本文選題：Tumor + associated　； 參考：《河北醫(yī)科大學(xué)》2016年博士論文

【摘要】：胃癌在全球最常見的惡性腫瘤中排第四位,雖然發(fā)病率較前有所下降,但我國胃癌人數(shù)仍占全世界的42%,位居?xùn)|亞地區(qū)常見癌癥的首位。目前胃癌患者大多可選擇化學(xué)治療及行手術(shù)治療,但其5年生存率仍低于50%,導(dǎo)致其死亡的主要原因之一是癌組織的浸潤和轉(zhuǎn)移。因此,研究胃癌的發(fā)生、發(fā)展和轉(zhuǎn)移相關(guān)的具體機(jī)制,并對(duì)其進(jìn)行早期預(yù)測和干預(yù),對(duì)于制定治療方案、改善患者預(yù)后是非常有益的。胃癌的發(fā)生、發(fā)展與腫瘤相關(guān)巨噬細(xì)胞的關(guān)系日益密切,而腫瘤相關(guān)巨噬細(xì)胞促進(jìn)胃癌增殖、遷移及侵襲的機(jī)制目前研究較少,Wu H等報(bào)道了腫瘤相關(guān)巨噬細(xì)胞通過VEGF和VEGF-C的表達(dá)促進(jìn)胃癌的血管新生及淋巴結(jié)轉(zhuǎn)移。一些證據(jù)表明,趨化因子及其受體在調(diào)節(jié)腫瘤局部炎癥及抑制免疫系統(tǒng)抗腫瘤反應(yīng)中發(fā)揮一定作用,趨化因子配體5(CCL5/RANTES)屬于CC類趨化因子家族,其受體有CCR1、CCR3和CCR5。CCL5主要表達(dá)于T淋巴細(xì)胞、巨噬細(xì)胞、血小板、滑膜成纖維細(xì)胞、腎小管上皮細(xì)胞和一定類型的腫瘤細(xì)胞,CCL5在腫瘤和炎性疾病中發(fā)揮重要作用。最近的研究發(fā)現(xiàn)乳腺癌病人中CCL5的表達(dá)與疾病進(jìn)展直接相關(guān),另外,在宮頸癌、卵巢癌、前列腺癌、胰腺癌、肺癌和黑色素瘤患者的組織和血清中CCL5的表達(dá)與疾病進(jìn)展具有相關(guān)性。而目前對(duì)CCL5的表達(dá)與胃癌的關(guān)系的研究尚未見報(bào)道�；谏鲜隼碚�,我們進(jìn)行了如下研究,首先我們對(duì)比了CCL5在胃癌組織和癌旁組織及胃癌患者和健康志愿者血清中的表達(dá)差異,并對(duì)組織中CCL5和腫瘤相關(guān)巨噬細(xì)胞的標(biāo)志物CD68關(guān)系進(jìn)行關(guān)聯(lián)分析,同時(shí)對(duì)胃癌組織和患者血清中CCL5的表達(dá)與胃癌的病理因素及生物學(xué)行為之間的關(guān)系進(jìn)行分析,結(jié)果發(fā)現(xiàn)位于胃癌腫瘤間質(zhì)的TAMs高表達(dá)CCL5,而且其表達(dá)水平與胃癌的分期、預(yù)后及遠(yuǎn)處轉(zhuǎn)移密切相關(guān)。因此我們還利用體外細(xì)胞實(shí)驗(yàn)對(duì)腫瘤相關(guān)巨噬細(xì)胞通過分泌CCL5對(duì)胃癌細(xì)胞發(fā)揮作用的具體機(jī)制進(jìn)行了研究,為臨床治療胃癌提供了新的思路和靶點(diǎn),對(duì)抗癌方案的設(shè)計(jì)具有非常重要的價(jià)值。具體研究內(nèi)容和結(jié)果如下:第一部分ccl5在胃癌腫瘤相關(guān)巨噬細(xì)胞中的表達(dá)及其相關(guān)性研究目的:對(duì)胃癌中腫瘤相關(guān)巨噬細(xì)胞(tam)及其與ccl5表達(dá)的相關(guān)性進(jìn)行研究。方法:選擇臨床病理資料完整的胃癌蠟塊標(biāo)本48例,取癌組織和癌旁組織,采用sp免疫組化法檢測cd68和ccl5在胃癌組織及癌旁組織中的表達(dá),并采用spearmann相關(guān)性統(tǒng)計(jì)方法對(duì)其相關(guān)性進(jìn)行統(tǒng)計(jì)。結(jié)果:1cd68在胃癌組織的巨噬細(xì)胞中呈陽性表達(dá),胞漿內(nèi)含清晰的黃色或黃褐色顆粒者視為陽性。在癌旁組織中僅1例呈弱陽性表達(dá),根據(jù)病理評(píng)分,行配對(duì)t檢驗(yàn),具有明顯統(tǒng)計(jì)學(xué)差異(p0.01),cd68的陽性表達(dá)程度與胃癌組織大小、浸潤深度、分化程度、淋巴結(jié)轉(zhuǎn)移情況、tnm分期情況、遠(yuǎn)處轉(zhuǎn)移呈正相關(guān)性(p0.05),與年齡、性別無相關(guān)性(p0.05)。2ccl5在胃癌組織中呈陽性表達(dá),胞漿內(nèi)含清晰的黃色或黃褐色顆粒者視為陽性,癌旁組織中為陰性表達(dá)。根據(jù)病理評(píng)分,行配對(duì)t檢驗(yàn),具有明顯統(tǒng)計(jì)學(xué)差異(p0.01),ccl5的陽性表達(dá)程度與胃癌組織浸潤深度、淋巴結(jié)轉(zhuǎn)移情況、tnm分期情況及腫瘤分化程度呈正相關(guān)性(p0.05),與年齡、性別無相關(guān)性(p0.05)。3經(jīng)spearmann相關(guān)分析,胃癌組織中ccl5與cd68的表達(dá)有顯著的相關(guān)性,且呈正相關(guān)關(guān)系(r=0.759,p0.01)。結(jié)論:cd68和ccl5可能對(duì)胃癌的發(fā)生、侵襲和轉(zhuǎn)移起重要的促進(jìn)作用,且ccl5與cd68的表達(dá)呈明顯正相關(guān),證明ccl5的分泌可能是tam促進(jìn)胃癌侵襲轉(zhuǎn)移的重要原因之一。第二部分ccl5在胃癌患者血清中的表達(dá)及臨床意義目的:檢測胃癌患者血清中的ccl5表達(dá)并探討其臨床意義。方法:抽取新診斷胃癌患者50例(均未行手術(shù)及放化療)及健康志愿者16例的血清標(biāo)本,采用elisa檢測ccl5在胃癌患者及健康志愿者血清中的表達(dá),比較兩者的表達(dá)差異,研究ccl5的表達(dá)水平與胃癌的病理因素及生物學(xué)行為之間的關(guān)系。結(jié)果:與對(duì)照組相比,胃癌患者血清的ccl5表達(dá)水平明顯高于對(duì)照組(p0.001),ccl5的表達(dá)水平在患者的年齡及性別比較中無明顯差別。反之,腫瘤越大、浸潤越深、分化程度越低、伴淋巴結(jié)及遠(yuǎn)處轉(zhuǎn)移的、瘤栓形成、病理分期晚的患者的血清ccl5的表達(dá)水平較腫瘤小的、浸潤淺的、分化程度高的、無淋巴結(jié)及遠(yuǎn)處轉(zhuǎn)移的、無瘤栓形成的、病理分期早的患者的血清ccl5的表達(dá)水平偏高,具有統(tǒng)計(jì)學(xué)差異(p0.01)。結(jié)論:ccl5在胃癌患者血清中高表達(dá),血清中ccl5的表達(dá)水平與胃癌組織浸潤深度、淋巴結(jié)轉(zhuǎn)移情況、tnm分期情況及腫瘤分化程度均具有相關(guān)性,與患者的性別及年齡無相關(guān)性。第三部分腫瘤相關(guān)巨噬細(xì)胞通過高表達(dá)ccl5對(duì)胃癌細(xì)胞生物學(xué)行為的影響目的:觀察腫瘤相關(guān)巨噬細(xì)胞(tumorassociated-macrophagestam)分泌的ccl5對(duì)胃癌細(xì)胞生物學(xué)行為的影響,探討tams在胃癌侵襲轉(zhuǎn)移中的作用及機(jī)制。方法:將人單核細(xì)胞株thp-1體外經(jīng)il-4誘導(dǎo)為類m2型巨噬細(xì)胞,并與胃癌細(xì)胞株ags共培養(yǎng)形成胃癌微環(huán)境,體外細(xì)胞實(shí)驗(yàn)驗(yàn)證tams通過分泌ccl5促進(jìn)胃癌細(xì)胞的增殖、侵襲和轉(zhuǎn)移,rt-pcr檢測趨化因子及其受體的表達(dá)情況,westernblot檢測stat3信號(hào)及下游目的蛋白的表達(dá)變化。結(jié)果:1mts細(xì)胞增殖實(shí)驗(yàn)顯示,培養(yǎng)5天后,用il-4誘導(dǎo)的巨噬細(xì)胞上清液培養(yǎng)的胃癌細(xì)胞增殖率為(397.57±28.82)%,高于與thp-1上清共培養(yǎng)組(385.95±32.56)%和未共培養(yǎng)的胃癌細(xì)胞組(320.49±7.62)%,(p0.05),具有統(tǒng)計(jì)學(xué)意義。用il-4誘導(dǎo)的巨噬細(xì)胞上清液培養(yǎng)的胃癌細(xì)胞數(shù)量高于與thp-1上清共培養(yǎng)組和單純胃癌細(xì)胞組,具有統(tǒng)計(jì)學(xué)意義(p0.05)。2與未共培養(yǎng)的胃癌細(xì)胞組相比,與thp-1上清共培養(yǎng)組和與thp-1上清共培養(yǎng)經(jīng)il-4誘導(dǎo)組穿出小室的細(xì)胞數(shù)明顯增多(p0.01),經(jīng)il-4誘導(dǎo)組胃癌細(xì)胞遷移能力增強(qiáng),穿出小室的細(xì)胞數(shù)多于用thp-1上清共培養(yǎng)組(p0.01),具有統(tǒng)計(jì)學(xué)意義。3與未共培養(yǎng)的胃癌細(xì)胞組相比,與thp-1共培養(yǎng)組和與thp-1共培養(yǎng)經(jīng)il-4誘導(dǎo)組的胃癌細(xì)胞的克隆形成數(shù)明顯增多(p0.01),分別為38.00±7.21/高倍視野和58.33±3.79/高倍視野,與THP-1共培養(yǎng)經(jīng)IL-4誘導(dǎo)組的胃癌細(xì)胞的克隆形成數(shù)較與THP-1共培養(yǎng)組明顯增多(P0.01),具有統(tǒng)計(jì)學(xué)意義。4 THP-1組、THP-1與AGS共培養(yǎng)組、THP-1與AGS共培養(yǎng)經(jīng)IL-4誘導(dǎo)組THP-1細(xì)胞中CCL2、CCL4、CCL17、CCL22的mRNA相對(duì)表達(dá)量比較無明顯差異(P0.05),而CCL5、MMP2、MMP9mRNA的相對(duì)表達(dá)量明顯增多(P0.01),CCL5mRNA的表達(dá)量較MMP2和MMP9明顯增多(P0.01)。CCL5、MMP2、MMP9mRNA的相對(duì)表達(dá)量在THP-1與AGS共培養(yǎng)組分別為2.32±0.94、1.59±0.34、1.46±0.13,THP-1與AGS共培養(yǎng)經(jīng)IL-4誘導(dǎo)組細(xì)胞中的表達(dá)分別為3.98±0.34、2 2.4±0.26、2.07±0.45均高于THP-1組(P0.01),且THP-1與AGS共培養(yǎng)經(jīng)IL-4誘導(dǎo)組中表達(dá)最多(P0.01),具有統(tǒng)計(jì)學(xué)意義。5與未共培養(yǎng)的胃癌細(xì)胞組相比,用THP-1上清共培養(yǎng)組和用THP-1上清共培養(yǎng)經(jīng)IL-4誘導(dǎo)組胃癌細(xì)胞表面CCL5受體CCR5mRNA的相對(duì)表達(dá)量明顯增多(P0.01),以用THP-1上清共培養(yǎng)經(jīng)IL-4誘導(dǎo)組的表達(dá)量最多(P0.01),具有統(tǒng)計(jì)學(xué)意義。6在AGS中加入不同濃度的CCL5(0μM/ml、10μM/ml、50μM/ml),隨著CCL5濃度的增加發(fā)生遷移的細(xì)胞數(shù)量也隨之增加,以CCL5(50μM/ml)濃度時(shí)發(fā)生遷移的細(xì)胞數(shù)最多,達(dá)28.33±5.13/高倍視野(P0.01),具有統(tǒng)計(jì)學(xué)意義。7與THP-1來源的巨噬細(xì)胞共培養(yǎng)后Stat3和p-Stat3(p-S727和p-Y705)的表達(dá)明顯增加,高于胃癌細(xì)胞組和與THP-1共培養(yǎng)組(P0.01),p-Stat3(p-S727和p-Y705)的變化趨勢(shì)與Stat3呈現(xiàn)一致性(P0.05)。結(jié)論:與THP-1來源的巨噬細(xì)胞上清共培養(yǎng)后胃癌細(xì)胞增殖和克隆形成能力增強(qiáng),而且共培養(yǎng)后THP-1細(xì)胞中CCL5mRNA的表達(dá)量最高,而AGS表面CCR5mRNA相對(duì)表達(dá)量最高。隨著CCL5濃度的增加發(fā)生遷移的胃癌細(xì)胞數(shù)量也隨之增加。共培養(yǎng)后AGS中Stat3信號(hào)及其目的蛋白表達(dá)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.2

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