本文選題：臍血 + 細胞因子��； 參考：《廣州醫(yī)學院》2009年碩士論文

【摘要】： 臍血作為一種造血干細胞資源越來越受到廣泛重視,但由于單份臍血中造血干細胞數(shù)量不足,不能滿足大多數(shù)成人和高體重兒童造血干細胞移植重建造血及免疫功能的需要,故限制了其廣泛應用。體外擴增雖然可以增加造血細胞數(shù)量,但可能導致造血干細胞分化并降低其歸巢和長期造血重建能力,因此提高臍血造血干細胞體外擴增質(zhì)量仍是臍血移植中亟待解決的問題。 間充質(zhì)干細胞作為造血微環(huán)境主要細胞成分基質(zhì)細胞的前體細胞,可以分泌與造血干細胞生長發(fā)育相關的黏附分子、細胞外基質(zhì)和多種細胞因子,為造血干細胞體外擴增提供適宜的微環(huán)境。隨著培養(yǎng)技術的提高,臍血間充質(zhì)干細胞有著與其他來源的間充質(zhì)干細胞所無法比擬的優(yōu)勢。本實驗研究臍血間充質(zhì)干細胞對人臍血單個核細胞體外擴增的支持作用。 目的探討臍血來源間充質(zhì)干細胞(UCB-MSCs)體外分離、培養(yǎng)和初步鑒定的方法,以及UCB-MSCs聯(lián)合外源性細胞因子對人臍血單個核細胞(UCB-MNCs)體外擴增的支持作用和最佳收獲時間,為配合造血干細胞移植的臨床應用提供實驗方法。 方法用羥乙基淀粉(HES)和人淋巴細胞分離液(Ficoll-Hypaque)以密度梯度離心法分離臍血單個核細胞,采用貼壁篩選法以MesencultTM專用培養(yǎng)基培養(yǎng)出臍血間充質(zhì)干細胞,并通過流式細胞儀檢測其細胞表面抗原。將新鮮臍血標本分離出的臍血單個核細胞接種于無血清培養(yǎng)體系(Stem spanTM)中培養(yǎng)18天,實驗分三組,A組:為空白對照組(培養(yǎng)體系中無外源性細胞因子和UCB-MSCs滋養(yǎng)層);B組:為因子組(培養(yǎng)體系中有外源性細胞因子但無UCB-MSCs滋養(yǎng)層);C組:為實驗組(培養(yǎng)體系中既有外源性細胞因子又有UCB-MSCs滋養(yǎng)層)。在第0、7、10、14及18天檢測有核細胞總數(shù)(MNCs)、CD34+細胞數(shù)、CD133+細胞數(shù)、集落形成單位數(shù)(CFU)和細胞在周期(G2+M+S期)含量的變化。 結果①從臍血中分離、培養(yǎng)的出間充質(zhì)干細胞,穩(wěn)定表達CD29、CD105和CD44,不表達CD34和CD133。②在體外培養(yǎng)過程中,外源性細胞因子及UCB-MSCs均對臍血單個核細胞的擴增起支持作用,但以UCB-MSCs聯(lián)合外源性細胞因子組效果最好,該組各項指標在同一時間點較其它兩組高,有統(tǒng)計學意義(P㩳0.05),且維持造血至少達18天。③以UCB-MSCs聯(lián)合外源性細胞因子對臍血單個核細胞擴增,第10天上述各項指標達到最高峰,有統(tǒng)計學意義(P㩳0.05)。 結論①采用密度梯度離心聯(lián)合貼壁篩選法并通過流式細胞檢測技術,可以成功地實現(xiàn)從臍血中分離、培養(yǎng)出間充質(zhì)干細胞,并完成其細胞表型的初步鑒定。②UCB-MSCs聯(lián)合外源性細胞因子可有效擴增臍血單個核細胞。③UCB-MSCs聯(lián)合外源性細胞因子體外擴增臍血單個核細胞細胞收獲的最佳時間為第10-14天。
[Abstract]:As a kind of hematopoietic stem cell resource, umbilical cord blood has been paid more and more attention. However, due to the insufficient number of hematopoietic stem cells in a single cord blood, it can not meet the needs of most adult and high-weight children with hematopoietic stem cell transplantation and reconstitution of hematopoietic and immune function.Therefore, its wide application is limited.Although in vitro amplification can increase the number of hematopoietic cells, it may lead to differentiation of hematopoietic stem cells and decrease their homing and long-term hematopoietic reconstitution ability. Therefore, improving the expansion quality of umbilical cord blood hematopoietic stem cells in vitro is still an urgent problem to be solved in umbilical cord blood transplantation.Mesenchymal stem cells, as precursors of stromal cells, which are the main cell components of hematopoietic microenvironment, can secrete adhesion molecules, extracellular matrix and many cytokines related to the growth and development of hematopoietic stem cells.To provide a suitable microenvironment for the expansion of hematopoietic stem cells in vitro.With the improvement of culture technology, umbilical cord blood mesenchymal stem cells have unparalleled advantages over other mesenchymal stem cells.The purpose of this study was to investigate the supporting effect of umbilical cord blood mesenchymal stem cells on the expansion of human umbilical cord blood mononuclear cells in vitro.Objective to investigate the methods of isolation, culture and identification of UCB-MSCs derived from umbilical cord blood in vitro, and the supporting effect of UCB-MSCs combined with exogenous cytokines on the expansion of human umbilical cord blood mononuclear cells (UCB-MNCs) in vitro and the optimal harvest time.To provide experimental methods for clinical application of hematopoietic stem cell transplantation.Methods umbilical cord blood mononuclear cells were isolated with hydroxyethyl starch (HES) and human lymphocyte isolate (Ficoll-Hypaque) by density gradient centrifugation. Mesenchymal stem cells from umbilical cord blood were cultured in MesencultTM medium by adherent screening method.The cell surface antigen was detected by flow cytometry.Cord blood mononuclear cells isolated from fresh cord blood samples were cultured in serum-free culture system Stem span TM1 for 18 days.The experiment was divided into three groups: blank control group (no exogenous cytokines in culture system and UCB-MSCs trophoblastic trophoblast group B): factor group (exogenous cytokines in culture system but no trophoblastic layer in UCB-MSCs group C): experimental group (culture group)Both exogenous cytokines and UCB-MSCs trophoblast were found in the system.The number of CD133 cells, the number of colony forming units (CFU) and the changes of cell contents in G 2 M S phase were detected on the 14th and 18th day of the 7th day. The total number of nucleated cells and the number of CD34 cells and the number of colony forming units (CFU) were measured.Results 1 Mesenchymal stem cells isolated from umbilical cord blood expressed CD29, CD105 and CD44 stably, but not CD34 and CD133.2. Exogenous cytokines and UCB-MSCs played a supporting role in the expansion of cord blood mononuclear cells in vitro.But the effect of UCB-MSCs combined with exogenous cytokines was the best, and the indexes of the group were higher than those of the other two groups at the same time point.At least 18 days after maintenance, UCB-MSCs combined with exogenous cytokines was used to amplify umbilical cord blood mononuclear cells. On the 10th day, the above indexes reached the highest peak, with statistical significance.Conclusion 1 using density gradient centrifugation combined with adherent screening and flow cytometry, mesenchymal stem cells can be successfully isolated and cultured from umbilical cord blood.The results showed that UCB-MSCs combined with exogenous cytokines could effectively amplify cord blood mononuclear cells. 3UCB-MSCs combined with exogenous cytokines could obtain the best harvest time of UCB-MSCs combined with exogenous cytokines in vitro. The best time for the harvest of UCB-MSCs combined with exogenous cytokines was 10-14 days.
【學位授予單位】：廣州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

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本文編號：1764800