發(fā)布時間：2018-05-03 21:24

  本文選題：宣威肺癌 + 十字花科蔬菜��； 參考：《昆明醫(yī)科大學》2017年博士論文

【摘要】：[目的]探討宣威地區(qū)居民膳食結構中十字花科蔬菜與肺癌的相關性,探討十字花科蔬菜富含的蘿卜硫素對宣威肺癌細胞凋亡的誘導作用和對宣威肺癌生長的抑制作用,為宣威地區(qū)肺癌的膳食預防研究提供線索,并從膳食營養(yǎng)的角度為宣威地區(qū)肺癌防治措施的制定提供參考。[方法]采用以人群為基礎的1:1配對病例對照研究方法,在宣威地區(qū)肺癌高發(fā)的2個鄉(xiāng)鎮(zhèn)招募107例原發(fā)性肺癌幸存者作為研究病例。以年齡、性別、民族、婚姻狀況、受教育程度、職業(yè)類型為配對條件,選擇與研究病例居住在同一村寨的107名非腫瘤患者作為對照。采用調(diào)查問卷和食物頻率表,由培訓合格的營養(yǎng)專業(yè)人員入戶進行面對面調(diào)查,測量并計算體質(zhì)指數(shù)(BMI)。采用SPSS進行統(tǒng)計分析,用條件Logistic回歸分析宣威地區(qū)肺癌的影響因素。利用宣威肺腺癌XWLC-05細胞,通過體外實驗探討西蘭花提取物蘿卜硫素(十字花科蔬菜有效化學成分之一)對宣威肺癌細胞增殖和凋亡的作用。用MTT法檢測蘿卜硫素對XWLC-05細胞增殖的抑制作用,繪制細胞生長曲線。采用流式細胞術檢測細胞周期。在透射電子顯微鏡下觀察細胞凋亡形態(tài)學,采用TUNEL染色的流式細胞術定量評估細胞凋亡程度,qPCR和Western Blot分析Bcl-2、Bax mRNA和蛋白表達,Western Blot檢測P53、P73及其下游調(diào)控因子PUMA、凋亡蛋白酶caspase-9和caspase-3的表達水平。建立宣威肺腺癌XWLC-05細胞裸小鼠皮下移植瘤模型,隨機分為接種癌細胞前后均給藥組、成瘤后給藥組、不給藥組,每組小鼠隔天1次灌胃給予50 mg/kg或100mg/kg或150mg/kg蘿卜硫素,對照組給予生理鹽水,觀察小鼠的精神、活動,每2天稱量體重、飲食和飲水量,成瘤后每2天測量腫瘤長徑和短徑,計算腫瘤體積,繪制腫瘤生長曲線。末次干預(接種后第31天)的次日處死小鼠,觀察離體腫瘤及腫瘤遠處轉(zhuǎn)移情況,稱量瘤重,計算腫瘤抑制率。TUNEL法檢測腫瘤組織的細胞凋亡情況,計算凋亡指數(shù)。為了考量用藥劑量的安全性,離體肝臟進行病理切片、HE染色、鏡下觀察。[結果]107對病例對照接受問卷調(diào)查,有效問卷214份。病例組營養(yǎng)不良率(13人,12%)高于對照組(7人,7%)(P0.05)。經(jīng)單因素統(tǒng)計分析,病例組和對照組在肺癌相關危險因素上差異無統(tǒng)計學意義(P0.05),經(jīng)條件Logistic回歸分析,生活煙煤暴露、吸煙、吸煙指數(shù)、二手煙暴露、腫瘤家族史、農(nóng)藥使用史、烹飪史均未留在Logistic回歸模型中。總體膳食情況:病例組和對照組奶類基本不食用,禽肉、蛋類、魚蝦類每日平均食用量均低于25g,維生素A、維生素B2、鈣、鋅、硒攝入量低于推薦量,經(jīng)單因素統(tǒng)計分析,病例組和對照組在谷薯類、水果、畜禽肉、魚蝦類、蛋類、奶及奶制品、大豆及堅果類、烹調(diào)油、鹽的攝入量上差異均無統(tǒng)計學意義(P0.05),在熱量、蛋白質(zhì)、脂肪、碳水化合物、膳食纖維、維生素A、維生素B1、維生素B2、維生素C、鈣、鐵、鋅、硒攝入量上差異均無統(tǒng)計學意義(P0.05)。但病例組的蔬菜攝入量(407.6±175.2)g低于對照組(460.0±159.2)g(P0.05),十字花科蔬菜攝入量(297.7±160.6)g低于對照組(351.4±153.1)g(P0.05)。病例組與對照組在能量的食物來源、能量的營養(yǎng)素來源、蛋白質(zhì)的食物來源、脂肪的食物來源、理想膳食模式(DDP)評分上差異均無統(tǒng)計學意義(P0.05),但病例組的國際膳食質(zhì)量指數(shù)(DQI-I)總分(43.0±6.1)差于對照組(46.3±3.2)(P0.05)。條件Logistic回歸分析結果顯示,蔬菜攝入量與肺癌呈負相關,OR值為0.740(95%CI:0.577-0.949),十字花科蔬菜攝入量與肺癌呈負相關,OR值為0.718(95%CI:0.558-0.924),家庭總收入與肺癌呈邊際負相關,OR值為 0.843(95%CI:0.703-1.010)(P=0.064)。體外實驗發(fā)現(xiàn),十字花科蔬菜主要成分之一——蘿卜硫素對宣威肺腺癌細胞XWLC-05的生長具有抑制作用,隨蘿卜硫素濃度增加和作用時間延長,細胞生長抑制逐漸增強,72小時的半數(shù)抑制濃度為3.Oμg/mL。蘿卜硫素1μg/mL作用于XWLC-05細胞48h后,G2/M期細胞比例升高,透射電鏡下可見凋亡小體和凋亡細胞,細胞凋亡率為27.6%(v.生理鹽水對照P0.05)。蘿卜硫素使XWLC-05細胞Bcl-2mRNA和蛋白下調(diào),BaxmRNA和蛋白上調(diào)(v.生理鹽水對照P0.05)。實驗組細胞P53表達明顯下降(v.生理鹽水對照組P0.05)。實驗組細胞P53的同源基因P73蛋白、P73下游的調(diào)控因子PUMA、凋亡蛋白酶caspase-9,caspase-3表達增加(v.生理鹽水對照組P0.05)。動物體內(nèi)實驗發(fā)現(xiàn),XWLC-05肺腺癌皮下移植瘤小鼠模型在給予不同濃度的蘿卜硫素(50mg/kg,100mg/kg,150mg/kg)干預后,與未用蘿卜硫素干預的小鼠相比,腫瘤體積和腫瘤重量均有不同程度降低,并隨蘿卜硫素濃度的增高,腫瘤抑制作用增強。相同濃度下,接種癌細胞前后均給藥組的腫瘤抑制率(60%-70%)大于成瘤后給藥組(18%-50%)。關于蘿卜硫素的安全性實驗,灌胃給藥小鼠出現(xiàn)飲食量下降,肝臟病理檢查未發(fā)現(xiàn)異常。[結論]膳食中十字花科蔬菜攝入量與宣威地區(qū)居民肺癌呈負相關;十字花科蔬菜的主要化學成分之一——蘿卜硫素可誘導宣威肺腺癌細胞凋亡,對小鼠體內(nèi)腫瘤生長有抑制作用,呈劑量效應關系;接種前后均給藥的抑制效果優(yōu)于成瘤后給藥。十字花科蔬菜對宣威地區(qū)肺癌發(fā)生發(fā)展的作用有必要做進一步的前瞻性研究。
[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.

【學位授予單位】：昆明醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R734.2

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8 謝述瓊;何s，

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