發(fā)布時間：2018-04-05 01:33

  本文選題：胚胎干細胞　切入點：腎上皮細胞　出處：《第二軍醫(yī)大學》2006年博士論文

【摘要】：研究背景和目的:由于抗生素以及抗腫瘤藥物的廣泛應(yīng)用,而腎臟又是一個藥物毒性頻繁作用的器官,因此近年來藥物性腎損傷發(fā)病率逐年提高,已經(jīng)成為危害人們身體健康的一大疾病。由于腎小管上皮細胞對于許多藥物具有分泌和重吸收功能,因而細胞內(nèi)藥物濃度較高,所以這一類疾病中,腎小管上皮細胞變性壞死發(fā)生率較高。藥物性腎損害程度較輕時,主要表現(xiàn)為急性腎小管損傷,損傷較重時表現(xiàn)為急性腎小管壞死。前者通常是可逆性的加上腎臟本身也具有一定的修復功能,因此可以通過適當?shù)膶ΠY治療得到治愈;而后者常由于治療不及時而引起腎功能衰竭,最終導致患者死亡。目前對于腎功能衰竭常用的透析療法,如持續(xù)性的腎臟替補療法對于總的致死率沒有什么影響,而最有效的治療手段-腎臟移植,常常由于供體腎臟來源少,且引起免疫排斥反應(yīng)從而導致治療效果較差,上述原因使得人們越來越關(guān)注細胞治療。ES細胞是從動物早期胚胎的內(nèi)細胞團或原始生殖細胞分離出來的具有發(fā)育全能性的一種未分化的無限增殖的細胞系,具有向三個胚層分化的潛能。胚胎干細胞在體內(nèi)體外均能分化成各種類型的細胞,而在特定微環(huán)境中能向特定類型的細胞分化。這些顯著的特性使得ES細胞成為細胞治療的首選供體來源。到目前為止,誘導ES細胞定向分化的研究已取得了有意義的進展,但尚未見到誘導其分化為腎上皮細胞及其相關(guān)分化機制探討的報道。同時對于ES細胞在體內(nèi)對藥物性腎損傷的治療作用也無報道。本研究將腎上皮細胞與胚胎干細胞共同培養(yǎng)并在培養(yǎng)基中添加不同的化學或生物試劑,其目的在于體外誘導胚胎干細胞向腎小管上皮樣細胞分化,并探討分化過程中所涉及的分子機制;同時利用順鉑建立腎損傷小鼠模型,利用此模型初步探討胚胎干細胞對損傷腎的修復作用以及ES細胞在體內(nèi)的分化情況。這一部分主要側(cè)重于修復作用的觀察。 第一部分:體外誘導胚胎干細胞向腎小管上皮樣細胞分化模型的建立 方法:(1)條件培養(yǎng)基收集及配制:腎小管上皮細胞(TCMK-1)單層培養(yǎng)48小時后,收集培養(yǎng)基,離心,過濾,備用。將不含LIF的ES細胞培養(yǎng)基與腎小管上皮細胞培養(yǎng)基按3:1混和,配制成條件培養(yǎng)基,4℃保存?zhèn)溆谩?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，

本文編號：1712646