| Acute Graft - Versus - Host - Disease ( aGVHD ) is common complication after allogeneic bone marrow transplantation (BMT) , still contributing substantially to morbidity and mortality in this therapeutic procedure. Over the past decade , many advances have been made with regard to the prevention and treatment of GVHD using various drugs such as cyclosporin A ( CsA) , FK506, methotrex-ate ( MTX) , mycophenolate mofetil and /or monoclonal IL - 2 receptor antagonists , At present, immunosuppressive agents are broadly adopted for inhibiting aGVHD in clinic, especially the combination of cyclosporin A (CsA) with meth-otrexate(MTX) , but their side - effect and toxic effect are serious ,and they can lead to severe infection and tumor. Therefor,it is an actute problem to seek a new effetive measure to prevent aGVHD. The Glucosidorum Tripterygii Tororum (GTT) is a chinese medicine, it has being adopted as immunosuppressive drug in clinic. So we observe the effects of GTT on aGVHD mice and try to further study on the mechanism of preventing aGVHD by GTT.Materials and MethodsC57BL/6 mice were purchased at 8 - 12 weeks ages and were used within Iwk of arrival. The mice were given autoclaved acidified acidified water and lab food ad libitum and were housed in sterile microisolator boxes.The recipient mice(C57BL/6 mice) were lethally total body irradiated by a linear accelerat ( SIMANS, Germany, total dose is 1800cGy, rate of dose is 200Gy/min)24h before transplantaed . Then they were given the single cell suspensions which were isolated from bone marrow (1 x 106) and spleens (5 x 106) of the BABC/C mice , so the model of aGVHD were duplicated . Starting on the day of transplantation, the mice of syn - BMT group and allo - BMT were treated i. p. with 0. 9% N. S. 0. 2ml; the mice of CsA + MTX group were treated i. p.with CsA 2.5mg/kg and MTX 7mg/M2; the mice of GTT group were treated i. p. with CTT 5mg/kg;the mice of GTT + CsA were treated i. p. with CTT 2.5mg/ kg and CsA2.5mg/kg,for 11 days.The tissue samples of skin, lung and colon were taked from recipient mice, and immediately placed in 10% buffered formaldehyde , Fixed tissues were embedded in paraffin,cut into 6um tissues sections,mounted on glass slides,then stained with a standard hematoxylin and eosin(H & E) procedure;or using im-munohistochemistry stained with 8ul FITC conjugated monoclonal anti - mous-eCD3,CD4,CD8 and PE conjugated monoclonal anti - mouseCD11a, CD18. then calculate fluorescence dimension ( x 105um2,LUZEX F,Japan)to observe the changes of CD3+,CD4+,CD8+,CD11a,CD18+ lymphocytes in skin, lung and colon.To determine the number of CD3+ , CD4+ , CD8+ , CD/CD11a, CD4+CD18+, CD8+CD11a+,CD8+CD18+ lymphocytes in spleen in recipient mice with flow cy-tometry ,the spleens were removed and single - cell suspensions were prepared. The spleen cell suspensions were depleted of erythrocytes by lysis with 0. 83% Tris - buffered ammonium choride, then washed twice in NaN3 - Hank' Spleep cells (1 x 107/ml) were incubated with FITC - conjugated monoclonal anti -mouseCD3,CD4, CD8 and PE conjugated monoclonal anti - mouseCD11a, CD18. Surface expression was analyzed on a FACStar flow cytometer( Becton Dickinson San Diego,CA)using the LYSIS program( Becton Dickinson). Valures shown are percent positive cells in the lymphocyte - gated cell population.Blood from recipient mice was collected in heparincoated syringes, the plasma was collected and analyzed for the presence of IL -2,TNFa,IL -4 and IL -10 using ELISA kits. ( DIACLONE)Total RNA was isolated from the spleens using Trizol reagent ( Gibcobrl). One microgram of RNA from each group was reverse transcribed into cDNA u-sing a reverse transcription system(Promega. Madison,WI). PCR reactions were then performed using primers for B - action, IL - 2, TNFa, IL -4 and IL - 10 (Takara Japan). (IL-2:sense 5' - AACAGCGCACCCACTTCAA -3' ,anti-sense 5' -TTGAGATGATGCTTTGACA-3';443bp. IL-4:sense5' -AGAT-CATCGGCATTTTGAACGAGGTC - 3' , antisense 5' - TGCTGATGCTCTTTAG-GCTTTCC -3' ,313bp. IL - 10:sense...
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