【作者】 刘杰；

【导师】 孙玉萍；

【作者基本信息】 山东大学 ， 肿瘤学（专业学位）， 2013， 博士

【摘要】 研究背景及目的近15年来我国乳腺癌的发病率和死亡率呈上升趋势,且发病年龄越来越趋于年轻化,严重威胁女性的健康。研究表明免疫系统的功能状态可直接影响乳腺癌的发生、发展和转移。明确影响免疫系统状态关键分子,不仅可丰富肿瘤发生的理论基础,还可为乳腺癌患者提供个体化的预防和治疗方案。所以,寻找和研究促进乳腺癌发生发展的免疫系统功能性分子已成为目前肿瘤免疫领域研究的热点。目前认为,免疫逃逸是肿瘤发生和发展过程中的重要环节,肿瘤细胞通过表达抑制性分子促进肿瘤的免疫逃逸越来越受到重视。免疫球蛋白样转录子(Immunoglobulin-like transcript, ILT)4(又称为LILRB2、MIR10、LIR-2、 CD85d)是ILT家族中的一员,因其蛋白胞外区含有4个免疫球蛋白区,所以又属于免疫球蛋白超家族。其胞内区为含有免疫受体酪氨酸抑制基序(ITIM)的长胞质尾部,可募集含SH2结构的酪氨酸磷酸激酶SHP-1和SHP-2发挥细胞外信号向细胞内传递的作用,因而被认为是一种免疫抑制性受体。以往的研究已证实,ILT4主要表达于树突状细胞和单核巨噬细胞上,在维持母胎免疫耐受、炎症和移植耐受方面发挥重要的免疫抑制作用。迄今为止,对ILT4及同类分子在肿瘤细胞中的表达情况知之甚少,其在肿瘤细胞上表达的作用及机制更不清楚。初步研究发现ILT4在非小细胞肺癌中高表达,ILT2在皮肤T细胞淋巴瘤中高表达,ILT3、ILT4在慢性淋巴性白血病中的恶性B细胞上高表达,且ILT3与淋巴组织中恶性B细胞的浸润有关。ILT3在胃癌细胞和产蛋雌鸡的自发性卵巢癌细胞中高表达,且与癌症进展密切相关,但其在乳腺癌中的表达情况和机制尚不明确。树突状细胞(DC)与CD8+Ts细胞共培养后发现其表面ILT4的表达增高,共刺激分子B7-1(CD80)、B7-2(CD86)的表达减少,刺激CD4+Th细胞活化、增殖的能力减弱。无独有偶,在敲除小鼠同源性配对免疫球蛋白样受体PIRB的巨噬细胞中发现,NF-κB途径被活化,其下游分子B7-1、B7-2表达增高,上述研究表明在抗原递呈细胞中ILT4可通过某个信号途径下调B7家族共刺激分子B7-1和B7-2,减弱T细胞活化所需的第2信号。B7-H3是B7共刺激分子家族中的一员,最初被认定为共刺激分子,然而近来越来越多的研究证实了它在肿瘤细胞中高表达,可以抑制T细胞活化,促进肿瘤进展。研究发现B7-H3在乳腺癌细胞上高表达,本课题组前期研究发现ILT4也可高表达于乳腺癌,同时也发现ILT4和B7-H3可共同表达于乳腺癌细胞上。那么乳腺癌中ILT4和B7-H3的表达之间是否存在相关性?在乳腺癌细胞中ILT4是否可以调节B7-H3的表达?基于上述研究目的,本课题通过免疫组化检测117例原发性乳腺癌组织中ILT4和B7-H3的表达水平,分析二者与患者临床病理因素、肿瘤浸润性淋巴细胞(TILs)及IL-10的相关性,特别是二者之间的相关性；通过RT-PCR和WesternBlot检测乳腺癌细胞株中ILT4及B7-H3的mRNA及蛋白表达情况；在体外通过基因转染和shRNA干扰两种技术使ILT4过表达或ILT4基因沉默,检测ILT4对乳腺癌细胞中B7-H3的调节作用,以期探讨ILT4在乳腺癌的发生、发展中的作用,为乳腺癌的治疗提供新思路。研究方法1.采用免疫组化法检测117例原发性乳腺癌患者中ILT4、B7-H3及IL-10在蛋白水平的表达情况；检测CD45RO的表达,并计数肿瘤浸润性淋巴细胞的数量。2.pEZ-ILT4质粒瞬时转染SK-BR-3细胞株,使ILT4过表达。通过RT-PCR和Western Blot检测该细胞株中B7-H3的mRNA及蛋白的表达。3.PGPU6-shILT4质粒瞬时转染MDA-MB-231细胞株,使ILT4基因沉默。通过RT-PCR和Western Blot检测该细胞株中B7-H3的mRNA及蛋白的表达。4.采用SPSS统计学分析软件15.0进行统计学分析,认为p<0.05有显著性差异。乳腺癌组织ILT4、B7-H3及IL-10与临床病理因素的相关性采用chi-square检验。ILT4、B7-H3与IL-10的相关性及二者之间的相关性用Spearman相关分析。TILs的数量用x±s表示,两样本均数的比较采用独立样本不配对t检验。乳腺癌细胞株mRNA和蛋白的相对表达量以x±s表示,两样本间均数比较采用独立样本t检验。结果1.ILT4在人乳腺癌组织及转移的腋窝淋巴结中的表达及其与临床病理因素的相关性在原发性乳腺癌组织中ILT4可表达在胞浆、胞膜上或二者均表达,而在周围正常的乳腺组织中不表达。在117例乳腺癌标本中,71例表达ILT4(60.7%)。形态学上也发现ILT4可表达于肿瘤间质中的巨噬细胞、成纤维细胞和浆细胞上。在转移的腋窝淋巴结中,72枚(74.2%)转移淋巴结中癌细胞上表达ILT4。ILT4表达在胞浆、胞膜上或二者均表达；其对应的原发肿瘤组织均有ILT4表达,在25枚ILT4表达阴性的转移淋巴结中,24枚相应的原发肿瘤组织ILT4表达为阴性,另一枚相应的原发肿瘤组织中ILT4的表达强度为10-40%。同时在转移淋巴结的间质细胞上也发现ILT4的阳性表达。乳腺癌组织中ILT4的表达与淋巴结转移显著相关(p=0.046),与年龄、组织学类型、肿瘤大小、肿瘤学分期、分化程度及受体表达情况均无明显的相关性。2.B7-H3在人乳腺癌组织中的表达及其与临床病理因素的相关性在117例原发性乳腺癌患者中106例(90.6%)表达B7-H3。B7-H3主要表达于胞浆,胞膜或二者同时表达,而在间质及癌旁正常乳腺组织中不表达。B7-H3的阳性表达率与年龄、组织学类型、肿瘤大小、分化程度及受体表达情况均无关,而与淋巴结转移和临床分期明显相关(p值分别为0.018和0.011)。3.乳腺癌细胞中ILT4与B7-H3表达的相关性ILT4和B7-H3均在乳腺癌细胞中高表达,本研究通过Spearman相关分析发现二者之间存在明显正相关(R=0.41；p<0.01)。4.乳腺癌细胞中ILT4及B7-H3的表达与IL-10表达的相关性IL-10主要表达在乳腺癌细胞胞浆中,为弥漫性、棕黄色颗粒。形态学发现IL-10也可表达在肿瘤间质内淋巴细胞上。IL-10在乳腺癌细胞中的阳性表达率为80.34%(94/117)。其表达与淋巴结转移和临床分期显著相关,而与年龄、组织学类型、肿瘤大小、分化程度及受体表达情况均无明显的相关性。ILT4及B7-H3的表达与1L-10的表达之间存在明显的正相关(R分别为：0.577和0.545,p值均<0.01)。5.乳腺癌细胞中ILT4及B7-H3的表达与肿瘤浸润性淋巴细胞(TILs)的相关性ILT4阳性表达组中TILs的数量为26.71±11.85,明显低于ILT4阴性表达组：33.58±13.73,p=0.004。B7-H3阳性表达组中TILs的数量为27.41±11.17,而阴性表达组中的数量为33.71±12.57,同样有显著性差异,p=0.005。IL-10阳性表达组及阴性表达组中TILs的数量分别为：27.65±12.91和34.80±14.49,两组之间存在显著差异,p=0.018。6.ILT4和B7-H3在乳腺癌细胞株中的表达通过RT-PCR和Western Blot发现,ILT4和B7-H3mRNA及蛋白在MDA-MB-453、MDA-MB-231、MCF-7和SK-BR-3等4株乳腺癌细胞株中均有表达。本研究选用SK-BR-3细胞株作为后续过表达ILT4的实验细胞株,而MDA-MB-231细胞株作为后续shRNA干扰的实验细胞株。7. pEZ-ILT4质粒鉴定通过SpeI限制性内切酶进行双酶切方法和测序鉴定pEZ-ILT4质粒,结果显示目的片段完全符合。8. pGPU6-shILT4质粒的筛选4组pGPU6-shILT4质粒瞬时转染MDA-MB-231细胞株,48小时后,RT-PCR和Western Blot分别检测其ILT4表达水平。与对照组相比,4个干扰组ILT4的mRNA及蛋白水平表达均明显下调,p值均小于0.01；尤以pGPU6-shILT4-853组下调明显,p<0.001。将pGPU6-shILT4-853定义为pGPU6-shILT4用于后续实验。9. pEZ-ILT4瞬时转染SK-BR-3细胞株后上调B7-H3mRNA和蛋白的表达ILT4过表达组中B7-H3的mRNA相对表达量(146.7±15.28)%明显高于对照组(74.2±4.01)%,p=0.001。ILT4过表达组及对照组中B7-H3蛋白的相对表达量分别为(136.4±10.1)%和(70.9±4.95)%,两者之间存在明显差异,p=0.001。10. pGPU6-shILT4瞬时转染MDA-MB-231细胞株后下调B7-H3mRNA和蛋白的表达ILT4干扰组中B7-H3的mRNA的相对表达量(54.3±3.9)%明显低于对照组(78.2±6.1)%,p=0.005。ILT4干扰组及对照组中B7-H3蛋白的相对表达量分别为(34.1±4.2)%,(62±4.6)%,两者之间存在明显差异,p=0.008。结论1.ILT4分子高表达于肿瘤组织和转移的淋巴结组织,而不表达于正常乳腺组织,且原发肿瘤组织中ILT4的表达与腋窝淋巴结转移相关,提示ILT4可能参与了肿瘤的发生发展过程。乳腺癌组织中1LT4与IL-10的表达明显正相关,与TILs数量明显负相关,表明在乳腺癌中ILT4和/或IL-10的表达抑制了TILs的活化和增殖,促进了TILs的凋亡,改变了肿瘤微环境,逃避免疫攻击,促进了肿瘤的侵袭和转移。2.乳腺癌组织中B7-H3和IL-10的表达明显正相关,且与TILs数量负相关,表明B7-H3和/或IL-10的表达可能是通过抑制T细胞活化,逃避免疫监视,促进乳腺癌的发生、发展和转移。3.乳腺癌组织中癌细胞上ILT4与B7-H3的表达呈正相关,且体外研究表明,在乳腺癌细胞株过表达ILT4可使B7-H3的表达增高,干扰ILT4表达后,B7-H3的表达明显下降,表明ILT4在体外可通过某个信号途径直接调节B7-H3的表达,诱导免疫耐受,逃避免疫攻击,从而促进了肿瘤进展。创新性及意义1.本研究首次检测了乳腺癌患者转移淋巴结中ILT4的表达情况,并分析了乳腺癌组织中ILT4的表达与临床病理因素的相关性,发现腋窝淋巴结转移组ILT4的阳性表达率明显高于无腋窝淋巴结转移组,表明ILT4可能参与了肿瘤的发生和发展过程,从而为以ILT4为靶点的靶向治疗和防治策略提供了理论依据。2.本研究首次分析了乳腺癌组织中ILT4和B7-H3的表达与IL-10表达的相关性,发现ILT4和B7-H3的表达与IL-10的表达呈显著正相关,为更进-步探讨ILT4和B7-H3在肿瘤免疫逃逸中的机制拓展新思路。3.本研究首次通过体外实验对ILT4调节乳腺癌细胞B7-H3的表达情况进行了研究,提示ILT4在体外可通过某个信号途径直接调节B7-H3的表达,为下一步进行ILT4对B7-H3的调节机制研究提供了理论依据。更多还原

【Abstract】 Background and objectiveBreast cancer is one of the most common female malignancies. It has been known that immune system controlscarcinogenesis. So it is important to detect a molecule to affect the immune system in the tumor.Immune escape is an important part of immunoediting, which can occur through many different mechanisms. More and more attentions have been drawn on the inhibitory molecules.Immunoglobulin-like transcript4(ILT4; also referred to LILRB2、MIR10、LIR-2、CD85d) belongs to a novel immunoglobulin superfamily involved in immune surveillance. ILT4is closely related to the killer-cell inhibitory receptor family, which also maps to the leukocyte receptor complex.ILT4has a long cytoplasmic tail containing immunoreceptor tyrosine-based inhibitory motifs, mediating the inhibition of cell activation by recruiting tyrosine phosphatase SHP-1and SHP-2. Previous studies demonstrate that ILT4plays an important role in maintenance of the maternal-fetal immune tolerance by tranducing a negative signal and inducing the transplantation tolerance in clinical practice.The expression of ILTs in cancer cells has been more and more attractive these days.ILT4is detected expressed in non-small cell lung cancer (NSCLC) cells with less number of infiltrating lymphoid cells. And the expression of ILTs, ILT3or ILT4, in neoplastic B cells is associated with lymphoid tissue involvement in chronic lymphocytic leukemia. The high expression of ILT3in gastric cancer is correlated with cancer progression. However, little is known about the expression and function of ILT4in other human carcinomas, for example, breast cancers.Previous study detects that co-cultured with CD8+suppressor T cells, dendritic cells express ILT4increasingly, with costimulatory molecules B7-1(CD80), B7-2(CD86) expression decreasing. Another study demonstrated that in the PIRB-/-macrophage, NF-κB pathway was activated, and its downstream molecules B7-1and B7-2expression was increased. These researches indicate that ILT4may regulate B7family in a certain signal pathway. B7-H3is a previously identified member of the B7family. It was initially identified as a co-stimulatory molecule. However, recent studies suggested an inhibitory role of B7-H3in the stimulation of T-cell response. Recent studies showed that B7-H3protein was of clinical significance in several different cancers and it inhibited T-lymphocyte proliferation and enhanced tumor progression. However, no studies were conducted to detect the role of ILT4expression in tumor cells and whether there were any relationships between ILT4and B7-H3expression in tumor tissues. Thus, we here aimed to evaluate the immunohistochemistry expression of ILT4in a series of invasive ductal and lobular breast carcinomas; and analyze the association of ILT4expression with B7-H3, IL-10, TILs and prognostic factors in breast cancer. Finally, we will detect the regulation of ILT4on B7-H3in vitro.Materials and Methods1. Immunohistochemistry was used to detect the expression of ILT4, B7-H3, IL-10and CD45RO in117primary breast cancer. CD45RO+cells were considered as tumor infiltrating cells. 2. B7-H3mRNA and protein expression in SK-BR-3transfected with pEZ-ILT4were detected by RT-PCR and Western Blot.3. B7-H3mRNA and protein expression in MDA-MB-231transfected with pGPU6-shILT4were detected by RT-PCR and Western Blot.4. Correlation of ILT4, B7-H3or IL-10expression and clinicopathological variables was analyzed by chi-square test. The correlation between number of TILs and ILT4/B7-H3/IL-10expression was compared by independent samples t test. With Spearman correlation analysis, the relationship of expression between ILT4or B7-H3and IL-10was evaluated. Student t test was used for comparison of the relative expression in different groups. p<0.05was considered statistically significant. Statistical analysis was performed by using SPSS v15.0.Results1. ILT4expression in human breast cancer tissues and lymph node metastasis and its association with clinical parameters.The expression of ILT4in117primary tumor specimens was determined by immunohistochemistry. ILT4were identified in tumor cell cytoplasm, membrane, or both. Their expression were absent in adjacent normal breast tissues. In total,71of the117specimens (60.7%) expressed ILT4. Expression of ILT4was also seen in some stromal macrophages, fibroblasts and plasma cells.ILT4was expressed by tumor cells in metastatic lymph nodes with74.2%(72/97) positive staining from12patients. Within the metastatic foci, ILT4expression was cytoplasmic, membranous, or both. The patterns were always identical among individual lymph nodes from the same patient. Furthermore, ILT4expression in tumor cells of nodal metastasis was identical to that observed in the corresponding primary tumors. Within ILT4-negative tumor metastatic lymph nodes (n=25), almost all corresponding primary tumors showed negative ILT4expression in the tumor cells. Only one showed ILT4expression in10%-40%of the tumor cells. Focal membranous and cytoplasmic ILT4expression was also detected in scattered stromal cells of metastatic lymph nodes. Expression level of ILT4was much higher in cases with lymph node metastasis (p=0.046).2. B7-H3expression in human breast cancer tissuesB7-H3was expressed in106(90.6%) of117specimens, and it was identified in the tumor cell cytoplasm, membrane, or both. Little staining of B7-H3was identified in the stromal cells. Expression level of B7-H3was much higher in cases with lymph node metastasis (p=0.018). In addition, patients with B7-H3positive expression were more likely to have advanced disease (stage Ⅱ-Ⅳ; p=0.011) compared to those with negative expression.3. Correlation between the expression of ILT4and B7-H3.A positive correlation was found between ILT4and B7-H3expression (R=0.41;p<0.01).4. Correlation between the expression of ILT4or B7-H3and IL-10IL-10was expressed in94of117specimens, and microscopically, it was identified in tumor cell cytoplasm. Patients with high IL-10expression were more likely to have lymph node metastasis (p=0.035) and advanced disease (p=0.039).The results indicated a positive correlation between ILT4or B7-H3and IL-10(R=0.577or0.545respectively; p<0.01).5. Correlation between expression of ILT4, B7-H3or IL-10and the number of TILsWe assessed the relationships between the expression of ILT4, B7-H3or IL-10and the number of TILs in all tumor specimens. The amount of TILs was compared between the groups expressing ILT4, B7-H3or IL-10or not. Tumor tissues with positive expression of ILT4, B7-H3or IL-10seemed to have less infiltration of lymphocytes. The mean number of TILs in ILT4-positive group was 26.71±11.85and was significantly lower than that in the ILT4-negative group,33.58±13.73(p=0.004). The mean TILs number in B7-H3-positive group was27.41±11.17, whereas that was33.71±12.57in B7-H3-negative group (p=0.005). In addition, the mean number of TILs in IL-10-positive group was27.65±12.91, and was significantly lower than that in the IL-10-negative group,34.80±14.49(p=0.018).6. ILT4and B7-H3expression in human breast cancer cell linesThe mRNA expression of ILT4and B7-H3were detected in all of the4human breast cancer cell lines by RT-PCR. These cell lines all expressed ILT4and B7-H3protein demonstrated by Western blot analysis.7. Identification of plasmid pEZ-ILT4.The recombinant vector was identified by Restriction endonuclease analysis and DNA sequence analysis.The results demonstrated that ILT4gene was identical to the sequence recently reported on pubmed. We concluded that the recombinant plasmid vectors pEZ-ILT4were constructed successfully.8. Identification of plasmid pGPU6-shILT4.ILT4mRNA and protein expression in MDA-MB-231transfected with pGPU6-shILT4was detected by RT-PCR and Western Blot. Compared with the non-targeting control plasmid, four shRNA sequences significantly decreased ILT4expression, and pGPU6-shILT4-853achieved more interference efficiency than others at both mRNA and protein level, p<0.001. So pGPU6-shILT4-853as pGPU6-shILT4participated in the following studies.9. B7-H3expression was up-regulated in SK-BR-3cells transfected with pEZ-ILT4We examined the effect of pEZ-ILT4transfection in SK-BR-3cells on B7-H3expression, and we detected that B7-H3mRNA and protein was induced dramatically high levels. 10. B7-H3expression was down-regulated in MDA-MB-231cells transfected with pGPU6-shILT4For further study we detected the effect of pGPU6-shILT4transfection in MDA-MB-231cells on B7-H3expression. Down-regulation of B7-H3mRNA and protein expression was shown.Conclusion1. ILT4was highly expressed in the tumor specimens and in the lymph node metastasis, but not in adjacent normal breast tissue, and ILT4expression in primary tumor tissues were correlated with the prevalence of lymph node metastasis, which demonstrated that ILT4protein may be involved in the tumor progression. In our study, a positive correlation between ILT4and IL-10was shown.What’s more, the number of CD45RO+TILs in ILT4-or lL-10-positive cancer tissues was less than that of negative groups. ILT4and/or IL-10expression might promote tumor growth and progression by inhibiting the proliferation of CD45RO+TILs and/or inducing the apoptosis of CD45RO+TILs in the tumor environment.2. In addition, we found a significant association between B7-H3and IL-10expression. We postulate that B7-H3or IL-10-expressing cancer tissues may escape the immune defenses and promote tumor growth.3. In our study, a significant correlation was shown between ILT4and B7-H3. We also found that in vitro, tumor-associated B7-H3expression was up-regulated in SK-BR-3cell line transfected with pEZ-ILT4and down-regulated in MDA-MB-231cells transfected with pGPU6-shILT4. We postulate that ILT4may regulate B7-H3by a certain signal pathway,which can inhibit the antigen presentation, induce immune tolerance and promote tumor progression.Originality 1. For the first time, we detected the expression of ILT4in the lymph node metastasis and analyzed its association with B7-H3expression in breast cancer. We first found that ILT4expression in primary tumor tissues were correlated with the prevalence of lymph node metastasis, which demonstrated that ILT4protein may be involved in the tumor progression.2. We analyzed the correlation between ILT4or B7-H3and IL-10expression for the first time.3. For the first time, we detected the regulation of inhibitory ILT4on tumor-associated B7-H3in vitro, which can provide a basis for further study on the regulation mechanism.更多还原