树突状细胞抗原负载及MAGE-3 DNA瘤苗研制
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摘要
树突状细胞(DCs)是目前已知功能最强的专职性抗原提呈细胞,可以通过多种作用方式行使其抗肿瘤免疫作用。研究表明，DCs是连接抗原与免疫应答细胞以激发有效的抗肿瘤免疫应答反应的桥梁。人类MAGE（melanoma antigen）基因家族编码的肿瘤排斥抗原（tumor rejection antigen）在细胞内经加工产生抗原肽，并与HLA-Ⅰ类分子结合形成复合物，被自体细胞毒性T细胞（CTL）识别，诱导其对相应肿瘤细胞的特异性杀伤。MAGE基因在许多肿瘤组织中有较高的表达，但是在正常组织（除了睾丸和胚胎外）均不表达，因而MAGE基因是一种CTL介导的肿瘤特异性免疫治疗的理想靶位。
1. 鼠骨髓来源的树突状细胞体外扩增及诱导成熟
我们应用GM-CSF及IL-4体外诱导扩增了小鼠骨髓来源的树突状细胞。小鼠骨髓细胞与细胞因子IL-4（500 U/ml）及GM-CSF（1000U/ml）共同培养第2d可见部分悬浮细胞；第3d悬浮细胞增多且细胞有短的毛刺状突起；第5d，绝大部分细胞悬浮，毛刺状突起拉长，培养瓶底部大圆形及梭形细胞减少。观察结果显示，此细胞为典型树突状细胞形态特征。
应用TNF-α刺激或冻融抗原负载，诱导DCs成熟，各组DCs表面标志CD83、CD86与空白对照组相比均具有显著差异（P＜0.05）；IL-4+GM-CSF+TNF-α组、IL-4+GM-CSF+肿瘤抗原组与IL-4+GM-CSF组比差异显著（P＜0.05）；而IL-4+GM-CSF+TNF-α组与IL-4+GM-CSF+肿瘤抗原组比无差异。结果表明，树突状细胞经抗原负载或TNF-α刺
激后，CD83及CD86表达上调。
应用上述成熟DCs进行混合淋巴细胞培养，以刺激T细胞增殖。结果表明，IL-4+GM-CS+TNF-α组及IL-4+GM-CS+冻融抗原组DCs与T细胞混合培养，可以明显刺激T细胞的增殖，cpm值显著高于空白对照组及IL-4+GM-CSF组（P<0.001）；IL-4+GM-CS+冻融抗原组DCs与T细胞混合培养，cpm值亦明显高于IL-4+GM-CS+TNF-α组（P<0.001）；按1∶5混合各组明显高于1∶10混合培养各组，但无统计学意义。
2. 肿瘤总RNA装载DC
我们诱导扩增了肺癌患者外周血单核细胞来源的DCs，形态学鉴定发现，PBMC培养3d后，倒置显微镜下观察细胞呈集簇生长，部分细胞的细胞膜有突起；培养至第6d时，大部分悬浮生长，可见较多细胞胞体大且外形不规则，细胞膜呈树枝状突起，胞浆丰富，具有典型的树突状细胞形态。透射电镜下可见，细胞膜有大量皱褶和突起，核大、偏位，胞内线粒体丰富。流式细胞仪检测诱导前后DCs表面标志的变化，结果显示，PBMC经GM-CSF和IL-4诱导，培养第5 d时，CD1a的阳性率为11.10±1.08（%），CD83的阳性率为7.24±2.25（%），同时伴有人类主要组织相容性抗原HLA-ABC和协同刺激分子CD86（B7-2）的上调表达，HLA-DR的水平基本不变，代表单核细胞的表面标志CD14下降（P<0.05），诱导第7 d时，上述结果变化更明显，与5 d时的结果相比差异显著（P<0.05），表明PBMC在GM-CSF和IL-4的作用下已向DCs转化。
用肺癌细胞的总RNA装载上述DCs，以检验DCs的抗原提呈能力。我们提取了肺癌细胞的总RNA，变性凝胶电泳显示完整的28S和18S两条带，证实RNA完整未降解。紫外分光光度计测RNA浓度为1.69μg/μl，OD260/OD280>1.8。参照基因?-actin的RT-PCR电泳结果可见1000bp处的条带，亦证明总RNA中有翻译必需的mRNA存在。将此肺癌细胞总RNA经脂质体包裹，负载肺癌患者外周血单核细胞诱导扩增的DCs，流式细胞仪分析结果显示，转染总RNA 3d后，DCs的特
异性表面标志如CD83表达上调，其它功能相关的表面标志如HLA-ABC、HLA-DR和共刺激分子CD86（B7-2）表达也上调（P<0.05），而代表单核细胞的CD14表达则下调（P<0.05）；混合淋巴细胞培养结果显示，未转染RNA的DCs与转染RNA后的DCs都能刺激自身T淋巴细胞增殖活性（P<0.05），对同种异体T淋巴细胞则无此作用；转染RNA的DCs比未转染的DCs刺激自身淋巴细胞增殖的能力增强（P<0.05）。
3. MAGE-3基因的提取及转染LA795细胞
为进一步研制肿瘤疫苗，MAGE-3基因转染LA795细胞。首先，利用TRIzol Reagent提取Mel526细胞株总RNA，应用MAGE-3特异引物进行RT-PCR，获得MAGE-3基因；1%琼脂糖凝胶电泳分析，出现一1000bp的DNA条带，与预想的长度相符。以TA克隆方式将此产物克隆入pMD18-T载体，经酶切鉴定插入片段存在，长度约1000bp。将此重组质粒命名为pMD-MAGE3。对此基因进行测序，测序结果与MAGE-3 RNA参考序列（U03735）进行同源性比较，发现第162位碱基突变T→C，但并未引起氨基酸残基突变（CCT → CCC，均编码脯氨酸）。再将此基因亚克隆入荧光载体pEGFP-C1，经SacⅠ和SalⅠ双酶切，1%琼脂糖凝胶电泳显示一1000 bp左右的DNA条带，表明插入片段存在，命名pEGFP-MAGE3。
载体pEGFP-C1上的EGFP与其下游的外源基因融合表达，为确保此融合基因正确表达，对重组质粒pEGFP-MAGE3进行测序，结果显示阅读框正确。质粒pEGFP-C1含有荧光蛋白基因EGFP，此基因表达可以使转染细胞发出绿色荧光，便于观察转染效果；为此，pEGFP-MAGE3质粒转染LA795细胞24、48和72h，荧光显微镜下，可见绿色荧光的细胞。镜下随机计数10个视野的细胞，计算出转染效率（绿色荧光细胞所占的比例）为20％~30％。将1(2×105个LA795细胞种植于6?
Dendritic cells (DCs) have the strong function of specific duty antigen presenting cells (APC) in now. It can be achieve immunofunction of antigen by some ways. Some study showed that DCs is a bridge linked antigen and immune response cells to activate the immuneresponse of effective antitumor. Tumor rejection antigen codded by MAGE family produces antigen peptided proceseed in cells. Tumor rejection antigen combined with HLA-I formed a compplex and it distinguished by self-cytotoxic T lymphocytes (CTL), induce it specially killing relevant tumor cells. MAGE more highly expresses in many tumor tissues but can’t expresss in normal tissues (except testis and fetal tissue), it is a kind of ideal target to special immune mediated by CTL.
1. The expansion and maturation of inducing dendritic cells derived from mouse bone marrow in vitro
The expansion and maturation of inducing dendritic cells derived from mouse bone marrow in vitro. The dendritic cells derived from mouse bone marrow were proliferated by using GM-CSF and IL-4 in vitro. A part of suspension cells were abserved on 2 days after mouse bone marrow cells were cocultured with IL-4 (500u/ml) and GM-CSF (1000u/ml), the suspension cells increased with short process with burr 3 days later, and most of cells were floated with long process with burr, at the same time large round and fusiform cells decreased on the bottom of bottles 5 days later. The results showed that these cells have the characteristic of typical morphology of dentritic cells. The expressions of surface marker CD83 and CD86 of CD maturation induced by TNF-α stimulating or freezing and thawing antigen increased significantly as
compared with that in blank control group (P<0.05). There was significantly different between IL-4+GM-CSF+TNF-α group, IL-4+GM-CSF+carcinoma antigen group and IL-4+GM-CSF group, while there was not different between IL-4+GM-CSF+TNF-α and IL-4+GM-CSF+caicinoma antigen groups. The results showed that the expressions of CD83 and CD86 increased after dentritic cells were loaded with antigen and stimulated with TNF-α. Then the above mentioned mature DCs were used to perform mixture lymphocyte culture to stimulate T cells proliferation. The results showed that the proliferation of T cells was stimulated after DCs in IL-4+GM-CSF+TM-α group, IL-4+GM-CSF+freezing and thawing antigen group were cultured with T cells, CPM in which were significantly higher than that in blank control group and IL-4+GM-CSF group (P<0.001). The cpm of DCs cultured with T cells in IL-4+GM-CSF+freezing and thawing antigen group was significantly higher than that in IL-4+GM-CSF+TNF-α group (P<0.001). And the cpm in all the groups of mixture ratio of 1 to 5 was higher than that in ratio of 1:10 groups, but there was not significantly different.
2. DCs loaded with total RNA of tumor
The induction of DCsorigined from mononuclear leukocytes of periphery blood of lung cancer patient increased, DCs morphologically grew in a cluster of clony under invereted microscope, for 3 days in the culture of cells, the membrane in a part of them processed; for 6 days in culture, most of them grew in suspension, cell body was biger and irregular, cell membrane processed with branch tower, cytoplasm enriched, the cells present a typical DCs in morphology. There were a lot of microfolds and processes in the cell membrane, cell nucleus was biger and inclined to one side, mitochondria enriched in cells. After PBMC inducted with GM-CSF and IL-4 for 5 days in the culture, the positive rates of surface maker CD1a and CD83 detected with FCM were 11.10±1.08(%) and 7.24±2.25(%). Respectively, accompanied with the up-regulting expressions of HLA-ABC and CD86 (B7-2), down-regulating expression of CD14 and unclchange of HLA-DR. The results suggest that PBMC differentiated into DCs in the effecfs of GM-CSF and IL-4.
The above-mentioned DCs loaded with total RNA of lung cancer cells to detect antigen presenting ability of DCs. We extracted the total RNA with two bands of 28 S and 18 S detected with dematured gel electr
1. 鼠骨髓来源的树突状细胞体外扩增及诱导成熟
我们应用GM-CSF及IL-4体外诱导扩增了小鼠骨髓来源的树突状细胞。小鼠骨髓细胞与细胞因子IL-4（500 U/ml）及GM-CSF（1000U/ml）共同培养第2d可见部分悬浮细胞；第3d悬浮细胞增多且细胞有短的毛刺状突起；第5d，绝大部分细胞悬浮，毛刺状突起拉长，培养瓶底部大圆形及梭形细胞减少。观察结果显示，此细胞为典型树突状细胞形态特征。
应用TNF-α刺激或冻融抗原负载，诱导DCs成熟，各组DCs表面标志CD83、CD86与空白对照组相比均具有显著差异（P＜0.05）；IL-4+GM-CSF+TNF-α组、IL-4+GM-CSF+肿瘤抗原组与IL-4+GM-CSF组比差异显著（P＜0.05）；而IL-4+GM-CSF+TNF-α组与IL-4+GM-CSF+肿瘤抗原组比无差异。结果表明，树突状细胞经抗原负载或TNF-α刺
激后，CD83及CD86表达上调。
应用上述成熟DCs进行混合淋巴细胞培养，以刺激T细胞增殖。结果表明，IL-4+GM-CS+TNF-α组及IL-4+GM-CS+冻融抗原组DCs与T细胞混合培养，可以明显刺激T细胞的增殖，cpm值显著高于空白对照组及IL-4+GM-CSF组（P<0.001）；IL-4+GM-CS+冻融抗原组DCs与T细胞混合培养，cpm值亦明显高于IL-4+GM-CS+TNF-α组（P<0.001）；按1∶5混合各组明显高于1∶10混合培养各组，但无统计学意义。
2. 肿瘤总RNA装载DC
我们诱导扩增了肺癌患者外周血单核细胞来源的DCs，形态学鉴定发现，PBMC培养3d后，倒置显微镜下观察细胞呈集簇生长，部分细胞的细胞膜有突起；培养至第6d时，大部分悬浮生长，可见较多细胞胞体大且外形不规则，细胞膜呈树枝状突起，胞浆丰富，具有典型的树突状细胞形态。透射电镜下可见，细胞膜有大量皱褶和突起，核大、偏位，胞内线粒体丰富。流式细胞仪检测诱导前后DCs表面标志的变化，结果显示，PBMC经GM-CSF和IL-4诱导，培养第5 d时，CD1a的阳性率为11.10±1.08（%），CD83的阳性率为7.24±2.25（%），同时伴有人类主要组织相容性抗原HLA-ABC和协同刺激分子CD86（B7-2）的上调表达，HLA-DR的水平基本不变，代表单核细胞的表面标志CD14下降（P<0.05），诱导第7 d时，上述结果变化更明显，与5 d时的结果相比差异显著（P<0.05），表明PBMC在GM-CSF和IL-4的作用下已向DCs转化。
用肺癌细胞的总RNA装载上述DCs，以检验DCs的抗原提呈能力。我们提取了肺癌细胞的总RNA，变性凝胶电泳显示完整的28S和18S两条带，证实RNA完整未降解。紫外分光光度计测RNA浓度为1.69μg/μl，OD260/OD280>1.8。参照基因?-actin的RT-PCR电泳结果可见1000bp处的条带，亦证明总RNA中有翻译必需的mRNA存在。将此肺癌细胞总RNA经脂质体包裹，负载肺癌患者外周血单核细胞诱导扩增的DCs，流式细胞仪分析结果显示，转染总RNA 3d后，DCs的特
异性表面标志如CD83表达上调，其它功能相关的表面标志如HLA-ABC、HLA-DR和共刺激分子CD86（B7-2）表达也上调（P<0.05），而代表单核细胞的CD14表达则下调（P<0.05）；混合淋巴细胞培养结果显示，未转染RNA的DCs与转染RNA后的DCs都能刺激自身T淋巴细胞增殖活性（P<0.05），对同种异体T淋巴细胞则无此作用；转染RNA的DCs比未转染的DCs刺激自身淋巴细胞增殖的能力增强（P<0.05）。
3. MAGE-3基因的提取及转染LA795细胞
为进一步研制肿瘤疫苗，MAGE-3基因转染LA795细胞。首先，利用TRIzol Reagent提取Mel526细胞株总RNA，应用MAGE-3特异引物进行RT-PCR，获得MAGE-3基因；1%琼脂糖凝胶电泳分析，出现一1000bp的DNA条带，与预想的长度相符。以TA克隆方式将此产物克隆入pMD18-T载体，经酶切鉴定插入片段存在，长度约1000bp。将此重组质粒命名为pMD-MAGE3。对此基因进行测序，测序结果与MAGE-3 RNA参考序列（U03735）进行同源性比较，发现第162位碱基突变T→C，但并未引起氨基酸残基突变（CCT → CCC，均编码脯氨酸）。再将此基因亚克隆入荧光载体pEGFP-C1，经SacⅠ和SalⅠ双酶切，1%琼脂糖凝胶电泳显示一1000 bp左右的DNA条带，表明插入片段存在，命名pEGFP-MAGE3。
载体pEGFP-C1上的EGFP与其下游的外源基因融合表达，为确保此融合基因正确表达，对重组质粒pEGFP-MAGE3进行测序，结果显示阅读框正确。质粒pEGFP-C1含有荧光蛋白基因EGFP，此基因表达可以使转染细胞发出绿色荧光，便于观察转染效果；为此，pEGFP-MAGE3质粒转染LA795细胞24、48和72h，荧光显微镜下，可见绿色荧光的细胞。镜下随机计数10个视野的细胞，计算出转染效率（绿色荧光细胞所占的比例）为20％~30％。将1(2×105个LA795细胞种植于6?
Dendritic cells (DCs) have the strong function of specific duty antigen presenting cells (APC) in now. It can be achieve immunofunction of antigen by some ways. Some study showed that DCs is a bridge linked antigen and immune response cells to activate the immuneresponse of effective antitumor. Tumor rejection antigen codded by MAGE family produces antigen peptided proceseed in cells. Tumor rejection antigen combined with HLA-I formed a compplex and it distinguished by self-cytotoxic T lymphocytes (CTL), induce it specially killing relevant tumor cells. MAGE more highly expresses in many tumor tissues but can’t expresss in normal tissues (except testis and fetal tissue), it is a kind of ideal target to special immune mediated by CTL.
1. The expansion and maturation of inducing dendritic cells derived from mouse bone marrow in vitro
The expansion and maturation of inducing dendritic cells derived from mouse bone marrow in vitro. The dendritic cells derived from mouse bone marrow were proliferated by using GM-CSF and IL-4 in vitro. A part of suspension cells were abserved on 2 days after mouse bone marrow cells were cocultured with IL-4 (500u/ml) and GM-CSF (1000u/ml), the suspension cells increased with short process with burr 3 days later, and most of cells were floated with long process with burr, at the same time large round and fusiform cells decreased on the bottom of bottles 5 days later. The results showed that these cells have the characteristic of typical morphology of dentritic cells. The expressions of surface marker CD83 and CD86 of CD maturation induced by TNF-α stimulating or freezing and thawing antigen increased significantly as
compared with that in blank control group (P<0.05). There was significantly different between IL-4+GM-CSF+TNF-α group, IL-4+GM-CSF+carcinoma antigen group and IL-4+GM-CSF group, while there was not different between IL-4+GM-CSF+TNF-α and IL-4+GM-CSF+caicinoma antigen groups. The results showed that the expressions of CD83 and CD86 increased after dentritic cells were loaded with antigen and stimulated with TNF-α. Then the above mentioned mature DCs were used to perform mixture lymphocyte culture to stimulate T cells proliferation. The results showed that the proliferation of T cells was stimulated after DCs in IL-4+GM-CSF+TM-α group, IL-4+GM-CSF+freezing and thawing antigen group were cultured with T cells, CPM in which were significantly higher than that in blank control group and IL-4+GM-CSF group (P<0.001). The cpm of DCs cultured with T cells in IL-4+GM-CSF+freezing and thawing antigen group was significantly higher than that in IL-4+GM-CSF+TNF-α group (P<0.001). And the cpm in all the groups of mixture ratio of 1 to 5 was higher than that in ratio of 1:10 groups, but there was not significantly different.
2. DCs loaded with total RNA of tumor
The induction of DCsorigined from mononuclear leukocytes of periphery blood of lung cancer patient increased, DCs morphologically grew in a cluster of clony under invereted microscope, for 3 days in the culture of cells, the membrane in a part of them processed; for 6 days in culture, most of them grew in suspension, cell body was biger and irregular, cell membrane processed with branch tower, cytoplasm enriched, the cells present a typical DCs in morphology. There were a lot of microfolds and processes in the cell membrane, cell nucleus was biger and inclined to one side, mitochondria enriched in cells. After PBMC inducted with GM-CSF and IL-4 for 5 days in the culture, the positive rates of surface maker CD1a and CD83 detected with FCM were 11.10±1.08(%) and 7.24±2.25(%). Respectively, accompanied with the up-regulting expressions of HLA-ABC and CD86 (B7-2), down-regulating expression of CD14 and unclchange of HLA-DR. The results suggest that PBMC differentiated into DCs in the effecfs of GM-CSF and IL-4.
The above-mentioned DCs loaded with total RNA of lung cancer cells to detect antigen presenting ability of DCs. We extracted the total RNA with two bands of 28 S and 18 S detected with dematured gel electr
引文
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