本文選題：骨髓間質(zhì)干細胞 + VEGF-C156s��； 參考：《山東大學》2007年博士論文

【摘要】： 背景 國際淋巴學會前主席JR. Casley Smith教授曾指出：“Treatment of lymphedema is a notorious difficulty”，淋巴水腫的治療的卻是一個世界性的難題，至今尚未找到良好的治療方法，。因此，研究探討淋巴水腫lymphoedema的治療，促進淋巴水腫組織內(nèi)的淋巴管內(nèi)皮細胞增生和淋巴管形成lymphngiogenesis，改善淋巴引流途徑，從根本上治療淋巴水腫，已成為擺在淋巴學工作者面前的極其艱巨而迫切的任務，具有重大的學術理論價值和和臨床意義。 另一方面，腫瘤tumor的淋巴轉(zhuǎn)移是造成腫瘤病人死亡的主要原因，，因此，研究抑制腫瘤的轉(zhuǎn)移途徑對控制腫瘤擴散也有十分重要的臨床意義。 基于這兩方面的目的，我們試圖利用干細胞stem cells首先在體外構建人淋巴管，進而在此基礎上開展淋巴水腫的治療研究和阻斷腫瘤轉(zhuǎn)移途徑的研究。 1981年Evans等首次成功地分離培養(yǎng)出小鼠胚胎干細胞embryonic stem cell；1998年，Thomson等首次培養(yǎng)出人的胚胎干細胞；從而為器官、組織移植的長遠目標打下良好基礎。 繼而成體干細胞可以分化成多種干細胞，特別是骨髓內(nèi)含有多能干細胞，又稱為骨髓間質(zhì)干細胞(Mesenchymal Stem Cell)。這種干細胞有許多優(yōu)點：易于從自身取材，無免疫排斥反應之憂，因而也就更易為病人所接受；成體干細胞，涉及較少倫理問題；體外易于擴增、易分離、以及體外操作簡便。因此，在組織器官缺損性疾病，組織器官退行性疾病，遺傳缺陷性疾病等多方面有重要的應用前景。 目前的研究表明VEGF-C具有多種生物學特性和功能，可廣泛作用于血管、淋巴管內(nèi)皮細胞。VEGF-C156s是一種點突變型VEGF-C，其同源二聚體只能激活VEGFR-3，不能與VEGFR-2結合，因此，選用VEGF-C156s對骨髓間質(zhì)干細胞進行誘導分化成淋巴管樣內(nèi)皮是最佳的方案，然而必須探討、確定誘導分化的最佳VEGF-C156s濃度和時間，這也必定涉及到大量的試驗。 目的 1．分離、培養(yǎng)出具有一定的純度的骨髓間質(zhì)干細胞，并且用流式細胞儀flow cytometry檢測純化后細胞的表面抗體surface-antibody進行鑒定appraisement，為后續(xù)試驗做好細胞儲備。 2．用VEGF-C156s對骨髓間質(zhì)干細胞進行誘導分化，確定誘導分化的最佳VEGF-C156s濃度和時間。 3．研究骨髓間質(zhì)干細胞誘導后形成的內(nèi)皮細胞在三維基質(zhì)中形成管狀結構的能力，對骨髓間質(zhì)干細胞來源淋巴管內(nèi)皮細胞的功能進行檢測，在體外構建新生淋巴管。 材料和方法 1．用密度為1.073g／ml的淋巴細胞分離液以及貼壁培養(yǎng)分離骨髓間質(zhì)干細胞，培養(yǎng)、傳代并凍存儲備細胞。然后用流式細胞儀檢測純化后細胞的表面抗體CD14，CD34，CD44，CD105和CD166進行鑒定。 2．在24孔板中用不同濃度的VEGF-C156s對骨髓間質(zhì)干細胞進行誘導分化，然后分別在不同的時間對誘導分化后的骨髓間質(zhì)干細胞進行Ⅷ因子染色(內(nèi)皮細胞標志物)，LYVE-1染色(淋巴管內(nèi)皮細胞標志物)鑒定，觀測染色后陽性細胞比例，確定誘導骨髓間質(zhì)干細胞分化的最佳VEGF-C156s濃度，同時確定誘導分化時間，為實際應用中的濃度和時間打下參考基礎。 3．在冰盒上制備膠原凝膠，然后置培養(yǎng)箱中37℃中使其凝固成為膠原凝膠。選取生長狀態(tài)良好的細胞吹打成單細胞懸液，然后接種在膠原凝膠表面。選取3，6，9，12天的膠原塊，用倒置相差顯微鏡從底面觀取表面單層細胞之下的不同平面，同時從垂直斷面觀察并照相。 結果 1．通過淋巴細胞分離液以及貼壁培養(yǎng)分離的方法，得到骨髓間質(zhì)干細胞。到第三代以后，雜細胞甚少。用流式細胞儀檢測細胞表面抗體，CD44，CD105和CD166呈陽性，CD14和CD34呈陰性。 2．用50ng／ml濃度的VEGF-C156s誘導骨髓間質(zhì)干細胞，在第5天開始出現(xiàn)Ⅷ因子染色陽性細胞，10天后，幾乎所有的細胞都Ⅷ因子染色陽性。50ng／ml濃度組和100ng／ml濃度組差異不大，而10ng／ml和20ng／ml組VEGF-C156s誘導骨髓間質(zhì)干細胞后，效果不明顯，10天后，染色陽性細胞不多。所以選擇50ng／ml的VEGF-C156s濃度作為最佳誘導濃度。 3．誘導后形成的內(nèi)皮細胞在三維基質(zhì)中形成管狀結構，我們成功地在體外用骨髓間質(zhì)干細胞誘導分化后構建了人淋巴管。 結論 1．成功分離培養(yǎng)出具有一定的純度的骨髓間質(zhì)干細胞，并且進行了用流式細胞儀檢測細胞表面抗體進行鑒定，結果跟文獻中的描述吻合，儲備的骨髓間質(zhì)干細胞為后續(xù)試驗做好了準備。 2．可以用50ng／ml濃度的VEGF-C156s進行誘導分化。骨髓間質(zhì)干細胞被誘導分化為淋巴管內(nèi)皮細胞。 3．在體外成功構建了新生人淋巴管，模擬了淋巴管的新生過程，同時證明了骨髓間質(zhì)干細胞來源的淋巴管內(nèi)皮細胞具有成管功能。 創(chuàng)新性： 1．首次證實骨髓間質(zhì)干細胞可以被誘導分化為淋巴管內(nèi)皮細胞，并且確定了誘導時間和最佳誘導濃度。 2．首次把骨髓間質(zhì)干細胞來源的淋巴管內(nèi)皮細胞在三維培養(yǎng)基質(zhì)中模擬了淋巴管的新生過程，在體外成功構建了人新生淋巴管。
[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.

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R322

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