Comparative Studies on the In Vitro: RESULTS(4)
IGFBP-1 mRNA expression in cells was demonstrated by RT-PCR (Fig. 4). In human cells, bands corresponding to the IGFBP-1 PCR product were evident in hormone-treated cells and dbcAMP-treated cells, and, most prominently, in cells treated with hormones plus dbcAMP (Fig. 4B). Baboon stromal cells also expressed IGFBP-1 mRNA in response to either dbcAMP or hormones only (Fig. 4A). However, much higher levels of IGFBP-1 mRNA were produced in response to dbcAMP and hormones. Given that RT-PCR is a very sensitive technique, it was not surprising to detect mRNA in response to either dbcAMP or hormones alone, since IGFBP-1 protein was also detected by RIA with these treatments. buy ampicillin
Our in vivo studies showed that cells expressing aSMA did not express IGFBP-1, whereas cells that produced high levels of IGFBP-1 did not express aSMA (Fig. 1; ). Figure 4A shows that baboon cells expressing high levels of IGFBP-1 mRNA after long-term hormone and dbcAMP treatment (+/+ Day 12 and 17) do not express aSMA. Human stromal cells that produced high levels of IGFBP-1 also showed a decrease in aSMA expression (Fig. 4B).
FIG. 4. Time course of IGFBP-1 mRNA expression using RT-PCR. Confluent stromal cells from baboon (A) and human (B) endometrium were treated as described for Figure 3. At each time point, cells were lysed with TriReagent. Total RNA was isolated and subjected to RT-PCR using primers for aSMA, IGFBP-1, and the internal standard H3.3. Complementary DNA products were run on 1.5% agarose gels. The data shown are representative autoradiograms of 3 baboon and 3 human tissues. Linear curves of densitometric values versus number of cycles for H3.3, aSMA, and IGFBP-1 are presented in C. Primers for H3.3, aSMA, and IGFBP-1 were added together in the same tube, and the sample was amplified for 20, 22, 24, 26, 28, and 30 cycles.