癌症严重威胁着人类生存和健康，迄今都是世界难题。恶性黑色素瘤发端于黑色素细胞，具有严重的致命性，目前临床治疗多采用达卡巴嗪（DTIC）化疗和手术切除等方法，但见效慢且缓解率低。如何引入新的抗肿瘤疗法具有极其重要的意义。膜联蛋白Annexin A5（ANV）广泛应用于细胞凋亡检测，但有较多研究表明，ANV参多种肿瘤的发生发展，可能发挥抗肿瘤效应。Lebestatin（LBT）是从钝鼻蝰属 (Macrovipera lebetina) 蛇毒中分离得到的具有41个氨基酸的小肽，是整合素α₁β₁的受体拮抗剂，能有效抑制细胞粘附和迁移，发挥抗血管生成等作用从而抑制肿瘤生长。基于此，为了增强对肿瘤细胞的抵抗作用，实验室将LBT和ANV融合构建形成新型的重组蛋白LbtA5，探究其对黑色素瘤的生物效应及相关作用机制，并为其他抗肿瘤研究提供参考信息。具体研究内容如下：

（1）目的蛋白的制备与纯化

基于生物工程蛋白质下游纯化技术，我们将构建的重组LbtA5、ANV及LBT在大肠杆菌细胞中表达，并通过亲和层析进行纯化。后期又纯化获得PP酶，通过优化酶切反应条件及参数，对相关重组蛋白质进行了制备，获得高纯度目的蛋白，为后续开展细胞实验和动物实验奠定基础。

（2）LBT、LbtA5及ANV理化性质分析

采用圆二色谱法检测ANV和LbtA5的二级结构，结果体现二者具有相同的结构组成和近似比例，说明ANV和LbtA5有相似的生化特性。利用分子生物学手段，构建LBT重组表达载体，并成功纯化出LBT蛋白。粘附实验表明，外源添加ANV对于HT29细胞与配体LN结合、PC12细胞与配体Col-Ⅳ结合无显著影响；融合蛋白LbtA5对于HT29细胞粘附LN无影响，但对于PC12细胞粘附Col-Ⅳ，表现出和LBT一样的较强的抑制性。说明融合蛋白LbtA5与LBT可以通过特异性结合整合素α₁β₁从而抑制其与配体的结合。

（3）LbtA5及ANV对B16-F10细胞抑制作用的机制研究

以B16-F10细胞为对象，运用MTT检测LbtA5及ANV对细胞活力的影响，结果表明，二者在作用48h时均能显著降低B16-F10细胞活力，且在时间和剂量呈现依赖性，相同浓度LbtA5的抑制效果较ANV有极显著差异。通过观察B16-F10细胞形态学，发现LbtA5和ANV不会改变细胞形态。采用流式细胞术检测显示，LbtA5和ANV不能诱导体外培养B16-F10细胞凋亡。划痕实验表明，LbtA5和ANV能阻碍B16-F10细胞迁移。进一步通过Western blotting研究LbtA5和ANV抗B16-F10细胞机制，结果显示LbtA5和ANV并不是通过下调B16-F10细胞的VEGF表达来发挥作用。

（4）LbtA5及ANV对小鼠黑色素瘤的作用机制研究

建立黑色素瘤荷瘤小鼠模型，根据连续给药13天期间小鼠体重和肿瘤体积变化，发现蛋白干预下的小鼠肿瘤生长速度缓慢，LbtA5组效果优于ANV组。利用HE染色观察黑色素瘤组织形态，发现LbtA5组和ANV组肿瘤组织细胞松散，坏死区域胞核消失，有中性粒细胞聚集，胞核溶解消失，甚至成破碎的细胞残片，同一注射量下，LbtA5对肿瘤组织的破坏作用较ANV更强。运用免疫组化法检测小鼠黑色素瘤肿瘤组织血管密度，结果表明，LbtA5和ANV能有效降低肿瘤组织血管密度，且呈剂量依赖性，LbtA5相较于ANV有更显著的抑制体内肿瘤血管生成效果。

（5）LbtA5和ANV在黑色素瘤中的组织定位检测

我们制备并纯化了FITC与ANV或LbtA5的偶联产物fANV和fLbtA5，通过尾静脉注射的方式作用荷瘤小鼠，结果显示，fANV和fLbtA5均可定位于黑色素瘤肿瘤组织中，且强度呈现剂量依赖性，LbtA5具有更强的肿瘤靶向性。表明LbtA5与ANV均通过定位于小鼠肿瘤组织发挥抗肿瘤生物学效应，且极有可能是通过影响小鼠黑色素瘤肿瘤组织中的血管增生方式，发挥拮抗-肿瘤的生物学效应。

上述实验结果表明，无论是体外培养的B16-F10细胞，还是小鼠黑色素瘤，ANV和LbtA5均能在一定程度上起到抑制作用。更重要的是，融合蛋白LbtA5的抑制效应要强于ANV。肿瘤生长调控是一个复杂精细的过程，需要从不同角度更加深入地探究。

Cancer is a serious threat to human survival and health. So far, it is a difficult problem in the world. Malignant melanoma originates from melanocytes and has serious lethality. At present, dacarbazine (DTIC) chemotherapy and surgical resection are mostly used in clinical treatment, but the effect is slow and the remission rate is low. How to introduce new anti-tumor therapy is of great significance. Annexin A5 (ANV) is widely used in the detection of apoptosis, but many studies have shown that ANV is involved in the occurrence and development of a variety of tumors and may play an anti-tumor effect. Lebestatin (LBT) is a small peptide with 41 amino acids isolated from the venom of macrovipera lebetina. It is a receptor antagonist of integrin α₁β₁ and can effectively inhibit cell adhesion and migration, play an anti angiogenesis role, and inhibit tumor growth. Based on this, in order to enhance the resistance to tumor cells, a new recombinant protein LbtA5 was constructed based on LBT and ANV and its biological effects on melanoma in mice were detected, and related anti-tumor mechanism was also analyzed and explored of. The research contents are shown as following:

      (1) Production and purification of target proteins

Based on the downstream technology of protein engineering, we expressed the constructed recombinant LbtA5, ANV and LBT in E. coli cells and purified them mainly by affinity chromatography. On the other hand, PPase protease was also prepared and purified. By optimizing the conditions and parameters of the enzymatic reaction, the relevant recombinant proteins were prepared with high-level purity, which laid the foundation for subsequent cell experiments and animal experiments.

      (2) Analysis of physical and chemical properties of LBT, LbtA5 and ANV

Circular dichroism was used to detect the secondary structure of ANV and LbtA5. The results showed that they had the same structural composition and approximate proportion, indicating that ANV and LbtA5 had similar biochemical characteristics. LBT recombinant expression vector was constructed by means of molecular biology, and lebestatin protein was successfully purified. The adhesion experiment showed that the exogenous addition of ANV had no significant effect on the binding of HT29 cells to ligand LN and PC12 cells to ligand col-Ⅳ; The fusion protein LbtA5 had no effect on the adhesion of HT29 cells to LN, but had strong inhibition on the adhesion of PC12 cells to col-Ⅳ as well as LBT. It shows that the fusion protein LbtA5 and LBT can bind integrin α₁β₁specifically to inhibit its binding to ligands.

      (3) Study on the inhibitory mechanism of LbtA5 and ANV on B16-F10 cells

Taking B16-F10 cells as the object, the effects of LbtA5and ANV on cell viability were detected by MTT. The results showed that both of them could significantly reduce the viability of B16-F10 cells in a time-dependent and dose-dependent manner, and the inhibitory effect of the same concentration of LbtA5 was significantly different from that of ANV. By observing the morphology of B16-F10 cells, it was found that LbtA5 and ANV did not change the cell morphology. Flow cytometry detection showed that LbtA5 and ANV could not induce apoptosis of B16-F10 cells in vitro. Scratch test showed that a certain concentration of LbtA5 and ANV could inhibit the migration of B16-F10 cells. The mechanism of LbtA5and ANV against B16-F10 cells was further studied by Western blotting. The results showed that LbtA5 and ANV did not play a role by down regulating the expression of VEGF in B16-F10 cells.

      (4) Study on the mechanism of LbtA5 and ANV on mouse melanoma

The melanoma bearing mouse model was established. According to the changes of mouse weight and tumor volume during continuous administration for 13 days, it can be found that the tumor growth rate of mice under protein intervention is slow, and the effect of LbtA5 group is better than that of ANV group. HE staining was used to observe the tissue morphology of melanoma. It was found that the tumor cells in LbtA5 group and ANV group were loose, the nuclei in necrotic areas disappeared, neutrophils gathered, the nuclei dissolved and even became broken cell fragments. Under the same injection amount, the destructive effect of LbtA5 on tumor tissue was stronger than that of ANV. The results showed that LbtA5 and ANV could effectively reduce the vascular density of tumor tissue, and LbtA5 had a more significant inhibitory effect on tumor angiogenesis than ANV.

      (5) Tissue localization of LbtA5 and ANV in melanoma

We prepared and purified the coupling products of FITC with ANV or LbtA5, fANV and fLbtA5, and treated tumor bearing mice by tail vein injection. The results showed that both fANV and fLbtA5 could be localized in melanoma tumor tissue in a dose-dependent manner, and LbtA5 had stronger tumor targeting. It shows that both LbtA5 and ANV exert anti-tumor biological effects by locating in mouse tumor tissues, and most likely by affecting the mode of angiogenesis.

       The above experimental results show that ANV and LbtA5can inhibit B16-F10 cultured in vitro and mouse melanoma to a certain extent. More importantly, the inhibitory effect of fusion protein LbtA5 may be stronger than ANV. Tumor growth regulation is a complex and delicate process, which needs to be further explored from different angles.