背景：恶性肿瘤严重威胁着人们的生命健康，是医学领域亟待解决的重要难题。除手术切除外，化学治疗和放射治疗是抗肿瘤治疗的重要手段。绝大多数实体肿瘤存在缺氧微环境，缺氧会改变肿瘤细胞的基因表型。HIF-1α是肿瘤细胞缺氧应答的关键调控因子，高表达HIF-1α的肿瘤对放化疗更加耐受，影响了肿瘤综合治疗的成功率。因此，增加缺氧肿瘤的氧气供应，降低HIF-1α表达是提高肿瘤治疗敏感性的有效方法。聚合人脐带血红蛋白(polymerized human cord hemoglobin，PolyCHb）是一种以人脐带血为原料，经过修饰而制备的纳米级携释氧载体。本课题组前期研究表明，其在重度失血性休克大鼠的救治、离体器官保存、肝癌移植瘤化疗增敏等方面均取得较好效果。为了进一步研究PolyCHb在肿瘤治疗的潜在应用，本课题通过体外细胞实验和动物实验，初步探究PolyCHb对改善乳腺癌和胰腺癌缺氧以及增加放化疗的有效性，以作为一种潜在的增敏剂为肿瘤治疗提供参考。

目的：（1）探究PolyCHb对肿瘤细胞活性的影响。（2）通过建立裸鼠皮下移植瘤模型，探究PolyCHb增加放化疗的敏感性及其可能机制。

方法：（1）按照本课题组前期工艺流程制备PolyCHb，并对其主要理化性质进行表征。（2）在常氧（20%O₂）和低氧（5%O₂）条件下，采用不同浓度的PolyCHb和DOX与肿瘤细胞共培养，通过MTT法检测其对肿瘤细胞活性的影响。（3）建立人乳腺癌MCF-7细胞裸鼠皮下移植瘤模型，成瘤后将裸鼠随机分为生理盐水组、多柔比星组和多柔比星+PolyCHb组，经尾静脉注射给药治疗，通过测量肿瘤体积，观察肿瘤生长情况；治疗结束后，完整剥取肿瘤并称重，通过免疫组化检测肿瘤相关分子指标的表达情况，结合病理切片、TUNEL凋亡及ROS含量检测，分析PolyCHb对肿瘤化疗的影响。（4）另建立人胰腺癌MIApaca-2细胞裸鼠皮下移植瘤模型，成瘤后将裸鼠随机分为对照组、放疗组和放疗+PolyCHb组，进行放射治疗。通过测量肿瘤体积，观察肿瘤生长变化，结合病理切片、TUNEL凋亡、ROS含量以及免疫组化检测，分析PolyCHb对肿瘤放疗的影响。

结果：（1）按照前期工艺流程制备出的PolyCHb，经检测其主要理化指标基本符合标准。（2）低浓度的PolyCHb对肿瘤细胞的活性没有显著性影响，高浓度的PolyCHb可以抑制肿瘤细胞的活性；PolyCHb能增强化疗药物DOX对肿瘤细胞的毒性作用。（3）实验结束时，生理盐水组、多柔比星组和多柔比星+PolyCHb组的肿瘤体积分别为519.42±177.33vs316.29±62.88vs196.35±103.45mm³。多柔比星+PolyCHb组肿瘤内部ROS含量和肿瘤细胞凋亡增多，HIF-1α和Ki67表达降低， E-cad表达增高。（4）实验结束时，对照组、放疗组和放疗+PolyCHb组的肿瘤体积分别为1046.58±333.88vs422.89±40.78vs284.34±136.45 mm³。放疗+PolyCHb组肿瘤内部ROS含量和肿瘤细胞凋亡增多，HIF-1α和VEGF表达降低，Ki67表达无明显变化。

结论：一定浓度的PolyCHb可以抑制肿瘤细胞的活性，并增强化疗药物DOX对肿瘤细胞的毒性作用；PolyCHb可以增加裸鼠皮下移植瘤放化疗的敏感性，其作用机制可能与增加肿瘤内部的氧含量，下调HIF-1α的表达，诱导ROS生成，促进肿瘤细胞凋亡有关。

关键词：聚合人脐带血红蛋白；皮下移植瘤；放化疗；HIF-1α；ROS 

Background: Malignant tumor seriously threatens people's life and health, and is an important problem to be solved urgently in the medical field. In addition to surgical excision, chemotherapy and radiotherapy are also the main means of antitumor therapy. Hypoxia microenvironment exists in most solid tumors, and hypoxia can change the gene phenotype of tumor cells. HIF-1α is a key regulator of tumor cell hypoxia response. Tumors with high expression of HIF-1α are more tolerant to radiotherapy and chemotherapy, which affects the success rate of tumor comprehensive therapy. Therefore, increasing oxygen supply and decreasing HIF-1α expression of hypoxic tumors are effective methods to improve tumor sensitivity to treatment. Polymerized human cord hemoglobin (PolyCHb) is a nano-sized oxygen carrier prepared from human cord hemoglobin by modification. The preliminary study of our group has shown that it has achieved good effects in the treatment of severe hemorrhagic shock rats, in vitro organ preservation, chemotherapy sensitization of liver cancer transplanted tumors, and many other aspects. In order to further study the potential application of PolyCHb in tumor therapy, this study conducted in vitro cell experiments and animal experiments to preliminarially explore the effectiveness of PolyCHb in improving hypoxia and increasing chemoradiotherapy in breast cancer and pancreatic cancer, so as to provide reference for tumor therapy as a potential sensitizer.

Objective: (1) to investigate the effect of PolyCHb on the proliferation of tumor cells. (2) The sensitivity of PolyCHb to chemoradiotherapy and its possible mechanism were investigated by establishing subcutaneous xenografts tumor in nude mice.

Methods: (1) PolyCHb was prepared according to the preliminary technological process of our research group, and its main physical and chemical properties were characterized. (2) PolyCHb and DOX were co-cultured with tumor cells at different concentrations in normoxic (20%O₂) and hypoxic (5%O₂) condition, and their effects on tumor cell viability were detected by MTT assay. (3) Human breast cancer MCF-7 cells subcutaneous tumor model was established in nude mice. After tumor formation, nude mice were randomly divided into normal saline group, doxorubicin group and doxorubicin +PolyCHb group, and the drug was administered through a tail vein. The tumor growth was observed by measuring tumor volume. Tumor bearing nude mice were completely sacrificed after the experiment, and the expression of tumor-related molecular indicators was detected by immunohistochemistry. Combined with pathological sections, TUNEL apoptosis and ROS content detection, the effect of PolyCHb on tumor chemotherapy was analyzed. (4)Human pancreatic cancer MIApaca-2 cells subcutaneous tumor model was established in nude mice. After tumor formation, the nude mice were randomly divided into control group, radiotherapy group and radiotherapy+PolyCHb group for radiotherapy, and the tumor growth was observed by measuring tumor volume. Tumor bearing nude mice were completely sacrificed after the experiment. The effect of PolyCHb on tumor radiotherapy was analyzed by observing tumor growth in each group, combining with pathological section, TUNEL apoptosis, ROS content and immunohistochemical detection.

Results: (1) The main physical and chemical indexes of PolyCHb prepared according to the previous technological process basically met the standards. (2) low concentrations of PolyCHb had no significant effect on the activity of tumor cells, while high concentrations of PolyCHb could inhibit the activity of tumor cells. PolyCHb could increase the toxic effect of DOX on tumor cells (3) At the end of the experiment,the tumor volume of the saline group, doxorubicin group and doxorubicin+ PolyCHb group was 519.42±177.33 vs 316.29±62.88 vs 196.35±103.45mm³, respectively. In the doxorubixin+PolyCHb group, the content of ROS and tumor cells apoptosis increased, and the expression of HIF-1α and Ki67 decreased, while the expression of E-cad increased. (4) At the end of the experiment ,the tumor volume of the control group, radiotherapy group and radiotherapy+PolyCHb group was 1046.58±333.88 vs 422.8±40.78 vs 284.34±136.45mm³, respectively. In the radiotherapy +PolyCHb group, the content of ROS and tumor cell apoptosis increased, and the expression of HIF-1α and VEGF decreased, while the expression of Ki67 did not change significantly.

Conclusion: PolyCHb at a certain concentration can inhibit the activity of tumor cells and enhance the toxic effect of DOX on tumor cells. PolyCHb can increase the chemoradiotherapy sensitivity of subcutaneous xenograft in nude mice, the mechanism may be related to the increase of oxygen content in tumor, down-regulation of HIF-1α expression, induction of ROS generation and promotion of tumor cell apoptosis.

Key words: polymerized human cord hemoglobin; subcutaneous xenografts; chemoradiotherapy; HIF-1α; ROS