The Founction And Mechinism Of The MDQ Treating The Splenomegalia Of Mice Model Caused By FLV

1. the purpose of study:1.1 Build up the rats` modle of FLV infection, using the Zidovudine as control medicine, choose the indexes as the spleen indexes, spleen weight, rat body weight, to investigate the therapeutical effect of the MDQ on the FLV infected rats modle, and make evaluation on the therapeutical effect of the MDQ.1.2 Build up the rats` modle of FLV infection, using the Zidovudine (AZT) as control medicine, choose the indexes as CD4T, CD8T, CD4T/CD8T, to investigate the influencial effect of the MDQ on the FLV infected rats modle, and make evaluation on the therapeutical effect of the MDQ on the FLV infected modle and made and make a rudment discussion on the mechenisam of the MDQ effect.1.3 Build up the rats` modle of FLV infection, using the Zidovudine as control medicine, choose the indexes as serum IL-1β , IL-2, IL-6 and INF- γ, to investigate the influencial effect of the MDQ on the FLV infected rats modle, and make evaluation on the therapeutical effect of the MDQ on the FLV infected modle and make a rudment discussion on the mechenisam of the MDQ effect, prepare for the further study and development of that medicine.1.4 Use optimal FLV-liposome to transfect cell PA317(NIH/Swiss, embryo, retorviruses packaging cell). After 5 transfers of culture, get PA317-FLV which express FLV stabilizingly. Use the RT-PCR method to identify this new cell line and use the NIH3T3 cell as the target cell, to estimate the anti-retrovirusfunction of MDQ. The indexes chosen as following: the number of the focues which was counted by the focus-forming experiment, the virus tilter;using the CLSM(confocal laser scanning microscope) to investigate the changes of Ca²⁺ passage before and after the MDQ is administrated.2. method2.1 The constuction of the animal model and the test of the indexes: After the rats were adaptably feed for 1 day, O^O^mlxlO1 FLV was intraperitoneal injected to them. Then randomly divided these rats into 6 groups : MDQ big dose group(MB), MDQ middle dose group (MM), MDQ low dose group (ML), AZT group and blank control group, there are 10 rats in each group. Rats were feed conventionally.Drug administration began at the same day as the virus attacking. Blank control group was administrated pure water.The dosage of MDQ was given to the rats is equal to 5, 10, 20 times of the dosage of an 60kg-adult by the body weight, the dosages are as following: 5 times,2.166g/kg;10 times ,4.33g/kg and 20 times,8.67g/kg;AZT group was given AZT dissolved in pure water. The dosage is lOOmg/kg. Drug administrated everyday and for 21 days.Each rat was administrated 0.4ml drug or water once per day. After 21 days of drug administration, all the rats were sacrificeed by picking off the eyeball. Collect the blood and the organs needed in the following experiment.2.2 The test of the spleen index: rats were weighted before they were sacrificed. Open the abdomen by an eye scissors along the abdomenal middle line. Spleen lies at the left side in the abdomenal cavity, blunt dissect off the spleen, cut off the adipose and connective tissue, weigh the spleen wet weight of the rats. Calculate the spleenSpleen weightindexes by the following equation sP|een lndex = l)ody weight 100% After weighting the spleen was cut into 5mm size pieces and put it into 10% formaldehyde solution,routine anhydration,paraffin imbedding,HE dyeing,slicing and observing.2.2CD4T,CD8T,CD4T/CD8T examination:The method of the building up of the animal model is the same as in 2.1. After the sacrificing of the rats, the blood was collected by the EDTA treated tube, the blood was treated with anticoagulation agents. 50 fi\ rat blood sample mixed with CD3T, CD4T, CD8T monoclonal antibody each 2.5 /dwhich were bought from Jinmei Biotechinich company. After the samples were incubated in room teperature for 30 minutes, add haemolysin, 10 minutes later, centrifuge the samples at the speed of 1000G for 5-10 minutes, and wash the sample with PBS, centrifuge the sample for5 minutes. Test the sample on the flow cytometry.2.3 Detection of the serum IL-1B, IL-2, IL-6 and INF-y: The method of the building up of the animal model is the same as in 2.1. After the sacrificing of the rats, the blood was collected by normal tube. And the blood don't need anticoagulate treatment. Standing the blood for 4 hours, and centrifuge the blood at 4000G, collect the serum. Examine the 4 indexes at the same time. All the preparation and procedure were following the test protocol. 2.4 FLV transfect cell PA317PA317 cells in exponential phase of growth, one day before transfection,treated by trysinization and count the number of the cells, and then plant the cells on the 24 well plate, the nutrient medium is the same in every well: 0.5ml culture medium with serum but without antibiotic, observing under the microscop, to make sure on the day of transfection the cell culture density is 90%. For transfection, each well 50 n 1 OPTI-MEM I attenuate FLVvirus storage to the 10"1, 10"2, 10³, 10"4concentration, and each concentration 6 wells. Each well use 50 w 1 OPTI-MEM I dilute 1 u 1-3 u 1 LIPOFECTAMINE 2000 agent, standing 30 minutes in room temperature then move it into the cell incubator.After 8 hours of transfection, the 10"1 group cells are dead, cells fall off the plate, 24 hours after transfection, 10² group cells fall off from the plate, after 72 hours, only 10"^ 10"4 group cells adherent on the plate. Observe under the scope, 10"3 10"4group cells are still in typical shape and adherent to the plate. So chose the 10"4 dilution as the transinfect concentration. After 2-3 times of passages, freezen the cells in thenitrogen canister, and step into the next experiment. 2. 5 plaque forming experiment:Treat in logarithmic growth NIH3T3 cells with pancreatic enzyme, and move the cells into plate, high glucosenutrient medium, 1% fetal bovine serum, 5%CO2 37°C incubator. After 48 hours, the cells are growing into a layer, use the haustorial tube to change the medium to 10ml medium without serum, standing in 37°C for lh. Suet medium, add 200 U lPA317supernatant to the NIH3T3 plate, put the plate into 5%CO2 37 °C incubator for lhour to make sure the viruses will be adherented on the cells. Add 44°C neutral rednutrient agari into the plate. Put the plate into 5%CO 2 37°C incubator. After 24 hours count the plaque. 2. 6 fluorescent quantitation PCR test the virus tilter after the MDQ treating:In logarithmic growth PA317-FLV was digested by 0.125pancreatic enzyme. Plant on 6 wells plate, the cell concentration is 1X 106,each well lml. Incubate in 5% CO 2 37 °C incubator. After 24 hours change the medium to MDQ medium, each well lml. The concentration of MDQ is 10, 20, 40mg/L, positive control is AZT 200mg/ml, then culture for 48 hours ,extract the total RNA from these cells by Tizol and give it to He Jingyang to finish the fluorescent quantitation PCR experiment.2. 7 confocal laser scanning microscope experiment:PA317-FLV was digested by 0.125 pancreatic enzyme one day before the experiment. Imbibe lml on the one well plate which is going to be used specially for the confocal laser scanning microscope experiment:.one hour before the experiment began, fix and stain the cells;put the wells away from light for 30 minutes. Observe under the confocal laser scanning microscope., and taking pictures. 10 s, add lg/1 MDQ which was resolved with the auxiliary solvent DMSO, the blank control group use the same concentration of DMSO.3. Result3.1 Body weight, spleen weight, spleen indexes and pathological section analysisWeight: make comparison between AZT and MB group , the body weight of the AZT group is almost the same as MB group but the spleen weight is lighter than that of the MB group (P<0.05).Spleen weight: the spleen weight between the AZT group and the normal control group is little difference, and MB showed the founction of curing the splenomegaly, MS group has little difference with the model group. In the index of the spleen index, the AZT group shows the best result in treating the splenomegaly, and followed by MB group.The pathological report of the spleen is as the following: the normal group shows : integrate histological structure , The white pulp is circular in structure and is made up mainly of lymphocytes. It functions in a manner similar to the nodules of thelymph node, The red pulp surrounds the white pulp and contains mainly red blood cells and macrophages. The main function of the red pulp is to phagocytize old red blood cells, the small lymphocytes centered around the splenic arteriole at the center, forming the white pulp. Around this is the red pulp comprised of many splenic sinusoids. The model group : splenic architecture is remodeled. With amyloidosis involving the spleen and resulting in splenomegaly, large hyaline masses are seen as lesions occupying the white pulp space. Two forms exist, including the "sago spleen," in which amyloid deposits are limited to follicles, and the "lardaceous spleen," in which amyloid is deposited in the walls of the splenic sinusoids. In a rare complication of typhoid fever, reactive splenic vasculitis may develop, erythroid hyperplasia of spleen cells, prominent dyserythropoiesis characterized by megaloblastoid cells, karyorrhexis and gigantoblasts with multiple nuclei;leukemic erythroblasts may contain cytoplasmic vacuoles;dysplastic platelets and megakaryocytes often present. In the AZT group , the typical imige is , in the in the spleen medullary sinus, there are a little of leukemia cells, and the white and red pulp have normal structure;for MB group,the red and white pulp are the normal structure and a little leukemia cells in the medullary sinus;for the MM group, the anatomical structure of the spleen was destroyed with a lot of leukemia cells;and for the MS group, the anatomical structure of the spleen was totally destroyed and a lot of leuckemia cells infiltrating in the spleen cells. 2CD4T, CD8T, CD4T/CD8T:MB group and the AZT group showed the raising CD4T function in the experiment compared with the Model group, and the AZT shows stronger effect than the MB group,(P<0.05). The CD8T has little differences between these groups;AZT group has the best CD4T/CD8T, but the MB group also showed the function to raise the CD4T/CD8T. 3. 3IL-1P, IL-2, IL-6andINF-YIn this experiment the IL-1 of the rats are in the following order from the higher to the lower: model group, AZT group, MB group,MM group, Ms group and the blank control group. Among them MB,MM and AZT showed significance with the modelgroup.( P<0.05) for IL-2, they are in the order as following: the blank control group, MB,AZT,MM, MS and model group. Among them, MB has significance when compared with the model group(P<0.01);and the blank control group, MM,and AZT compared with the Model group showing difference^ P<0.05) for the IL-6, the order is as following: model group, AZT group, MS,MM and MB. Compard with the model group, MB and MM group show significant difference.(P<0.05), the normal group, AZT and MS group showed difference(P<0.01).the order from higher to lower of IFN- Y is the normal group, MB. MM.AZT MS and the model group. Among them the MB,MS show difference with the model group (P<0.05) . 3. 4The result of plaque forming experiment:MDQ shows the function to lower the vitality of the FLV.3. 5 The result of fluorescent quantitation PCR test the virus tilter after the MDQ treating:After the applying of MDQ, the FLV tilter was reduced. 3. 6 the result of confocal laser scanning microscope experiment:MDQ has the function to reduce the calcium ion inflowing. 4.Conclusion4.1 MDQ has the curative function to treat the FLV caused murine lekemia, because it can reduce the size of the splenomegaly, spleen protection, and raise the CD4T number.4.2 The mechanism of the MDQ theriputic funcion maybe the following: regulation of the immunity system, protect the body from the contamination of the extra-activation of the immunal system;regulating the exterior and interior ion concentration of the cells so that regulating the mini-biosurrundings of the virus to prevent the virus come into the active phase.