【作者】 张莉；

【导师】 秦志海；

【作者基本信息】 郑州大学 ， 肿瘤学， 2022， 硕士

【摘要】 研究背景恶性肿瘤是人类生命健康的重大威胁之一,是大多数国家人口死亡的主要原因。针对恶性肿瘤,目前已报道的治疗方式主要包括手术治疗、放化疗、生物治疗等。虽然基础和临床研究的进展使得肿瘤的治疗策略越来越多,但是化疗仍然是治疗恶性肿瘤的主要策略之一。不管是手术治疗还是其他的治疗手段,都需要辅助化疗。但是临床上有部分病人对化疗不敏感,经化疗刺激后可能会有不好的结局。研究发现化疗对原位肿瘤进行杀伤的同时也会促进肿瘤转移。化疗药物紫杉醇(paclitaxel,PTX)是一种经典的紫杉烷类化疗药物,已被用于多种恶性肿瘤的治疗,包括乳腺癌和黑色素瘤等,然而研究发现PTX除抑制肿瘤生长外,在某些情况下还能促进乳腺癌转移。肿瘤转移是大多数肿瘤患者死亡的主要原因,90%的肿瘤患者都死于肿瘤的恶性转移。肿瘤转移一般通过直接侵犯周围组织、血行转移和淋巴转移三种途径进行。其中,大多数肿瘤转移主要是通过淋巴系统进行的,淋巴转移发生在肿瘤转移的初始阶段,也是决定临床治疗和预测肿瘤患者预后的重要依据。淋巴转移主要是通过肿瘤细胞分泌血管内皮生长因子C(Vascular endothelial growth factor-C,VEGF-C)和血管内皮生长因子 D(Vascular endothelial growth factor-D,VEGF-D)等促进肿瘤淋巴管生成,此外研究表明淋巴管内皮细胞分泌的趋化因子如CC趋化因子配体21、CXC趋化因子配体12和CC趋化因子配体1等可以招募表达相应受体的肿瘤细胞到淋巴管。根据最新的统计数据显示,恶性黑色素瘤的发生率和死亡率逐年升高,已成为发病率增长最快的恶性肿瘤之一。PTX可以通过诱导乳腺癌淋巴管生成和诱导乳腺癌淋巴内皮细胞自噬增强通透性等不同机制来促进淋巴转移。然而PTX对黑色素瘤淋巴转移的影响目前并不清楚。CC趋化因子受体7(CC chemokinereceptor 7,CCR7)是一种G蛋白偶联受体,在树突状细胞(Dendritic cells,DCs)、T细胞和B细胞等免疫细胞上都有表达。CC 趋化因子配体 21(CC chemokine ligand 21,CCL21)是 CCR7 的配体,主要在淋巴管内皮细胞表达。CCL21/CCR7信号轴最初主要介导免疫细胞的淋巴结归巢。近年来的研究证明CCR7在多种恶性肿瘤细胞中均有表达,包括乳腺癌、肺癌、食管鳞状细胞癌和黑色素瘤等,并且与肿瘤转移和预后不良有关。研究表明淋巴管内皮细胞分泌的CCL21可以招募表达CCR7的肿瘤细胞进入淋巴管参与肿瘤淋巴转移。CCL21/CCR7信号轴在化疗后黑色素瘤淋巴转移中的作用目前尚不清楚。研究目的本文拟探讨CCL21/CCR7信号轴是否参与化疗后黑色素瘤淋巴转移的过程,以及CCL21/CCR7信号轴在黑色素瘤化疗抵抗中的分子机制。进一步地,研究阻断CCL21/CCR7信号轴是否可以改善化疗后黑色素瘤淋巴转移,提高化疗效果,为黑色素瘤的临床治疗提供了新的思路和治疗靶点。研究方法1.使用RNA测序检测化疗药紫杉醇对黑色素瘤细胞B16F10基因表达的影响。2.使用实时荧光定量PCR检测紫杉醇刺激B16F10细胞后CCR7的表达量。3.免疫荧光检测注射紫杉醇对小鼠B16F10黑色素瘤淋巴管、血管及CCR7的影响。4.Western Blot检测注射紫杉醇后对小鼠B16F10肿瘤组织CCR7的影响;紫杉醇对B16F10细胞、B16F1细胞以及4T1细胞CCR7的影响;顺铂和阿霉素对B16F10细胞CCR7的影响。5.利用划痕实验分析紫杉醇对CCL21/CCR7信号轴介导的B16F10黑色素瘤细胞迁移的影响。6.使用siRNA转染将B16F10细胞中CCR7降低,划痕实验检测降低CCR7后紫杉醇对细胞迁移的影响。7.Western Blot检测紫杉醇对丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)家族信号通路蛋白的影响。8.划痕实验检测MAPK家族蛋白对化疗后B16F10细胞迁移的影响。9.足垫接种B16F10-mCherry细胞,尾静脉注射紫杉醇,观察肿瘤的生长情况以及淋巴转移情况。10.足垫接种B16F10-mCherry细胞,尾静脉注射紫杉醇和CCR7抗体,观察紫杉醇和CCR7抗体联合治疗对B16F10黑色素瘤淋巴转移的影响。11.ELISA检测紫杉醇对淋巴管内皮细胞CCL21的影响。12.CCK-8检测化疗药对B16F10细胞的毒性作用以及IC50。13.使用小动物活体成像检测B16F10黑色素瘤淋巴转移情况。研究结果1.紫杉醇能够延缓B16F10-mCherry荷瘤小鼠的肿瘤生长,但促进了黑色素瘤足垫荷瘤小鼠的肿瘤淋巴转移。2.RNA测序结果表明紫杉醇促进黑色素瘤淋巴转移与B16F10细胞CCR7的表达有关。体内外结果均表明紫杉醇能够增强B16F10细胞CCR7的表达。3.紫杉醇促进CCL21/CCR7轴介导的B16F10细胞迁移。4.紫杉醇通过上调CCR7表达,促进CCL21介导的B16F10细胞迁移。5.紫杉醇能够增强CCL21/CCR7信号轴下游MAPK家族蛋白ERK、JNK和p38的磷酸化水平。6.紫杉醇通过激活CCL21/CCR7信号轴下游JNK和p38信号通路促进B16F10细胞迁移。7.阻断CCL21/CCR7信号轴能够有效的抑制紫杉醇诱导的B16F10黑色素瘤淋巴转移。研究结论本研究在小鼠足垫模型中发现紫杉醇抑制了 B16F10黑色素瘤生长,同时,紫杉醇治疗促进了黑色素瘤发生淋巴转移。进一步的研究表明:紫杉醇上调了B16F10细胞CCR7的表达,通过激活JNK和p38信号通路促进B16F10细胞迁移。阻断CCL21/CCR7信号轴可以有效地阻断紫杉醇治疗后B16F10黑色素瘤的淋巴转移。更多还原

【Abstract】 BackgroundMalignant tumors pose a serious threat to human health and are the first or second leading cause of death in most countries.For the treatment of malignant tumors,the main cancer treatments that have been reported include surgery,radiotherapy,chemotherapy,targeted therapy,immunotherapy and so on.Although the progress of basic and clinical research has made more and more tumor treatment strategies,chemotherapy is still one of the main treatment strategies for malignant tumors.Adjuvant chemotherapy is required regardless of surgery or other treatments.However,some patients are insensitive to chemotherapy,and may have a bad outcome after chemotherapy stimulation.Studies have found that while chemotherapy kills tumors in situ,tumor metastasis will also occur.As a classic taxane chemotherapeutic drug,paclitaxel(PTX)has been used in the treatment of various malignant tumors,including breast cancer and melanoma.However,studies have found that in addition to inhibiting tumor growth,PTX can also promote breast cancer metastasis in some cases.Tumor metastasis is the main cause of death in cancer patients,90%of cancer patients die due to malignant metastasis.Tumor metastasis is usually carried out by direct invasion of surrounding tissues,hematogenous metastasis and lymphatic metastasis.Lymphatic metastasis is the main metastasis pathway of most tumors,and it is also an important basis for determining clinical treatment and predicting the prognosis of tumor patients.Regarding the mechanism of lymphatic metastasis,the current mainly involves the secretion of growth factors such as vascular endothelial growth factor-C(VEGF-C)and vascular endothelial growth factor-D(VEGF-D)to promote tumor lymphangiogenesis.In addition,studies have shown that chemokines secreted by lymphatic endothelial cells,such as CC chemokine ligand 21,CXC chemokine ligand 12 and CC chemokine ligand 1,can recruit tumor cells expressing corresponding receptors to lymphatic vessels.According to the latest statistics,the incidence and mortality of malignant melanoma are increasing year by year,and it has become one of the fastest growing malignant tumors.PTX can promote lymphatic metastasis of breast cancer through different mechanisms including induction of lymphangiogenesis and induction of autophagy of lymphatic endothelial cells to enhance permeability.However,the effect of PTX on lymphatic metastasis of melanoma is not clear.CC chemokine receptor 7(CCR7)is a G protein-coupled receptor,which is expressed in Dendritic cells(DCs),T cells and B cells.CC chemokine ligand 21(CCL21)is a ligand of CCR7 and is mainly expressed in lymphatic endothelial cells.CCL21/CCR7 signal axis can mediate lymphatic migration and homing of immune cells such as DCs and T cells.Recent studies have shown that CCR7 is also expressed on the surface of various malignant tumor cells such as melanoma,esophageal squamous cell carcinoma and breast cancer,and is associated with tumor metastasis and poor prognosis.Studies have shown that CCL21 secreted by lymphatic endothelial cells can recruit tumor cells expressing CCR7 into lymphatic vessels and participate in lymphatic metastasis.The role of CCL21/CCR7 signaling axis in lymphatic metastasis of melanoma after chemotherapy is still unclear.PurposeIn this paper,we investigated whether CCL21/CCR7 signaling axis is involved in the lymphatic metastasis of melanoma after chemotherapy,and the molecular mechanism of CCL21/CCR7 signaling axis in chemotherapy resistance of melanoma.Whether blocking CCL21/CCR7 signaling axis can improve lymphatic metastasis of melanoma after chemotherapy and improve the efficacy of chemotherapy.This provides a new idea and therapeutic target for clinical treatment of melanoma.Methods1.Using RNA sequencing to detect the effect of chemotherapy drug paclitaxel on gene expression of B16F10 melanoma cells.2.The expression of CCR7 in B16F10 cells stimulated by paclitaxel was detected by real-time fluorescence quantitative PCR.3.The effects of paclitaxel injection on lymphatic vessels,blood vessels and CCR7 in B16F10-mCherry-bearing mice were detected by immunofluorescence.4.Western Blot was used to detect the effect of paclitaxel on CCR7 in tumor tissue of B16F10-mCherry-bearing mice;the effect of paclitaxel on CCR7 of B16F10 cells,B16F1 cells and 4T1 cells;the effect of cisplatin and doxorubicin on CCR7 of B16F10 cells.5.Wound healing assay was used to detect the effect of paclitaxel on CCL21mediated cell migration.6.The CCR7 in B16F10 cells was reduced by siRNA transfection,and the effect of paclitaxel on cell migration was detected by scratch assay after reducing CCR7.7.Western Blot was used to detect the efifect of paclitaxel on mitogen-activated protein kinase(MAPK)family signaling pathway proteins.8.Using wound healing assay to detect the effect of MAPK family proteins on the migration of B16F10 cells after chemotherapy.9.B16F10-mCherry cells were injected through the footpad,and paclitaxel was injected into the tail vein to observe the tumor growth and lymphatic metastasis.10.B16F10-mCherry cells were implanted in the footpad,and paclitaxel and CCR7 antibody were injected into the tail vein,and observe the effect of paclitaxel and CCR7 antibody combined therapy on lymphatic metastasis of B16F10 melanoma.11.The efifect of paclitaxel on CCL21 secreted by lymphatic endothelial cells was detected by ELISA.12.Using CCK-8 to detect the toxicity of chemotherapy drugs on B16F10 cells and IC50 of chemotherapy drugs on B16F10 cells.13.Small animals imaging was used to detect lymphatic metastasis of B16F10mCherry-bearing mice.Results1.Although paclitaxel can delay tumor growth in B16710-mCherry-bearing mice,it can promote lymphatic metastasis in tumor-bearing mice.2.RNA-sequencing results indicate that paclitaxel-induced lymphatic metastasis of B16710 melanoma is associated with CCR7.Paclitaxel can enhance the expression of CCR7 in B16F10 in vitro and in vivo.3.Paclitaxel promotes CCL21/CCR7 axis-mediated migration of B16710 cells.4.Paclitaxel promotes CCL21-mediated migration of B16F10 cells by up-regulating the expression of CCR7.5.Paclitaxel can enhance the phosphorylation levels of MAPK family proteins ERK,JNK and p38,downstream of CCL21/CCR7 signaling axis.6.Paclitaxel promotes the migration of B16F10 cells by activating JNK and p38 signaling pathways,downstream of CCL21/CCR7 signaling axis.7.Blocking the CCL21/CCR7 signaling axis can effectively inhibit the lymphatic metastasis of B16F10 melanoma induced by paclitaxel.ConclusionIn this study,we found that paclitaxel inhibited the tumor growth of B16F10 melanoma in tumor-bearing mice.Meanwhile,paclitaxel therapy promoted lymphatic metastasis of melanoma.It was further found that paclitaxel up-regulated the expression of CCR7 in B16F10 cells and promoted the migration of B16F10 cells by activating JNK and P38 signaling pathways.Blocking CCL21/CCR7 signaling axis can effectively inhibit lymphatic metastasis of B16F10 melanoma.更多还原