The Influence Of Growth Factors Withdrawal Upon The Proliferation And Expracellular Matrix Gene Expression Of HUCMSCs

Objective:Human umbilical cord mesenchymal stem cells (hUCMSCs) have multi-cell differenttiation potential and high self-renewal capacity. The implantation of hUCMSCs is expected to become a widely used treatment. In order to obtain large quantities of hUCMSCs, researchers often add growth factors in the culture medium. But when hUCMSCs enter the human body, without added growth factors, will their proliferation, migration and colonization ability be affected? As extracellular matrix is closely related to these ability, this study examined the expression of extracellular matrix.The purpose of this study was to explore the effects of removing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) on the proliferation and extracellular matrix expression of hUCMSCs and to observe the effects of half amount of the growth factors on hUCMSCs. Method:1. Isolation culture and identification of hUCMSCs Human umbilical cords were processed within 6 hours with explantation technique. The medium was changed every 3-4 days. When the cells reached 80% confluency, they were passaged at a density of 5 X 104/cm2. The phenotype of the 5th generation cells were identified with flow cytometry.2. Experimental groups When hUCMSCs of the 5th generation reached 70% confluency, the growth factors were removed and the cells were divided into three groups. The group without removing growth factors was named as the control group. The half dose withdrawal group was named as experimental group 1. The growth factors completely removed group(The medium does not contain any added growth factors, with only normal levels of serum) was named as experimental group 2.3. Observe the cells with Wright-Giemsa staining The 5th generation cells were passaged to 24-well plate. When the cells reached 70% confluency, the medium was changed with different concentration of growth factors and the cells were cultured for 30 hours. The cells were observed under microscope with Wright-Giemsa staining.4. Detect cell proliferation and growth by MTT assay The 5th generation cells were cultured in 96-well plate at 5×103 cells/well(200μL) with different medium for 30 hours. The cell proliferation was detected by MTT assay.5. The expression of apoptosis-related and extracellular matrix genes was detected with Real-time quantitative PCR.5.1 RNA extraction The 5th generation cells were passaged to 6-well plate. When the cells reached 70% confluency, the medium was changed with different concentration of growth factors and the cells were cultured for 15 hours.1 ml trizol was added each well and the total RNA was extracted following the instruction.5.2 The concentration of the extracted RNA was detected with UV spectrophotometer and the cDNA was prepared by reverse transcription according to the instruction.5.3 The gene expression of Collagen-Ⅰ(Col-Ⅰ), Collagen-Ⅳ(Col-Ⅳ), Fibronectin (FN), Laminin (LN), Integrin, Tenascin-C, Matrix Metalloproteinase-2 (MMP-2), Matrix Metalloproteinase-7 (MMP-7), Matrix Metalloproteinase-9 (MMP-9), BCL-2 and BAX was detected with Real-time quantitative PCR.Results:1. Identification of the phenotypeCD44, CD105, CD71 and CD90 were positive while CD34 and CD45 were negative, which coincided with the characteristics of hUCMSCs.2. Morphological observation2.1 In control group the cells were in flat or long spindle shape, showing processes. The nucleus were large, flat oval. The chromatin was thin with light color and the nucleoli were clear. The cytoplasm was abundant and weakly basophilic, containing basic particles.2.2 In experimental group 1, some cells became larger and rounder than in control group.2.3 In experimental group 2, the majority of cells became larger and rounder, and the nuclear-cytoplasmic ratio became smaller compared with the cells in experimental group 1.3. Cell proliferation detected by MTT assayCompared with the control group, the number of cells in experimental group 2 reduced but not significantly (P=0.16). The number of cells in experimental group 1 slightly rose, still not significantly (P=0.49).4. Real-time quantitative PCR results4.1 Expression of apoptosis-related gene BCL-2 and BAX After the removal of growth factors, the expression of BCL-2 increased, the expression of BAX decreased, and the BCL-2/BAX ratio increased. The BCL-2/BAX ratio was larger in experimental group 1 than in experimental group 2.4.2 Gene expression of extracellular matrix The expression of Col-1, FN, LN, Integrin and Tenascin-C increased in experimental group 1, while in experimental group 2 the expression of LN, Integrin, Tenascin-C decreased. The expression of ColIV, MMP-2, MMP-7, MMP-9 decreased after the removal of growth factors and it decreased much more when the growth factors were removed completely.Conclusion:1. Removing the growth factors in the medium inhibits the proliferation of hUCMSCs, and half dose of the growth factors could maintain the proliferation.2. Removal of EGF, VEGF and bFGF leads to larger cell size and smaller nuclear-cytoplasmic ratio.3. After the removal of EGF, VEGF and bFGF, hUCMSCs upregulate the expression of Bcl-2 and reduce the expression of Bax to enhance the anti-apoptosis ability. The anti-apoptosis ability is stronger when only half amount of the growth factors are removed than they are removed completely.4. Removal of growth factors could affect the expression of extracellular matrix gene. Half dose of the growth factors could maintain them.