| Background The pathogenesis of lung cancer is tissue cells uncontrolled growth, and it’s has the character of morbidity and mortality increased very fast. According to WHO statistics, lung cancer is the leading cause of cancer death worldwide, account to about 19% of all malignant tumors, especially lung adenocarcinoma, when first diagnosis is metastatic and likely to cause the development of thrombosis. The etiology of lung cancer has not entirely been understood. Protein Z(PZ) is a vitamin K-dependent(VKD) glycoprotein that acts as a potent cofactor for the enzyme protein Z-dependent protease inhibitor(ZPI). Together, these proteins mediate thrombin activation and blood coagulation by strongly inhibitingthe activation of Factor Xa(FXa). In fact, the presence of PZ enhances the inhibition of FXa by ZPI by 1000-fold.When PZ deficiency, inhibiting FXa effect decreased, lead to clotting activity increased, thereby increasing the risk of thrombosis.Our previous studies have found that as malignant tumors progress, plasma levels of PZ significantly decrease,whichindicates that PZ may bea factor contributing to poor prognosis in cancer patients. It remains unclear, however, whether circulating concentrations or local tissue PZ expression is primarily responsible forthe downstream increase in thrombotic episodes in patients.Abnormal tissue expression of PZ has been observed in human cancers of the lung, breast, colon, and stomach; interestingly, patients with these types of cancers are also more prone to developing thrombosis, which suggest that PZ may play a role in the anti-clotting intumor tissue, thereby affect tumor angiogenesis, invasion and metastasis. In order to explore the relation between PZ and tumorigenesis, development and metastasis, we choose adenocarcinoma as our model, because adenocarcinoma has the character of early metastasis and thrombosis.Purpose 1.To investigate the expression of PZ and PZ m RNA in lung adenocarcinoma. 2. To obtain experimental evidence of PZ play an important role in the growth and metastasis of lung adenocarcinoma. 3. To explore the role of PZ in the pathogenesis of lung adenocarcinoma, development, and prognosis significance.Methods1. Immunohistochemistry to detect the expression of PZ in normal lung tissue and lung adenocarcinoma.A total of 45 normal lung tissues, 45 lung adenocarcinoma specimens, and 45 lung adenocarcinoma tissue adjacent tissues were collected. Informed consent was obtained from all subjects before the study. Complete statistical analysis, to explore PZ expression in lung adenocarcinoma and its relationship with gender, differentiation and staging.2. Western blot method to detect the expression of PZ in lung adenocarcinoma tissues, lung adenocarcinoma tissue adjacent.The experiment was divided into 2 groups: 45 samples of lung adenocarcinoma and 45 samples of normal lung tissues. After each sample was collected, immersed in liquid nitrogen for 30 minutes, and then put to-70 ℃ refrigerator. After all samples were collected, frozen tissue samples for block cracking and use glass homogenate slurry and overspeed centrifugal extraction tissue protein in low temperature. Protein concentration was determined by BCA method. Accomplished statistical analysis, to further explore PZ expression in lung adenocarcinoma and its relationship with gender, differentiation and staging.3. Western blot to detect the expression of PZ protein in normal bronchial cells and lung adenocarcinoma cells.Cultured normal human bronchial epithelial cells and lung adenocarcinoma cells, take the logarithmic growth phase cells, trypsinized cells, medium and transferred to a centrifuge tube, discard the supernatant after overspeed centrifugal extraction, PBS washing liquid precipitation, RIPA lysis buffer cleavage, after sufficiently cleavage, low temperature ultracentrifugation extracted cellular proteins. Protein concentration was determined by BCA method, to investigate whether PZ overexpression in lung adenocarcinoma cells.4. Using RT-PCR method to detect the expression of PZ m RNA in lungadenocarcinoma tissues and lung adenocarcinoma tissue adjacentThe experiment was divided into 2 groups:5 samples of lung adenocarcinoma and 5 samples of normal lung tissues. Pick the samples from-70 ℃ refrigerator, use glass homogenate slurry and overspeed centrifugal extraction tissue protein in low temperature. Protein concentration was determined by BCA method. Accomplished statistical analysis, to further confirm PZ expression in lung adenocarcinoma and its relationship with differentiation and staging.5. Using RT-PCR method to detect the expression of PZ in lung adenocarcinoma cells and normal human bronchial epithelial cells.The experiment was divided into 2 groups: normal human bronchial epithelial cells and lung adenocarcinoma cells. Cultured normal human bronchial epithelial cells and lung adenocarcinoma cells, take the logarithmic growth phase cells, extract total DNA, reverse transcriptase synthetic c DNA, RT-PCR detected the expression of PZ m RNA in each cell, Accomplished statistical analysis, to investigatethe expression of PZ m RNA in lung adenocarcinoma cells.ResultsPart 1 The expression of PZ in normal lung tissue, lung adenocarcinoma and adjacent normal tissues.1. the expression of PZ in cancerous lung tissue was increased. In contrast, healthy lung tissues showed minimal expression of PZ. Upon close examination, PZ protein appears to be expressed in macrophages and endothelial cells in the newly formed vessels.2. With the stage increased, the expression of PZ in lung adenocarcinoma increased significantly(P<0.05).3. The PZ expression in human lung adenocarcinoma tissues has no difference between female group and male group.Part 2 The PZ expression and clinical data analysisin human lung tissue1. Compared with lung adenocarcinoma tissue adjacent and normal lung tissue, PZ in lung adenocarcinoma tissue is overexpression,the difference was statistically significant(P<0.001). The expression of in lung adenocarcinoma tissue adjacent and normal lung tissue has no significant difference(P>0.05).2. With the stage increased, the expression of PZ in lung adenocarcinoma increased significantly(P<0.05),3. The PZ expression in human lung adenocarcinoma tissues has no difference between female group and male group(P> 0.05).Part3. the expression of PZ protein in normal bronchial cells and lung adenocarcinoma cells.Expression of PZ in A549, GLC-82 cells was significantly higher than that in 16-HBE-T cells, in which A549 is significant, the differences were statistically significant(P<0.0005); Difference of the expression of PZ in212102 cells and 16-HBE-T cellswas not statistically significant(P> 0.05).Part4. the expression of PZ protein in normal lung tissues and lung adenocarcinoma tissues.Due to insufficient samples, the degree of dispersion of data is large, there was nosignificant statistic, the subsequent need to further increase the samples for testingPart 5. The expression of PZ m RNA in normal bronchial cells and lung adenocarcinoma cellsThe expression of PZ m RNA between human normal bronchial epithelial cells and lung adenocarcinoma cells has no significant difference(P>0.05).Conclusions:1.The expression of PZ in lung adenocarcinoma group is significantly higher than normal lung tissue, which suggest that PZ may play an important role with the onset of lung adenocarcinoma.2.With the stage increased, the expression of PZ in lung adenocarcinoma increased significantly, which means that in advanced lung adenocarcinoma, the expression of PZ may be higher.3. The expression of PZ in low differentiation group is visibly higher than high differentiation group, which suggest that its expression may be associated with the differentiation of lung adenocarcinoma, namely the lower the degree of tumor differentiation, the expression of PZ may be higher.4. The PZ expression in human lung adenocarcinoma tissues has no difference between female group and male group, which may indicate that the expression of PZ in lung adenocarcinoma has no correlation with gender.5. 1)PZ protein is overexpression in lung adenocarcinoma tissues and cells.2)We just explore PZ m RNA in 5 lung adenocarcinoma tissues and 5 normal lung tissues, due to insufficient samples, the degree of dispersion of data is large, there was no significant statistic, the subsequent need to further increase the samples for testing.3)The expression of PZ m RNA between human normal bronchial epithelial cells and lung adenocarcinoma cells has no significant difference, this kind of contradiction for unknown reasons. |
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