Association between malignant pleural mesothelioma and oncogenic simian virus SV40 in crocidolite contaminated area of Dayao County, Yunnan Province
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摘要:
背景 我国云南省楚雄彝族自治州大姚县 5% 的地表散布着蓝色的青石棉，是恶性胸膜间皮瘤（malignant pleural mesothelioma, MPM）的高发区。致瘤性猿猴病毒SV40（simian virus 40, SV40）是一种小型环状双链DNA多瘤病毒，可使人和动物多种组织类型的正常细胞发生恶性转化并促进肿瘤生长。早年的流行病学调查显示，大姚县青石棉污染区恶性间皮瘤发病率高出一般人群几十倍，该地区MPM的发生是否与致瘤性SV40相关尚未见报道。 目的 本研究旨在探讨致瘤性猿猴病毒SV40是否与云南省大姚县青石棉污染区MPM的发生相关。 方法 收集大理大学第四附属医院（楚雄彝族自治州第一人民医院）和大姚县人民医院胸外科2014年1月-2019年12月诊治的51例（其中40例有石棉暴露史）MPM患者肿瘤组织和12例非MPM患者的胸膜组织（包括肺大疱、肺结核等疾病）。同时，体外培养人正常胸膜间皮细胞LP9、Met5A（SV40转化的间皮细胞）和MPM细胞系NCI-H28（上皮型）、NCI-H2052（肉瘤型）、NCI-H2452（双相混合型）。提取各组细胞和组织基因组DNA后，用3组低污染风险引物（SVINT、SVfor2和SVTA1）对SV40 大T抗原（TAg） 的基因片段分别进行聚合酶链式反应（PCR）扩增检测，用2种SV40 TAg相关抗体（PAb101和PAb416）分别进行Western blotting和免疫组织化学染色，检测MPM肿瘤组织和MPM细胞系中是否存在SV40 TAg蛋白表达。 结果 PCR、Western blotting和免疫组化染色结果均显示Met5A细胞系中SV40为阳性，含有SV40 TAg基因和蛋白质。而各种MPM细胞系NCI-H28、NCI-H2052和NCI-H2452中SV40均为阴性。在12例非MPM组织和51例MPM组织中，3组低污染风险引物的PCR反应均为阴性。在12例非MPM组织和51例MPM组织中，2种抗体的免疫组化染色均未检测出SV40 TAg。 结论 云南省青石棉污染区MPM的发生与SV40病毒感染的关系可能不密切，青石棉暴露可能才是导致MPM发生的主要原因。 -
关键词:
- 恶性胸膜间皮瘤(MPM) /
- 青石棉 /
- 猿猴病毒SV40 /
- 聚合酶链式反应 /
- 免疫组织化学
Abstract:Background In Dayao County, Chuxiong Yi Autonomous Prefecture, Yunnan Province, China, 5% of the surface is scattered with blue asbestos, which is a high incidence area of malignant pleural mesothelioma (MPM). Simian virus 40 (SV40) is a small circular double-stranded DNA polyomavirus that can cause malignant transformation of normal cells of various human and animal tissue types and promote tumor growth. Early epidemiological surveys show that the incidence of malignant mesothelioma in crocidolite contaminated areas of Dayao County is dozens of times higher than that of the general population. Objective To investigate whether the oncogenic SV40 is associated with the occurrence of MPM in the crocidolite contaminated area of Dayao County, Yunnan Province. Methods Tumor tissues from 51 patients with MPM (40 of whom had a history of asbestos exposure) and pleural tissues from 12 non-MPM patients (including diseases such as pulmonary maculopathy and pulmonary tuberculosis) treated from January 2014 to December 2019 at the Department of Thoracic Surgery of the Fourth Affiliated Hospital of Dali University (the First People's Hospital of Chuxiong Yi Autonomous Prefecture) and People's Hospital of Dayao County were collected. Meanwhile, human normal pleural mesothelial cell line Met5A (SV40-transformed mesothelial cells) and MPM cell lines NCI-H28 (epithelial-like type), NCI-H2052 (sarcoma-like type) and NCI-H2452 (biphasic mixed type) were cultured in vitro. After extraction of genomic DNA from each group of cells and tissues, the gene fragments of SV40 large T antigen (TAg) were amplified by polymerase chain reaction (PCR) with three sets of low contamination risk primers (SVINT, SVfor2 and SVTA1). In addition, the presence of SV40 TAg in MPM tumour tissue and MPM cell lines was detected by Western blotting and immunohistochemical staining with 2 SV40-related antibodies (Pab l01 and PAb416), respectively. Results PCR, Western blotting and immunohistochemical staining results showed that the Met5A cell line was positive for SV40 and contained the SV40 TAg gene and protein. In contrast, various MPM cell lines NCI-H28, NCI-H2052 and NCI-H2452 were negative for SV40. PCR reactions were negative for all three sets of low contamination risk primers in 12 non-MPM tissues and 51 MPM tissues. SV40 TAg was not detected in 12 non-MPM tissues and 51 MPM tissues by immunohistochemical staining. Conclusion The occurrence of MPM in crocidolite contaminated areas in Yunnan Province may not be closely related to SV40 virus infection, and crocidolite exposure may be the main cause of MPM. -
图 1 SV40 TAg引物组和抗体的位置
黑色框对应SV40 TAg四个区域的DNA。SV40基因组的编号方向与TAg的转录方向相反。本研究中使用的3对低污染风险的特异性引物已用箭头标示。
Figure 1. Location of SV40 Tag primer sets and antibodies schematic representation of the SV40 genomic DNA encoding TAg.
图 2 MPM组织和非MPM胸膜间皮组织的HE染色结果（200 × ）
A：非MPM胸膜间皮组织；B：上皮型MPM；C：肉瘤型MPM；D：双相混合型MPM；标尺：100 μm
Figure 2. HE staining results of MPM tissue and non-MPM pleural mesothelial tissue (200 × ).
图 3 3对低污染风险PCR引物的扩增特异性验证
Figure 3. 3 validation of amplification specificity for low contamination risk PCR primers
图 4 PCR检测MPM组织中SV40的表达情况
M：DNA Marker；1-10：实验组（分组分别对2例非MPM组织和51例MPM组织进行检测）；11：无DNA阴性对照组；12：293T细胞 DNA 阳性对照组
Figure 4. PCR detection of SV40 expression in MPM tissues
图 5 Western blotting检测正常胸膜间皮细胞系和MPM细胞系中SV40 TAg蛋白的表达
Figure 5. Western blotting detection of SV40 TAg protein expression in normal pleural mesothelial cell lines and MPM cell lines
图 6 免疫组织化学检测正常胸膜间皮细胞和MPM组织中SV40 TAg表达情况(400 × )
A：LP9细胞；B：Met5A细胞；C：MPM样本1；D： MPM样本2；标尺：50 μm
Figure 6. Immunohistochemical detection of SV40 TAg expression in MPM tissues and MPM cell lines
表 1 引物序列
Table 1. Primer sequences
引物 序列(5’-3’) 碱基数 Tm值 PCR片段长度 SVINT. for AAGTAAGGTTCCTTCACAAAG 21 51.71℃ 190 SVINT. rev AACTGAGGTATTTGCTTCTTC 21 51.71℃ SV.for2 CTTTGGAGGCTTCTGGGATGCAACT 25 61.22℃ 574 SV.rev GCATGACTCAAAAAACTTAGCAATTCTG 28 56.69℃ TA1.for GACCTGTGGCTGAGTTTGCTCA 22 59.54℃ 255 TA2.rev GGGACTGTGAATCAATGCCTGT 22 57.67℃ β-actin.for ACTGTCGAGTCGCGTCC 17 56.57℃ 227 β-actin.rev CTGACCCATTCCCACCATCA 20 55.48℃ 
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表 2 PCR反应条件
Table 2. PCR reaction conditions
SVINT.for /SVINT.rev SV.for2/SV.rev TA1.for/TA2.rev 温度 时间 温度 时间 温度 时间 预变性 94℃ 3 min 95℃ 10 min 95℃ 7 min 循环次数 50 50 50 变性 94℃ 60sec 94℃ 30sec 95℃ 30sec 退火 58℃ 60sec 58℃ 30sec 58℃ 30sec 延伸 72℃ 60sec 72℃ 60sec 72℃ 30sec
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表 3 12例非MPM组和51例MPM组患者一般资料和临床病理资料
Table 3. General data and clinicopathological data of 12 non-MPM patients and 51 MPM patients
临床指标 非MPM组（12例） MPM组（51例） 数量/例 构成比/% 数量/例 构成比/% 年龄 <50 4 33.33 6 11.76 ≥50 8 66.67 45 88.24 性别 男 6 50.00 38 74.51 女 6 50.00 13 25.49 职业 有 10 83.33 43 84.31 无 2 16.67 8 15.69 石棉接触 是 1 0.83 40 78.43 否 11 91.67 11 21.57 吸烟 是 5 41.67 22 43.14 否 7 58.33 29 56.86 病理类型 上皮型 - - 44 86.27 肉瘤型 - - 3 5.88 双相混合型 - - 4 7.84 发病部位 - 左侧 - - 22 43.14 右侧 - - 26 50.98 双侧 - - 3 5.88 临床分期 I - - 20 39.22 Ⅱ - - 10 19.61 Ⅲ - - 15 29.41 Ⅳ - - 6 11.76
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表 4 PCR 和免疫组织化学检测各种MPM细胞中TAg表达结果
Table 4. Results of PCR and immunohistochemistry assays
细胞 病毒基因组 DNA质控(β-actin) SV.for2/SV.rev SVINT. for/SV INT. rev TA1/TA2 免疫组织化学(PAb101/PAb416) HEK293 无 + - - - -/- HEK293JCT JC病毒TAg + - - - -/ + HEK293T SV40 TAg + + + + + / + LP9 无 + - - - -/- Met5A SV40 TAg + + + + + / + NCI-H2052 无 + - - - -/- NCI-H2452 无 + - - - -/- NCI-H28 无 + - - - -/-
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