巨噬细胞—多囊肾囊肿衬里上皮细胞相互作用及其机制研究

发布时间：2018-04-13 11:41

本文选题：ADPKD + 乳酸　；参考：《第二军医大学》2017年硕士论文

【摘要】：研究目的:本课题研究在常染色体显性多囊肾病(Autosomal Dominant Polycystic Kidney Disease,ADPKD)的微环境中,囊肿衬里上皮细胞趋化巨噬细胞募集至囊肿周围并使其发生表型转化的机制,并进一步探讨巨噬细胞刺激囊肿衬里上皮细胞增殖的机制,两者相互影响,进而促进多囊肾病的疾病进展。实验方法:1.在Pkd1诱导敲除小鼠肾脏组织中,观察病程的进展与肾组织中乳酸浓度的关系。2.在Pkd1诱导敲除小鼠肾脏组织中,观察肾组织中乳酸水平和囊肿指数的相关性。3.收集OX161和UCL93细胞的条件培养液,检测条件培养液中乳酸含量,酶联免疫吸附法检测两种细胞的条件培养液中MCP-1的浓度变化。4.予以乳酸刺激RAW264.7细胞,用RT-PCR法、蛋白免疫印迹法和细胞免疫荧光法分别从基因、蛋白等水平检测RAW264.7细胞中ARG1的表达量。5.予以乳酸刺激RAW264.7细胞,收集RAW264.7条件培养液,用酶联免疫吸附法检测条件培养液中ARG1的浓度变化。6.予以乳酸刺激RAW264.7细胞,收集RAW264.7细胞,用ARG1活性检测法检测RAW264.7细胞内ARG1的活性变化。7.建立稳定的二维共培养体系:即OX161/UCL93+RAW264.7细胞Transwell共培养,并在该体系中加入ARG1竞争性抑制剂—Nor-NOHA,用Ed U法检测增殖期的阳性细胞核数目,计算处于增殖期细胞的百分比;流式细胞术检测细胞周期所占百分比。8.在OX161细胞培养液中加入重组的人ARG1细胞因子和ARG1竞争性抑制剂—Nor-NOHA,用Ed U法检测增殖期的阳性细胞核数目,计算处于增殖期细胞的百分比;流式细胞术法检测细胞周期所占百分比,用蛋白免疫印迹法检测细胞增值通路蛋白的表达变化。结果:1.Pkd1诱导敲除小鼠,随多囊肾疾病进展,肾脏囊肿指数增大,肾组织中乳酸浓度增高。2.囊肿衬里上皮细胞分泌趋化因子MCP-1,募集巨噬细胞至囊肿周围;同时产生乳酸,诱导巨噬细胞转化为M2表型,表达并分泌有活性的ARG1。3.ARG1能够刺激囊肿衬里上皮细胞增殖,进而促进囊肿的增大和多囊肾病疾病进展。结论:多囊肾病疾病进程中,囊肿衬里上皮细胞分泌趋化因子MCP-1募集巨噬细胞分布在囊肿周围。随着囊肿进行性增大,肾组织缺氧加重,乳酸浓度增加,诱导巨噬细胞向M2型分化,表达ARG1;ARG1不仅作为M2型巨噬细胞的表型标记物和精氨酸代谢途径的关键酶,也可作为可溶性因子被分泌到周围环境中,促进囊肿衬里上皮细胞增殖。巨噬细胞和囊肿衬里上皮细胞相互作用,形成恶性循环链,导致疾病进展迅速。因此,抑制巨噬细胞表达ARG1或直接抑制ARG1的促增殖作用可能成为新的ADPKD疾病治疗靶点。
[Abstract]:Objective: to study the mechanism of chemotactic macrophages in the microenvironment of autosomal Dominant Polycystic Kidney ADPKD, which induces the phenotypic transformation of macrophages around the cysts in autosomal dominant polycystic kidney disease (autosomal Dominant Polycystic Kidney DiseaseADPKD).Experimental method: 1.In the kidney tissue of Pkd1 induced knockout mice, the relationship between the progression of disease course and the concentration of lactic acid in renal tissue was observed.The correlation between lactate level and cyst index was observed in Pkd1 induced knockout mice.The content of lactic acid in conditioned medium and the concentration of MCP-1 in conditioned medium of OX161 and UCL93 were detected by enzyme-linked immunosorbent assay (Elisa).RAW264.7 cells were stimulated with lactic acid. The expression of ARG1 in RAW264.7 cells was detected by RT-PCR, Western blot and Cellular immunofluorescence, respectively.RAW264.7 cells were stimulated with lactic acid and RAW264.7 conditioned medium was collected. The concentration of ARG1 in conditioned medium was detected by enzyme-linked immunosorbent assay (Elisa).RAW264.7 cells were stimulated with lactic acid and RAW264.7 cells were collected. The changes of ARG1 activity in RAW264.7 cells were detected by ARG1 assay.A stable two-dimensional co-culture system was established: Transwell co-culture of OX161/UCL93 RAW264.7 cells was carried out, and ARG1 competitive inhibitor -Nor-NOHA was added to the system. The number of positive nuclei in proliferative phase was detected by Ed U method, and the percentage of cells in proliferative phase was calculated.Percentage of cell cycle detected by flow cytometry.Recombinant human ARG1 cytokines and ARG1 competitive inhibitor -Nor-NOHA were added to OX161 cell culture medium. The number of positive nuclei in proliferative phase was detected by Ed U method, and the percentage of cells in proliferative phase was calculated.The percentage of cell cycle was detected by flow cytometry and the expression of cell proliferation pathway protein was detected by Western blot.Results 1. Pkd1 induced knockout mice. With the progression of polycystic kidney disease, the renal cyst index increased and the concentration of lactic acid in renal tissue increased.Cyst-lining epithelial cells secrete chemokine MCP-1, recruit macrophages around the cyst, produce lactic acid, induce macrophages to transform into M2 phenotype, express and secrete active ARG1.3.ARG1 can stimulate the proliferation of cyst-lining epithelial cells.In turn, it promotes the enlargement of cyst and the progression of polycystic kidney disease.Conclusion: in the process of polycystic kidney disease, the epithelial cells secreting chemokine MCP-1 recruitment macrophages are distributed around the cyst.With the progressive enlargement of cysts, anoxia in renal tissues and the increase of lactate concentration, macrophages were induced to differentiate into M2 type, and ARG1- ARG1 was not only the key enzyme of phenotypic markers and arginine metabolism pathway of M2 macrophages.It can also be secreted into the surrounding environment as a soluble factor to promote the proliferation of cyst lining epithelial cells.Macrophages interact with epithelial cells lining cysts, forming a vicious circle that leads to rapid disease progression.Therefore, inhibiting the expression of ARG1 in macrophages or directly inhibiting the proliferation of ARG1 may become a new target for the treatment of ADPKD disease.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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