胚胎干细胞体外分化为肾小管上皮细胞分子机制及其在肾损伤修复中作用的实验研究

发布时间：2018-04-05 01:33

  本文选题：胚胎干细胞　切入点：肾上皮细胞　出处：《第二军医大学》2006年博士论文

【摘要】：研究背景和目的:由于抗生素以及抗肿瘤药物的广泛应用,而肾脏又是一个药物毒性频繁作用的器官,因此近年来药物性肾损伤发病率逐年提高,已经成为危害人们身体健康的一大疾病。由于肾小管上皮细胞对于许多药物具有分泌和重吸收功能,因而细胞内药物浓度较高,所以这一类疾病中,肾小管上皮细胞变性坏死发生率较高。药物性肾损害程度较轻时,主要表现为急性肾小管损伤,损伤较重时表现为急性肾小管坏死。前者通常是可逆性的加上肾脏本身也具有一定的修复功能,因此可以通过适当的对症治疗得到治愈;而后者常由于治疗不及时而引起肾功能衰竭,最终导致患者死亡。目前对于肾功能衰竭常用的透析疗法,如持续性的肾脏替补疗法对于总的致死率没有什么影响,而最有效的治疗手段-肾脏移植,常常由于供体肾脏来源少,且引起免疫排斥反应从而导致治疗效果较差,上述原因使得人们越来越关注细胞治疗。ES细胞是从动物早期胚胎的内细胞团或原始生殖细胞分离出来的具有发育全能性的一种未分化的无限增殖的细胞系,具有向三个胚层分化的潜能。胚胎干细胞在体内体外均能分化成各种类型的细胞,而在特定微环境中能向特定类型的细胞分化。这些显著的特性使得ES细胞成为细胞治疗的首选供体来源。到目前为止,诱导ES细胞定向分化的研究已取得了有意义的进展,但尚未见到诱导其分化为肾上皮细胞及其相关分化机制探讨的报道。同时对于ES细胞在体内对药物性肾损伤的治疗作用也无报道。本研究将肾上皮细胞与胚胎干细胞共同培养并在培养基中添加不同的化学或生物试剂,其目的在于体外诱导胚胎干细胞向肾小管上皮样细胞分化,并探讨分化过程中所涉及的分子机制;同时利用顺铂建立肾损伤小鼠模型,利用此模型初步探讨胚胎干细胞对损伤肾的修复作用以及ES细胞在体内的分化情况。这一部分主要侧重于修复作用的观察。 第一部分:体外诱导胚胎干细胞向肾小管上皮样细胞分化模型的建立 方法:(1)条件培养基收集及配制:肾小管上皮细胞(TCMK-1)单层培养48小时后,收集培养基,离心,过滤,备用。将不含LIF的ES细胞培养基与肾小管上皮细胞培养基按3:1混和,配制成条件培养基,4℃保存备用。(2)建立胚胎干
[Abstract]:Background and objective: because of the widespread use of antibiotics and antitumor drugs, the kidney is a frequently toxic organ, so the incidence of drug-induced renal injury has been increasing year by year in recent years.It has become a major disease that harms people's health.Because renal tubular epithelial cells have the function of secretion and reabsorption of many drugs, the intracellular drug concentration is higher, so the incidence of degeneration and necrosis of renal tubular epithelial cells is higher in this kind of diseases.When the degree of drug induced renal damage was mild, the main manifestations were acute tubular injury and acute tubular necrosis.The former is usually reversible and the kidney itself has a certain repair function, so it can be cured by proper symptomatic treatment, while the latter often causes renal failure due to untimely treatment and eventually leads to the death of the patient.At present, dialysis therapy commonly used for renal failure, such as continuous renal replacement therapy, has little effect on the overall mortality rate, and the most effective treatment, kidney transplantation, is often due to a small donor kidney source.And it causes immune rejection, which leads to poor therapeutic effect.For these reasons, there is growing concern that cell therapy. Es cells are an undifferentiated, infinite proliferative cell line, separated from the inner cell mass of an early animal embryo or primordial germ cells.It has the potential to differentiate into three embryo layers.Embryonic stem cells can differentiate into various types of cells in vitro and in vivo, but can differentiate into specific types of cells in specific microenvironments.These remarkable characteristics make es cells the preferred donor source for cell therapy.Up to now, significant progress has been made in the research of inducing es cells to differentiate into renal epithelial cells, but there are no reports on inducing es cells to differentiate into renal epithelial cells and their related differentiation mechanisms.At the same time, there is no report on the therapeutic effect of es cells on drug induced renal injury in vivo.In this study, renal epithelial cells and embryonic stem cells were co-cultured and different chemical or biological reagents were added to the culture medium to induce the differentiation of embryonic stem cells into renal tubular epithelioid cells in vitro.The molecular mechanism involved in the process of differentiation was discussed, and the model of kidney injury in mice was established by cisplatin. The effects of embryonic stem cells on the repair of damaged kidney and the differentiation of es cells in vivo were preliminarily studied.This part mainly focuses on the observation of the effect of restoration.Part one: establishment of model of differentiation of embryonic stem cells into renal tubular epithelioid cells induced in vitroMethods the conditional medium was collected and prepared: after 48 hours of monolayer culture of TCMK-1, the culture medium was collected, centrifuged, filtered and set aside.Embryonic stem was established by mixing es cell medium without LIF with renal tubular epithelial cell culture medium at 3:1 and preparing conditional medium for preservation at 4 鈩，

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