| 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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