摘要
研究背景和目的：
肺癌是呼吸系统的常见恶性肿瘤,已成为各种肿瘤死亡原因之首。肺癌中80%为非小细胞肺癌,已经成为基础医学和临床医学研究的热点之一肺癌由于具有高侵袭性、高转移性的生物学特性,早期症状不明显,缺乏简单有效的早期、无创检查方法,从而造成约70%的肺癌就诊患者就诊时已经进入疾病的晚期,手术切除率不足30～40%,总的5年生存率不超过30%,治疗效果差。导致肺癌患者失去手术条件主要原因并非是肿瘤生长过大,而是肿瘤转移导致手术效果差。淋巴结转移程度是影响肺癌病理分期和治疗后生存时间的主要因素。淋巴结转移在肺癌的分期、诊断、治疗和预后判定中具有重要的临床意义。所以,探索肺癌淋巴转移的具体机制己成为当今研究热点之一。转移程度是影响肺癌病理分期和治疗后生存时间的主要因素。淋巴结转移在肺癌的分期、诊断、治疗和预后判定中具有重要的临床意义。所以,探索肺癌淋巴转移的具体机制已成为当今研究热点之一。肿瘤细胞通过淋巴管扩散到局部淋巴结是临床分期的重要指征。在发生淋巴结转移的肿瘤基质中,常能发现淋巴管的增生和扩张,但其分子机制至今尚未充分阐明,淋巴管形成在淋巴结转移中的作用还不清楚。在过去的几十年里,由于缺乏明确的淋巴管生成因子及特异的淋巴管标记因子,因此对淋巴结转移的研究进展缓慢,大多集中在对血管系统的研究上。近年来发现了血管内皮生长因子C(vascular endothelial growth factor C, VEGF-C)及其受体VEGFR-3,研究认为VEGF-C是相对特异的淋巴管内皮生长因子,与VEGFR-3结合后可发生淋巴管内皮扩张,促使淋巴管形成,从而为研究淋巴结转移机制开辟了新的途径。血管内皮生长因子C与其受体VEGFR-3结合后,具有强大特异的淋巴管生成作用,并认为与肿瘤的淋巴结转移有密切关系。淋巴结转移是肺癌患者尤其是非小细胞肺癌患者预后的十分重要的不良因素,淋巴结转移是非小细胞肺癌主要扩散途径,所以肺癌淋巴结转移的诊断尤为重要。而目前主要的临床诊断手段-胸部CT却对肺癌淋巴结转移存在与否的预测缺乏特异性和敏感性。尽管常规病理HE染色对于淋巴结转移是金标准,但是,对于早期的淋巴结微转移却缺乏敏感性,对肺癌淋巴结转移的基础实验研究则可能有助于我们找到解决问题的金钥匙。而随着VEGF-C作为特异性淋巴管生成因子,促进淋巴结转移机制的深入研究,对于非小细胞肺癌(non-small cell lung cancer, NSCLC)患者早期诊断、准确分期、改善手术方式和术后治疗方案,甚至研制相关新药,促进肿瘤治疗进展将具有重大意义。
本研究通过实时荧光定量RT-PCR、免疫组化和酶联免疫法检测不同组织中VEGF-C表达水平及微淋巴管密度(micro lymphatic vessel density, MLVD)和淋巴结中CK19mRNA的表达,结合临床病理资料进行统计分析,旨在探讨VEGF-C的表达与非小细胞肺癌淋巴结转移和淋巴结微转移的相关性,以及在非小细胞肺癌患者肿瘤组织和外周血中的表达有无相关性,明确能否用外周血中的表达水平来预测肿瘤组织中的表达,寻找一种更简便的检测方法,并研究VEGF-C表达水平和肿瘤治疗之间的关系,分析其作为肿瘤标记物的意义。
研究材料和方法：
本研究取青岛大学医学院胸外科2008年3月～2008年10月手术前没有施行放疗和化疗、施行了根治性肺叶切除、系统性淋巴结清扫、随访资料确实的全部NSCLC患者标本资料129例的癌组织、癌旁肺组织(为距肿块边缘5 cm以上肺组织)、肺门第10组淋巴结和同期20例行良性疾病肺切除术患者正常肺组织作为对照,术中取新鲜标本组织直径约为0.5cm,用液氮快速冷冻,-80℃冰箱保存。另取标本直径约0.5cm行病理科常规固定石蜡包埋等处理。采取全部NSCLC患者术前1天,手术后第7天清晨空腹血5ml,离心保留血清置于-80℃冰箱保存。选择确诊为NSCLC行标准化疗(未行手术治疗)患者69例,取化疗前1天和第3周期化疗前(化疗后)1天清晨空腹血5mI,处理同上。本研究应用实时荧光定量RT-PCR法检测肺癌组织、癌旁组织、部分淋巴结组织和肺对照组织VEGF-CmRNA表达水平,并进行相对定量计算；应用免疫组化S-P法检测肺癌组织、癌旁组织和肺对照组织中D2-40标记微淋巴管,并计数MLVD;应用固相夹心法酶联免疫吸附法(enzyme linked immune sorbent assay, ELISA)检测手术患者手术前、手术后和化疗患者化疗前和化疗后空腹血清VEGF-C蛋白含量,应用实时荧光定量RT-PCR检测淋巴结中CK19mRNA含量,并对所得数据结合患者临床病理特征进行统计分析。
结果：
在NSCLC癌组织中,93.0%检测到VEGF-CmRNA,在不同性别、年龄、吸烟史、病理类型、肿瘤大小、病理分化程度和病理类型等方面VEGF-CmRNA含量无明显差别(P>0.05); VEGF-CmRNA相对含量与淋巴结转移密切相关,在有淋巴结转移的癌组织中,VEGF-CmRNA相对含量较没有淋巴结转移的癌组织中显著增高(P<0.01),并且淋巴结转移越远,VEGF-CmRNA含量增高越明显(p<0.01)。在全部癌组织中均可见到D2-40标记的微淋巴管,MLVD与患者性别、年龄、吸烟史、病理类型、肿瘤大小、病理分化程度和病理类型未见显著差异(P>0.05)。MLVD与淋巴结转移密切相关,随淋巴结转移程度的增加而相应增加(p<0.01)。MLVD随VEGF-CmRNA含量增加而增加,两者成线性相关(r=0.831,p<0.01)。血清ELISA测定VEGF-C蛋白含量是一种有效的检测方法,其检测准确性低于肿瘤组织实时荧光定量RT-PCR,没有显著差异(P>0.05),但是具有较好的一致性(r=0.848,p=<0.01),可以在NSCLC患者术前和治疗前后随时监测,可以使VEGF-C在肿瘤检测、肿瘤标志物等意义方面提供有效的临床推广实用的检测手段。血清VEGF-C蛋白含量可以推测肿瘤化疗的敏感性,患者在手术前和手术后血清VEGF-C蛋白含量变化明显,具有显著差异(p=<0.01),相关分析显示手术后血清VEGF-C蛋白含量与手术前其含量存在线性相关(r=0.78,p<0.01),化疗效果好的患者化疗前血清VEGF-C蛋白含量高,且化疗后降低更加明显,反之,血清VEGF-C蛋白含量较低的患者,化疗效果相对较差,化疗后随着癌症病变进展,化疗后血清VEGF-C蛋白含量反而会升高。淋巴结CK19mRNA检测较常规病理检测有更高的阳性率(P<0.01),并且病理阳性淋巴结中均有CK19mRNA表达,在病理阴性淋巴结中有16(16/56,28.6%)例CK19阳性,因而具有更高的敏感性。癌组织中VEGF-C高度表达、MLVD明显增高时,淋巴结微转移可能最大(25/26,96.2%)；而在癌组织中VEGF-C低表达、MLVD较低时,淋巴结微转移的可能小(0/26,0.0%)
结论：
1、VEGF-C在NSCLC癌组织中过度表达,VEGF-C在癌组织中的表达与淋巴结转移和淋巴结微转移密切相关,与患者性别、年龄、吸烟史、肿瘤大小、肿瘤分化程度和肿瘤病理性质无明显关系。
2、VEGF-C在NSCLC癌组织中过度表达可能刺激微淋巴管形成,导致微淋巴管密度增加,从而促进肿瘤细胞向淋巴结转移,首先形成微转移灶,微转移灶生长、发展逐渐成为病理阳性的显性转移。
3、ELISA检测NSCLC患者血清VEGF-C蛋白含量是一种可行、有效,简单、易行且易于推广应用的方法,与癌组织VEGF-C表达具有明显的相关性,在一定程度上可以代替肿瘤组织的检测,从而为无法得到肿瘤组织或得到肿瘤组织有困难的病人提供一种更加简便的检测方法。
4、VEGF-C过度表达的NSCLC患者,淋巴结微转移出现可能大,淋巴结转移相对较早,提示手术应当强化淋巴结清扫；VEGF-C过度表达的NSCLC患者,化疗敏感性高,提示术后应该尽早施行化疗等辅助治疗,并且提示化疗效果较好。
意义：
1、本研究通过快速高效,定量准确、重复性好以及特异性和敏感性更高的实时荧光定量RT-PCR方法检测组织中的VEGF-CmRNA,并通过检测淋巴结CK19来检测病理淋巴结阳性前的微转移,分析其与VEGF-C过度表达的关系,使结果更加准确、可靠、可信。2、本研究证实了作为临床广泛应用的酶联免疫吸附法测定血清VEGF-C蛋白含量是可行和有效的,从而为非小细胞肺癌患者提供了一种容易的对肿瘤淋巴结分期的预测办法,从而指导手术的施行和治疗方案的选择,并且预知化疗敏感性,从而使患者可以选择更好的、更有效的治疗方案,具有较高的临床实用价值。
Background and Objective:
Lung cancer has become the world's highest incidence of malignant tumors and death rate is rising rapid to be the first in all kinds of tumor. Of about 80% lung cancer is non-small cell lung cancer(NSCLC), and it is become to be one of the hottest spots to preclinical medicine and clinical medicine. Because the lung cancer has the biologicalcharacteristics of sever invasive, easier to metastasis and there always has not obviously clinical symptoms, even a simple easier effective examination taken to find it, so about 70% patients with lung cancer couldn't be find out before it was a later stage to the cancer. Only 30% to 40% of all cases with lung cancer could be operated which is the best way to deal with lung cancer. The five years survival rate is no more than 30% for all the cases with lung cancer. There is not an effective matter to do with the lung cancer when is it in last stage due to a bad survival rate. The main reasons caused the patients with lung cancer lost the chances to be operated is not the tumor get bigger, but it is the metastases of the tumor. Most metastases way is via lymph node pass way. The stage of lymph metastasis is the main reasons to effect the pathological staging and survival time after treated for lung cancer, so the metastasis has important significance in the lung cancer pathological staging, diagnosis, treatment and predictor to lung cancer. So it came to be the hot pot study areas for study on substantial mechanism of lymph metastases of lung cancer. tumor cells through lymphatic spread to local lymph nodes are an important indication of the clinical stage. In the tumor matrix of lymph node metastasis, lymphatic proliferation and expansion Can often be found, but the molecular mechanism has not yet been fully clarified, and the role of the formation of lymphatic vessels to lymph node metastasis remains unclear. In the past few decades, because of the lack of specific lymph angiogenesis factor and specific lymphatic marker factor, the study on lymph node metastasis makes less progress, and most of them are focused on the vascular system. In recent years, the vascular endothelial growth factor C(VEGF-C)and its receptor VEGFR-3 are found, and VEGF-C is known that it is a relatively specific lymphatic endothelial growth factor. The expansion of lymphatic endothelial to promote the formation of lymphatic vessels is occurred after VEGF-C and VEGFR-3 combine together, and thus to study the mechanism of lymph node metastasis has opened new avenues. They have strong special function of promoting the formation of lymphatic vessels after VEGF-C combined with VEGFR-3. And was known keep a close relationship with lymph node metastases. Lymph node metastasis is one of the most important negative predictor to patients with lung cancer especially to non-small cell lung cancer. Because the lymph node metastasis is the major metastases way for NSCLC, it's especially important to be diagnosis for metastases of lymph node. The most common clinical device used to diagnosis about lung cancer is chest computer tomography. But the chest computer tomography cannot give us a little useful advice about lymph node metastases. There is no specificity or sensitivity to lymph node metastases in chest computer tomography. To study the basic theory about lymph node metastases in lung cancer can be helpful to look for the gold key to see the essential qualities of lymph metastases. With the study progresses on VEGF-C—which is a special lymphageogenesis factor, we can gradually maste the matter used on early diagnosis, correct stage, improve operation style, select better treatment plan after surgery and investigate new drugs resist tumor. It will be improve the treatment about lung cancer and have great significance.
In this study, the VEGF-C expression level was detected by RT-PCR, immunohistochemical streptavidin peroxidase method, and ELISA method; the MLVD was counted, CK19mRNA in lymph nodes were detected and all the datas was annalized statitically with Clinical and Pathological features. So we can investigate the relationship between the expression of VEGF-C and the lymphatic metastasis or micro metastasis in NSCLC. To study the correlationship between the expression level in cancer tissue and VEGF-C protein in peripheral blood serum, so identify if it can be taken the place of detect VEGF-C expression in tissue by VEGF-C protein detected in serum, so we can find a easier method detection way for VEGF-C. The relationgship between the VEGF-C expression's level and tumor treatment way selected. The clinical significance of VEGF-C expression as a tumor marker was analized together.
Methods and materials:
129 cases in the affiliated hospital of Qingdao university chest surgery department from March,2008 to October,2008 with primary NSCLC were included in this study. All of them didn't get any radiotherapy or chemotherapy befor operation; operated with complete lobectomy with systemical lymphadectomy. All of the follow-up data was foll and correct. The tissue in cancer, paracancer tissue which is 5 centimeters part from tumor, lymph node of 10th group also named lung pilar and normal lung tissue got from 20 cases without malignant tumor who treatd with lung resection as contrast samples. Fresh tissue about 0.5 centimeters in adiameter was cut down and rapid deepfreeze with liquid nitrogen and kept in deep temperature refrigerator at-80℃. Another tissue about 0.5 centimeters in adiameter was cut down and fixed in 10% neutral formalin and embedded with paraffin just like pathologic division applied normally. All the cases of NSCLC were hemospasia in the morning with stomach empty in the 1st day befor operation and 7th day after operation. All these blood was entrifuged and the serum was kept in deep temperature refrigerator at-80℃.69 cases without operation and treated only with standard chemotherapy who was diagnosised NSCLC was selected in the same hospital tumor department. With the same method we got them serum of 1st day befor chemotherapy and 1st day befor the 3rd cycle of chemotherapy which was signed as the serum after chemotherapy. In this study the VEGF-C expression level in tissue of cancer, para cancer, and normal contrast was detected with real time quantitative RT-PCR method, and the relative quantitation was calculated to be analized. The MLVD signed with D2-40 were detected with immunohistochemical streptavidin peroxidase method in all of the tissues. ELIS A methods were used to detect the serum VEGF-C protein in all the serum samples. The CK19mRNA in lymph nodes were detected with real time quantitative RT-PCR method. Then the rsults were statistically annalized with clinical pathological features of all cases.
Results:
In the tissue of NSCLC cancer, VEGF-CmRNA was detected in about 93.0% of all cases. There was not statisitcal significace with the gender, age, smoking habit, pathological differentiation, the diameter of tumor and histologyof the tumor (P>0.05). There was a close relationship between quantitaty of VEGF-CmRNA and lymph node metastasis, quantitaty of VEGF-CmRNA was higher in cases with lymph node metastasis than those without lymph node metastasis (P<0.01). With the degree of lymph node metastasis, the VEGF-CmRNA got more obviously (p<0.01). the micro lymphatic vessel stained by D2-40 could be find in all cancer tissues and There was not statisitcal significace with the gender, age, smoking habit, pathological differentiation, the diameter of tumor and histologyof the tumor (P>0.05). There was a close relationship between MLVD and lymph node metastasis. It got high with lymph node metastasis distant (p<0.01) and there was a ling relationgship between them (r=0.831, p<0.01). it was confirmed to be an effective method to detect VEGF-C protein in serum with ELISA. Although the sensitivity of ELISA method was lower than that of real time quantitative polymerase chain reaction method, the result showed no statisitcal significace. Both of the methods was reliable in these tests (r=0.848, p=<0.01). Then we could detect VEGF-C all time like befor and after operation then showed its clinical significance on tumor diagnosis as tumor marker. We could know the senitivity of a NSCLC to chenmotheropy bu detect VEGF-C in serum. The difference of VEGF-C bofore operation and after operation was significant and has statistically means (p=<0.01). there was a line relationgship between them with corelation analyze (r=0.78, p<0.01) Effective chemotherapy before chemotherapy in patients with serum VEGF-C protein content is high, and decreases more apparent after chemotherapy, on the contrary, serum VEGF-C lower protein content, effect of chemotherapy in patients with relatively poor, chemotherapy with cancer progression, chemotherapy serum protein content than VEGF-C. High sensitivity to chemotheropy was higher VEGF-C than they others. The positive rate of lymph node metastasis was higher with method of CK19mRNA detection than normal pathological method (P<0.01). CK19mRNA could be detectd in all of the positive lymph nodes identified by pathological method. Because 16 lymph nodes in all 56 negative lymph nodes identified by nomal method could be detected CK19, so we conclude that the method to detect CK19 has more sensitivity than nomal pathological method. The property of lymph nodes micro metastasis may be high in those with VEGF-C over expression, MLVD obviously superior (25/26,96.2%). On the contrary, the property of lymph nodes micro metastasis would be little in those with hypoexpression of VEGF-C and MLVD lower (0/26,0.0%)
Conclusions:
1. The VEGF-C in cancer tissue was over expressed and has a close relation to lymph node metastasis or micro metastasis with the NSCLC. But there was not statisitcal significace with the gender, age, smoking habit, the diameter of tumor, pathological differentiation and histologyof the tumor.
2. The over expression of VEGF-C in cancer tissue may stimulate the new lymphangiogienesis. Causes lymphatic density increases, thereby promoting tumor cells to form the first lymph node metastasis, micrometastasis, micrometastasis in growth, development on their way to becoming a dominant transfer of positive pathology.
3. The method using ELIS A to detect VEGF-C in serum is a feasible, effectively, simple, easier and easy to be generalized method on VEGF-C detection. There is an obvious correlation with the over expression of VEGF-C in tissue. Thereby the method using ELIS A to detect VEGF-C in serum can stand for the method to detect in tissue, then we can provid a good method to detect VEGF-C for those couldn't get tumor tissue and difficult to detect on tissue.
4. The lymph node metastasis would be earlier and micro metastasis property would be higher on those patients with over expression of VEGF-C than they others, so suggest us emphasis on lymphadectomy in an operation. Its higher sencitivity to chemotheropy for those with over expression fo VEGF-C prompt us a better result to chemotheropy and chemotheropy should taken earlier after operation.
Study significance:
1. This study quantitative by fast and efficient, accurate, repetitive and specificity and sensitivity higher real time quantitative RT-PCR detection of VEGF-CmRNA, in the cancer tissue and by detecting lymph node CK19 to detect pathological lymph node-positive front of micrometastasis, analyze the relationships with VEGF-C overexpression, make the results more accurate, reliable, trusted.
2. The present study confirmed the method of using ELISA to detect VEGF-C protein in serum a good effective way and the ELISA is a widespread testing method in clinical work. So it will provide a easier method to detect VEGF-C in NSCLC and will be helpful for staging of tumor and give advice for operation and select better treatmeng plan. We evev can predict the sensiticity of chemotheropy so make a new plan to deal with the tumor. It's useful in clinical work and theat with NSCLC.
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