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目的:探讨人乳头状瘤病毒16E7蛋白(HPV16E7)、细胞周期调节蛋白D1(Cyclin D1)和视网膜母细胞瘤蛋白(pRb)的异常表达对喉癌的影响及其机制。方法:取喉癌组织标本(喉癌组)、喉声带息肉组织标本(息肉组)和喉癌前病变组织标本(癌前组)各69例。采用过氧化物酶标记的链酶卵蛋白素染色免疫组织化学技术和Western Blot技术检测各组喉组织标本中HPV16E7、Cyclin D1和pRb的表达水平。结果:喉癌组HPV16E7和Cyclin D1阳性表达出现于细胞核,为大量黄褐色粗大颗粒,其阳性表达率分别为44.93%和52.17%,明显强于息肉组(分别为2.90%和8.70%)和癌前组(分别为10.14%和18.84%)(P均<0.01)。pRb的阳性表达发生于细胞核,为少而小的黄色颗粒,其阳性表达率为10.14%,显著小于息肉组的92.75%和癌前组的59.42%(P均<0.01)。癌前组HPV16E7和Cyclin D1阳性表达率较息肉组提高,pRb阳性表达率较息肉组明显下降(P均<0.005)。在喉癌肿瘤临床分期中,HPV16E7阳性表达率在Ⅰ-Ⅳ期中随着分期增加呈上升趋势,与临床Ⅲ期比较,HPV16E7在Ⅳ期的阳性表达率显著加重(P<0.025);Cyclin D1阳性表达率逐渐增多,pRb阳性表达率逐渐减少(P均<0.005)。在喉癌组织病理分级中,HPV16E7、Cyclin D1和pRb的阳性表达率随着分化程度减弱,HPV16E7和Cyclin D1阳性表达率分别为Ⅲ级>Ⅱ级>Ⅰ级,pRb阳性表达率为Ⅲ级<Ⅱ级<Ⅰ级(P均<0.005)。喉癌组中HPV16E7和Cyclin D1蛋白量均强烈增加,pRb蛋白定量显著减小;HPV16E7阳性表达率的增加与Cyclin D1阳性表达率增加呈显著的正相关(r=0.48,P<0.001),与pRb阳性表达率下降呈严重的负相关(r=-0.41,P<0.005);而Cyclin D1的阳性表达率增加与pRb阳性表达率下降也呈明显的负相关(r=-0.44,P<0.002)。结论:喉癌肿瘤的发生和发展与HPV严重感染宿主细胞造成HPV16E7过度表达,并刺激Cyclin D1恶性增加和pRb严重降低密切相关。通过对HPV16E7、Cyclin D1和pRb的检测有利于提高临床早期诊断和防治喉癌的发生和发展,对患者喉癌恶性程度和生存预后评判均具有重要的临床意义。
Objective: To investigate the effects of abnormal expression of human papillomavirus 16E7 protein (HPV16E7), cyclin D1 and retinoblastoma protein (pRb) on laryngeal carcinoma and its mechanism. Methods: Totally 69 laryngeal cancer specimens (laryngocarcinoma group), throat polyp tissue specimens (polyp group), and laryngeal precancerous lesion specimens (precancerous group). Peroxidase-labeled streptavidin-ovalbumin immunohistochemistry and Western Blot were used to detect the expression of HPV16E7, Cyclin D1 and pRb in each group of laryngeal tissues. Results: The positive expression rate of HPV16E7 and Cyclin D1 in laryngeal carcinoma was large in the nucleus, with a large number of coarse brown granules. The positive rates of HPV16E7 and Cyclin D1 were 44.93% and 52.17%, respectively, which were significantly higher than those in polyps (2.90% and 8.70%, respectively) The former group (10.14% and 18.84% respectively) (P <0.01). The positive expression rate of pRb in the nucleus, small and small yellow granules, was 10.14%, which was significantly lower than that in the polyp group (92.75%) and the precancerous group (59.42%, P <0.01). The positive rate of HPV16E7 and Cyclin D1 in precancerous lesions was higher than that in polyps and the positive rate of pRb was significantly lower than that in polyps (all P <0.005). In the clinical stage of laryngeal cancer, the positive rate of HPV16E7 in stage Ⅰ-Ⅳ increased with the staging. Compared with stage Ⅲ, the positive rate of HPV16E7 in stage Ⅳ was significantly increased (P <0.025); the positive rate of Cyclin D1 The expression rate of pRb gradually decreased and the positive rate of pRb decreased gradually (all P <0.005). The positive rates of HPV16E7, Cyclin D1 and pRb were weakened with the degree of differentiation, the positive rates of HPV16E7 and Cyclin D1 were grade Ⅲ> grade Ⅱ> grade Ⅰ, pRb positive expression rate was grade Ⅲ < Grade Ⅱ <Ⅰ (P <0.005). The protein levels of HPV16E7 and Cyclin D1 were significantly increased in laryngeal squamous cell carcinoma group, while the expression of pRb protein was significantly decreased. The positive rate of HPV16E7 positive expression was positively correlated with the increase of Cyclin D1 expression (r = 0.48, P <0.001) (r = -0.41, P <0.005). However, the positive expression rate of Cyclin D1 was negatively correlated with the decline of pRb positive expression (r = -0.44, P <0.002) ). CONCLUSIONS: The occurrence and development of laryngeal carcinoma are closely related to HPV16E7 overexpression caused by HPV infection in host cells and stimulation of malignant increase of Cyclin D1 and severe decrease of pRb. The detection of HPV16E7, Cyclin D1 and pRb is helpful to improve the early diagnosis and prevention of laryngeal cancer in clinic, and has an important clinical significance in evaluating the malignancy and prognosis of laryngeal cancer.