摘要
研究背景：
胰腺癌是消化系统常见的恶性肿瘤,在所有外分泌恶性肿瘤中占80%-90%,其发病率在全球范围内呈逐年上升趋势。手术治疗是目前唯一有可能治愈胰腺癌的方法,由于胰腺解剖位置较深,早期症状不典型,同时胰腺癌生物学特性为早期胰外侵犯,具体表现为淋巴、神经及血液转移出现早；而且胰腺癌细胞具有高侵犯及转移能力,肿瘤进展迅速,同时缺乏敏感性高的早期诊断方法,多数患者发现时已属晚期,仅有10%-15%的患者能有手术切除的机会,其中仅5%能根治。即使行根治性手术治疗,因诊断时可能已经存在手术难以根除的微转移,故而难以根治性地切除肿瘤,故而胰腺癌术后复发率高,预后差,术后5年生存率小于15%。目前认为胰腺癌进展迅速,细胞高侵犯及转移能力是造成其预后差的原因。趋化因子及其相应受体在越来越多的研究中被发现能促进肿瘤细胞增殖、侵袭、迁移。目前已经发现50余种趋化因子及20余种趋化因子受体,具体在胰腺癌的肿瘤进展中,目前研究较多涉及的是基质衍生因子(stromal derived factor-1, SDF-1,即CXCL12)及其受体CXCR4构成的CXCL12/CXCR4生物学轴和胸腺表达趋化因子(thymus expressed chemokine, TECK,即CCL25)及其受体CCR9组成的CCL25/CCR9轴,认为其在胰腺癌发展过程中发挥主要作用。本课题组曾用人低分化胰腺癌转移瘤细胞株MiaPaCa-2建立了裸鼠皮下及原位移植瘤模型,并通过cDNA芯片检测出其未转移与已转移原位移植肿瘤表达差异的趋化因子CCL25及受体CCR9对胰腺癌细胞的促增殖,促迁移,促侵袭作用,初步探讨了趋化因子及其受体在胰腺癌发展过程中发挥的相关作用。本研究着眼在不同侵袭潜能胰腺癌细胞系的趋化因子及受体轴的表达情况及其功能。
研究目的：
1)了解临床胰腺癌术后预后不良(生存<6个月)和预后较好(生存≥18个月)的病例肿瘤及周围组织中CXCL12/CXCR4和CCL25/CCR9轴的表达和临床特征之间关系。
2)了解不同侵袭能力的胰腺癌细胞系中CXCL12/CXCR4和CCL25/CCR9轴的表达,及其对胰腺癌细胞的增殖和侵袭能力的影响；并阻断相应趋化因子及其受体的相互作用,观察胰腺癌细胞的增殖和侵袭能力的变化。
研究方法：
1)选择预后不良(生存<6个月)和预后较好(生存≥18个月)的胰腺癌术后病例,分别为32例和30例,以免疫组织化学方式检测肿瘤及周围组织中CXCL12/CXCR4和CCL25/CCR9轴的表达和临床特征之间关系。
2)选择三系侵袭能力分别为低、中、高的胰腺癌细胞系：Capan-1、Aspc-1和Mia PaCa-2。以Rt-PCR, SQ-PCR, Western Blot等方法检测各系细胞中的趋化因子受体表达水平。
3)通过平板克隆形成实验、MTT实验、流式细胞仪检测细胞周期变化、及Transwell侵袭实验来验证不同趋化因子对不同侵袭能力胰腺癌细胞的促增殖作用和促侵袭作用,并运用相应趋化因子受体的单克隆抗体阻断趋化因子与受体之间的相互作用后,再观察趋化因子对胰腺癌细胞上述作用的变化。
研究结果：
1)对于临床病例标本的免疫组化结果显示：CCL25/CCR9和CXCL12/CXCR4蛋白主要在胰腺癌细胞胞浆中表达,胰腺癌癌组织中的趋化因子配体的表达与临床特征无明显关联(p>0.05),但受体的高表达和疾病进展尤其是淋巴结转移相关(p<0.001,p<0.001),并造成患者预后不佳(p<0.001,p=0.001)。此外,转移淋巴结中的受体高表达且与配体高表达相同步(r=0.31p=0.023,r=0.69p=0.002),也进一步验证了趋化因子受体/配体组合高表达与胰腺癌淋巴结转移及预后不佳相关。
2)对于不同侵袭能力的胰腺癌细胞系的受体定量检验显示：趋化因子受体CCR9和CXCR4在不同的胰腺癌细胞系中表达不同,说明不同分化程度的胰腺癌细胞具有各自不同的趋化因子配体及受体体系(CXCR4p=0.681,0.148,0.082, CCR9p=0.047,<0.001,<0.001)。
3)关于重组人TECK或者SDF-1对三系胰腺癌细胞促增殖(影响克隆形成和细胞周期)和促侵袭作用的验证试验显示：趋化因子对胰腺癌细胞系的上述作用呈浓度依赖型,并可以被受体特异性抗体所阻断,证明趋化因子和胰腺癌细胞中相应受体相互作用实现上述促增殖和促侵袭效果。此外,结合趋化因子受体在不同的分化程度的胰腺癌细胞系中表达定量结果,可以发现胰腺癌细胞系的增殖侵袭潜能和受体的种类及表达水平紧密相关：受体表达量越高,细胞系对于趋化因子的促增殖和侵袭的作用反应越敏感,而且高侵袭潜能的细胞系具有较高的受体表达量。
结论：
胰腺癌癌组织中存在趋化因子受体的表达,而不同侵袭能力的胰腺癌细胞系中趋化因子受体的表达种类和量不同；相应的趋化因子配体可以和受体共同作用,促进胰腺癌细胞增殖(影响克隆形成和细胞周期)和侵袭,并且该作用和受体的种类和量紧密相关。胰腺癌转移淋巴结及胰腺癌旁组织中的受体高表达且与配体高表达相同步,说明在临床病例中胰腺癌的进展也和趋化因子配体和受体的相互作用有关。
Background:
Pancreatic cancer (PaCa) is one of the most common malignant tumors in the digestive system, which constitutes80%-90%of all the exocrine malignancies, with an still increasing trend of incidence worldwide. The curative surgery remains the only effective treatment to cure PaCa potentially. However, the early diagnosis for PaCa is difficult due to its anatomically profumdus location and asymptom in early stage. The advance of PaCa can occur early via lymphatic, nervous or vessel systems during onset. Without available sensitive measures of early detection, most cases are advanced at the time of first detection. About10%-15%patients have the opportunity for resections, only5%of which is curative. Even if the radical resection can be achieved, the undetectable micrometastasis could also jeopardize the effect of surgery. The recurrent rate of pancreatic cancer is high after surgery, with a postoperative five-year survival rate less than15%. The current understanding attributes the poor prognosis of PaCa to the high invasive and metastatic potential of its tumor cells. Increasing researches suggested that chemokines and their receptors functioned as key factors in the proliferation, invasion and migration of malignant tumor cells. Over50chemokines and20receptors have been indentified to date, in the progression of pancreatic cancer specifically, the role of the biological axis of CXCL12(stromal derived factor-1, SDF-1)/CXCR4and CCL25(thymus expressed chemokine, TECK)/CCR9are frequently investigated. The TECK/CCR9was screened out as differentially expressed gene across stages of PaCa by cDNA microarray of the specimen from subcutaneous and orthotopic transplantation tumor model in our prior research. The present study will look into the expression and function of different axis of chemokines and receptors in pancreatic cancer cell lines with diverse potential of progression.
Purpose:
1) To compare the expression of CXCL12/CXCR4and CCL25/CCR9in the tumor and adjacent tissue between the groups of better prognosis and poor prognosis, and also the correlation with the clincpathological features, of the PaCa patients who undergone curative resections.
2) To investigate the expression and functions of CXCL12/CXCR4and CCL25/CCR9in PaCa cell lines with different potential of proliferation and invasion, the effect of chemokines will be further verified by blockade of corresponding receptors by specific antibodies.
Methods:
1) The patients undergone curative resections of primary PaCas were categorized according to postoperative survivals into two groups:better prognosis (survival no less than18months,30cases) and poor prognosis (survival less than6months,32cases). The expression of CXCL12/CXCR4and CCL25/CCR9in tumor and adjacent tissue was detected by immunohistochemistry (IHC), its correlation with the clincpathological features was also analyzed.
2) The expression of CXCR4and CCR9in three PaCa cell lines with different potential of proliferation and invasion, Capan-1, Aspc-1and Mia PaCa-2, was evaluated by Rt-PCR, SQ-PCR and Western Blot.
3) The effect of CXCL12and CCL25of promoting the proliferation and invasion on PaCa cell lines with different progression potential were verified by colony formation assay, MTT proliferation assay, Transwell invasion assay and cell cycle by flow cytometry analysis (FCM) associated with the blockade by corresponding antibodies of receptors.
Results:
1) The IHC of tumor and adjacent tissue indicated that CCL25/CCR9and CXCL12/CXCR4are mainly detected in the cytoplasm of PaCa cells. The density of positive staining of chemokine ligand didn't demonstrate significant correlations with clincpathological features (p>0.05), whereas the strongly positive staining of chemokine receptors was associated with disease progression and poor prognosis (p<0.001, p<0.001), which was also synchronous with the staining of ligands in involved lymph nodes (r=0.31p=0.023, r=0.69p=0.002). This result supported that the interaction of ligands and receptors played certain role in the progression, especially lymph node metastasis, of PaCa.
2) The quantitative analysis of CCR9and CXCR4showed significant differences of expressions among the three PaCa cell lines with different potential of progression (CXCR4p=0.681,0.148,0.082, CCR9p=0.047,<0.001,<0.001), which suggested that PaCa cell lines with diverse differentiation have different ligand/receptors axis..
3) The recombinant human TECK or SDF-1presented with the effect of promoting the proliferation and invasion of PaCa cells, the sensitivity of which was determined by the density of corresponding chemokine receptors. This effect can be neutralized by blockade of the chemokine receptors. This result also implied that the progression potential of PaCa cell lines was partly determined by the expression of receptors:cell line with high expression of receptors is more sensitive to the ligands and more invasive.
Conclusion:
The PaCa tumor cells express chemokine receptors, which varied from PaCa cell lines with different progression potentials. The chemokine ligands can promote the proliferation, by effecting the clone formation and cell cycles, and invasion by interacts with corresponding receptors in a concentration-dependent manner. The high expression of receptors, synchronous with the expression of ligands, in metastatic lymph nodes and adjacent tissue of PaCa patients with poor prognosis suggested that the progression of PaCa was related to the interaction of chemokine ligands and receptors.
引文
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