肿瘤相关巨噬细胞分泌的CCL5对胃癌的作用及机制研究

发布时间：2018-06-21 16:37

本文选题：Tumor + associated　；参考：《河北医科大学》2016年博士论文

【摘要】：胃癌在全球最常见的恶性肿瘤中排第四位,虽然发病率较前有所下降,但我国胃癌人数仍占全世界的42%,位居东亚地区常见癌症的首位。目前胃癌患者大多可选择化学治疗及行手术治疗,但其5年生存率仍低于50%,导致其死亡的主要原因之一是癌组织的浸润和转移。因此,研究胃癌的发生、发展和转移相关的具体机制,并对其进行早期预测和干预,对于制定治疗方案、改善患者预后是非常有益的。胃癌的发生、发展与肿瘤相关巨噬细胞的关系日益密切,而肿瘤相关巨噬细胞促进胃癌增殖、迁移及侵袭的机制目前研究较少,Wu H等报道了肿瘤相关巨噬细胞通过VEGF和VEGF-C的表达促进胃癌的血管新生及淋巴结转移。一些证据表明,趋化因子及其受体在调节肿瘤局部炎症及抑制免疫系统抗肿瘤反应中发挥一定作用,趋化因子配体5(CCL5/RANTES)属于CC类趋化因子家族,其受体有CCR1、CCR3和CCR5。CCL5主要表达于T淋巴细胞、巨噬细胞、血小板、滑膜成纤维细胞、肾小管上皮细胞和一定类型的肿瘤细胞,CCL5在肿瘤和炎性疾病中发挥重要作用。最近的研究发现乳腺癌病人中CCL5的表达与疾病进展直接相关,另外,在宫颈癌、卵巢癌、前列腺癌、胰腺癌、肺癌和黑色素瘤患者的组织和血清中CCL5的表达与疾病进展具有相关性。而目前对CCL5的表达与胃癌的关系的研究尚未见报道。基于上述理论,我们进行了如下研究,首先我们对比了CCL5在胃癌组织和癌旁组织及胃癌患者和健康志愿者血清中的表达差异,并对组织中CCL5和肿瘤相关巨噬细胞的标志物CD68关系进行关联分析,同时对胃癌组织和患者血清中CCL5的表达与胃癌的病理因素及生物学行为之间的关系进行分析,结果发现位于胃癌肿瘤间质的TAMs高表达CCL5,而且其表达水平与胃癌的分期、预后及远处转移密切相关。因此我们还利用体外细胞实验对肿瘤相关巨噬细胞通过分泌CCL5对胃癌细胞发挥作用的具体机制进行了研究,为临床治疗胃癌提供了新的思路和靶点,对抗癌方案的设计具有非常重要的价值。具体研究内容和结果如下:第一部分ccl5在胃癌肿瘤相关巨噬细胞中的表达及其相关性研究目的:对胃癌中肿瘤相关巨噬细胞(tam)及其与ccl5表达的相关性进行研究。方法:选择临床病理资料完整的胃癌蜡块标本48例,取癌组织和癌旁组织,采用sp免疫组化法检测cd68和ccl5在胃癌组织及癌旁组织中的表达,并采用spearmann相关性统计方法对其相关性进行统计。结果:1cd68在胃癌组织的巨噬细胞中呈阳性表达,胞浆内含清晰的黄色或黄褐色颗粒者视为阳性。在癌旁组织中仅1例呈弱阳性表达,根据病理评分,行配对t检验,具有明显统计学差异(p0.01),cd68的阳性表达程度与胃癌组织大小、浸润深度、分化程度、淋巴结转移情况、tnm分期情况、远处转移呈正相关性(p0.05),与年龄、性别无相关性(p0.05)。2ccl5在胃癌组织中呈阳性表达,胞浆内含清晰的黄色或黄褐色颗粒者视为阳性,癌旁组织中为阴性表达。根据病理评分,行配对t检验,具有明显统计学差异(p0.01),ccl5的阳性表达程度与胃癌组织浸润深度、淋巴结转移情况、tnm分期情况及肿瘤分化程度呈正相关性(p0.05),与年龄、性别无相关性(p0.05)。3经spearmann相关分析,胃癌组织中ccl5与cd68的表达有显著的相关性,且呈正相关关系(r=0.759,p0.01)。结论:cd68和ccl5可能对胃癌的发生、侵袭和转移起重要的促进作用,且ccl5与cd68的表达呈明显正相关,证明ccl5的分泌可能是tam促进胃癌侵袭转移的重要原因之一。第二部分ccl5在胃癌患者血清中的表达及临床意义目的:检测胃癌患者血清中的ccl5表达并探讨其临床意义。方法:抽取新诊断胃癌患者50例(均未行手术及放化疗)及健康志愿者16例的血清标本,采用elisa检测ccl5在胃癌患者及健康志愿者血清中的表达,比较两者的表达差异,研究ccl5的表达水平与胃癌的病理因素及生物学行为之间的关系。结果:与对照组相比,胃癌患者血清的ccl5表达水平明显高于对照组(p0.001),ccl5的表达水平在患者的年龄及性别比较中无明显差别。反之,肿瘤越大、浸润越深、分化程度越低、伴淋巴结及远处转移的、瘤栓形成、病理分期晚的患者的血清ccl5的表达水平较肿瘤小的、浸润浅的、分化程度高的、无淋巴结及远处转移的、无瘤栓形成的、病理分期早的患者的血清ccl5的表达水平偏高,具有统计学差异(p0.01)。结论:ccl5在胃癌患者血清中高表达,血清中ccl5的表达水平与胃癌组织浸润深度、淋巴结转移情况、tnm分期情况及肿瘤分化程度均具有相关性,与患者的性别及年龄无相关性。第三部分肿瘤相关巨噬细胞通过高表达ccl5对胃癌细胞生物学行为的影响目的:观察肿瘤相关巨噬细胞(tumorassociated-macrophagestam)分泌的ccl5对胃癌细胞生物学行为的影响,探讨tams在胃癌侵袭转移中的作用及机制。方法:将人单核细胞株thp-1体外经il-4诱导为类m2型巨噬细胞,并与胃癌细胞株ags共培养形成胃癌微环境,体外细胞实验验证tams通过分泌ccl5促进胃癌细胞的增殖、侵袭和转移,rt-pcr检测趋化因子及其受体的表达情况,westernblot检测stat3信号及下游目的蛋白的表达变化。结果:1mts细胞增殖实验显示,培养5天后,用il-4诱导的巨噬细胞上清液培养的胃癌细胞增殖率为(397.57±28.82)%,高于与thp-1上清共培养组(385.95±32.56)%和未共培养的胃癌细胞组(320.49±7.62)%,(p0.05),具有统计学意义。用il-4诱导的巨噬细胞上清液培养的胃癌细胞数量高于与thp-1上清共培养组和单纯胃癌细胞组,具有统计学意义(p0.05)。2与未共培养的胃癌细胞组相比,与thp-1上清共培养组和与thp-1上清共培养经il-4诱导组穿出小室的细胞数明显增多(p0.01),经il-4诱导组胃癌细胞迁移能力增强,穿出小室的细胞数多于用thp-1上清共培养组(p0.01),具有统计学意义。3与未共培养的胃癌细胞组相比,与thp-1共培养组和与thp-1共培养经il-4诱导组的胃癌细胞的克隆形成数明显增多(p0.01),分别为38.00±7.21/高倍视野和58.33±3.79/高倍视野,与THP-1共培养经IL-4诱导组的胃癌细胞的克隆形成数较与THP-1共培养组明显增多(P0.01),具有统计学意义。4 THP-1组、THP-1与AGS共培养组、THP-1与AGS共培养经IL-4诱导组THP-1细胞中CCL2、CCL4、CCL17、CCL22的mRNA相对表达量比较无明显差异(P0.05),而CCL5、MMP2、MMP9mRNA的相对表达量明显增多(P0.01),CCL5mRNA的表达量较MMP2和MMP9明显增多(P0.01)。CCL5、MMP2、MMP9mRNA的相对表达量在THP-1与AGS共培养组分别为2.32±0.94、1.59±0.34、1.46±0.13,THP-1与AGS共培养经IL-4诱导组细胞中的表达分别为3.98±0.34、2 2.4±0.26、2.07±0.45均高于THP-1组(P0.01),且THP-1与AGS共培养经IL-4诱导组中表达最多(P0.01),具有统计学意义。5与未共培养的胃癌细胞组相比,用THP-1上清共培养组和用THP-1上清共培养经IL-4诱导组胃癌细胞表面CCL5受体CCR5mRNA的相对表达量明显增多(P0.01),以用THP-1上清共培养经IL-4诱导组的表达量最多(P0.01),具有统计学意义。6在AGS中加入不同浓度的CCL5(0μM/ml、10μM/ml、50μM/ml),随着CCL5浓度的增加发生迁移的细胞数量也随之增加,以CCL5(50μM/ml)浓度时发生迁移的细胞数最多,达28.33±5.13/高倍视野(P0.01),具有统计学意义。7与THP-1来源的巨噬细胞共培养后Stat3和p-Stat3(p-S727和p-Y705)的表达明显增加,高于胃癌细胞组和与THP-1共培养组(P0.01),p-Stat3(p-S727和p-Y705)的变化趋势与Stat3呈现一致性(P0.05)。结论:与THP-1来源的巨噬细胞上清共培养后胃癌细胞增殖和克隆形成能力增强,而且共培养后THP-1细胞中CCL5mRNA的表达量最高,而AGS表面CCR5mRNA相对表达量最高。随着CCL5浓度的增加发生迁移的胃癌细胞数量也随之增加。共培养后AGS中Stat3信号及其目的蛋白表达增加。
[Abstract]:Gastric cancer is the fourth most common malignant tumor in the world, although the incidence is lower than before, but the number of gastric cancer in China still accounts for 42% of the world, ranking first in the common cancer in East Asia. At present, most of the patients with gastric cancer can choose chemical treatment and operation treatment, but their 5 year survival rate is still lower than 50%, leading to the main cause of death. One of these is the invasion and metastasis of cancer tissue. Therefore, the study of the specific mechanisms related to the occurrence, development and metastasis of gastric cancer, and the early prediction and intervention of the cancer are very beneficial to the formulation of the treatment scheme and the improvement of the prognosis of the patients. The development of gastric cancer is increasingly closely related to the tumor related macrophages, and the tumor related megagocytosis is very thin. The mechanism of cell promoting gastric cancer proliferation, migration and invasion is currently less. Wu H and other reports suggest that tumor related macrophages promote angiogenesis and lymph node metastasis through VEGF and VEGF-C expression. Some evidence suggests that chemokine and its receptors play a role in regulating local inflammation and inhibiting the anti-tumor response of the immune system. Chemokine ligand 5 (CCL5/RANTES) belongs to the CC chemotactic factor family. Its receptors are CCR1, CCR3 and CCR5.CCL5 are mainly expressed in T lymphocytes, macrophages, platelets, synovial fibroblasts, renal tubular epithelial cells and certain types of tumor cells. CCL5 plays an important role in cancer and inflammatory diseases. Recent research has been developed. The expression of CCL5 in the patients with breast cancer is directly related to the progression of the disease. In addition, the expression of CCL5 in the tissues and serum of the patients with cervical, ovarian, prostate, pancreatic, lung and melanoma is related to the progression of the disease. However, there is no report on the relationship between the expression of CCL5 and the gastric cancer. Based on the above theory, I We conducted the following studies. First, we compared the expression differences of CCL5 in the serum of gastric cancer tissue, para cancer tissue, gastric cancer patients and healthy volunteers, and related analysis of the relationship between CCL5 and tumor related macrophages in tissue, and the expression of CCL5 in gastric cancer tissues and patients and the pathological causes of gastric cancer. The relationship between TAMs and biological behavior was analyzed. The results showed that the high expression of CCL5 in the stroma of gastric cancer was closely related to the stage of gastric cancer, the prognosis and the distant metastasis. Therefore, we also used in vitro cell experiment to express the effect of CCL5 on cancer cells in tumor related macrophages. The mechanism has been studied to provide new ideas and targets for the clinical treatment of gastric cancer. The design of anti cancer scheme is of great value. The specific research content and results are as follows: the first part of CCL5 expression in tumor related macrophages in gastric cancer and its correlation study: the tumor related macrophage (TAM) and its relationship with cancer in gastric cancer The correlation of CCL5 expression was studied. Methods: 48 specimens of gastric paraffin specimens with complete clinicopathological data were selected to take cancer tissue and para cancer tissue. The expression of CD68 and CCL5 in gastric cancer tissue and para cancer tissues were detected by SP immunohistochemical method. The correlation was statistically analyzed by spearmann correlation statistical method. Results: 1cd68 was in gastric cancer. The positive expression of macrophages in the tissue was positive. Only 1 cases of clear yellow or yellowish brown granules in the cytoplasm were positive. Only 1 cases were weakly positive in the para cancerous tissues. According to the pathological score, the paired t test was performed with significant statistical difference (P0.01). The positive expression of CD68 was associated with the size of gastric cancer, depth of invasion, degree of differentiation, lymph node. Metastasis, TNM staging, distant metastasis positive correlation (P0.05), and age, sex without correlation (P0.05).2ccl5 positive expression in gastric cancer tissue, cytosol with clear yellow or yellowish brown granules in the positive, negative expression in the para cancerous tissue. According to the pathological score, the paired t test, with significant statistical difference (p0.0 1) the positive expression of CCL5 was positively correlated with the depth of gastric cancer tissue infiltration, lymph node metastasis, TNM staging and the degree of tumor differentiation (P0.05), and the correlation with age, sex and sex (P0.05).3 through spearmann correlation analysis, and the correlation between CCL5 and CD68 in gastric carcinoma and positive correlation (r=0.759, P0.01). CD68 and CCL5 may play an important role in promoting the occurrence, invasion and metastasis of gastric cancer, and the expression of CCL5 is positively correlated with the expression of CD68. It is proved that the secretion of CCL5 may be one of the important reasons for Tam to promote the invasion and metastasis of gastric cancer. Second part of the expression of CCL5 in the serum of gastric cancer patients and its clinical significance: the detection of CC in the serum of gastric cancer patients. L5 expression and its clinical significance. Methods: the serum samples of 50 patients with gastric cancer (all without operation and radiotherapy) and 16 healthy volunteers were selected. The expression of CCL5 in the serum of gastric cancer patients and healthy volunteers was detected by ELISA, and the expression difference between the two patients was compared. The expression level of CCL5 and the pathological factors and biological factors of gastric cancer were studied. Results: compared with the control group, the level of CCL5 expression in the serum of the patients with gastric cancer was significantly higher than that of the control group (p0.001). The expression level of CCL5 was not significantly different in the age and sex comparison of the patients. On the contrary, the greater the tumor, the deeper the infiltration, the lower the differentiation, the formation of the lymph node and distant metastasis, the formation of the tumor thrombus and the pathological stage. The expression level of serum CCL5 in the late patients was smaller than that of tumor, shallow and highly differentiated, without lymph node and distant metastasis, without tumor thrombus, and high expression level of serum CCL5 in patients with early pathological stages (P0.01). Conclusion: CCL5 is highly expressed in serum of patients with gastric cancer and the expression level of CCL5 in serum There was a correlation between the depth of gastric carcinoma, lymph node metastasis, TNM staging and the degree of tumor differentiation. There was no correlation with the sex and age of the patients. The effect of the third part of tumor related macrophages on the biological behavior of gastric cancer cells through high expression of CCL5 was to observe the tumor related macrophages (tumorassociated-macrop Hagestam) the effect of the secreted CCL5 on the biological behavior of gastric cancer cells, and to explore the role and mechanism of TAMs in the invasion and metastasis of gastric cancer. Methods: human mononuclear cell line THP-1 was induced by IL-4 into M2 like macrophages in vitro and co cultured with gastric cancer cell line AGS to form gastric microenvironment. In vitro cell experiment verified that TAMs promoted the stomach by secreting CCL5. The proliferation, invasion and metastasis of cancer cells, RT-PCR detection of chemokine and its receptor expression, Westernblot detection of STAT3 signal and the expression of downstream target protein expression. Results: 1mts cell proliferation test showed that 5 days after culture, the proliferation rate of gastric cancer cells cultured in macrophage supernatant induced by IL-4 was (397.57 + 28.82)%, higher than that of t. Hp-1 supernatant co culture group (385.95 + 32.56)% and non co cultured gastric cancer cell group (320.49 + 7.62)%, (P0.05), with statistical significance. The number of gastric cancer cells cultured in IL-4 induced macrophage supernatant was higher than that in co culture group with THP-1 supernatant and simple gastric cancer cell group, which had statistical significance (P0.05).2 and non co cultured gastric cancer cells. Compared with the THP-1 supernatant co culture group and the co culture of THP-1 supernatant, the number of cells in the IL-4 induced group increased significantly (P0.01). The migration ability of gastric cancer cells was enhanced by IL-4 induced group, and the number of cells through the chamber was more than that of the THP-1 supernatant co culture group (P0.01). The statistically significant.3 was compared with the non co cultured gastric cancer cell group. The number of clones in the THP-1 co culture group and the coculture of THP-1 co cultured with IL-4 was significantly increased (P0.01), which was 38 + 7.21/ high fold and 58.33 + 3.79/ high times. The number of clones of gastric cancer cells induced by IL-4 in IL-4 induced group was significantly increased (P0.01) than that in the THP-1 co culture group (P0.01), with a statistically significant.4. THP-1 group, THP-1 and AGS co culture group, THP-1 and AGS co culture CCL2 in THP-1 cells induced by IL-4, CCL4, CCL17, CCL22 mRNA relative expression is no significant difference. THP-1 and AGS co culture groups were 2.32 + 0.94,1.59 + 0.34,1.46 + 0.13 respectively. The expressions of THP-1 and AGS in the cells induced by IL-4 were 3.98 + 0.34,2 2.4 + 0.26,2.07 + 0.45 respectively higher than those of THP-1 group (P0.01). Compared with the gastric cancer cell group, the relative expression of CCL5 receptor CCR5mRNA on the surface of gastric cancer cells induced by the co culture of THP-1 supernatant and the co culture of THP-1 supernatant increased significantly (P0.01), and the expression of IL-4 induced group was the most (P0.01) in the co culture of THP-1 supernatant (P0.01), and.6 in AGS was added to AGS (0 mu, 1). 0 mu M/ml, 50 mu M/ml), the number of cells migrated with the increase of CCL5 concentration also increased. The number of cells migrated at the concentration of CCL5 (50 mu M/ml) was the most, reaching 28.33 + 5.13/ high fold field of vision (P0.01). The expression of Stat3 and p-Stat3 (p-S727 and p-S727) increased significantly after co culture of.7 and THP-1 source macrophages. In the gastric cancer cell group and the THP-1 co culture group (P0.01), the change trend of p-Stat3 (p-S727 and p-Y705) was concordant with Stat3 (P0.05). Conclusion: the proliferation and cloning ability of gastric cancer cells were enhanced after co culture with macrophage supernatant from THP-1, and the expression of CCL5mRNA in THP-1 cells was the highest after co culture, while AGS surface CCR5mRNA The number of gastric cancer cells increased with the increase of CCL5 concentration. The expression of Stat3 signal and its target protein in AGS increased after co culture.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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