摘要
原发性及继发性肾脏疾病晚期均会出现慢性肾功能衰竭,伴有肾性贫血。由于此时肾脏结构的严重破坏,导致肾脏产生促红细胞生成素(EPO)相对不足,是其肾性贫血的主要原因。然而,血清EPO水平检测发现,肾性贫血患者血清EPO水平并不下降,反而高于正常人数倍。肾性贫血患者何以产生如此高水平的EPO呢?EPO水平正常或升高而病人却出现贫血,其EPO来源和肾性贫血机制又是如何呢?此外,EPO是红细胞生成的调节因子,通过结合红系祖细胞表面的EPO受体(EPOR)来发挥作用。肾性贫血时EPOR的数量和结构是否出现了变化,EPOR结合的敏感性和亲和力会不会降低?以及EPO-EPOR介导的细胞内信号转导通路是否出现了异常?为了弄清楚上述的问题我们进行了系列的研究。
在实验中,采用5/6肾切除法制备大鼠慢性肾衰竭模型;通过免疫组化方法,观察了EPO及EPOR在慢性肾衰竭时肾、心、肝、肺及脑组织的表达,通过RT-PCR和放免技术检测了肾、肝组织EPOmRNA的表达及血清EPO含量;并通过流式细胞仪、125I-EPO结合实验、RT-PCR和免疫杂交检测了肾性贫血时EPOR数量、与EPO结合的亲和力、EPOR的mRNA和蛋白质的表达水平以及EPOR的功能,即细胞内信号传导通路;并通过骨髓细胞集落培养和3H-TdR掺入实验,观察了肾性贫血时骨髓红系祖细胞增殖和凋亡情况。
结果显示:肾性贫血时EPOmRNA及EPO在残肾组织的表达与正常肾组织无明显变化,肝组织EPOmRNA及EPO表达均明显增强,血清EPO水平较正常升高;骨髓有核细胞EPOR数量减少、与EPO结合的亲和力增加;EPOR的mRNA水平及蛋白质水平均降低,信号蛋白Jak2、Stat5在总蛋白水平接近正常组,但EPO刺激后EPOR及Jak2、Stat5的磷酸化水平减弱;骨髓红系祖细胞增殖减弱和凋亡增加;EPOR在慢性肾衰竭时肾、心、肝、肺及脑组织的表达均增强。
从实验结果中得出以下结论:①采用5/6肾切除法可成功获得慢性肾衰竭及肾性贫血的大鼠疾病模型;②肾性贫血时,肝脏是代偿性产生EPO,提高血清EPO水平的主要器官;③肾性贫血时,肾功能的改变是引起EPOR数量减少的重要原因,EPOR数量减少及功能变化是肾性贫血发病机制之一;④肾性贫血时,EPOR表达的降低导致EPOR受体数量降低;再加上EPOR及其下游Jak2-Stat5通路介导的细胞内信号转导减弱,可能是造成骨髓有核细胞对EPO反应性降低从而导致肾性贫血的分子机制之一;⑤肾性贫血时,骨髓红系祖细胞对EPO反应性降低,增殖减弱,凋亡增加,需要高浓度EPO刺激才能出现增殖高峰,为重组EPO治疗肾性贫血提供了确切的理论依据。
Renal anemia is secondary which occurs when the glomerular rate(GFR) falls below 30mL/min, roughly corresponding to a serum creatinine above 300μmol/L.It is one of the complications of CRF.The main cause of renal anemia is the deficency of EPO synthesis.However,the serum level of EPO in CRF patients is much higher than the normal.The high level of EPO,but the patients are anemia.What is the cause?Why do we use hrEPO in clinic for renal anemia patients?
EPO is the regulatory factor of erythrocyte development. It can promote the erythrocyte series of bone marrow proliferation and differentiation by combination with the EPOR on the surface of erythrocyte. It also suppress the series apoptosis by delay the DNA division. The erythrocyte progenitor reach the summit in the CFU-E phase, about 1000EPOR/per cell. With the maturation process, the EPOR is decreasing and disappear in reticulocyte.
EPO binding to the receptor changes the conformation of the EPO receptor (erythropoietin receptor, EPOR), which is necessary for JAK2 activation by a mechanism of self dimerization. Since the EPO-R, like other hematopoietic cytokine receptors, lacks intrinsic enzymatic activity, the receptor must associate with and activate protein tyrosine kinases in order to transmit a signal. EPO-induced intracellular signaling occurs through a rapid tyrosine phosphorylation of several proteins even though the EPO receptor does not possess endogenous tyrosine kinase activity. First, intracellular signaling occurs by activating the JAK2 tyrosine kinase, which is associated with the EPO receptor constitutively. JAK2 is associated with the EPO receptor in the transmembrane region. After EPO activates the receptor, eight tyrosine residues in the cytoplasmic domain of the EPO receptor are phosphorylated .
Now EPOR structure and its function have been popular accepted, but most works are focus on some normal tissues and cell.There is little resear on the CRF patients.We are interesting whether there is EPOR and its signal pathway changes in bone marrow cell in CRF patiens.In our research, we discussed the tissue-specific EPOR expression.
We use the CRF model of mice according to the international standard of 5/6 nephrectomy.The biochemical parameters were obviously increased after 12 weeks and the count of erythrocyte and Hb were decreased.There was significance between the normal , the persudo-operation group and the operation group.The more severe deficiency of renal function,the more severe of anemia could be found.On the basis of the former study,our research was divided into four parts.
The serum level of EPO in CRF model was studied by radioimmunology. We found that the EPO level is much higher than the normal and persudo-operation group and there was significance. But there is no direct relation between the EPO level and anemia. To further exploer the mechanism of renal anemia, we observed the EPO mRNA level and EPO expression by RT-PCR and immunochemistry. We found there were no changes between the normal and the residul renal tissue. But we found the more expression in liver and suspected that liver maybe the main organ of EPO in CRF model.
In the study of EPOR, we observed the affinity and count of EPOR by FACS and 125-I combination test. The results showed that the number of EPOR was decreased and negative relation with the renal function, right relation with the hemoglobin. We concluded that the severer of renal function, the severe of anemia. So improve the renal function is the main effect mesure. Our experiments also showed that the affinity of EPOR was much lower than the normal and persudo-operation group. There was significance. The results confirmed that the body took effect by strengthen the affinity of EPOR in face of the decreased number of EPOR and EPO level. We also observed the RNA and protein changes of EPOR. The signal transduction protein, Jak2 and Stat5 also were studied. The mRNA level and expression of EPOR were decreased. The phosphorylation of Jak2 and Stat5 was decreased. The results showed that the signal pathway was weakened.
We also observed the erythrocyte series proliferation and apoptosis in CRF model. The number of BFU-E, CFU-E were decreased compared with the normal group in the same level of EPO.3-H combination test and flow cytometry confirmed that the proliferation weakened and apoptosis increased.
On the basis of former study, we also observed the EPOR expression in renal, liver, brain, heart and lung. he results showed that the increasing expression could be found when CRF. Whether the phenomenon is reactive or the protective function in local region should be furthered studied. The relationship between the encephorypathy ,myopahy and lung injure should be explored in later. The former research set foundation for studying the EPOR in non-hematopoiesis organ.
From our works we can conclude: 1. EPO is probably produced by the liver in CRF. 2. The number of EPOR was decreased and affinity ability improved of renal anemia. 3. The expression of EPO mRNA was increased liver in CRF. 4.Jak2-Stat5 pathway weakened in CRF. 5.The proliferation of erythrocyte decreased, especially in low level of EPO condition.6.The apoptosis of erythrocyte series of bone marrow increased in CRF condition.
We first explored the EPOR and Jak2-Stat5 pathway in renal anemia, and observed the site of EPO synthesis ,the affinity of EPO and EPOR, the pathway involved in the process by molecular technichs. The research made a foundation for the renal anemia treatment. There is no similar report now.
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