本文選題：內皮祖細胞 + 臍帶組織�。� 參考：《第二軍醫(yī)大學》2012年碩士論文

【摘要】：內皮祖細胞(endothelial progenitor cells, EPCs)是指出生后機體中存在能特異性歸巢至血管新生組織并分化成內皮細胞的一群干細胞，包括從成血一血管干細胞到完全分化的內皮細胞之間一定階段的細胞群體，它不僅參與胚胎時期血管生成，還與損傷的血管內皮的愈合和血管新生密切相關。內皮祖細胞是血管內皮細胞的前體細胞，在骨髓中與周圍基質細胞緊密接觸，在生理或病理因素刺激下，可從骨髓中動員而出，此后循環(huán)內皮祖細胞經(jīng)過進一步的增殖和分化歸巢至外周組織進行損傷的修復。近來的多項研究顯示，EPC是參與出生后生理性及病理性血管形成最主要的細胞。因此，內皮祖細胞在心腦血管疾病、腫瘤血管形成及創(chuàng)傷愈合等方面均發(fā)揮重要作用，具有廣闊的應用前景。 內皮祖細胞應用于臨床治療主要包括三個領域，包括損傷血管壁的修復、缺血組織新血管形成與再生以及人造血管的包被。限制其臨床應用的一個最主要的原因是從骨髓和外周血中分離培養(yǎng)出EPC數(shù)量都非常有限，不能滿足實驗研究及臨床應用的需要。解決這個問題的途徑包括應用臍帶血或臍帶組織培養(yǎng)人內皮祖細胞，或者用細胞因子、生長因子、藥物來促進人內皮祖細胞動員。 因此，對人內皮祖細胞培養(yǎng)方法的研究仍然很有必要。為了培養(yǎng)擴增出大量穩(wěn)定的內皮祖細胞，本研究參照國內外多家實驗室的培養(yǎng)及鑒定方案，在實驗中反復摸索，對影響EPC培養(yǎng)擴增的部分重要因素進行比較，通過對人骨髓、臍帶血、臍帶組織培養(yǎng)內皮祖細胞的比較，以及分別用不同濃度VEGF與EPC共培養(yǎng)研究VEGF對人內皮祖細胞動員的促進作用，進而選擇培養(yǎng)獲得率高、祖細胞特征穩(wěn)定以及增殖能力、成血管能力都較強的人內皮祖細胞培養(yǎng)方案，為下一步移植內皮祖細胞用于治療心血管疾病及多器官功能障礙綜合癥奠定良好的基礎。 本實驗研究共分三部分： 第一部分：不同組織來源的人內皮祖細胞的分離、培養(yǎng) 目的：通過對人骨髓、臍帶血、臍帶組織培養(yǎng)內皮祖細胞的比較，建立起人EPC的標準化分離、培養(yǎng)和擴增的方法，為內皮祖細胞的移植奠定基礎。 方法：采用密度梯度差速貼壁培養(yǎng)法及組織塊植塊法，分別從骨髓、臍帶血及臍帶組織中分離培養(yǎng)內皮祖細胞，按照不同接種密度、換液時間、基礎培養(yǎng)液、血清濃度等進行分組，比較原代EPC細胞集落個數(shù)及EPC獲得率，成熟后EPC通過比較細胞的生長曲線及增殖倍數(shù)，利用SPSS統(tǒng)計軟件，對相關數(shù)據(jù)進行總結、分析、比較。 結果：通過各種不同培養(yǎng)條件及方法得到的EPC細胞形態(tài)基本相似。骨髓來源的人內皮祖細胞在連續(xù)傳代3代以后，細胞形態(tài)即發(fā)生明顯改變，而臍帶血及臍帶組織來源的人內皮祖細胞可穩(wěn)定傳代10代以上，細胞形態(tài)不發(fā)生明顯改變。臍帶組織及臍帶血來源的人內皮祖細胞較骨髓來源的人內皮祖細胞祖細胞形態(tài)特征穩(wěn)定且增殖能力較強。 結論：從骨髓、臍帶血及臍帶組織中能成功分離培養(yǎng)出內皮祖細胞，并能傳代擴增。臍帶組織及臍帶血來源的人內皮祖細胞較骨髓來源的人內皮祖細胞細胞形態(tài)特征穩(wěn)定且增殖能力較強。臍帶組織來源的人內皮祖細胞獲得率較高且培養(yǎng)方法簡便、可靠，可為最終臨床應用于防治多器官功能障礙做準備。 第二部分內皮祖細胞鑒定與功能檢測 目的：鑒定培養(yǎng)擴增出的人內皮祖細胞并測定其功能，保證所分離及培養(yǎng)擴增的細胞的性質及純度。 方法：通過采用細胞形態(tài)和細胞超微結構觀察，用攝取Dil標記的人乙酰化低密度脂蛋白（Dil-ac-LDL）與FITC標記的特異性荊豆凝集素（FITC-UEA-1）的雙色熒光染色法和用CD133、CD34、CD31、KDR等表型表達行免疫組化鑒定EPC及流式細胞儀(FACS)檢測EPC純度以及體外血管生成功能試驗進行鑒定，對人骨髓、臍帶血、臍帶組織培養(yǎng)的人內皮祖細胞進行比較。 結果：細胞形態(tài)及細胞超微結構符合內皮祖細胞特征，Dil-ac-LDL、FITC-UEA-1雙染超過80%，免疫組化、流式細胞儀檢測CD133、CD34、CD31、KDR等的陽性率均符合EPC表型特征；體外具有成血管能力。 結論：本研究采用綜合的鑒定體系（形態(tài)特征、流式細胞儀、雙色熒光、體外血管生成功能試驗）所得結果完全可證明我們培養(yǎng)擴增的細胞為人內皮祖細胞。臍帶組織來源的人內皮祖細胞及臍帶血來源的人內皮祖細胞與骨髓來源的人內皮祖細胞相比祖細胞特征穩(wěn)定且成血管能力較強。 第三部分VEGF促進臍帶組織來源的人內皮祖細胞增殖作用機制的研究 目的：研究VEGF促進人臍帶組織來源的內皮祖細胞增殖作用機制。 方法：比較不同濃度VEGF與人臍帶組織來源的內皮祖細胞共培養(yǎng)后內皮祖細胞數(shù)量變化及細胞培養(yǎng)液一氧化氮(NO)含量。用PI3K的特異性抑制劑LY294002處理臍帶EPCs，同時設空白組和溶劑（DMSO）對照組和10ng/mlVEGF+抑制劑LY294002組，用western blot檢測磷酸化Akt蛋白的表達，硝酸還原酶法測定細胞培養(yǎng)液一氧化氮(NO)含量，，檢測加入抑制劑后細胞增殖能力及成血管能力。 結果：10ng/ml濃度的VEGF與人臍帶組織來源的內皮祖細胞共培養(yǎng)后內皮祖細胞數(shù)量明顯增加，細胞培養(yǎng)液一氧化氮(NO)含量明顯提高，而20ng/ml濃度的VEGF與人臍帶組織來源的內皮祖細胞共培養(yǎng)后內皮祖細胞數(shù)量及細胞培養(yǎng)液一氧化氮(NO)含量與10ng/ml濃度相比變化不明顯。加入PI3K的特異性抑制劑LY294002抑制PI3K后，磷酸化Akt蛋白表達也明顯減少；細胞培養(yǎng)液NO含量明顯減少；細胞增殖能力及成血管能力明顯下降。 結論：10ng/ml的VEGF濃度對人內皮祖細胞的增殖具有明顯促進作用，同時培養(yǎng)液中NO濃度明顯增高。而20ng/ml濃度的VEGF與10ng/ml濃度的VEGF相比對人內皮祖細胞增殖的促進作用不明顯，可能與受體飽和有關。加入PI3K的特異性抑制劑LY294002抑制PI3K后，磷酸化Akt蛋白表達也明顯減少；細胞培養(yǎng)液NO含量明顯減少；細胞增殖能力及成血管能力明顯下降，提示VEGF通過PI3K/Akt/eNOS通路促進人內皮祖細胞的增殖。 總之，通過對人骨髓、臍帶血、臍帶組織培養(yǎng)人內皮祖細胞的比較發(fā)現(xiàn)臍帶組織及臍帶血來源的人內皮祖細胞較骨髓來源的人內皮祖細胞細胞形態(tài)及祖細胞特征穩(wěn)定且臍帶組織及臍帶血來源的人內皮祖細胞增殖能力及成血管能力均強于骨髓來源的人內皮祖細胞，且臍帶組織來源的人內皮祖細胞獲得率較高且培養(yǎng)方法簡便、可靠。通過分別用不同濃度VEGF與人臍帶組織來源的EPC共培養(yǎng)研究VEGF對人內皮祖細胞增殖的促進作用發(fā)現(xiàn)10ng/ml的VEGF濃度對人內皮祖細胞的增殖具有明顯促進作用，同時培養(yǎng)液中NO濃度明顯增高，加入PI3K的特異性抑制劑LY294002抑制PI3K后，磷酸化Akt蛋白表達也明顯減少；細胞培養(yǎng)液NO含量明顯減少；細胞增殖能力及成血管能力明顯下降，提示VEGF通過PI3K/Akt/eNOS通路促進人內皮祖細胞的增殖。這一結論為為下一步研究臨床應用移植人內皮祖細胞防治MODS提供確實的細胞學基礎并且為下一步研究VEGF促進臍帶組織來源的人內皮祖細胞增殖的作用機制打下良好基礎。
[Abstract]:Endothelial progenitor cells (EPCs) is a group of stem cells in the postnatal body that can be specifically homed to neovascularization and differentiate into endothelial cells, including a group of cells from a blood - derived stem cell to a fully differentiated endothelial cell. It is not only involved in embryonic angiogenesis. It is also closely related to the healing of vascular endothelial cells and angiogenesis. Endothelial progenitor cells are the precursor cells of vascular endothelial cells, which are closely contacted with the surrounding matrix cells in the bone marrow, and can be mobilized from the bone marrow under the stimulation of physiological or pathological factors. After that, the circulating endothelial progenitor cells are returned to the outside by further proliferation and differentiation. A number of recent studies have shown that EPC is the most important cell to participate in the physiological and pathological angiogenesis after birth. Therefore, endothelial progenitor cells play an important role in cardiovascular and cerebrovascular diseases, tumor angiogenesis and wound healing, which have broad prospects for application.
The application of endothelial progenitor cells to clinical treatment mainly consists of three fields, including repair of damaged vascular walls, new vascular formation and regeneration of ischemic tissue, and inclusion of artificial blood vessels. One of the main reasons for limiting its clinical application is that the number of EPC isolated from bone marrow and peripheral blood is very limited and cannot satisfy experimental research and The needs of clinical applications include the use of umbilical cord blood or umbilical cord tissue to cultivate human endothelial progenitor cells, or the use of cytokines, growth factors, and drugs to promote the mobilization of human endothelial progenitor cells.
Therefore, it is still necessary to study the culture method of human endothelial progenitor cells. In order to develop a large number of stable endothelial progenitor cells, this study, referring to the training and identification schemes of many laboratories at home and abroad, has been repeatedly explored in the experiment, and compared some important factors affecting the amplification of EPC culture, through the human bone marrow, umbilical cord blood, and umbilical cord. The comparison of tissue culture endothelial progenitor cells and the effects of VEGF on the mobilization of human endothelial progenitor cells were studied with different concentrations of VEGF and EPC, and the culture of human endothelial progenitor cells with high acquisition rate, stable progenitor cell characteristics and proliferation ability and strong vascular ability were selected for the next transplantation of endothelial progenitor cells. The cells laid a good foundation for the treatment of cardiovascular diseases and multiple organ dysfunction syndrome.
The study is divided into three parts:
Part one: isolation and culture of human endothelial progenitor cells from different tissue sources
Objective: to establish a standardized method of isolation, culture and amplification of human EPC by comparison of human bone marrow, umbilical cord blood and umbilical cord tissue culture endothelial progenitor cells, and lay a foundation for the transplantation of endothelial progenitor cells.
Methods: the endothelial progenitor cells were isolated and cultured from bone marrow, umbilical cord blood and umbilical cord tissue by density gradient differential adhesion culture method and tissue block method. According to different inoculation density, change time, basic culture fluid, serum concentration and so on, the number of primary EPC cell colonies and the EPC acquisition rate were compared. After the maturation, the EPC was comparatively fine. Cell growth curve and proliferation multiplier, using SPSS statistical software, the relevant data were summarized, analyzed and compared.
Results: the morphology of EPC cells obtained by various culture conditions and methods was basically similar. The cell morphology of human endothelial progenitor cells derived from bone marrow changed obviously after 3 generations of continuous passage, while human endothelial progenitor cells from umbilical cord blood and umbilical cord tissue were stable over 10 generations, and the cell morphology did not change obviously. Human endothelial progenitor cells derived from tissue and umbilical cord blood are more stable and proliferative than human bone marrow derived progenitor progenitor cells.
Conclusion: endothelial progenitor cells can be successfully isolated and cultured from bone marrow, umbilical cord blood and umbilical cord tissue. Human endothelial progenitor cells from umbilical cord tissue and umbilical cord blood are more stable and more proliferating than human endothelial progenitor cells derived from bone marrow. Human endothelial progenitor cells from umbilical cord tissue derived from human umbilical cord tissue have higher rate and culture. The method is simple, reliable, and can be used for the final clinical application in the prevention and treatment of multiple organ dysfunction syndrome.
Identification and functional detection of endothelial progenitor cells in second parts
Objective: to identify cultured and expanded human endothelial progenitor cells and to determine their functions, and to ensure the properties and purity of the cells isolated and cultured.
Methods: through the observation of cell morphology and cell ultrastructure, Dil labeled human acetylated LDL (Dil-ac-LDL) and FITC labeled specific bingbean agglutinin (FITC-UEA-1) were used as a double color fluorescence staining method and CD133, CD34, CD31, KDR and other phenotypic tables with CD133, CD34, CD31 and KDR to identify EPC and flow cytometry (FACS). Purity and in vitro vascular function tests were carried out to compare human bone marrow, umbilical cord blood and umbilical cord tissue cultured human endothelial progenitor cells.
Results: the cell morphology and cell ultrastructure conformed to the characteristics of endothelial progenitor cells. Dil-ac-LDL, FITC-UEA-1 double staining was more than 80%. The positive rates of CD133, CD34, CD31 and KDR were all conformed to the EPC phenotypic characteristics, and the ability of angiogenesis in vitro.
Conclusion: the results of the comprehensive identification system (morphological characteristics, flow cytometry, double color fluorescence, and in vitro angiogenesis test) can fully demonstrate that the cultured cells are human endothelial progenitor cells, human endothelial progenitor cells from umbilical cord tissue and human umbilical cord blood derived human endothelial progenitor cells and human bone marrow sources Compared with progenitor cells, the progenitor cells are stable and have strong blood vessel capability.
The third part is about the mechanism of VEGF promoting the proliferation of human umbilical cord tissue derived endothelial progenitor cells.
Objective: To study the mechanism of VEGF promoting proliferation of human umbilical cord derived endothelial progenitor cells.
Methods: To compare the number of endothelial progenitor cells and the content of nitric oxide (NO) of endothelial progenitor cells after co culture of endothelial progenitor cells from different concentrations of VEGF and human umbilical cord tissue. The umbilical cord EPCs was treated with the specific inhibitor LY294002 of PI3K, and the blank group and solvent (DMSO) control group and the 10ng/mlVEGF+ inhibitor LY294002 group were set up, and Western blot was used. The expression of phosphorylated Akt protein was detected, the content of nitric oxide (NO) in cell culture liquid was measured by nitrate reductase method, and the cell proliferation ability and vascular ability after the addition of the inhibitor were detected.
Results: the number of VEGF in 10ng/ml concentration and the endothelial progenitor cells of human umbilical cord tissue derived from endothelial progenitor cells increased significantly, and the content of nitric oxide (NO) in cell culture fluid increased significantly. The number of VEGF in 20ng/ml concentration and the number of inner progenitor cells of endothelial progenitor cells from human umbilical cord tissue derived endothelial progenitor cells and the nitric oxide (NO) in cell culture fluid. There was no obvious change in content compared with 10ng/ml concentration. After the addition of PI3K specific inhibitor LY294002 to PI3K, the expression of phosphorylated Akt protein decreased obviously, the content of NO in cell culture fluid decreased obviously, and the proliferation ability and vascular ability of cells decreased significantly.
Conclusion: the concentration of VEGF in 10ng/ml has a significant effect on the proliferation of human endothelial progenitor cells, and the concentration of NO in the culture medium is significantly higher, while the VEGF of the 20ng/ml concentration is not obvious to the 10ng/ml concentration of VEGF and is not obvious to the proliferation of human endothelial progenitor cells, and may be related to the receptor saturation. After PI3K, the expression of phosphorylated Akt protein decreased obviously, the content of NO in cell culture fluid decreased obviously, and the proliferation ability and vascular ability of cell decreased obviously, suggesting that VEGF promoted the proliferation of human endothelial progenitor cells through PI3K/Akt/eNOS pathway.
In summary, a comparison of human endothelial progenitor cells from human umbilical cord tissue and umbilical cord blood from human bone marrow, umbilical cord blood and umbilical cord tissue shows that human endothelial progenitor cells from umbilical cord and umbilical cord blood are more stable than those of bone marrow derived human endothelial progenitor cells and the proliferation and vascular ability of human endothelial progenitor cells from umbilical cord tissue and umbilical cord blood from human umbilical cord tissue and umbilical cord blood. Human endothelial progenitor cells, which are stronger than bone marrow, have high rate of acquisition of human endothelial progenitor cells from umbilical cord tissue, and the culture method is simple and reliable. EPC co culture of different concentrations of VEGF and human umbilical cord tissue was used to study the effect of VEGF on the proliferation of human endothelial progenitor cells and to present the VEGF concentration of 10ng/ml to human endothelial progenitor cells The proliferation has an obvious promoting effect, while the concentration of NO in the culture medium is obviously increased, and the expression of phosphorylated Akt protein is obviously reduced after the addition of PI3K specific inhibitor LY294002 to PI3K, the content of NO in the cell culture solution decreases obviously, and the cell proliferation ability and the capacity of blood vessel descend obviously, suggesting that VEGF promotes human within the PI3K/Akt/eNOS pathway. This conclusion provides a good basis for the next step in the study of the clinical application of transplanted human endothelial progenitor cells to the prevention and treatment of MODS and to further study the mechanism of VEGF to promote the proliferation of human endothelial progenitor cells from the umbilical cord tissue.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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