Influence Of Dendritic Cell In The Pathogenesis Of Chronic Viral Hepatitis

PARTIIn vitro Culture of Dendritic Cell Induced from Peripheral Blood MonocyteDendritic cell (DC) is important for the elimination of pathogens such asvirus, bacteria or parasites. And DC is also prospective in the gene therapy of tumor or immune diseases. In vitro proliferation is the way to get enough cells for the rare amount of DC in body. Monocyte in peripheral blood is the precursor cell of DC and it can be differentiated into DC by the stimulation of some pathogens or inflammatory factors such as lipopolysaccharide. DC induced from monocyte is homogenous in phenotype and function for gene therapy, and it is possible to12separate monocyte from peripheral blood repeatedly. We aimed to establish a simple method to induce lots of DCs from monocyte.1. Materials and methodsWe used the basic method to induce DC from monocyte by stimulation of GM-CSF and IL-4, and simplified the method. The steps are as follows: ?Separate the mononuclear cells from white blood cell (WBC) obtained by blood separator. And purify the monocyte by the patching method, ヽulture the monocyte by RPMI 1640 with 10% FBS, hGM-CSF (30ng/mL) andrhIL-4 (20ng/mL), in the condition of 37"C 5%CO2. Take away half of the culture medium gently and add the same volume of medium with the same concentration of cytokine as before. Gather the suspension cells on the sixth day (DC6). nalyze the culture duration by differ phase microscope and analyze the DC6 by transmission electronic microscope, calculate the harvest rate of DC6, analyze the phenotypes of DC6 by flow cytometry; ㏒timulate DC6 by TNF a (30ng/mL) for 48 hours, and analyze the phenotype of cells, un-stimulated cell is used for negative control.2. Results(1) Morphologic analysis Observing the growth duration of cells by differ phase microscope, and it was found that: ﹕everal hours after the addition of 30ng/mL rhGM-CSF and 20ng/mL rhIL-4 into the monocytes, cellular clusters could be found; ﹐n the 3"1 day, lots of proliferation cluster loosely adhere to the bottom of culture plate and suspension irregular cell began to occur; n the 6 day, there were lots of suspension cells with irregular shape and abundant cytoplasm projections, and the cells were dendritic-like.13Collect DC6 and observed the morphologic characteristics by transmission electronic microscope. The cell is abundant of cytoplasm projections with irregular nucleus. There are incisions on the nuclear membrane, lots of membrane apparatus (such as endoplasm, Golgi's complex) in the cytoplasm, Birbeck-like granules and rare mitochondria.(2 ) Phenotype analysis of DC6 DC6 were stained with the monoclinic antibodies and analyzed by flow cytometry. More than 10, 000 cells were analyzed per time. According to FS/SS gating, the cells were crowded together very well (Figure 1-3A), it is suggested the good homogeneity of DC6.The phenotype of DC6 was CDla+, CD80+, CD86+, CD83-, CD14+ (the MnX was lower than that of monocyte) , CD56-, CD3-, CD 19-. The results were similar for culture 3 times. CD la is the specific cellular sign of DC derived from peripheral blood monocyte, the percentage of CDla+ cell were 98.5%, 98.8% and 99.8% separately. So it is suggested the high purity of DC6.(3) Harvest rate of DC6 according to the harvest rate formula DC counts /monocyte counts x 100%, the harvest rate were 66.2% 57.3% and 62.2% separately for 3 times culture, and the average rate was 61.9%.It was found that the appropriate concentration of mononucleocyte is l~2xl07/mL. The higher concentration means more loss of monocyte and decrease of the harvest rate, the lower concentration means the less cluster and inhibition of DC proliferation. The suspension cells will be gently washed away to avoid the loss of monocyte, but the lymphocyte should be washed completely to increase the purity of DC6.(4 ) TNF a influencing the phenotype of DC6 DC