Study Of The Effect Of Osteopontin On Proliferation And Migration Of Bone Mesenchymal Stem Cell

Objective:This experiment will test the change of the ability of proliferation and migration ofthe bone mesenchymal stem cells, according to the expression of osteopontin inducedby endogenous and exogenous, witch are the result of the infecting cells with lentiviousand osteopontin protein.Methods:1.Isolate and culture bone mesenchymal stem cells from OPN knock-out andwild-type C57BL/6new-born mice, aging of one day. Authenticate with flowcytometry,Western Blot and PCR.2.Get OPN gene from wild C57BL/6adult mice, and build a OPN plasmid with it.Then package a lentivirus containing OPN gene with calcium chloride-chloroquinemethod.3.Infect the bone mesenchymal stem cells with lentivirus. Authenticate theexpression of OPN in cells with Realtime PCR and Western Blot.4.Test the ability of proliferation of cells,which are disposed with OPN protein andlentivious, with CCK8.5.As the group upon, test the capability of migration of bone mesenchymal stemcells in each group with transwell boyden chamber.Results:1.As we can see from the microscope that the cells getting from the bone marrowof new-born mice are in a vigorous growth, arranging as a vortex. The flow cytometrytest shows that the cells have a high purity. On the other hand, both PCR and WB testsshow that the cells isolate from OPN knock-out mice do not express OPN in both geneand protein level, while the wild-type mice can express as normal.2.The length of OPN gene getting from the kidney of wild adult mice is900bp. Weinsert the OPN gene into a lentivirus carrier. And then package lentivious, including the OPN type and empty one, with calcium chloride-chloroquine method. According to thetest, we get a titer of2.5×108TU/ml.3.According to the Realtime PCR and western blot tests, the expression of OPN isa change of gradient in the mesenchymal stem cells infected with lentivious.4.As we can see from the fluorescence microscope that the cells infected withlentivious can express the fluorescent light. And both of the Realtime PCR and WB testsshow that the expression of OPN is like a gradient.5.CCK8tests show that the functions of endogenous and exogenous on the abilityof proliferation of bone mesenchymal stem cells come the the same. In one word, theyboth improve it.6.The transwell boyden chamber also show that the more expression of OPN, nomatter induced by endogenous or exogenous, the stronger of capability of migration ofbone mesenchymal stem cells.Conclusion:Osteopontin can improve the abilities of proliferation and migration of bonemesenchymal stem cells in both endogenous or exogenous ways.