摘要
本文研究了小鼠骨髓基质细胞的生物学特性,探索了骨髓造血微环境的特征;还研究了基因工程重组的人血液血管细胞生成素对骨髓基质细胞的生物学活性的影响;并以人血液血管细胞生成素基因修饰骨髓基质细胞,用于体外造血的支持。
第一章为了研究骨髓基质细胞(间充质干细胞)的生物学性状和多系分化潜能,我们取Balb/c 小鼠骨髓单个核细胞在低糖的培养液中培养出贴壁生长的细胞,进行形态学观察、细胞周期和免疫表型分析;在不同的细胞因子作用下诱导向成骨细胞、软骨细胞和脂肪细胞分化,并检测诱导后细胞相应的基因表达。结果发现,小鼠骨髓间充质干细胞贴壁生长后形态较均一,增殖能力随着传代逐渐增强,但从第8 代后增殖能力明显减退。细胞表达CD29, CD38, CD44,CD106 等抗原,但CD34 和H-2k 表达阴性。在不同的诱导培养体系里间充质干细胞能分化为成骨细胞、软骨细胞和脂肪细胞,相应细胞的骨钙蛋白基因、Ⅱ型胶原基因和脂蛋白脂酶基因表达都明显增强。
第二章由于血液血管细胞生成素是一种新近被认识的细胞因子,其诸多生物学活性,包括对骨髓基质细胞的作用,还有待于深入探讨。因此,我们研究了基因工程重组的人血液血管细胞生成素(rhHAPO)对原代骨髓基质细胞的增殖和成纤维集落形成单位的影响;对骨髓基质细胞系黏附和增殖及其细胞表面黏附分子的作用;此外,还利用RT-PCR 研究了骨髓基质细胞的细胞因子基因表达情况。结果表明,rhHAPO 通过上调基质细胞的S 期细胞比例,促进骨髓基质细胞的增殖;通过上调VCAM 和ICAM 等细胞表面黏附分子,促进骨髓基质细胞的总黏附能力;而骨髓基质细胞除了表达M-CSF 和c-mpl 等因子基因外,并不表达HAPO 和
This academic thesis has exhibited its essences of research onbiological characteristics of murine marrow-derived mesenchymal stemcells and bone marrow microenvironment, and biological effects ofrecombined human hemangiopoietin upon bone marrow stromal cells, as wellas the potential impact of stromal cells transduced with humanhemangiopoietin gene on in vitro hematopoiesis.
Section 1. The aim of this section is to explore the biologicalcharacteristics and in vitro differentiation potential of murinemarrow-derived mesenchymal stem cells. The techniques for isolation ofmurine marrow-derived mesenchymal stem cells have been described, as wellas the methods to directing their differentiation into osteogenic,chondrogenic, and adipogenic lineages. Their characteristics regardingmorphology, surface marker profiles and growth kinetics have also beenexplored. These cells were positive for the surface markers CD29, CD38,CD44, and CD106, and exhibited a homogeneous, distinctive morphology, andwere shown to differentiate along osteogenic, chondrogenic, andadipogenic pathways. After the 8th passage, MSC had a markedly diminishproliferation rate.
Section 2. The objective of this section is to survey the biological
effects of a novel cytokine, recombined human hemangiopoietin (rhHAPO),on bone marrow stromal cells. The biological effects of rhHAPO on theproliferartion of primary bone marrow stromal cells, the formulation offibroblast-colony-forming unit, and the regulation of cell adhesionmolecules on the stromal cell line HESS-5, have been explored by by Typanblue exclusion, MTT assay, colony assay and flow cytometry analysisrespectively. The expression of cytokine gene of HESS-5 cells has beendetermined by reverse transcription polymerase chain reaction (RT-PCR).The rhHAPO stimulated the proliferation of stromal cells by increasingtheir proportion of S-phase cells, and enhanced the total adhesive abilityby up-regulating the expression of adhesion molecules (VCAM and ICAM) onHESS-5 cells. However, HESS-5 cells expressed M-CSF and c-mpl genes, butnot HAPO or G-CSF gene. Section 3.and 4. In these two sections, the main subjects areconstructing the eukaryotic expression vector containing humanhemangiopoietin gene and expressing it in mouse bone marrow stromal cellline HESS-5, then supporting hematopoiesis in vitro with gene modifiedHESS-5 (HAPO-HESS-5). The PCR products of HAPO were digested with BamHⅠand BgI Ⅱ. Then the HAPO gene segment obtained was cloned intopIRES2-EGFP to construct recombinant eukaryotic expression vectorHAPO-pIRES2-EGFP. The recombinant vector was identified by enzymedigestion analysis, PCR, and sequencing. HESS-5 cells were transfectedby recombinant vector and positive clones were selected with G418. Theexpression of HAPO gene in the transformed cells was detected by studyingEGFP expression, RT-PCR, and Western blotting analysis. Support of humanhematopoiesis by HAPO-HESS-5 cells was evaluated in co-culture systemswith human CD34+ cells. Enzyme digestion analysis and sequencing showedthat target gene had been cloned into recombinant vector. The expressionof HAPO gene in the transformed stromal cells had been demonstrated by
fluoro-microscopy and RT-PCR analysis. HAPO protein was also detected inthe supernatant of HAPO-HESS-5 by Western blotting analysis. As expected,stably transfected HAPO-HESS-5 cells significantly increased in bothrelative and absolute numbers of CD34+ cells after 7 days of culture. ThePKH26 study demonstrated that cell division and self-regeneration werepromoted faster in CD34+ cells cocultured with HAPO-HESS-5 cells than incells cocultured with vector-HESS-5 cells. The HAPO-HESS-5 cells alsosupported human hematopoiesis in vitro more efficiently than controlvector-HESS-5 cells. Conclusions and Perspectives: 1, The methodology for the culture and induced differentiation ofmouse mesenchymal stem cells, as well as a comprehensive understandingof their biology, will make it possible to the development of cellularand genetic therapy protocols in murine models, and provide newperspectives in the field of adult stem cells research. 2, The data suggest for the first time that rhHAPO is a novel growthfactor stimulating the proliferation and adhesion of stromal cells, andthat it may have a clinical potential in the reconstitution of bone marrowmicroenvironment as well as the homing of stem cells after hematopoieticstem cell transplantation. 3, The recombinant eukaryotic expression vector has been constructedand expressed successfully in the transformed cells. The HAPO-HESS-5cells support rapid generation of primitive progenitor cells and maintainreconstituting ability of hematopoietic stem cells in vitro. Therefore,it is possible to use the stromal cells expression HAPO gene as subsidiarycells in the hematopoietic stem cell transplantation.
引文
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