基于抑制狼疮小鼠B淋巴细胞分化的Blimp-1 SiRNA防治狼疮肾炎的机制研究
摘要
SLE患者由于免疫耐受的异常和B淋巴细胞发育过程中存在轻重不一的缺陷,表现为身体内明显增多的对自身组织反应的B淋巴细胞,从而会生成以抗核抗体和双链DNA抗体为代表的多种多样的自身抗体,随时间发展越来越多的免疫复合物沉积最后临床出现结缔组织病[1-3]。同时B淋巴细胞内及细胞间也存在各种异常：1.信号转导异常如细胞内酪氨酸磷酸化及激酶水平升高,涉及细胞分化、存活、迁移和细胞周期异常等。2.B细胞相关的细胞因子失调如B淋巴细胞刺激因子(B-lymphocyte stimulator, BLyS)活性明显增加远远超过正常[4,5]。3.B细胞相关亚群异常如调节性B细胞功能缺陷。针对自身的B淋巴细胞免疫反应被多种因素增强。
作为锌指蛋白家族成员之一的B淋巴细胞诱导成熟蛋白-1(B lymphocyte induced maturation protein-1, Blimp-1)是由Turner于1994年报道[6，7]。作为DNA结合蛋白的Blimp-1由PR区锌指蛋白1(PRDMl)基因编码,主要功能是抑制转录。其中B淋巴细胞相关转录因子是被抑制的重要对象,它决定B细胞终末分化,促进B细胞分化为能够分泌免疫球蛋白的浆细胞,并影响它的存活。B1imp-1表达异常会导致自身免疫功能失调,诱发系统性红斑狼疮等自身免疫相关疾病[8,9]。
研究认为Blimp-1促进B细胞分化主要通过调节三类因子表达：1.Blimp-1下调c-Myc、E2F1抑制B细胞分裂和增殖；2.Blimp-1促进J链、XBP-1表达及Ig重链和轻链重排,增强抗体分泌；3.Blimp-1下调生发中心B细胞Pax-5、 bcl-6表达。Blimp-1表达而发挥促B淋巴细胞分化作用需要抑制因子Bcl-6、 Pax-5. MITF和诱导因子XBP-1、 IRF4、 AP-1、 NF-KB、 STAT3之间平衡[10,11]。Blimp-1基因是狼疮性肾炎的一个易感基因,其可能在系统性红斑狼疮发生发展中起关键性作用。Garaud JC12]等研究发现红斑狼疮患者外周血B细胞Blimp-1mRNAs呈高表达。Panchanathan R13实验中Blimp-1基因表达缺失雌性小鼠产生高滴度的狼疮样自身抗体。国内牛晓昶[14]研究狼疮病B1imp-1蛋白高表达,成熟浆细胞数明显增加。总之目前实验证据表明Blimp-1与包括红斑狼疮在内的多种自身免疫性疾病有密切关联。
RNA干扰(RNA interference, RNAi)现象[15]朝是把体外与自身mRNA互补的双链RNA通过各种基因转移手段导入细胞,导致与自身序列互补相同的mRNA降解。产生结果是阻止了相关mRNA翻译,也就是关于此mRNA的基因不再表达而变为沉默基因,即通常说的转录后基因沉默。1990年约根森(Jorgensen)[16]研究小组在研究查尔酮合成酶对花青素合成速度的影响时发现RNA干扰现象。之后这种现象逐渐被验证并于1998年由Andrew Z. Fire17]等详细阐述。目前,以核酸为基础RNAi技术在癌症和自身免疫相关疾病治疗中开始展现良好前景。
成熟浆细胞产生的多种自身抗体在系统性红斑狼疮患者病情中起决定性作用[3]。Blimp-1决定B淋巴细胞终末分化和多种自身反应性抗体分泌,Blimp-1异常与红斑狼疮的发生密切相关[14]。基于以上背景为了探讨Blimp-1在狼疮发展中的潜在作用,我们构建了编码Blimp-1SiRNA的腺病毒载体。探讨Blimp-1SiRNA对Blimp-1表达及CD138+B细胞比例影响,进而观察B淋巴细胞分化情况。另外从SLE临床角度研究了Blimp-1SiRNA对系统性红斑狼疮病情作用,包括(抗双链DNA抗体、尿蛋白、肾功能、血清白蛋白等)。
方法：基因库获得Blimp-1互补DNA,合成Blimp-1SiRNA序列。扩增鉴定后与表达质粒PDC315载体连接,获得阳性克隆质粒命名为PDC315-RNAi。 Adeno-XTM载体系统与PDC315-RNAi及骨架质粒pBHGloXEl.3Cre经酶切后重组,获得阳性克隆质粒命名为pAd-Blimp-1/siRNAt18-20。 Lipofectamine2000试剂将pAd-Blimp-1/siRNA载体转染293细胞,3次感染得第四代重组腺病毒pAd-Blimp-1/siRNA。同法获得第四代重组腺病毒pAd-siRNA/LacZ作为阴性对照。12周BWF1小鼠尾静脉注射pAd-siRNA/Blimp-1(1*109空斑形成单位)。对照组分别注射pAd-siRNA/LacZ(1*109空斑形成单位)和PBS溶液100ul。每三周处死小鼠用流式细胞仪分析CD138+细胞比率。对腺病毒注射21天小鼠分别应用RT-PCR检测Blimp-1mRNA表达Western-Blot检钡Blimp-1蛋白、XBP-1蛋白、BCMA蛋白及C-myc蛋白表达。每三周对小鼠酶联免疫分析抗双链DNA抗体滴度(u/m1),自动生化仪检测尿素氮、肌酐、血清白蛋白,考马斯亮蓝法测定尿蛋白含量[21,22],同时观察各组小鼠生存期,腺病毒注射18周时分别观察各组小鼠肾组织病理变化。
结果：
1.构建了编码B1imp-1SiRNA重组腺病毒载体。
2.对照组12周后CD138+B细胞比例开始上升,24周达到高峰8.51±3.08%。而Ad-siRNA/Blimp-1组一直保持相对稳定(p<0.05)。
3. Ad-siRNA/Blimp-1组RT-PCR示脾脏B细胞Blimp-1mRNA表达抑制率68%,Blimp-170.6％、XBP-1.BCMA蛋白表达下降,C-Myc蛋白表达上升,与对照组比较p<0.05.
4.Ad-siRNA/Blimp-1组抗双链DNA抗体滴度维持在基线,对照组抗体水平从200—3000u/ml逐步上升(p<0.05),15周后白蛋白下降缓慢。
5.模型鼠自第3周开始出现尿蛋白升高,Ad-SiRNA/Blimp-1干扰组从12周尿蛋白排泄明显减少,较对照组明显下降(p＜0.05),Ad-SiRNA/LacZ组与PBS组比较差异无统计学意义p＞0.05(12周p＜0.05)。
6.各组模型小鼠血清肌酐24周观察期呈现升高趋势,12周前三组肾功能均正常,变化不明显,组间无统计学差异p＞0.05。15周对照组血清肌酐开始明显升高,之后9周发展较快。与对照组比较p＜0.01。
7.Ad-siRNA/Blimp-1组小鼠15周无死亡,对照组15周(造模6月)开始死亡,PBS组2只,Ad-SiRNA/LacZ3只,直至24周(造模9月)时对照组小鼠死亡PBS组7只,Ad-SiRNA/LacZ死亡8只,Ad-siRNA/Blimp-1组存活8只,以存活比例和尾静脉注射后死亡时间作Kaplan-meier生存曲线,对照组小鼠生存期明显缩短,经Log rank检验Ad-siRNA/Blimp-1组和对照组比较有统计学差异,Log值6.80,P=0.03,(p<0.05).三组半数生存期分别为：>24W(Ad-siRNA/Blimp-1组),21W(Ad-SiRNA/LacZ),23W(PBS组).
8.Ad-s iRNA/Blimp-1组肾小球体积轻度增大,仅有轻度系膜细胞基质增生,小球炎症细胞浸润轻,无间质纤维化,血管壁轻度增厚,小管周围炎症细胞轻度增生。模型对照组肾小球体积增大,弥漫性增生,系膜基质增加,炎症细胞侵润重,PBS组小球节段结构不清,血管壁结构破坏。肾小球和小管损伤计分与Ad-siRNA/Blimp-1组比较p<0.05。
结论：
1.Blimp-1SiRNA解除了对C-myc途径抑制,使其上调表达,下调了B淋巴细胞Blimp-1、 XBP-1、 BCMA蛋白及Blimp-1mRNA表达,抑制活体B细胞向浆细胞分化。
2.Blimp-1SiRNA降低了狼疮鼠双链DNA抗体滴度,进而降低了蛋白尿,升高血清白蛋白,保护狼疮鼠肾脏功能,减轻炎性细胞在肾组织沉积,阻止狼疮发展。
3. Blimp-1SiRNA可能给我们提供一条通过阻止B细胞分化治疗狼疮的新道路。
Background
due to the abnormal of B-lymphocytes and the defect of immune tolerance,there are all kinds of autoantibodies and large amounts of self reactive B-cells in SLE patients that will result in autoimmunity[1-3]. There are various intracellular and intercellular abnormalities of B-lymphocytes cells,such as the increasing tyrosine phosphorylation and phosphate tyrosine kinase, excessive activation of B-lymphocyte stimulator,the defect of B lymphocyte subpopulation, cell survival, migration and differentiation, generation cycle[4,5]. These factors promotes B-cell immune responses.
Blimpl(B-lymphocyte-induced maturation protein1) is one of the family that contains five zinc finger motifs[6,7]. As a DNA-binding protein which is encoded by zinc finger protein1(PRDM1) gene, the main function of blimp-1were to enhance the terminal differentiation of B-lymphocytes into plasma cells that secrete immunoglobulin by it's inhibition of transcription. The expression of Blimp-1can cause auto-immune dysfunction and autoimmune-related diseases such as Systemic Lupus Erythematosus[8,9].
Related studies suggest that Blimp-1promotes B-cell differentiation bydown-regulation of c-Myc and E2F1to inhibit the proliferation of B cells, up-regulation of j chain and XBP-1to enhance Secretory capacity and down-regulation of Pax-5and bcl-6in germinal center.To play a correct roles requires the balance of Bcl-6, Pax-5, MITF,XBP-1, IRF4, AP-1, NF-KB and STAT3[10,11].
Blimp-1which is a susceptibility gene may play a key role in the development of Systemic Lupus Erythematosus. The study by Garaud JC [l2］found that the expression of Blimp-1mRNAs is high in B cells of peripheral blood in patients with Systemic Lupus In the experiment by Panchanathan R [13]there were high titer autoantibodies in female mice with systemic lupus erythematosus that was knocked Blimp-1gene. Domestic study of Niu Xiaochang[14] reported Blimp-1protein and mature plasma cells increased significantly in Lupus patients.In short,the current experimental evidences showed that there are closely interrelated between Blimp-1and various autoimmune diseases, including Lupus Erythematosus.
RNA interference (RNA interference,RNAi) is a phenomena that the exogenous double-stranded RNA was import into the cells and cause mRNA degradation with similar sequences[15]. This expression by the same sequence of mRNA shut downed is post-transcriptional gene silencing. In1990, RNA interfere was discovered by Joergensen (Jorgensen)[16] research group during the study about the synthesis speed of chalcone synthase on anthocyanin. This phenomenon was gradually verified and elaborated in1998by Andrew Z.Fire[17]. At present, based on RNAi technology it shows a good prospect in the diseases of cancer and autoimmune.
Methods:
To explore the potential role of Blimp-1in development of lupus, we constructed the adenovirus encoding Blimp-1siRNA. We observe effects on the expression of Blimp-1and the percentage of CD138+ B cells in order to Understand differentiation of B lymphocytes. Besides we study clinical indicators of lupus mice including antibody against double-stranded DNA, proteinuria, renal function and serum albumin.The complementary DNA sequence of Blimp-1was obtained from GenBank. The potential target sequence for RNA interference (RNAi) was scanned and subcloned into shuttle vector pDC315and sequenced. The desired replication-deficient adenovirus containing the full-lengthcDNA of siRNA was generated by homologous recombination of plasmids pDC315-RNAi and pBHG1oXE1.3Cre[18-20]。 Through co-transfection pAd-Blimp-1/siRNA was infected into293cells by Lipofectamine2000. After several rounds of plaque purification, the adenovirus was amplified and purified from cell lysates by banding twice in CsCl density gradients. Using same measure the fourth-generation adenovirus pAd- siRNA/LacZ was obtained as a negative control.
12-week-old BWF1mice received an intravenous tail vein injection of Ad-siRNA/Blimp-1(1 *109plaque forming units [Pfu]). And the control mice received Ad-siRNA/LacZ(1*109Pfu) or100ul PBS. The mice were sacrificed and the B cells were isolated and analyzed for CD45RCD138+ plasma cell percentage by flow cytometry assay with3weeks interval, mRNA expression of Blimp-1detected by RT-PCR, Protein expression ofBlimp-1, XBP-1, BCMA and c-myc was detected by Western blotting. The anti-dsDNA Ab concentrations in mouse blood serum were determined by ELISA assay with3weeks interval. Bun,cr,albumin were estimated by automatic biochemical Analyzer, proteinuria was detected by Bradford method with3weeks interval[121,22]. In addition, kidney tissue of lupus mice after18weeks and the lifespan of lupus mice was observed.
Results:
1.The recombinant adenovirus vector encoding Blimp-1siRNA was constructed and delivered to the BWF1mice Successfully.
2.1n the control groups, the CD138+ plasma cell percentage of B cells began to increase after12weeks of administration,and reached a plateau at8.5% of B cells after24weeks.However, after administration of Ad-siRNA/Blimp-1, CD138+ plasma cell percentage remained constant (P<0.05).
3.Blimp-1mRNA and protein were strongly inhibited in B cells as detected by RT-PCR and Western blot. The inhibition rates of Blimp-1mRNA after infection with specific Blimp-1siRNA was68%. The inhibition rate of Blimp-1protein after.infection with specific Blimp-1siRNA was70.6%. The results demonstrated that Blimp-1expression was decreased significantly after infection with specific Blimp-1siRNA compared with control groups.(P<0.05) The western blot demonstrated that Ad-siRNA/Blimp-1decreased XBP-1, BCMA and increased c-myc expression.Compared with controls, P<0.05.
4.the anti-dsDNA Ab level remained at baseline levels after administer-ation of Ad-siRNA/Blimp-1. However,the anti-dsDNA Ab level began to increase steadily from200to3000U/ml in the control groups (P<0.05).
5. Elevated urinary protein in mouse models began to appear since the3rd week, compared with the control groups there were Significant decrease in proteinuria from12weeks (p<0.05), but there wasn't difference in the controls.p>0.05.
6. Serum creatinine apears a rising trend in the whole observation period of each group.Before12weeks kidney functions are normal and there were no significant differences between the three groups p>0.05. After12weeks serum creatinine rapidaly increased and got faster, it had shown a clear distinction Compared with the control group p<0.01.
7. At15weeks it began to appear death in the control group s(modeled6months),2in PBS and3in Ad-SiRNA/LacZ group, at24weeks (modeled9months) the survival mice was8,but3in PBS group and2in other control group.The survival time of control groups were clearly shorter by Kaplan-meier analyse.Through Log rank test there was difference in the three groups.the value was6.08,p=0.03. Median survival time respectively were24W(Ad-siRNA/Blimp-1),21W(Ad-SiRNA/LacZ),23W(PBS).
8. In Ad-siRNA/Blimp-1group glomerular volume slightly increased, with only mild hyperplasia of mesangial cell and matrix, inflammatory cell infiltration in glomerular and tubular. However in control group Glomerular volume increases, diffuse hyperplasia in mesangial matrix, inflammatory cell invasion, in PBS group the structure of glomerular is not clear, the same structural damage appered in the vessel wall. compared with the Ad-siRNA/Blimp-1group by glomerular and tubular injury score p<0.05.
Conclusion:
1. Ad-siRNA/Blimp-1could inhibit B cells differentiation to plama cell by regulating the XBP-1,BCMA and c-myc expression.
2. Ad-siRNA/Blimp-1could prevent anti-dsDNA Abproduction, decrease proteinuria, increase albumin,protect the function prevent thedevelopment of lupus.
3. Ad-siRNA/Blimp-1may provided the new clue to the restore differentiation therapeutical strategy in lupus disease.
作为锌指蛋白家族成员之一的B淋巴细胞诱导成熟蛋白-1(B lymphocyte induced maturation protein-1, Blimp-1)是由Turner于1994年报道[6，7]。作为DNA结合蛋白的Blimp-1由PR区锌指蛋白1(PRDMl)基因编码,主要功能是抑制转录。其中B淋巴细胞相关转录因子是被抑制的重要对象,它决定B细胞终末分化,促进B细胞分化为能够分泌免疫球蛋白的浆细胞,并影响它的存活。B1imp-1表达异常会导致自身免疫功能失调,诱发系统性红斑狼疮等自身免疫相关疾病[8,9]。
研究认为Blimp-1促进B细胞分化主要通过调节三类因子表达：1.Blimp-1下调c-Myc、E2F1抑制B细胞分裂和增殖；2.Blimp-1促进J链、XBP-1表达及Ig重链和轻链重排,增强抗体分泌；3.Blimp-1下调生发中心B细胞Pax-5、 bcl-6表达。Blimp-1表达而发挥促B淋巴细胞分化作用需要抑制因子Bcl-6、 Pax-5. MITF和诱导因子XBP-1、 IRF4、 AP-1、 NF-KB、 STAT3之间平衡[10,11]。Blimp-1基因是狼疮性肾炎的一个易感基因,其可能在系统性红斑狼疮发生发展中起关键性作用。Garaud JC12]等研究发现红斑狼疮患者外周血B细胞Blimp-1mRNAs呈高表达。Panchanathan R13实验中Blimp-1基因表达缺失雌性小鼠产生高滴度的狼疮样自身抗体。国内牛晓昶[14]研究狼疮病B1imp-1蛋白高表达,成熟浆细胞数明显增加。总之目前实验证据表明Blimp-1与包括红斑狼疮在内的多种自身免疫性疾病有密切关联。
RNA干扰(RNA interference, RNAi)现象[15]朝是把体外与自身mRNA互补的双链RNA通过各种基因转移手段导入细胞,导致与自身序列互补相同的mRNA降解。产生结果是阻止了相关mRNA翻译,也就是关于此mRNA的基因不再表达而变为沉默基因,即通常说的转录后基因沉默。1990年约根森(Jorgensen)[16]研究小组在研究查尔酮合成酶对花青素合成速度的影响时发现RNA干扰现象。之后这种现象逐渐被验证并于1998年由Andrew Z. Fire17]等详细阐述。目前,以核酸为基础RNAi技术在癌症和自身免疫相关疾病治疗中开始展现良好前景。
成熟浆细胞产生的多种自身抗体在系统性红斑狼疮患者病情中起决定性作用[3]。Blimp-1决定B淋巴细胞终末分化和多种自身反应性抗体分泌,Blimp-1异常与红斑狼疮的发生密切相关[14]。基于以上背景为了探讨Blimp-1在狼疮发展中的潜在作用,我们构建了编码Blimp-1SiRNA的腺病毒载体。探讨Blimp-1SiRNA对Blimp-1表达及CD138+B细胞比例影响,进而观察B淋巴细胞分化情况。另外从SLE临床角度研究了Blimp-1SiRNA对系统性红斑狼疮病情作用,包括(抗双链DNA抗体、尿蛋白、肾功能、血清白蛋白等)。
方法：基因库获得Blimp-1互补DNA,合成Blimp-1SiRNA序列。扩增鉴定后与表达质粒PDC315载体连接,获得阳性克隆质粒命名为PDC315-RNAi。 Adeno-XTM载体系统与PDC315-RNAi及骨架质粒pBHGloXEl.3Cre经酶切后重组,获得阳性克隆质粒命名为pAd-Blimp-1/siRNAt18-20。 Lipofectamine2000试剂将pAd-Blimp-1/siRNA载体转染293细胞,3次感染得第四代重组腺病毒pAd-Blimp-1/siRNA。同法获得第四代重组腺病毒pAd-siRNA/LacZ作为阴性对照。12周BWF1小鼠尾静脉注射pAd-siRNA/Blimp-1(1*109空斑形成单位)。对照组分别注射pAd-siRNA/LacZ(1*109空斑形成单位)和PBS溶液100ul。每三周处死小鼠用流式细胞仪分析CD138+细胞比率。对腺病毒注射21天小鼠分别应用RT-PCR检测Blimp-1mRNA表达Western-Blot检钡Blimp-1蛋白、XBP-1蛋白、BCMA蛋白及C-myc蛋白表达。每三周对小鼠酶联免疫分析抗双链DNA抗体滴度(u/m1),自动生化仪检测尿素氮、肌酐、血清白蛋白,考马斯亮蓝法测定尿蛋白含量[21,22],同时观察各组小鼠生存期,腺病毒注射18周时分别观察各组小鼠肾组织病理变化。
结果：
1.构建了编码B1imp-1SiRNA重组腺病毒载体。
2.对照组12周后CD138+B细胞比例开始上升,24周达到高峰8.51±3.08%。而Ad-siRNA/Blimp-1组一直保持相对稳定(p<0.05)。
3. Ad-siRNA/Blimp-1组RT-PCR示脾脏B细胞Blimp-1mRNA表达抑制率68%,Blimp-170.6％、XBP-1.BCMA蛋白表达下降,C-Myc蛋白表达上升,与对照组比较p<0.05.
4.Ad-siRNA/Blimp-1组抗双链DNA抗体滴度维持在基线,对照组抗体水平从200—3000u/ml逐步上升(p<0.05),15周后白蛋白下降缓慢。
5.模型鼠自第3周开始出现尿蛋白升高,Ad-SiRNA/Blimp-1干扰组从12周尿蛋白排泄明显减少,较对照组明显下降(p＜0.05),Ad-SiRNA/LacZ组与PBS组比较差异无统计学意义p＞0.05(12周p＜0.05)。
6.各组模型小鼠血清肌酐24周观察期呈现升高趋势,12周前三组肾功能均正常,变化不明显,组间无统计学差异p＞0.05。15周对照组血清肌酐开始明显升高,之后9周发展较快。与对照组比较p＜0.01。
7.Ad-siRNA/Blimp-1组小鼠15周无死亡,对照组15周(造模6月)开始死亡,PBS组2只,Ad-SiRNA/LacZ3只,直至24周(造模9月)时对照组小鼠死亡PBS组7只,Ad-SiRNA/LacZ死亡8只,Ad-siRNA/Blimp-1组存活8只,以存活比例和尾静脉注射后死亡时间作Kaplan-meier生存曲线,对照组小鼠生存期明显缩短,经Log rank检验Ad-siRNA/Blimp-1组和对照组比较有统计学差异,Log值6.80,P=0.03,(p<0.05).三组半数生存期分别为：>24W(Ad-siRNA/Blimp-1组),21W(Ad-SiRNA/LacZ),23W(PBS组).
8.Ad-s iRNA/Blimp-1组肾小球体积轻度增大,仅有轻度系膜细胞基质增生,小球炎症细胞浸润轻,无间质纤维化,血管壁轻度增厚,小管周围炎症细胞轻度增生。模型对照组肾小球体积增大,弥漫性增生,系膜基质增加,炎症细胞侵润重,PBS组小球节段结构不清,血管壁结构破坏。肾小球和小管损伤计分与Ad-siRNA/Blimp-1组比较p<0.05。
结论：
1.Blimp-1SiRNA解除了对C-myc途径抑制,使其上调表达,下调了B淋巴细胞Blimp-1、 XBP-1、 BCMA蛋白及Blimp-1mRNA表达,抑制活体B细胞向浆细胞分化。
2.Blimp-1SiRNA降低了狼疮鼠双链DNA抗体滴度,进而降低了蛋白尿,升高血清白蛋白,保护狼疮鼠肾脏功能,减轻炎性细胞在肾组织沉积,阻止狼疮发展。
3. Blimp-1SiRNA可能给我们提供一条通过阻止B细胞分化治疗狼疮的新道路。
Background
due to the abnormal of B-lymphocytes and the defect of immune tolerance,there are all kinds of autoantibodies and large amounts of self reactive B-cells in SLE patients that will result in autoimmunity[1-3]. There are various intracellular and intercellular abnormalities of B-lymphocytes cells,such as the increasing tyrosine phosphorylation and phosphate tyrosine kinase, excessive activation of B-lymphocyte stimulator,the defect of B lymphocyte subpopulation, cell survival, migration and differentiation, generation cycle[4,5]. These factors promotes B-cell immune responses.
Blimpl(B-lymphocyte-induced maturation protein1) is one of the family that contains five zinc finger motifs[6,7]. As a DNA-binding protein which is encoded by zinc finger protein1(PRDM1) gene, the main function of blimp-1were to enhance the terminal differentiation of B-lymphocytes into plasma cells that secrete immunoglobulin by it's inhibition of transcription. The expression of Blimp-1can cause auto-immune dysfunction and autoimmune-related diseases such as Systemic Lupus Erythematosus[8,9].
Related studies suggest that Blimp-1promotes B-cell differentiation bydown-regulation of c-Myc and E2F1to inhibit the proliferation of B cells, up-regulation of j chain and XBP-1to enhance Secretory capacity and down-regulation of Pax-5and bcl-6in germinal center.To play a correct roles requires the balance of Bcl-6, Pax-5, MITF,XBP-1, IRF4, AP-1, NF-KB and STAT3[10,11].
Blimp-1which is a susceptibility gene may play a key role in the development of Systemic Lupus Erythematosus. The study by Garaud JC [l2］found that the expression of Blimp-1mRNAs is high in B cells of peripheral blood in patients with Systemic Lupus In the experiment by Panchanathan R [13]there were high titer autoantibodies in female mice with systemic lupus erythematosus that was knocked Blimp-1gene. Domestic study of Niu Xiaochang[14] reported Blimp-1protein and mature plasma cells increased significantly in Lupus patients.In short,the current experimental evidences showed that there are closely interrelated between Blimp-1and various autoimmune diseases, including Lupus Erythematosus.
RNA interference (RNA interference,RNAi) is a phenomena that the exogenous double-stranded RNA was import into the cells and cause mRNA degradation with similar sequences[15]. This expression by the same sequence of mRNA shut downed is post-transcriptional gene silencing. In1990, RNA interfere was discovered by Joergensen (Jorgensen)[16] research group during the study about the synthesis speed of chalcone synthase on anthocyanin. This phenomenon was gradually verified and elaborated in1998by Andrew Z.Fire[17]. At present, based on RNAi technology it shows a good prospect in the diseases of cancer and autoimmune.
Methods:
To explore the potential role of Blimp-1in development of lupus, we constructed the adenovirus encoding Blimp-1siRNA. We observe effects on the expression of Blimp-1and the percentage of CD138+ B cells in order to Understand differentiation of B lymphocytes. Besides we study clinical indicators of lupus mice including antibody against double-stranded DNA, proteinuria, renal function and serum albumin.The complementary DNA sequence of Blimp-1was obtained from GenBank. The potential target sequence for RNA interference (RNAi) was scanned and subcloned into shuttle vector pDC315and sequenced. The desired replication-deficient adenovirus containing the full-lengthcDNA of siRNA was generated by homologous recombination of plasmids pDC315-RNAi and pBHG1oXE1.3Cre[18-20]。 Through co-transfection pAd-Blimp-1/siRNA was infected into293cells by Lipofectamine2000. After several rounds of plaque purification, the adenovirus was amplified and purified from cell lysates by banding twice in CsCl density gradients. Using same measure the fourth-generation adenovirus pAd- siRNA/LacZ was obtained as a negative control.
12-week-old BWF1mice received an intravenous tail vein injection of Ad-siRNA/Blimp-1(1 *109plaque forming units [Pfu]). And the control mice received Ad-siRNA/LacZ(1*109Pfu) or100ul PBS. The mice were sacrificed and the B cells were isolated and analyzed for CD45RCD138+ plasma cell percentage by flow cytometry assay with3weeks interval, mRNA expression of Blimp-1detected by RT-PCR, Protein expression ofBlimp-1, XBP-1, BCMA and c-myc was detected by Western blotting. The anti-dsDNA Ab concentrations in mouse blood serum were determined by ELISA assay with3weeks interval. Bun,cr,albumin were estimated by automatic biochemical Analyzer, proteinuria was detected by Bradford method with3weeks interval[121,22]. In addition, kidney tissue of lupus mice after18weeks and the lifespan of lupus mice was observed.
Results:
1.The recombinant adenovirus vector encoding Blimp-1siRNA was constructed and delivered to the BWF1mice Successfully.
2.1n the control groups, the CD138+ plasma cell percentage of B cells began to increase after12weeks of administration,and reached a plateau at8.5% of B cells after24weeks.However, after administration of Ad-siRNA/Blimp-1, CD138+ plasma cell percentage remained constant (P<0.05).
3.Blimp-1mRNA and protein were strongly inhibited in B cells as detected by RT-PCR and Western blot. The inhibition rates of Blimp-1mRNA after infection with specific Blimp-1siRNA was68%. The inhibition rate of Blimp-1protein after.infection with specific Blimp-1siRNA was70.6%. The results demonstrated that Blimp-1expression was decreased significantly after infection with specific Blimp-1siRNA compared with control groups.(P<0.05) The western blot demonstrated that Ad-siRNA/Blimp-1decreased XBP-1, BCMA and increased c-myc expression.Compared with controls, P<0.05.
4.the anti-dsDNA Ab level remained at baseline levels after administer-ation of Ad-siRNA/Blimp-1. However,the anti-dsDNA Ab level began to increase steadily from200to3000U/ml in the control groups (P<0.05).
5. Elevated urinary protein in mouse models began to appear since the3rd week, compared with the control groups there were Significant decrease in proteinuria from12weeks (p<0.05), but there wasn't difference in the controls.p>0.05.
6. Serum creatinine apears a rising trend in the whole observation period of each group.Before12weeks kidney functions are normal and there were no significant differences between the three groups p>0.05. After12weeks serum creatinine rapidaly increased and got faster, it had shown a clear distinction Compared with the control group p<0.01.
7. At15weeks it began to appear death in the control group s(modeled6months),2in PBS and3in Ad-SiRNA/LacZ group, at24weeks (modeled9months) the survival mice was8,but3in PBS group and2in other control group.The survival time of control groups were clearly shorter by Kaplan-meier analyse.Through Log rank test there was difference in the three groups.the value was6.08,p=0.03. Median survival time respectively were24W(Ad-siRNA/Blimp-1),21W(Ad-SiRNA/LacZ),23W(PBS).
8. In Ad-siRNA/Blimp-1group glomerular volume slightly increased, with only mild hyperplasia of mesangial cell and matrix, inflammatory cell infiltration in glomerular and tubular. However in control group Glomerular volume increases, diffuse hyperplasia in mesangial matrix, inflammatory cell invasion, in PBS group the structure of glomerular is not clear, the same structural damage appered in the vessel wall. compared with the Ad-siRNA/Blimp-1group by glomerular and tubular injury score p<0.05.
Conclusion:
1. Ad-siRNA/Blimp-1could inhibit B cells differentiation to plama cell by regulating the XBP-1,BCMA and c-myc expression.
2. Ad-siRNA/Blimp-1could prevent anti-dsDNA Abproduction, decrease proteinuria, increase albumin,protect the function prevent thedevelopment of lupus.
3. Ad-siRNA/Blimp-1may provided the new clue to the restore differentiation therapeutical strategy in lupus disease.
引文
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