MK调节MICA/B表达参与胃肿瘤免疫逃逸的机制及MK靶向疗效的评价
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摘要
中期因子(Midkine, MK)是一种肝素结合的细胞因子或生长因子,它的基因主要在胚胎期表达,随着发育的进展其表达组织逐渐局限,表达量逐渐减少：成年期个体仅在肺、结肠、胃、肾和脾等器官中极低地表达。近年来的研究发现,MK高表达于多种肿瘤组织。我们实验室先前的研究已表明,MK在癌组织中高表达(表达率为94.6%),且与癌旁组织和正常组织的表达有显著差异；进一步发现,MK的表达与肿瘤的临床分期及远处转移相关,而与肿瘤的组织分化、大小及淋巴结转移情况无关；免疫组化定位分析发现,MK蛋白广泛存在于肿瘤细胞的细胞质,且明显表达细胞外组织的腺泡处。这些结果提示MK有望成为新型的肿瘤标志物。可是,MK的检测体系还不完善；MK是否参与肿瘤的免疫逃逸尚不清楚；MK靶向治疗肿瘤的新方法需要探索。因此,本论文在以下方面进行了研究：
1、人中期因子(Midkine)单克隆抗体和ELISA检测试剂盒的研制：
为探讨MK作为肿瘤标志的可能性,本研究用MK重组蛋白免疫Balb/c小鼠,3次免疫后取脾细胞与SP2/0细胞融合、筛选得到3株抗人MK单克隆抗体杂交瘤细胞株,其中1C5杂交瘤细胞株较好。进一步利用1C5杂交瘤细胞株的腹水纯化获得单抗,HRP标记后,运用交叉配对法建立了ELISA检测体系,并通过比较24例肿瘤患者血清与正常人血清中的MK浓度进行了检测,初步评价了该检测体系。另外,运用筛选得到的抗MK单克隆抗体进行免疫印记试验,证实在胃癌组织和癌旁组织中MK的蛋白含量高于正常的胃粘膜组织。
2、MK参与肿瘤免疫逃逸机制的探讨：
目前的研究认为,MK主要具有诱发瘤细胞发生、促纤溶和细胞趋化、促进肿瘤血管形成及增强肿瘤细胞耐药性等方面的作用。可是,在肿瘤微环境组织中也有许多免疫细胞浸润,MK是否参与肿瘤的免疫逃逸尚不清楚。因此,为了探索MK在肿瘤免疫逃逸中的作用,利用基因芯片技术对MK高表达肿瘤细胞株BGC-MK进行相关基因分析,结果表明,当MK高表达时,肿瘤杀伤细胞(主要为NK细胞和CD8+T细胞)的活化配体MIC A/B也高表达,并经过RT-PCR、Q-PCR、FCM和ELISA确证,BGC-MK细胞的细胞膜表面和可溶性的MIC A/B表达都增加。进一步经过免疫共沉淀和染色体免疫共沉淀等试验发现,MK通过P38信号通路提高转录因子CHOP的表达,CHOP能与AP-1结合加强MIC A/B的表达。这些结果首次揭示了MK参与肿瘤免疫逃逸的新机制。
3、MK靶向治疗肿瘤的新方法的探索：
基于在胃癌组织和癌旁组织中MK的蛋白含量高于正常的胃粘膜组织的发现,我们认为MK可作为肿瘤靶向治疗的生物靶标。通常认为,与单克隆抗体相比,单链抗体(SCFV)可将药物、毒素、放射性核素等导向肿瘤,具很大的临床应用潜能等特性。为此,我们设计通用引物,采用RT-PCR方法从1C5杂交瘤细胞株中扩增抗体轻链可变区基因VL和重链可变区基因VH,用重叠PCR把VH和VL用短肽(Ser4Gly)4连接起来,在c端引入6×His标签,与pMD18 T-vector连接测序正确后,将该片段克隆入大肠杆菌表达载体pET30a(+)中,转化大肠杆菌表达菌株BL21(DE3),筛选得到可诱导表达MK的SCFV重组蛋白工程菌株,经IPTG诱导及镍亲和柱纯化获得SCFV重组蛋白。进一步通过氧化葡聚糖的醛基与SCFV和阿霉素(DOX)的NH3反应,把SCFV和DOX连接起来形成靶向药物SCFV-DOX。体内外试验证明,SCFV-DOX能在MK高表达的肿瘤组织中有效富集,提高DOX抑制肿瘤生长的效应。这种MK靶向治疗肿瘤的新方法可望用于临床治疗。
总之,本研究制备了人中期因子(Midkine)单克隆抗体,建立了MK的ELISA检测体系；首次揭示了MK通过调节MICA/B表达参与胃肿瘤免疫逃逸的新机制；利用MK单链抗体(SCFV)导向抗肿瘤药物的特性,探索了MK靶向治疗肿瘤的新方法。本研究为阐明MK在肿瘤发生用于发展中的作用机制及有效利用MK作为肿瘤靶向治疗的生物靶标都具有重要的意义。
Midkine (MK), a heparin-binding growth factor, is most strongly expressed in midgestation, with the progress of embryonic development, the expression of tissue gradually limitations, and is weakly expressed in the tissues of the lung, colon, stomach, kidney, and spleen, and not at all in the liver. Recently research found that MK expression in tumor tissues was higher than the corresponding adjacent normal tissues. Our previous research by RT-PCR and immunohistochemical analysis showed that compared with the healthy tissues, MK is highly expressed in gastric carcinoma tissues in Chinese patients. Which is related with the clicical stage and the far metastasis and mainly locate in the matrix of tumor cells. The research of MK expression characteristics and mechanism in tumor tissue of cancer patients, have provide new ways for tumor diagnosis and treatment to us.
To explore the possibility of MK as a tumor marker, we have immunized Balb/c mice three times with the MK recombinant protein, and then, taked the mice's spleen cells to fuse with the SP2/0 cells. We got three anti-human-MK hybridoma cell lines by screening with HAT and HT, three kinds of monoclonal antibody and those labeled with HRP by these cells. By using hybrid matching method with those antibodies and the HRP labeled's, we established the ELISA detection system to detect the MK concentrations in the 24 cancer patient's serum and in the health's serum. Conclusion showed that this ELISA kits can be used to detect the level of MK in the patient serum and urine of patient in clinical.
The current study showed that the roles of MK during the tumor formation, mainly were related to the occurrence of induced tumor cells, promote fibrinolysis and a chemotactic role in promoting tumor angiogenesis, increased drug resistance of tumor cells, have not MK and tumor immunity relevant aspects of the reported escape. In order to explore and study the occurrence of MK in the role of tumor immunity, using DNA microarray analysis of genes associated with the MK expression analysis found that high expression of the MK, the tumor killer cells (mainly NK cells and CD8+T cells) activation ligand MIC A/B high expression, and after RT-PCR, Q-PCR and FCM and ELISA tests to prove that MK expression of tumor cells, the cell surface and soluble MIC A/B expression were increased. After further study found, MK can increased the expression of transcription factor CHOP through the P38 signaling pathway, and then CHOP was combined with AP-1 together to promote the MIC A /B expression. We first found the new regulation mechanism of MIC A/B expression. We concluded that MK promot the MIC A/B expression in the tumor cells through the P38 signaling pathway to make tumor cells to escape from the immunal killing effect of NK cell and T cell killing effect.
MK-SCFV-DOX, which is an immunoconjugate of doxorubicin (DOX) and a single-chain Fv fragment against human midkine (MK-SCFV), was used to target chemotherapy to a mouse tumor model expressing high levels of the human MK antigen. The recombinant MK-SCFV expressed in bacteria was purified by metal affinity chromatography and then conjugated to the DOX by using oxidative dextran (Dex) as a linker. The molecular formula of this immunoconjugate was MK-SCFV(Dex)1.3(DOX)20. Apoptosis assay showed that the MK-SCFV-DOX had higher cytotoxicity against MK-over-expressed BGC823 cells (BGC823-MK cells) as compared to BGC823-3.1 cells. When given intravenously to tumor-bearing mice implanted with BGC823-MK cells, MK-SCFV-DOX suppressed tumor growth more effectively than free DOX (tumor growth inhibition rate of 51.83% as compared to 40.81%). Histologic analysis of the tumor tissue revealed that the MK-SCFV-DOX promoted more DOX accumulation in tumors and more tumor cell death than did free DOX. These results showed that MK-SCFV-DOX is more effective in the targeted inhibition of tumor growth and that MK-SCFV may be a competent drug-carrier.
1、人中期因子(Midkine)单克隆抗体和ELISA检测试剂盒的研制：
为探讨MK作为肿瘤标志的可能性,本研究用MK重组蛋白免疫Balb/c小鼠,3次免疫后取脾细胞与SP2/0细胞融合、筛选得到3株抗人MK单克隆抗体杂交瘤细胞株,其中1C5杂交瘤细胞株较好。进一步利用1C5杂交瘤细胞株的腹水纯化获得单抗,HRP标记后,运用交叉配对法建立了ELISA检测体系,并通过比较24例肿瘤患者血清与正常人血清中的MK浓度进行了检测,初步评价了该检测体系。另外,运用筛选得到的抗MK单克隆抗体进行免疫印记试验,证实在胃癌组织和癌旁组织中MK的蛋白含量高于正常的胃粘膜组织。
2、MK参与肿瘤免疫逃逸机制的探讨：
目前的研究认为,MK主要具有诱发瘤细胞发生、促纤溶和细胞趋化、促进肿瘤血管形成及增强肿瘤细胞耐药性等方面的作用。可是,在肿瘤微环境组织中也有许多免疫细胞浸润,MK是否参与肿瘤的免疫逃逸尚不清楚。因此,为了探索MK在肿瘤免疫逃逸中的作用,利用基因芯片技术对MK高表达肿瘤细胞株BGC-MK进行相关基因分析,结果表明,当MK高表达时,肿瘤杀伤细胞(主要为NK细胞和CD8+T细胞)的活化配体MIC A/B也高表达,并经过RT-PCR、Q-PCR、FCM和ELISA确证,BGC-MK细胞的细胞膜表面和可溶性的MIC A/B表达都增加。进一步经过免疫共沉淀和染色体免疫共沉淀等试验发现,MK通过P38信号通路提高转录因子CHOP的表达,CHOP能与AP-1结合加强MIC A/B的表达。这些结果首次揭示了MK参与肿瘤免疫逃逸的新机制。
3、MK靶向治疗肿瘤的新方法的探索：
基于在胃癌组织和癌旁组织中MK的蛋白含量高于正常的胃粘膜组织的发现,我们认为MK可作为肿瘤靶向治疗的生物靶标。通常认为,与单克隆抗体相比,单链抗体(SCFV)可将药物、毒素、放射性核素等导向肿瘤,具很大的临床应用潜能等特性。为此,我们设计通用引物,采用RT-PCR方法从1C5杂交瘤细胞株中扩增抗体轻链可变区基因VL和重链可变区基因VH,用重叠PCR把VH和VL用短肽(Ser4Gly)4连接起来,在c端引入6×His标签,与pMD18 T-vector连接测序正确后,将该片段克隆入大肠杆菌表达载体pET30a(+)中,转化大肠杆菌表达菌株BL21(DE3),筛选得到可诱导表达MK的SCFV重组蛋白工程菌株,经IPTG诱导及镍亲和柱纯化获得SCFV重组蛋白。进一步通过氧化葡聚糖的醛基与SCFV和阿霉素(DOX)的NH3反应,把SCFV和DOX连接起来形成靶向药物SCFV-DOX。体内外试验证明,SCFV-DOX能在MK高表达的肿瘤组织中有效富集,提高DOX抑制肿瘤生长的效应。这种MK靶向治疗肿瘤的新方法可望用于临床治疗。
总之,本研究制备了人中期因子(Midkine)单克隆抗体,建立了MK的ELISA检测体系；首次揭示了MK通过调节MICA/B表达参与胃肿瘤免疫逃逸的新机制；利用MK单链抗体(SCFV)导向抗肿瘤药物的特性,探索了MK靶向治疗肿瘤的新方法。本研究为阐明MK在肿瘤发生用于发展中的作用机制及有效利用MK作为肿瘤靶向治疗的生物靶标都具有重要的意义。
Midkine (MK), a heparin-binding growth factor, is most strongly expressed in midgestation, with the progress of embryonic development, the expression of tissue gradually limitations, and is weakly expressed in the tissues of the lung, colon, stomach, kidney, and spleen, and not at all in the liver. Recently research found that MK expression in tumor tissues was higher than the corresponding adjacent normal tissues. Our previous research by RT-PCR and immunohistochemical analysis showed that compared with the healthy tissues, MK is highly expressed in gastric carcinoma tissues in Chinese patients. Which is related with the clicical stage and the far metastasis and mainly locate in the matrix of tumor cells. The research of MK expression characteristics and mechanism in tumor tissue of cancer patients, have provide new ways for tumor diagnosis and treatment to us.
To explore the possibility of MK as a tumor marker, we have immunized Balb/c mice three times with the MK recombinant protein, and then, taked the mice's spleen cells to fuse with the SP2/0 cells. We got three anti-human-MK hybridoma cell lines by screening with HAT and HT, three kinds of monoclonal antibody and those labeled with HRP by these cells. By using hybrid matching method with those antibodies and the HRP labeled's, we established the ELISA detection system to detect the MK concentrations in the 24 cancer patient's serum and in the health's serum. Conclusion showed that this ELISA kits can be used to detect the level of MK in the patient serum and urine of patient in clinical.
The current study showed that the roles of MK during the tumor formation, mainly were related to the occurrence of induced tumor cells, promote fibrinolysis and a chemotactic role in promoting tumor angiogenesis, increased drug resistance of tumor cells, have not MK and tumor immunity relevant aspects of the reported escape. In order to explore and study the occurrence of MK in the role of tumor immunity, using DNA microarray analysis of genes associated with the MK expression analysis found that high expression of the MK, the tumor killer cells (mainly NK cells and CD8+T cells) activation ligand MIC A/B high expression, and after RT-PCR, Q-PCR and FCM and ELISA tests to prove that MK expression of tumor cells, the cell surface and soluble MIC A/B expression were increased. After further study found, MK can increased the expression of transcription factor CHOP through the P38 signaling pathway, and then CHOP was combined with AP-1 together to promote the MIC A /B expression. We first found the new regulation mechanism of MIC A/B expression. We concluded that MK promot the MIC A/B expression in the tumor cells through the P38 signaling pathway to make tumor cells to escape from the immunal killing effect of NK cell and T cell killing effect.
MK-SCFV-DOX, which is an immunoconjugate of doxorubicin (DOX) and a single-chain Fv fragment against human midkine (MK-SCFV), was used to target chemotherapy to a mouse tumor model expressing high levels of the human MK antigen. The recombinant MK-SCFV expressed in bacteria was purified by metal affinity chromatography and then conjugated to the DOX by using oxidative dextran (Dex) as a linker. The molecular formula of this immunoconjugate was MK-SCFV(Dex)1.3(DOX)20. Apoptosis assay showed that the MK-SCFV-DOX had higher cytotoxicity against MK-over-expressed BGC823 cells (BGC823-MK cells) as compared to BGC823-3.1 cells. When given intravenously to tumor-bearing mice implanted with BGC823-MK cells, MK-SCFV-DOX suppressed tumor growth more effectively than free DOX (tumor growth inhibition rate of 51.83% as compared to 40.81%). Histologic analysis of the tumor tissue revealed that the MK-SCFV-DOX promoted more DOX accumulation in tumors and more tumor cell death than did free DOX. These results showed that MK-SCFV-DOX is more effective in the targeted inhibition of tumor growth and that MK-SCFV may be a competent drug-carrier.
引文
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