十字花科蔬菜和萝卜硫素对云南宣威高发肺癌的影响

发布时间：2018-05-03 21:24

本文选题：宣威肺癌 + 十字花科蔬菜　；参考：《昆明医科大学》2017年博士论文

【摘要】：[目的]探讨宣威地区居民膳食结构中十字花科蔬菜与肺癌的相关性,探讨十字花科蔬菜富含的萝卜硫素对宣威肺癌细胞凋亡的诱导作用和对宣威肺癌生长的抑制作用,为宣威地区肺癌的膳食预防研究提供线索,并从膳食营养的角度为宣威地区肺癌防治措施的制定提供参考。[方法]采用以人群为基础的1:1配对病例对照研究方法,在宣威地区肺癌高发的2个乡镇招募107例原发性肺癌幸存者作为研究病例。以年龄、性别、民族、婚姻状况、受教育程度、职业类型为配对条件,选择与研究病例居住在同一村寨的107名非肿瘤患者作为对照。采用调查问卷和食物频率表,由培训合格的营养专业人员入户进行面对面调查,测量并计算体质指数(BMI)。采用SPSS进行统计分析,用条件Logistic回归分析宣威地区肺癌的影响因素。利用宣威肺腺癌XWLC-05细胞,通过体外实验探讨西兰花提取物萝卜硫素(十字花科蔬菜有效化学成分之一)对宣威肺癌细胞增殖和凋亡的作用。用MTT法检测萝卜硫素对XWLC-05细胞增殖的抑制作用,绘制细胞生长曲线。采用流式细胞术检测细胞周期。在透射电子显微镜下观察细胞凋亡形态学,采用TUNEL染色的流式细胞术定量评估细胞凋亡程度,qPCR和Western Blot分析Bcl-2、Bax mRNA和蛋白表达,Western Blot检测P53、P73及其下游调控因子PUMA、凋亡蛋白酶caspase-9和caspase-3的表达水平。建立宣威肺腺癌XWLC-05细胞裸小鼠皮下移植瘤模型,随机分为接种癌细胞前后均给药组、成瘤后给药组、不给药组,每组小鼠隔天1次灌胃给予50 mg/kg或100mg/kg或150mg/kg萝卜硫素,对照组给予生理盐水,观察小鼠的精神、活动,每2天称量体重、饮食和饮水量,成瘤后每2天测量肿瘤长径和短径,计算肿瘤体积,绘制肿瘤生长曲线。末次干预(接种后第31天)的次日处死小鼠,观察离体肿瘤及肿瘤远处转移情况,称量瘤重,计算肿瘤抑制率。TUNEL法检测肿瘤组织的细胞凋亡情况,计算凋亡指数。为了考量用药剂量的安全性,离体肝脏进行病理切片、HE染色、镜下观察。[结果]107对病例对照接受问卷调查,有效问卷214份。病例组营养不良率(13人,12%)高于对照组(7人,7%)(P0.05)。经单因素统计分析,病例组和对照组在肺癌相关危险因素上差异无统计学意义(P0.05),经条件Logistic回归分析,生活烟煤暴露、吸烟、吸烟指数、二手烟暴露、肿瘤家族史、农药使用史、烹饪史均未留在Logistic回归模型中。总体膳食情况:病例组和对照组奶类基本不食用,禽肉、蛋类、鱼虾类每日平均食用量均低于25g,维生素A、维生素B2、钙、锌、硒摄入量低于推荐量,经单因素统计分析,病例组和对照组在谷薯类、水果、畜禽肉、鱼虾类、蛋类、奶及奶制品、大豆及坚果类、烹调油、盐的摄入量上差异均无统计学意义(P0.05),在热量、蛋白质、脂肪、碳水化合物、膳食纤维、维生素A、维生素B1、维生素B2、维生素C、钙、铁、锌、硒摄入量上差异均无统计学意义(P0.05)。但病例组的蔬菜摄入量(407.6±175.2)g低于对照组(460.0±159.2)g(P0.05),十字花科蔬菜摄入量(297.7±160.6)g低于对照组(351.4±153.1)g(P0.05)。病例组与对照组在能量的食物来源、能量的营养素来源、蛋白质的食物来源、脂肪的食物来源、理想膳食模式(DDP)评分上差异均无统计学意义(P0.05),但病例组的国际膳食质量指数(DQI-I)总分(43.0±6.1)差于对照组(46.3±3.2)(P0.05)。条件Logistic回归分析结果显示,蔬菜摄入量与肺癌呈负相关,OR值为0.740(95%CI:0.577-0.949),十字花科蔬菜摄入量与肺癌呈负相关,OR值为0.718(95%CI:0.558-0.924),家庭总收入与肺癌呈边际负相关,OR值为 0.843(95%CI:0.703-1.010)(P=0.064)。体外实验发现,十字花科蔬菜主要成分之一——萝卜硫素对宣威肺腺癌细胞XWLC-05的生长具有抑制作用,随萝卜硫素浓度增加和作用时间延长,细胞生长抑制逐渐增强,72小时的半数抑制浓度为3.Oμg/mL。萝卜硫素1μg/mL作用于XWLC-05细胞48h后,G2/M期细胞比例升高,透射电镜下可见凋亡小体和凋亡细胞,细胞凋亡率为27.6%(v.生理盐水对照P0.05)。萝卜硫素使XWLC-05细胞Bcl-2mRNA和蛋白下调,BaxmRNA和蛋白上调(v.生理盐水对照P0.05)。实验组细胞P53表达明显下降(v.生理盐水对照组P0.05)。实验组细胞P53的同源基因P73蛋白、P73下游的调控因子PUMA、凋亡蛋白酶caspase-9,caspase-3表达增加(v.生理盐水对照组P0.05)。动物体内实验发现,XWLC-05肺腺癌皮下移植瘤小鼠模型在给予不同浓度的萝卜硫素(50mg/kg,100mg/kg,150mg/kg)干预后,与未用萝卜硫素干预的小鼠相比,肿瘤体积和肿瘤重量均有不同程度降低,并随萝卜硫素浓度的增高,肿瘤抑制作用增强。相同浓度下,接种癌细胞前后均给药组的肿瘤抑制率(60%-70%)大于成瘤后给药组(18%-50%)。关于萝卜硫素的安全性实验,灌胃给药小鼠出现饮食量下降,肝脏病理检查未发现异常。[结论]膳食中十字花科蔬菜摄入量与宣威地区居民肺癌呈负相关;十字花科蔬菜的主要化学成分之一——萝卜硫素可诱导宣威肺腺癌细胞凋亡,对小鼠体内肿瘤生长有抑制作用,呈剂量效应关系;接种前后均给药的抑制效果优于成瘤后给药。十字花科蔬菜对宣威地区肺癌发生发展的作用有必要做进一步的前瞻性研究。
[Abstract]:[Objective] to explore the correlation between the cruciferous vegetables and lung cancer in the diet structure of Xuanwei residents, and to explore the induction of the apoptosis of the lung cancer cells and the inhibitory effect on the growth of Xuanwei lung cancer cells, which are rich in cruciferous vegetables, and provide a clue for the dietary prevention of lung cancer in Xuanwei area. To provide reference for the prevention and treatment of lung cancer in Xuanwei area. [Methods] a population based 1:1 matched case-control study was used to recruit 107 cases of primary lung cancer survivors in 2 villages and towns with high incidence of lung cancer in Xuanwei area. Age, sex, nationality, marital status, education, and occupational types were matched, 107 non tumor patients who lived in the same village were selected as control. The questionnaire and food frequency table were used to investigate and calculate the body mass index (BMI) by training qualified nutrition professionals. SPSS was used to analyze and analyze the effects of lung cancer in Xuanwei area with conditional Logistic regression. The effect of carhanus sulforaphane (one of the effective chemical components of cruciferae vegetables) on the proliferation and apoptosis of Xuanwei lung cancer cells was investigated in vitro by using the XWLC-05 cells of lung adenocarcinoma of Xuanwei. The inhibitory production of sulforaphane on XWLC-05 cell proliferation was detected by MTT method and the cell growth curve was drawn. Flow cytometry was used. The cell cycle was detected. The apoptosis morphology was observed under the transmission electron microscope. The apoptosis degree was evaluated by flow cytometry with TUNEL staining. QPCR and Western Blot were used to analyze Bcl-2, Bax mRNA and protein expression. Western Blot detected P53, P73 and downstream regulatory factor PUMA, apoptotic protease and expressed water. A model of subcutaneous transplantation of XWLC-05 cells in nude mice of Xuanwei lung adenocarcinoma was set up, randomly divided into each group before and after the inoculation of cancer cells, and the drug group was given after the tumor. The mice in each group were given 50 mg/kg or 100mg/kg or 150mg/kg sulforaphane 1 times a day, and the control group was given saline, observed the spirit and activity of mice every 2 days. Weight, diet and drinking water, measured tumor length and short diameter every 2 days after tumor formation, calculate tumor volume and plot tumor growth curve. The last day after intervention (thirty-first days after inoculation), the mice were killed, distant metastasis of tumor and tumor in vitro were observed, tumor weight was weighed, and tumor inhibition rate.TUNEL method was used to calculate the cell apoptosis in tumor tissue. Apoptosis index. In order to examine the safety of dosage, pathological section of isolated liver, HE staining, microscopic observation. [results]107 to case control questionnaire survey, 214 effective questionnaires (13 people, 12%) higher than the control group (7, 7%) (P0.05). A single factor statistical analysis, case group and control group in lung cancer related risk There was no statistically significant difference in risk factors (P0.05). After conditional Logistic regression analysis, living bituminous coal exposure, smoking, smoking index, second-hand smoke exposure, family history of cancer, pesticide use history, and cooking history did not remain in the Logistic regression model. The average consumption of food was lower than 25g, vitamin A, vitamin B2, calcium, zinc, and selenium intake were lower than the recommended amount. By single factor analysis, there was no statistical difference between the case group and the control group in the valley, fruit, animal meat, fish and shrimp, eggs, milk and dairy products, soya and nuts, cooking oil and salt (P0.05), in heat, protein, Fat, carbohydrates, dietary fiber, vitamin A, vitamin B1, vitamin B2, vitamin C, calcium, iron, zinc, and selenium intake were not statistically significant (P0.05). But the intake of vegetables in the case group (407.6 + 175.2) g was lower than that of the control group (460 + 159.2) G (P0.05), and the intake of cruciferous vegetables (297.7 + 160.6) g was lower than the control group (351.4 + 153.1) g. (P0.05). There was no significant difference between the case group and the control group in the food source, the source of the energy, the food source of the protein, the food source of the fat, the ideal dietary pattern (DDP) score (P0.05), but the total score of the international dietary quality index (43 + 6.1) of the case group was worse than the control group (46.3 + 3.2) (P0.05). The condition Log Istic regression analysis showed that the intake of vegetables was negatively correlated with lung cancer, OR was 0.740 (95%CI:0.577-0.949), and the intake of cruciferous vegetables was negatively correlated with lung cancer, and the OR value was 0.718 (95%CI:0.558-0.924). The total family income was negatively correlated with lung cancer, and the value of OR was 0.843 (95%CI:0.703-1.010) (P=0.064). In vitro experiments found, Cruciferae, Cruciferae. One of the main ingredients of vegetables, sulforaphane, could inhibit the growth of XWLC-05 in the lung adenocarcinoma cell of Xuanwei. With the increase of sulforaphane concentration and the prolongation of the action time, the inhibition of cell growth gradually increased, and the 72 hour half inhibitory concentration was 3.O g/mL. sulforaphin 1 g/mL after XWLC-05 cell 48h, and the proportion of G2/M phase cells increased. Apoptotic bodies and apoptotic cells were observed under transmission electron microscopy, the apoptosis rate was 27.6% (v. physiological saline control P0.05). Sulforaphane reduced Bcl-2mRNA and protein in XWLC-05 cells, BaxmRNA and protein up regulation (v. physiological saline control P0.05). The expression of P53 in the experimental group decreased significantly (v. normal saline control group P0.05). Gene P73 protein, regulatory factor PUMA downstream of P73, apoptotic protease caspase-9, caspase-3 expression increased (v. physiological saline control group P0.05). In vivo experiments in animals found that the mice model of XWLC-05 lung adenocarcinoma subcutaneous xenografts was given to different concentrations of sulforaphane (50mg/kg, 100mg/kg, 150mg/kg), and mice with unused sulforaphane The tumor volume and tumor weight were reduced in varying degrees, and the tumor inhibition increased with the increase of sulforaphane concentration. Under the same concentration, the tumor inhibition rate (60%-70%) before and after the inoculation of cancer cells was greater than that of the post tumor drug delivery group (18%-50%). No abnormalities were found in the liver pathological examination. [Conclusion] the intake of cruciferous vegetables in the diet was negatively correlated with lung cancer in Xuanwei area. One of the main chemical components of the cruciferous vegetables, sulforaphane, could induce apoptosis of lung adenocarcinoma cells in Xuanwei, and had a inhibitory effect on the growth of tumor in mice. The inhibitory effect of the latter is better than that after the tumor formation. The role of cruciferous vegetables in the occurrence and development of lung cancer in Xuanwei area is necessary for further prospective study.

【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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