| Objective:To compare the effect of different way adding human leukemia inhibitory factor (hLIF) on self-renewal and homing ability of cord blood hematopoietic stem / progenitor cells in vitro. Study the activation of hLIF on JAK / STAT signaling pathway and its molecular mechanisms in the maintenance of hematopoietic stem cell proliferation and self-renewal process.Methods:1. The recombinant adenovirus vector of hLIF or GFP gene was infected the human embryo lung fibroblasts cells WI-38(One group is infecting the WI-38cells directly, another is infecting the WI-38 cells which were microbiomationed on silk fibroin), and the objective gene was detected by RT-PCR and ELISA .2. The WI-38cells which were infected with Ad-hLIF and Ad-GFP were used as feeder layer cells to culture the cord blood hematopoietic stem / progenitor cells in vitro(or adding hLIF directly), and also take the cytokine group and other groups as contrast. Observe the split apart and homing ability about the stem cells which were cultured in each group for 7 days through FCM (flow cytometry) and Transwell. Then culturing the stem cells in each experiment group for 30 days to compare its ability in amplification.3. Adding the specificity blocking agent AG490 of the JAK/STAT signaling pathway to the co-culturing system of gene transfected feeder layer cell group and cytokine group, and also take the groups without AG490 as contrast. Observe the split apart and homing ability about the cord blood stem cells which were cultured in experiment and contrast groups for 7 days through FCM (flow cytometry) and Transwell.4. Observe the transcription and espression of STAT3 by RT-PCR and ELISA.5. Observe the expression change of p-STAT3 and p-JAK1/JAK2 in every co-culture system stem cells by Western blot. Then culturing the stem cells in each experiment and contrast group for 28 days to count cell numbers and also draw growth curve. Detecting the CD34+ ratio of stem cells by FCM at different time point to compare its ability in self-renewal before and after interruption.Results:1. The hLIF could be detected in WI-38 cells which were infected by Ad-hLIF through RT-PCR and ELISA.2. The way putting cord blood hematopoietic stem cells directly on feeder layer cells or suspensioning feeder layer cells in co-culture system both have important effect in amplification and self-renewal of HSCs in vitro, and there was no significant deviation between the two. The way adding buying hLIF factor directly was no better than the two ways before.3. The expression of adhesion molecule and homing ability of HSCs are both down after adding the specificity blocking agent of JAK/STAT signaling pathway. There was no significant change of the transcription and espression of STAT3 before and after interruption through the method of RT-PCR and immunocytochemistry.4. The activation in stem cells of STAT3 and JAK1/JAK2 was suppressed after adding AG490 detecting by Western blot, also the velocity of stem cell increase was obviously slower than before. The CD34+ ratio of stem cells culturing in systems adding AG490 was lower than without it detecting at the same time point. The discrepancy between the two have statistical significance.Conclution:1. The way putting stem cells directly on feeder layer cells or suspensioning feeder layer cells in co-culture system both have special effect in amplification and self-renewal of HSCs in vitro, and there was no significant deviation between the two. 2. The way feeder layer supplying hLIF was obviously better than the way adding buying hLIF factor directly.3. The hLIF factor that feeder layer supplying could active JAK/STAT signaling pathway successfully and affected the self-renewal and functions related to homing ability of HSCs through this signaling pathway. |
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