【作者】 王平；

【导师】 陈江华；

【作者基本信息】 浙江大学 ， 内科学， 2006， 硕士

【摘要】 研究背景:急慢性肾脏功能衰竭是临床常见的肾脏疾病,二十年来其主要治疗方法之一就是以血液透析或血液滤过方式为主的肾脏替代疗法。虽然它明显延缓了肾衰患者的疾病进展,但长期血液透析带来的一系列的并发症以及二十年来居高不下的死亡率仍然无法得到根本解决。研究者认为,目前的肾脏替代疗法仅仅替代了肾小球滤过水溶性中小分子物质的功能,而无法提供肾小管的重吸收、代谢、及内分泌等重要生理功能。众所周知,肾小管细胞在糖原再生、谷胱甘肽合成、以及其他中分子物质重吸收、活化VitD3等方面有着重要作用,从而有助于机体应激状态下的防卫。但肾脏及其肾小管细胞潜在的免疫学功能,还较少为人认识。同时,人们逐渐认识到,丧失肾功能不再仅仅是疾病严重性的标志,而已成为患者死亡率的独立性预测因素。ARF患者向SIRS和脓毒症发展的倾向性也提示肾功能,尤其是发生ATN后的肾小管细胞的功能在个体应激状态下起着关键的免疫调节作用。另外,研究发现在败血症休克早期就出现肾小管上皮细胞的损害。因此,人们猜测如果及早地替代在脓毒症休克中受损的肾小管上皮细胞的生理活性,或许可以改善SIRS阶段机体的免疫调节功能,从而减轻进一步破坏性发展的过程。而目前倍受关注的细胞疗法的特点就是利用细胞和组织培养技术扩增某种特定细胞,通过体外循环或者植入体更多还原

【Abstract】 BackgroudAcute and chronic renal failure are familiar diseases in clinic. Mostly, the treatment is the renal substitution therapy with hemodialysis or hemofiltration treament. Although current renal substitution therapy has had a dramatic life-sustaining effect in patients suffering from both acute or chronic renal failure, these clinical disorders are still serious medical conditions. This persistently poor prognosis of these disorders may be due to the fact that hemodialysis and hemofiltrations are not complete renal therapies. These techniques provide only a clearence or filtration function for small solute but not replace the lost transport ,metabolic, and endocrine functions of the kidney, which are predominantly found in the tubular elements of the organ.The renal tubule cell’s role in gulutathione reclamation, glutathione peroxidase synthesis, other middle molecule metabolism, and activatin of vitamin D3,are well recongnized pathways to maintain import tissue integrity and host defense under stress conditions. A less recongnized role of the kidney and the renal tubule cells is its potential immunoregulatory functionl.Loss of renal function is an independent predictor of mortality in hospitalized patients weiht ARE At the same time , Loss of renal function not only is the sign of seriousness of disease, but also is anindependent predictor of mortality in hospitalized patients with ARF.The propensity of patients with ARF to develop SIRS and sepsis suggests that renal function, specifically renal tubule cell function secondary to ATN, plays a critical immunomodunlatory role in individuals under stress states. Additional,renal tubule cells are injured early in septic shock. So, we hypothesis that replacement of their activity with cell therapy will improve immunoregulation of the SIRS of sepsis and thereby provide additional physiologic activities that improve the current natural history of this disease process. Cell therapy is dependent on cell and tissue culture methodologies to expand specific cells to replace important differentiated processes deranged or lost in various disease states. Bioartificial renal tubule is just the applacations in renal disease based on cell therapy.In order to replace the renal tubule funtion, people has developed an extracorporeal device using a standard hemofiltratin cartridge containing many renal tubule cells ,which has the function of renal tubule. According to the studies of Humes HD et al, In vitro studies of the renal tubule assist device(RAD)have shown that the cells retain differntiated active transport properties, differntiated metabolic activities,and important endocrine proceses. Further studies have shown that the RAD, when incorporated in series with a hemofiltration cartridge in an extra-corporeal blood perfusin circuit,replaces filtrations, transport, metabolic, and endocrine functions of the kidney in acutely uremic dogs. There is in the growing appreciation that most disease processes are not due to the lack of a single protein but develop due to alterations in complex interactions of a variety of cell products. Bioartificial renal tubule based on cell therapy is an exciting approach to the treament of acute and chronic renal disease. Based on these abundance backgrounds, this study focus on the building of bioartifical renal tubule assist device, in order to creating a working flat for the further investigation correlated animal and clinical studies.ObjectiveTo study the buildup of the bioartificial renal tubule asisit device, simulate the proliferation condition of the implantation cells, and investigate the method of building bioartificial renal tubule asisit device.Methods—. Renal Tubule Asisit Device. Design and Cell SeedingF60 high-fix hemofiltration cartridge containing polysulfone hollow fibers was choosen as bioreactor. Then connected with tubings for the next work.Firstly,50ml serum free RPMI 1640 medium was perfused into the intracapillary. To prepare the intraluminal surface of the hollow fiber for epithelial attachment and confluent growth, serum free RPMI 1640 medium containing laminin (2ng/m 2 )was perfused into the intracapillary and allowed to remain for 60min.secondly,renal tubule cells were dislodged from the culture plates with hosphate buffered saline containing 0.25%trypsin. the cells were suspended in 80ml culture medium with 1% fetal calf serum at a total number of 6.2 X 107.Thirdly, 150ml culutre medium with 10% fetal calf serum was perfused into the extracapillary. Fourthly, the cell suspension was introduced into the intracapillary form two ports of the filtration cartridge, and allowed to sit for 90 min. After this time, the bioreactor was rotated 90°,and another seeding of cells was performed. This process was repeated two more times to complete a 360° seeding procedure. The final cell suspension was flushed from the intracapillary, and the bioreactor was placed in a cell incubator .After 24 hr, the bioreactor was perfused with culture media through the extracapillary space(ECS) at 150nl/min.Four days after cell seeding, the bioreactor was perfused intraluminally with culture media at a rate of 10^/min,lasting for about 10 days.—-. Histologic ProcessingDrew out the polysulfone hollow fibers from the hemofiltration cartridge onthe lth,4th, and 14th day after seeding cells, respectively. Samples were fixed with 95 alcohol or 4% fonnaldehyd. For light microscopy, samples were embedded in paraffin, and sectioned and processed with HE staining methods.ResultsIn the sections of fibers drew out from the hemofiltration cartridge on the lth day after seeding LLC-PKl,we found some attached cells in the light microscopy. While on the 4th and 14th day, there was no cells to be observed. Counting the cells in the outflewed medium from intracapillary on the 5th, 10th, 14th day, respectively, the outcome of the cell counting was: 25.2X 10\ 28.4X 10\ 7.6X 106, amount to about 6.1 X 107, which was not obvious difference with the total of seeding cells at first.ConclusionIn the construction of RAD, the initial steps in the construction of a RAD had already finished to optimize renal substitution therapy . The method of building RAD had explored initially. Summarized some problems in the construction, pointed out the reasons which influenced the success of model to build up, also had accumulated some experiences for the next work.更多还原