Effects Of Renal Ischemia-reperfusion Time On Proliferation And Differentiation Of Bone Marrow Mesenchymal Stem Cells In Rats

OBJECTIVE: To simulate the microenvironment of acute kidney injury in vitro by inducing renal homogenate supernatant at different time points of ischemia-reperfusion in rats,to induce proliferation and transdifferentiation of bone marrow mesenchymal stem cells,and to explore renal ischemia.After reperfusion,it is most suitable for the proliferation and differentiation of bone marrow mesenchymal stem cells.METHODS: Rat BMSCs were isolated and cultured in vitro by adherent culture method and divided into negative control group(stem cell culture medium);normal kidney homogenate group(stem cell culture medium +10% rat normal kidney homogenate supernatant);ischemia-reperfusion12 h group(stem cell culture medium + 10% rat ischemia-reperfusion 12 h kidney homogenate supernatant);ischemia-reperfusion 24 h group(stem cell culture medium + 10% rat ischemia-reperfusion 24 h kidney homogenate Supernatant);ischemia-reperfusion 48 h group(stem cell culture medium + 10% rat ischemia-reperfusion 48 h kidney homogenate supernatant);ischemia-reperfusion 72 h group(stem cell culture medium+ 10% rat ischemia-reperfusion 72 h kidney homogenate supernatant);ischemia-reperfusion 96 h group(stem cell culture medium + 10% rat ischemia-reperfusion 96 h kidney homogenate supernatant).The effects of renal homogenate on the proliferation of BMSCs were observed byCCK8 method and cell plate counting method.Inverted microscopes were used on the 1st,3d,5d,7d,9d,11 d,13d,15 d,17d,19 d,21d,23 d,25d,and 27 d.The morphological changes of the cells were observed.The expression of CK-18 was evaluated by Western Blot and reverse transcriptase polymerase chain reaction(RT-PCR),and the effects of each group on the differentiation of BMSCs were observed.RESULTS: 1.BMSCs cultured to the third passage showed densely arranged,vortex-like,relatively uniform morphology of long spindle-shaped cell populations;flow cytometry identified cell phenotype CD29,CD90 expression positive,CD34,CD45 expression is negative;BMSCs can differentiate into adipocytes after adipogenic induction.2.Cell proliferation was detected by cell plate counting method and CCK8 method.Statistical analysis was performed in each group.The supernatant of kidney homogenate was the best for BMSCs proliferation after 72 h ischemia-reperfusion.3.Morphological changes of cells were observed under microscope.Normal kidney homogenate supernatant,ischemia-reperfusion 12 h,24h,48 h,96h kidney homogenate supernatant was used to intervene BMSCs,and kidney homogenate supernatant was treated with ischemia-reperfusion for 72 h.Compared with BMSCs,the number of cells differentiated at 72 h after ischemia-reperfusion was the highest;the morphology of BMSCs in negative control group was longspindle-shaped,and a few cell morphology changed.4.Western blotting and quantitative reverse transcriptase polymerase chain reaction(RT-PCR)were used to evaluate the expression of keratin18(CK-18)in cells after induction.The expression of CK-18 was the most obvious in the supernatant of kidney homogenate after ischemia and reperfusion for 72 h.The expression of CK-18 in the control group may be affected by the external environment.Conclusion: 1.Ischemia-reperfusion injury rat kidney homogenate can induce the differentiation of bone marrow mesenchymal stem cells into renal tubular epithelial cells in vitro.2.Different time of renal ischemia-reperfusion,there are different damage microenvironments.3.There may be a time point in the process of acute injury repair that is conducive to stem cell repair.Stem cell therapy at 72 h after ischemia-reperfusion may help to maximize stem cell function.