Non-small cell lung cancer with BMP-2 expression and significance of CD105
|Keywords||Non-small cell lung cancer Bone morphogenetic protein-2 Microvessel density Immunohistochemistry Western blotting|
Background and Purpose: Lung cancer is a serious threat to human health diseases, so far, have yet to find a satisfactory treatment, so look for new anti-tumor approach is particularly important. Tumor invasion and metastasis is an important feature, but also difficult to cure cancer, leads to death of the key issues. Malignant tumor growth is angiogenesis, invasion and metastasis foundation. Not only tumor angiogenesis and tumor cell nutrition and oxygen degree, but also reflects the tumor invasion and metastasis, is to determine the tumor size, metastasis, recurrence and an important prognostic factor. Most of including lung cancer growth and metastasis of malignant tumors are angiogenesis-dependent characteristics. Microvessel density (micro vascular density, MVD) is recognized as the most effective tumor angiogenesis reflect objective indicators, has been widely considered to be an important factor in determining tumor prognosis. Transmembrane glycoprotein (CD105) is expressed mainly in proliferating vascular endothelial cells, is the neovascular endothelial cell specific markers. Furthermore CD105 by regulating TGF-β activity, and promote vascular endothelial cell proliferation, differentiation and migration, in angiogenesis. Bone morphogenetic proteins (BMPs) belong (transforming growth factor-β) transforming growth factor-β superfamily of cytokines, is involved in regulating a wide variety of cell proliferation, differentiation and apoptosis of functional protein biological processes , precursor cells can induce migration and differentiation, regulation of cell growth, proliferation, migration, and embryonic stem cell self-renewal. At present, most studies agree that BMP-2 in tumor growth, invasion, migration has a very important role in promoting. Currently on BMP-2 expression in lung cancer research very little domestic has not been reported. BMP-2 and lung cancer is also not very clear. To further explore the BMP-2 and CD105 MVD signs and lung cancer invasion, metastasis and angiogenesis, seek early detection of lung cancer invasion and metastasis inhibition index and look for the invasion and metastasis of lung cancer and effective way. The research method of immunohistochemistry and immunoblotting to detect lung cancer cells in lung tissues of BMP-2 and BMP-2 in CD105 expression of MVD flags while using RT-PCR method for determination of non-small cell lung cancer (NSCLC) in BMP-2 mRNA expression levels, and analyze the expression levels of both the clinical and pathological features of lung cancer between. Method: 1. Immunohistochemical Methods: BMP-2 and CD105 in 50 cases of non-small cell lung cancer and 10 cases of normal tissues circumstances, the choice of cases without any preoperative chemotherapy and radiotherapy. 2 immunoblotting (Western Blot) detected 20 cases of non-small cell lung cancer and 10 cases of normal tissue expression of BMP-2, and the relationship between clinical staging of lung cancer. 3 reverse transcription-PCR (RT-PCR) detection of mRNA. Step extraction using guanidine isothiocyanate total cellular RNA. 1% agarose gel electrophoresis identified its integrity. By RT-PCR: reverse transcribed cDNA, established PCR system, 94 ℃ denaturation for 5min, then 94 ℃ deformation 45s, 52 ℃ annealing 45s, 72 ℃ extension 60s, a total of 30 cycles, 72 ℃ for 10min. 30 cycles of amplification, PCR products on 1.5% agarose gel electrophoresis (90V, 35mi n). UV analyzer observed and photographed. Statistical analysis: All results were analyzed using SPSS13.0 software, immunohistochemical expression of BMP-2 protein analysis and comparison of positive rates X2 test; tumor MVD sign CD105 BMP-2 and its analysis of the relationship between t test, test level α = 0.05; Western Blot results and RT-PCR results of analysis of variance, P lt; 0.05 statistically significant results: 1.BMP-2 in tissues: lung cancer and normal tissues in the group show BMP-2 in non-small cell lung cancer group was 68% positive rate, significantly higher than the normal rate of lung tissues 0% (P lt; 0.05). In non-small cell lung cancer clinical and pathological features group show: Ⅰ Ⅱ, positive rate of 53.6%, lower than the stage Ⅲ - Ⅳ stage, the positive expression rate of 86.4% (P lt; 0.05); BMP-2 protein in poorly The positive expression of lung cancer was 85.0%, significantly higher than the scores of positive expression rate of lung cancer 56.7% (P lt; 0.05); expression with lymph node metastasis was 78.1%, higher than that without lymph node metastasis group, 50%% (P lt; 0.05); however, gender, age and different histological types of expression between groups was not statistically significant difference (P gt; 0.05). 2.MVD sign of CD105 in tissues: NSCLC tissues MVD value of 41.32 ± 4.52, normal lung tissue, MVD value of 30.12 ± 3.21, the difference was statistically significant (P lt; 0.05). In NSCLC, I 11 patients with MVD in the group (35.63 ± 4.31) than patients with stage Ⅲ Ⅳ MVD was 44.80 ± 4.61 (P lt; 0.05); MVD with lymph node metastasis group is 47.31 ± 4.94 higher than those without MVD in lymph node metastasis group 34.61Q4.19 (P lt; 0.05). In moderately differentiated NSCLC group is 38.63 ± 4.8 MVD than MVD in poorly differentiated group 41.27 ± 4.23 (p gt; 0.05); however, gender, age and different tissue types as well as the expression of tumor differentiation was no statistical significance (P gt; 0.05). 3 non-small cell lung cancer signs of MVD BMP-2 expression of CD105 and relationships: BMP-2 positive group MVD values ??(42.46 ± 3.75) than BMP-2 negative group (33.28 ± 3.21), the difference between the two There was statistically significant (P lt; 0.05). 4. Western Blot assay visible BMP-2 in non-small cell lung cancer tissues showed high expression levels in normal tissues is very low. BMP-2 protein relative OD values ??of each group: squamous cell carcinoma group was 1.869 ± 0.032; adenoma carcinoma was 2.278 ± 0.041; normal tissue group was 0.199 ± 0.027. Univariate analysis of variance found that squamous cell carcinoma and adenocarcinoma of the BMP-2 protein levels showed no significant difference (p gt; 0.05), respectively, by squamous cell carcinoma and adenocarcinoma compared with normal tissue group differences were There was statistically significant (P lt; 0.01), test results and immunohistochemical method was consistent (Table 5). 5. RT-PCR assay BMP-2 mRNA in non-small cell lung carcinoma cases. 10 cases of normal lung tissues and 20 cases, respectively, two cases and 17 cases can be detected BMP-2mRNA positive expression rates were 20% and 85%. After χ2 test, normal tissue and non-small cell lung cancer and BMP-2 gene mRNA expression rate was statistically significant (p lt; 0.05). Conclusions: (1) The results showed that BMP-2 mRNA and protein are low in normal lung tissue expression, whereas in non-small cell lung cancer tissues showed high expression; and BMP-2 protein expression levels correlated with the presence or absence of non-small cell lung cancer lymph node metastasis, clinical stage, differentiation degree. BMP-2 protein expression increased with non-small cell lung cancer is closely related to, but its pathological types. Showed that BMP-2 protein and gene expression may be associated with a high in non-small cell lung cancer invasion, metastasis and angiogenesis play a catalytic role in the process. (2) signs of MVD CD105 in non-small cell lung cancer tissues showed high expression in normal lung tissue with low expression; MVD values ??were correlated with non-small cell lung cancer clinical stage and lymph node metastasis, and the patient's age , gender and degree of differentiation of NSCLC, pathological type irrelevant. Tip CD105 possible in non-small cell lung tumor angiogenesis, invasion and metastasis play an intermediary role. (3) BMP-2 and MVD in non-small cell lung cancer showed a positive correlation between the expression of that both in non-small cell lung cancer invasion and metastasis, especially angiogenesis interrelated effects may exist.