Preparative irradiation for hepatocyte transplantation

描述(由申请人提供)： 肝细胞移植在遗传性和获得性肝病的治疗中具有重要价值。然而，目前这一过程的好处受到一次安全移植的肝细胞数量的限制。一种允许移植细胞优先于宿主肝细胞增殖的方法将对肝脏的重新填充具有重要价值。为了开发一种临床可行的肝移植准备方案，我们探索了肝再生的制备性肝照射(HIR)。虽然预备性照射已被常规用于骨髓移植，但我们是第一个将其应用于促进HT的。适形、三维和调强放射治疗的进展使得在不发生肝功能衰竭的情况下提供更高的辐射剂量。我们的初步结果表明，预备性HIR和部分肝切除(PH)，然后是HT，在12周内几乎所有宿主肝细胞都被移植的未受照射的肝细胞所取代。尽管PH提供了强大的有丝分裂刺激，但它是一种侵入性手术，在临床上适用于有限的情况，例如需要肝切除的肝癌患者。为了扩大制备HIR在临床上的适用性，我们需要(I)提高供体细胞的获取以及肝脏再增殖的速度和程度，(Ii)设计非侵入性的PH替代品，(Iii)减少所需的HIR剂量或为部分肝脏提供HIR以供选择性叶再生长，以及(Iv)建立一种无创的方法来监测肝脏辐射损伤和供体细胞在肝脏中的增殖。我们设计的实验旨在验证以下假设：(1)HIR对肝窦内皮细胞的氧化损伤应促进移植肝细胞的植入。(2)由于PH+HIR后的HT可使肝脏放射性损伤恢复正常，我们假设PH+HIR+HT后的肝脏代谢状态(用ATP/PI指数评估)将会恢复，对肝脏的31P-MRS分析将使我们能够评估受照射宿主肝脏中供体细胞的增殖情况。(3)促肝细胞生长因子或甲氨蝶呤+HIR诱导的肝细胞坏死可替代PH作为移植肝细胞的增殖刺激。由于PH+HIR方案在我们实验室已经成为一种标准化和可重复性的制备方案，在特定的目标1A中，我们将使用PH+HIR方案作为模型系统来测试旨在改善供体细胞植入、评估各种类型供体细胞(例如大肝细胞和小肝细胞)的增殖潜力以及标准化基于磁共振的无创性代谢恢复和供体细胞增殖评估的变量。一旦这些变量在我们的PH+HIR方案中得到优化，我们将在基于HIR的无创HT方案中使用优化的参数。特定目的IB将检查MRS是否可以评估受照射动物的肝脏能量代谢和评估供体细胞的再繁殖。在特定的目标2A中，我们将研究是否可以使用肝生长因子、HGF和甲状腺激素等伴生因素来替代PH作为供体细胞的有丝分裂刺激。在特定的目标2B中，将测试由氨甲喋呤+HIR诱导的肝坏死提供的代偿性再生刺激是否可以作为PH的替代品。

DESCRIPTION (provided by applicant): Hepatocyte transplantation (HT) could be of major value in the treatment of both inherited and acquired liver diseases. However, benefits of this procedure are currently limited by the number of hepatocytes that can be transplanted safely at a time. A method that would permit preferential proliferation of the engrafted cells over host hepatocytes would be of great value in repopulating the liver. In order to develop a clinically feasible preparative regimen for HT, we have explored preparative hepatic irradiation (HIR) for liver repopulation. Although preparative irradiation has been used routinely for bone marrow transplantation, we were the first to apply it to facilitate HT. Advances in conformal 3-dimensional and intensity-modulated radiation therapy permit the delivery higher radiation doses without liver failure. Our preliminary results demonstrate that preparative HIR and partial hepatectomy (PH), followed by HT results in the replacement of virtually all host hepatocytes by the transplanted non-irradiated hepatocytes in 12 weeks. Although PH provides a robust mitogenic stimulus, it is an invasive procedure, which is clinically applicable in limited situations, such as in patients requiring hepatic resection for liver cancer. To broaden the applicability of preparative HIR in the clinic, we need to (i) enhance the enqraftment and the rapidity and extent of hepatic repopuiation of the donor cells, (ii) design non-invasive alternatives to PH, (iii) reduce the required HiR dose or provide HIR to a portion of the liver for selective lobar repopulation, and (iv) develop a noninvasive method to monitor hepatic radiation injury and donor cell proliferation in the liver. We have designed our experiments to test the following Hypotheses - (1) HIR-induced oxidative damage to the sinusoidal endothelium of the liver should enhance engraftment of transplanted hepatocytes. (2) Since HT after PH+HIR normalizes hepatic radiation injury, we, hypothesize that hepatic metabolic state (assessed by the ATP/Pi index) would be restored in animals treated with PH+HIR+HT and 31P-MRS analyses of the liver would enable us to evaluate donor cell proliferation in the irradiated host liver. (3) Hepatotropic growth factors or methotrexate+HIR-induced necrosis should substitute for PH as a proliferative stimuli to transplanted hepatocytes. Since PH + HIR has become a standardized and reproducible preparative regimen in our laboratory, in Specific aim 1A, we will use the PH+HIR regimen as a model system to test variables aimed at improving donor cell engraftment, evaluation of the proliferative potential of various types of donor cells (e.g. large vs. small hepatocytes) and standardization of a noninvasive magnetic resonance-based assessment of metabolic recovery and donor cell proliferation. Once these variables are optimized in our PH+HIR regimen, we will use the optimized parameters in our HIR-based noninvasive regimens of HT. Specific aim IB will examine whether MRS can assess hepatic energy metabolism in irradiated animals and evaluate donor cell repopulation. In Specific aim 2A, we will examine whether hepatic growth factors, HGF and comitogens such as thyroid hormone can be used to substitute PH as a mitogenic stimulus for donor cells. In specific aim 2B, will test whether compensatory regenerative stimuli, provided by methotrexate + HIR-induced liver necrosis, can be used as a substitute to PH.