益气解毒方对TgN(p53mt-LMP1)/HT转基因小鼠鼻腔和鼻咽黏膜上皮细胞 增殖及凋亡相关基因表达的影响******★

背景：益气解毒中药复方在体外能通过下调端粒酶的活性抑制细胞生长和诱导细胞凋亡，还可能通过抑制细胞内重要的核转录因子、细胞分裂增殖、细胞周期、DNA修复和诱导细胞凋亡、促进坏死等信号通路，从而抑制细胞增殖和诱导细胞死亡。 目的：实验拟观察益气解毒方对TgN(p53mt-LMP1)/HT转基因小鼠鼻腔和鼻咽黏膜上皮细胞增殖及凋亡相关基因bax、bcl-2、增殖细胞核抗原和p53mt基因表达的影响。 设计、时间及地点：随机对照动物实验，于2006-01/2007-12在湖南中医药大学中西医结合学院基础研究室和中医五官重点学科实验室完成。 材料：G4代8月龄TgN(p53mt-LMP1)/HT转基因阳性小鼠和阴性小鼠各24只，体质量（25.4±2.3）kg，用于制备鼻咽和鼻腔癌前病变模型；益气解毒颗粒方药液为自制。 方法：48只小鼠随机分为4组，转基因阳性中药组和转基因阴性中药组小鼠灌胃益气解毒颗粒方药液，给药剂量为12.91 g/(kg? d)；转基因阳性生理盐水组和转基因阴性生理盐水组小鼠灌等体积生理盐水。 主要观察指标：苏木精-伊红染色观察鼻腔和鼻咽黏膜病理组织学变化，免疫组织化学染色法检测其bax、bcl-2、增殖细胞核抗原和p53mt基因表达水平。 结果：纳入转基因阳性小鼠和阴性小鼠各24只，转基因阳性生理盐水组和转基因阴性生理盐水组于实验结束前各死亡1只， 转基因阴性中药组死亡1只，转基因阳性中药组死亡2只小鼠，共43只小鼠进入结果分析。①转基因阴性生理盐水组、转基因阴性中药组小鼠的鼻腔/鼻咽黏膜组织均无明显病变；转基因阳性中药组有1只小鼠表现中度非典型性增生，转基因阳性生理盐水组有10只小鼠鼻腔和/或鼻咽黏膜上皮细胞呈现中度或中度以上非典型增生或灶性鳞状化生。转基因阴性生理盐水组、转基因阴性中药组、转基因阳性中药组和转基因阳性生理盐水组小鼠鼻腔或鼻咽黏膜上皮癌前病变率分别为0、0、10%和90.9%，差异具有显著性意义（P < 0.01）。②与转基因阳性生理盐水组相比，其他各组小鼠鼻腔和鼻咽黏膜上皮细胞中p53mt、bcl-2和增殖细胞核抗原表达水平均下降（P < 0.01），bax表达水平显著增高，bcl-2/bax比值降低（P < 0.01）。 结论：益气解毒方中药可有效抑制或逆转TgN(p53mt-LMP1)/HT阳性小鼠鼻腔或鼻咽黏膜上皮的癌前病变表型，其作用机制之一可能通过下调p53mt基因和增殖细胞核抗原的表达，降低bcl-2/bax比值，使细胞增殖和凋亡速度达到平衡，进而促使鼻腔和鼻咽黏膜上皮细胞正常增长。

Effects of Yiqijiedu compound on proliferation of nasal and nasopharyngeal epithelial cells and expressive activities of apoptosis-related genes in TgN(p53mt-LMP1)/HT transgenic mice

BACKGROUND: Yiqijiedu compound can inhibit cell growth and induce cell apoptosis through down-regulating telomerase activity, and can inhibit cell proliferation and induce cell death through signal pathways of inhibiting nuclear factor, cell division and proliferation, cell cycle, DNA repair and inducing cell apoptosis to promote necrosis. OBJECTIVE: To investigate the effects of Yiqijiedu compound on cell proliferation of nasal and/or nasopharyngeal epithelial cells and expressive activities of apoptosis-related genes bax, bcl-2, p53mt and cell proliferation-related gene proliferating cell nuclear antigen (PCNA) in TgN(p53mt-LMP1)/HT transgenic mice. DESIGN, TIME AND SETTING: Randomized controlled animal trial was performed at the Basic Laboratory and Key Laboratory of Integrative Chinese and Western Medicine, Hunan University of Chinese Medicine between January 2006 and December 2007. MATERIALS: Twenty-four generation G4 of TgN(p53mt-LMP1)/HT transgenic positive and negative mice, aged 8 months, weighing (25.4±2.3) kg, were selected to prepare models of nasopharyngeal and nasal cavity precancerosis. Yiqijiedu compound was prepared by our team. METHODS: Forty-eight mice were randomly divided into 4 groups. Positive and negative Yiqijiedu compound groups were infused with Yiqijiedu compound at dose of 12.91 g/kg per day; while positive and negative normal saline groups were infused with normal saline. MAIN OUTCOME MEASURES: Epithelial tissue samples were taken from nasal cavity and nasopharynx of mice for pathohistological evaluation by HE staining; expressions of p53mt, bax, bcl-2 and PCNA genes were assayed by immunohistochemistry. RESULTS: During the experiment, one mouse from negative and one from positive normal saline groups died, one from negative and two from positive Yiqijiedu compound groups died. Finally, 43 mice were included in the final analysis. No obvious pathological changes were found in negative normal saline and Yiqijiedu compound groups. One mouse in positive Yiqijiedu compound group developed moderate atypical hyperplasia, and 10 mice in positive normal saline group developed moderate atypical hyperplasia or squamous metaplasia of nasal and/or nasopharyngeal epithelial cells. The occurrence rates of precancerous lesions in nasal and/or nasopharyngeal epithelia in negative normal saline and Yiqijiedu compound groups and positive Yiqijiedu compound and normal saline groups were 0, 0, 10% and 90.9%, respectively (P < 0.01). Compared with those of positive normal saline group, the expressive activities of p53mt, bcl-2 and PCNA were significantly decreased in the other groups (P < 0.01), and expression of bax was increased obviously, with the ratio of bcl-2/bax elevated (P < 0.01). CONCLUSION: The occurrence and development of precancerous lesions of nasal and/or nasopharyngeal epithelia in TgN(p53mt-LMP1)/HT transgenic positive mice are reversed or inhibited by Yiqijiedu compound, and one of its mechanisms maybe down-regulate the expression of p53mt and PCNA, decrease ratio of bcl-2/bax, then balance the speeds of cell proliferation and apoptosis, resulting in normal growth of nasal and/or nasopharyngeal epithelial cells.