Gene Expression Profiles, Proliferation And Extracellular Matrix Production Of HUCMSCs In Response To TGF-β1

Objective:1. To examine the effect of 2ng/mL EGF,bFGF,HGF,PDGF and TGF-β1 on proliferation and migration of hUCMSCs.2. To investigate gene expression profiles, proliferation and ECM production of hUCMSCs in response to TGF-β1Methods:1. The hUCMSCs were isolated by explanation technique from umbilical cords and cultured under appropriate conditions. Surface antigens of the fifth passage cultured cells were detected by flow cytometry. The fifth to seventh passage cells were assessed for their potency by inducing their differentiation into osteoblasts and adipocytes under appropriate conditions for each lineage.2. hUCMSCs of P5 were studied with Cell Counting Kit -WST-8 for cellular growth and survival to investigate the effects of different factors on proliferatin of hUCMSCs.And the influences on hUCMSCs'migration ability were assessed by in vitro wound healing model and transwell inserts technique.3. hUCMSCs of P5 were cultured in medium with 0.1ng/ml TGF-β1 for 24h and then collected to prepare total RNA with Trizol reagent, the gene expression profiles were examined by cDNA microarray.4. The effects of TGF-β1 with different concentrations (0.1,0.2,0.5,1.0,2.0,5.0,10.0 and 20.0ng/ml) on cell proliferation was determined by Cell Counting Kit-8 at 24h,48h and 72h afrer incubation. The mRNA expression of ECM components (CollagenⅠ,CollagenⅣ,Fibronectin,Laminin,Integrin and Tenascin-C) and components(MMP-1,MMP-2,MMP-9,TIMP-1 and TIMP-2) may involved in the mechanism of ECM production regulation were detected by qRT-PCR. And CollagenⅠ,CollagenⅣ,Fibronectin and Laminin expression were observed by immunocytochemi stry.Results:1.1 Some fibroblast-like cells with prominent nucleolus and pseudopods were observed as early as one week after transferring fragments into plate, and they reached 70%-80% confluence with uniform fusiform appearance within 15 days. After 2-3 times'passaging the cells formed a monolayer of homogeneous spindle-like cells with a whirlpool like array.1.2 Characterization by flow cytometry revealed that the isolated cells were negative for CD34 and CD45 and positive for CD44,CD71,CD90 and CD105。1.3 The osteogenic potential of the cells were evidenced by alkaline phosphatase (ALP) staining and Alizarin red staining and the adipogenic potential were identified at the presence of lipid-rich vacuoles stained with oil-red O.2.1 All the experimental groups treated with different growth factors showed increases in proliferation in compare with the control group. According to the OD values, total number of MSCs in the HGF group was the largest.2.2 In wound healing model, the migration distances of all the experimental groups were increased compared to the control group, in which the migration distance of the HGF group was the longest.2.3 In transwell assay, the number of hUCMSCs through the martrigel-coated transwell for all the experimental groups were more than the control group, in which the cell number of the FGF group was the most.3. Among the 296 differentially expressed genes (2 times and 1.5times difference), up-regulated and down-regulated genes were 153 and 143, respectively. There were 9 up-regulated genes closely related to cell movement and migration. Analysis by MAS system indicated differentially expressed genes involved transcription,translation biosynthesis,metabolism,signal transduction,migration and Adherens junction ect. 4.1 In the proliferation assay, the OD values of the experimental groups changed as "dent wave" with the increasing dose of TGF-β1.Compared with the control group, the number of cells in experimental group didn't change significantly. Overall the OD value of 0.1ng/ml group was relatively at a peak at the three time point.4.2 The mRNA of Col-Ⅰ,MMP-2,MMP-9 and TIMP-2 expression increased to maximum in 0.5ng/ml group and Co1-Ⅳ,FN,LN,Integri,Tenascin-C,MMP-1 and TIMP-1 reached peak expression in 0.1ng/ml group.4.3 Immunocytochemical analysis for Col-Ⅰand Col-Ⅳrevealed slightly production in 0.5ng/ml and 0.1ng/ml group respectively while immunostaining for FN showed diffuse cell membrane and cytoplasmic staining randomly distributed in cells of both groups but LN staining was absent in both groups.Conclusions:1.EGF,bFGF,HGF,PDGF and TGF-β1 all stimulated the proliferative ability and the migration ability of hUCMSCs at the concentration of 2ng/ml.2. TGF-β1 played important roles in various aspects of hUCMSCs including transcription translation,biosynthesis,metabolism signal transduction,migration and adherens junction ect. TGF-β1 promoted migration ability of hUCMSCs by up-regulating the gene expression of actin,tubulin and RhoA.4. TGF-β1 with concentration of 0.1ng/ml promoted proliferation of hUCMSCs at 24,48 and 72h. TGF-β1 upregulated the expression of different ECM components with different concentrations.