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

【摘要】： 背景 國(guó)際淋巴學(xué)會(huì)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(gè)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(jìn)淋巴水腫組織內(nèi)的淋巴管內(nèi)皮細(xì)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(xué)工作者面前的極其艱巨而迫切的任務(wù)，具有重大的學(xué)術(shù)理論價(jià)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(duì)控制腫瘤擴(kuò)散也有十分重要的臨床意義。 基于這兩方面的目的，我們?cè)噲D利用干細(xì)胞stem cells首先在體外構(gòu)建人淋巴管，進(jìn)而在此基礎(chǔ)上開(kāi)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(xì)胞embryonic stem cell；1998年，Thomson等首次培養(yǎng)出人的胚胎干細(xì)胞；從而為器官、組織移植的長(zhǎng)遠(yuǎn)目標(biāo)打下良好基礎(chǔ)。 繼而成體干細(xì)胞可以分化成多種干細(xì)胞，特別是骨髓內(nèi)含有多能干細(xì)胞，又稱為骨髓間質(zhì)干細(xì)胞(Mesenchymal Stem Cell)。這種干細(xì)胞有許多優(yōu)點(diǎn)：易于從自身取材，無(wú)免疫排斥反應(yīng)之憂，因而也就更易為病人所接受；成體干細(xì)胞，涉及較少倫理問(wèn)題；體外易于擴(kuò)增、易分離、以及體外操作簡(jiǎn)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(yīng)用前景。 目前的研究表明VEGF-C具有多種生物學(xué)特性和功能，可廣泛作用于血管、淋巴管內(nèi)皮細(xì)胞。VEGF-C156s是一種點(diǎn)突變型VEGF-C，其同源二聚體只能激活VEGFR-3，不能與VEGFR-2結(jié)合，因此，選用VEGF-C156s對(duì)骨髓間質(zhì)干細(xì)胞進(jìn)行誘導(dǎo)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(dǎo)分化的最佳VEGF-C156s濃度和時(shí)間，這也必定涉及到大量的試驗(yàn)。 目的 1．分離、培養(yǎng)出具有一定的純度的骨髓間質(zhì)干細(xì)胞，并且用流式細(xì)胞儀flow cytometry檢測(cè)純化后細(xì)胞的表面抗體surface-antibody進(jìn)行鑒定appraisement，為后續(xù)試驗(yàn)做好細(xì)胞儲(chǔ)備。 2．用VEGF-C156s對(duì)骨髓間質(zhì)干細(xì)胞進(jìn)行誘導(dǎo)分化，確定誘導(dǎo)分化的最佳VEGF-C156s濃度和時(shí)間。 3．研究骨髓間質(zhì)干細(xì)胞誘導(dǎo)后形成的內(nèi)皮細(xì)胞在三維基質(zhì)中形成管狀結(jié)構(gòu)的能力，對(duì)骨髓間質(zhì)干細(xì)胞來(lái)源淋巴管內(nèi)皮細(xì)胞的功能進(jìn)行檢測(cè)，在體外構(gòu)建新生淋巴管。 材料和方法 1．用密度為1.073g／ml的淋巴細(xì)胞分離液以及貼壁培養(yǎng)分離骨髓間質(zhì)干細(xì)胞，培養(yǎng)、傳代并凍存儲(chǔ)備細(xì)胞。然后用流式細(xì)胞儀檢測(cè)純化后細(xì)胞的表面抗體CD14，CD34，CD44，CD105和CD166進(jìn)行鑒定。 2．在24孔板中用不同濃度的VEGF-C156s對(duì)骨髓間質(zhì)干細(xì)胞進(jìn)行誘導(dǎo)分化，然后分別在不同的時(shí)間對(duì)誘導(dǎo)分化后的骨髓間質(zhì)干細(xì)胞進(jìn)行Ⅷ因子染色(內(nèi)皮細(xì)胞標(biāo)志物)，LYVE-1染色(淋巴管內(nèi)皮細(xì)胞標(biāo)志物)鑒定，觀測(cè)染色后陽(yáng)性細(xì)胞比例，確定誘導(dǎo)骨髓間質(zhì)干細(xì)胞分化的最佳VEGF-C156s濃度，同時(shí)確定誘導(dǎo)分化時(shí)間，為實(shí)際應(yīng)用中的濃度和時(shí)間打下參考基礎(chǔ)。 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(zhǎng)狀態(tài)良好的細(xì)胞吹打成單細(xì)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(xì)胞之下的不同平面，同時(shí)從垂直斷面觀察并照相。 結(jié)果 1．通過(guò)淋巴細(xì)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(xì)胞。到第三代以后，雜細(xì)胞甚少。用流式細(xì)胞儀檢測(cè)細(xì)胞表面抗體，CD44，CD105和CD166呈陽(yáng)性，CD14和CD34呈陰性。 2．用50ng／ml濃度的VEGF-C156s誘導(dǎo)骨髓間質(zhì)干細(xì)胞，在第5天開(kāi)始出現(xiàn)Ⅷ因子染色陽(yáng)性細(xì)胞，10天后，幾乎所有的細(xì)胞都Ⅷ因子染色陽(yáng)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(dǎo)骨髓間質(zhì)干細(xì)胞后，效果不明顯，10天后，染色陽(yáng)性細(xì)胞不多。所以選擇50ng／ml的VEGF-C156s濃度作為最佳誘導(dǎo)濃度。 3．誘導(dǎo)后形成的內(nèi)皮細(xì)胞在三維基質(zhì)中形成管狀結(jié)構(gòu)，我們成功地在體外用骨髓間質(zhì)干細(xì)胞誘導(dǎo)分化后構(gòu)建了人淋巴管。 結(jié)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(xì)胞，并且進(jìn)行了用流式細(xì)胞儀檢測(cè)細(xì)胞表面抗體進(jìn)行鑒定，結(jié)果跟文獻(xiàn)中的描述吻合，儲(chǔ)備的骨髓間質(zhì)干細(xì)胞為后續(xù)試驗(yàn)做好了準(zhǔn)備。 2．可以用50ng／ml濃度的VEGF-C156s進(jìn)行誘導(dǎo)分化。骨髓間質(zhì)干細(xì)胞被誘導(dǎo)分化為淋巴管內(nèi)皮細(xì)胞。 3．在體外成功構(gòu)建了新生人淋巴管，模擬了淋巴管的新生過(guò)程，同時(shí)證明了骨髓間質(zhì)干細(xì)胞來(lái)源的淋巴管內(nèi)皮細(xì)胞具有成管功能。 創(chuàng)新性： 1．首次證實(shí)骨髓間質(zhì)干細(xì)胞可以被誘導(dǎo)分化為淋巴管內(nèi)皮細(xì)胞，并且確定了誘導(dǎo)時(shí)間和最佳誘導(dǎo)濃度。 2．首次把骨髓間質(zhì)干細(xì)胞來(lái)源的淋巴管內(nèi)皮細(xì)胞在三維培養(yǎng)基質(zhì)中模擬了淋巴管的新生過(guò)程，在體外成功構(gòu)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(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R322

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