RANKL-TNF样区多肽疫苗对胶原诱导关节炎小鼠的保护作用研究
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摘要
目的：研究人RANKL-TNF样区多肽疫苗能否刺激小鼠产生特异性抗体，并检测该抗体拮抗肿瘤坏死因子α（Tumor necrosis factor-α，TNF-α）的致炎和核因子-κB受体活化因子配体（Receptor activator of nuclear factor-κB ligand，RANKL)的致骨破坏效果，并评价疫苗对胶原诱导关节炎（Collagen inducedarthritis，CIA）小鼠的保护作用。
方法：选取人RANKL-TNF样区与人TNF-α高度相似区域为疫苗候选表位，非相似区域用Th2细胞表位取代，重组PCR法拼接上述基因片段，构建pET-28a-RTFP-2原核表达载体；经异丙基-β-D-硫代半乳糖苷（Isopropy-β-D-thiogalactoside，IPTG）诱导表达目的蛋白并鉴定，大量提取，纯化，去除内毒素（多肽疫苗命名为RTFP-2）；于0d，14d和28d应用100μg该疫苗免疫BALB/c雌性小鼠，间断收集血清，间接ELISA法检测血清中产生的双靶向抗体识别结合RANKL和TNF-α的能力，动态监测抗体滴度变化及持续时间；二次加强免疫后10d，腹腔注射人TNF-α使小鼠产生恶液质，监测死亡率和体重下降率；静脉注射人RANKL使小鼠血钙升高和破骨细胞活化，监测血钙升高水平，一周后处死，抗酒石酸酸性磷酸酶染色计数各组小鼠胫骨的破骨细胞数目；二次加强免疫后1周，各组DBA/1小鼠间隔三周尾根部皮内免疫100μg牛Ⅱ型胶原，制备CIA小鼠，监测各组小鼠发病率，记录关节评分至二次免疫后50d，处死小鼠，四肢骨行组织学和Micro-CT检测，分析炎症浸润和骨破坏程度，ELISA法检测血清中RANKL和TNF-α的变化。
结果：RTFP-2多肽疫苗在pET-28a原核体系中表达良好，经检测为包涵体表达，RANKL和TNF-α抗体可识别纯化后的多肽疫苗；与佐剂对照组小鼠相比，疫苗免疫的小鼠可产生高滴度的抗体，停止免疫后抗体滴度可持续约2个月，再次加强免疫后抗体滴度可在短期内达到峰值；疫苗免疫的小鼠产生的抗血清可拮抗TNF-α和RANKL的生物学活性，使TNF-α所致的L929细胞的凋亡率下降50%，使RANKL所致的破骨细胞的分化成熟受阻；疫苗免疫后的小鼠，恶液质症状减轻，体重下降减缓，死亡率下降28%，血钙水平升高延缓，小鼠股骨的破骨细胞数量维持在正常水平；疫苗免疫的小鼠关节炎发病率下降，反应疾病严重程度的关节评分降低，与阳性对照组小鼠相比，组织学评估显示疫苗组小鼠关节面光滑，关节腔中炎症细胞浸润减少，软骨面完整，厚度正常，骨髓腔中未见炎性细胞，影像学Micro-CT扫描显示疫苗组小鼠的关节软骨和骨结构相对完整，骨破坏程度明显减轻，血清学检测显示疫苗组小鼠血清中RANKL和TNF-α的分泌水平也相应下降。
结论：人RANKL-TNF样区多肽疫苗可刺激机体产生特异性抗体，该抗体可有效中和TNF-α和RANKL的生物学活性，减轻恶液质和高钙血症小鼠的症状，降低CIA小鼠的发病率和疾病严重程度，为炎症性骨病的治疗提供了新的思路和方向。
Objective Combination of two agents targeting tumor necrosis factor-α (TNF-α) andthe receptor activator nuclear of NF-κB ligand (RANKL) has been proved highlysuccessful in experimental autoimmune arthritis models and rheumatoid arthritis(RA) patients. This raises a possibility whether a single agent simultaneouslytargeting TNF-α and RANKL proves a potential therapeutic opportunity. This studyaimed to design a vaccine and evaluate its therapeutic effects reducing inflammationand bone resorption through inhibiting overexpression of both TNF-α and RANKLin RA mice model.
Methods Standard molecular biological techniques were used to establish thevaccine to generate human RANKL-TNF-like core fusion protein (RTFP-2). Theimmunogenicity of RTFP-2vaccine was determined by measuring the titer ofspecific antibodies using sandwich enzyme linked immunosorbant assay (ELISA).The neutralizing effects of specific antibodies were detected by TNF-α mediatedcytotoxicity and RANKL induced osteoclastogenesis assay in vitro. The dualfunctions of vaccine against TNF-α and RANKL in vivo were assessed using theexperimental cachexia and hypercalcemia models. The therapeutic effects of vaccinewere evaluated using collagen induced arthritis (CIA) mice.
Results High titers of antibodies against human TNF-α and RANKL were elicited atfour weeks after immunization of the vaccine in mice. The antiserum induced byRTFP-2vaccine decreased TNF-α mediated apoptosis of L929cells to41%incomparison with90%in positive controls. In addition, the antiserum completely abrogated the differentiation of bone marrow progenitors to mature osteoclasts invitro. Immunization with the RTFP-2also reduced the mortality of TNF-α inducedcachexia in mice from56%to28%. The RANKL-mediated hypercalcemic effectswere significantly attenuated in RTFP-2vaccined mice compared with control mice.Furthermore, RTFP-2vaccine significantly mitigated the incidence and severity ofCIA via inhibition of inflammation and bone resorption.
Conclusion Our results showed that the immunization of RTFP-2ameliorates thesymptoms of CIA mice through targeting TNF-α and RANKL, suggesting that thevaccine may provide a potential possibility to treat inflammatory bone diseases suchas RA.
方法：选取人RANKL-TNF样区与人TNF-α高度相似区域为疫苗候选表位，非相似区域用Th2细胞表位取代，重组PCR法拼接上述基因片段，构建pET-28a-RTFP-2原核表达载体；经异丙基-β-D-硫代半乳糖苷（Isopropy-β-D-thiogalactoside，IPTG）诱导表达目的蛋白并鉴定，大量提取，纯化，去除内毒素（多肽疫苗命名为RTFP-2）；于0d，14d和28d应用100μg该疫苗免疫BALB/c雌性小鼠，间断收集血清，间接ELISA法检测血清中产生的双靶向抗体识别结合RANKL和TNF-α的能力，动态监测抗体滴度变化及持续时间；二次加强免疫后10d，腹腔注射人TNF-α使小鼠产生恶液质，监测死亡率和体重下降率；静脉注射人RANKL使小鼠血钙升高和破骨细胞活化，监测血钙升高水平，一周后处死，抗酒石酸酸性磷酸酶染色计数各组小鼠胫骨的破骨细胞数目；二次加强免疫后1周，各组DBA/1小鼠间隔三周尾根部皮内免疫100μg牛Ⅱ型胶原，制备CIA小鼠，监测各组小鼠发病率，记录关节评分至二次免疫后50d，处死小鼠，四肢骨行组织学和Micro-CT检测，分析炎症浸润和骨破坏程度，ELISA法检测血清中RANKL和TNF-α的变化。
结果：RTFP-2多肽疫苗在pET-28a原核体系中表达良好，经检测为包涵体表达，RANKL和TNF-α抗体可识别纯化后的多肽疫苗；与佐剂对照组小鼠相比，疫苗免疫的小鼠可产生高滴度的抗体，停止免疫后抗体滴度可持续约2个月，再次加强免疫后抗体滴度可在短期内达到峰值；疫苗免疫的小鼠产生的抗血清可拮抗TNF-α和RANKL的生物学活性，使TNF-α所致的L929细胞的凋亡率下降50%，使RANKL所致的破骨细胞的分化成熟受阻；疫苗免疫后的小鼠，恶液质症状减轻，体重下降减缓，死亡率下降28%，血钙水平升高延缓，小鼠股骨的破骨细胞数量维持在正常水平；疫苗免疫的小鼠关节炎发病率下降，反应疾病严重程度的关节评分降低，与阳性对照组小鼠相比，组织学评估显示疫苗组小鼠关节面光滑，关节腔中炎症细胞浸润减少，软骨面完整，厚度正常，骨髓腔中未见炎性细胞，影像学Micro-CT扫描显示疫苗组小鼠的关节软骨和骨结构相对完整，骨破坏程度明显减轻，血清学检测显示疫苗组小鼠血清中RANKL和TNF-α的分泌水平也相应下降。
结论：人RANKL-TNF样区多肽疫苗可刺激机体产生特异性抗体，该抗体可有效中和TNF-α和RANKL的生物学活性，减轻恶液质和高钙血症小鼠的症状，降低CIA小鼠的发病率和疾病严重程度，为炎症性骨病的治疗提供了新的思路和方向。
Objective Combination of two agents targeting tumor necrosis factor-α (TNF-α) andthe receptor activator nuclear of NF-κB ligand (RANKL) has been proved highlysuccessful in experimental autoimmune arthritis models and rheumatoid arthritis(RA) patients. This raises a possibility whether a single agent simultaneouslytargeting TNF-α and RANKL proves a potential therapeutic opportunity. This studyaimed to design a vaccine and evaluate its therapeutic effects reducing inflammationand bone resorption through inhibiting overexpression of both TNF-α and RANKLin RA mice model.
Methods Standard molecular biological techniques were used to establish thevaccine to generate human RANKL-TNF-like core fusion protein (RTFP-2). Theimmunogenicity of RTFP-2vaccine was determined by measuring the titer ofspecific antibodies using sandwich enzyme linked immunosorbant assay (ELISA).The neutralizing effects of specific antibodies were detected by TNF-α mediatedcytotoxicity and RANKL induced osteoclastogenesis assay in vitro. The dualfunctions of vaccine against TNF-α and RANKL in vivo were assessed using theexperimental cachexia and hypercalcemia models. The therapeutic effects of vaccinewere evaluated using collagen induced arthritis (CIA) mice.
Results High titers of antibodies against human TNF-α and RANKL were elicited atfour weeks after immunization of the vaccine in mice. The antiserum induced byRTFP-2vaccine decreased TNF-α mediated apoptosis of L929cells to41%incomparison with90%in positive controls. In addition, the antiserum completely abrogated the differentiation of bone marrow progenitors to mature osteoclasts invitro. Immunization with the RTFP-2also reduced the mortality of TNF-α inducedcachexia in mice from56%to28%. The RANKL-mediated hypercalcemic effectswere significantly attenuated in RTFP-2vaccined mice compared with control mice.Furthermore, RTFP-2vaccine significantly mitigated the incidence and severity ofCIA via inhibition of inflammation and bone resorption.
Conclusion Our results showed that the immunization of RTFP-2ameliorates thesymptoms of CIA mice through targeting TNF-α and RANKL, suggesting that thevaccine may provide a potential possibility to treat inflammatory bone diseases suchas RA.
引文
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