【作者】 李荣良；

【导师】 孙凌云；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2010， 博士

【摘要】 目的：探讨系统性红斑狼疮(SLE)患者OAZ通路相关基因的功能。方法：实验一：收集SLE患者和正常对照骨髓前体细胞(各5例),提取RNA,逆转录为cDNA,加入相应的引物、SYBR Green I在ABI Prism 7500型高通量荧光定量仪上进行实时荧光定量PCR,采用看家基因酸性核糖体磷蛋白大亚基(RPLPO)进行标化,观察OAZ、Id3、BMP4、BMP6、Smad6、EHZF、LY6E基因的表达量与SLE疾病活动相关指标关系。同时收集30例SLE患者和20例正常人外周血细胞,对上述结果进行验证。实验二：采集并分离SLE患者外周血单个核细胞,分为实验组、阳性对照组、阴性对照组和空白对照组进行细胞培养。实验组和阳性对照组分别用OAZ和GAPDH小干扰RNA(siRNA)在体外对培养细胞进行干扰,阴性对照组采用无关RNA序列,空白对照组为培养基对照。72小时后分离细胞和上清液,从细胞中提取RNA,逆转录为cDNA,用实时荧光定量PCR分析不同分组OAZ信号通路相关基因OAZ、Id1、Id2、Id3、Id4mRNA表达水平的变化。ELISA法测定上清液中IFN-γ、IL4、IL10、IL12、IL21、CCL2、ANA浓度,并分析这些因子分泌水平与OAZ基因表达的相关性。实验三：从骨髓或脐带血中分离培养间充质干细胞(MSC)移植治疗10例SLE患者,采集并分离移植前、移植后1周、4周患者外周血有核细胞,从细胞中提取RNA,逆转录为cDNA,用实时荧光定量PCR技术分析不同分组OAZ信号通路相关基因OAZ、Id1、Id2、Id3 mRNA表达水平的变化。ELISA法测定移植前后患者血清中IL10、IL12、IL21、CCL2、ANA浓度,并分析这些因子分泌水平与OAZ基因表达的相关性。结果：实验一：SLE患者骨髓前体细胞OAZ、Id3基因的mRNA表达水平(△Ct分别为10.6±0.5、5.8±3.2)较正常对照组(△Ct分别为16.5±0.9、10.4±2.6)显著增高,且表达水平高于外周血细胞；BMP6、BMP4基因表达水平虽较正常对照有高表达趋势,但差异无统计意义。SLE患者外周血细胞OAZ、Id3、BMP4、BMP6mRNA基因表达(△Ct分别为14.1±2.7、7.5±1.8、7.6±3.3、5.2±2.2)均显著高于正常(△Ct分别为16.1±2.2、9.5±1.7、9.7±0.3、6.4±1.2)。骨髓前体细胞OAZ、BMP6、BMP4、Id3的表达水平ACt值与SLEDAI积分、肾脏损伤指数、抗双链DNA (dsDNA)抗体、RNA相关抗体水平呈负相关；与补体C3呈正相关。实验二：实验组OAZ、Id1、Id2、Id3基因的mRNA表达水平(△Ct分别为12.5±1.4、8.9±1.5、4.3±0.8、8.0±1.1)较阴性对照组(△Ct分别为10.2±1.1、6.5±1.2、2.4±1.3、6.2±1.2)显著降低(P<0.05)；Id4表达水平虽较阴性对照组有低表达趋势,但差异无统计学意义。实验组IFN-γ、IL10、IL12、IL21、ANA的水平明显低于阴性对照组；而CCL2高于阴性对照组(P<0.05)。实验组与阴性对照组OAZ mRNA表达水平△△Ct值与ANA的OD比值、IL21浓度比值呈负相关；与Th1/Th2、CCL2比值呈正相关。实验三：移植后1周、4周OAZ、Id1、Id3基因的mRNA表达水平[ACt(1周)分别为12.4±1.1、9.7±1.9、9.7±1.9、2.1±1.0；△Ct(4周)分别为13.3±1.2、10.4±1.5、10.8±1.2、2.1±1.2]较移植前(△Ct分别为11.0±0.9、7.4±2.1、7.8±2.1、0.1±1.5)均显著降低(P<0.05)；Id2表达水平虽较移植前有低表达趋势,但差异无统计学意义。移植4周后IL10、IL21、ANA的水平明显低于移植前,IL12／IL10、CCL2高于移植前(P<0.05),但移植后1周的水平较移植前没有差异。移植前与移植后4周OAZ mRNA表达水平变化与ANA的OD值和IL21浓度比值呈负相关；与IL12/IL10、CCL2比值呈正相关。结论：一：OAZ通路相关基因在SLE中异常表达,与疾病发生相关。二：OAZ基因沉默可有效降低狼疮外周血单个核细胞中OAZ信号通路相关基因表达,同时减低多种细胞因子及抗核抗体水平,推测OAZ可能通过Id基因影响细胞因子的水平并导致ANA异常表达。三：同种异体MSC移植治疗SLE可降低患者外周血中OAZ信号通路相关基因表达,同时减低多种细胞因子及抗核抗体水平,推测OAZ信号通路是MSC移植治疗SLE有效机制之一。更多还原

【Abstract】 Objective To investigate the function of genes involved in OAZ signaling pathway in the patients with systemic lupus erythematosus (SLE)Methods Part one:The expression levels of OAZ, BMP6, BMP4, Id3, Smad6, EHZF、LY6E genes were valuated in bone marrow progenitor cells of 5 SLE patients and 5 normal subjects and replicated in peripheral blood cells of 30 SLE patients and 20 normal individuals by real-time quantitative PCR technique. Relationships of the expression levels of OAZ, BMP6, BMP4, Id3、LY6E mRNA with disease activity and other clinical index were analyzed.Part two:Peripheral blood mononuclear cells (PBMC) from SLE patients were collected. Each sample was equally divided into four groups for the cell culture in 96 well plates. Specific siRNA for OAZ and GAPDH were concordantly added to experimental group and positive control group, while nonspecific siRNA was added to negative control group and only culture medium was added to Mock control group. Cells and supernatant were harvested after culturing for 72 hours, then RNA was extracted and reverse transcripted to cDNA. OAZ, Id1, Id2, Id3, Id4, LY6E mRNA expression levels were analyzed by real-time PCR. Levels of IFN-y, IL4, IL10, IL12, IL21, CCL2, ANA in supernatant were tested by ELISA. Relationships of the expression levels of OAZ mRNA with levels of cytokines and ANA were analyzed.Part three:Human MSCs isolating and expanding from bone marrow cells or umbilical cord of healthy donors were infused into SLE patients. Peripheral blood cells were collected from 10 patients pre-MSCT as well as 1 week and 4 week post-MSCT, and RNA was extracted and reverse transcripted to cDNA. mRNA expression levels of OAZ and Idl-3 were measured by real-time PCR. Serum levels of IL10, IL12, IL21, CCL2 and ANA were tested by ELISA. Relationships of the gene expression levels with the levels of cytokines and ANA were analyzed.Results Part one:The expression levels of OAZ, Id3, LY6E mRNA(ACt) in the bone marrow (10.6±0.5,5.8±3.2) and peripheral blood (14.1±2.7,7.5±1.8) of SLE patients were significantly increased than those observed in normal controls (16.5±0.9,10.4±2.6, 16.1±2.2，9.5±1.7), which was found to negatively correlate with SLEDAI score, renal lesion index, titers of anti-dsDNA and anti-RNA antibodies, but positively correlate with serum complement C3. Expression levels of BMP4 and BMP6 were differentially expressed in peripheral blood cells but not bone marrow progenitor cells.Part two:OAZ, Id1, Id2, Id3, LY6E gene mRNA expression levels (△Ct:12.5±1.4, 8.9±1.5,4.3±0.8,8.0±1.1) in experimental group were significantly decreased comparing to those in negative control group (△Ct:10.2±1.1,6.5±1.2,2.4±1.3,6.2±1.2 respectively, p< 0.05). Levels of IFN-y, IL10, IL12, IL21 and ANA in experimental group were significantly lower than those in negative control group (p< 0.05); while level of CCL2 was higher than the negative control group (p<0.05). Difference of OAZ mRNA expression levels (△△Ct) between experimental group and negative control group were negatively correlated with changes of ANA, IL21 levels, but positively correlated with changes of Thl/Th2, CCL2.Part three:mRNA expression levels of OAZ, Idl and Id3 gene in patients with SLE were significantly decreased 1 week (△Ct:12.4±1.1、9.7±1.9、9.7±1.9、2.1±1.0) and 4week (△Ct: 13.3±1.2,10.4±1.5、10.8±1.2、2.1±1.2) after MSCT comparing to those pre-MSCT (△Ct: 11.0±0.9、7.4±2.1、7.8±2.1、0.1±1.5 respectively, p all< 0.05). Levels of IL10, IL21 and ANA were significantly lower 4 week post-MSCT than those before (p< 0.05); while level of CCL2 was higher than pre-MSCT (p<0.05). Cytokines and ANA levels 1 week after MSCT were not differentially changed comparing to those pre-MSCT. Alteration of OAZ mRNA expression levels pre-and post-MSCT were negatively correlated with changes of ANA, IL21 levels and positively correlated with changes of IL12/IL10 and CCL2 levels.Conclusion 1. OAZ pathway is involved in the pathogenesis of SLE.2:OAZ siRNA can effectively reduce the expression of genes involving in the OAZ signaling pathway in SLE. OAZ may lead to abnormal production of ANA via regulating Id genes and cytokines.3:The expression of genes involving in the OAZ signaling pathway was effectively reduced along with the alteration of several cytokines and ANA after allogeneic MSCT in SLE patients. OAZ signaling pathway could play an important role in MSCT treatment for SLE.更多还原