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Study on the Relationship between CD4~+/CD8~+ T Subsets, T-bet/GATA3 Gene Expression and Type 1 Diabetes Mellitus

Author: ZouXiaoLei
Tutor: XiangMing
School: Huazhong University of Science and Technology
Course: Pharmacology
Keywords: Type 1 diabetes mellitus (T1DM) CD4 ~ T cells CD8 ~ T cells Flow cytometry (FACS) Adoptive transfer of T-bet GATA-3 Reverse transcription - polymerase chain reaction (RT-PCR)
CLC: R587.1
Type: Master's thesis
Year: 2008
Downloads: 197
Quote: 1
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Abstract


The first part of STZ-induced mice with type 1 diabetes model CD4 / CD8 T lymphocyte subsets and analytical purposes: research STZ-induced mice with type 1 diabetes mellitus (T1DM) model CD4 / CD8 T lymphocyte subsets changes, type 1 diabetes pathogenesis study provides a theoretical basis. : 40mg/kg streptozotocin (STZ) for 5d intraperitoneal injection of the preparation of mouse type 1 diabetes model. FACS detection of mouse spleen CD4 T cells, CD8 T cells, CD4 CD25 T cells positive rate, MTT assay of mouse spleen lymphocyte proliferative responses, ELISA detection of serum cytokines IL-2, IL-4, IL-10 and IFN- γ levels in mouse pancreatic pathological changes by HE staining. Results: after the last injection of STZ 2wk, diabetes model mouse spleen CD4 T and CD8 T-cell ratio decreased significantly (P lt; 0.05), CD4 / CD8 ratio increased, CD4 CD25 T cell decline, but comparison with normal mice, Both was no significant difference (P gt; 0.05). 4wk, diabetic mice spleen CD4 T cell count and CD4 / CD8 ratio was significantly higher than in normal mice (P lt; 0.05, P lt; 0.01), CD8 T cells and CD4 CD25 T cells was significantly lower than that of normal mice (P lt; 0.01), a statistically significant difference. MTT assay showed that diabetic mice spleen lymphocyte proliferation in the last STZ after 2wk and 4wk see significantly enhanced (P lt; 0.05). ELISA showed that serum cytokines IFN-γ were in the STZ model the 2wk groups and 4wk group were significantly higher than the normal control group (P lt; 0.05, P lt; 0.01), while IL-2, IL-4 and IL-10 The levels in each group was no significant difference. HE staining found 2wk mice pancreas of STZ model can be seen apparent infiltration of inflammatory cells, islet structure is still intact; while the overt hyperglycemia of STZ model 4wk mice islet see a large number of inflammatory cell infiltration, islet structure severely damaged. Conclusion: the type 1 diabetes disease process there is always the imbalance of lymphocyte subsets, CD4 Th1 cells abnormal activation of CD8 T cell defects play an important role in pathological changes in type 1 diabetes. The role of the second part of the pathogenic CD4 T cells in the induction of type 1 diabetes research purposes: Adoptive transfer of purified CD4 T cell induced diabetic pathogenic CD4 T cells in the induction of the incidence of type 1 diabetes mellitus (T1DM) role. Methods: five consecutive times, the low dose of STZ (STZ, 40mg/kg) injected intraperitoneally (ip) Preparation of type 1 diabetes mellitus (T1DM) mouse model. The 4wk incidence of mice were sacrificed, take the spleen cell suspension was prepared after the pathogenic CD4 T cell culture 7d collected after induced diabetic spleen cells and preparation of purified IL-2 stimulation. Diabetic spleen cells and purified the pathogenicity CD4 T cells were induced by intravenous injection (iv) adoptive transfer to STZ2 pretreatment BALB / c mice, the conventional observation 4wk. FACS detection spleen cell phenotype induced diabetic MTT assay the activity of cell proliferation, ELISA detection of cytokines (IL-2, IFN-γ, IL-4, IL-10) content, HE staining of pancreatic pathological changes. Results: 3 × 106 spleen cells induced diabetic adoptive transfer recipient mice successfully induced diabetes, while the same amount of pathogenic CD4 T cells purified adoptive transfer did not cause the recipient mice hyperglycemia. The FACS detection diabetic spleen cells on T cells accounted for 85.6% of the CD4 + T cells accounted for 81.7%. MTT assay demonstrated that diabetic spleen cells and purified caused by pathogenic CD4 T cells with high proliferative activity of lymphocytes (P lt; 0.05), spleen cells adoptive mice spleen lymphocyte proliferation was significantly enhanced (P lt; 0.05 ), while the CD4 T cell adoptive spleen lymphocyte proliferation in mice with the normal control group did not differ significantly (P gt; 0.05). The ELISA detected, induced diabetic spleen cells and purified pathogenic CD4 T cells in the supernatant contained high concentrations of IL-2 and IFN-gamma, the differences in comparison with normal spleen cells have a significant resistance (P LT; 0.05) ; spleen cells adoptive serum levels of IFN-γ concentration was significantly higher (P lt; 0.01), IL-2, IL-4 and IL-10 levels did not change significantly; CD4 T cell adoptive serum levels of various cell factor content similar to the normal control group, the difference was not statistically significant (P gt; 0.05). HE examination revealed the spleen cell adoptive mice islets see a large number of infiltration of lymphocytes and severe pancreatic β cell damage and the CD4 T cell adoptive apparent inflammatory infiltration mice islets, islet structure is still intact. Conclusion: pathogenic CD4 T cells alone adoptive transfer caused by the recipient mice pancrentitis emergence of blood glucose to maintain normal, indicating that pathogenic CD4 T-cell-mediated inflammatory infiltration of the islets in the process of induction of T1DM onset, rather than final effector cells, play an important role early in T1DM onset. T-bet/GATA-3 mRNA expression of the third part of the transcription factor correlation to the onset of type 1 diabetes research purposes: analysis of type 1 diabetes mellitus (T1DM) incidence mouse transcription factor T-bet/GATA-3 mRNA expression of Th1/Th2 two class of cytokines related to research from the perspective of transcription factor type 1 diabetes Th1/Th2 cell differentiation trend, to explore T-bet/GATA-3 role in the pathogenesis of type 1 diabetes. Continuous 5d: 40mg/kg dose streptozotocin (STZ) intraperitoneal injection (ip) in BALB / c mice to establish a mouse model of STZ-induced T1DM incidence of mice were sacrificed after the last STZ 4wk experiment; another non-obese diabetic (NOD) mice to 22wk age, reared in SPF environment after random incidence mouse line correlation detection. Mice in each group were sacrificed using Trizol step extraction spleen total RNA, reverse transcriptase (RT) reaction of cDNA polymerase chain reaction (PCR) detection spleen T-bet and GATA-3 transcription factor of IFN-γ, IL- 4 cytokine gene expression status, and enzyme-linked immunosorbent assay (ELISA), the serum cytokine IFN-γ, IL-4 levels. Results: STZ-induced T1DM model mice and NOD mice spleen T-bet the IFN-γmRNA expression was significantly increased in diabetes GATA-3 mRNA expression was significantly decreased, IL-4 mRNA expression in each experimental group were not seized a. ELISA showed that diabetes mice (STZ-induced T1DM model mice and NOD mice) serum IFN-γ concentration was significantly higher than that of control mice (P lt; 0.01), IL-4 levels declined, but not statistically significant. Conclusions: T1DM onset, of the mouse spleen transcription factor T-bet/GATA-3 mRNA expression and serum Th1/Th2 type cytokines content corresponding, indicating the transcription factor T-bet/GATA-3 by affecting Th1/Th2 cell differentiation closely associated with type 1 diabetes.

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CLC: > Medicine, health > Internal Medicine > Endocrine diseases and metabolic diseases > Islet disease > Diabetes
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