NAD对抗X射线诱导L02细胞损伤的初步研究
发布时间：2018-09-12 17:54
【摘要】： 研究背景 放射治疗是恶性肿瘤治疗的重要手段之一,但放射治疗在杀死肿瘤细胞的同时,不可避免地损伤正常组织,也因此限制了肿瘤放射治疗的照射剂量强度,导致肿瘤细胞不能被完全杀灭而使病人生存质量的下降。同时,随着医疗保健事业的发展和核工业技术的广泛应用,医源性辐射和非医源性辐射对正常组织细胞辐射损伤也正日益受到放射学家的关注。因而,细胞辐射损伤的机理及辐射损伤防护方面的研究已成为辐射分子生物学和辐射流行病学研究的的热点和重点之一。 国内外学者研究提示,辐射致细胞损伤的机理主要集中在如下几个方面：1)改变细胞信号传递途径：细胞经受辐射后,细胞内或细胞外多种信号分子受其诱导,引起信号传递途径的改变,最终导致细胞凋亡。其中,p53具有中心地位作用。2)DNA损伤：辐射引起DNA损伤,包括单链断裂(SSB)、双链断裂(DSB)、碱基损伤和蛋白质交联等多种形式。3)细胞周期调控：主要通过调控细胞周期G0／G1、S、G2／M调控点,调节细胞辐射敏感性和辐射抗性。细胞DNA损伤后,野生型p53基因诱导细胞进入G1期,直到损伤DNA修复,若损伤不被修复,p53基因就活化诱导细胞凋亡的基因转录,使细胞发生凋亡。4)微环境变化：细胞的生存离不开其微环境,包括细胞氧供应、pH值、营养物质、代谢产物、离子平衡、细胞因子等,其微环境改变可引起相关基因表达和细胞对辐射的反应。 随着细胞辐射损伤分子机制的深入研究,辐射损伤防护方面的研究也取得了较大进展。其中,有关辐射损伤防护剂的研究,近年来有较多报道,主要涉及以下几类：1)抗氧化剂,如Amifostine、NAC、VitC等；2)细胞因子,如IL-2、IL-3、IL-6、TGF等；3)微量元素,如硒、锌等；4)中草药成分,如人参皂甘、迷迭香等。但由于目前研究较多的抗氧化剂Amifostine、NAC毒副作用大,细胞因子作用网络化特点以及中草药成分难以提纯,还没有一种较为理想的辐射防护剂应用于临床。寻找一种新的有效的毒副作用小的辐射防护药物对辐射防护仍具重要意义。 NAD’,化学名为烟酰胺腺嘌呤二核苷酸,是细胞能量代谢重要辅酶,参与细胞氧化还原反应和呼吸链电子传递,在线粒体内NAD+接受电子传递还原成NADH,通过电子传递能够抑制自由基生成,同时生成细胞代谢所需的ATP,因此可能起到保护细胞免遭辐射损伤的作用。 本研究采用细胞生物学和分子生物学方法,初步探讨NAD+辐射损伤防护作用及其机制,这对于进一步研究正常组织细胞的放射损伤机理和探寻一种新的有效的、毒副作用小的辐射防护药物具有理论价值和现实意义。 目的 本研究通过观察氧化型辅酶Ⅰ(NAD+)对辐射损伤的人正常肝细胞株L02细胞的影响,初步探讨氧化型辅酶Ⅰ(NAD+)抗辐射损伤作用及其机制。进而为研究医源性和非医源性辐射损伤防护、寻求新型放射防护剂提供一种新的思路和手段。 方法 1细胞培养及分组正常人肝细胞株L02细胞培养在含10%胎牛血清的PRMI1640培养基中,置于37℃、5%C02培养箱中培养。将L02细胞分为3组：处理组和照射组细胞照射后分别加入含和不含NAD(1000μg/ml)的RPMI-1640培养基；对照组细胞未照射,仅加入RPMI-1640培养基。 2 X射线照射采用Varian 6-MV X线直线加速器,照射剂量为5 Gy,剂量率为500cGy/min,照射源距靶中心距离100cm。 3 MTT法检测不同浓度NAD对细胞增殖的影响细胞以每孔3×105～5×105／ml接种于96孔组织培养板,100 ul／孔,细胞贴壁后去上清,加入0.01mol／L PBS(pH 7.4),按照上述条件进行X线照射。照射后去上清,加入RPMI 1640(含10%胎牛血清)稀释的不同浓度的NAD,分别为0、200、400、600、800、1000、1200、1400 ug/ml,100 ul/孔,在37℃、5%CO2培养箱中培养24h。用MTT比色法检测各浓度NAD对L02细胞增殖的影响。 4流式细胞仪检测细胞凋亡率L02细胞经胰蛋白酶消化后制备成单细胞悬液,以每孔3×105～5×105／ml接种于6孔组织培养板,1ml／孔,待细胞贴壁后去上清,加入0.01mol／L PBS(pH 7.4),按照上述条件进行X线照射。照射后去上清,处理组和照射组分别加入含和不含NAD (1000μg/ml)的RPMI 1640培养基,在37℃、5%CO2孵箱中培养24h。收集各组细胞,调节细胞浓度为1×106～6×106／ml,采用Annexin V/PI染色,检测细胞凋亡率。 5流式细胞仪检测细胞周期细胞以每孔3×105～5×105／ml接种于6孔组织培养板,1ml／孔,待细胞贴壁后去上清,加入0.01mol/L PBS (pH7.4),按照上述条件进行X线照射。照射后去上清,处理组和照射组分别加入含和不含NAD(1000μg／ml)的RPMI 1640培养基,在37℃、5%CO2孵箱中培养24h。收集各组细胞,调节细胞浓度为3×106～6×106／ml,离心后PBS洗涤,加入冰冷的70%乙醇固定,用流式细胞仪检测细胞周期各时相细胞百分比。 6流式细胞仪检测p53、bax、bcl-2蛋白表达百分率分别收集经过上述处理的三组细胞,以0.5%多聚甲醛溶液1ml固定30min,破膜剂裂解细胞后分管、洗涤、离心后去上清,各管分别加入鼠抗人p53、bax、bcl-2单克隆抗体,混匀,37℃孵育1h。洗涤离心后分别加入FITC标记的抗鼠二抗,37℃孵育1h,在流式细胞仪上检测蛋白表达百分率。 7 Caspase-3、Caspase-8、Caspase-9活性测定分别收集5×106个经过上述处理的三组细胞,按试剂盒说明书进行每步操作,另设未加细胞而其他操作一样的孔为空白对照组,通过酶标仪405nm处测定各孔吸光度OD值。 8透射电镜观察L02细胞形态用3%琼脂糖在锥形离子管中制造中间带锥形孔的模具,加入收集处理的细胞,离心,2.5%戊二醛固定、1%锇酸固定双重固定,酒精梯度脱水,环氧丙烷浸透,脂包埋,超薄切片,电子染色电镜观察。 9统计学处理实验所得数据应用SPSS13.0统计软件进行处理。实验数据用x±s表示,行单因素方差分析,P0.05为差异具有统计学意义。 结果 1 MTT法检测不同浓度NAD对细胞增殖的影响L02细胞在接受5.0Gy X线照射后,加入RPMI 1640(含10%胎牛血清)稀释的不同浓度的NAD,分别为0、200、400、600、800、1000、1200、1400 ug/ml,在37℃、5%CO2培养箱中培养24h。MTT检测细胞增殖活性,随着NAD浓度的增加,细胞增殖活性升高,在NAD浓度为1000～1400 ug/ml时,照射后细胞增殖活性的增加达到平台期。因此我们把NAD浓度为1000 ug/ml作为以后的实验条件。 2各组细胞凋亡率细胞照射后培养24h检测各组细胞凋亡率,对照组(1.50±0.67)与处理组(12.85±1.59)均显著低于照射组(31.72±3.07)(P0.05)。说明NAD能明显降低X线照射后的L02细胞的凋亡率,具有抗X线诱导的细胞凋亡作用。 3 NAD对受照细胞周期的调节L02细胞在X线照射后24h时间点G0／G1、S、G2／M期细胞数,照射组与对照组相比G1、S期细胞数明显增加,G2／M期细胞数减少,表现为G1期阻滞；处理组与照射组相比,G1期细胞数减少,S期和G2／M期细胞数增加,表现为处于细胞分裂期和细胞DNA合成期细胞数增加。 4 NAD对X射线诱导的细胞凋亡的凋亡相关蛋白的调节L02细胞经5.0GyX线照射后培养24h,检测三组细胞的凋亡相关蛋白发现。处理组细胞p53、bax表达低于照射组(P0.05),而照射组高于对照组(P0.05),表明细胞受照射后p53、bax表达增强,NAD能够减少细胞受照射后p53、bax表达。三组细胞的bcl-2表达情况则恰恰相反,处理组细胞bcl-2表达高于照射组(P0.05),而照射组低于对照组(P0.05),表明细胞受照射后bcl-2表达减少,NAD能够上调细胞受照射后bcl-2的表达。 5 NAD对受照射细胞Caspase-3、Caspase-8、Caspase-9活性的影响细胞经照射培养24h后,NAD+处理组L02细胞Caspase-3、Caspase-8、Caspase-9的活性较照射组降低,差异有统计学意义(P0.05)。表明NAD能抑制Caspase-3、Caspase-8、Caspase-9的活性,从而抑制X线照射引起的L02细胞凋亡 结论 一、NAD+能够抑制X射线诱导的L02细胞增殖活性的下降,能够降低X射线引起的L02细胞凋亡。 二、X射线引起L02细胞停滞于G0／G1期,NAD+使L02细胞进入S期进行DNA复制。三、NAD+能够减少细胞受照射后p53、bax表达,上调bcl-2的表达。能够降低细胞受照射后Caspase-3、Caspase-8、Caspase-9活性的表达。 本实验通过观察氧化型辅酶Ⅰ(NAD+)对辐射损伤的人正常肝细胞株L02细胞的影响表明：NAD+能够对抗X射线引起的L02细胞凋亡的增加,恢复细胞增殖活性,其途径可能与下调p53、bax,上调bcl-2的表达,降低Caspase-3、Caspase-8、Caspase-9活性的表达有关。
[Abstract]:Research background
Radiotherapy is one of the most important methods for the treatment of malignant tumors. However, while killing tumor cells, radiation therapy inevitably damages normal tissues, which limits the radiation dose intensity of tumor radiation therapy, resulting in the inability of tumor cells to be completely destroyed and the decline of patients'quality of life. With the development of nuclear industry and the extensive application of nuclear technology, the radiation damage of normal tissue cells caused by iatrogenic and non-iatrogenic radiation has been paid more and more attention by radiologists. One of the points.
Researchers at home and abroad have shown that the mechanism of radiation-induced cell injury mainly focuses on the following aspects: 1) altering the cell signal transduction pathway: after radiation, a variety of intracellular or extracellular signal molecules are induced by radiation, resulting in the alteration of signal transduction pathway and eventually leading to apoptosis. Among them, p53 plays a central role. DNA damage: DNA damage caused by radiation, including single strand break (SSB), double strand break (DSB), base damage and protein cross-linking and other forms of cell cycle regulation: mainly through the regulation of cell cycle G0/G1, S, G2/M regulatory points, regulate cell radiosensitivity and radiation resistance. After DNA damage, wild-type p53 gene induces cell entry G1 phase, until the damaged DNA is repaired, if the damage is not repaired, p53 gene activates the gene transcription that induces apoptosis and causes apoptosis. 4) microenvironment changes: cell survival is inseparable from its microenvironment, including cell oxygen supply, pH value, nutrients, metabolites, ion balance, cytokines, and so on. Gene expression and cell response to radiation.
With the in-depth study of the molecular mechanism of cell radiation injury, great progress has been made in the study of radiation injury protection. In recent years, there have been many reports on radiation injury protective agents, mainly involving the following categories: 1) antioxidants, such as Amifostine, NAC, VitC, etc.; 2) cytokines, such as IL-2, IL-3, IL-6, TGF, etc. Elements, such as selenium, zinc, etc. 4) Chinese herbal ingredients, such as ginseng saponin, rosemary, etc. However, due to the current research more antioxidants Amifostine, NAC toxic side effects, cytokine network characteristics and Chinese herbal ingredients are difficult to purify, there is no ideal radiation protection agent used in clinical. Radiation protection drugs with little toxic side effects are still important for radiation protection.
NAD', chemically known as nicotinamide adenine dinucleotide, is an important coenzyme in cell energy metabolism. It participates in cell redox reactions and electron transfer in the respiratory chain. NAD + receives electron transfer and is reduced to NADH in mitochondria. Electron transfer inhibits free radical production and produces ATP required for cell metabolism. Therefore, it may play a protective role. Cells are immune to radiation damage.
In this study, cell biology and molecular biology methods were used to preliminarily explore the protective effect and mechanism of NAD + radiation injury, which is of theoretical value and practical significance for further studying the mechanism of radiation injury in normal tissues and cells and searching for a new effective radiation protection drug with little side effects.
objective
In this study, the effects of oxidative coenzyme I (NAD+) on radiation-induced injury of human normal hepatocyte line L02 were observed, and the anti-radiation effect and mechanism of oxidative coenzyme I (NAD+) were preliminarily investigated.
Method
1 Cell culture and grouping normal human hepatocyte line L02 cells were cultured in PRMI1640 medium containing 10% fetal bovine serum and placed in 37 C_ 02 incubator. L02 cells were divided into three groups: treatment group and irradiation group were irradiated with RPI-1640 medium containing or without NAD (1000 ug/ml), control group cells were not irradiated, only RPI-1640 medium was added. MI-1640 medium.
2 X-ray irradiation was performed with a Varian 6-MV linear accelerator at a dose of 5 Gy, a dose rate of 500 cGy/min, and a distance of 100 cm from the source to the target center.
3 MTT assay was used to detect the effect of different concentrations of NAD on cell proliferation. The cells were inoculated into 96-well tissue culture plate with a dose of 3 105-5 65507 NAD, 0,200,400,600,800,1000,1200,1400 ug/ml, 100 ul/hole, respectively, were cultured in a 37 C, 5% CO2 incubator for 24 hours. MTT colorimetric assay was used to detect the effects of various concentrations of NAD on the proliferation of L02 cells.
The apoptosis rate of L02 cells was detected by flow cytometry. After trypsin digestion, L02 cells were prepared into a single cell suspension. The cells were inoculated into 6-well tissue culture plate with 3 65 RPMI 1640 medium containing or without NAD (1000 ug/ml) was added and cultured at 37 C for 24 hours in 5% CO2 incubator. Cells in each group were collected and the cell concentration was regulated from 1 106 to 6 106/ml. The apoptosis rate was detected by Annexin V/PI staining.
Cell cycle cells were inoculated into 6-well tissue culture plate with 3 65507 Medium was incubated in a 5% CO2 incubator at 37 C for 24 hours. Cells in each group were collected and the concentration of cells was regulated from 3 106 to 6 106 / ml. After centrifugation, PBS was washed and fixed with 70% ethanol. The percentage of cells in each phase of cell cycle was measured by flow cytometry.
The expression percentage of p53, Bax and bcl-2 protein was detected by 6-flow cytometry. The cells were immobilized in 0.5% paraformaldehyde solution for 30 minutes. The cells were lysed by membrane breaker, washed, centrifuged and supernatant was removed. Monoclonal antibodies against human p53, Bax and Bcl-2 were added to the tubes, mixed and incubated at 37 C for 1 h. FITC labeled anti mouse two antibody was added, incubated with 1H at 37 degrees centigrade, and the percentage of protein expression was detected by flow cytometry.
Caspase-3, Caspase-8 and Caspase-9 activity assay were used to collect 5 65507
The morphology of L02 cells was observed by transmission electron microscopy. The mold with conical hole was made by 3% agarose in the conical ion tube. The collected cells were centrifuged, fixed by 2.5% glutaraldehyde, fixed by 1% osmium acid, double fixed by alcohol gradient dehydration, epoxypropane immersion, lipid embedding, ultrathin section and electron staining electron microscopy.
9. Statistical data were processed by SPSS13.0 statistical software. The experimental data were expressed by X + s and analyzed by one-way ANOVA. The difference was statistically significant in P 0.05.
Result
1 MTT assay was used to detect the effect of different concentrations of NAD on cell proliferation. L02 cells were irradiated with 5.0 Gy X-ray and diluted with different concentrations of NAD (including 10% fetal bovine serum) of RPMI 1640 (0,200,400,600,800,1000,1200,1400 ug/ml), respectively. The proliferation activity of L02 cells was detected by MTT at 37 C and in a 5% CO2 incubator for 24 hours. The proliferative activity increased. When the concentration of NAD was 1000-1400 ug/ml, the proliferative activity increased to plateau stage after irradiation.
The apoptosis rate of L02 cells in each group was detected 24 hours after irradiation. The apoptosis rate of L02 cells in control group (1.50.67) and treatment group (12.85.59) was significantly lower than that in irradiation group (31.72.07) (P 0.05).
The number of L02 cells in G0/G1, S, G2/M phase at 24 hours after X-ray irradiation was significantly increased in irradiation group compared with control group, while the number of G2/M phase cells was decreased in treatment group, and the number of G1 phase cells and G2/M phase cells was increased in irradiation group. The number of cells increased during cell division and DNA synthesis.
The expression of p53 and Bax in the treatment group was lower than that in the irradiation group (P 0.05), but the expression of p53 and Bax in the irradiation group was higher than that in the control group (P 0.05). The expression of Bcl-2 was higher in the treatment group than in the irradiation group (P 0.05), but lower in the irradiation group than in the control group (P 0.05).
The activity of Caspase-3, Caspase-8 and Caspase-9 in irradiated L02 cells treated with NAD+ was significantly lower than that in irradiated L02 cells (P 0.05). The results showed that NAD could inhibit the activity of Caspase-3, Caspase-8 and Caspase-9 in irradiated L02 cells, thereby inhibiting the activity of L02 cells induced by X-ray irradiation. Apoptosis
conclusion
First, NAD+ could inhibit the X-ray-induced decrease of L02 cell proliferation and apoptosis.
Second, X-ray induced L02 cells to stagnate in G0/G1 phase, NAD + caused L02 cells to enter S phase for DNA replication. Third, NAD + could reduce the expression of p53, Bax and up-regulate the expression of Bcl-2 after irradiation, and reduce the expression of Caspase-3, Caspase-8 and Caspase-9 after irradiation.
The effect of oxidative coenzyme I (NAD+) on radiation-induced apoptosis and cell proliferation of human normal hepatocyte line L02 was observed. The results showed that NAD+ could inhibit the increase of apoptosis and restore the proliferation activity of L02 cells induced by X-ray. The pathway might be down-regulation of p53, bax, up-regulation of Bcl-2 expression, down-regulation of Caspase-3, Caspase-8 and Caspase-9 expression. Close.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
本文编号：2239792
[Abstract]:Research background
Radiotherapy is one of the most important methods for the treatment of malignant tumors. However, while killing tumor cells, radiation therapy inevitably damages normal tissues, which limits the radiation dose intensity of tumor radiation therapy, resulting in the inability of tumor cells to be completely destroyed and the decline of patients'quality of life. With the development of nuclear industry and the extensive application of nuclear technology, the radiation damage of normal tissue cells caused by iatrogenic and non-iatrogenic radiation has been paid more and more attention by radiologists. One of the points.
Researchers at home and abroad have shown that the mechanism of radiation-induced cell injury mainly focuses on the following aspects: 1) altering the cell signal transduction pathway: after radiation, a variety of intracellular or extracellular signal molecules are induced by radiation, resulting in the alteration of signal transduction pathway and eventually leading to apoptosis. Among them, p53 plays a central role. DNA damage: DNA damage caused by radiation, including single strand break (SSB), double strand break (DSB), base damage and protein cross-linking and other forms of cell cycle regulation: mainly through the regulation of cell cycle G0/G1, S, G2/M regulatory points, regulate cell radiosensitivity and radiation resistance. After DNA damage, wild-type p53 gene induces cell entry G1 phase, until the damaged DNA is repaired, if the damage is not repaired, p53 gene activates the gene transcription that induces apoptosis and causes apoptosis. 4) microenvironment changes: cell survival is inseparable from its microenvironment, including cell oxygen supply, pH value, nutrients, metabolites, ion balance, cytokines, and so on. Gene expression and cell response to radiation.
With the in-depth study of the molecular mechanism of cell radiation injury, great progress has been made in the study of radiation injury protection. In recent years, there have been many reports on radiation injury protective agents, mainly involving the following categories: 1) antioxidants, such as Amifostine, NAC, VitC, etc.; 2) cytokines, such as IL-2, IL-3, IL-6, TGF, etc. Elements, such as selenium, zinc, etc. 4) Chinese herbal ingredients, such as ginseng saponin, rosemary, etc. However, due to the current research more antioxidants Amifostine, NAC toxic side effects, cytokine network characteristics and Chinese herbal ingredients are difficult to purify, there is no ideal radiation protection agent used in clinical. Radiation protection drugs with little toxic side effects are still important for radiation protection.
NAD', chemically known as nicotinamide adenine dinucleotide, is an important coenzyme in cell energy metabolism. It participates in cell redox reactions and electron transfer in the respiratory chain. NAD + receives electron transfer and is reduced to NADH in mitochondria. Electron transfer inhibits free radical production and produces ATP required for cell metabolism. Therefore, it may play a protective role. Cells are immune to radiation damage.
In this study, cell biology and molecular biology methods were used to preliminarily explore the protective effect and mechanism of NAD + radiation injury, which is of theoretical value and practical significance for further studying the mechanism of radiation injury in normal tissues and cells and searching for a new effective radiation protection drug with little side effects.
objective
In this study, the effects of oxidative coenzyme I (NAD+) on radiation-induced injury of human normal hepatocyte line L02 were observed, and the anti-radiation effect and mechanism of oxidative coenzyme I (NAD+) were preliminarily investigated.
Method
1 Cell culture and grouping normal human hepatocyte line L02 cells were cultured in PRMI1640 medium containing 10% fetal bovine serum and placed in 37 C_ 02 incubator. L02 cells were divided into three groups: treatment group and irradiation group were irradiated with RPI-1640 medium containing or without NAD (1000 ug/ml), control group cells were not irradiated, only RPI-1640 medium was added. MI-1640 medium.
2 X-ray irradiation was performed with a Varian 6-MV linear accelerator at a dose of 5 Gy, a dose rate of 500 cGy/min, and a distance of 100 cm from the source to the target center.
3 MTT assay was used to detect the effect of different concentrations of NAD on cell proliferation. The cells were inoculated into 96-well tissue culture plate with a dose of 3 105-5 65507 NAD, 0,200,400,600,800,1000,1200,1400 ug/ml, 100 ul/hole, respectively, were cultured in a 37 C, 5% CO2 incubator for 24 hours. MTT colorimetric assay was used to detect the effects of various concentrations of NAD on the proliferation of L02 cells.
The apoptosis rate of L02 cells was detected by flow cytometry. After trypsin digestion, L02 cells were prepared into a single cell suspension. The cells were inoculated into 6-well tissue culture plate with 3 65 RPMI 1640 medium containing or without NAD (1000 ug/ml) was added and cultured at 37 C for 24 hours in 5% CO2 incubator. Cells in each group were collected and the cell concentration was regulated from 1 106 to 6 106/ml. The apoptosis rate was detected by Annexin V/PI staining.
Cell cycle cells were inoculated into 6-well tissue culture plate with 3 65507 Medium was incubated in a 5% CO2 incubator at 37 C for 24 hours. Cells in each group were collected and the concentration of cells was regulated from 3 106 to 6 106 / ml. After centrifugation, PBS was washed and fixed with 70% ethanol. The percentage of cells in each phase of cell cycle was measured by flow cytometry.
The expression percentage of p53, Bax and bcl-2 protein was detected by 6-flow cytometry. The cells were immobilized in 0.5% paraformaldehyde solution for 30 minutes. The cells were lysed by membrane breaker, washed, centrifuged and supernatant was removed. Monoclonal antibodies against human p53, Bax and Bcl-2 were added to the tubes, mixed and incubated at 37 C for 1 h. FITC labeled anti mouse two antibody was added, incubated with 1H at 37 degrees centigrade, and the percentage of protein expression was detected by flow cytometry.
Caspase-3, Caspase-8 and Caspase-9 activity assay were used to collect 5 65507
The morphology of L02 cells was observed by transmission electron microscopy. The mold with conical hole was made by 3% agarose in the conical ion tube. The collected cells were centrifuged, fixed by 2.5% glutaraldehyde, fixed by 1% osmium acid, double fixed by alcohol gradient dehydration, epoxypropane immersion, lipid embedding, ultrathin section and electron staining electron microscopy.
9. Statistical data were processed by SPSS13.0 statistical software. The experimental data were expressed by X + s and analyzed by one-way ANOVA. The difference was statistically significant in P 0.05.
Result
1 MTT assay was used to detect the effect of different concentrations of NAD on cell proliferation. L02 cells were irradiated with 5.0 Gy X-ray and diluted with different concentrations of NAD (including 10% fetal bovine serum) of RPMI 1640 (0,200,400,600,800,1000,1200,1400 ug/ml), respectively. The proliferation activity of L02 cells was detected by MTT at 37 C and in a 5% CO2 incubator for 24 hours. The proliferative activity increased. When the concentration of NAD was 1000-1400 ug/ml, the proliferative activity increased to plateau stage after irradiation.
The apoptosis rate of L02 cells in each group was detected 24 hours after irradiation. The apoptosis rate of L02 cells in control group (1.50.67) and treatment group (12.85.59) was significantly lower than that in irradiation group (31.72.07) (P 0.05).
The number of L02 cells in G0/G1, S, G2/M phase at 24 hours after X-ray irradiation was significantly increased in irradiation group compared with control group, while the number of G2/M phase cells was decreased in treatment group, and the number of G1 phase cells and G2/M phase cells was increased in irradiation group. The number of cells increased during cell division and DNA synthesis.
The expression of p53 and Bax in the treatment group was lower than that in the irradiation group (P 0.05), but the expression of p53 and Bax in the irradiation group was higher than that in the control group (P 0.05). The expression of Bcl-2 was higher in the treatment group than in the irradiation group (P 0.05), but lower in the irradiation group than in the control group (P 0.05).
The activity of Caspase-3, Caspase-8 and Caspase-9 in irradiated L02 cells treated with NAD+ was significantly lower than that in irradiated L02 cells (P 0.05). The results showed that NAD could inhibit the activity of Caspase-3, Caspase-8 and Caspase-9 in irradiated L02 cells, thereby inhibiting the activity of L02 cells induced by X-ray irradiation. Apoptosis
conclusion
First, NAD+ could inhibit the X-ray-induced decrease of L02 cell proliferation and apoptosis.
Second, X-ray induced L02 cells to stagnate in G0/G1 phase, NAD + caused L02 cells to enter S phase for DNA replication. Third, NAD + could reduce the expression of p53, Bax and up-regulate the expression of Bcl-2 after irradiation, and reduce the expression of Caspase-3, Caspase-8 and Caspase-9 after irradiation.
The effect of oxidative coenzyme I (NAD+) on radiation-induced apoptosis and cell proliferation of human normal hepatocyte line L02 was observed. The results showed that NAD+ could inhibit the increase of apoptosis and restore the proliferation activity of L02 cells induced by X-ray. The pathway might be down-regulation of p53, bax, up-regulation of Bcl-2 expression, down-regulation of Caspase-3, Caspase-8 and Caspase-9 expression. Close.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
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