基于HPV16的新型伪病毒对DNA特异性B细胞的杀伤研究

目的 构建基于人乳头状病毒(human papilloma virus,HPV)16型的新型伪病毒,并检测其对DNA特异性B细胞(R4A)的杀伤效应.方法 利用昆虫杆状病毒表达系统表达病毒蛋白,在体外变性与复性过程中将病毒蛋白包装B细胞特异性启动子IgK控制下的白喉毒素(Diphtheriatoxin,DT)A链(DT-A)基因表达型质粒DNA,形成伪病毒.转染R4A细胞,荧光显微镜和流式细胞仪检测其杀伤效应.酶联免疫法检测R4A细胞分泌抗ds-DNA抗体的滴度.结果 转染R4A细胞48 h后,荧光显微镜和流式细胞仪成功检测到新型伪病毒对R4A细胞的杀伤效应,R4A细胞分泌抗ds-DNA抗体的滴度明显降低.结论 基于HPV16的新型伪病毒能有效特异杀伤R4A细胞,并降低抗ds-DNA抗体的滴度,为系统性红斑狼疮的治疗提供了理想的策略.     

Gallium nitrate suppresses lupus in MRL/lpr mice

Gallium (Ga) nitrate, a drug which prevents a variety of experimental autoimmune diseases, was investigated in a murine model of systemic lupus erythematosus (SLE). In one experiment, female MRL/Mp lpr/lpr (MRL/lpr) mice were randomized into 2 groups of 6:1) vehicle (trisodium citrate) and 2) Ga. Subcutaneous injections began at 3 weeks of age and continued weekly until the mice were euthanized a week after the thirteenth injection. The loading dose of Ga (calculated as elemental Ga) was 45 mg/kg, followed by 15 mg/kg/week. In another experiment (n = 18) with 3 males and 3 females per group, mice received 1) vehicle, 2) Ga × 1 (one 45 mg/kg dose), and 3) Ga × 13. In the experiment with 12 mice, axillary lymph nodes from Ga-treated mice were significantly smaller than those from vehicle-treated mice (91 ± 42 and 360 ± 358 mg respectively, mean ± SD), and spleens as well as lymph nodes from the former showed significantly less lymphoid infiltrate. In the experiment with 18 mice, prescapular lymph nodes weighed 312 ± 98, 217 ± 52, and 42 ± 34 mg, and spleens weighed 732 ± 492, 409 ± 164, and 192 ± 93 mg in the groups which received vehicle, Ga × 1, and Ga × 13 respectively. Control mice had significantly more lymphoid infiltrates in the lungs, spleen, and lymph nodes and, unlike Ga × 13 mice, exhibited glomerulitis and renal vasculitis. Within groups, females developed more severe disease than males. The Ga × 13 group had increased percentages of CD4-bearing and CD8-bearing lymphocytes in lymph nodes and increased CD4-bearing lymphocytes in the spleen, with an increased proliferative response to mitogen stimulation in vitro in lymph nodes, although not in the spleen. The Ga × 13 group also gained less weight and developed osteosclerosis. Although preliminary, our findings suggest that clinical trials with Ga in SLE are merited. Received: 9 January 1997 / Accepted: 30 May 1997     

商陆皂苷甲对MRL/lpr小鼠iTr35细胞及其相关细胞因子表达的影响

摘要：目的 观察商陆皂苷甲（EsA）对MRL/lpr小鼠的治疗作用和体内iTr35（CD4+Foxp3-IL-12p35+IL-27EBI3+） 细胞、白细胞介素（IL）-35及IL-17表达的影响,探讨EsA治疗狼疮性肾炎的可能机制。方法 将24只16周龄、雌性 MRL/lpr小鼠随机分为模型对照组、EsA组和EsA+IL-12p35抗体组,分别腹腔注射给药,每日1次,4周后处死小鼠, 进行尿蛋白/肌酐值、血肌酐浓度、IL-35、IL-17、iTr35细胞比例、肾脏病理的检测。结果 3组小鼠尿蛋白/肌酐值、血 肌酐浓度、IL-35、IL-17、iTr35差异有统计学意义（P＜0.05）,其中模型对照组尿蛋白/肌酐值、血肌酐浓度、IL-17含量 最高,其次为EsA+IL-12p35抗体组,EsA组最低;相反,EsA组中IL-35、iTr35水平最高,其次为EsA+IL-12p35抗体 组,模型对照组最低;与模型对照组相比,EsA组和EsA+IL-12p35抗体组小鼠肾脏病理表现有一定改善。结论 狼 疮性肾炎小鼠血清中存在IL-35、IL-17的表达异常,EsA对狼疮性肾炎有效,其机制可能是通过调节IL-35、IL-17及 iTr35的表达而发挥作用的。     

三氧化二砷对MRL/lpr小鼠免疫功能和肾脏组织病理变化的影响(英文)

目的研究三氧化二砷(As2O3)对MRL/lpr小鼠免疫功能和肾脏组织病理变化的影响。方法45只MRL/lpr狼疮小鼠ip给予环磷酰胺50 mg·kg-1(每周1次)和As2O30.8 mg·kg-1,每天1次,共2个月。用ELISA法检测血清抗双链DNA(dsDNA)抗体、干扰素γ(IFN-γ)和白细胞介素12(IL-12)浓度;用流式细胞术测定脾CD3+,CD19+,CD3+CD4+和CD3+CD8+细胞亚群的百分比;用PAS染色法观察肾组织病理变化;用免疫荧光方法检测肾组织IgG和补体C3的表达。结果与给药前比较,给药2个月后,正常对照组血清抗dsDNA抗体水平升高,由给药前1.18±0.26升高至1.80±0.26(P<0.01),As2O3和环磷酰胺组该抗体水平明显降低,分别由给药前1.14±0.58和1.09±0.22降低至0.92±0.06和0.67±0.14(P<0.05,P<0.01)。与正常对照组比较:①As2O3和环磷酰胺组血清抗ds-DNA抗体、IFN-γ和IL-12浓度明显降低(P<0.05),环磷酰胺组抗ds-DNA抗体比As2O3组显著降低(P<0.01);②As2O3组CD3+,CD3+CD4+和CD19+细胞百分率明显降低(P<0.01),环磷酰胺组CD3+,CD3+CD8+和CD19+细胞百分率明显降低(P<0.01);As2O3组CD3+CD4+细胞百分率明显降低(P<0.01);③As2O3和环磷酰胺组小鼠肾小球细胞计数和活动度积分明显降低(P<0.05,P<0.01),As2O3和环磷酰胺组无显著差异;④As2O3和环磷酰胺组肾IgG表达明显降低(P<0.05),补体C3表达无明显差异,As2O3和环磷酰胺组之间无显著性差异。结论As2O3能降低MRL/lpr狼疮小鼠血清抗ds-DNA抗体水平,抑制T,B和Th细胞活化和增殖,降低血清IFN-γ和IL-12水平,从而缓解狼疮肾炎的病理变化。     

HPV16的新型伪病毒对DNA特异性B细胞的靶向实验研究

目的:构建人乳头状病毒(human papilloma virus,HPV)16型的新型伪病毒,并检测其对DNA特异性B细胞(R4A)的靶向性。方法:利用昆虫杆状病毒表达系统表达病毒蛋白,在体外变性与复性过程中将病毒蛋白包装绿色荧蛋白EGFP表达型质粒,形成伪病毒。透射电镜观察病毒样颗粒(virus-like particle,VLP)的结构,并转染R4A细胞,荧光显微镜和流式细胞仪检测其转染效率。结果:透射电镜观察证实病毒蛋白可自我组装成VLP,在转染R4A细胞后,荧光显微镜和流式细胞仪成功检测到荧光的表达。结论:HPV16的新型伪病毒能成功转染R4A细胞,为系统性红斑狼疮(SLE)的靶向治疗提供了理想的策略。     

解毒祛瘀滋阴药对MRL／lpr小鼠外周血淋巴细胞凋亡及线粒体跨膜电位的影响

目的探讨解毒祛瘀滋阴中药对MRL／lpr小鼠外周血淋巴细胞凋亡及其线粒体跨膜电位的影响。方法80只MRL／lpr小鼠随机分为：模型组：20只,灌胃0．9％氯化钠注射液;中药组：20只,灌胃解毒祛瘀滋肾中药煎剂;西药组：20只,灌胃强的松混悬液;中西药组：20只,中西药分别灌胃;正常组：昆明小鼠选用20只,0．9％氯化钠注射液灌胃,均每日1次,每次0．5ml。连续饲养用药12周后取血,以梯度离心法分离提纯外周血淋巴细胞（PBLC）,培养48h,流式细胞仪检测凋亡率及其线粒体跨膜电位水平。结果正常组、中药组、西药组和中西药组PBLC0和48h凋亡率高于模型组,差异有显著性（P〈0．05,P〈0．01）。0小时西药组、正常组与中西药组的凋亡率无明显差异（P〉0．05）,但中药组和模型组与中西药组比较,差异有显著性（P〈0．05）;48h西药组与中西药组的凋亡率有明显差异（P〈0．05）,中药组和模型组与中西药组比较,差异有显著性（P〈0．01）;正常组、中药组、西药组和中西药组PBLC线粒体跨膜电位水平水平均明显低于模型组,差异有显著性（P〈0．05和P〈0．01）;西药组与中西药组低于正常组,模型组高于正常组,且比较均差异有显著性（P〈0．05和P〈0．01）;中药组与正常组之间差异无显著性（P〉0．05）;西药组与中西药组之间差异无显著性（P〉0．05）。结论解毒祛瘀滋阴药能增加MRL／Ipr小鼠的PBLC凋亡率并下调其线粒体跨膜电位水平。     

Ac-SDKP ameliorates the progression of lupus nephritis in MRL/lpr mice

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is an endogenous tetrapeptide which can inhibit the differentiation, migration and activation of macrophages and suppress the proliferation of fibroblast. This study examined the effects of Ac-SDKP on the progression of lupus nephritis (LN). MRL/lpr mice received subcutaneous infusion of Ac-SDKP (1.0 mg kg^− 1 d^− 1) or vehicle through implanted osmotic mini-pumps from 12 to 20 weeks until being euthanized. MRL/MpJ mice served as normal controls. The data indicative of renal inflammation and fibrosis were evaluated before and after treatment. Ac-SDKP-treated MRL/lpr mice showed reduced proteinuria and improved renal function compared with vehicle-treated controls. Ac-SDKP-treated mice demonstrated decreased inflammatory infiltrates of T cells and macrophages in the kidneys as compared to vehicle-treated animals. The treatment also inhibited the activation of NF-κB and production of TNF-α. Despite this, immune complex deposition and plasma anti-dsDNA levels were not statistically different between the two groups. In addition, the treatment inhibited renal expression of TGF-β1, α-SMA and fibronectin as well as the phosphorylation of Smad2/3. Ac-SDKP treatment ameliorated LN through exerting anti-inflammatory and anti-fibrotic effects on MRL/lpr mice, providing therapeutic potential for halting the progression of LN.     

黄芪多糖对系统性红斑狼疮小鼠免疫调节的影响

目的 探讨黄芪多糖对系统性红斑狼疮MRL/lpr小鼠免疫功能的影响及作用机制.方法 将20只MRL/lpr狼疮小鼠依据随机数字表法分为模型组和药物组,每组各10只,另选择10只雌性C57BL/6小鼠作为对照组.药物组采用黄芪多糖治疗,对照组和模型组均采用同等剂量生理盐水治疗.比较各组小鼠的自身抗体水平.采用流式技术法测定各组脾脏组织辅助性T细胞1(Th1)和辅助性T细胞2(Th2)细胞比例,使用蛋白质印迹法(Western blotting)和q-PCR技术测定T盒转录因子(T-bet)和GATA结合蛋白3(GATA-3)的表达情况.本次研究起止时间为2019年2—6月.结果 模型组小鼠血清抗核抗体(ANA)、抗双链脱氧核糖核酸抗体(Anti-dsDNA)和抗小核糖核蛋白/Sm抗体(Anti-snRNP/Sm)水平分别为(38.11±3.84)ng/L、(524.63±52.69)μmol/L、(23.05±2.31)ng/L,与对照组(24.23±2.41)ng/L、(340.26±34.37)μmol/L、(15.01±1.51)ng/L相比均明显升高(P<0.05);药物组小鼠血清ANA抗体、Anti-dsDNA抗体和Anti-snRNP/Sm抗体水平分别为(29.45±2.95)ng/L、(468.14±46.88)μmol/L、(18.44±1.83)ng/L,与模型组相比明显降低(P<0.05).模型组小鼠Th1亚群比例、Th2亚群比例分别为(11.32±1.13)％、(2.84±0.29)％,与对照组小鼠(18.95±1.71)％、(2.21±0.22)％相比,模型组小鼠Th1亚群比例明显降低,Th2亚群比例明显升高(P<0.05);与模型组相比,药物组小鼠Th1亚群比例(14.69±1.47)％明显升高,Th2亚群比例(2.49±0.24)％明显降低(P<0.05).与对照组小鼠比较,模型组小鼠T-bet蛋白表达和mRNA水平显降低,GATA-3蛋白和mRNA表达水平均显著升高(P<0.05);与模型组小鼠相比,药物组小鼠T-bet蛋白和mRNA表达水平明显升高,GATA-3蛋白和mRNA表达水平显著降低(P<0.05).结论 黄芪多糖可影响MRL/lpr狼疮小鼠T-bet、GATA3的表达水平,调节Th1/Th2细胞的平衡,进而下调自身抗体水平发挥治疗作用.