【作者】 王平；

【导师】 熊思东；

【作者基本信息】 复旦大学 ， 免疫学， 2009， 博士

【摘要】 肿瘤是一类严重威胁人类健康的疾病,近年的研究表明：通过合理的综合治疗,可以提高恶性肿瘤的治疗效果。肿瘤生物治疗作为继传统的手术、放疗和化疗之后的第四种治疗模式在肿瘤的综合治疗中发挥愈来愈重要的作用。在肿瘤患者重建有效的免疫应答及抑制肿瘤的血管形成,是肿瘤治疗的两个重要策略。趋化因子不仅可引导淋巴细胞定向迁移,同时又能抑制血管形成,阻断肿瘤的血液供应,因此是肿瘤生物治疗的理想选择。已有的研究表明,CXC亚家族趋化因子IFN诱导的T细胞α趋化因子(ITAC)和IFN-y诱导蛋白10(IP10)既具有募集表达CXC趋化因子受体3(CXCR3)淋巴细胞的功能,诱导Thl型免疫应答,又能够抑制血管生成。功能分析表明,ITAC是CXCR3最强的激动剂；IP10具有较ITAC更显著的抗血管作用。结构分析表明,趋化因子的N端和N-LOOP区是结合及活化受体的关键部位,主要通过C端结合在血管内皮细胞表面发挥抗血管生成的作用。我们实验室的前期工作根据小鼠ITAC和IP10基因序列设计构建了一个新的分子,由ITAC的N端、N-LOOP区与IP10的C端融合而成,命名为ITIP。计算机模拟结果表明：新分子ITIP能形成稳定的趋化因子样三维空间构象。我们期望ITIP功能上具有类似趋化因子的特性,更重要的是保留ITAC和IP10各自的功能优势,从而发挥更显著的抗肿瘤作用。为验证我们的设想是否成立,本研究表达纯化得到ITIP蛋白,通过体外功能实验检测其活性；在不同的肿瘤模型,观察ITIP抗肿瘤效果；最后探讨ITIP的抗肿瘤机制。第一部分新分子ITIP的原核表达、纯化及其特异性抗体制备为了大量制备有活性的ITIP蛋白,我们采用原核系统表达融合蛋白的策略：在目的基因的N端融合硫氧还蛋白标记、6组氨酸标记以及编码肠激酶特异识别多肽的碱基序列。硫氧还蛋白的融合表达,有助于提高表达产物的稳定性；6组氨酸标记有利于后续融合蛋白的纯化；肠激酶的识别位点便于酶切去除N端标签蛋白得到ITIP目的蛋白。我们首先以实验室前期构建的pcDNA3-ITIP载体为模板PCR扩增得到编码ITIP成熟肽的基因片段,插入原核表达载体pET32a(+)构建pET32a-ITIP的重组质粒。将构建成功的pET32a-ITIP转化大肠杆菌BL21(DE3)经异丙基硫代-β-D-半乳糖苷(IPTG)诱导,结果在分子量27KDa可见明显的条带,与预期的融合蛋白分子量大小一致(标签蛋白约19KDa,ITIP目的蛋白约8.7KDa),提示成功诱导表达ITIP融合蛋白。经蛋白表达条件优化,最后确定0.1mMIPTG37℃诱导4h为最适条件。SDS聚丙烯酰氨凝胶电泳(SDS-PAGE)分析蛋白分布,结果融合蛋白在上清和包涵体均有表达,主要以可溶性形式表达。融合蛋白以镍离子亲和层析法纯化,SDS-PAGE鉴定纯度>90%。低温冷冻干燥浓缩融合蛋白后经二辛可宁酸(BCA)法蛋白定量,以重组肠激酶(rEK)酶切去除N端融合的标签蛋白,再过镍离子亲和层析柱,收集穿透液得到ITIP蛋白。目的蛋白的Tricine-SDS-PAGE分析结果显示,分子量8.7KDa处可见明显条带,与ITIP蛋白预期分子量大小一致,纯度>90%。以ITAC的N端抗体、IP10的C端抗体分别进行Western blot鉴定ITIP蛋白,均可见单一条带。以ITIP蛋白作为免疫原免疫新西兰白兔,免疫后血清经饱和硫酸铵沉淀、QAE-Sephadex A50离子交换层析纯化得到ITIP多克隆抗体(pAb)。酶联免疫吸附实验(ELISA)测定效价为1：100000,Western blot鉴定得到单一条带。同时为后续动物实验对照蛋白的需要,我们也构建了pET32a-ITAC及pET32a-IP10重组质粒,以相同条件诱导表达、纯化相应蛋白,经Tricine-SDS-PAGE和Western blot鉴定,制备得到ITAC和IP10蛋白。第二部分ITIP的体外功能特性鉴定为确定构建表达的新分子ITIP是否具有趋化因子样的功能特性,更重要的是检测是否具有我们所期望的ITAC更为优势的激动受体CXCR3的能力,及IP10更好的抗血管作用。我们分别应用趋化、受体内化、钙离子内流实验检测ITIP是否保留了与ITAC类似的活性,应用血管内皮细胞的划痕修复、碱性成纤维细胞生长因子(bFGF)诱导的小鼠腹壁Matrigel种植体实验鉴定ITIP是否具有与IP10相当的抑制血管形成的特性。鉴于ITAC和IP10均为CXCR3的配体,而ITIP由ITAC和IP10的结构域组成,我们推测ITIP也通过CXCR3受体发挥作用。已有的研究表明,CXCR3主要高表达在活化的T淋巴细胞表面,我们首先以荧光激活细胞分类术(FACS)确定Con A与IL-2刺激活化的淋巴细胞CXCR3的表达,然后应用此细胞检测ITIP的趋化活性。结果显示：1、10、100、1000ng/ml ITIP对活化的淋巴细胞均有不同程度的趋化作用,其中以100ng/ml趋化活性最强。以CXCR3阻断抗体与活化淋巴细胞孵育后,再以100n/ml ITIP进行趋化实验,结果趋化活性明显受到抑制,提示ITIP对于CXCR3+淋巴细胞具有趋化活性。以市售的ITAC、IP10蛋白为对照,比较ITIP的趋化能力,结果表明浓度10、100、1000ng/ml ITIP的趋化活性与ITAC相当,较IP10显著,也优于ITAC与IP10联合使用(P<0.05),提示新分子基本保留了ITAC的N端和N-LOOP区的功能特性。为进一步证实ITIP的活性,我们构建了pcDNA3.1-mCXCR3重组质粒,电转CHO细胞,经G418筛选,RT-PCR、FACS鉴定稳定高表达mCXCR3的细胞株。然后分别应用活化的淋巴细胞、稳定转染mCXCR3的CHO(CHO/mCXCR3)细胞进行CXCR3受体内化实验。结果与趋化实验一致：ITIP与ITAC活性类似,在100ng/ml使细胞表面CXCR3表达降低55-65%, IP10使其降低30-35%,ITAC与IP10联合使用降低35-40%。最后分别应用活化的淋巴细胞和CHO/mCXCR3细胞进行钙离子内流实验进一步证实ITIP的活性：ITIP可引起短暂而迅速的钙离子内流,ITIP、ITAC激发的平均荧光强度(MFI)高于IP10、ITAC与IP10联合使用所检测到的MFI。通过上述几种不同的检测方法,我们均得到一致的结果,提示ITIP保留了ITAC的N端和N-LOOP区所具有的功能优势,具有与ITAC类似的更强的激动受体CXCR3的活性,优于IP10单独或与ITAC合用的效果。血管内皮细胞划痕修复实验结果显示：ITIP对bFGF诱导血管内皮细胞迁移的抑制作用与IP10相当,迁移入划痕区的细胞数,较ITAC处理组明显减少(P<0.01),与ITAC和IP10简单混合组比较具有统计学差异(P<0.05),提示ITIP保留了IP10的功能特性。为进一步确证ITIP抑制血管的活性,我们采用检测体内血管生成的常用方法Matrigel种植体技术,观察ITIP对bFGF诱导的小鼠体内血管生成的抑制效果。结果显示：ITIP处理组与IP10处理组类似,与ITAC处理组、ITAC与IP10联合处理组比较,迁移入Matrigel种植体内的血管内皮细胞的数量减少,形成的血管管腔的数目降低,结果均有统计学差异(P<0.05),提示ITIP保留了IP10更有效的抑制血管形成的作用。总结以上结果：本研究构建表达的新分子ITIP是有生物活性的,更重要的是具有我们预期得到的ITAC和IP10各自的功能优势,可用于下一步的动物实验研究。第三部分ITIP在不同肿瘤模型中抗肿瘤效果研究本部分工作,旨在通过建立不同的肿瘤模型,观察融合了ITAC和IP10功能优势的新分子ITIP是否可发挥更显著的抗肿瘤的作用,有效抑制肿瘤的生长乃至促进肿瘤消退。我们分别建立BALB/c小鼠CT26结肠癌、4T1乳腺癌和C57BL/6小鼠3LL肺癌三种肿瘤模型,应用ITIP蛋白肿瘤局部注射,同时以PBS、ITAC、IP10、ITAC联合IP10蛋白治疗组为对照。ITAC与IP10蛋白为第一部分制备得到的蛋白,经趋化实验、bFGF诱导的小鼠腹壁Matrigel种植体实验鉴定与市售ITAC、IP10蛋白具有相近的活性。三种肿瘤模型的治疗结果显示：ITIP蛋白可抑制肿瘤生长,较PBS对照组统计学差异显著(P<0.001)。应用ITAC、IP10蛋白治疗,可以延缓肿瘤的生长,一定程度上延长小鼠的生存期,结果与已有报道一致。应用ITIP蛋白治疗,与单独应用ITAC、IP10蛋白或是联合应用两种蛋白治疗比较,能够更为有效的抑制肿瘤生长、延长小鼠生存期,结果均有统计学差异(P<0.05)。尤其在CT26结肠癌、4T1乳腺癌模型,具有明显的抗肿瘤效果,ITIP治疗组小鼠肿瘤完全消退,小鼠生存期明显延长,90天依然100%存活。同时我们观察了ITIP蛋白可能对小鼠潜在的副作用,重要器官心、肝、脾、肺、肾的苏木素-伊红(H&E)染色结果显示：各组织未见异常的形态学变化。ELISA检测不同时间点收集的小鼠血清未检测到抗ITIP的抗体。第四部分ITIP抗肿瘤机制探讨我们以CT26结肠癌模型为研究基础,分别检测各组治疗后24h脾脏淋巴细胞亚群的比例变化、肿瘤局部的淋巴细胞浸润、荷瘤小鼠细胞免疫应答水平及肿瘤局部的血管形成,初步探讨ITIP可能的抗肿瘤机制。首先基于已有的报道：应用趋化因子进行肿瘤局部治疗,不仅可以抑制治疗侧肿瘤的生长,同时也可一定程度延缓远侧未治疗肿瘤的生长。对ITIP也进行了同样的检测,结果发现ITIP治疗具有这样的作用。考虑ITIP局部治疗可能激发了全身性的免疫应答,我们应用FACS检测各治疗组脾脏淋巴细胞比例的变化。结果显示：ITIP治疗组CXCR3+淋巴细胞比例与ITAC治疗组比较无明显差异,但较其它各组均有增加(P<0.05)。对表达CXCR3的主要淋巴细胞亚群进行分析,发现其中CD3+T细胞增加最为显著；Mφ、NK、DC细胞的比例变化各蛋白治疗组之间比较无明显差异。进一步检测结果显示：ITIP治疗组CD4+和CD8+的淋巴细胞比例,与PBS、IP10、ITAC联合IP10蛋白治疗组比较均有上调(P<0.05)。肿瘤组织的H&E染色结果显示：ITIP与ITAC治疗组,可见大面积的坏死,大量的淋巴细胞浸润；其他蛋白治疗组只有散在的坏死灶和部分淋巴细胞浸润。肿瘤组织的免疫荧光染色结果显示：CXCR3+淋巴细胞浸润明显,其中CD3+T细胞为主,CD4+、CD8+的淋巴细胞均有不同程度浸润,CD8+淋巴细胞浸润更为明显。肿瘤组织的CD31免疫荧光染色结果可见：ITIP与IP10治疗组肿瘤的血管密度明显降低,与其它各蛋白治疗组相比,有统计学差异(P<0.05)。荷瘤小鼠细胞免疫应答检测结果：ITIP治疗组脾脏淋巴细胞以肿瘤特异性抗原刺激后,体外增殖能力与ITAC组相当,较IP10、ITAC联合IP10治疗组增强；对CT26靶细胞的特异性杀伤活性增强,与IP10、ITAC联合IP10治疗组比较有统计学意义(P<0.05),提示ITIP增强了机体细胞免疫应答水平。综上所述,基于ITAC和IP10的功能优势,通过理论预测而构建的新分子ITIP,不仅形成了趋化因子样的空间结构,功能上也具有趋化因子样的特性,更重要的是保留了两个分子各自的优势功能,在抗肿瘤免疫中体现了各自的优点：不仅有效趋化淋巴细胞,增强特异性免疫应答,同时显著抑制血管形成,发挥了更有效的抗肿瘤作用。为应用于肿瘤的生物学防治提供了理论基础和实验依据；同时也为利用不同分子的优势结构域构建功能更强的新分子,进一步应用于疾病的防治提供了新的思路和数据支持。更多还原

【Abstract】 A common devastating disease is malignant tumor, which causing great insult to the health and the life quality of human. In recent years many different approaches to anticancer therapies have been developed. Some of them rely on the enhancement of an antitumor immune response through the attraction and activation of immune cells into the surrounding area of a developing tumor. Others are focused on disturbing proper energy supply through inhibition of angiogenesis. Chemokines are small, secreted proteins that activate G protein-coupled receptors, resulting in the activation of several distinct signaling cascades and leading most importantly, to directed migration of cells along the chemokine gradient. Based on either of these two mechanisms, several chemokines have been shown to elicit strong antitumor responses.ITAC and IP 10, members of the CXC chemokine family, have the same receptor CXCR3 which is predominantly expressed on activated T lymphocytes. Increasing evidence suggests that the ITAC and IP10 may be important in the development of type 1 cytokine-induced cell-mediated antitumor immunity, and at the same time inhibit tumor associated angiogenesis leading to suppression of tumor growth. ITAC is the most potent agonist of CXCR3 compared to the other ligands of CXCR3. Relative to ITAC, IP10 exert more effective angiogenesis activity. Previous studies of structure-activity relations have shown that both the NH2 terminus and N-loop region contribute to the higher receptor-binding affinity and activity of ITAC compared with IP10 and suggest that these two domains are the major functional determinants to interact with CXCR3. While other studies have suggested that the chemokines exert angiostatic effects directly on endothelial cells via the C-terminal domain. Chemokines adopt a remarkably conserved structure. The regions of chemokines with similar structure are interchangeable. On the basis of the functional differences of ITAC and IP10, combination of the two chemokines may result in a marked antitumor effect. However, they that act at the common receptor CXCR3 block the other activity. Thus ITAC and IP 10 in combination could not exhibit enhanced antitumor activity compared with either chemokine alone. Our previous study designed a novel molecular which was constructed by substituting the N-terminal and N-Loop region of mouse IP10 with those of ITAC, named ITIP. Analyzed with Insight II work station, ITIP could form a stable dimensional structure. The homogeneity of amino acid with IP 10 is 66%. We hypothesis that ITIP may exert chemokine like activity, moreover it retain the function superiority of both chemokines. And the new molecule ITIP would be expected to lead to enhanced antitumor responses as compared to either chemokine alone.In the present study, the novel protein was expressed and purified in Escherichia coli. The biological properties of that were assessed in vitro and in vivo. It was demonstrated that the novel protein displayed more potent antitumor activity than ITAC or IP10 in vivo in three tumor models. Further, the mechanisms of ITIP-mediated anti-tumor effects were explored on the CT26 tumor model.Part I Preparation and purification ITIP protein and its specific polyclonal antibodyThe nucleotide sequence encoding the mature protein region of ITIP cDNA was amplified with PCR and then cloned into an expression plasmid pET-32a. The final recombinant expression plasmid (pET32a-ITIP) was sequenced to ensure that the insert was in the correct reading frame and subsequently the protein was expressed in Escherichia coli BL21(DE3), under 0.1 mM ITPG. This system produces a recombinant thioredoxin fusion protein. Compared with before induction, a new protein band in SDS-PAGE appeared in the expected site with a predicted molecular mass of 27 KDa. To determine the location of the fusion protein in the cellular fractions of E. coli, the supernatant and inclusion body fractions were tested. The recombinant protein was found mainly in the soluble fraction. The recombinant fusion protein was purified by immobilized metal affinity chromatography (IMAC) using a nickel-chelating column according to the manufacturer’s recommendations. The N-terminal fusion partner was cleaved from the fusion protein with enterokinase. Analysis of purity by Tricine-SDS-PAGE showed a single band with a predicted molecular mass of 8.7 KDa. The novel molecular ITIP was also examined by Western blot using both N-terminal Ab of ITAC and C-terminal Ab of IP10. The protein concentration was determined by the BCA protein assay with bovine serum albumin as standard. The pAb were prepared against ITIP and preliminarily purified. The titer was 1:100 000 assayed by ELISA. The specificity of pAb was identified by Western blot. For the need of the control protein in the animal study, we also constructed pET32a-ITAC and pET32a-IP10 recombinant expression plasmid. And the ITAC and IP10 protein were prepared with similar process as ITIP.PartⅡFunctional characterization of ITIPThe functional activities of ITIP were first assessed by measuring chemotactic migration of Con A/IL-2-treated lymphocytes expressing CXCR3. Commercially available recombinant murine ITAC, IP10 were used as standard control. A dose-dependent chemotactic effect was obtained with ITIP on Con A/IL-2-treated T cells, which was significantly blocked by mCXCR3 antibody at 100ng/ml (p<0.001) but not by isotype IgG (p>0.05). As intended, similar capacity of chemotaxis was observed with ITIP compared to ITAC. Importantly, the chemotactic activity of ITIP was more potent than IP10 alone or in combination with ITAC at 10,100, and 1000ng/ml (p<0.05). These results suggested that the new molecule ITIP containing the N-terminal and N-Loop regions of ITAC retained its potent lymphocyte chemotactic activity against activated lymphocytes. To further dissect whether ITIP contained similar features of ITAC, the receptor internalization assay was performed by FACS before and after agent exposure. We initially used Con A/IL-2-treated lymphocytes to investigate the down-regulation of CXCR3 following incubation with different agent for 30 min. At higher concentrations, ITIP as ITAC was much more potent than IP 10 or ITAC plus IP10. To confirm this, the CHO/mCXCR3 cell line was established. The expression of CXCR3 was detected by RT-PCR, and FACS. A similar attenuated pattern was also found with CHO/mCXCR3 cells. For both cell types, ITIP as ITAC was more potent than IP10 or ITAC plus IP10, and the maximum level of receptor down-regulation observed was reduced to<50% of starting levels at 100ng/ml. Further, the Con A/IL-2-treated lymphocytes and CHO/mCXCR3 cells were used to investigate whether ITIP could desensitize calcium signaling through CXCR3. Upon stimulation with ITIP, ITAC, IP10, or ITAC plus IP10, there were evidences of calcium mobilization on Con A/IL-2-treated lymphocytes and CHO/mCXCR3 cells. Importantly, ITIP and ITAC were more efficient to induce an [Ca+]i compared with IP10, or ITAC plus IP 10.We postulated that ITIP generated from IP10 may also be an inhibitor of angiogenesis. To test this hypothesis, an in vitro wound-healing assay was performed. The capacity of ITIP to block bEND-3 cell regrowth after wounding was assessed by quantifying cell numbers in the denuded zone. The results suggested ITIP was a more-potent inhibitor compared with ITAC (P<0.01) or ITAC plus IP 10 (P<0.05), whereas no statistically significant difference was found between the inhibitory potency of ITIP and IP10 (P=0.5347). This in vitro potency of ITIP to inhibit endothelial cell migration was finally confirmed using the in vivo Matrigel plug assay for angiogenesis. Addition of ITIP to Matrigel plus bFGF resulted in marked reduction in the number of endothelial cells invading the plug and the absence of blood vessels. In this assay, ITIP was also more effective than ITAC or ITAC plus IP10 (P<0.05) but has similar potency as IP10. These results indicated that the novel molecular ITIP retained the angiostatic effect of IP 10.Part III Anti-tumor effects of ITIPThe antitumor efficacy of ITIP was evaluated in BALB/c mice with established CT26 tumors,4T1 tumors and in C57BL/6 mice with established 3LL tumors. Mice were intratumoral administrated of ITIP, while the control groups were injected with PBS solution, ITAC, IP10, or in combination of both at the same time and points. In the three tumor models, tumor volume and life span of mice assay showed that both ITAC and IP 10 individually or in combination resulted in reduction of tumor growth and led to tumor stasis. Remarkably, ITIP treatment had a superior antitumor effect, versus either chemokine alone or in combination with both (P<0.05). More importantly, in the CT26 and 4T1 tumor models, ITIP resulted in complete tumor eradication and the mice survived 90 days.ITIP-treated animals without tumor burden were particularly investigated for potential toxicity of ITIP for>2 months. None of pathologic changes in liver, lung, kidney, etc. were found by microscopic examination after administration of ITIP. No adverse consequences were showed in gross measures, such as weight loss, ruffling of fur, life span, behavior, or feeding. No autoreactive antibody in serum can be determined by ELISA. Part IV The anti-tumor mechanisms of ITIPThe mechanisms of ITIP-mediated anti-tumor effects were explored in the CT26 tumor model. Firstly, to analyze distant effects of ITIP intratumoral treatment, we injected CT26 tumor cells into mice at two separate sites, and treated one of the resultant tumors. For mice that had received ITIP injections in the right tumors, the reduction was observed for the untreated left tumors through day 15. These data suggest that ITIP treatment initiates immune responses capable of acting both locally against the treated primary tumor and systemically against a spatially distant tumor.The lymphocyte subsets of splenocytes from tumor-bearing mice that had been treated intratumorally with ITIP or other control were analyzed by FACS. The results showed that the CXCR3-expressing lymphocytes of ITIP treated mice increased versus IP10, ITAC plus IP10 group (P<0.05). The increased cells were mainly CD3+ T cells, including both CD4+ and CD8+ lymphocytes. Histological examination of tumor sections by H&E staining showed that there were remarkable response with necrosis and lymphocyte infiltration in ITIP and ITAC treated group. To identify tumor-infiltrating leukocytes, we performed immunofluorescent (IF) on cryosections from treated tumors. IP10 alone or combine with ITAC-treated tumor sections had few infiltrating CD3+ T cells. In contrast, ITIP and ITAC treated tumor sections had increased numbers of CD3+ T cells spread diffusely throughout the entire section, including both CD4+ and CD8+ lymphocytes.We further quantified vessel density as measures of angiogenesis by immunolabeling of CD31 in tissue sections. The ITIP apparently reduced the number of vessels compared with control groups, including ITAC alone or in combination with IP 10 (P<0.05). No statistically significant differences were obtained between ITIP and IP 10 group.The cellular immune response was examined in tumor bearing mice. After stimulation with CT26 cells in vitro, splenocytes from the ITIP and ITAC mice exhibited vigorous specific proliferation and cytotoxic activity.In summary, the novel molecule ITIP constructed by combining the N-terminal, N-LOOP region of ITAC with the C-terminal of IP10 was demonstrated to have anti-tumor effects. Recruiting CXCR3-expressing lymphocytes, inducing enhanced cellular immune response and anti-angiogenesis might be involved in ITIP-mediated anti-tumor effects.更多还原