| AIDS ( Acquired Immunodeficiency Syndrome ) , is a disease caused by HIV ( Human Immunodeficiency Virus ) . - It transfers by blood ^sexual contact and vertical method. Since the I8t AIDS case was found in 1985, there is 600,000 HIV - infected patient in china now in Sep. 2001. The effect of cytokines and the mainly type of immune reaction to the process of HIV infection is not defined right now. However, most of researchers think type I cytokine and cellular immune reaction is the main factor to interfere the development of HIV infection. As a result, regulating the cytokine balance, create a cellular immune reaction environment is an important part of control the infection of HIV.Lentinan can regulate the immune function of tumor patient. Its regulation effect has something to do with changing some cytokine level in serum or cytokine receptor. By co - culture the normal and HIV infected peripheral blood mononuclear cell (PBMC) with lentinan, we measure the IL -2,IL -12 ,IFN -r,IL -4 ,IL - 10,TNF - a level in the supernatant, and research the effect of lentinan to cytokine secreting. At last, to explore a new way to cure HIV/AIDS is our final object.Materials and Method1. Materials; PBMC is from eight healthy donor, extracted by Fi-coll - hypaque (Tianjin, China) density gradient centrifugation. HIV-1 SF33 has a p24 quantity of 500ng/ml. Lentinan is from medical factory in Nanjing, and is diluted to defined concentration. IL-2,IL - 12,IFN -r JL -4 ,IL -10,TNF - AELISA kit is from Jingmei Co. , Shenzhen.2. The concentration of lentinan in vitro; Calculate the peak concentration of lentinan in human plasma : 0. 4ug/ml. Dilute the concentration by 1,2,4,8,16 times , measure the toxicity to PBMC by MTT method. Co - culture the diluted lentinan and PBMC for 60 hours in 37: 5%CO2 incubator. Add MlT(5mg/ml,40ul) , then centrifuge at 400g 6 hours later. Discard the supernatant and add DMSO 200ul in each well. Read the OD value on 570nm in 10 minute until the purple crystal dissolved. Calculate the ratio of lentinan group to normal control group. If the ratio is between 0. 9 and 1.0, it is defined that the lentinan is asepsis to human PBMC. And, the most concentrated non ?toxic concentration and its 0. 1 times concentration is chosen as our experiment concentration.3. Measure the cytokine in supernatant by ELISA: PBMC is extracted from 8 healthy donor in 24 hours. The surviving ratio is more than 95% by trypanblue staining. One part is for normal control, the other is infected by HIV -1 SF33, for HIV - 1 infected PBMC, and both are in a cell concentration 2 * 106. Add PHA (concentration 5ug/ml) , then add the blank control group, the lower lentinan group and the higher lentinan group. Normal PBMC and HIV infected PBMCis added into each group. Culture the mixture in 37TI 5% C02 incubator. After 60 hours , the supernatants are harvested . Measure the cytokine IL -2 ,IL -12 ,IFN - r JL -4 ,IL - 10,TNF - a level in the supernatant by ELISA kit.Results1. The concentration of lentinan in vitro: In MTT experiment , 4ug/ml lentinan concentration is asepsis to human PBMC. The 1,2, 4, 8, 16 times dilutions also are non ?toxic. So 0. 4ug/ml and 0. 04ug/ml are chosen as the concentration of lentinan experiment.2. The cytokine shift of PBMC secretion before and after HIV -1 infection; When infected by HIV - 1, healthy donor's PBMC secrete more IL - 4 JL -10,TNF - a, and less IL - 2 JL - 12 JFN - r.3. The Effect of lentinan on healthy PBMC: Lentinan promote the IL -2 JL -12 JFN - r secretion of healthy PBMC, and inhibit the IL-4,IL -10,TNF - asecretion. And the result is concentration dependent.4. The Effect of lentinan on HIV - 1 infected PBMC: Lentinan promote the IL -2 JL -12,IFN - r secretion of HIV - 1 infected PBMC , and inhibit the IL -4 ,IL - 10,TNF - asecretion. And the result is concentration dependent.DiscussionCytokine is a bio - active substance secreted by immune cell (T cell , B cell, et al. )and non -immune cell(lymphoblast cell, endo-thelial cell,...
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