本文選題：單核細胞 + 轉(zhuǎn)分化�。� 參考：《山東大學(xué)》2010年碩士論文

【摘要】： 研究背景：迄今為止,淋巴水腫尚無理想的治療措施。特別是近些年來乳腺癌發(fā)病率逐年上升,乳腺癌根治術(shù)引起的患側(cè)上肢繼發(fā)性淋巴水腫病例隨之增多。該并發(fā)癥一旦發(fā)生,常逐漸發(fā)展成整個上肢水腫甚至硬化。促進淋巴管新生無疑是治愈淋巴水腫的關(guān)鍵措施,其中,促進淋巴管內(nèi)皮細胞增生是重要環(huán)節(jié)。研究發(fā)現(xiàn),單核巨噬細胞在組織修復(fù)、免疫排斥反應(yīng)、惡性腫瘤淋巴轉(zhuǎn)移等許多病理生理過程中,通過分泌VEGF-C/D等細胞因子促進淋巴管增生,或直接整合到淋巴管壁上,參與淋巴管新生。另外,研究發(fā)現(xiàn),單核細胞不僅是巨噬細胞的前體細胞,經(jīng)體外誘導(dǎo)能分化成多潛能細胞(MOMCs)或內(nèi)皮祖細胞(EPCs),后者在VEGF等作用下能分化為血管內(nèi)皮樣細胞。因此,近些年來,將EPCs用于血管再生、治療缺血性疾病的研究一直是近些年來非�；钴S的課題。然而,單核細胞來源的MOMCs或EPCs能否轉(zhuǎn)分化為淋巴管內(nèi)皮細胞,國內(nèi)外均未見報道。本文從CD14+單核細胞獲得MOMCs之后,進行誘導(dǎo)培養(yǎng),通過RT-PCR和熒光免疫細胞化學(xué)技術(shù),檢測不同階段細胞的淋巴管內(nèi)皮特異性標志物L(fēng)YVE-1、Podoplanin、Prox-1、VEGFR-3及內(nèi)皮細胞標志物VEGFR-2、CD144、vWF、CD34的基因和蛋白表達,觀察其向淋巴管內(nèi)皮細胞分化的可能性。 目的：本研究旨在探討在體外單核細胞分化成淋巴管內(nèi)皮細胞的可能性,為進一步研究淋巴管新生提供理論依據(jù)。 方法：取健康成人新鮮外周血,提取單個核細胞：①未用FN鋪板,用L-DMEM加10%FBS培養(yǎng)8-12h后,收集貼壁細胞,用流式細胞術(shù)和RT-PCR技術(shù)檢測；②將單個核細胞接種到鋪有FN的培養(yǎng)板,用L-DMEM加10%FBS培養(yǎng)7-10d后,獲得MOMCs;另外一組細胞,在同時收細胞前24h,添加10ng/ml的腫瘤壞死因子α[(TNF-α)刺激；用熒光免疫細胞化學(xué)和RT-PCR技術(shù)檢測細胞對淋巴管內(nèi)皮特異性標志物L(fēng)YVE-1、Podoplanin、Prox1、VEGFR-3以及內(nèi)皮細胞標志物vWF、CD144、VEGFR-2、CD34的基因和蛋白表達；③將MOMCs換用EGM-2繼續(xù)培養(yǎng)3-7d,用RT-PCR和熒光免疫細胞化學(xué)法,分別檢測細胞對淋巴管內(nèi)皮特異性標志物和內(nèi)皮細胞標志物的基因和蛋白表達；對用TNF-a刺激24h的MOMCs,采用同樣方法繼續(xù)培養(yǎng)3d,用RT-PCR方法檢測細胞對上述標志物的表達情況。 結(jié)果：①未用FN鋪板,8-12h短期培養(yǎng)的貼壁細胞約有96%為CD14+單核細胞；RT-PCR結(jié)果顯示,細胞對于VEGFR-3表達呈弱陽性,LYVE-1、Podoplanin、Prox1、vWF、CD144、VEGFR-2、CD34的表達均呈陰性；②MOMCs:RT-PCR結(jié)果顯示對于VEGFR-3、Podoplanin、Prox-1、CD34、vWF的表達均呈陽性,而LYVE-1、CD144、VEGFR-2表達呈陰性；熒光免疫細胞化學(xué)染色檢測VEGFR-3、VEGFR-2的蛋白表達,結(jié)果均呈陽性；TNF-α刺激24h的MOMCs,其RT-PCR結(jié)果顯示,VEGFR-3、Podoplanin、Prox-1、CD34、vWF的表達亦均呈陽性,而LYVE-1、CD144、VEGFR-2表達呈陰性；③EGM-2誘導(dǎo)MOMCs 3d后,RT-PCR結(jié)果顯示,除了VEGFR-3表達呈陰性,LYVE-1、Podoplanin、Prox-1、CD34、CD144、VEGFR-2及vWF均呈陽性；采用熒光免疫細胞化學(xué)染色,結(jié)果顯示對于VEGFR-3、LYVE-1、Podoplanin、VEGFR-2及vWF均不同程度的呈陽性；繼續(xù)培養(yǎng)7d后,RT-PCR結(jié)果表明,細胞對于LYVE-1、Podoplanin、Prox-1、CD34、vWF的表達呈陽性,但與前者相比,相應(yīng)標志物的表達強度均減弱,而VEGFR-3、CD144、VEGFR-2呈陰性；對用TNF-a刺激24h的MOMCs,繼續(xù)培養(yǎng)3d后,RT-PCR結(jié)果顯示,Podoplanin、Prox-1、CD34、vWF呈陽性,但與由MOMCs誘導(dǎo)3d的細胞相比,相應(yīng)標志物的表達強度均明顯減弱,而對VEGFR-3、LYVE-1、CD144、VEGFR-2表達均呈陰性。 結(jié)論：①實驗所用的貼壁細胞絕大部分是CD14+單核細胞；②單核細胞來源的MOMCs經(jīng)誘導(dǎo)后,不僅表達血管內(nèi)皮標志物,同時也表達淋巴管內(nèi)皮特異性標志物,而且對后者的表達較穩(wěn)定,認為能分化為淋巴管內(nèi)皮樣細胞；③單核細胞來源的MOMCs及其誘導(dǎo)而來的內(nèi)皮樣細胞對VEGFR-2和VEGFR-3的表達短暫,細胞不能增殖傳代。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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相關(guān)期刊論文 前2條

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本文編號：1881682