人淋巴管的体外构建研究

发布时间：2018-05-08 18:09

本文选题：骨髓间质干细胞 + VEGF-C156s　；参考：《山东大学》2007年博士论文

【摘要】： 背景国际淋巴学会前主席JR. Casley Smith教授曾指出：“Treatment of lymphedema is a notorious difficulty”，淋巴水肿的治疗的却是一个世界性的难题，至今尚未找到良好的治疗方法，。因此，研究探讨淋巴水肿lymphoedema的治疗，促进淋巴水肿组织内的淋巴管内皮细胞增生和淋巴管形成lymphngiogenesis，改善淋巴引流途径，从根本上治疗淋巴水肿，已成为摆在淋巴学工作者面前的极其艰巨而迫切的任务，具有重大的学术理论价值和和临床意义。另一方面，肿瘤tumor的淋巴转移是造成肿瘤病人死亡的主要原因，，因此，研究抑制肿瘤的转移途径对控制肿瘤扩散也有十分重要的临床意义。基于这两方面的目的，我们试图利用干细胞stem cells首先在体外构建人淋巴管，进而在此基础上开展淋巴水肿的治疗研究和阻断肿瘤转移途径的研究。 1981年Evans等首次成功地分离培养出小鼠胚胎干细胞embryonic stem cell；1998年，Thomson等首次培养出人的胚胎干细胞；从而为器官、组织移植的长远目标打下良好基础。继而成体干细胞可以分化成多种干细胞，特别是骨髓内含有多能干细胞，又称为骨髓间质干细胞(Mesenchymal Stem Cell)。这种干细胞有许多优点：易于从自身取材，无免疫排斥反应之忧，因而也就更易为病人所接受；成体干细胞，涉及较少伦理问题；体外易于扩增、易分离、以及体外操作简便。因此，在组织器官缺损性疾病，组织器官退行性疾病，遗传缺陷性疾病等多方面有重要的应用前景。目前的研究表明VEGF-C具有多种生物学特性和功能，可广泛作用于血管、淋巴管内皮细胞。VEGF-C156s是一种点突变型VEGF-C，其同源二聚体只能激活VEGFR-3，不能与VEGFR-2结合，因此，选用VEGF-C156s对骨髓间质干细胞进行诱导分化成淋巴管样内皮是最佳的方案，然而必须探讨、确定诱导分化的最佳VEGF-C156s浓度和时间，这也必定涉及到大量的试验。目的 1．分离、培养出具有一定的纯度的骨髓间质干细胞，并且用流式细胞仪flow cytometry检测纯化后细胞的表面抗体surface-antibody进行鉴定appraisement，为后续试验做好细胞储备。 2．用VEGF-C156s对骨髓间质干细胞进行诱导分化，确定诱导分化的最佳VEGF-C156s浓度和时间。 3．研究骨髓间质干细胞诱导后形成的内皮细胞在三维基质中形成管状结构的能力，对骨髓间质干细胞来源淋巴管内皮细胞的功能进行检测，在体外构建新生淋巴管。材料和方法 1．用密度为1.073g／ml的淋巴细胞分离液以及贴壁培养分离骨髓间质干细胞，培养、传代并冻存储备细胞。然后用流式细胞仪检测纯化后细胞的表面抗体CD14，CD34，CD44，CD105和CD166进行鉴定。 2．在24孔板中用不同浓度的VEGF-C156s对骨髓间质干细胞进行诱导分化，然后分别在不同的时间对诱导分化后的骨髓间质干细胞进行Ⅷ因子染色(内皮细胞标志物)，LYVE-1染色(淋巴管内皮细胞标志物)鉴定，观测染色后阳性细胞比例，确定诱导骨髓间质干细胞分化的最佳VEGF-C156s浓度，同时确定诱导分化时间，为实际应用中的浓度和时间打下参考基础。 3．在冰盒上制备胶原凝胶，然后置培养箱中37℃中使其凝固成为胶原凝胶。选取生长状态良好的细胞吹打成单细胞悬液，然后接种在胶原凝胶表面。选取3，6，9，12天的胶原块，用倒置相差显微镜从底面观取表面单层细胞之下的不同平面，同时从垂直断面观察并照相。结果 1．通过淋巴细胞分离液以及贴壁培养分离的方法，得到骨髓间质干细胞。到第三代以后，杂细胞甚少。用流式细胞仪检测细胞表面抗体，CD44，CD105和CD166呈阳性，CD14和CD34呈阴性。 2．用50ng／ml浓度的VEGF-C156s诱导骨髓间质干细胞，在第5天开始出现Ⅷ因子染色阳性细胞，10天后，几乎所有的细胞都Ⅷ因子染色阳性。50ng／ml浓度组和100ng／ml浓度组差异不大，而10ng／ml和20ng／ml组VEGF-C156s诱导骨髓间质干细胞后，效果不明显，10天后，染色阳性细胞不多。所以选择50ng／ml的VEGF-C156s浓度作为最佳诱导浓度。 3．诱导后形成的内皮细胞在三维基质中形成管状结构，我们成功地在体外用骨髓间质干细胞诱导分化后构建了人淋巴管。结论 1．成功分离培养出具有一定的纯度的骨髓间质干细胞，并且进行了用流式细胞仪检测细胞表面抗体进行鉴定，结果跟文献中的描述吻合，储备的骨髓间质干细胞为后续试验做好了准备。 2．可以用50ng／ml浓度的VEGF-C156s进行诱导分化。骨髓间质干细胞被诱导分化为淋巴管内皮细胞。 3．在体外成功构建了新生人淋巴管，模拟了淋巴管的新生过程，同时证明了骨髓间质干细胞来源的淋巴管内皮细胞具有成管功能。创新性： 1．首次证实骨髓间质干细胞可以被诱导分化为淋巴管内皮细胞，并且确定了诱导时间和最佳诱导浓度。 2．首次把骨髓间质干细胞来源的淋巴管内皮细胞在三维培养基质中模拟了淋巴管的新生过程，在体外成功构建了人新生淋巴管。
[Abstract]:background
Professor JR. Casley Smith, former chairman of the international lymphatic society, has pointed out that "Treatment of lymphedema is a notorious difficulty", the treatment of lymphedema is a worldwide problem and has not yet been found good treatment. Therefore, the treatment of lymphoedema lymphedema has been studied to promote lymphedema tissue. It has become an extremely arduous and urgent task in front of the lymphologists to improve lymphatic endothelial cell proliferation and lymphatic formation of lymphngiogenesis, improve lymphatic drainage and radically treat lymphedema, which has great academic value and clinical significance.
On the other hand, lymphatic metastasis of tumor tumor is the main cause of cancer death. Therefore, it is also of great clinical significance to study the inhibition of tumor metastasis to control tumor proliferation.
Based on these two aspects, we attempt to construct human lymphatics in vitro by using stem cell stem cells in vitro, and then study on the treatment of lymphedema and block the pathway of tumor metastasis.
In 1981, the Evans and other embryonic stem cells embryonic stem cell were successfully isolated and cultured for the first time. In 1998, the human embryonic stem cells were cultured for the first time by Thomson and so on, which laid a good foundation for the long-term objective of organ transplantation.
Then adult stem cells can differentiate into a variety of stem cells, especially in the bone marrow, which contains pluripotent stem cells, also called Mesenchymal Stem Cell. The stem cells have many advantages, which are easy to take from themselves, have no worries of immune rejection, and are more easily accepted by the patients; adult stem cells involve less Lenten. It is easy to expand in vitro, easy to be separated and easy to operate in vitro. Therefore, it has important application prospects in many aspects, such as tissue and organ defect, organ and organ degenerative disease, genetic defect disease and so on.
The current research shows that VEGF-C has a variety of biological characteristics and functions, which can be widely used in blood vessels. Lymphatic endothelial cell.VEGF-C156s is a point mutant VEGF-C. Its homologous two polymer can only activate VEGFR-3 and can not bind to VEGFR-2. Therefore, VEGF-C156s is used to induce bone marrow mesenchymal stem cells to differentiate into lymphatic endothelium. The best scheme, however, must be explored to determine the optimal VEGF-C156s concentration and time for inducing differentiation, which also involves a large number of experiments.
objective
1., the bone marrow mesenchymal stem cells with certain purity were cultured, and the surface antibody surface-antibody of the purified cells was detected by flow cytometry flow cytometry to identify the appraisement, and the cell reserve was done for the follow-up test.
2. the induction and differentiation of bone marrow mesenchymal stem cells by VEGF-C156s were used to determine the optimal VEGF-C156s concentration and time for inducing differentiation.
3. the ability of endothelial cells formed by bone marrow mesenchymal stem cells to form a tubular structure in the three-dimensional matrix was studied. The function of the bone marrow mesenchymal stem cells from the lymphatic endothelial cells derived from the bone marrow mesenchymal stem cells was detected and the new lymphatic vessels were constructed in vitro.
Materials and methods
1. the bone marrow mesenchymal stem cells were isolated with the density of 1.073g / ml and adherent culture. The cells were cultured, passaged and frozen, and the surface antibody CD14, CD34, CD44, CD105 and CD166 were detected by flow cytometry.
2. the bone marrow mesenchymal stem cells were induced and differentiated with different concentrations of VEGF-C156s in 24 orifice plates, and then the bone marrow mesenchymal stem cells were stained with factor VIII (endothelial cell marker), LYVE-1 staining (lymphatic endothelial cell marker), and the proportion of positive cells after staining, and the proportion of positive cells after staining was determined. The optimal VEGF-C156s concentration inducing the differentiation of bone marrow mesenchymal stem cells and determining the time for inducing differentiation can provide a reference basis for the concentration and time in practical application.
3. the collagen gel was prepared on the ice box, then the collagen gel was solidified in the incubator at 37 C. The cells with good growth state were blown into single cell suspension and then inoculated on the surface of collagen gel. The collagen blocks of 3,6,9,12 days were selected and the surface of the surface monolayer cells under the surface of the surface were observed by inverted phase contrast microscope. Observe and photograph from the vertical section.
Result
1. the bone marrow mesenchymal stem cells were obtained by the method of lymphocyte separation and adherent culture. After third generations, the heterocells were very small. The cell surface antibodies were detected by flow cytometry, CD44, CD105 and CD166 were positive, and CD14 and CD34 were negative.
2. 50NG / mL concentration of VEGF-C156s was used to induce bone marrow mesenchymal stem cells. The positive cells of factor VIII staining began to appear on the fifth day. After 10 days, almost all of the cells with factor VIII staining positive.50ng / mL concentration group and 100ng / mL concentration group were not different, while 10NG / ml and 20ng / ml VEGF-C156s induced bone marrow mesenchymal stem cells were not effective. Obviously, 10 days later, there were not many positive staining cells. Therefore, the VEGF-C156s concentration of 50NG / ml was chosen as the best induction concentration.
3. induced endothelial cells formed a tubular structure in the three-dimensional matrix. We successfully constructed the human lymphoid tube after differentiation of bone marrow mesenchymal stem cells in vitro.
conclusion
1. the bone marrow mesenchymal stem cells with a certain purity were successfully isolated and cultured, and the cell surface antibodies were detected by flow cytometry. The results were consistent with the description in the literature. The stored bone marrow mesenchymal stem cells were prepared for the follow-up test.
2., VEGF-C156s can be induced by 50NG / mL concentration. Bone marrow mesenchymal stem cells are induced to differentiate into lymphatic endothelial cells.
3. the new lymphoid tubes were successfully constructed in vitro, which simulated the process of lymphatic angiogenesis and demonstrated that the lymphatic endothelial cells derived from bone marrow mesenchymal stem cells have tubular function.
Innovation:
1. it is the first time that bone marrow mesenchymal stem cells can be induced to differentiate into lymphatic endothelial cells, and the induction time and optimal induction concentration have been determined.
2. for the first time, the lymphatic endothelial cells derived from bone marrow mesenchymal stem cells were used to simulate the new process of lymphatic vessels in the three-dimensional culture matrix, and the new lymphoid tubes were successfully constructed in vitro.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R322

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