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IntroductionLung cancer is a common malignancy, current domestic and international morbidity and mortality of lung cancer increased year by year. Despite the combination of surgery, chemotherapy, radiotherapy and biological therapy and other means, but the prognosis is not improved, in-depth study molecular biological characteristics of lung cancer early diagnosis of lung cancer, treatment and improving the prognosis. Metallothionein (metallothionein, MT) participate in many pathological processes, including metal ion homeostasis and detoxification, protection of oxidative damage, cell proliferation and apoptosis, drug and radiotherapy resistance and certain carcinogenic process. In different human tumor, such as breast cancer, kidney cancer, nasopharyngeal cancer, ovarian cancer, testicular cancer, thyroid cancer and bladder cancer, and so expression, but in some tumors, such as hepatocellular carcinoma (HCC), prostate cancer and colorectal cancer downregulated, conclusions are not the same. MT expression in lung cancer at present is rarely reported. Our immunohistochemical method to detect MT in squamous cell carcinoma, adenocarcinoma, and its relationship with the relationship between clinicopathological factors of lung cancer.Materials and methods1、The source of specimen(1) Collection of the First Affiliated Hospital of China Medical University, Department of Pathology from January 1999 to March 2009 surgical specimens, including 66 cases of primary tumor and corresponding normal lung tissue samples, of which 42 patients with paired the lymph nodes. All cases no preoperative radiotherapy, chemotherapy, and clinical information. Specimens were fixed in 10% formalin. Embedded in paraffin after conventional treatment. By the International Union Against Cancer (UICC) 1999 Amendment pTNM staging of lung cancer is divided into 26 cases:1-2,3-4 40. Aged between 34-82 years, male 37 cases,29 females, median age 58 years, mean age 55 years.24 cases of squamous cell carcinoma (high grade squamous cell carcinoma in 20 cases,4 cases of poorly differentiated squamous cell carcinoma), adenocarcinoma 42 cases (38 cases of high differentiated adenocarcinoma, poorly differentiated adenocarcinoma in 4 cases). Adjacent normal lung tissue from the same patient more than 5cm from the edge of tumor normal lung tissue, a total of 15 cases.(2) Two types of lung cancer cell lines were:lung adenocarcinoma cell line A549, lung giant cell carcinoma cell line BE1.2、ReagentsReagents MT rabbit anti-human monoclonal antibody (FL-61, sc-11377) purchased the company from three towers, sp kit (kit-9701) were purchased from Fuzhou, the taking of new bio-technology Development Co., Ltd..3、Experimental Method(1) Cell cultureLung adenocarcinoma cell line A549 with DAAM culture medium, BE1 cell line containing 10% fetal calf serum culture medium, after 1640, with 0.5% Co2 at 37℃hatched box of culture,0.25% trypsin, passage.(2) Immunohistochemistry (s-p) methodAction major steps:slice conventional dewaxing, hydration,3% hydrogen peroxide to remove endogenous peroxidase, high temperature high pressure hot fix, repair citrate antigen retrieval solution 2min,5×3 times distilled water, PBS wash 5×3 times, non-immune serum at room temperature incubation 10min, to close non-specific sites. Inclined to add an anti-serum (1:100),4℃in refrigerator overnight incubation, distilled water, PBS washed 5×3 times, plus biotin-labeled secondantibody and incubated at room temperature 20min, PBSwashed 5×3 times, plus the broad-spectrum kit with type C solution, incubated at room temperature 20min. Distilled water, PBS washed 5×3 times, plus the use of broad-spectrum kit type D liquid at room temperature for 25min. Distilled water, PBS washed 5×3 times, DAB color. Hematoxylin, gradient alcohol dehydration, xylene, neutral gum Fengpian. With PBS instead of primary antibody as negative control.Evaluation criteria results in lung cancer cells of brown particles appeared positive, score, combined with the percentage of positive cells and staining intensity of positive cells in both score and assessment standards. MT expression according to the percentage of positive cells and staining determined the extent of semi-quantitative results:no positive cell marked 0, the percentage of positive cells≤50% recorded 1 min,51% to 75% of the recorded 2 points,≥75% note 3; cell pulp and/or the nucleus of the basic non-colored marked 0, pale-brown staining recorded 1 minute, stained brown, marked 2,3 stain brown in mind. Percentage of positive cells per slice staining score for two scores and adding a final score. Total score of 0-1 points recorded as negative (a),2-3 recorded as weakly positive (+),4-5 recorded as positive (++), more than 6 points recorded as strongly positive (+++); to lower negative to weakly positive expression, positive for the expression of positive Xeon.(3) lung cancer cell lines immunohistochemical staining (sp) lawA bottle of good cell growth state selected until the cells adhered to the wall and stretched out pseudopodia, restore normal shape drained medium, making cells climbing film.5×3 times PBS wash, add fixative fixed for 20 minutes.5×3 times PBS wash, add 0.2% Triton-100 Punch,37℃incubation of 15 min, PBS wash after 5×3 times by adding A solution, incubated at room temperature 20 minutes, PBS washed after 5×3 times by adding B solution, incubated at room temperature 20 minutes, tilting to the serum without washing, add diluted primary antibody (1:100),4℃refrigerator overnight, PBS, after washing 5×3 times by adding C solution, incubated at room temperature 15 minutes, PBS washed 5×3 times. Join the D liquid were incubated at room temperature 15 minutes, PBS washed 5×3 times. DAB color (brown color reaction product on behalf of antigen localization), hematoxylin,gradient alcohol dehydration, xylene, neutral gum Fengpian. With PBS instead of primary antibody as negative control.(4) Statistical analysisThe statistical software SPSS 13.0 was applied for data analysis. T-test were used while P<0.05 was significative in statistic.Results1、MT in the lung and adjacent normal tissuesSp method using immunohistochemical staining 66 cases of squamous cell carcinoma, adenocarcinoma of MT expression. Lung cancer and adjacent normal tissues can be seen only see lumen bronchial epithelial cells express membrane MT (2 /15,13%), negative expression in alveolar epithelial cells; MT in the cytoplasm of lung cancer,54 were positive (54/66,81.81%), increased light yellow to brown granular matter, in which the expression of 21 cases of squamous cell carcinoma (see Figure A) (21/24,87.5%).33 cases of adenocarcinoma of the expression (see Figure B), (33/42,78.57%),.2、Metallothionein gene expression and clinical pathologic parameters (Table 1)In 66 cases of human lung squamous cell carcinoma, adenocarcinoma of the expression of MT lineχ2 test. The results showed that MT expression with age (p= 0.479), gender (p=0.079), histological (p=1.002), histological type (p=0.366) correlated with staging (p=0.028),3,4 The positive expression rate of lung cancer was sign-ificantly higher than 1,2 positive rate of lung cancer. MT expression in lung cancer rates of lung cancer tissues was significantly higher than the positive expression rate P=0.001.3、MT in the lung cancer and lymph node metastasis (Table 2)In 42 patients with matched lymph node metastasis, the primary cancer positive MT expression was 75.7%(28/37), the expression of metastasis was 100 MT%(5/ 5), MT expression was correlated with lymph node metastasis (p=0.002), primary tumors and metastases compared, MT positive intensity in the lung did not change significantly.4、MT in the lung cancer cell line A549, BE1 expressionSp method using immunohistochemical staining MT in the lung cancer cell line A549, BE1 expression, the expression of both the cytoplasm.ConclusionMT in squamous cell carcinoma, adenocarcinoma with high expression, and with squamous cell carcinoma, adenocarcinoma of the TNM stage and lymph node metastasis.
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