【作者】 钟洁希；

【导师】 陈锐深；

【作者基本信息】 广州中医药大学 ， 中西医结合临床， 2012， 博士

【摘要】 鼻咽癌是我国常见的恶性肿瘤之一,在头颈部肿瘤中占首位,发病率有明显的地区分布。我国及东南亚地区较多见,其中广东中西部发病率最高。近年来NPC发病率逐年攀升,因其具体发病机制尚不明确,联合化疗近期效果显著,但毒副反应严重,容易导致不同程度的后遗症,严重影响患者的生活质量及预后。近年来中医药治疗鼻咽癌的优势备受国内外关注,已有实验和临床资料表明,中医药在提高肿瘤病人生活质量,延长生存时间,降低肿瘤复发和转移等方面具有独特的优势。“清鼻方”是陈锐深教授临床治疗鼻咽癌的临床经验方,广泛用于晚期鼻咽癌的治疗及鼻咽癌放疗的辅助治疗,在临床使用多年,具有良好的临床疗效。本课题将探讨“清鼻方”对免疫力低下小鼠非特异性免疫功能的影响及通过体外实验探讨“清鼻方”对CNE细胞增殖的影响,旨在从不同层面、不同靶点探讨“清鼻方”对鼻咽癌的作用机理,为临床使用提供科学的依据。目的：在建立免疫力低下模型小鼠基础上,应用清鼻方干预模型小鼠,研究清鼻方在增强机体免疫力的作用机理；在己分离出的鼻咽癌细胞株(CNE细胞)及血清药理学的基础上,通过细胞生物学、分子生物学技术,探讨清鼻方对CNE细胞的抑制作用。方法：(1)清鼻方对环磷酰胺诱导所致免疫力低下小鼠非特异性免疫功能的影响取健康雌雄各半的BALB/c种小白鼠70只,按随机区组法分为5组：模型对照组、清鼻方高剂量组、清鼻方低剂量组、干扰素阳性药对照药组及正常对照组。除正常对照组外,其余各组皮下注射环磷酰胺,按80mg/kg体重给药,连续给药3d造模。正常对照组、模型对照组、清鼻方高剂量组、清鼻方低剂量组均采取每天灌胃给药,干扰素组则采用隔天肌注给药,试验周期均为30天。以小鼠体重、脾脏称重、脾脏悬液细胞计数、血清IgA、IgG、IgM含量、T细胞亚群CD3/4/8表达水平进行评价。(2)清鼻方治疗效应对大鼠鼻咽上皮细胞增殖的影响研究制备中药复方清鼻方浓缩煎液。取健康雌雄各半SD大鼠12只,随机分为模型组6只和实验组6只。模型组采用生理盐水灌胃,而实验组采用中药清鼻方浓缩煎液灌胃,处理时长为5天。在大鼠腹主动脉取血,离心取上清制成含药血清直接作用于CNE细胞,并采用MTT法、流式细胞术,电子显微镜观察细胞生长曲线来研究清鼻方分别作用于CNE细胞后对肿瘤细胞生长的抑制情况,以及清鼻方诱导肿瘤细胞发生凋亡的情况。结果：(1)与空白组比较,各组小鼠体重、脾质量、脾脏指数、脾细胞总数均明显下降(P<0.01),其中模型组各数值均最低。骨髓有核细胞数各组与空白组比较,差异无统计学意义(P>0.05)。模型组IgA水平明显降低,干扰素组、低剂量组和高剂量组IgA水平明显升高(P<0.01); IgG水平方面,模型组、干扰素组均明显降低(P<0.01),清鼻方低剂量组和高剂量组变化均不明显(P>0.05)；模型组、干扰素组、低剂量组IgM水平均升高,高剂量组反而降低,但均无统计学差异(P<0.05)。(2)与模型组比较,各用药组骨髓有核细胞数变化不明显(P>0.05)。低剂量组在体重、脾质量、脾脏指数、脾细胞总数方面与之比较,两者均无统计学意义(P>0.05)；高剂量组在小鼠体重、脾质量、脾脏指数方面明显高于模型组(P<0.01)；干扰素组体重、脾质量方面与模型组对比,差异均明显具有统计学意义(P<0.01)。干扰素组、低剂量组和高剂量组IgA水平均明显升高(P<0.01)；干扰素组IgG变化不明显,清鼻方低剂量组和高剂量组IgG水平俊明显升高(P<0.05)；干扰素组和低剂量组均无统计学差异(P<0.05),高剂量组明显低于模型组,两者相比差异有统计学意义(P<0.05)。(3)小鼠外周血CD3、CD4、CD8的整体表达水平相对脾脏较低。小鼠造模后外周血CD3、 CD4、CD8表达水平无明显下降,而脾脏CD3、CD4、CD8表达水平则明显下降；给予干扰素和清鼻方低高剂量后CD3、CD4、CD8表达水平均有一定程度的提升,其中干扰素组接近正常组水平,清鼻方低、高剂量组则均未达到正常组水平。(4)MTT法比色结果显示,在作用24h后,不同浓度清鼻方含药血清对CNE细胞的抑制作用不明显。作用48h后,血清浓度5%、10%、15%对CNE细胞的抑制作用明显；作用72h后,血清浓度5%、10%、对CNE细胞的抑制作用明显。(5)CNE细胞的增殖曲线结果显示：未灭活的正常大鼠血清或含药血清(10%和15%)均具有具有抑制细胞增殖的效果。其中15%未灭活含药血清较10%未灭活含药血清具有更强的抑制CNE细胞增殖的作用。灭活的正常大鼠血清或含药血清(10%和15%)也出现类似的情况,但整体效应低于未灭活血清组。(6)细胞凋亡率方面,用10%含药血清和正常大鼠血清处理CNE细胞后,正常大鼠血清出现凋亡细胞(23%),含药血清出现更高的凋亡率31.0%；用15%含药血清和正常大鼠血清处理CNE细胞后,正常大鼠血清也出现凋亡细胞(25.3%),而15%含药血清凋亡率达33.9%。结论：清鼻方能减轻化疗药物所致的免疫抑制状态,调节机体的细胞免疫与体液免疫,使紊乱免疫机能趋于正常,这将于鼻咽癌患者恢复；清鼻方含药血清能抑制CNE细胞的增殖,诱导细胞凋亡,这可能是其重要作用机理。更多还原

【Abstract】 Nasopharyngeal carcinoma (NPC for short), one of the common malignant tumors in China, is ranked as top above all head and neck cancer, with a significant regional distribution of incidence rate. It is more common in China and Southeast Asia, and among all, Guangdong is the highest. In recent years, NPC incidence rate has increased steadily, because its’ specific pathogenesis is not certain. Although recent combination chemotherapy showed significant effect, but its’serious toxicity of side effect, easily lead to sequelae of different degree, severely affecting the quality of life and prognosis of the patients. Chinese medicine, as an effective adjuvant therapy, not only could alleviate radiation therapy side effects, enhance immunity, improve the tolerance of chemotherapy and radiation therapy, but also could improve patients’ quality of life. Thus it has been increasingly used in clinical, and has become an important research topic in medical field. At the moment, Chinese medicine research has been more in-depth in China and overseas, and the researchers found that single herbs or Chinese herbal formula is indeed therapeutic, especially in immune regulation and tumor suppressor. Domestic Chinese Medicine treatment of NPC normally according to the treatment with syndrome differentiation and prescription studies, its exact mechanism of action remains to be elucidated."Nasal Cleaning Formula"(NCF for short), is formulated by Professor Chen Ruishen based on the "Qi" deficiency and contamination, as well as years of clinical practice experience, and has performed excellent clinical efficacy. Current research of NPC pathogenesis in immunology has progressed rapidly; the treatment of diseases in Chinese medicine is applying multi-target synergy. The author has studied the clinical efficacy and its mechanism of action of "Nasal Cleaning Formula" in the treatment of nasopharyngeal carcinoma.1、Clinical Research Objective:To observe the clinical efficacy of the adjunctive therapy of "Nasal Cleaning Formula" in radiation therapy of nasopharyngeal cancer. Method:76cases of NPC patients were randomly divided into treatment group ("Nasal Cleaning Formula"+radiation therapy) and the control group (radiation therapy),38cases respectively. Control group using conventional radiation therapy, while treatment group using additional "Nasal Cleaning Formula", with a dose every day, a total of seven weeks. Skin mucosal damage and adverse events before and after treatment is observed. T cell subsets, IL-2, TNF-alpha and etc. before and after treatment are determined. Result:(1)No explicit difference (P<0.05) in term of age, gender, radiation dose, pathological staging, radiation treatment and other general information between two groups.(2) Oropharyngeal injury appeared mainly in1,2degrees among patients of treatment group after three weeks of radiation therapy, incidence rate of grade Ⅲ reaction was44.7%,57.9%in the control group; incidence of grade Ⅳ reaction in treatment group was0,15.8%in the control group, the difference is significant(P<0.05);after7weeks of radiation therapy, incidence of grade Ⅲ reaction in treatment group was52.63%,71.05%in the control group; incidence of grade IV reaction in treatment group was2.6%,31.57%in the control group, the difference is significant(P<0.01).(3)Reaction time for radioactive oropharyngeal to be appeared in the treatment group (14.3±1.76) d lower than the control group (19.3±1.56) d (P <0.01).(4) IL-2与TNF-α of treatment group after treatment higher than control group(P<0.01); CD3、CD4&CD4/CD8of treatment group significantly higher than control group.(5) The toxicity in skin, mucous membranes, gastrointestinal and bone marrow of the treatment group is lighter than the control group.Conclusion:"Nasal Cleaning Formula" has the effect of supplementing vital energy and nourishing yin, clearing away heat, detoxication and resolving blood stasis. It could alleviate and delay the injury of skin and mucus due to radiation therapy, and could also reduce the gastrointestinal, bone marrow suppression and other adverse reactions. Besides, it could mitigate the injury of immunity made by radiation therapy and regulate the immune function, leading to a better protection effect against injury after radiation therapy. Therefore, it could increase patients’quality of life by increasing the effect and reducing the toxins.2、Laboratory Research Objectives:On the basis of the establishment of the rat model with low immunity, applying "Nasal Cleaning Formula" in interventions of rat model, to study the mechanism of "Nasal Cleaning Formula" in enhancing immunity of organism. Based on cell line (CNE cells) isolated from the nasopharyngeal carcinoma in China and serum pharmacology, explore the anticancer effect of "Nasal Cleaning Formula" against CNE cells through cell biology, molecular biology techniques. Method:(1)The effect of "Nasal Cleaning Formula"(NCF for short) on non-specific immune functions of immunosuppressive rats induced by cyclophosphamide70healthy BALB/c rats (both male&female50%) were randomly divided into5groups:model control group,"NCF" High Dosage Group,"NCF" Low Dosage Group, interferon positive control medicine group, normal control(blank) group. Every group except normal control group is given subcutaneous injection of cyclophosphamide, with multiple-doses of80mg/kg according to weight. Model control group,"NCF" High Dosage Group,"NCF" Low Dosage Group and normal control group were given medicine by intragastric administration, while interferon group were given medicine by intramuscular administration every other day, all with the testing cycle of30days. Evaluate by the body weight of rat, spleen weight, spleen suspension cell count, IgA, IgG and IgM level of serum and T-cell subsets CD3/4/8expression levels.(2)"NCF" treatment effects on the proliferation of nasopharyngeal epithelial cells in rats Preparation of Chinese herbal formula "NCF" concentrated decoction12healthy SD rats (both male&female50%) were randomly divided into6model groups and6experimental groups. Model groups were given normal saline through intragastric administration, while experimental groups were given "NCF" concentrated decoction through intragastric administration, with the testing cycle of5days. Blood was collected from rat’s abdominal aorta, supernatant was made through centrifugation to make up medicine-contained serum to be act upon CNE cells, and by using MTT method, flow cytometry, electron microscope for growth curve observation, we can study the inhibition of the growth of tumor cells when "NCF" act on CNE cells, and also tumor cells apoptosis induced by "NCF". Results:(1)Compared to blank group, each group has the body weight of rats, spleen weight, spleen index, spleen cell count significantly decreased (P<0.01), while model control group hits the lowest for all values. When comparing the quantity of bone marrow cells (BMC) of each group with the blank group, there is no statistical significance (P>0.05). IgA level for model control group decreased significantly, but IgA level of interferon group,"NCF" Low Dosage Group and "NCF" High Dosage Group increased significantly (P<0.01). For IgG level, model control group and interferon group showed significant decrease (P<0.01), but "NCF" High Dosage Group and "NCF" Low Dosage Group showed insignificant change (P>0.05). Although IgM level of model control group, interferon group and "NCF" Low Dosage Group increased, level of "NCF" High Dosage Group decreased, but none of them showed statistical discrepancy(P<0.05).(2) Compared to model control group, the quantity of bone marrow cells (BMC) of each group with medication changed insignificantly (P>0.05). If we compared the body weight, spleen weight, spleen index, spleen cells count between "NCF" Low Dosage Group and model control group, both showed no statistical significance (P>0.05); Whereas the body weight, spleen weight, spleen index of "NCF" High Dosage Group significantly higher than model control group (P<0.01); The difference of the body weight, spleen weight between interferon group and model control group both showed obvious statistical significance(P<0.01). IgA level of interferon group,"NCF" Low Dosage Group and "NCF" High Dosage Group have increased significantly (P<0.01); IgG level of interferon group changed insignificantly, while IgG level of "NCF" Low Dosage Group and "NCF" High Dosage Group showed significant increase(P<0.05); There is no statistical discrepancy (P<0.05) between interferon group and "NCF" Low Dosage Group, while "NCF" High Dosage Group significantly lower than model control group, difference between them are statistical significant (P<0.05).(3)The overall expression levels of rat peripheral blood CD3, CD4, CD8are relatively lower than spleen, and showed no significant drop after modeling, but expression levels of spleen CD3, CD4, CD8decreased significantly. After giving interferon, high and low dosage of "NCF", CD3, CD4, CD8levels indicated certain degrees of increment. Among this, interferon group nearly reached the level of normal control group, while both "NCF" Low and High Dosage Group did not achieve.(4) Color comparison result of MTT method showed, after acting on for24hours, the inhibition of "NCF" medicine-contained serum of different concentration towards CNE cells was not significant. But after48hours, the inhibition effect of serum of5%,10%and15%concentration upon CNE cells was significant. After72hours, inhibition effect of serum of5%,10%concentration upon CNE cells was significant.(5) The growth curve of CNE cells showed:Activated serums of normal rat or medicine-contained (10%and15%) both possess cells growth inhibition effect. Activated medicine-contained serum of15%concentration are stronger than activated medicine-contained serum of10%in inhibiting CNE cells growth. Inactivated serums of normal rat or medicine-contained (10%and15%) also showed similar effect, but overall performance was lower than activated serum group.(6) For cells apoptosis rate, after using serum of normal rat and of medicine-contained (10%) to process CNE cells, there is apoptotic cells (23%) in serum of normal rat, whereas the apoptosis rate in medicine-contained serum is much higher (31.0%). By processing CNE cells using15%concentrated serum of normal rat and medicine-contained, apoptotic cells appeared in the normal rat serum (25.3%), while medicine-contained serum reached a total of33.9%apoptosis rate. Conclusion:"Nasal Cleaning Formula" could alleviate the immunosuppression caused by chemotherapy, regulate the cellular immunity and humoral immunity of organism, so as to reverse the immunity disorder, and promotes NPC patients recovery;"NCF" medicine-contained serum could inhibit the growth of CNE cells, inducing cells apoptosis, and this could possibly be its important mechanism.更多还原