. The remaining animals survive to term, but die perinatally mainly from abdominal or intracranial hemorrhage. We have rescued FX-deficient mice by transplanting liver cells from FX+/+ Rosa26 fetuses into midgestation embryos-derived from FX+/- heterozygous crosses. FX-/- embryos were born at the expected frequency and 50% of the FX-/- neonates survived longer than 4 months. FX-/- embryos receiving saline injections that survived to term died perinataily, similar to untreated FX-/- mice. The plasma levels of FX in the rescued 16-week genotypically FX-/- mice were approximately 2-5% of age-matched wild-type levels. Beta-galactosidase-staining cells, derived from the donor Rosa26 fetal liver cells, were detected in approximately 45% of the livers of adult mice. The observations suggest that this rescue approach can be used to treat potential hemophiliacs detected in utero. The aims of this proposal are to better define and modify the procedure to develop: 1) a therapeutic strategy to treat deficiencies of liver-expressed proteins; 2) a research protocol for conditional rescue of critical hemostatic factors. This latter aim involves characterizing the donor cell population to identify the cell subpopulations possessing liver-colonizing properties. It is also proposed to develop cultured cell lines with colonizing activity, thus eliminating the need for primary cells. Furthermore, the donor cells will be modified genetically to increase their efficacy for this strategy. This will involve increasing the levels of expression of the factor of interest, or increasing the liver-colonizing potential of the donor cell lines. The introduction of negative selection markers into the donor cell population would enable the eradication of the donor cell population at a chosen time. This permits the approach to be used as a conditional rescue strategy to study the physiological consequences of the deficiency of particular genes in adult animals, when the same deficiency would normally result in embryonic or perinatal lethality. In particular, this strategy will be used to study the pathophysiological consequences of FX deficiency in vivo in older mice, a feat that cannot be accomplished at present.

因子X（FX）缺陷胚胎遭受部分胚胎致死，约30%的胚胎在妊娠中期停止。其余动物存活至足月，但主要死于围产期腹部或颅内出血。我们通过将来自FX +/+ Rosa26胎儿的肝细胞移植到来自FX +/-杂合子杂交的妊娠中期胚胎中，拯救了FX缺陷小鼠。FX-/-胚胎以预期的频率出生，50%的FX-/-新生儿存活超过4个月。接受盐水注射的FX-/-胚胎存活至围产期死亡，与未处理的FX-/-小鼠相似。在获救的16周基因型FX-/-小鼠中，FX的血浆水平约为年龄匹配的野生型小鼠的2 - 5 程度.在大约45%的成年小鼠肝脏中检测到源自供体Rosa 26胎肝细胞的β-半乳糖苷酶染色细胞。观察结果表明，这种拯救方法可用于治疗子宫内检测到的潜在血友病患者。该提案的目的是更好地定义和修改程序，以开发：1）治疗肝脏表达蛋白缺陷的治疗策略; 2）有条件挽救肝脏表达蛋白缺陷的研究方案。 关键止血因素。后一个目的涉及表征供体细胞群以鉴定具有肝定殖特性的细胞亚群。还提出开发具有定殖活性的培养细胞系，从而消除对原代细胞的需要。此外，供体细胞将被遗传修饰，以增加其对该策略的功效。这将涉及增加感兴趣的因子的表达水平，或增加供体细胞系的肝定殖潜力。将阴性选择标记物引入供体细胞群将能够根除供体细胞 人口在一个特定的时间。这使得该方法可以用作一种有条件的拯救策略，来研究成年动物特定基因缺陷的生理后果，而同样的缺陷通常会导致胚胎或围产期死亡。特别是，这一策略将被用来研究FX缺乏症在老年小鼠体内的病理生理后果，这是目前无法完成的壮举。