【摘要】： 研究背景 放射治療是惡性腫瘤治療的重要手段之一,但放射治療在殺死腫瘤細(xì)胞的同時(shí),不可避免地?fù)p傷正常組織,也因此限制了腫瘤放射治療的照射劑量強(qiáng)度,導(dǎo)致腫瘤細(xì)胞不能被完全殺滅而使病人生存質(zhì)量的下降。同時(shí),隨著醫(yī)療保健事業(yè)的發(fā)展和核工業(yè)技術(shù)的廣泛應(yīng)用,醫(yī)源性輻射和非醫(yī)源性輻射對(duì)正常組織細(xì)胞輻射損傷也正日益受到放射學(xué)家的關(guān)注。因而,細(xì)胞輻射損傷的機(jī)理及輻射損傷防護(hù)方面的研究已成為輻射分子生物學(xué)和輻射流行病學(xué)研究的的熱點(diǎn)和重點(diǎn)之一。 國(guó)內(nèi)外學(xué)者研究提示,輻射致細(xì)胞損傷的機(jī)理主要集中在如下幾個(gè)方面：1)改變細(xì)胞信號(hào)傳遞途徑：細(xì)胞經(jīng)受輻射后,細(xì)胞內(nèi)或細(xì)胞外多種信號(hào)分子受其誘導(dǎo),引起信號(hào)傳遞途徑的改變,最終導(dǎo)致細(xì)胞凋亡。其中,p53具有中心地位作用。2)DNA損傷：輻射引起DNA損傷,包括單鏈斷裂(SSB)、雙鏈斷裂(DSB)、堿基損傷和蛋白質(zhì)交聯(lián)等多種形式。3)細(xì)胞周期調(diào)控：主要通過(guò)調(diào)控細(xì)胞周期G0／G1、S、G2／M調(diào)控點(diǎn),調(diào)節(jié)細(xì)胞輻射敏感性和輻射抗性。細(xì)胞DNA損傷后,野生型p53基因誘導(dǎo)細(xì)胞進(jìn)入G1期,直到損傷DNA修復(fù),若損傷不被修復(fù),p53基因就活化誘導(dǎo)細(xì)胞凋亡的基因轉(zhuǎn)錄,使細(xì)胞發(fā)生凋亡。4)微環(huán)境變化：細(xì)胞的生存離不開其微環(huán)境,包括細(xì)胞氧供應(yīng)、pH值、營(yíng)養(yǎng)物質(zhì)、代謝產(chǎn)物、離子平衡、細(xì)胞因子等,其微環(huán)境改變可引起相關(guān)基因表達(dá)和細(xì)胞對(duì)輻射的反應(yīng)。 隨著細(xì)胞輻射損傷分子機(jī)制的深入研究,輻射損傷防護(hù)方面的研究也取得了較大進(jìn)展。其中,有關(guān)輻射損傷防護(hù)劑的研究,近年來(lái)有較多報(bào)道,主要涉及以下幾類：1)抗氧化劑,如Amifostine、NAC、VitC等；2)細(xì)胞因子,如IL-2、IL-3、IL-6、TGF等；3)微量元素,如硒、鋅等；4)中草藥成分,如人參皂甘、迷迭香等。但由于目前研究較多的抗氧化劑Amifostine、NAC毒副作用大,細(xì)胞因子作用網(wǎng)絡(luò)化特點(diǎn)以及中草藥成分難以提純,還沒有一種較為理想的輻射防護(hù)劑應(yīng)用于臨床。尋找一種新的有效的毒副作用小的輻射防護(hù)藥物對(duì)輻射防護(hù)仍具重要意義。 NAD’,化學(xué)名為煙酰胺腺嘌呤二核苷酸,是細(xì)胞能量代謝重要輔酶,參與細(xì)胞氧化還原反應(yīng)和呼吸鏈電子傳遞,在線粒體內(nèi)NAD+接受電子傳遞還原成NADH,通過(guò)電子傳遞能夠抑制自由基生成,同時(shí)生成細(xì)胞代謝所需的ATP,因此可能起到保護(hù)細(xì)胞免遭輻射損傷的作用。 本研究采用細(xì)胞生物學(xué)和分子生物學(xué)方法,初步探討NAD+輻射損傷防護(hù)作用及其機(jī)制,這對(duì)于進(jìn)一步研究正常組織細(xì)胞的放射損傷機(jī)理和探尋一種新的有效的、毒副作用小的輻射防護(hù)藥物具有理論價(jià)值和現(xiàn)實(shí)意義。 目的 本研究通過(guò)觀察氧化型輔酶Ⅰ(NAD+)對(duì)輻射損傷的人正常肝細(xì)胞株L02細(xì)胞的影響,初步探討氧化型輔酶Ⅰ(NAD+)抗輻射損傷作用及其機(jī)制。進(jìn)而為研究醫(yī)源性和非醫(yī)源性輻射損傷防護(hù)、尋求新型放射防護(hù)劑提供一種新的思路和手段。 方法 1細(xì)胞培養(yǎng)及分組正常人肝細(xì)胞株L02細(xì)胞培養(yǎng)在含10%胎牛血清的PRMI1640培養(yǎng)基中,置于37℃、5%C02培養(yǎng)箱中培養(yǎng)。將L02細(xì)胞分為3組：處理組和照射組細(xì)胞照射后分別加入含和不含NAD(1000μg/ml)的RPMI-1640培養(yǎng)基；對(duì)照組細(xì)胞未照射,僅加入RPMI-1640培養(yǎng)基。 2 X射線照射采用Varian 6-MV X線直線加速器,照射劑量為5 Gy,劑量率為500cGy/min,照射源距靶中心距離100cm。 3 MTT法檢測(cè)不同濃度NAD對(duì)細(xì)胞增殖的影響細(xì)胞以每孔3×105～5×105／ml接種于96孔組織培養(yǎng)板,100 ul／孔,細(xì)胞貼壁后去上清,加入0.01mol／L PBS(pH 7.4),按照上述條件進(jìn)行X線照射。照射后去上清,加入RPMI 1640(含10%胎牛血清)稀釋的不同濃度的NAD,分別為0、200、400、600、800、1000、1200、1400 ug/ml,100 ul/孔,在37℃、5%CO2培養(yǎng)箱中培養(yǎng)24h。用MTT比色法檢測(cè)各濃度NAD對(duì)L02細(xì)胞增殖的影響。 4流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率L02細(xì)胞經(jīng)胰蛋白酶消化后制備成單細(xì)胞懸液,以每孔3×105～5×105／ml接種于6孔組織培養(yǎng)板,1ml／孔,待細(xì)胞貼壁后去上清,加入0.01mol／L PBS(pH 7.4),按照上述條件進(jìn)行X線照射。照射后去上清,處理組和照射組分別加入含和不含NAD (1000μg/ml)的RPMI 1640培養(yǎng)基,在37℃、5%CO2孵箱中培養(yǎng)24h。收集各組細(xì)胞,調(diào)節(jié)細(xì)胞濃度為1×106～6×106／ml,采用Annexin V/PI染色,檢測(cè)細(xì)胞凋亡率。 5流式細(xì)胞儀檢測(cè)細(xì)胞周期細(xì)胞以每孔3×105～5×105／ml接種于6孔組織培養(yǎng)板,1ml／孔,待細(xì)胞貼壁后去上清,加入0.01mol/L PBS (pH7.4),按照上述條件進(jìn)行X線照射。照射后去上清,處理組和照射組分別加入含和不含NAD(1000μg／ml)的RPMI 1640培養(yǎng)基,在37℃、5%CO2孵箱中培養(yǎng)24h。收集各組細(xì)胞,調(diào)節(jié)細(xì)胞濃度為3×106～6×106／ml,離心后PBS洗滌,加入冰冷的70%乙醇固定,用流式細(xì)胞儀檢測(cè)細(xì)胞周期各時(shí)相細(xì)胞百分比。 6流式細(xì)胞儀檢測(cè)p53、bax、bcl-2蛋白表達(dá)百分率分別收集經(jīng)過(guò)上述處理的三組細(xì)胞,以0.5%多聚甲醛溶液1ml固定30min,破膜劑裂解細(xì)胞后分管、洗滌、離心后去上清,各管分別加入鼠抗人p53、bax、bcl-2單克隆抗體,混勻,37℃孵育1h。洗滌離心后分別加入FITC標(biāo)記的抗鼠二抗,37℃孵育1h,在流式細(xì)胞儀上檢測(cè)蛋白表達(dá)百分率。 7 Caspase-3、Caspase-8、Caspase-9活性測(cè)定分別收集5×106個(gè)經(jīng)過(guò)上述處理的三組細(xì)胞,按試劑盒說(shuō)明書進(jìn)行每步操作,另設(shè)未加細(xì)胞而其他操作一樣的孔為空白對(duì)照組,通過(guò)酶標(biāo)儀405nm處測(cè)定各孔吸光度OD值。 8透射電鏡觀察L02細(xì)胞形態(tài)用3%瓊脂糖在錐形離子管中制造中間帶錐形孔的模具,加入收集處理的細(xì)胞,離心,2.5%戊二醛固定、1%鋨酸固定雙重固定,酒精梯度脫水,環(huán)氧丙烷浸透,脂包埋,超薄切片,電子染色電鏡觀察。 9統(tǒng)計(jì)學(xué)處理實(shí)驗(yàn)所得數(shù)據(jù)應(yīng)用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行處理。實(shí)驗(yàn)數(shù)據(jù)用x±s表示,行單因素方差分析,P0.05為差異具有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1 MTT法檢測(cè)不同濃度NAD對(duì)細(xì)胞增殖的影響L02細(xì)胞在接受5.0Gy X線照射后,加入RPMI 1640(含10%胎牛血清)稀釋的不同濃度的NAD,分別為0、200、400、600、800、1000、1200、1400 ug/ml,在37℃、5%CO2培養(yǎng)箱中培養(yǎng)24h。MTT檢測(cè)細(xì)胞增殖活性,隨著NAD濃度的增加,細(xì)胞增殖活性升高,在NAD濃度為1000～1400 ug/ml時(shí),照射后細(xì)胞增殖活性的增加達(dá)到平臺(tái)期。因此我們把NAD濃度為1000 ug/ml作為以后的實(shí)驗(yàn)條件。 2各組細(xì)胞凋亡率細(xì)胞照射后培養(yǎng)24h檢測(cè)各組細(xì)胞凋亡率,對(duì)照組(1.50±0.67)與處理組(12.85±1.59)均顯著低于照射組(31.72±3.07)(P0.05)。說(shuō)明NAD能明顯降低X線照射后的L02細(xì)胞的凋亡率,具有抗X線誘導(dǎo)的細(xì)胞凋亡作用。 3 NAD對(duì)受照細(xì)胞周期的調(diào)節(jié)L02細(xì)胞在X線照射后24h時(shí)間點(diǎn)G0／G1、S、G2／M期細(xì)胞數(shù),照射組與對(duì)照組相比G1、S期細(xì)胞數(shù)明顯增加,G2／M期細(xì)胞數(shù)減少,表現(xiàn)為G1期阻滯；處理組與照射組相比,G1期細(xì)胞數(shù)減少,S期和G2／M期細(xì)胞數(shù)增加,表現(xiàn)為處于細(xì)胞分裂期和細(xì)胞DNA合成期細(xì)胞數(shù)增加。 4 NAD對(duì)X射線誘導(dǎo)的細(xì)胞凋亡的凋亡相關(guān)蛋白的調(diào)節(jié)L02細(xì)胞經(jīng)5.0GyX線照射后培養(yǎng)24h,檢測(cè)三組細(xì)胞的凋亡相關(guān)蛋白發(fā)現(xiàn)。處理組細(xì)胞p53、bax表達(dá)低于照射組(P0.05),而照射組高于對(duì)照組(P0.05),表明細(xì)胞受照射后p53、bax表達(dá)增強(qiáng),NAD能夠減少細(xì)胞受照射后p53、bax表達(dá)。三組細(xì)胞的bcl-2表達(dá)情況則恰恰相反,處理組細(xì)胞bcl-2表達(dá)高于照射組(P0.05),而照射組低于對(duì)照組(P0.05),表明細(xì)胞受照射后bcl-2表達(dá)減少,NAD能夠上調(diào)細(xì)胞受照射后bcl-2的表達(dá)。 5 NAD對(duì)受照射細(xì)胞Caspase-3、Caspase-8、Caspase-9活性的影響細(xì)胞經(jīng)照射培養(yǎng)24h后,NAD+處理組L02細(xì)胞Caspase-3、Caspase-8、Caspase-9的活性較照射組降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。表明NAD能抑制Caspase-3、Caspase-8、Caspase-9的活性,從而抑制X線照射引起的L02細(xì)胞凋亡 結(jié)論 一、NAD+能夠抑制X射線誘導(dǎo)的L02細(xì)胞增殖活性的下降,能夠降低X射線引起的L02細(xì)胞凋亡。 二、X射線引起L02細(xì)胞停滯于G0／G1期,NAD+使L02細(xì)胞進(jìn)入S期進(jìn)行DNA復(fù)制。三、NAD+能夠減少細(xì)胞受照射后p53、bax表達(dá),上調(diào)bcl-2的表達(dá)。能夠降低細(xì)胞受照射后Caspase-3、Caspase-8、Caspase-9活性的表達(dá)。 本實(shí)驗(yàn)通過(guò)觀察氧化型輔酶Ⅰ(NAD+)對(duì)輻射損傷的人正常肝細(xì)胞株L02細(xì)胞的影響表明：NAD+能夠?qū)筙射線引起的L02細(xì)胞凋亡的增加,恢復(fù)細(xì)胞增殖活性,其途徑可能與下調(diào)p53、bax,上調(diào)bcl-2的表達(dá),降低Caspase-3、Caspase-8、Caspase-9活性的表達(dá)有關(guān)。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 陳如松;輻射的低劑量生物效應(yīng)及分子流行病學(xué)研究現(xiàn)狀[J];輻射防護(hù)通訊;2003年01期

2 高月;馬增春;;輻射損傷防治藥物發(fā)展歷史與展望[J];輻射防護(hù)通訊;2009年05期

3 吳德昌;輻射防護(hù)的生物學(xué)基礎(chǔ)[J];輻射防護(hù);1998年Z1期

4 杜澤吉,田海林,鳴海一成,渡邊宏;抗輻射菌Deinococcus radiodurans一種新的DNA修復(fù)基因pprA的分子克隆測(cè)序及特性研究(英文)[J];輻射研究與輻射工藝學(xué)報(bào);1998年04期

5 劉戈飛;李一凡;宋旭紅;杜昆;李蕊;謝建平;黃東陽(yáng);;神經(jīng)母細(xì)胞瘤輔酶Ⅱ依賴性視黃醇脫氫/還原酶的功能性表達(dá)與生物信息學(xué)分析[J];醫(yī)學(xué)分子生物學(xué)雜志;2006年02期

6 姚莉;輻射損傷與細(xì)胞周期[J];國(guó)外醫(yī)學(xué)(放射醫(yī)學(xué)核醫(yī)學(xué)分冊(cè));2004年01期

7 馬秋蘭,陳彪,邵明,董秀敏,楊靜芳;NAD(P)H:醌氧化還原酶基因多態(tài)性與阿爾茨海默病遺傳易感性的關(guān)系[J];中華老年醫(yī)學(xué)雜志;2001年02期

8 賈克明;細(xì)胞凋亡的研究[J];中華內(nèi)科雜志;1999年03期

9 宋旭紅;梁斌;劉戈飛;李蕊;謝建平;黃東陽(yáng);;宮頸鱗癌組織中NRDR選擇性剪接新亞型的鑒定及意義[J];腫瘤;2006年12期

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