金属硫蛋白在肺鳞癌、腺癌中的表达及其临床意义
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摘要
目的
肺癌是常见的恶性肿瘤,目前国内外肺癌的发病率和死亡率呈逐年上升趋势。尽管已经联合应用手术、化疗、放疗和生物治疗等各种手段,但其预后并无明显改善,深入研究肺癌分子生物学特征对肺癌的早期诊断、治疗及改善预后具有重要意义。金属硫蛋白(metallothionein, MT)参与许多病理过程,包括金属离子稳态和解毒、保护氧化损伤、细胞增殖和凋亡、药物和放射治疗耐受和致癌的某些过程。在人体多种肿瘤,如乳腺癌、肾癌、鼻咽癌、卵巢癌、睾丸癌、甲状腺癌及膀胱癌等表达增加,但在某些肿瘤,如肝细胞癌(HCC),前列腺癌和大肠癌表达下调,结论不尽一致。MT在肺癌组织中的表达目前研究报道较少。我们应用免疫组化方法来检测MT在肺鳞癌、腺癌的表达,并探讨其与肺癌临床病理因素间关系。
材料与方法
1、标本来源
(1)石蜡切片收集中国医科大学第一附属医院病理科1999年1月至2009年3月手术切除的标本,包括66例原发灶及相应正常肺组织标本,其中42例有配对的淋巴结转移癌。所有病例术前均未行放疗、化疗,并且临床资料完整。标本均经10%福尔马林固定。常规处理后石蜡包埋。按国际抗癌联盟(UICC)1999年修订的肺癌pTNM分期标准分为：1-2期26例,3-4期40例。年龄在34岁-82岁之间,男性37例,女性29例,中位年龄58岁,平均年龄55岁。肺鳞癌24例(高中分化鳞癌20例,低分化鳞癌4例),肺腺癌42例(高中分化腺癌38例,低分化腺癌4例)。癌旁正常肺组织取自同一患者距肿块边缘5cm以上的正常肺组织,共15例。
(2)肺癌细胞系二种分别为：肺腺癌细胞系A549、肺巨细胞癌细胞系BE1。
2、试剂
实验试剂兔抗人MT单克隆抗体(FL-61,sc-11377)购自三塔公司,s-p试剂盒(kit-9701)购自福州迈新生物技术开发有限公司。
3、实验方法
(1)细胞培养
肺腺癌细胞系A549用DAAM培养基培养、BE1细胞系用含10%的胎牛血清的1640培养基培养后,于含0.5%Co2的37℃孵箱内培养,0.25%胰蛋白酶消化、传代。
(2)免疫组织化学(s-p)法
操作主要步骤：切片常规脱蜡、水化,3%过氧化氢去除内源性过氧化物酶,高温高压热修复,柠檬酸盐抗原修复液修复2min,蒸馏水冲洗5＇×3遍、PBS冲洗5＇×3遍,非免疫动物血清室温孵育10min,以封闭非特异性位点。倾去血清加一抗(1：100),4℃于冰箱孵育过夜,蒸馏水冲洗、PBS冲洗5’×3遍,加生物素标记的二抗,室温孵育20min,PBS冲洗5＇×3遍,加即用型广谱试剂盒C液,室温孵育20min。蒸馏水冲洗、PBS冲洗5’×3遍,加即用型广谱试剂盒D液,室温25min。蒸馏水冲洗、PBS冲洗5＇×3遍,DAB显色。苏木素复染,梯度酒精脱水,二甲苯透明,中性树胶封片。用PBS代替一抗作为阴性对照。
结果评定标准以肺癌细胞胞浆内出现棕黄色颗粒为阳性表达,评分参考文献,结合阳性细胞百分比及阳性细胞染色强弱两个方面评分结果评定标准。MT阳性表达根据阳性细胞百分率和染色程度进行半定量结果判定：无阳性细胞记0分,阳性细胞百分率≤50%记1分,51%一75%记2分,≥75%记3分；胞浆和／或胞核基本不着色记0分,染色棕黄记1分,染色棕色记2分,染色褐色记3分。每张切片的阳性细胞百分率得分和染色程度得分两项相加作为最后得分。总分为0-1分记为阴性(一),2-3分记为弱阳性(+),4-5分记为阳性(++),6分以上记为强阳性(+++)；以阴性至弱阳性为低表达,阳性至强阳性为高表达。
(3)肺癌细胞系的免疫化学染色(s-p)法：
选择生长状态好的细胞一瓶,待细胞贴壁生长且伸开伪足,形态恢复正常时倒掉培养基,制作细胞爬片。PBS洗5＇×3遍后,加入固定液固定20分钟。PBS洗5’×3遍后,加入0.2%的Triton-100打孔,370C孵育15分,PBS洗5＇×3遍后加入A液,室温孵育20分,PBS洗5’×3遍后加入B液,室温孵育20分,倾去血清不必冲洗,加稀释的一抗(1：100),4℃冰箱过夜,PBS洗5’×3遍后加入C液,室温孵育15分,PBS洗5＇×3遍。加入D液,室温孵育15分,PBS洗5＇×3遍。DAB显色(棕褐色反应产物代表抗原定位),苏木素复染,梯度酒精脱水,二甲苯透明,中性树胶封片。用PBS代替一抗作为阴性对照。
结果
1、MT在肺癌、癌旁正常组织的表达
用s-p法进行免疫组织化学染色检测66例鳞癌、腺癌MT的表达。可见肺癌旁正常组织仅见支气管上皮细胞腔膜面MT表达(2/15,13%),肺泡上皮细胞阴性表达；MT在肺癌胞浆中54例阳性表达(54/66,81.81%),出现浅黄色至棕褐色颗粒样物,其中鳞癌21例表达(见图A) (21/24,87.5%)。腺癌33例表达(见图B),(33/42,78.57%)。
2、金属硫蛋白基因表达与临床病理参数的关系(见表1)
对66例人肺鳞癌、腺癌组织中MT的表达情况行X2检验。结果显示MT表达与年龄(p=0.479)、性别(p=0.079)、组织学分化(p=1.002)、组织学类型(p=0.366)无关,与分期有关(p=0.028),3、4期肺癌组织的阳性表达率显著高于1、2期肺癌组织的阳性表达率。MT在肺癌组织的阳性表达率显著高于肺癌旁组织的阳性表达率P=0.001
3、MT在肺癌中的表达与淋巴结转移的关系(表2)
在42例配对淋巴结转移组中,原发癌MT的阳性表达率为75.7%(28/37),转移灶MT的表达率为100%(5/5),MT的表达与淋巴结转移有相关性(p=0.002),原发灶与转移灶相比较,MT在肺癌中的阳性强度没有明显改变。
4、MT在肺癌细胞系A549、BE1的表达
采用s-p法进行免疫组织化学染色检测MT在肺癌细胞系A549、BE1表达,均为胞浆表达。
结论
MT在肺鳞癌、腺癌中高表达,并与肺鳞癌、腺癌的TNM分期、淋巴结转移有关。
Introduction
Lung 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 methods
1、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、Reagents
Reagents 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 culture
Lung 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) method
Action 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 second
antibody 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) law
A 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 analysis
The statistical software SPSS 13.0 was applied for data analysis. T-test were used while P<0.05 was significative in statistic.
Results
1、MT in the lung and adjacent normal tissues
Sp 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 expression
Sp method using immunohistochemical staining MT in the lung cancer cell line A549, BE1 expression, the expression of both the cytoplasm.
Conclusion
MT in squamous cell carcinoma, adenocarcinoma with high expression, and with squamous cell carcinoma, adenocarcinoma of the TNM stage and lymph node metastasis.
肺癌是常见的恶性肿瘤,目前国内外肺癌的发病率和死亡率呈逐年上升趋势。尽管已经联合应用手术、化疗、放疗和生物治疗等各种手段,但其预后并无明显改善,深入研究肺癌分子生物学特征对肺癌的早期诊断、治疗及改善预后具有重要意义。金属硫蛋白(metallothionein, MT)参与许多病理过程,包括金属离子稳态和解毒、保护氧化损伤、细胞增殖和凋亡、药物和放射治疗耐受和致癌的某些过程。在人体多种肿瘤,如乳腺癌、肾癌、鼻咽癌、卵巢癌、睾丸癌、甲状腺癌及膀胱癌等表达增加,但在某些肿瘤,如肝细胞癌(HCC),前列腺癌和大肠癌表达下调,结论不尽一致。MT在肺癌组织中的表达目前研究报道较少。我们应用免疫组化方法来检测MT在肺鳞癌、腺癌的表达,并探讨其与肺癌临床病理因素间关系。
材料与方法
1、标本来源
(1)石蜡切片收集中国医科大学第一附属医院病理科1999年1月至2009年3月手术切除的标本,包括66例原发灶及相应正常肺组织标本,其中42例有配对的淋巴结转移癌。所有病例术前均未行放疗、化疗,并且临床资料完整。标本均经10%福尔马林固定。常规处理后石蜡包埋。按国际抗癌联盟(UICC)1999年修订的肺癌pTNM分期标准分为：1-2期26例,3-4期40例。年龄在34岁-82岁之间,男性37例,女性29例,中位年龄58岁,平均年龄55岁。肺鳞癌24例(高中分化鳞癌20例,低分化鳞癌4例),肺腺癌42例(高中分化腺癌38例,低分化腺癌4例)。癌旁正常肺组织取自同一患者距肿块边缘5cm以上的正常肺组织,共15例。
(2)肺癌细胞系二种分别为：肺腺癌细胞系A549、肺巨细胞癌细胞系BE1。
2、试剂
实验试剂兔抗人MT单克隆抗体(FL-61,sc-11377)购自三塔公司,s-p试剂盒(kit-9701)购自福州迈新生物技术开发有限公司。
3、实验方法
(1)细胞培养
肺腺癌细胞系A549用DAAM培养基培养、BE1细胞系用含10%的胎牛血清的1640培养基培养后,于含0.5%Co2的37℃孵箱内培养,0.25%胰蛋白酶消化、传代。
(2)免疫组织化学(s-p)法
操作主要步骤：切片常规脱蜡、水化,3%过氧化氢去除内源性过氧化物酶,高温高压热修复,柠檬酸盐抗原修复液修复2min,蒸馏水冲洗5＇×3遍、PBS冲洗5＇×3遍,非免疫动物血清室温孵育10min,以封闭非特异性位点。倾去血清加一抗(1：100),4℃于冰箱孵育过夜,蒸馏水冲洗、PBS冲洗5’×3遍,加生物素标记的二抗,室温孵育20min,PBS冲洗5＇×3遍,加即用型广谱试剂盒C液,室温孵育20min。蒸馏水冲洗、PBS冲洗5’×3遍,加即用型广谱试剂盒D液,室温25min。蒸馏水冲洗、PBS冲洗5＇×3遍,DAB显色。苏木素复染,梯度酒精脱水,二甲苯透明,中性树胶封片。用PBS代替一抗作为阴性对照。
结果评定标准以肺癌细胞胞浆内出现棕黄色颗粒为阳性表达,评分参考文献,结合阳性细胞百分比及阳性细胞染色强弱两个方面评分结果评定标准。MT阳性表达根据阳性细胞百分率和染色程度进行半定量结果判定：无阳性细胞记0分,阳性细胞百分率≤50%记1分,51%一75%记2分,≥75%记3分；胞浆和／或胞核基本不着色记0分,染色棕黄记1分,染色棕色记2分,染色褐色记3分。每张切片的阳性细胞百分率得分和染色程度得分两项相加作为最后得分。总分为0-1分记为阴性(一),2-3分记为弱阳性(+),4-5分记为阳性(++),6分以上记为强阳性(+++)；以阴性至弱阳性为低表达,阳性至强阳性为高表达。
(3)肺癌细胞系的免疫化学染色(s-p)法：
选择生长状态好的细胞一瓶,待细胞贴壁生长且伸开伪足,形态恢复正常时倒掉培养基,制作细胞爬片。PBS洗5＇×3遍后,加入固定液固定20分钟。PBS洗5’×3遍后,加入0.2%的Triton-100打孔,370C孵育15分,PBS洗5＇×3遍后加入A液,室温孵育20分,PBS洗5’×3遍后加入B液,室温孵育20分,倾去血清不必冲洗,加稀释的一抗(1：100),4℃冰箱过夜,PBS洗5’×3遍后加入C液,室温孵育15分,PBS洗5＇×3遍。加入D液,室温孵育15分,PBS洗5＇×3遍。DAB显色(棕褐色反应产物代表抗原定位),苏木素复染,梯度酒精脱水,二甲苯透明,中性树胶封片。用PBS代替一抗作为阴性对照。
结果
1、MT在肺癌、癌旁正常组织的表达
用s-p法进行免疫组织化学染色检测66例鳞癌、腺癌MT的表达。可见肺癌旁正常组织仅见支气管上皮细胞腔膜面MT表达(2/15,13%),肺泡上皮细胞阴性表达；MT在肺癌胞浆中54例阳性表达(54/66,81.81%),出现浅黄色至棕褐色颗粒样物,其中鳞癌21例表达(见图A) (21/24,87.5%)。腺癌33例表达(见图B),(33/42,78.57%)。
2、金属硫蛋白基因表达与临床病理参数的关系(见表1)
对66例人肺鳞癌、腺癌组织中MT的表达情况行X2检验。结果显示MT表达与年龄(p=0.479)、性别(p=0.079)、组织学分化(p=1.002)、组织学类型(p=0.366)无关,与分期有关(p=0.028),3、4期肺癌组织的阳性表达率显著高于1、2期肺癌组织的阳性表达率。MT在肺癌组织的阳性表达率显著高于肺癌旁组织的阳性表达率P=0.001
3、MT在肺癌中的表达与淋巴结转移的关系(表2)
在42例配对淋巴结转移组中,原发癌MT的阳性表达率为75.7%(28/37),转移灶MT的表达率为100%(5/5),MT的表达与淋巴结转移有相关性(p=0.002),原发灶与转移灶相比较,MT在肺癌中的阳性强度没有明显改变。
4、MT在肺癌细胞系A549、BE1的表达
采用s-p法进行免疫组织化学染色检测MT在肺癌细胞系A549、BE1表达,均为胞浆表达。
结论
MT在肺鳞癌、腺癌中高表达,并与肺鳞癌、腺癌的TNM分期、淋巴结转移有关。
Introduction
Lung 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 methods
1、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、Reagents
Reagents 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 culture
Lung 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) method
Action 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 second
antibody 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) law
A 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 analysis
The statistical software SPSS 13.0 was applied for data analysis. T-test were used while P<0.05 was significative in statistic.
Results
1、MT in the lung and adjacent normal tissues
Sp 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 expression
Sp method using immunohistochemical staining MT in the lung cancer cell line A549, BE1 expression, the expression of both the cytoplasm.
Conclusion
MT in squamous cell carcinoma, adenocarcinoma with high expression, and with squamous cell carcinoma, adenocarcinoma of the TNM stage and lymph node metastasis.
引文
1 Soslow RA, Dannenberg AJ, Rush D, et al. COX-2 is expressed in human pulmonary, colonic, and mammary tumors. Cancer.2000.89(12):37-45.
2 West A K,Stallings R,Hildebrand C E,etal.Human metallothionein genes:structure of the functional locus at 16ql3[J].Genomics.1990.8(3):513-518.
3 Floriczyk B, Grzybowska L. Metallothionein in levels in cell fractions from breast cancer tissues[J].Acta Oncol.2000.39(2):141-143.
4 Cardoso SV, et al.Expression of metallothionein and p53 antigens are correlated in oral squamous cell carcinoma. Anticancer Res.2009 (4):1189-93.
5 Pontes HA, et al. Metallothionein and p-Akt proteins in oral dysplasia and in oral squamous cellcarcinoma:an immunohistochemical study. J Oral Pathol Med.2009.38(8):644-50.
6 Schmitz KJ, Lang H, Kaiser G, et al. Metallothionein overexpression and its prognostic relevance in intrahepaticcholangiocarcinoma and extrahepatic hilar cholangiocarcinoma (Klatskin tumors). Hum Pathol.2009.40(12):1706-14.
7 Sliwinska-Mosson M, Milnerowicz H, Rabczynski J,etal. Immunohistochemical localization of metallothionein and p53 protein in pancreaticserous cystadenomas.Arch Immunol Ther Exp (Warsz).2009.57(4):295-301.
8 Jasani B. Schmid KW. Significance of metallothionein overexpression in human tumors Histopathology.1997.31:211.
9 Oyama T, Take H, Hikino T, et al. lmmunohistochemical expression of metallothionein in invasive breast cancer in relation to proliferative activity, histology and prognosis. Oncology 1996.53:112.
10 Haerselv T, Jacobsen GK. Zedeler K.The prognosticsignificance of immunohistochemically detectable metallothionein in primary breast carcinemas. APMIS.1995.103:279.
11 Fresno M, Wu W, Rodriguez JM, et al. Localization of metallothinein in breast carcinomas, an immunohistochemical study. Virchows Archir Pathol Anat.1993.34:151.
12 Mccluggage WG,Maxwell P,Hamilton PW, etal. High metallthionein expression is associated with features predictive of aggressive behavior in endemetrial Carcinoma histopathology.1999.34:51-55.
13 Hishikawa Y, Kohno H, Ueda S, etal.Expression of metallothionein in colorectal cancers and synchronous liver metastases. Oncology.2001.61(2):162-7.
1 Romeroy Isart N, Vasak M. Advances in the structure anchemistry of metallothioneins [J].JinorgBiochem.2002.88(3/4):388-396.
2 Ron D, Chen CH, Caldwell J, Jamieson L, Orr E, Mochly-RosenD (1994) Cloning of an intracellular receptor for protein kinaseC:a homolog of the beta subunit of G proteins. Proc Natl AcadSci USA.1994.91:839-843.
3 Rupp H. Bioehem Biophys Acta.1978.533:209-226.
4 Nielson K B, J boil Chem.1985.260:5342-5350.
5 Andrews G K. Regulation of metallothionein gene expressionby oxidative stress and metalions[J]. Biochem Pharmacol.2000.59(1):95-104.
6 闰海亮,张晶.丁洪浩,等.金属硫蛋白及其诱导、分离纯化研究进展[J].饲斟工业.2007.28(24)：52-54.
7 Sehmidt C, Beyersmann D. Transient peaks in zinc and metal-liothionein levels during differentiation of 3TaL1 cells[J]. Arch Biochem Biophys.1999.364:91-98.
8 Coyle P, Philcox J C, Rofe A M. Metallothionein null mice absorbless Zn from an egg white diet, but asimilar amount fromsolutions, although site altered intertissue Zn distr ution[J]. J Nutr.1999.129:372-379.
9 Conrad C C, Wa[ter C A, Richardson A, et al. Cadmium toxicity and distribution in metallothionein-I and 11 deficient transgenic mice[J]. J Toxicol Environ Health.1997.52: 527-543.
10 Liu Y, Liu J, Habeebu S S, et al. Susceptibility of MT null mice to chronic CdCI2-induced nephrotoxicity indicates that renal injury is not mediated by the CdMT complexf.ToxicolSci.1998.46:197-203.
11 Berns H, Humar R, Hengerer B, Kiefer FN, Battegay EJ (2000)RACK1 is up-regulated in angiogenesis and human carcinomas.FASEB.2000.14:2549-2558.
12 王婷,郑玉建,吴顺华,等.锌、硒对砷诱导金属硫蛋白合成影响的研究[J].新疆医科大学学报.2008.37(7)：799-801.
13 Han HJ, Russo J, Kohwi Y, Kohwi-Shigematsu T (2008) SATBlreprogrammes gene expression to promote breast tumor growthand metastasis. Natur.2008.e 452:187-193.
14 Alonso ME, Bello Mj, Gonzalez-Gomez P et al. Aberrant promot-er methylation of multiple genes in oligedendrogliomas and ependymomas[J]. Cancer Genet Cytogenet.2003.
144(2):134-142.
15 RiichirohM, Shinichi T, Kiyomi OT et al. Aberrant promotermethylation profile of prostate cancers and its relationship to chili-copathological features[J]. Clin Cancer Res.2002.8(2): 514-519.
16 安萍,于波,李世拥,等.组织蛋白酶B与金属硫蛋白在结直肠癌中的表达及其临床意义[J].解放军医学杂志.20083.3(5)：498-499。
17 Alonso ME, Bello Mj, Gonzalez-Gomez P et al. Aberrant promoter methylation of multiple genes in oligedendrogliomas and ependymomas[J]. Cancer Genet Cogenet.2003.14 (2): 134-142.
18 Bo Ai hua, Lu Guang ping, Zhang Xiaoli, et al. Clinical Significations on the Expressions of MetallOthionein in ThreeTypes Cancer of Woman. Chinese-German J Clin Oncol.2003.7(1): 59-61.
19 兰永昊,薄爱华,王晓静,等.金属硫蛋白与P-糖蛋白在乳腺癌中表达的临床意义EJ].中国老年学杂志.2009.29(2)：207--208。
20 Pastuszewski W, Dziegiel P, Krecicki T et al. Prognosticsignificance of metallothionein. p53 protein and Ki-67. antigen expression in laryngeal cancer[J]. Anticancer Res.2007.27(1): 335-342.
21 段杨平,濮德敏,李天,等.金属硫蛋白在子宫内膜癌中的表达及意义.中国妇幼保健.2008.23(17)：2428-2430.
22 Shimoda R, Achanzar WE, Qu W, et al. Metallothionein is apotential negative regulator of apoptosis[J]. Taxicol Sci.2003.73(2):294-300.
23 Mccluggage WG, Stracl K, Abdulkadir A. Immunohistochemical localization of metallothionein in beniyn and malignant epithelial ovarian tumors[J]. Int J Gynecol Cancer.2002.12(1):62-65.
24 Belinsky SA, Palmisano WA, Gillilan d FD et al. Aberrant promotermethylation in bronchial epithelium an d sputum from current and former smokers[J]. Cancer Res,2002.62(8): 2370-2377.
2 West A K,Stallings R,Hildebrand C E,etal.Human metallothionein genes:structure of the functional locus at 16ql3[J].Genomics.1990.8(3):513-518.
3 Floriczyk B, Grzybowska L. Metallothionein in levels in cell fractions from breast cancer tissues[J].Acta Oncol.2000.39(2):141-143.
4 Cardoso SV, et al.Expression of metallothionein and p53 antigens are correlated in oral squamous cell carcinoma. Anticancer Res.2009 (4):1189-93.
5 Pontes HA, et al. Metallothionein and p-Akt proteins in oral dysplasia and in oral squamous cellcarcinoma:an immunohistochemical study. J Oral Pathol Med.2009.38(8):644-50.
6 Schmitz KJ, Lang H, Kaiser G, et al. Metallothionein overexpression and its prognostic relevance in intrahepaticcholangiocarcinoma and extrahepatic hilar cholangiocarcinoma (Klatskin tumors). Hum Pathol.2009.40(12):1706-14.
7 Sliwinska-Mosson M, Milnerowicz H, Rabczynski J,etal. Immunohistochemical localization of metallothionein and p53 protein in pancreaticserous cystadenomas.Arch Immunol Ther Exp (Warsz).2009.57(4):295-301.
8 Jasani B. Schmid KW. Significance of metallothionein overexpression in human tumors Histopathology.1997.31:211.
9 Oyama T, Take H, Hikino T, et al. lmmunohistochemical expression of metallothionein in invasive breast cancer in relation to proliferative activity, histology and prognosis. Oncology 1996.53:112.
10 Haerselv T, Jacobsen GK. Zedeler K.The prognosticsignificance of immunohistochemically detectable metallothionein in primary breast carcinemas. APMIS.1995.103:279.
11 Fresno M, Wu W, Rodriguez JM, et al. Localization of metallothinein in breast carcinomas, an immunohistochemical study. Virchows Archir Pathol Anat.1993.34:151.
12 Mccluggage WG,Maxwell P,Hamilton PW, etal. High metallthionein expression is associated with features predictive of aggressive behavior in endemetrial Carcinoma histopathology.1999.34:51-55.
13 Hishikawa Y, Kohno H, Ueda S, etal.Expression of metallothionein in colorectal cancers and synchronous liver metastases. Oncology.2001.61(2):162-7.
1 Romeroy Isart N, Vasak M. Advances in the structure anchemistry of metallothioneins [J].JinorgBiochem.2002.88(3/4):388-396.
2 Ron D, Chen CH, Caldwell J, Jamieson L, Orr E, Mochly-RosenD (1994) Cloning of an intracellular receptor for protein kinaseC:a homolog of the beta subunit of G proteins. Proc Natl AcadSci USA.1994.91:839-843.
3 Rupp H. Bioehem Biophys Acta.1978.533:209-226.
4 Nielson K B, J boil Chem.1985.260:5342-5350.
5 Andrews G K. Regulation of metallothionein gene expressionby oxidative stress and metalions[J]. Biochem Pharmacol.2000.59(1):95-104.
6 闰海亮,张晶.丁洪浩,等.金属硫蛋白及其诱导、分离纯化研究进展[J].饲斟工业.2007.28(24)：52-54.
7 Sehmidt C, Beyersmann D. Transient peaks in zinc and metal-liothionein levels during differentiation of 3TaL1 cells[J]. Arch Biochem Biophys.1999.364:91-98.
8 Coyle P, Philcox J C, Rofe A M. Metallothionein null mice absorbless Zn from an egg white diet, but asimilar amount fromsolutions, although site altered intertissue Zn distr ution[J]. J Nutr.1999.129:372-379.
9 Conrad C C, Wa[ter C A, Richardson A, et al. Cadmium toxicity and distribution in metallothionein-I and 11 deficient transgenic mice[J]. J Toxicol Environ Health.1997.52: 527-543.
10 Liu Y, Liu J, Habeebu S S, et al. Susceptibility of MT null mice to chronic CdCI2-induced nephrotoxicity indicates that renal injury is not mediated by the CdMT complexf.ToxicolSci.1998.46:197-203.
11 Berns H, Humar R, Hengerer B, Kiefer FN, Battegay EJ (2000)RACK1 is up-regulated in angiogenesis and human carcinomas.FASEB.2000.14:2549-2558.
12 王婷,郑玉建,吴顺华,等.锌、硒对砷诱导金属硫蛋白合成影响的研究[J].新疆医科大学学报.2008.37(7)：799-801.
13 Han HJ, Russo J, Kohwi Y, Kohwi-Shigematsu T (2008) SATBlreprogrammes gene expression to promote breast tumor growthand metastasis. Natur.2008.e 452:187-193.
14 Alonso ME, Bello Mj, Gonzalez-Gomez P et al. Aberrant promot-er methylation of multiple genes in oligedendrogliomas and ependymomas[J]. Cancer Genet Cytogenet.2003.
144(2):134-142.
15 RiichirohM, Shinichi T, Kiyomi OT et al. Aberrant promotermethylation profile of prostate cancers and its relationship to chili-copathological features[J]. Clin Cancer Res.2002.8(2): 514-519.
16 安萍,于波,李世拥,等.组织蛋白酶B与金属硫蛋白在结直肠癌中的表达及其临床意义[J].解放军医学杂志.20083.3(5)：498-499。
17 Alonso ME, Bello Mj, Gonzalez-Gomez P et al. Aberrant promoter methylation of multiple genes in oligedendrogliomas and ependymomas[J]. Cancer Genet Cogenet.2003.14 (2): 134-142.
18 Bo Ai hua, Lu Guang ping, Zhang Xiaoli, et al. Clinical Significations on the Expressions of MetallOthionein in ThreeTypes Cancer of Woman. Chinese-German J Clin Oncol.2003.7(1): 59-61.
19 兰永昊,薄爱华,王晓静,等.金属硫蛋白与P-糖蛋白在乳腺癌中表达的临床意义EJ].中国老年学杂志.2009.29(2)：207--208。
20 Pastuszewski W, Dziegiel P, Krecicki T et al. Prognosticsignificance of metallothionein. p53 protein and Ki-67. antigen expression in laryngeal cancer[J]. Anticancer Res.2007.27(1): 335-342.
21 段杨平,濮德敏,李天,等.金属硫蛋白在子宫内膜癌中的表达及意义.中国妇幼保健.2008.23(17)：2428-2430.
22 Shimoda R, Achanzar WE, Qu W, et al. Metallothionein is apotential negative regulator of apoptosis[J]. Taxicol Sci.2003.73(2):294-300.
23 Mccluggage WG, Stracl K, Abdulkadir A. Immunohistochemical localization of metallothionein in beniyn and malignant epithelial ovarian tumors[J]. Int J Gynecol Cancer.2002.12(1):62-65.
24 Belinsky SA, Palmisano WA, Gillilan d FD et al. Aberrant promotermethylation in bronchial epithelium an d sputum from current and former smokers[J]. Cancer Res,2002.62(8): 2370-2377.
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