靶向基因修饰树突状细胞瘤苗治疗移植性肝癌的实验研究

【作者】 李媛媛；

【导师】 谢裕安；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 目的建立体外诱导和扩增小鼠骨髓来源树突状细胞（DC）的方法,并对其形态和表型进行鉴定。方法在无菌条件下提取C57BL/6J小鼠骨髓细胞,利用细胞因子rmGM-CSF（50ng/ml）和rmIL-4（2ng/ml）联合诱导,于体外培养第6天收集部分疏松贴壁细胞,行流式细胞术（FACS）检测其表型,其余细胞加入rmTNF-a（50ng/ml）继续培养,刺激其成熟,并于48小时后行流式细胞术（FACS）检测其表型。在光镜和电镜下观察DC的形态特征。结果小鼠骨髓单核细胞经细胞因子（rmGM-CSF和rmIL-4）体外诱导培养24小时后,可见增殖性细胞集落,3天后集落增多明显,培养至第7天,收集悬浮细胞在光镜和电镜下均显示细胞表面不规则,呈树突状突起,具有DC的典型形态学特征。流式细胞术检测结果显示:经细胞因子（rmGM-CSF和rmIL-4）诱导的细胞表面均表达CD11c、MHCⅡ、CD80和CD86等DC表面分子,而经rmTNF-a刺激48小时后的细胞,其表达率明显提高,分别为[（81.60±10.02）%],[（78.70±11.35）%],[（65.30±5.73）%]和[（79.65±8.66）%],提示经rmTNF-a刺激后DC趋于成熟。结论细胞因子rmGM-CSF和rmIL-4可在体外联合诱导小鼠骨髓单核细胞发育成DC,体外诱导的DC经rmTNF-a刺激后,可迅速发育成熟,采用这种方法可以体外诱导扩增大量DC。目的构建小鼠甲胎蛋白（mAFP）基因转染的树突状细胞瘤苗（pAdBM5-mAFP-DC）。方法应用HEK293A细胞作为包装细胞,大量扩增含目的基因mAFP的重组腺病毒pAdBM5-mAFP;利用改良的CsCl超速离心法对其进行纯化;用TCID₅₀法测定重组腺病毒滴度。从C57BL/6J小鼠骨髓分离单个核细胞,用重组小鼠白细胞介素4（rmIL-4）和重组小鼠粒细胞-巨噬细胞集落刺激因子（rmGM-CSF）诱导和扩增DC。将重组腺病毒颗粒转染DC,用流式细胞术（FACS）检测转染前后DC细胞表面分子MHCⅠ、MHCⅡ、CD18a（LEA）、CD54（ICAM）、CD80（B7.1）、和CD86（B7.2）等的变化。结果获得了50μl纯化病毒液,病毒滴度达0.88×10⁸ PFU/ml。将纯化的重组腺病毒pAdBM5-mAFP转染体外培养的DC,经mAFP基因转染后的DC分子表面高表达MHCⅠ、MHCⅡ、CD18a、CD54、CD80和CD86等分子,其分子表达率分别为69.3%、41.0%、42.1%、63.2%、39.4%和38.6%,与转染前比较差异有统计学意义（p＜0.05）。结论重组腺病毒pAdBM5-mAFP在HEK293A细胞中可成功扩增,所得病毒液量可满足体内基因转染实验的需要。所扩增的重组腺病毒pAdBM5-mAFP可成功转染DC而构建出pAdBM5-mAFP-DC转基因瘤苗;mAFP基因修饰后的DC能表达高水平的MHCⅠ、MHCⅡ、CD18a、CD54、CD80和CD86分子。目的探讨pAdBM5-mAFP-DC瘤苗对C57BL/6J小鼠移植性肝癌发生发展的阻断作用。方法40只C57BL/6J小鼠随机分为A、B、C、D和E组,每组8只。以免疫表达mAFP基因的重组腺病毒转染DC（pAdBM5-mAFP-DC）为A组;免疫表达mAFP基因的质粒DNA（pAdBM5-mAFP）为B组,免疫表达mAFP基因的另一种质粒DNA（pcDNA3.1（+）-mAFP）为C组;免疫单纯DC为D组,以注射单纯PBS为对照组E组。免疫方法:A组和D组在每只小鼠的右腋下注射5×10⁵个细胞（0.1ml/次）,B组和C组在每只小鼠的右腋下注射10.0μg重组质粒（0.1ml/次）,E组,仅注射0.1ml PBS,每1周注射1次,连续免疫4次。在初次免疫后的第3周给所有小鼠移植Hepal-6肝癌细胞。观察小鼠移植瘤的生长情况,于移植肿瘤第14天取血,用ELISA法检测血清TNF-a和IFN-γ的水平,并处死小鼠,检查成瘤情况。同时对肿瘤组织进行组织病理学检查。结果pAdBM5-mAFP-DC瘤苗免疫可诱导强烈的免疫保护作用,瘤苗免疫小鼠能有效抵抗Hepal-6肝癌细胞的攻击,表现为肿瘤生长缓慢（至实验结束,A组有3只小鼠未长出移植瘤）。A、B、C、D和E组的瘤体重量分别为（0.05475±0.029148）g、（0.25425±0.092698）g、（0.14025±0.081074）g、（0.10400±0.027092）g、（0.29300±0.134784）g,A组瘤体重量最轻,与B、C、E组瘤体重量比较有显著性差异（P＜0.01）。计算肿瘤抑制率结果为:A组肿瘤抑制率为81.31%,显著高于其它组。各组小鼠血清中的TNF-a、IFN-γ含量结果表明:A组和D组小鼠血清中TNF-a、IFN-γ水平显著升高,A组血清中TNF-a、IFN-γ水平分别为（180.25±30.25,155.45±24.37）,D组血清中TNF-a、IFN-γ水平分别为（105.32±12.57,90.33±9.70）;A组和D组之间比较有统计学差异（p＜0.05）。组织病理学检查显示:各组肿瘤组织有不同程度的炎细胞浸润,各组比较,以A组最为明显,并以淋巴细胞浸润为主;除A组外,其他各组均见肿瘤因生长过快而血供相对不足所导致的缺血性坏死。结论将构建的pAdBM5-mAFP-DC瘤苗免疫小鼠,可以刺激小鼠体内产生强烈的抗肿瘤免疫反应,使荷瘤小鼠血清中细胞因子TNF-a、IFN-γ水平显著提高,体内诱导产生细胞毒性T细胞,向表达AFP的肝癌细胞趋化,并向肿瘤组织浸润,杀伤肿瘤细胞,从而抑制肿瘤的发生、发展。更多还原

【Abstract】 OBJECTIVE To establish a method to generate and amplify dendritic cells（DC）from mouse bone marrow using the cytokine in vitro and identify their morphology and immunological phenotype.METHODS The dendritic cells（DC）were gotten after inducing the mouse bone marrow cells with granulocyte-macrophage colony-stimulating factor（GM-CSF,50ng/ml）and interleukin-4（IL-4,2ng/ml）at the sixth day,which were loosely adherent cells. The DCs were divided into two groups,one of them were analysised with Flow cytometry（FACS）.The others were stimulated with tumor necrosis factor alpha （TNF-a,50ng/ml）of the culture and analysised with Flow cytometry 48 hours later,then the morphology of DCs was observed by light microscopy and electron microscopy.RESULTS The clusters of cells were gotten after inducing the mouse marrow mononuclear cells using rmGM-CSF and rmlL-4 for 24 hours in vitro,which increased significantly 3 days later.The suspension cells were collected at the seventh day.The cells were branching-like under inverted light microscope and electron microscopy,which was typical morphology of DC.The express of CD11c,MHCⅡ,CD80 and CD86 of the cells induced with rmGM-CSF and rmlL-4 were detected by fluorescence activation cell sorting.The expressing rate of them of DCs were elevated after stimulating with rmTNF-a 48 hours.The expressing rate of them were[（81.60±10.02）%],[（78.70±11.35）%],[（65.30±5.73）%]and[（79.65±8.66）%]separately, which demonstrated that the DCs were maturate gradually after stimulating with rmTNF-a.CONCLUSION A large number of DCs can be gotten by inducing the mouse marrow mononuclear cells with rmGM-CSF and rmlL-4 in vitro,which were stimulated to mature by rmTNF-a. OBJECTIVE To construct a murine alpha-fetoprotein gene-trasfected dendritic cells vaccine（pAdBM5-mAFP-DC）.METHODS The cell 293,a human embryonic kidney cell line as a packaging cell was used for culture of recombinant adenovirus vectors containing mAFP gene.The recombinant adenovirus was reproduced and purified.Virus titer was detected by TCID₅₀ method.DCs were differentiated from C57BL/6J murine bone marrow progenitor cells and induced and augmented by recombinant murine IL-4（rmIL-4）and recombinant routine GM-CSF（rmGM-CSF）.DCs were transfected by recombinant adenovirus engineered with murine alpha-fetoprotein gent.MHCⅠ,MHCⅡ,CD18a（LFA）,CD54（ICAM）,CD80（B7.1） and CD86（BT.2）molecule of the DCs were detected by FACS analysis before and after gene transfection.RESULTS The purified recombinant adenovirus pAdBM5-mAFP was obtained with total volume of 50μl and titer of 0.88×10⁸ PFU/ml.The DCs were transfected by recombinant adenovirus pAdBM5-mAFP. The expressing rate of MHCⅠ,MHCⅡ,CD18a（LFA）,CD54（ICAM）,CDS0（B7.1） and CD86（B7.2）molecule of DCs were elevated significantly after transfection of mAFP gene,which were 69.3%,41.0%,42.1%,63.2%,39.4%and 38.6% separately（p＜0.05）.CONCLUSION Culturing and reproduction of cell 293 can reproduce recombined adenovirus pAdBM5-mAFP which can be used to establish the tumour vaccine of pAdBM5-mAFP-DC after transfection of the DCs.The volume of pAdBM5-mAFP can meet the needs of gene transfection in vivo at laboratory study.DC vaccine engineered with mAFP had been constructed successfully,and it had a high level expression of MHCⅠ,MHCⅡ, CD18a,CD54,CD80 and CD86 molecule after transduction compared with before transduction. OBJECTIVE To investigate the effect of pAdBM5-mAFP-DC tumour vaccine on the inhibition of implanted liver cancer in C57BL/6J mice. METHODS 40 C57BL/6J mice were randomly divided into group A,B,C,D and E（8 mice each group）.Group A was inoculated mAFP gene-transfected DC（pAdBM5-mAFP-DC）;group B was inoculated with simple pAdBM5-mAFP plasmid DNA;group C was inoculated with simple pcDNA3.1（+）-mAFP plasmid DNA.Group D was inoculated with simple DC,and group E is simply injected with PBS.Each mouse of group A and D was immunized with 5×10⁵ cells（0.1ml per one time）administered s.c.in the rignt axillary.Each mouse of group B and C was immunized with 10.0μg plasmid DNA（0.1ml per one time） administered s.c.in the rignt axillary.Each mouse of group E was injected with 0.1ml PBS.Each mouse was accepted vaccination vaccine one time per 1w,and immuned continually 4 times.All mice were injected with Hepal-6 cells at the third week after initial immunity.The growth of implanted tumor in mice was observed.Serum were collected from tumor-bearing mice and measured for TNF-a and IFN-γby ELISA at the 14^thdays after being implanted tumor cells.At the same time all mice were killed and tumor tissues were assessed by histopathology examination.RESULTS The mAFP transgenic DC tumor vaccine could induce powerful immunoprotection,immunized mice could effectively resist the attack of Hepal-6 hepatocarcinoma cell,the tumor’s growth was delayed（three mice of group A never generate tumor）.The tumor weight of group A,group B,group C,group D and group E were （0.05475±0.029148）g,（0.25425±0.092698）g,（0.14025±0.081074）g,（0.10400±0.027092）g and（0.29300±0.134784）g.The tumor weight of group A were the lowest among all groups.It has statistics significance that the tumor weight of group A compared with group B,group C and group E（p＜0.05）.The tumor suppression rate of group A was 81.31%which was higher than other groups signifcantly.The levels of TNF-a and IFN-γin the A and D groups increased signifcantly which were（180.25±30.25,155.45±24.37）pg/ml and （105.32±12.57,90.33±9.70）pg/ml separately.And it has statistics significance that group A and D compared with each other（p＜0.05）.The histopathology inspection indicated that inflammatory cells infiltration in tumor tissues were found in all groups,especially for group A,which is chiefly infiltrated by lympholeukocytes.Morevere,duc（?）the tumor tissues grown too fast without enough blood supply,the ischemic necrosis occured in most of the gruops except group A.CONCLUSION The mAFP transgenic DC tumor vaccine can inhibit the growth of tumor by inducing the T cells which can infiltrate and kill tumor cells through sitmulating the anti-tumor immune response,which can elevate the level of TNF-a and IFN-γin serum of the tumor-bearing mice.更多还原