| Objective:It's a complexity which is of many factors and multi-stage process included that the occurrence and development of malignant tumor, its very nature is reflected in the genetic changes,such as activation of oncogenes and inactivation of tumor suppressor gene,which maketumor escape the body's immune surveillance system, and the body loseits role of monitoring and killing cells which are of abnormal changes,resulting in invasion and metastasis of malignant tumor.In recent years, the inhibition of the spleen tyrosine kinase(Syk) in the occurrence and metastasis of malignant tumors is wide and deep, and syk(L) has the inhibition by the existence of the functional structure of inter-domain domain (IDB) nuclear localization signal functions, while Syk (S) has no thisfeature.As many scholars at home and abroad have been doing broad and deep research on the relationship between the deletion of Syk geneand many sorts of tumors, which associated with breast cancer, gastric cancer, pancreatic cancer, lympho hematopoietic system, some confirmed the expression of Syk in the serum of lung adenocarcinoma was reducedcompared with normal people, and was negatively correlated with lymph node metastasis, which provided a theoretical basis for the monitoringof lung adenocarcinoma patients with lymph node metastasis, but no detailed analysis to explore; However, there is still lack of Syk gene prognosis of lung cancer in different pathological types, and other influencing factors in-depth verification.The monoclonal antibody plays an important role in the early diagnosis of malignant tumors, provides a new molecular target for antibody-dependent cell-mediated cytotoxicity therapyand prognostic monitoring because of high purity, high specificity, sensitivity, and other characteristics. This experiment aided to prepare high titer anti-Syk (L) monoclonalantibody; to verify the differences of protein expression in different pathological types of lung cancer using immunohistochemical staining method to detected lung cancer tissues,to explore the role of Syk (L) gene inlung cancer development, prognosis, efficacy evaluation, to provide a new way of thinking about early diagnosis of lung cancer, determining the pathological type, guiding treatment, monitoring efficacy, evaluation and prognosis molecular markers.Methods:1Preparation of anti-syk(L) monoclonal antibodySyk protein of high purity as the antigen, immunized of BALB/c mice routinely, and detected of anti-serum with indirect ELISA methods, separated from homologous murine macrophages as feeder cells, which were tiled in96-well culture plates; separated of syngeneic mice spleen cells, using cell fusion of spleen cells of immunized animals with SP2/0myeloma cells fused into hybridoma cell culture with HAT medium; indirected ELISA method to filter out specific secretion of anti-Syk (L)antibody positive clone; limited dilution method of positive hybridoma cells were cloned three times culture; these which were verified multipletimes can secrete pre-antibody positive clones were inoculated into homologous BALB/c mice intra-abdominal collection of ascites further purified, and prepared a large number of monoclonal antibodies; immunohistochemistry was used to detect the Syk protein expression positive anddeficient cells (breast cancer cell line MCF-7, gastric adenocarcinoma cell line MGC-803positive expression standards; breast cancer MDA-MB-231cells were negativestandard), to verify the accuracy of the preparation of monoclonal antibody in this room, and agility.2Using immunohistochemical staining method to detect lung cancertissues:Detected the expression of Syk (L) protein in60cases of differentpathological types of lung cancer tissues and adjacent normal tissues wh ich were initial treatment without preoperative radiotherapy and chemotherapy by immunohistochemistry staining method; compared with the differences of Syk (L) protein expression, and explored the relationship between Syk (L) protein and the clinical features.Results:1The titer of anti-Syk antibody was1:10000by ELISA methods,which is of high sensitivity and specificity.2The expression of Syk (L) is masculine not only in lung cancertissues but also in normal lung tissues,and the positive expression rate in lung cancer (53.3%) lower than the normal lung tissues (61.7%), which result is statistically significant.Syk polyclonal antibody which was prepared in the previous studyhas also been equally meaningful conclusions, but there are no significant differences in the expression of normal lung tissue with the two antibodies.3The expression of Syk (L) in lung cancer tissues is different: cytoplasm\nucleus masculine, cytoplasm masculine\nucleus negative, cytoplasm\nucleus negative.4The relationship between the Syk (L) protein and the pathological type of lung cancer tissues:The positive expression of Syk (L) protein in non-small cell lung cancer (including adenocarcinoma and squamous cell carcinoma) is71.1%,higher than25%of small cell lung cancer, the results statistics significance (P <0.05).The positive expression rate of Syk (L) in adenocarcinoma was67.8%lower than75%of squamous cell carcinomas, two pathological types have different, but the difference was not statistically significant (P>0.05).5The relationship between the Syk (L) protein and clinical features of lung cancer: The expression of the Syk(L) protein is related with pathological types of lung cancer and clinical TNM stage (P<0.05),which is no related with tumor size and lymph node metastasis (P＞0.05).Conclusion:1Anti-Syk(L) monoclonal antibody with high-purity, high-sensitivity,high specificity has been prepared successfully, which titer was greaterthan1:10000, and which provides a basis for the detection of Syk (L)protein and further research.2The rate and expression level of Syk (L) protein in lung cancer tissues was below the normal lung tissues, indicating that Syk (L) is associated with lung cancer, and may be one of tumor suppressor factors about invasion and metastasis of malignant tumor.3The expression of Syk (L) in lung cancer tissues was different: cytoplasm\nucleus masculine, cytoplasm masculine\nucleus negative, cytoplasm\nucleus negative.Putative tumor suppressor gene silencing or downregulation of themechanism, and Syk (L) gene promoter methylation, demethylation drugs may make application of reprogramming of gene expression, inhibitionof tumor cell proliferation, which maybe provide a new idea for lungcancer targeted therapy.4Syk (L) protein expression of different pathological types of lungcancer, squamous cell carcinoma, adenocarcinoma, small cell lung cancer, followed by decline; the expression rate in small cell lung cancer islower than which is in non-small cell lung cancer (including lung adenocarcinoma and squamous cell carcinoma), consistent with the clinical features of small cell lung cancer the higher degree of malignancy, the worse prognosis; differentially expressed in lung squamous cell carcinomaand adenocarcinoma, but not statistically significant, which need to expand the number of cases and combine with other molecular markers tofurther validate.5The expression of the Syk(L) protein is related with pathological types of lung cancer and clinical TNM stage,which is no related with tumor size and lymph node metastasis, but it still needs further lymph node lesion of the subgroup analysisIn summary, this experiment was successfully prepared the anti-Syk(L) monoclonal antibody, and to verify the different pathological typesof lung cancer, and to provide a theoretical basis for the study of Syk(L) as a tumor suppressor gene, in the early diagnosis of lung cancer, pathological type,determine disease staging, prognosis and clinical evaluation, targeted therapy for lung cancer of new tumor markers. |
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