基因与蛋白水平抑制B7/CD28信号通路对狼疮样肾炎模型的逆转效应及分子机制研究
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摘要
B7分子是表达于抗原提呈细胞（antigen-presenting cell，APC）表面的重要协同刺激分子。B7与CD28结合介导的协同刺激作用对初次免疫应答是必需的，若缺乏该信号，T细胞将进入无反应状态（Anergy）或免疫耐受（Tolerance），甚至引起程序性死亡（Apoptosis）。B7/CD28信号通路的过度活化与自身免疫性疾病的发生密切相关。
系统性红斑狼疮（systemic lupus erythematosus，SLE）是一种自身免疫性疾病，病因至今尚无明确定论。机体以出现T、B细胞活化增强，产生抗核抗体(antinuclearantibody，ANA)、抗双链DNA(double stranded DNA，dsDNA)抗体、形成免疫复合物（immune complex，IC）以及多种组织器官损伤为其特征。其中狼疮性肾炎（lupus nephritis，LN）是系统性红斑狼疮最常见和最严重的并发症，是导致患者死亡的主要原因。研究表明，B7/CD28信号通路在SLE的发病及病理损伤过程中发挥了重要的作用。
RNA干扰(RNA interference，RNAi)是由长约21-23个核苷酸的双链RNA分子(small interfering RNA，siRNA)介导,以序列特异的方式抑制同源基因表达的一项技术，属于转录后基因沉默(post-transcriptional gene silencing，PTGS)，能够迅速、特异、有效地抑制特定基因的表达。
特异性抗体与相应抗原的结合，可影响抗原分子介导的生物信号。B7分子的抑制性抗体通过封闭抗原分子，可阻止或削弱其与CD28结合而转导B7/CD28协同刺激信号，进而抑制T、B细胞的活化与应答。
本课题运用化学法（Pristane）诱导类似人类SLE发病过程及临床表现的小鼠（C57BL/6J）狼疮样肾炎模型，对其进行生物学鉴定的基础上，采用B7-1shRNA慢病毒表达载体及单克隆抗体进行整体干预，通过免疫学、血清学及病理损伤评价等指标的动态变化，探究在基因与蛋白水平抑制B7/CD28信号通路，对狼疮样肾炎模型的形成及病理损伤的逆转效应及其分子机制，以期对SLE疾病模型形成中免疫细胞活化及病理损伤的演变过程进行研究，同时为该类疾病寻找新的特异、高效、低毒的生物干预手段。
第一部分小鼠B7-1基因RNA干扰慢病毒载体的构建及对受体细胞膜型B7-1分子表达的干扰效应研究
目的：构建小鼠B7-1shRNA慢病毒表达载体，根据其对受体细胞膜型B7-1分子表达的干扰效应挑选最佳靶序列。方法：选择四条B7-1基因RNA干扰有效靶序列，合成靶序列的双链DNA，接入LV3（pGLV-H1-GFP+Puro）载体，再与pLV/helper-SL3、pLV/helper-SL4、pLV/helper-SL5质粒共转染293T细胞，包装产生慢病毒，依据绿色荧光蛋白（green fluorescent protein，GFP）的表达水平测定病毒滴度。慢病毒感染L929细胞，运用免疫荧光及流式细胞术，筛选对受体细胞膜型B7-1分子表达干扰效应较高的靶序列。结果：经过测序证实，LV3-B7-1shRNA慢病毒表达载体构建正确，病毒滴度达1×10~8TU/ml，适合感染L929细胞的复合感染（multiplicity of infection，MOI）值为60，此时感染效率为88.7%。四条候选的shRNA模板序列中，B7-1shRNA-256，对L929细胞膜型B7-1分子的沉默效率最高，达73.2%，以此作为最佳靶序列。结论：成功构建了小鼠B7-1基因RNA干扰慢病毒载体，其能有效沉默L929细胞膜型B7-1分子的表达。
第二部分鼠抗人B7-1分子功能性单克隆抗体的制备及生物学特性研究
目的：制备鼠抗人B7-1分子功能性单克隆抗体，并对其生物学特性进行研究。方法：运用本科室已成功获取的分泌鼠抗人B7-1单克隆抗体杂交瘤细胞株，采用体内诱生腹水法进行单抗的制备；ProteinG免疫亲和层析法纯化抗体；小鼠IgG亚类快速定性试纸鉴定单抗的亚类；间接免疫荧光法测定抗体效价；流式细胞术分析单抗对人及小鼠细胞膜型B7-1分子的识别；MTT法分析抗体对协同刺激信号的拮抗作用。结果：经体内诱生腹水法制备单克隆抗体，小鼠腹水形成阳性率约为80%，腹水收获量平均为5.9ml/只小鼠；经免疫亲和层析法纯化，腹水中抗体蛋白的得率为2.3mg/ml；单抗与细胞株Daudi、Raji、H1299以及小鼠脾脏细胞的阳性结合率分别为89.2%、92.7%、20.6%及48.5%；经MTT法分析，抗体能够阻断B7-1介导的协同刺激信号，从而抑制L929-B7-1刺激PBTCs的增殖效应。结论：成功制备了鼠抗人B7-1分子功能性单克隆抗体，体外研究结果显示，其能够有效阻断B7-1介导的协同刺激信号。
第三部分小鼠狼疮样肾炎模型的建立及生物学鉴定
目的：运用化学法建立小鼠（C57BL/6J）狼疮样肾炎模型，并对其进行生物学鉴定。方法：6-8周龄雌性C57BL/6J小鼠30只，随机分为造模组和正常对照组。造模组予一次性腹腔注射Pristane0.5ml/只，正常对照组予一次性腹腔注射生理盐水0.5ml/只。建模后第10天，每组处死5只小鼠，免疫荧光及流式细胞术(flowcytometry，FCM)分析脾脏中巨噬细胞（CD11b+）、树突状细胞（CD11c+）、粒细胞（Gr1+）及B细胞（CD21+）的阳性表达率，同时检测CD21+B细胞膜型协同刺激分子CD86及MHC-II分子的表达；每月定期活体眶经脉丛取血，检测血清中ANA及抗dsDNA抗体水平及采用Albustix试纸法检测尿蛋白量；持续观察至8个月时处死小鼠，HE染色观察小鼠肾脏病理损伤，直接免疫荧光法分析肾脏免疫复合物（IC）的沉积。结果：1.建模后第10天，脾脏中Mφ、DC、粒细胞及B细胞均出现不同程度的活化，其阳性表达率分别为7.27±0.85%、4.72±0.68%、9.82±1.26%及17.79±0.65%，与对照组相比明显升高，具有统计学差异（p<0.05）；同时，CD21+B细胞膜型协同刺激分子CD86及MHC-II分子表达上调，分别为38.69±3.14%及55.33±2.58%，与对照组具有统计学差异（p<0.05）。2.动态观察至3个月时，造模组30%小鼠血清ANA阳性，4个月时达80%，8个月时ANA阳性率为100%；建模后4个月，20%造模组小鼠可检测到抗dsDNA抗体，6个月时达60%，8个月时抗dsDNA抗体阳性率为80%。3.经动态分析发现，建模后4个月，20%小鼠开始出现蛋白尿，尿蛋白含量为+～++（300～1000mg/L），随着时间的推移，蛋白尿的阳性率和尿蛋白含量逐渐增加，8个月时100%小鼠出现蛋白尿，含量为++～++++（1000～20000mg/L）。4.肾脏冰冻切片直接免疫荧光法分析显示，造模组小鼠肾小球出现强阳性的IC沉积。5.肾脏组织病理学分析显示，造模组肾小球体积增大，细胞数量增多，肾小球血管充血明显，间质明显淋巴细胞浸润。结论：Pristane诱导的小鼠狼疮样肾炎模型作为研究人类SLE的疾病模型具有可靠性。
第四部分基因与蛋白水平抑制B7/CD28信号通路对狼疮样肾炎模型的逆转效应及分子机制研究
目的：运用B7-1shRNA慢病毒载体及B7-1单克隆抗体分别在基因与蛋白水平抑制B7/CD28信号通路，比较不同水平的干预，对狼疮样肾炎模型的形成及病理损伤的逆转效应及其分子机制，同时考察特异性抗体对该类疾病的治疗作用。方法：1.实验动物的分组处理：6-8周龄雌性C57BL/6J小鼠随机分为8组。A：正常对照组，一次性腹腔注射生理盐水0.5ml/只；B：造模组，一次性腹腔注射Pristane0.5ml/只；C：B7-1抗体早期干预组，腹腔注射Pristane0.5ml/只后，第1、3、5、8及15天分别尾静脉注射B7-1单抗200μg/只，然后每隔1个月注射一次，连续注射3个月；D：抗体同型对照组，注射等剂量的小鼠IgG1；E：慢病毒干扰组，腹腔注射Pristane0.5ml/只后，第1、60天分别尾静脉注射0.4x10~8TU的LV-B7-1shRNA；F：慢病毒空载组，腹腔注射Pristane0.5ml/只后，第1、60天分别尾静脉注射LV-NCshRNA；G：化学药物阳性对照（CTX）组，腹腔注射Pristane0.5ml/只后，第1、8、22、29、43、50、64、71、85、92天分别尾静脉注射CTX0.5ml（60mg/kg/次）；H：B7-1抗体延迟干预（治疗）组，腹腔注射Pristane0.5ml/只后第4个月，待出现蛋白尿及自身抗体时运用B7-1抗体，注射次数及剂量同C组。2.B7-1shRNA慢病毒感染后对脾脏APC膜型B7-1分子表达的干预效应：首次感染后第10天，取小鼠脾脏细胞，采用免疫荧光及FCM分析CD11b+B7-1+、CD11c+B7-1+及CD21+B7-1+双阳性细胞的百分率，以考察B7-1shRNA慢病毒载体干扰后对靶分子表达的沉默效应。3.脾脏细胞中免疫细胞的活化分析：各实验组小鼠在首次处理后第10天取小鼠脾脏细胞，采用免疫荧光及FCM分析其中Mφ（CD11b+）、DC（CD11c+）、粒细胞（Gr1+）及B细胞（CD21+）的阳性表达率，同时检测CD21+B细胞膜型协同刺激分子CD86及MHC-II分子的表达。4.ANA和抗dsDNA抗体检测：每月定期活体眶经脉丛取血，检测血清中ANA和抗dsDNA抗体水平。5.尿蛋白的检测：每月定期采用Albustix试纸法检测尿蛋白量。6.血清细胞因子的含量分析：采用ELISA法检测小鼠血清IL-4及IFN-γ水平。7.肾脏IC的沉积分析：采用直接免疫荧光法分析。8.肾脏组织病理改变分析：运用光镜及透射电镜进行分析。结果：1. B7-1shRNA慢病毒干扰后，脾脏细胞中CD11b+B7-1+、CD11c+B7-1+及CD21+B7-1+双阳性细胞的阳性表达率分别为58.43±2.87%、60.18±3.08%及52.85±2.75%，与对照组相比明显下降，差异具有显著性（p<0.05）。提示B7-1shRNA慢病毒干扰可有效抑制APC上靶分子的表达。2.运用慢病毒及抗体早期干预后，脾脏细胞中Mφ、DC、粒细胞及B细胞的活化下调，与造模组比较具有统计学差异（p<0.05），抗体组的下调作用强于慢病毒干扰组（p<0.05）。3.对ANA的分析结果表明，实验3个月时，慢病毒干扰组及抗体早期干预组小鼠，其血清稀释度在1:100时均为阴性，造模组出现1:100-1:300的ANA阳性结果。实验观察至8个月时，造模组ANA阳性率100%，抗体滴度在1:1000-1:3000之间，而抗体早期干预组、慢病毒干扰组及抗体延迟干预组小鼠，其ANA的滴度均出现下降，与造模组相比具有统计学差异（P<0.05），提示上述处理均可减少ANA的产生，但抗体早期干预组优于另两组（P<0.05）。4.实验进行4个月时，造模组出现抗dsDNA抗体，但滴度较低，约为1:10，8个月时，抗dsDNA抗体阳性率为90%，滴度升至1:100左右；抗体早期干预组、慢病毒干扰组及抗体延迟干预组抗dsDNA抗体滴度较造模组均降低，但无统计学差异（P>0.05）。5.血清中细胞因子的水平：B7-1抗体早期干预组及慢病毒干扰组小鼠血清IL-4水平分别为0.84±0.52pg/ml及0.88±0.64pg/ml，IFN-γ水平为3.06±0.38pg/ml及3.02±0.55pg/ml，均较造模组降低，差异有统计学意义（P<0.05）；而B7-1抗体延迟干预组小鼠血清IL-4及IFN-γ水平与造模组相比，无统计学差异（P>0.05）。6.尿蛋白生成及动态变化:造模组建模4个月时，20%小鼠开始出现蛋白尿，尿蛋白含量为+～++，随着时间的推移，蛋白尿的阳性率和尿蛋白含量增加，8个月时100%小鼠出现蛋白尿，尿蛋白含量为++～++++（1000～20000mg/L），随着时间的延长，蛋白尿程度加重；慢病毒干扰组及B7-1抗体延迟干预组蛋白尿程度减轻，尿蛋白含量为±～+++（≤3000mg/L），与造模组相比，差异有统计学意义（P<0.05）；B7-1抗体早期干预组蛋白尿程度最轻，尿蛋白含量为±～++（≤1000mg/L），与慢病毒干扰组及B7-1抗体延迟干预组相比，亦具有统计学差异（P<0.05）。7.肾脏IC沉积分析显示：造模组肾小球出现强阳性的IC沉积，慢病毒干扰组及抗体干预组小鼠肾脏IC沉积减少，B7-1抗体早期干预组IC沉积最少。8.肾脏组织病理改变的光镜分析结果显示：造模组大部分小鼠肾小球体积中-重度增大，细胞数量明显增多，肾小球血管充血明显，间质淋巴细胞浸润严重，纤维组织增生；慢病毒干扰组及抗体干预组小鼠肾小球体积轻-中度增大，细胞数轻度增多，肾小球血管轻度充血，间质淋巴细胞浸润减少，B7-1抗体早期干预组光镜下病理损伤最轻。9.肾脏超微结构的透射电镜分析显示：造模组大部分小鼠肾小球内皮细胞下有大块状的电子致密物沉积，部分小鼠系膜内、上皮细胞下及基底膜内也有电子致密物沉积，足细胞间突起广泛融合，足细胞表面有假绒毛形成，基膜增厚；慢病毒干扰组及抗体干预组小鼠肾小球内皮细胞下有少量电子致密物沉积，足细胞间突起部分融合，基膜稍增厚，B7-1抗体早期干预组电镜下超微结构改变最少。结论：运用特异的RAN及抗体均能抑制B7-1分子介导的信号通路，从而降低免疫细胞的活化及逆转疾病模型病理损伤的程度，但抗体的干预效果优于RNA干扰；特异性抗体不仅对狼疮样肾炎的形成有预防作用，而且对已经形成的狼疮样肾炎具有治疗作用，且早期干预效果更好。
B7-1molecule is an important co-stimulatory molecule expressed on the surface ofantigen-presenting cell (APC), and its combination to the receptor CD28can generatecostimulatory signals, which is essential to the primary immune response. Without thiscostimulatory signal, T cells will enter to a state of anergy, tolerance even apoptosis.Thehyperreaction of B7/CD28signal is closely related to the occurrence of autoimmunediseases.
Systemic lupus erythematosus (SLE) is a common autoimmune disease whichetiology is uncertain characterized by T、B lymphocyte dysfunction, a variety ofantoantibodies, especially antinuclear antibody (ANA) and anti double-stranded DNA(dsDNA) antibody, immune complex formation and many organs damage.Lupusnephritis (LN) is one of the most common and serious complications of SLE, which isalso the primary cause leading to death of patients. Some studies have revealed thatB7/CD28signal participated in the pathogenesis of SLE.
RNA interference (RNAi) is a kind of technology mediated by double strandedRNA with21-23nucleotides-small interfering RNA (siRNA), which can suppresshomologene expression by specific sequence, belonging to post-transcriptional genesilencing (PTGS). It can suppress definite gene expression quickly, specificly andefficiently.
Specific antibody interaction with its ligand leads to inhibit or stimulate biologicsignal. Blocking antibody of B7molecule can prevent or down-regulate its interactionwith CD28, then inhibit the activation and response of T, B lymphocytes.
In the present study, we established a C57BL/6J mouse model with lupus-likedisease induced by Pristane, which is similar to clinical pathogenesy and manifestationof patients with SLE. Based upon the model, we apply the lentiviral expression vector B7-1shRNA and mouse anti-human B7-1monoclonal antibody to the model mouse inorder to evaluate its reverse effect and molecular mechanism by blocking B7/CD28signal pathway, which could provide some experimental and theoretical data, even finda biologic intervention which is specific, efficient and harmfulless for SLE.
Part Ⅰ Construction of lentiviral vector targeting mouse B7-1gene byRNA interference and evaluation its disturbing effect on theexpression of membrane B7-1molecule
Object: To construct lentiviral expression vector targeting mouse B7-1gene byshort hairpin RNA (shRNA), evaluate its disturbing effect on the expression ofmembrane B7-1molecule and select an optimal target sequence. Methods: Thecomplementary DNA containing both sense and antisense oligonucleotides of thetargeting sequence was designed, synthesized. After annealed, double-stranded DNAwas inserted into the LV3（pGLV-H1-GFP+Puro）vector.293T cells were cotransfectedwith pLV/helper-SL3、pLV/helper-SL4and pLV/helper-SL5. The titer of virus wastested according to the expression level of GFP. Mouse L929fibroblast cells wasinfected by the recombinant lentiviruses, the optimal target sequence was selectedaccording to the disturbing effect on the expression of membrane B7-1molecule byflow cytometry and immunofluorescence Results: DNA sequencing demonstrated thatlentivirus plasmid LV3-B7-1shRNA was constructed successfully. The titer of therecombinant lentivirus was1×10~8TU/ml, the best MOI for lentivirus infecting L929cell was60and infection efficiency was88.7%. As for the four shRNA templatesequence candidates, the optimal target sequence is B7-1shRNA-256, its silencingefficiency of the membrane B7-1molecule on L929cells reached the highest rate for73.2%. Conclusions: Lentiviral expression vectors targeting mouse B7-1gene by RNAinterference was constructed successfully. The recombinant lentivirus can effectivelysilence the membrane B7-1molecule on L929cells.
Part Ⅱ Preparation and assay of mouse anti-human B7-1antibody
Object: To obtain pure B7-1antibody, analyze its titer and recognition of membrane B7-1molecule. Methods: Using of the4E5cell line our institutesuccessfully established, ascites were induced to produce the antibody from BALB/cmouse and purified with ProteinG affinity chromatography method. The Ig isotype of4E5was identified with the rapid test paper. Flow cytometry assay was used to test theability of4E5to recognize the membranes B7-1molecule on different cells. Theblocking effect on costimulatory signals of4E5was detected by MTT assay. Results:The positive rate of ascites formation in mice was about80%. Ascites harvest was5.9ml from each BALB/c mouse on average. The purified4E5from ascites was about2.3mg/ml. The4E5could specifically recognize membrane B7-1on cells of Daudi、Raji、H1299、spleen cells of mouse with the positive rate of88.2%、92.7%、20.6%,48.5%,respectively. In addition,4E5could inhibit the growth and proliferation of L929-B7-1ce11s and PBTCs through blocking the B7-1costimulatory signals with MTT assay.Conclusions: Mouse anti-human B7-1monoclonal antibody which can efficiently blockthe B7-1costimulatory signals in vitro was successfully obtained.
Part Ⅲ Establishment and evaluation of mouse lupus nephritis model
Object: To establish and evaluate of lupus nephritis model in C57BL/6J miceinduced by Pristane. Methods: Thirty female mice aged6-8weeks were divided intotwo groups randomly. The model-making group was injected with0.5ml Pristane byintraperitoneal while the control group with0.5ml saline.The activation ofmacrophages、dendritic cells、granulocytes and B cells in spleens and the expression ofcostimulatory molecule CD86and MHC-II on B cells were measured byimmunofluorescence and FCM at day10after injection. ANA、anti-dsDNA antibodiesin serum and proteinuria were detected monthly after injection.8months after injection,all mice were killed and kidneys were slided and stained with H&E or FITC-labeledIgG to observe the evidence of glomerulonephritis histopathologically. Results:1.10days after injection, macrophages、dendritic cells、granulocytes and B cells in spleenswere activated, positive expression were7.27±0.85%、4.72±0.68%、9.82±1.26%and17.79±0.65%respectively, significantly higher than that of control group (p<0.05).Meanwhile, the expression of costimulatory molecule CD86and MHC-II in CD21+Bcells up-regulates to38.69±3.14%and55.33±2.58%, which was also significantly higher compared to the control group (p<0.05).2. The concentration of ANA andanti-dsDNA antibody in mice sera showed the same trend. The concentration of ANAwas up-regulated from30%to100%, after3months injection to8months by dynamicmonitoring. Anti-dsDNA antibody was up-regulated from20%to80%.3. Four monthsafter injection, the urine protein of model group was detected with+～++（300～1000mg/L）in20%model-making group mice. Finally++～++++（1000～20000mg/L）in100%model mice were tested as time went by.4. Immunofluorescence intensity ofimmune complex in model-making group mice was strong positive.5.Kidneyhistopathology analysis showed that glomerular volume enlarged severely, the numberof cell significantly increased, and fibrous tissue appeared, interstitial tissue wasinfiltrated by lymphocytes. Conclusions: The manifestation of mouse lupus nephritismodel induced by Pristane was similar to human SLE with characteristic immune cellsactivation and kidney damage.
Part Ⅳ Studyon the reverse effect and molecular mechanism ofinhibiting B7/CD28signal pathway from gene to protein level inlupus-like disease
Object: To explore the formation of the lupus like disease model and the reverseeffect in pathological injury and its molecular mechanism by different interventionmethods with the adoption of B7-1shRNA lentiviral vector and B7-1antibody to inhibitB7/CD28signal pathway on gene and protein level. The curing effects of the specificantibody on the related diseases were also studied. Methods:1.The treatment methodsof experimental animal groups: C57BL/6J female mice aged6-8weeks were randomlydivided into8groups. A: Negative control group, in which each mouse was injectedwith0.5ml saline by intraperitoneal. B: Model-making group, in which each wasinjected with0.5ml Pristane by intraperitoneal. C：B7-1antibody early interventiongroup, in which each was injected with0.5ml Pristane by intraperitoneal at first, theninjected B7-1monoclonal antibody200μg through tail intravenous on day1,3,5,8,15,and once a month in next three months. D: Antibody isotype control group, in whicheach was injected with same doses of IgG1. E: Lentivirus interference group, in whicheach was injected with0.5ml Pristane by intraperitoneal at first, then injected 0.4x10~8TU of LV-B7-1shRNA on day1,60through tail intravenous. F. Lentivirus blankgroup, in which each was injected with0.5ml Pristane by intraperitoneal at first, theninjected LV-NC shRNA through tail intravenous on day1,60. G: positive agent controlgroup（CTX）, in which each was injected with0.5ml Pristane by intraperitoneal at first,then injected with0.5ml CTX（counted by60mg/kg each time）on day1、8、22、29、43、50、64、71、85、92. H：B7-1antibody delayed intervention group, in which eachwas injected with0.5ml Pristane and injected same dosage and frequency of B7-1antibody after4month when proteinuria and autoantibody emerged.2. Disturbing effectof recombinant lentivirus on the expression of membrane B7-1molecule in spleenantigen present cells: on day10after first infection, spleen cells of mice were collected.The percentage of CD11b+B7-1+、CD11c+B7-1+and CD21+B7-1+double positivecells were analyzed by immumofluorescence method and FCM to explore thesilencing effect of B7-1shRNA lentivirus infection on target molecules.3. Activationanalysis of immune cells in spleen: spleen cells were collected from mice10days afterfirst treatment. The positive expression percentages of Mφ、DC、Gr1+and B cells aswell as the expressions of membrane costimulatory molecules CD86and MHC-II onCD21+B cells were analyzed by immumofluorescence method and FCM.4. Expressionof ANA、anti-dsDNA antibodies in serum were detected monthly after injection.5. Urineprotein analysis: urine protein was detected by Albustix test paper monthly.6.Concentration analysis of cytokines in serum: the concentration of IL-4and IFN-γ inserum was determined by ELISA.7. The immune complex deposit in kidney wasanalyzed by direct immumofluorescence method.8. Renal tissues from mice wereanalyzed by light microscope and transmission electron microscope. Results:1. AfterB7-1shRNA lentivirus interference,positive expression of CD11b+B7-1+,CD11c+B7-1+and CD21+B7-1+cells in spleens were58.43±2.87%,60.18±3.08%,52.85±2.75%,respectively, significantly lower than the control group (p<0.05), suggestion thatB7-1shRNA lentivirus could efficiently inhibit the target molecule expression on APC.2.After intervented by lentivirus and early monoclonal antibody, the Mφ, DC, granulocyteand B cell in spleens down-regulated, significantly lower than the model-making group(p<0.05). Meanwhile, antibody treatment group showed lower than B7-1shRNAlentivirus interference group.3. ANA was detected in part of mice both in B7-1shRNAlentivirus interference group and antibody early intervention group with titer lower than 1:00in all after3months, significantly lower than the model-making group(1:00-1:300). ANA down-regulated in all of B7-1shRNA lentivirus interference group,antibody early intervention group and antibody delayed intervention group, and ANA ofthe three groups was significantly lower than the model-making group (1:000-1:3000)(p<0.05), suggestion that ANA could decrease by B7-1shRNA lentivirus interference orantibody intervention, further more, antibody early intervention group showed lowerANA comparing with B7-1shRNA lentivirus interference group and antibody delayedintervention group (P<0.05).4. Anti-dsDNA antibody was detected in model-makinggroup in4months with low titer around1:10, another4months later, it changed into1:100in90%mouse, higher than the other3groups (B7-1shRNA lentivirus interferencegroup, antibody early intervention group and antibody delayed intervention group),butwithout significant statistical differences (P>0.05).5. IL-4showed0.84±0.52pg/ml and0.88±0.64pg/ml while IFN-γ was3.06±0.38pg/ml and3.02±0.55pg/inB7-1shRNA lentivirus interference group and antibody early intervention grouprespectively, all significant lower than model-making group(P<0.05),but withoutstatistical differences comparing to antibody delayed intervention group.6. In4th month,the urine protein was detected with+～++(300～1000mg/L) in20%mice ofmodel-making group. In8th month, it changed to++～++++（1000～20000mg/L）in allthe mice of model-making group.Proteinuria was±～+++（≤3000mg/L）inB7-1shRNA lentivirus interference group and antibody delayed intervention group,significant lower than model-making group (P<0.05). The urine protein was lowest inB7-1early intervention group, it’s significant lower than B7-1shRNA lentivirusinterference group and antibody delayed intervention group（P<0.05）with the urineprotein content±～++(≤1000mg/L).7. Immunofluorescence intensity of IC in theintervention group mice was weaker than the model-making ones. The kidneys ofcontrol group mice had no significant pathological changes and IC could not be detected.Comparing all the groups except control group, B7-1early intervention group showedleast IC.8. Kidney histopathologically detection showed less damage in B7-1earlyintervention group, B7-1shRNA lentivirus interference group was much severer, and themodel-making group was the severest.9. Ultrastructure of transmission electronmicroscopy analysis showed less change in B7-1early intervention group, B7-1shRNAlentivirus infection group was much severer, and the model-making group was the severest.Conclusions: Both specific RNA and antibody could inhibit the B7-1moleculemediated signal pathway, down-regulate immune cell activation and reversepathological injury. B7-1antibody had more intervention effect than B7-1shRNAlentiviral vector. Further, specific antibody had preventive effect as well as therapeuticeffect against lupus nephritis. Earlier antibody intervention had better therapeutic effectsthan delayed one.
系统性红斑狼疮（systemic lupus erythematosus，SLE）是一种自身免疫性疾病，病因至今尚无明确定论。机体以出现T、B细胞活化增强，产生抗核抗体(antinuclearantibody，ANA)、抗双链DNA(double stranded DNA，dsDNA)抗体、形成免疫复合物（immune complex，IC）以及多种组织器官损伤为其特征。其中狼疮性肾炎（lupus nephritis，LN）是系统性红斑狼疮最常见和最严重的并发症，是导致患者死亡的主要原因。研究表明，B7/CD28信号通路在SLE的发病及病理损伤过程中发挥了重要的作用。
RNA干扰(RNA interference，RNAi)是由长约21-23个核苷酸的双链RNA分子(small interfering RNA，siRNA)介导,以序列特异的方式抑制同源基因表达的一项技术，属于转录后基因沉默(post-transcriptional gene silencing，PTGS)，能够迅速、特异、有效地抑制特定基因的表达。
特异性抗体与相应抗原的结合，可影响抗原分子介导的生物信号。B7分子的抑制性抗体通过封闭抗原分子，可阻止或削弱其与CD28结合而转导B7/CD28协同刺激信号，进而抑制T、B细胞的活化与应答。
本课题运用化学法（Pristane）诱导类似人类SLE发病过程及临床表现的小鼠（C57BL/6J）狼疮样肾炎模型，对其进行生物学鉴定的基础上，采用B7-1shRNA慢病毒表达载体及单克隆抗体进行整体干预，通过免疫学、血清学及病理损伤评价等指标的动态变化，探究在基因与蛋白水平抑制B7/CD28信号通路，对狼疮样肾炎模型的形成及病理损伤的逆转效应及其分子机制，以期对SLE疾病模型形成中免疫细胞活化及病理损伤的演变过程进行研究，同时为该类疾病寻找新的特异、高效、低毒的生物干预手段。
第一部分小鼠B7-1基因RNA干扰慢病毒载体的构建及对受体细胞膜型B7-1分子表达的干扰效应研究
目的：构建小鼠B7-1shRNA慢病毒表达载体，根据其对受体细胞膜型B7-1分子表达的干扰效应挑选最佳靶序列。方法：选择四条B7-1基因RNA干扰有效靶序列，合成靶序列的双链DNA，接入LV3（pGLV-H1-GFP+Puro）载体，再与pLV/helper-SL3、pLV/helper-SL4、pLV/helper-SL5质粒共转染293T细胞，包装产生慢病毒，依据绿色荧光蛋白（green fluorescent protein，GFP）的表达水平测定病毒滴度。慢病毒感染L929细胞，运用免疫荧光及流式细胞术，筛选对受体细胞膜型B7-1分子表达干扰效应较高的靶序列。结果：经过测序证实，LV3-B7-1shRNA慢病毒表达载体构建正确，病毒滴度达1×10~8TU/ml，适合感染L929细胞的复合感染（multiplicity of infection，MOI）值为60，此时感染效率为88.7%。四条候选的shRNA模板序列中，B7-1shRNA-256，对L929细胞膜型B7-1分子的沉默效率最高，达73.2%，以此作为最佳靶序列。结论：成功构建了小鼠B7-1基因RNA干扰慢病毒载体，其能有效沉默L929细胞膜型B7-1分子的表达。
第二部分鼠抗人B7-1分子功能性单克隆抗体的制备及生物学特性研究
目的：制备鼠抗人B7-1分子功能性单克隆抗体，并对其生物学特性进行研究。方法：运用本科室已成功获取的分泌鼠抗人B7-1单克隆抗体杂交瘤细胞株，采用体内诱生腹水法进行单抗的制备；ProteinG免疫亲和层析法纯化抗体；小鼠IgG亚类快速定性试纸鉴定单抗的亚类；间接免疫荧光法测定抗体效价；流式细胞术分析单抗对人及小鼠细胞膜型B7-1分子的识别；MTT法分析抗体对协同刺激信号的拮抗作用。结果：经体内诱生腹水法制备单克隆抗体，小鼠腹水形成阳性率约为80%，腹水收获量平均为5.9ml/只小鼠；经免疫亲和层析法纯化，腹水中抗体蛋白的得率为2.3mg/ml；单抗与细胞株Daudi、Raji、H1299以及小鼠脾脏细胞的阳性结合率分别为89.2%、92.7%、20.6%及48.5%；经MTT法分析，抗体能够阻断B7-1介导的协同刺激信号，从而抑制L929-B7-1刺激PBTCs的增殖效应。结论：成功制备了鼠抗人B7-1分子功能性单克隆抗体，体外研究结果显示，其能够有效阻断B7-1介导的协同刺激信号。
第三部分小鼠狼疮样肾炎模型的建立及生物学鉴定
目的：运用化学法建立小鼠（C57BL/6J）狼疮样肾炎模型，并对其进行生物学鉴定。方法：6-8周龄雌性C57BL/6J小鼠30只，随机分为造模组和正常对照组。造模组予一次性腹腔注射Pristane0.5ml/只，正常对照组予一次性腹腔注射生理盐水0.5ml/只。建模后第10天，每组处死5只小鼠，免疫荧光及流式细胞术(flowcytometry，FCM)分析脾脏中巨噬细胞（CD11b+）、树突状细胞（CD11c+）、粒细胞（Gr1+）及B细胞（CD21+）的阳性表达率，同时检测CD21+B细胞膜型协同刺激分子CD86及MHC-II分子的表达；每月定期活体眶经脉丛取血，检测血清中ANA及抗dsDNA抗体水平及采用Albustix试纸法检测尿蛋白量；持续观察至8个月时处死小鼠，HE染色观察小鼠肾脏病理损伤，直接免疫荧光法分析肾脏免疫复合物（IC）的沉积。结果：1.建模后第10天，脾脏中Mφ、DC、粒细胞及B细胞均出现不同程度的活化，其阳性表达率分别为7.27±0.85%、4.72±0.68%、9.82±1.26%及17.79±0.65%，与对照组相比明显升高，具有统计学差异（p<0.05）；同时，CD21+B细胞膜型协同刺激分子CD86及MHC-II分子表达上调，分别为38.69±3.14%及55.33±2.58%，与对照组具有统计学差异（p<0.05）。2.动态观察至3个月时，造模组30%小鼠血清ANA阳性，4个月时达80%，8个月时ANA阳性率为100%；建模后4个月，20%造模组小鼠可检测到抗dsDNA抗体，6个月时达60%，8个月时抗dsDNA抗体阳性率为80%。3.经动态分析发现，建模后4个月，20%小鼠开始出现蛋白尿，尿蛋白含量为+～++（300～1000mg/L），随着时间的推移，蛋白尿的阳性率和尿蛋白含量逐渐增加，8个月时100%小鼠出现蛋白尿，含量为++～++++（1000～20000mg/L）。4.肾脏冰冻切片直接免疫荧光法分析显示，造模组小鼠肾小球出现强阳性的IC沉积。5.肾脏组织病理学分析显示，造模组肾小球体积增大，细胞数量增多，肾小球血管充血明显，间质明显淋巴细胞浸润。结论：Pristane诱导的小鼠狼疮样肾炎模型作为研究人类SLE的疾病模型具有可靠性。
第四部分基因与蛋白水平抑制B7/CD28信号通路对狼疮样肾炎模型的逆转效应及分子机制研究
目的：运用B7-1shRNA慢病毒载体及B7-1单克隆抗体分别在基因与蛋白水平抑制B7/CD28信号通路，比较不同水平的干预，对狼疮样肾炎模型的形成及病理损伤的逆转效应及其分子机制，同时考察特异性抗体对该类疾病的治疗作用。方法：1.实验动物的分组处理：6-8周龄雌性C57BL/6J小鼠随机分为8组。A：正常对照组，一次性腹腔注射生理盐水0.5ml/只；B：造模组，一次性腹腔注射Pristane0.5ml/只；C：B7-1抗体早期干预组，腹腔注射Pristane0.5ml/只后，第1、3、5、8及15天分别尾静脉注射B7-1单抗200μg/只，然后每隔1个月注射一次，连续注射3个月；D：抗体同型对照组，注射等剂量的小鼠IgG1；E：慢病毒干扰组，腹腔注射Pristane0.5ml/只后，第1、60天分别尾静脉注射0.4x10~8TU的LV-B7-1shRNA；F：慢病毒空载组，腹腔注射Pristane0.5ml/只后，第1、60天分别尾静脉注射LV-NCshRNA；G：化学药物阳性对照（CTX）组，腹腔注射Pristane0.5ml/只后，第1、8、22、29、43、50、64、71、85、92天分别尾静脉注射CTX0.5ml（60mg/kg/次）；H：B7-1抗体延迟干预（治疗）组，腹腔注射Pristane0.5ml/只后第4个月，待出现蛋白尿及自身抗体时运用B7-1抗体，注射次数及剂量同C组。2.B7-1shRNA慢病毒感染后对脾脏APC膜型B7-1分子表达的干预效应：首次感染后第10天，取小鼠脾脏细胞，采用免疫荧光及FCM分析CD11b+B7-1+、CD11c+B7-1+及CD21+B7-1+双阳性细胞的百分率，以考察B7-1shRNA慢病毒载体干扰后对靶分子表达的沉默效应。3.脾脏细胞中免疫细胞的活化分析：各实验组小鼠在首次处理后第10天取小鼠脾脏细胞，采用免疫荧光及FCM分析其中Mφ（CD11b+）、DC（CD11c+）、粒细胞（Gr1+）及B细胞（CD21+）的阳性表达率，同时检测CD21+B细胞膜型协同刺激分子CD86及MHC-II分子的表达。4.ANA和抗dsDNA抗体检测：每月定期活体眶经脉丛取血，检测血清中ANA和抗dsDNA抗体水平。5.尿蛋白的检测：每月定期采用Albustix试纸法检测尿蛋白量。6.血清细胞因子的含量分析：采用ELISA法检测小鼠血清IL-4及IFN-γ水平。7.肾脏IC的沉积分析：采用直接免疫荧光法分析。8.肾脏组织病理改变分析：运用光镜及透射电镜进行分析。结果：1. B7-1shRNA慢病毒干扰后，脾脏细胞中CD11b+B7-1+、CD11c+B7-1+及CD21+B7-1+双阳性细胞的阳性表达率分别为58.43±2.87%、60.18±3.08%及52.85±2.75%，与对照组相比明显下降，差异具有显著性（p<0.05）。提示B7-1shRNA慢病毒干扰可有效抑制APC上靶分子的表达。2.运用慢病毒及抗体早期干预后，脾脏细胞中Mφ、DC、粒细胞及B细胞的活化下调，与造模组比较具有统计学差异（p<0.05），抗体组的下调作用强于慢病毒干扰组（p<0.05）。3.对ANA的分析结果表明，实验3个月时，慢病毒干扰组及抗体早期干预组小鼠，其血清稀释度在1:100时均为阴性，造模组出现1:100-1:300的ANA阳性结果。实验观察至8个月时，造模组ANA阳性率100%，抗体滴度在1:1000-1:3000之间，而抗体早期干预组、慢病毒干扰组及抗体延迟干预组小鼠，其ANA的滴度均出现下降，与造模组相比具有统计学差异（P<0.05），提示上述处理均可减少ANA的产生，但抗体早期干预组优于另两组（P<0.05）。4.实验进行4个月时，造模组出现抗dsDNA抗体，但滴度较低，约为1:10，8个月时，抗dsDNA抗体阳性率为90%，滴度升至1:100左右；抗体早期干预组、慢病毒干扰组及抗体延迟干预组抗dsDNA抗体滴度较造模组均降低，但无统计学差异（P>0.05）。5.血清中细胞因子的水平：B7-1抗体早期干预组及慢病毒干扰组小鼠血清IL-4水平分别为0.84±0.52pg/ml及0.88±0.64pg/ml，IFN-γ水平为3.06±0.38pg/ml及3.02±0.55pg/ml，均较造模组降低，差异有统计学意义（P<0.05）；而B7-1抗体延迟干预组小鼠血清IL-4及IFN-γ水平与造模组相比，无统计学差异（P>0.05）。6.尿蛋白生成及动态变化:造模组建模4个月时，20%小鼠开始出现蛋白尿，尿蛋白含量为+～++，随着时间的推移，蛋白尿的阳性率和尿蛋白含量增加，8个月时100%小鼠出现蛋白尿，尿蛋白含量为++～++++（1000～20000mg/L），随着时间的延长，蛋白尿程度加重；慢病毒干扰组及B7-1抗体延迟干预组蛋白尿程度减轻，尿蛋白含量为±～+++（≤3000mg/L），与造模组相比，差异有统计学意义（P<0.05）；B7-1抗体早期干预组蛋白尿程度最轻，尿蛋白含量为±～++（≤1000mg/L），与慢病毒干扰组及B7-1抗体延迟干预组相比，亦具有统计学差异（P<0.05）。7.肾脏IC沉积分析显示：造模组肾小球出现强阳性的IC沉积，慢病毒干扰组及抗体干预组小鼠肾脏IC沉积减少，B7-1抗体早期干预组IC沉积最少。8.肾脏组织病理改变的光镜分析结果显示：造模组大部分小鼠肾小球体积中-重度增大，细胞数量明显增多，肾小球血管充血明显，间质淋巴细胞浸润严重，纤维组织增生；慢病毒干扰组及抗体干预组小鼠肾小球体积轻-中度增大，细胞数轻度增多，肾小球血管轻度充血，间质淋巴细胞浸润减少，B7-1抗体早期干预组光镜下病理损伤最轻。9.肾脏超微结构的透射电镜分析显示：造模组大部分小鼠肾小球内皮细胞下有大块状的电子致密物沉积，部分小鼠系膜内、上皮细胞下及基底膜内也有电子致密物沉积，足细胞间突起广泛融合，足细胞表面有假绒毛形成，基膜增厚；慢病毒干扰组及抗体干预组小鼠肾小球内皮细胞下有少量电子致密物沉积，足细胞间突起部分融合，基膜稍增厚，B7-1抗体早期干预组电镜下超微结构改变最少。结论：运用特异的RAN及抗体均能抑制B7-1分子介导的信号通路，从而降低免疫细胞的活化及逆转疾病模型病理损伤的程度，但抗体的干预效果优于RNA干扰；特异性抗体不仅对狼疮样肾炎的形成有预防作用，而且对已经形成的狼疮样肾炎具有治疗作用，且早期干预效果更好。
B7-1molecule is an important co-stimulatory molecule expressed on the surface ofantigen-presenting cell (APC), and its combination to the receptor CD28can generatecostimulatory signals, which is essential to the primary immune response. Without thiscostimulatory signal, T cells will enter to a state of anergy, tolerance even apoptosis.Thehyperreaction of B7/CD28signal is closely related to the occurrence of autoimmunediseases.
Systemic lupus erythematosus (SLE) is a common autoimmune disease whichetiology is uncertain characterized by T、B lymphocyte dysfunction, a variety ofantoantibodies, especially antinuclear antibody (ANA) and anti double-stranded DNA(dsDNA) antibody, immune complex formation and many organs damage.Lupusnephritis (LN) is one of the most common and serious complications of SLE, which isalso the primary cause leading to death of patients. Some studies have revealed thatB7/CD28signal participated in the pathogenesis of SLE.
RNA interference (RNAi) is a kind of technology mediated by double strandedRNA with21-23nucleotides-small interfering RNA (siRNA), which can suppresshomologene expression by specific sequence, belonging to post-transcriptional genesilencing (PTGS). It can suppress definite gene expression quickly, specificly andefficiently.
Specific antibody interaction with its ligand leads to inhibit or stimulate biologicsignal. Blocking antibody of B7molecule can prevent or down-regulate its interactionwith CD28, then inhibit the activation and response of T, B lymphocytes.
In the present study, we established a C57BL/6J mouse model with lupus-likedisease induced by Pristane, which is similar to clinical pathogenesy and manifestationof patients with SLE. Based upon the model, we apply the lentiviral expression vector B7-1shRNA and mouse anti-human B7-1monoclonal antibody to the model mouse inorder to evaluate its reverse effect and molecular mechanism by blocking B7/CD28signal pathway, which could provide some experimental and theoretical data, even finda biologic intervention which is specific, efficient and harmfulless for SLE.
Part Ⅰ Construction of lentiviral vector targeting mouse B7-1gene byRNA interference and evaluation its disturbing effect on theexpression of membrane B7-1molecule
Object: To construct lentiviral expression vector targeting mouse B7-1gene byshort hairpin RNA (shRNA), evaluate its disturbing effect on the expression ofmembrane B7-1molecule and select an optimal target sequence. Methods: Thecomplementary DNA containing both sense and antisense oligonucleotides of thetargeting sequence was designed, synthesized. After annealed, double-stranded DNAwas inserted into the LV3（pGLV-H1-GFP+Puro）vector.293T cells were cotransfectedwith pLV/helper-SL3、pLV/helper-SL4and pLV/helper-SL5. The titer of virus wastested according to the expression level of GFP. Mouse L929fibroblast cells wasinfected by the recombinant lentiviruses, the optimal target sequence was selectedaccording to the disturbing effect on the expression of membrane B7-1molecule byflow cytometry and immunofluorescence Results: DNA sequencing demonstrated thatlentivirus plasmid LV3-B7-1shRNA was constructed successfully. The titer of therecombinant lentivirus was1×10~8TU/ml, the best MOI for lentivirus infecting L929cell was60and infection efficiency was88.7%. As for the four shRNA templatesequence candidates, the optimal target sequence is B7-1shRNA-256, its silencingefficiency of the membrane B7-1molecule on L929cells reached the highest rate for73.2%. Conclusions: Lentiviral expression vectors targeting mouse B7-1gene by RNAinterference was constructed successfully. The recombinant lentivirus can effectivelysilence the membrane B7-1molecule on L929cells.
Part Ⅱ Preparation and assay of mouse anti-human B7-1antibody
Object: To obtain pure B7-1antibody, analyze its titer and recognition of membrane B7-1molecule. Methods: Using of the4E5cell line our institutesuccessfully established, ascites were induced to produce the antibody from BALB/cmouse and purified with ProteinG affinity chromatography method. The Ig isotype of4E5was identified with the rapid test paper. Flow cytometry assay was used to test theability of4E5to recognize the membranes B7-1molecule on different cells. Theblocking effect on costimulatory signals of4E5was detected by MTT assay. Results:The positive rate of ascites formation in mice was about80%. Ascites harvest was5.9ml from each BALB/c mouse on average. The purified4E5from ascites was about2.3mg/ml. The4E5could specifically recognize membrane B7-1on cells of Daudi、Raji、H1299、spleen cells of mouse with the positive rate of88.2%、92.7%、20.6%,48.5%,respectively. In addition,4E5could inhibit the growth and proliferation of L929-B7-1ce11s and PBTCs through blocking the B7-1costimulatory signals with MTT assay.Conclusions: Mouse anti-human B7-1monoclonal antibody which can efficiently blockthe B7-1costimulatory signals in vitro was successfully obtained.
Part Ⅲ Establishment and evaluation of mouse lupus nephritis model
Object: To establish and evaluate of lupus nephritis model in C57BL/6J miceinduced by Pristane. Methods: Thirty female mice aged6-8weeks were divided intotwo groups randomly. The model-making group was injected with0.5ml Pristane byintraperitoneal while the control group with0.5ml saline.The activation ofmacrophages、dendritic cells、granulocytes and B cells in spleens and the expression ofcostimulatory molecule CD86and MHC-II on B cells were measured byimmunofluorescence and FCM at day10after injection. ANA、anti-dsDNA antibodiesin serum and proteinuria were detected monthly after injection.8months after injection,all mice were killed and kidneys were slided and stained with H&E or FITC-labeledIgG to observe the evidence of glomerulonephritis histopathologically. Results:1.10days after injection, macrophages、dendritic cells、granulocytes and B cells in spleenswere activated, positive expression were7.27±0.85%、4.72±0.68%、9.82±1.26%and17.79±0.65%respectively, significantly higher than that of control group (p<0.05).Meanwhile, the expression of costimulatory molecule CD86and MHC-II in CD21+Bcells up-regulates to38.69±3.14%and55.33±2.58%, which was also significantly higher compared to the control group (p<0.05).2. The concentration of ANA andanti-dsDNA antibody in mice sera showed the same trend. The concentration of ANAwas up-regulated from30%to100%, after3months injection to8months by dynamicmonitoring. Anti-dsDNA antibody was up-regulated from20%to80%.3. Four monthsafter injection, the urine protein of model group was detected with+～++（300～1000mg/L）in20%model-making group mice. Finally++～++++（1000～20000mg/L）in100%model mice were tested as time went by.4. Immunofluorescence intensity ofimmune complex in model-making group mice was strong positive.5.Kidneyhistopathology analysis showed that glomerular volume enlarged severely, the numberof cell significantly increased, and fibrous tissue appeared, interstitial tissue wasinfiltrated by lymphocytes. Conclusions: The manifestation of mouse lupus nephritismodel induced by Pristane was similar to human SLE with characteristic immune cellsactivation and kidney damage.
Part Ⅳ Studyon the reverse effect and molecular mechanism ofinhibiting B7/CD28signal pathway from gene to protein level inlupus-like disease
Object: To explore the formation of the lupus like disease model and the reverseeffect in pathological injury and its molecular mechanism by different interventionmethods with the adoption of B7-1shRNA lentiviral vector and B7-1antibody to inhibitB7/CD28signal pathway on gene and protein level. The curing effects of the specificantibody on the related diseases were also studied. Methods:1.The treatment methodsof experimental animal groups: C57BL/6J female mice aged6-8weeks were randomlydivided into8groups. A: Negative control group, in which each mouse was injectedwith0.5ml saline by intraperitoneal. B: Model-making group, in which each wasinjected with0.5ml Pristane by intraperitoneal. C：B7-1antibody early interventiongroup, in which each was injected with0.5ml Pristane by intraperitoneal at first, theninjected B7-1monoclonal antibody200μg through tail intravenous on day1,3,5,8,15,and once a month in next three months. D: Antibody isotype control group, in whicheach was injected with same doses of IgG1. E: Lentivirus interference group, in whicheach was injected with0.5ml Pristane by intraperitoneal at first, then injected 0.4x10~8TU of LV-B7-1shRNA on day1,60through tail intravenous. F. Lentivirus blankgroup, in which each was injected with0.5ml Pristane by intraperitoneal at first, theninjected LV-NC shRNA through tail intravenous on day1,60. G: positive agent controlgroup（CTX）, in which each was injected with0.5ml Pristane by intraperitoneal at first,then injected with0.5ml CTX（counted by60mg/kg each time）on day1、8、22、29、43、50、64、71、85、92. H：B7-1antibody delayed intervention group, in which eachwas injected with0.5ml Pristane and injected same dosage and frequency of B7-1antibody after4month when proteinuria and autoantibody emerged.2. Disturbing effectof recombinant lentivirus on the expression of membrane B7-1molecule in spleenantigen present cells: on day10after first infection, spleen cells of mice were collected.The percentage of CD11b+B7-1+、CD11c+B7-1+and CD21+B7-1+double positivecells were analyzed by immumofluorescence method and FCM to explore thesilencing effect of B7-1shRNA lentivirus infection on target molecules.3. Activationanalysis of immune cells in spleen: spleen cells were collected from mice10days afterfirst treatment. The positive expression percentages of Mφ、DC、Gr1+and B cells aswell as the expressions of membrane costimulatory molecules CD86and MHC-II onCD21+B cells were analyzed by immumofluorescence method and FCM.4. Expressionof ANA、anti-dsDNA antibodies in serum were detected monthly after injection.5. Urineprotein analysis: urine protein was detected by Albustix test paper monthly.6.Concentration analysis of cytokines in serum: the concentration of IL-4and IFN-γ inserum was determined by ELISA.7. The immune complex deposit in kidney wasanalyzed by direct immumofluorescence method.8. Renal tissues from mice wereanalyzed by light microscope and transmission electron microscope. Results:1. AfterB7-1shRNA lentivirus interference,positive expression of CD11b+B7-1+,CD11c+B7-1+and CD21+B7-1+cells in spleens were58.43±2.87%,60.18±3.08%,52.85±2.75%,respectively, significantly lower than the control group (p<0.05), suggestion thatB7-1shRNA lentivirus could efficiently inhibit the target molecule expression on APC.2.After intervented by lentivirus and early monoclonal antibody, the Mφ, DC, granulocyteand B cell in spleens down-regulated, significantly lower than the model-making group(p<0.05). Meanwhile, antibody treatment group showed lower than B7-1shRNAlentivirus interference group.3. ANA was detected in part of mice both in B7-1shRNAlentivirus interference group and antibody early intervention group with titer lower than 1:00in all after3months, significantly lower than the model-making group(1:00-1:300). ANA down-regulated in all of B7-1shRNA lentivirus interference group,antibody early intervention group and antibody delayed intervention group, and ANA ofthe three groups was significantly lower than the model-making group (1:000-1:3000)(p<0.05), suggestion that ANA could decrease by B7-1shRNA lentivirus interference orantibody intervention, further more, antibody early intervention group showed lowerANA comparing with B7-1shRNA lentivirus interference group and antibody delayedintervention group (P<0.05).4. Anti-dsDNA antibody was detected in model-makinggroup in4months with low titer around1:10, another4months later, it changed into1:100in90%mouse, higher than the other3groups (B7-1shRNA lentivirus interferencegroup, antibody early intervention group and antibody delayed intervention group),butwithout significant statistical differences (P>0.05).5. IL-4showed0.84±0.52pg/ml and0.88±0.64pg/ml while IFN-γ was3.06±0.38pg/ml and3.02±0.55pg/inB7-1shRNA lentivirus interference group and antibody early intervention grouprespectively, all significant lower than model-making group(P<0.05),but withoutstatistical differences comparing to antibody delayed intervention group.6. In4th month,the urine protein was detected with+～++(300～1000mg/L) in20%mice ofmodel-making group. In8th month, it changed to++～++++（1000～20000mg/L）in allthe mice of model-making group.Proteinuria was±～+++（≤3000mg/L）inB7-1shRNA lentivirus interference group and antibody delayed intervention group,significant lower than model-making group (P<0.05). The urine protein was lowest inB7-1early intervention group, it’s significant lower than B7-1shRNA lentivirusinterference group and antibody delayed intervention group（P<0.05）with the urineprotein content±～++(≤1000mg/L).7. Immunofluorescence intensity of IC in theintervention group mice was weaker than the model-making ones. The kidneys ofcontrol group mice had no significant pathological changes and IC could not be detected.Comparing all the groups except control group, B7-1early intervention group showedleast IC.8. Kidney histopathologically detection showed less damage in B7-1earlyintervention group, B7-1shRNA lentivirus interference group was much severer, and themodel-making group was the severest.9. Ultrastructure of transmission electronmicroscopy analysis showed less change in B7-1early intervention group, B7-1shRNAlentivirus infection group was much severer, and the model-making group was the severest.Conclusions: Both specific RNA and antibody could inhibit the B7-1moleculemediated signal pathway, down-regulate immune cell activation and reversepathological injury. B7-1antibody had more intervention effect than B7-1shRNAlentiviral vector. Further, specific antibody had preventive effect as well as therapeuticeffect against lupus nephritis. Earlier antibody intervention had better therapeutic effectsthan delayed one.
引文
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