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Compairson of Capacity of Homing and Ifferentiation of Umbilical Cord Blood CD34~+When Xpanded by Different Cytokines Combinations

Author: WangCuiCui
Tutor: SunZiMin
School: Anhui Medical University,
Course: Internal Medicine
Keywords: cord blood CD34~+cells co-culture ex vivo cytokines expansion differentiation homing adhesion molecules
CLC: R329
Type: Master's thesis
Year: 2012
Downloads: 58
Quote: 0
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Abstract


Objective Cytokines combination: Stem cell factor(SCF), FLT3ligand(FL) andthrombopoietin (TPO) was one foundational method to culuture umbilical cord blood(UCB) CD34~+in vitro. Interleukin-3(IL-3)played a role in Inducing UCB intodifferentiating. Reserch on expansion of cord blood hematopoietic cell by differentculture system invitro is in hotpot, however, there was little information about changesof homing iapacity when expanded. In this dissertation, a study was performed aboutproliferation, homing and differentiation by different cytokins combinations in vitro.Methods we have attempted to expand selected CD34~+cells in2-week cultures in thepresence various cytokine combinations:Group A:SCF+FL+TPO、 GroupB:SCF+FL+TPO+IL-3,under serum-free and stromal free medium conditions and analysecolony forming cells(CFC) to study on cell proliferation. by flow cytometry,we canfound out changes of their homing-related molecules expression(CLA,CD11a,CD18,CD26,CD44,CD62L,CD162,CD184) and differentiation-related moleculesexpression (CD33, CD41, CD71) when expanded in vitro by different cytokinescombinations.Results1. Cells expansion:Fourteen days after culured, amplification of TNC, cellsnumber of groupA and B were206.40±32.05、340.00±82.45fold increased respectively,cell counts which group with IL-3is higher(P=0.01).Amplification of CD34~+, cellsnumber of groupA and B was27.78±4.15、21.56±6.09fold increased respectively, cellcounts which group without IL-3is little higher(P=0.096),however, there was nosignificant difference between them. Amplification of CFC, groupA and B was77.86±5.96、107.16±13.06fold increased respectively, counts which group with IL-3ishigher(P=0.005).2. The differentiation ability change of different groups: After thein vitro culture, both group A and B were all appeared differentiation, there wassignificant difference compared with pre-treatment. The expression of CD33(P=0.001)and CD44(P=0.004) on CD34~+cells of Group B were higher.,but CD71(0.002) eachgroup was gradually decreased and CD41remained a low lever, but the expression of CD71was lower than gourp A.3.The homing ability change The purity of theisolated CD34~+cells was (98.36±0.62)%. Fourteen days after in vitro culture, theexpression of homing related adhesion molecular CD11a,CD18were both increasedthan pre-treatment.(P<0.05),meanwhile, the expressing of gourp B was higher thanA(P=0.017), there was significant difference between two groups. The expression ofhoming-related adhesion molecules such as CD184,CD26were both increased thanpre-treatment(P<0.05),but there was no significant difference between groups. Theexpression of homing-related slection ligands such as CD62L, group B was decreasedafter cultured(P<0.05), meanwhile there was no significant difference betweenpre-treatment and groupsA(P=0.068)and there was no significant difference betweengroup A and B(P=0.269). The expression of CD162,group A was more higher thanB(0.041), decreased after cultured(P=0.004). The expression of CD44which was thereceptor for hyaluronan-mediated motility,both group A and B were decreased aftercultured(P<0.05), but there was no significant difference between group A and B.Conclusions1. This two different cytokine combination were both had capacity ofproliferation and differentiation of UCB CD34~+cells;2. The groupB:SCF+FL+TPO+IL-3was better than group A, and its alility of inducingdifferentiation to Megakaryocyte and myelod was more stronger;3.After in vitrocultured, the expression of homing related molecular changed differentenly. thoughsome may increased homing capacity, accompanied unfavorable affects.Cytokinscombination which contains IL-3not only could increased the capacity ofproliferation and differentiation UCB cells,but also had the trand of increasing homingcapacity.

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