锌指蛋白ZBTB20及B细胞异常活化与SLE发病的相关性研究

发布时间：2018-05-28 06:57

本文选题：SLE + ZBTB20　；参考：《郑州大学》2017年硕士论文

【摘要】：目的通过检测锌指蛋白ZBTB20在SLE患者B淋巴细胞中的表达情况并分析其与B细胞分化相关转录因子Blimp-1的关系,同时检测狼疮小鼠体内ZBTB20的表达,探究ZBTB20与SLE发病的相关性。方法(1)收集36例初诊未经治疗的SLE患者和30例正常对照者外周血,分离外周血单个核细胞(PBMCs)。(2)采用磁珠分选法提取PBMCs中的CD19+B细胞,并用RT-PCR技术检测CD19+B细胞中ZBTB20 m RNA与Blimp1 m RNA的表达水平(以-△△Ct值表示);采用流式细胞术检测另一管PBMCs中B细胞亚群比例。(3)分析SLE患者外周血B细胞ZBTB20 m RNA的表达与B细胞亚群(包括CD19+B细胞,CD19-CD138+浆细胞)比例变化的关系,与Blimp1m RNA表达及实验室指标抗ds-DNA抗体及免疫球蛋白Ig G的相关性。(4)狼疮模型MRL/lpr小鼠、NZB/WF1小鼠培养至25-28周发病最严重期,并以同周期的正常对照C57BL/6小鼠作为对照,ELISA法检测各组小鼠血清中抗ds-DNA抗体及免疫球蛋白Ig G水平。(5)取出小鼠的脾脏,淋巴结,及胸腺,各组织其中一部分用于制备石蜡切片用于免疫组化检测ZBTB20阳性表达,另一部分用于提取总RNA,RTPCR检测ZBTB20 m RNA、Blimp-1 m RNA的表达水平(以-△△Ct值表示)。结果(1)ZBTB20 m RNA在SLE患者组外周血CD19+B细胞中的表达水平明显高于正常对照组[(1.106±0.161)vs(2.527±0.355),P0.05];且SLE患者组Blimp1 m RNA的表达也明显高于正常对照组[(1.106±0.161)vs(2.527±0.355),P0.05];(2)与对照组相比,SLE患者外周血B细胞亚群比例发生变化,其中CD19+B细胞比例降低[(4.87±3.49)vs(6.92±3.85),P0.05],而CD19-CD138+浆细胞比例和浆细胞/B细胞之间的比值均较对照组显著增高[(0.49±0.41)vs(0.27±0.25),P0.05;(0.14±0.05)vs(0.03±0.03),P0.05],差异有统计学意义。(3)SLE患者B细胞ZBTB20 m RNA表达与CD19-CD138+浆细胞比例和浆细胞/B细胞比值呈正比[(r=0.165,P=0.028),(r=0.225,P=0.021)],与B细胞中Blimp1 m RNA的表达呈明显的正相关性(r=0.112,P=0.039);SLE患者ZBTB20 m RNA的表达与血清中抗ds-DNA抗体,免疫球蛋白Ig G水平呈明显正相关[(r=0.415,P=0.007),(r=0.507,P=0.005)]。(4)MRL/lpr小鼠、NZB/WF1小鼠处于发病严重期,脾脏、淋巴结及胸腺均有不同程度的肿大,血清中抗ds-DNA抗体,免疫球蛋白Ig G均较对照组增高[(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)],差异有统计学意义,说明构建的小鼠模型符合实验预期。(5)MRL/lpr小鼠、NZB/WF1小鼠脾脏中ZBTB20 m RNA表达明显高于正常对照组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)],而模型组小鼠淋巴结及胸腺中ZBTB20 m RNA表达与对照组相比差异无统计学意义;且免疫组化的结果显示,ZBTB20在狼疮小鼠MRL/lpr小鼠、NZB/WF1小鼠脾脏中的表达与对照组相比阳性表达率明显增高[(21.3%vs5.2%),(40.2%vs5.2%)];而在淋巴结和胸腺中,ZBTB20的阳性表达率与对照组相比差异无统计学意义。结论1.SLE患者外周血CD19+B细胞中ZBTB20 m RNA表达较对照组明显增高,且同患者外周血CD19-CD138+浆细胞比例成正相关,与血清中自身抗体水平呈正相关,因此,ZBTB20可能与SLE发病有关;2.SLE患者ZBTB20 m RNA的表达与同样较对照组表达增高的B细胞分化关键调节因子Blimp1 m RNA表达呈正相关,说明ZBTB20参与SLE发病可能与B细胞异常分化有关;3.狼疮鼠模型实验进一步说明ZBTB20的表达与B细胞异常过度活化并分化为浆细胞分泌抗体有关,ZBTB20可能是通过这一机制参与到SLE发病的。
[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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.241

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