Isolated Culture And Identify Of ImDCs And DCs Derived From Rat Bone Marrow In Vitro

Objective:The dendritic cell (DC) plays a key role in the process of transplantrejection.This experiment is designed to establish a simple and efficient process in vitroculture and purification way as the source of rat bone marrow precursor cells. And on thecell morphology, to identify the cell phenotype and function of imDC and mDC. Andidentification of the cell morphology, the cell phenotype and cell function of imDC andmDC, and provide experimental basis for the effect of DC on transplantation immunetolerance..Methods:There were imDC group (GM-CSF+IL-4) and mDC group(GM-CSF+IL-4+LPS)1.Got imDCs standby through separant induction of rat bone marrow hematopoieticprogenitor cell by combined use of GM-CSF+IL-4for7days. Use microscope identify cellmorphological.2.Use imDCs which had been trained for7days and purified by immunomagneticbeads, then use flow cytometry instrument test the positive expression rate of OX62which respectively expressed on the surface of imDCs before immunomagnetic beadspurification and imDCs after immunomagnetic beads purification.3.Use those imDCs after immunomagnetic beads purification,and add LPS which finalconcentration is1ug/ml to the original induction medium,then got mDC, Usemicroscope identify cell morphological.4.Collect imDCs after immunomagnetic beads purification and mDCs, use flowcytometry instrument test the positive expression rate of CD86、CD80、RT1B which respectively expressed on the surface of those former two sets cells and the secretion levelof IL-12were tested by ELISA method. measured both abilities of promoting lymphocyteproliferation by mixed lymphocyte reaction(MLR).Results: Identify these cells trained via the method which combined with cellmorphological observation、surface markers and functional status, and confirm these cellswere DCs. Through the test by microscope, we can observe the imDCs and mDCs wereshown as the typical immature and mature morphology, and there were amount of cellcolony can be observed through microscope in those imDCs which cultured in vitro to7thday and increased significantly protuberant burr can be observed through microscope inthose mDCs after stimulated by LPS for2day. Most of those cells were observed growingsuspended, and those cells distributed more equation than imDCs,. The positive expressionrate of OX62is82.8%and91.9%correspond to those cells before immunomagnetic beadspurification and those cells after immunomagnetic beads purification, and the purity ofthose cells after purification is higher than the other(P<0.05).The positive expression rate ofCD86、CD80、RT1B expressed on the surface of imDCs correspond to11.48%、6.25%、77.2%.The positive expression rate of CD86、CD80、RT1B expressed on the surface ofmDCs which stimulated by LPS correspond to91.38%、93.54%、98.6%（P<0.01）.UseELISA method test the secretion level of IL-12of those former two group cells, the level ofimDCs is36±9pg/ml，the mDCs is228±27pg/ml(P<0.01),we can observe that mDCs has amuch higher energy of stimulate T lymphocyte cell multiplication than that of imDCsthrough MLR method(P<0.01).Conclusion:1:Our experiment prove that high-purity and large amount of DC can beinduced by the united application of GM-CSF+IL-4in vitro.2.A much more relatively number、high-purity DC can be obtained byimmunomagnetic beads purification, and those cells can meet the experiment needs...