DC靶向性DNA疫苗抗肿瘤作用及机制研究

发布时间：2018-04-04 18:24

本文选题：DNA疫苗　切入点：DC靶向性　出处：《第二军医大学》2011年硕士论文

【摘要】：DNA疫苗在肿瘤免疫治疗中具有巨大的应用前景,临床研究表明接受DNA疫苗免疫的病人能够产生抗原特异性抗肿瘤免疫应答,然而,目前的DNA疫苗免疫极少能诱导持久高效的抗肿瘤免疫反应,这表明DNA疫苗仍需不断优化。DNA疫苗起作用的一个很关键的环节是其表达的蛋白抗原需要被抗原提呈细胞(antigen presenting cells,APC)摄取,然后加工处理并提呈给抗原特异性T淋巴细胞,诱导T细胞活化及增殖。树突状细胞(dendritic cells,DC)是目前体内已知的功能最强的抗原提呈细胞,其能够刺激初始(na(?)ve)T细胞增殖进而启动机体免疫应答,诱导抗原特异性细胞免疫反应。皮下或肌肉注射质粒DNA疫苗后,大部分质粒被注射部位的皮肤及肌肉细胞摄取,其编码的免疫抗原在这些细胞表达或释放。皮肤和肌肉细胞因缺乏MHC II和共刺激分子表达不能有效递呈抗原而不能致敏抗原特异性T细胞,因此DC的抗原摄取效率直接与诱导的免疫反应相关。在DNA疫苗免疫中,DC获取免疫抗原主要有两种途径,一是局部少量DC直接摄取DNA质粒并表达免疫抗原,这一途径相对次要,毕竟局部能够直接摄取质粒DNA的DC比例极低。其二是局部DC摄取皮肤或肌肉细胞释放的免疫抗原或吞噬表达免疫抗原的死亡皮肤肌肉细胞,这是主要途径。因此,我们设想:通过DC靶向性分子(DC表面分子特异抗体或配体)把免疫抗原直接递送至DC将能够有效提高DC摄取抗原效率,进而有效增强DNA疫苗诱导的免疫应答。为验证上述假设,本研究中我们制备了DC靶向性及对照的非靶向DNA疫苗scFv~(NLDC-145)-HER2/neu和HER2/neu,其中scFv~(NLDC-145)编码DEC-205分子特异单链抗体片段,DEC-205是DC表面特异表达的吞噬受体,能够有效介导抗原内吞从而进入抗原加工递送通路;HER2/neu为原癌基因,编码HER2/neu生长因子受体,在乳腺癌等多种肿瘤中过表达,其直接促进肿瘤生长及发展。实验表明:DC靶向性DNA疫苗能够把编码的免疫抗原有效的递送至DC进而被加工递呈;在HER2/neu阳性的移植乳腺癌动物模型中,与非靶向HER2/neu DNA疫苗相比,DC靶向疫苗诱导了显著增强的HER2/neu特异细胞和体内免疫应答,能够有效预防HER2/neu阳性小鼠肿瘤生长,并产生抗原特异性免疫记忆;更重要的是,DC靶向疫苗联合低剂量环磷酰胺介导的调节性T细胞短暂清除,能够消除已生成的HER2阳性移植乳腺肿瘤,延缓BALB-neuT转基因小鼠中自发性乳腺肿瘤生长。第一部分:DNA疫苗的构建及表达一、DNA疫苗的构建:单链抗体片段NLDC-145(scFv~(NLDC-145))通过参考其他学者前期发表的文章全基因合成。HER2/neu的胞外区片段分别从SK-BR-3或TUBO乳腺癌细胞系扩增出,测序正确后两胞外区分别与scFv~(NLDC-145)经overlapping PCR方法构建出scFv~(NLDC-145)-HER2和scFv~(NLDC-145)-neu,两PCR产物装入pGEM-Teasy载体扩增。我们用HindIII和XbaI双酶切上述载体分别获得scFv~(NLDC-145)-HER2和scFv~(NLDC-145)-neu片段,然后装入经相同酶切的pcDNA3.1(+)中成功构建出含上述两片段的表达载体。二、DNA疫苗的融合蛋白表达:抽提上述构建的表达载体质粒,瞬时转染293T细胞,72h后收获上清,用WB方法检测质粒疫苗的蛋白表达,两靶向疫苗表达的融合蛋白比预期的稍大,推测为融合蛋白在真核表达系统中存在糖基化的原因。第二部分:DNA疫苗的体内靶向性研究一、构建pcDNA3.1-scFv~(NLDC-145)-EGFP表达载体:从质粒载体pEGFP-N1中克隆出EGFP基因,然后用EGFP基因替代上述表达载体scFv~(NLDC-145)-HER2中HER2片段,构建成scFv~(NLDC-145)-EGFP表达载体。二、scFv~(NLDC-145)体内靶向性检测:用电击免疫的方法在BALB/c小鼠右后大腿肌肉注射scFv~(NLDC-145)-EGFP质粒,以PE标记的CD11c阳性细胞设门,用流式细胞术检测EGFP绿色荧光,结果显示scFv~(NLDC-145)具有良好的DC靶向性。第三部分:BALB/c小鼠体内抗肿瘤作用研究一、预防性抗肿瘤作用: BALB/c小鼠随机分为5组,对应每组分别用电击免疫的方法在小鼠右后大腿肌肉注射scFv~(NLDC-145)-HER2,scFv~(NLDC-145)-neu,HER2,neu和空载体pcDNA3.1,每只50μg/50μL。两周之后再用相同的方法免疫第二次。BALB/c小鼠经过两次免疫一周后,在相反侧背部皮下注射D2F2/E2细胞(D2F2/E2是稳定表达HER2的人的乳腺癌细胞株),后观察并记录肿瘤生长情况。结果显示,靶向的scFv~(NLDC-145)-HER2组可有效预防肿瘤的生长,此组在120天时100%小鼠都成活,而其他组在80天时已全部死亡。有意思的是scFv~(NLDC-145)-neu组比非靶向的HER2,neu和pcDNA3.1组稍微有效的延缓了肿瘤生长。二、治疗性抗肿瘤作用:首先验证DNA疫苗单独应用的抗肿瘤作用,BALB/c小鼠背部皮下接种D2F2/E2细胞,当肿瘤直径约40mm~3时,上述方法两次免疫小鼠,观察肿瘤生长情况。结果显示,scFv~(NLDC-145)-HER2组比其他组虽然有明显的抑瘤效果,但到120天时该组也才只有20%小鼠生存,所以scFv~(NLDC-145)-HER2单独应用作用有限。随后我们验证DNA疫苗联合应用环磷酰胺抗肿瘤作用,BALB/c小鼠接种D2F2/E2细胞,当肿瘤直径约3-4mm时,腹腔注射低剂量的环磷酰胺(100mg/kg),四天后小鼠按照上述方法免疫两次,后观察肿瘤生长情况。结果显示,scFv~(NLDC-145)-HER2联合环磷酰胺组在120时能显著抑制80%小鼠肿瘤生长并能使肿瘤体积减小至消除,其他各联合应用组也比对应的单独应用质粒免疫有好的抑瘤效果。第四部分:DNA疫苗的抗肿瘤机制研究一、诱导HER2特异性T细胞反应:上述方法两次免疫BALB/c小鼠,一周后,取脾单细胞分别用HER2或TRP2蛋白体外刺激3天,通过液相闪烁计数仪检测后可知scFv~(NLDC-145)-HER2组经HER2蛋白刺激的比TRP2蛋白刺激的有显著的T细胞增殖。同样都是经HER2蛋白刺激的各组比较,结果显示scFv~(NLDC-145)-HER2组也比其他组有显著的T细胞增殖,而scFv~(NLDC-145)-neu组也比其他各非靶向对照组的T细胞增殖稍显著。ELISA检测上述经HER2蛋白刺激的各组的培养脾细胞的血清,scFv~(NLDC-145)-HER2组可检测IFN-γ, TNF-α的高表达,同样scFv~(NLDC-145)-neu组IFN-γ, TNF-α的表达也略高于其他各非靶向对照组。流式检测经HER2肽段刺激的scFv~(NLDC-145)-HER2组有CD4+ IFN-γ+,CD4+ TNF-α+,CD8+ IFN-γ+和CD8+ TNF-α+ T细胞高表达。二、诱导HER2特异性抗体反应:收集上述各组的血清,ELISA可检测到scFv~(NLDC-145)-HER2组比其他各组有高滴度的HER2特异性抗体,且主要为IgG2a亚型。另上清与D2F2/E2细胞孵育,后与FITC标记的二抗结合孵育后流式检测scFv~(NLDC-145)-HER2组D2F2/E2特异性抗体明显高于其他组,而scFv~(NLDC-145)-neu也略高于各非靶向组。第五部分:scFv~(NLDC-145)-neu在BALB-neuT小鼠中抗肿瘤作用研究一、预防性抗肿瘤作用:建立稳定表达neu抗原的小鼠乳腺癌细胞株TUBO可移植肿瘤模型;在BALB-neuT小鼠经过两次免疫一周后,在相反侧背部皮下注射TUBO细胞,后观察肿瘤生长情况。结果显示,靶向的scFv~(NLDC-145)-neu组可预防肿瘤的生长,此组在120天时约90%小鼠都成活,而其他组在不到60天时已全部死亡。二、抗小鼠自发性乳腺癌作用:BALB-neuT小鼠是表达neu抗原的可自发性生长乳腺癌的小鼠,在小鼠8周和10周时用电击免疫的方法肌肉注射scFv~(NLDC-145)-neu+CTX , scFv~(NLDC-145)-neu , neu , pcDNA3.1和PBS ,其中scFv~(NLDC-145)-neu+CTX组在第一次免疫的4天前注射低剂量的环磷酰胺(100mg/kg),再肌肉注射scFv~(NLDC-145)-neu,后观察各组小鼠自发性肿瘤个数。结果显示,scFv~(NLDC-145)-neu联合环磷酰胺组可有效延缓自发性肿瘤生长,比其他组延缓约4周生长肿瘤。到第38周时,环磷酰胺联合应用组小鼠还都活着,而非靶向对照组在26周时就已死亡或肿瘤体积超过1500mm~3,另scFv~(NLDC-145)-neu单独应用组效果也好于其他各非靶向组,该组绝大部分小鼠在30周时还生存着。结论: 综上所述我们构建的DC靶向性的DNA疫苗在小鼠体内可以把肿瘤特异性抗原靶向到DC表面,从而引起抗原特异性的细胞和体液免疫,更重要的若联合低剂量的环磷酰胺短暂清除调节性T细胞后,其作为治疗性疫苗能在小鼠体内起到较好的抗肿瘤作用,从而为肿瘤治疗提供一个潜在可行的治疗策略。
[Abstract]:DNA vaccine has a great potential in tumor immunotherapy , and clinical research shows that patients immunized with DNA vaccine can produce antigen - specific anti - tumor immune response .

In order to validate the above hypothesis , we prepared a non - targeted DNA vaccine scFv ~ ( NLDC - 145 ) - HER2 / neu and HER2 / neu , wherein scFv ~ ( NLDC - 145 ) encodes a specific single - chain antibody fragment of DEC - 205 .

Part I : Construction and Expression of DNA Vaccine

1 . Construction of DNA vaccine : Single - chain antibody fragment NLDC - 145 ( scFv ~ ( NLDC - 145 )) was amplified from SK - BR - 3 or TUBO breast cancer cell line . The two extracellular domains were amplified from SK - BR - 3 or TUBO breast cancer cell lines . The two extracellular domains were amplified by overlapping PCR method . The scFv ~ ( NLDC - 145 ) - HER2 and scFv ~ ( NLDC - 145 ) - neu fragments were respectively obtained by overlapping PCR .

2 . Expression of the fusion protein of DNA vaccine : the expression vector plasmid constructed above was extracted , transiently transfected into 293 cells , the supernatant was harvested after 72 h , the protein expression of the plasmid vaccine was detected by WB method , the fusion protein expressed by the two target vaccines was slightly larger than expected , and it was speculated that the fusion protein had glycosylation in the eukaryotic expression system .

Second part : In vivo targeting of DNA vaccines

1 . Construction of pcDNA3.1 - scFv ~ ( NLDC - 145 ) - EGFP expression vector : The EGFP gene was cloned from the plasmid pcDNA3.1 - scFv ~ ( NLDC - 145 ) - EGFP , and then the expression vector scFv ~ ( NLDC - 145 ) - HER2 was replaced by EGFP gene to construct the scFv ~ ( NLDC - 145 ) - EGFP expression vector .

2 . In vivo targeting detection of scFv ~ ( NLDC - 145 ) : Using electric shock immunity method , the scFv ~ ( NLDC - 145 ) - EGFP plasmid was injected into the right posterior thigh muscle of BALB / c mice . The cells of CD11c - positive cells marked by PE were constructed . The green fluorescence of EGFP was detected by flow cytometry . The results showed that the scFv ~ ( NLDC - 145 ) had good DC targeting .

The third part : the anti - tumor effect of BALB / c mice

1 . Preventive anti - tumor effect : BALB / c mice were randomly divided into 5 groups . Each group was immunized with the same method after two weeks of immunization . The target scFv ~ ( NLDC - 145 ) - HER2 group was able to effectively prevent tumor growth . The results showed that the targeted scFv ~ ( NLDC - 145 ) - HER2 group could effectively prevent tumor growth . The target scFv ~ ( NLDC - 145 ) - HER2 group had died at 80 days . Interestingly , the scFv ~ ( NLDC - 145 ) - neu group had delayed tumor growth more effectively than non - targeted HER2 , neu and pcDNA3.1 groups .

The results showed that scFv ~ ( NLDC - 145 ) - HER2 combined with cyclophosphamide ( 100 mg / kg ) could significantly inhibit tumor growth of 80 % mice at 120 days , and then observe the growth of tumor . The results showed that the scFv ~ ( NLDC - 145 ) - HER2 combined cyclophosphamide group could significantly inhibit tumor growth of 80 % mice at 120 days and reduce tumor volume to elimination .

Part IV : Study on the Anti - tumor Mechanism of DNA Vaccine

The results showed that scFv ~ ( NLDC - 145 ) - HER2 group was significantly higher than that of other non - targeted control groups . The results showed that the scFv ~ ( NLDC - 145 ) - HER2 group was significantly higher than that of other non - targeted control groups .

2 . To induce HER2 specific antibody reaction : the serum of the above groups was collected , and the specific antibody of HER2 specific antibody with high titer was detected by ELISA . The specific antibody of scFv ~ ( NLDC - 145 ) - HER2 was significantly higher than that of other groups after incubation with D2F2 / E2 cells . The specific antibody of scFv ~ ( NLDC - 145 ) - HER2 was higher than that of other groups , and the scFv ~ ( NLDC - 145 ) - neu was also slightly higher than that of non - targeting groups .

The antitumor effect of scFv ~ ( NLDC - 145 ) - neu in BALB / neuT mice

1 . Preventive anti - tumor effect : A mouse breast cancer cell line TUBO transplantable tumor model stably expressing neu antigen was established . After two weeks of immunization in BALB / neuT mice , TUBO cells were injected subcutaneously on the opposite side , then the growth of tumor was observed . The results showed that the targeted scFv ~ ( NLDC - 145 ) - neu group could prevent tumor growth , and the target scFv ~ ( NLDC - 145 ) - neu group was alive at 120 days , while the other groups died in less than 60 days .

The results showed that scFv ~ ( NLDC - 145 ) - neu combined cyclophosphamide group could effectively delay the growth of spontaneous tumor . The results showed that scFv ~ ( NLDC - 145 ) - neu combined cyclophosphamide group could effectively delay the growth of spontaneous tumor . The results showed that scFv ~ ( NLDC - 145 ) - neu combined cyclophosphamide group could effectively delay the growth of spontaneous tumor . The results showed that scFv ~ ( NLDC - 145 ) - neu combined cyclophosphamide group could effectively delay the growth of spontaneous tumor . The results showed that scFv ~ ( NLDC - 145 ) - neu combined cyclophosphamide group could effectively delay the growth of spontaneous tumor .

Conclusion :

In conclusion , the DC - targeted DNA vaccine can target the tumor - specific antigen to the DC surface in vivo , thus causing antigen - specific cellular and humoral immunity , and more importantly , when the combined low - dose cyclophosphamide transiently clears the regulatory T cell , it can play a better anti - tumor effect in the mouse body as a therapeutic vaccine , thereby providing a potentially feasible treatment strategy for tumor treatment .

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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