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microRNA-21在溃疡性结肠炎患者中的表达及 临床意义
吴慧华
2016-11-15
导师王爱英
专业内科学
授予单位北京大学
授予地点北京大学第三临床医学院
学位硕士
关键词溃疡性结肠炎 miR-21 发病机制
其他题名Clinical significance of microRNA-21 expression in ulcerative colitis
分类号R574.62
摘要

【目的】初步探讨微小核糖核酸-21(microRNA-21,miR-21)的表达与溃疡性结肠炎发生的关系,进一步了解溃疡性结肠炎的发病机制,从而为溃疡性结肠炎(ulcerative colitis,UC)的临床诊断提供一种新的途径。

【方法】选取25例UC患者及20例健康自愿者。1.通过实时定量聚合酶链反应(Real-time quantitative polymerase chain reaction ,RT-PCR)方法检测血清和组织中的miR-21的表达。2. 原位杂交法检测组织中的miR-21的表达。3.通过免疫组织化学方法检测 B淋巴细胞瘤-6基因(B-cell lymphoma-6,bcl-6)的表达。4.通过免疫荧光方法测定组织中趋化因子受体3(CC chemokine receptor 3,CCR3) 和淋巴细胞活化基因 -3(lymphocyte activation gene-3,LAG-3)的表达。

【结果】

1. UC组中男性16例,女性9例,年龄19岁—65岁,平均年龄39.0±11.9岁;对照组男性14例,女性6例,年龄21岁—57岁,平均年龄37.7±9.8岁。两组比较,性别及年龄无统计学差异(P>0.05)。

2. 血清miR-21的表达量结果:正常对照组血清miR-21的水平为 1.11±0.17, UC组的血清miR-21的水平为1.24±0.41,UC组的血清miR-21水平与正常对照组无显著性差异(t=1.70,P>0.05)。组织miR-21的表达量结果:对照组组织miR-21的水平为1.05±0.29,UC组组织miR-21水平为1.82±0.73,UC组织中的miR-21水平较正常对照组有统计学差异(t=4.47,P<0.05)。

3. 原位杂交法检测组织中的miR-21的表达结果:对照组组织中miR-21的水平为0.24±0.05;UC组织中miR-21水平为0.34±0.08。UC组中组织的miR-21的水平较正常对照组有显著统计学差异(t=4.76,P<0.01)。

4.免疫组织化学方法检测BCL-6的结果:实验结果用每组的吸光度值来表示,对照组组织BCL-6的吸光度值0.51±0.12; UC组织中BCL-6的吸光度值0.35±0.12。UC组织中BCL-6的吸光度值较正常对照组有显著统计学差异(t=4.43,P<0.01)。

5.免疫荧光方法测定CCR3和LAG-3的结果:对照组组织CCR3光密度值0.04±0.02,UC组中CCR3光密度值0.09±0.04,UC组中CCR3光密度值较正常对照组有明显升高(t=4.34,P< 0.01)。对照组中LAG3光密度值0.11±0.05,UC组中LAG3光密度值0.15±0.07,较正常对照组有明显升高(t=2.51;P<0.05)。 计算LAG3/CCR3阳性细胞光密度比率:对照组为2.82±0.90;UC组为1.91±0.70,有统计学差异(t=4.43,P< 0.05)。

【结论】

1.miR-21的高表达与UC的发病有关。

2.miR-21可能通过调节BCL-6的表达来影响UC的发病。

3. LAG3 /CCR3阳性细胞比率在UC患者中明显降低,提示UC患者中存在着TH1/TH2免疫细胞失衡。

总之,本研究结果表明miR-21在UC组织中高表达,miR-21对UC的发病可能是通过下调BCL-6的表达或者影响TH1/TH2的失衡来发挥作用。

英文摘要

[Objective] The expression of miR-21 was detected in ulcerative colitis (UC), and the relationship between the expression of miR-21 and imbalance of inflammatory cells in UC were investigated.

[Methods] 25 UC patients and 20 healthy volunteers were involved. Optical density of CCR3 and LAG-3 were determined by immunofluorescence assay. MiR-21 were determined by situhybridization and real time quantitative PCR.The expression of bcl-6 was detected by immunohistochemistry.

[Results]

1. UC group of 16 cases of the men and women in 9 cases, age 19-65 years, mean age 39.0 ±11.9 years old . Control group of 16 cases of the men and women in 9 cases, age 19-65 years, mean age 37.7±9.8 years old .Comparing the two groups, gender and age is no statistical difference (P > 0.05).

2. The expression of serum miR-21 quantity results: Normal control group the levels of serum miR-21 was 1.11±0.17,and UC group was 1.24±0.41. Levels of serum miR-21 in UC group and normal control group were no significant difference (t= 1.70,P>0.05). The expression of tissue miR - 21 quantity results: Normal control group the levels of tissue miR-21 was 1.05±0.29,and UC group was 1.82±0.73. Levels of tissue miR-21 in UC group and normal control group were significant difference (t=4.47;P< 0.05).

3. Hybridization in situ were used to detect the expression of tissue miR-21 quantity: Normal control group the levels of tissue miR-21 was 0.24±0.05,and UC group was 0.34±0.08. Levels of tissue miR-21 in UC group and normal control group were significant difference (t=4.76,P< 0.01).

4. Immunohistochemistry were used to detect the expression of BCL-6 quantity: Image analysing system was used to analyse the image the result was expressed as absorbance. Normal control group the levels of tissue BCL-6 was 0.51±0.12,and UC group was 0.35±0.12. Levels of tissue BCL-6 in UC group and normal control group were significant difference(t=4.43,P<0.01).

5.  Methods the level of CCR3 and LAG3 was determined by fluorescence immunization:Normal control group the levels of tissue CCR3 was 0.04±0.02,and UC group was0.09±0.04. Levels of tissue CCR3 in UC group and normal control group were significant difference(t=4.34,P< 0.01). Normal control group the levels of tissue LAG3 was 0.11±0.05,and UC group was0.15±0.07. Levels of tissue LAG3 in UC group and normal control group were significant difference(t=2.51;P< 0.05).The LAG3/ CCR3 (Optical density) ratio in UC group and normal group were significant difference .(t=4.43,P<0.05).

[Conclusions]The increase of miR-21 in UC were obseved and the expression of miR21 may be related to the unbalance of immune cells in UC. Although the possible mechanisms of miR-21 involed in the pathogenesis of UC were still unclear, the expression of MiR21 in UC may be related to the unbalance of T cells and other immune cells which was considered to be involved in the pathogenesis of UC.

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文献类型学位论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/124737
专题北京大学第三临床医学院
作者单位北京大学第三临床医学院
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吴慧华. microRNA-21在溃疡性结肠炎患者中的表达及 临床意义[D]. 北京大学第三临床医学院. 北京大学,2016.
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