人淋巴管的体外构建研究
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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。
Background
Pro-chairman of International Lymphology Academy, Professor JR. Casley Smith indicate even : "Treatment of lymphedema is a notorious difficulty" , We adopt such conservative treatment according to patient's condition as massage acupuncture and so on., or we adopt operative treatment and drug therapy; however, conservative treatment is time-consuming; it only relieve symptom temporary and its effectiveness is not obvious, operation treatment is usually limited in range in treatment.and post-operation occur lymphatic vessel re-blockage .Its short-term effectiveness is satisfactory .long-term effectiveness is not satisfactory。operative treatment。As the aspect of drug treatment, emictory use in especial treatment of lymphedema, which threaten the function of heart, lung. But it did not adopt in common lymphedema. Therefore, there is not ideal treatment method to aim directly at lymphedema in clinic.
Since 1996, A critical advance came from the pioneering work by Joukov and colleagues, who identified a ligand of FLT-4 (VEGFR-3), the vascular endothelial growth factor VEGF-C, a member of VEGF/PDGF family and abundant aminothiopropionic acid in C'. After found it, in early stage, most research was developed on its genes, molecular structure, receptor and its structural feature, bionomics, and so on.
Mesenchymal Stem Cells (MSCs) developed from mesodermic desmohemoblast, and has the ability differentiate into osteoblasts, chondroblasts, and adipocytes in response to appropriate stimuli, it had been became a kinds of ideal cell in tissue and cell repair for its merits as followed:
1. Bone marrow is an alternative source of stem cells which are well suited for clinical application because they are easily obtained from patients and because autologous transplantation, which obviates immunologic incompatibilities, is possible.
2. Of the various progenitor cells that exist within bone marrow, mesenchymal stem cells (MSC) are particularly attractive for clinical use because they are easily isolated, can be expanded in culture, and can be genetically manipulated using currently available molecular techniques.
3. MSCs are adult stem cells, has no ethics questions
So, it is important for applicated MSCs into degenerative disease, genetic deficiency or others.
Objectives
1. To separate and culture human bone marrow mesenchymal stem cells. Then identify the stem cells and store cells for further study.
2. Induce the differentiation of human bone marrow mesenchymal stem cells by VEGF-C156s and decide the best concentration and time of VEGF-C156s to induce differentiation.
3. To study the function of lymphatic endothelial cells induced from MSCs in three dimensional substances.
Methods
1. Get the human bone marrow mesenchymal stem cells by lymphocyte separation medium (1.073g/ml) and adherent culture. Store cells for further study by passage and freeze cells. Identified the stem cells by flow cytometry to detect the surface antibody as CD14,CD34,CD44,CD105 and CD166.
2. Observe the effect of VEGF-C156s on differentiation of human bone marrow mesenchymal stem cells into lymphatic endothelial cells with different concentration and at different time. Count the ratio of positive stain cells after stained by VI factor and LYVE-1.
3. Prepares collagen gel on the ice box and then put into 37℃incubator. Inoculate the cells with good growth condition on the surface of the collagen gel. Then observe the growth of cultured cells by inverted phase contrast microscope. Vertical section was also observed.
Results
1. Human bone marrow mesenchymal stem cells were get by lymphocyte separation medium and adherent culture. The surface antibody identified by flow cytometry show results with positive CD44,CD105 and CD166 surface antibody. Negative results were get by CD14,CD34 surface antibody analysis. The results are coincidence with references, and proved the isolated cells are mesenchymal stem cells.
2. With concentration of 50ng/ml, VEGF-C156s can induce the differentiation of human bone marrow mesenchymal stem cells from the five day. The differentiated cells show positive stain of VI factor and LYVE-1.
3. Lymphatic endothelial cells induced from MSCs can grow with tubiform structure.
Conclusions
1. Isolated and cultured human bone marrow mesenchymal stem cells from bone marrow with high purity and get ready for further study.
2. Human bone marrow mesenchymal stem cells can be induced by VEGF-C156s and differentiated into lymphatic endothelial cells.
3. Lymphatic endothelial cells induced from MSCs have the function to form vessels and formed lymphatic vessels in vitro.
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。
Background
Pro-chairman of International Lymphology Academy, Professor JR. Casley Smith indicate even : "Treatment of lymphedema is a notorious difficulty" , We adopt such conservative treatment according to patient's condition as massage acupuncture and so on., or we adopt operative treatment and drug therapy; however, conservative treatment is time-consuming; it only relieve symptom temporary and its effectiveness is not obvious, operation treatment is usually limited in range in treatment.and post-operation occur lymphatic vessel re-blockage .Its short-term effectiveness is satisfactory .long-term effectiveness is not satisfactory。operative treatment。As the aspect of drug treatment, emictory use in especial treatment of lymphedema, which threaten the function of heart, lung. But it did not adopt in common lymphedema. Therefore, there is not ideal treatment method to aim directly at lymphedema in clinic.
Since 1996, A critical advance came from the pioneering work by Joukov and colleagues, who identified a ligand of FLT-4 (VEGFR-3), the vascular endothelial growth factor VEGF-C, a member of VEGF/PDGF family and abundant aminothiopropionic acid in C'. After found it, in early stage, most research was developed on its genes, molecular structure, receptor and its structural feature, bionomics, and so on.
Mesenchymal Stem Cells (MSCs) developed from mesodermic desmohemoblast, and has the ability differentiate into osteoblasts, chondroblasts, and adipocytes in response to appropriate stimuli, it had been became a kinds of ideal cell in tissue and cell repair for its merits as followed:
1. Bone marrow is an alternative source of stem cells which are well suited for clinical application because they are easily obtained from patients and because autologous transplantation, which obviates immunologic incompatibilities, is possible.
2. Of the various progenitor cells that exist within bone marrow, mesenchymal stem cells (MSC) are particularly attractive for clinical use because they are easily isolated, can be expanded in culture, and can be genetically manipulated using currently available molecular techniques.
3. MSCs are adult stem cells, has no ethics questions
So, it is important for applicated MSCs into degenerative disease, genetic deficiency or others.
Objectives
1. To separate and culture human bone marrow mesenchymal stem cells. Then identify the stem cells and store cells for further study.
2. Induce the differentiation of human bone marrow mesenchymal stem cells by VEGF-C156s and decide the best concentration and time of VEGF-C156s to induce differentiation.
3. To study the function of lymphatic endothelial cells induced from MSCs in three dimensional substances.
Methods
1. Get the human bone marrow mesenchymal stem cells by lymphocyte separation medium (1.073g/ml) and adherent culture. Store cells for further study by passage and freeze cells. Identified the stem cells by flow cytometry to detect the surface antibody as CD14,CD34,CD44,CD105 and CD166.
2. Observe the effect of VEGF-C156s on differentiation of human bone marrow mesenchymal stem cells into lymphatic endothelial cells with different concentration and at different time. Count the ratio of positive stain cells after stained by VI factor and LYVE-1.
3. Prepares collagen gel on the ice box and then put into 37℃incubator. Inoculate the cells with good growth condition on the surface of the collagen gel. Then observe the growth of cultured cells by inverted phase contrast microscope. Vertical section was also observed.
Results
1. Human bone marrow mesenchymal stem cells were get by lymphocyte separation medium and adherent culture. The surface antibody identified by flow cytometry show results with positive CD44,CD105 and CD166 surface antibody. Negative results were get by CD14,CD34 surface antibody analysis. The results are coincidence with references, and proved the isolated cells are mesenchymal stem cells.
2. With concentration of 50ng/ml, VEGF-C156s can induce the differentiation of human bone marrow mesenchymal stem cells from the five day. The differentiated cells show positive stain of VI factor and LYVE-1.
3. Lymphatic endothelial cells induced from MSCs can grow with tubiform structure.
Conclusions
1. Isolated and cultured human bone marrow mesenchymal stem cells from bone marrow with high purity and get ready for further study.
2. Human bone marrow mesenchymal stem cells can be induced by VEGF-C156s and differentiated into lymphatic endothelial cells.
3. Lymphatic endothelial cells induced from MSCs have the function to form vessels and formed lymphatic vessels in vitro.
引文
1. Z.liu, Z. Z Guo, P. Dong, Y. Fang, Y. Liu, H. Tian, Z. Ding, Y. Bi, J. Liu, Y. Yang. Experimental Study on Lymphoedema Therapy by Vascular Endothelial Growth Factor-C Gene. 16th International Congress of the IFAA Proceedings. 2004.
2. Zhiyu Liu, Jie Gao, Zhong Guo, Ping Dong Yunhai Fang Yanli Liu Hua Tian Zhaoxi Ding Yushun Bi Jinlan Liu Yuying Yang. Experimental Study on Lymphoedema Therapy by Vascular Endothelial Growth Factor-C Gene. The International Lymphology Symposium in Oita, Japan. 2004.
3. Yunhai Fang, Zhiyu Liu, Yao Chen, Nianming Gong, Yanli Liu. Expression of Cell Adhesion Molecules on Vascular Endothelial Cells in Human Rectal Cancer. Annual Scientific Meeting of the American College of Gastroenterology. USA. 2005.
4. Zhiyu Liu, Jie Gao. Construction of eukaryotic expression vector for human vascular endothelial growth factor C gene. Human Genome Meeting in Shanghai, China.2002.
5. Liu Zhiyu, Chen Yao, Fang Yunhai, Li Ruixiang, Tian Hua, Song Tao. Expression of CEA, ICAM-1 on lymphatic endothelial cells in rectum cancer of human. 26th Lymphology Symposium in Japan. 2002.
6. Sun Jinhao, Liu Zhiyu, Bi Yushun, Tian Hua, Ding Zhaoxi. Effects of Tumor Necrosis Factor- sand Interleukin-2 on Lymphocyte Migration in Mouse. International Symposium on Morphological Sciences. Beijing, China. 2000.
7. Zhaoxi Ding, Zhiyu. Liu, Yushun Bi at all: Morphological study of the interaction between M21 melanoma and lymphatic endothelium. Lymphology, 2005, 38:87-91.
8. J. sun, Zhiyu liu, Y. Bi at all: Effect of tumor necrosis factor-2 and iL-2 on spleen lymphocyte migration in mouse skin, lymphology, 1999, 32:166-170.
9. Yao Chen, Zhi-Yu. liu, Ruixiang Li at all: Structural studies of initial lymphatics adjacent to gastric and colonic malignant neoplasms, Lymphology 1999, 32:70-74.
10．李明焕、田铧、刘执玉、宋涛、李贵宝、田广平：ECV304细胞用于三维血管新生的研究，中国现代普通外科进展，2004，7：165-167．
11．董平、刘执玉、毕玉顺、田铧、丁兆习：VEGF-C真核表达载体的构建和体外表达，山东大学基础医学院学报，2004，18：129-132．
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