单核细胞来源的多潜能细胞向淋巴管内皮细胞转分化的可行性

发布时间：2018-05-13 04:29

本文选题：单核细胞 + 转分化　；参考：《山东大学》2010年硕士论文

【摘要】： 研究背景：迄今为止,淋巴水肿尚无理想的治疗措施。特别是近些年来乳腺癌发病率逐年上升,乳腺癌根治术引起的患侧上肢继发性淋巴水肿病例随之增多。该并发症一旦发生,常逐渐发展成整个上肢水肿甚至硬化。促进淋巴管新生无疑是治愈淋巴水肿的关键措施,其中,促进淋巴管内皮细胞增生是重要环节。研究发现,单核巨噬细胞在组织修复、免疫排斥反应、恶性肿瘤淋巴转移等许多病理生理过程中,通过分泌VEGF-C/D等细胞因子促进淋巴管增生,或直接整合到淋巴管壁上,参与淋巴管新生。另外,研究发现,单核细胞不仅是巨噬细胞的前体细胞,经体外诱导能分化成多潜能细胞(MOMCs)或内皮祖细胞(EPCs),后者在VEGF等作用下能分化为血管内皮样细胞。因此,近些年来,将EPCs用于血管再生、治疗缺血性疾病的研究一直是近些年来非常活跃的课题。然而,单核细胞来源的MOMCs或EPCs能否转分化为淋巴管内皮细胞,国内外均未见报道。本文从CD14+单核细胞获得MOMCs之后,进行诱导培养,通过RT-PCR和荧光免疫细胞化学技术,检测不同阶段细胞的淋巴管内皮特异性标志物LYVE-1、Podoplanin、Prox-1、VEGFR-3及内皮细胞标志物VEGFR-2、CD144、vWF、CD34的基因和蛋白表达,观察其向淋巴管内皮细胞分化的可能性。目的：本研究旨在探讨在体外单核细胞分化成淋巴管内皮细胞的可能性,为进一步研究淋巴管新生提供理论依据。方法：取健康成人新鲜外周血,提取单个核细胞：①未用FN铺板,用L-DMEM加10%FBS培养8-12h后,收集贴壁细胞,用流式细胞术和RT-PCR技术检测；②将单个核细胞接种到铺有FN的培养板,用L-DMEM加10%FBS培养7-10d后,获得MOMCs;另外一组细胞,在同时收细胞前24h,添加10ng/ml的肿瘤坏死因子α[(TNF-α)刺激；用荧光免疫细胞化学和RT-PCR技术检测细胞对淋巴管内皮特异性标志物LYVE-1、Podoplanin、Prox1、VEGFR-3以及内皮细胞标志物vWF、CD144、VEGFR-2、CD34的基因和蛋白表达；③将MOMCs换用EGM-2继续培养3-7d,用RT-PCR和荧光免疫细胞化学法,分别检测细胞对淋巴管内皮特异性标志物和内皮细胞标志物的基因和蛋白表达；对用TNF-a刺激24h的MOMCs,采用同样方法继续培养3d,用RT-PCR方法检测细胞对上述标志物的表达情况。结果：①未用FN铺板,8-12h短期培养的贴壁细胞约有96%为CD14+单核细胞；RT-PCR结果显示,细胞对于VEGFR-3表达呈弱阳性,LYVE-1、Podoplanin、Prox1、vWF、CD144、VEGFR-2、CD34的表达均呈阴性；②MOMCs:RT-PCR结果显示对于VEGFR-3、Podoplanin、Prox-1、CD34、vWF的表达均呈阳性,而LYVE-1、CD144、VEGFR-2表达呈阴性；荧光免疫细胞化学染色检测VEGFR-3、VEGFR-2的蛋白表达,结果均呈阳性；TNF-α刺激24h的MOMCs,其RT-PCR结果显示,VEGFR-3、Podoplanin、Prox-1、CD34、vWF的表达亦均呈阳性,而LYVE-1、CD144、VEGFR-2表达呈阴性；③EGM-2诱导MOMCs 3d后,RT-PCR结果显示,除了VEGFR-3表达呈阴性,LYVE-1、Podoplanin、Prox-1、CD34、CD144、VEGFR-2及vWF均呈阳性；采用荧光免疫细胞化学染色,结果显示对于VEGFR-3、LYVE-1、Podoplanin、VEGFR-2及vWF均不同程度的呈阳性；继续培养7d后,RT-PCR结果表明,细胞对于LYVE-1、Podoplanin、Prox-1、CD34、vWF的表达呈阳性,但与前者相比,相应标志物的表达强度均减弱,而VEGFR-3、CD144、VEGFR-2呈阴性；对用TNF-a刺激24h的MOMCs,继续培养3d后,RT-PCR结果显示,Podoplanin、Prox-1、CD34、vWF呈阳性,但与由MOMCs诱导3d的细胞相比,相应标志物的表达强度均明显减弱,而对VEGFR-3、LYVE-1、CD144、VEGFR-2表达均呈阴性。结论：①实验所用的贴壁细胞绝大部分是CD14+单核细胞；②单核细胞来源的MOMCs经诱导后,不仅表达血管内皮标志物,同时也表达淋巴管内皮特异性标志物,而且对后者的表达较稳定,认为能分化为淋巴管内皮样细胞；③单核细胞来源的MOMCs及其诱导而来的内皮样细胞对VEGFR-2和VEGFR-3的表达短暂,细胞不能增殖传代。
[Abstract]:Background: so far, there is no ideal treatment for lymphedema. Especially in recent years, the incidence of breast cancer has increased year by year. The incidence of secondary lymphedema in the lateral upper limbs caused by radical mastectomy is increasing. Once the complication occurs, it is often developed into the whole upper limb edema and even sclerosis. It is an important step to cure lymphedema. Among them, it is an important link to promote lymphatic endothelial cell proliferation. It is found that mononuclear macrophages can promote lymphatic hyperplasia by secreting VEGF-C/D and other fine cell factors during tissue repair, immune rejection, and lymphatic metastasis of malignant tumor. In addition, it has been found that monocytes are not only the precursor cells of macrophages, but are induced in vitro to differentiate into multipotential cells (MOMCs) or endothelial progenitor cells (EPCs), and the latter can be differentiated into vascular endothelial cells under the action of VEGF. In recent years, EPCs has been used for vascular regeneration to treat ischemic disease. The study of disease has been a very active topic in recent years. However, there are no reports on whether monocyte derived MOMCs or EPCs can turn into lymphatic endothelial cells at home and abroad. After obtaining MOMCs from CD14+ mononuclear cells, it was induced and cultured, and the cells of different stages were detected by RT-PCR and immunofluorescence cytochemical technology. The gene and protein expression of LYVE-1, Podoplanin, Prox-1, VEGFR-3 and endothelial cell markers VEGFR-2, CD144, vWF, CD34, and the possibility of differentiation into lymphatic endothelial cells.
Objective: To explore the possibility of differentiation of monocytes into lymphatic endothelial cells in vitro, and to provide theoretical evidence for further study of lymphangiogenesis.
Methods: to extract fresh peripheral blood from healthy adults and extract mononuclear cells: (1) unused FN paving, L-DMEM and 10%FBS for 8-12h, collecting adherent cells, using flow cytometry and RT-PCR technology; (2) inoculating mononuclear cells to FN culture plate and MOMCs with L-DMEM plus 10%FBS to cultivate 7-10d, and another group of cells, in the same group. 24h, 10ng/ml tumor necrosis factor alpha (TNF- alpha) was added to the cell, and the gene and protein expression of LYVE-1, Podoplanin, Prox1, VEGFR-3, and endothelial cells were detected by fluorescent immunocytochemistry and RT-PCR technique. 3-7d, RT-PCR and fluorescent immunocytochemical method were used to detect the gene and protein expression of the cell specific markers and endothelial markers in the lymphatic vessels, and the 3D was continued by the same method with the MOMCs of 24h stimulated by TNF-a, and the expression of the cells to the above markers was detected by RT-PCR method.
Results: (1) there were about 96% CD14+ mononuclear cells in 8-12h short term cultured adherent cells without FN pad, and RT-PCR results showed that the expression of VEGFR-3 was weak positive, LYVE-1, Podoplanin, Prox1, vWF, CD144, VEGFR-2, and CD34. Positive, but LYVE-1, CD144, VEGFR-2 expression was negative; fluorescent immunocytochemical staining detected VEGFR-3, the protein expression of VEGFR-2 was positive; TNF- alpha stimulated 24h MOMCs, and its RT-PCR results showed that VEGFR-3, Podoplanin, Prox-1, and negative expression were also negative; thirdly, induction induction After MOMCs 3D, RT-PCR results showed that the expression of LYVE-1, Podoplanin, Prox-1, CD34, CD144, VEGFR-2 and vWF were positive except for the negative VEGFR-3 expression. The expression of VE-1, Podoplanin, Prox-1, CD34, vWF was positive, but compared with the former, the expression intensity of the corresponding markers decreased while VEGFR-3, CD144 and VEGFR-2 were negative. The expression intensity of the substance decreased significantly, but the expression of VEGFR-3, LYVE-1, CD144 and VEGFR-2 was negative.
Conclusion: (1) most of the adherent cells used in the experiment are CD14+ mononuclear cells. (2) the MOMCs of mononuclear cells is induced not only to express vascular endothelial markers but also to express the specific markers in the lymphatic vessels, and the expression of the latter is more stable. It is considered that it can be divided into lymphatic endothelium like cells; and the source of monocyte is monocyte. MOMCs and its induced endothelium like cells express VEGFR-2 and VEGFR-3 transiently, and cells can not proliferate.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

【参考文献】