本文選題：SLE + ZBTB20　； 參考：《鄭州大學》2017年碩士論文

【摘要】：目的通過檢測鋅指蛋白ZBTB20在SLE患者B淋巴細胞中的表達情況并分析其與B細胞分化相關轉錄因子Blimp-1的關系,同時檢測狼瘡小鼠體內(nèi)ZBTB20的表達,探究ZBTB20與SLE發(fā)病的相關性。方法(1)收集36例初診未經(jīng)治療的SLE患者和30例正常對照者外周血,分離外周血單個核細胞(PBMCs)。(2)采用磁珠分選法提取PBMCs中的CD19+B細胞,并用RT-PCR技術檢測CD19+B細胞中ZBTB20 m RNA與Blimp1 m RNA的表達水平(以-△△Ct值表示);采用流式細胞術檢測另一管PBMCs中B細胞亞群比例。(3)分析SLE患者外周血B細胞ZBTB20 m RNA的表達與B細胞亞群(包括CD19+B細胞,CD19-CD138+漿細胞)比例變化的關系,與Blimp1m RNA表達及實驗室指標抗ds-DNA抗體及免疫球蛋白Ig G的相關性。(4)狼瘡模型MRL/lpr小鼠、NZB/WF1小鼠培養(yǎng)至25-28周發(fā)病最嚴重期,并以同周期的正常對照C57BL/6小鼠作為對照,ELISA法檢測各組小鼠血清中抗ds-DNA抗體及免疫球蛋白Ig G水平。(5)取出小鼠的脾臟,淋巴結,及胸腺,各組織其中一部分用于制備石蠟切片用于免疫組化檢測ZBTB20陽性表達,另一部分用于提取總RNA,RTPCR檢測ZBTB20 m RNA、Blimp-1 m RNA的表達水平(以-△△Ct值表示)。結果(1)ZBTB20 m RNA在SLE患者組外周血CD19+B細胞中的表達水平明顯高于正常對照組[(1.106±0.161)vs(2.527±0.355),P0.05];且SLE患者組Blimp1 m RNA的表達也明顯高于正常對照組[(1.106±0.161)vs(2.527±0.355),P0.05];(2)與對照組相比,SLE患者外周血B細胞亞群比例發(fā)生變化,其中CD19+B細胞比例降低[(4.87±3.49)vs(6.92±3.85),P0.05],而CD19-CD138+漿細胞比例和漿細胞/B細胞之間的比值均較對照組顯著增高[(0.49±0.41)vs(0.27±0.25),P0.05;(0.14±0.05)vs(0.03±0.03),P0.05],差異有統(tǒng)計學意義。(3)SLE患者B細胞ZBTB20 m RNA表達與CD19-CD138+漿細胞比例和漿細胞/B細胞比值呈正比[(r=0.165,P=0.028),(r=0.225,P=0.021)],與B細胞中Blimp1 m RNA的表達呈明顯的正相關性(r=0.112,P=0.039);SLE患者ZBTB20 m RNA的表達與血清中抗ds-DNA抗體,免疫球蛋白Ig G水平呈明顯正相關[(r=0.415,P=0.007),(r=0.507,P=0.005)]。(4)MRL/lpr小鼠、NZB/WF1小鼠處于發(fā)病嚴重期,脾臟、淋巴結及胸腺均有不同程度的腫大,血清中抗ds-DNA抗體,免疫球蛋白Ig G均較對照組增高[(5.27±3.79)vs(0.71±0.65),P0.05;(4.50±2.18)vs(0.71±0.65),P0.05)],[(12.35±7.74)vs(1.68±0.75),P0.05;(10.91±6.12)vs(1.68±0.75),P0.05)],差異有統(tǒng)計學意義,說明構建的小鼠模型符合實驗預期。(5)MRL/lpr小鼠、NZB/WF1小鼠脾臟中ZBTB20 m RNA表達明顯高于正常對照組C57BL/6小鼠[(0.49±0.41)vs(0.27±0.25),P0.05;(0.14±0.05)vs(0.03±0.03),P0.05)],而模型組小鼠淋巴結及胸腺中ZBTB20 m RNA表達與對照組相比差異無統(tǒng)計學意義;且免疫組化的結果顯示,ZBTB20在狼瘡小鼠MRL/lpr小鼠、NZB/WF1小鼠脾臟中的表達與對照組相比陽性表達率明顯增高[(21.3%vs5.2%),(40.2%vs5.2%)];而在淋巴結和胸腺中,ZBTB20的陽性表達率與對照組相比差異無統(tǒng)計學意義。結論1.SLE患者外周血CD19+B細胞中ZBTB20 m RNA表達較對照組明顯增高,且同患者外周血CD19-CD138+漿細胞比例成正相關,與血清中自身抗體水平呈正相關,因此,ZBTB20可能與SLE發(fā)病有關;2.SLE患者ZBTB20 m RNA的表達與同樣較對照組表達增高的B細胞分化關鍵調(diào)節(jié)因子Blimp1 m RNA表達呈正相關,說明ZBTB20參與SLE發(fā)病可能與B細胞異常分化有關;3.狼瘡鼠模型實驗進一步說明ZBTB20的表達與B細胞異常過度活化并分化為漿細胞分泌抗體有關,ZBTB20可能是通過這一機制參與到SLE發(fā)病的。
[Abstract]:Objective to detect the expression of zinc finger protein ZBTB20 in the B lymphocyte of patients with SLE and to analyze the relationship with B cell differentiation related transcription factor Blimp-1, and to detect the expression of ZBTB20 in lupus mice, and to explore the correlation between ZBTB20 and SLE. Methods (1) 36 cases of early diagnosis of untreated SLE patients and 30 normal controls were collected. Peripheral blood, isolated peripheral blood mononuclear cells (PBMCs). (2) the CD19+B cells in PBMCs were extracted by magnetic bead sorting, and the expression level of ZBTB20 m RNA and Blimp1 m RNA in CD19+B cells was detected by RT-PCR technique (expressed in the value of delta delta Ct), and the proportion of the subgroup in the other tube was detected by flow cytometry. (3) analysis of the peripheral blood of the patients. The relationship between the expression of ZBTB20 m RNA and the proportion of B cell subsets, including CD19+B cells, CD19-CD138+ plasma cells, and the correlation between the expression of Blimp1m RNA and the anti ds-DNA antibody and immunoglobulin Ig G in the laboratory. (4) lupus model MRL/lpr mice were cultured to the most severe period of the onset of the 25-28 weeks, and the normal pairs of the same cycle were in the same cycle. The anti ds-DNA antibody and immunoglobulin Ig G level in the serum of each group were detected by ELISA method. (5) the spleen, lymph nodes and thymus of mice were taken out and part of the tissues were used to prepare the paraffin section for immunohistochemical detection of ZBTB20 positive expression, and the other part was used to extract total RNA, and RTPCR to detect ZBTB20 m RNA, The expression level of Blimp-1 m RNA (with the value of delta delta Ct). Results (1) the expression level of ZBTB20 m RNA in the peripheral blood CD19+B cells of the patients with SLE was significantly higher than that of the normal control group [(1.106 + 0.161) vs (2.527 + 0.355), P0.05], and the expression of the SLE patients was significantly higher than that of the normal control group [(1.106 + 0.161) (2.527 + 0.355). 2) compared with the control group, the proportion of B cell subsets in peripheral blood of SLE patients was changed, and the proportion of CD19+B cells decreased [(4.87 + 3.49) vs (6.92 + 3.85), P0.05], and the ratio of CD19-CD138+ plasma cell ratio and plasma cell /B cell increased significantly [(0.49 + 0.41) vs (0.27 + 0.25), P0.05; (0.14 + 0.05) vs (0.03 + 0.03), P0.05], poor (3) the expression of ZBTB20 m RNA in B cells in SLE patients was proportional to the ratio of CD19-CD138+ pulp cells and the ratio of /B cells to plasma cells (r=0.165, P=0.028), and (r=0.225, P=0.021)). The level of pestig protein Ig G was obviously positive correlation [(r=0.415, P=0.007), (r=0.507, P=0.005)]. (4) NZB/WF1 mice were in severe stage of onset, the spleen, lymph nodes and thymus were enlarged in varying degrees, the anti ds-DNA antibodies in the serum and the G of immunoglobulin Ig G were higher than those of the control group [(5.27 + 3.79) vs (0.71 + 0.65), 4.50 + 2.18) (0.) 71 + 0.65), P0.05), [(12.35 + 7.74) vs (1.68 + 0.75), P0.05, (10.91 + 6.12) vs (1.68 + 0.75), P0.05)], the difference was statistically significant, indicating that the constructed mouse model was in conformity with the experimental expectation. (5) MRL/lpr mice, the expression of ZBTB20 m RNA in the spleen of NZB/WF1 mice was significantly higher than that of C57BL/6 mice in the normal control group [0.49 + 0.41) vs. .05) vs (0.03 + 0.03), P0.05)], and the expression of ZBTB20 m RNA in the lymph nodes and thymus of the model mice was no significant difference compared with the control group; and the immunohistochemical results showed that the expression rate of ZBTB20 in the spleen of lupus mice was significantly higher than that of the control group [(21.3%vs5.2%), (40.2%vs5.2%)). In the lymph nodes and thymus, the positive expression rate of ZBTB20 was not statistically significant compared with the control group. Conclusion the expression of ZBTB20 m RNA in the peripheral blood CD19+B cells of 1.SLE patients was significantly higher than that of the control group, and it was positively correlated with the proportion of CD19-CD138+ plasma cells in peripheral blood, and was positively correlated with the level of autoantibodies in the serum. Therefore, ZBTB20 could be found. The expression of ZBTB20 m RNA in patients with 2.SLE was also positively correlated with the RNA expression of Blimp1 m, a key regulator of B cell differentiation in the control group, indicating that ZBTB20 participation in SLE may be related to the abnormal differentiation of B cells. 3. the 3. lupus rat model experiment further indicated that the expression of ZBTB20 and the abnormal activation of the cells were abnormal. ZBTB20 may be involved in the pathogenesis of SLE through differentiation into plasma cells secreting antibodies.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R593.241

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