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Object:To evaluate the influence of insulin at different concentrations on the time-specific expression of cardiac specific gene in the early differentiation period of the suspension cultured embryoid body (EB).Method:EBs had been cultivated in suspention in vitro for11days to simulate the early stage of embryo development. Different concentrations of insulin (60pM,6nM respectively) was added to the cultivation medium. EBs were collected at day1,3,5,7,9,11respectively. And the expression levels of Gata4, Mef2c, Tbxl, Lrrc10were detected by Q-PCR.Results:1. The morphology of EB changed gradually during the period of observation. The capsule cavity of EB appeared at day5in60pM group and became more obvious at day7;however, this change delayed in6nM group.2. Gene Gata4is the early marker gene which suggests the start of heart development. Gata4expressed in both insulin groups. The first peak-expression was at day3in60pM group, which was at day5in6nM group, and then the trend was descended. However, the expression of gene Gata4reached the second peak at day7and day9(in60pM and6nM group, respectively).3. Mef2c is the cardiac specific gene, which expresses in both atrium and ventricle. The expression quantity of Mef2c ascended with time in both high and low concentration of insulin and its peak-expression was at day7. But comparing with60pM group, gene expression quantity was lower at day3,5,79in6nM group.4. Tbxl is the marker gene of second heart field. The Tbx1expression trend in EBS was ascend, the expression peak was at day7in60pM group. However, the expression of Tbx1showed no obvious peak in6nM group, and the expression quantity at day1,3,7,9was lower than that in group60pM.5. Lrrc10is the heart cyclization specific gene. The expression of Lrrc10ascended with time in both groups. On the9th day the peak-expression reached and then the trend descended. But in6nM group, the expression quantity is lower than that in60pM group. Conclusion:1. The expression pattern of gene expression, such as Gata4、 Mef2c、Tbx1、Lrrc10that related to heart development, is different in EBs cultured with varies concentration of insulin. Comparing with6nM insulin,60pM insulin is better for the expression of these four genes.2. Under the same insulin level, the expression patterns of the four genes (Gata4, Mef2c, Tbxl and Lrrc10) varies with different culture time. The peak-expression was at day3for Gata4, at day7for Mef2c and Tbx1, and at day9for Lrrc10. And for gene Gata4, there is a second peak-expression at day7. These patterns suggest that gene expression is time-specific during the development period.
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