【摘要】：中子輻射較γ射線(xiàn)損傷重,腸道是中子輻射高度敏感的靶器官,中子輻射腸道損傷重、難恢復(fù)。目前中子輻射腸道損傷的病理特點(diǎn)已基本明確,然而其損傷機(jī)制仍未完全闡明,尚缺乏有效的治療措施。為此,本課題在既往研究的基礎(chǔ)上,復(fù)制中子照射腸上皮細(xì)胞損傷整體和離體模型,以NF-κB信號(hào)通路在中子輻射腸道損傷中的作用為切入點(diǎn),開(kāi)展NF-κB信號(hào)通路在中子輻射致腸上皮細(xì)胞損傷中的調(diào)控作用研究,為闡明中子輻射腸上皮細(xì)胞損傷的分子機(jī)制、尋找新的防治措施和發(fā)現(xiàn)新的治療靶點(diǎn)提供依據(jù)。 材料與方法 一、實(shí)驗(yàn)動(dòng)物分組與模型制作方法 選取二級(jí)雄性BALB/c小鼠120只,隨機(jī)分為對(duì)照組(C組,n=30)、3Gy中子照射組(N組,n=60)和3Gy中子照射+姜黃素組(Cur組,n=30);采用3Gy中子全身均勻照射N(xiāo)組和Cur組小鼠;照射后即刻給予Cur組小鼠腹腔注射姜黃素混懸液1次,劑量為200 mg·kg-1·d-1,此后連續(xù)給藥5d(1次/d),N組和C組小鼠腹腔注射同體積的生理鹽水。 二、小鼠整體行為和空腸組織結(jié)構(gòu)光鏡和電鏡觀察 照射后每天觀察并記錄動(dòng)物整體行為、體重變化、腹瀉及死亡情況;分別于照射后6h、1d、3d和5d活殺C組和N組動(dòng)物,Cur組于照射后3d和5d活殺,留取各組各時(shí)間點(diǎn)小鼠空腸組織;通過(guò)光鏡和電鏡觀察3Gy中子照射及應(yīng)用Cur后小鼠空腸組織結(jié)構(gòu)以及超微結(jié)構(gòu)的變化。 三、小鼠空腸上皮細(xì)胞增殖與死亡的檢測(cè) 采用AgNOR和Feulgen染色方法檢測(cè)中子照射及應(yīng)用姜黃素后小鼠空腸上皮細(xì)胞的嗜銀蛋白及DNA含量變化;采用TUNEL方法檢測(cè)小鼠空腸上皮細(xì)胞凋亡改變。 四、小鼠空腸組織NF-κB通路中信號(hào)分子檢測(cè) 通過(guò)IHC和EMSA方法檢測(cè)中子照射及應(yīng)用姜黃素后小鼠空腸上皮細(xì)胞NF-κB的表達(dá)及活性變化;采用WB檢測(cè)小鼠空腸上皮細(xì)胞NF-κB、IKKβ、IκBα、PI3K和Akt的表達(dá)變化;通過(guò)Co-IP技術(shù)檢測(cè)小鼠空腸組織中Akt和IKKβ相互作用;通過(guò)Real-time PCR方法檢測(cè)IKKβmRNA的變化。 五、IEC-6細(xì)胞培養(yǎng)、分組、照射及處理 IEC-6細(xì)胞傳代培養(yǎng)后,隨機(jī)分為5組:對(duì)照組(C)、4Gy中子照射組(N)、4Gy+LY294002組(LY)、10Gyγ射線(xiàn)照射組(R)和10Gyγ射線(xiàn)+LY294002組(LY2);分別采用4Gy中子和10Gyγ射線(xiàn)對(duì)IEC-6細(xì)胞進(jìn)行均勻照射;LY294002處理方法:于中子和γ射線(xiàn)照射前24h,LY組和LY2組細(xì)胞更換為含LY294002終濃度為10μmol/L的培養(yǎng)液。 六、IEC-6細(xì)胞增殖與死亡檢測(cè) 于中子和γ射線(xiàn)照射后6h和24h,通過(guò)IPCM觀察IEC-6細(xì)胞的形態(tài)變化;于中子照射后6h和24h收集細(xì)胞留取樣品,γ射線(xiàn)照射后15min、30min、1h、6h和24h收集細(xì)胞留取樣品;分別采用MTT方法和FCM檢測(cè)IEC-6細(xì)胞的增殖活力以及凋亡與壞死率改變。 七、IEC-6細(xì)胞NF-κB通路中信號(hào)分子檢測(cè) 通過(guò)WB方法檢測(cè)中子和γ射線(xiàn)照射后IEC-6細(xì)胞中NF-κB、IKKα/β和IκBα的表達(dá)及其磷酸化水平改變。 八、圖像分析和定量方法 (一)小鼠空腸細(xì)胞AgNOR、DNA含量及NF-κB免疫組化結(jié)果,采用CMIAS-Ⅱ系列多功能真彩色病理圖像分析系統(tǒng),在光鏡10×40倍視野下,每組切片采集10個(gè)視野,檢測(cè)其MOD和IOD。 (二)WB結(jié)果采用Image Pro 5.0軟件進(jìn)行圖像分析,測(cè)定電泳條帶的IOD,將各目的蛋白條帶與相應(yīng)內(nèi)參GAPDH的IOD比值進(jìn)行定量分析。 (三)IKKβ和相應(yīng)GAPDH mRNA應(yīng)用7300 system SDS軟件進(jìn)行定量分析。 (四)Co-IP結(jié)果采用CMIAS-Ⅱ圖像分析系統(tǒng)進(jìn)行IOD測(cè)定和定量分析。 九、統(tǒng)計(jì)學(xué)處理 文中數(shù)據(jù)以均數(shù)和標(biāo)準(zhǔn)差(?X±s)表示;采用SPSS 13.0統(tǒng)計(jì)軟件進(jìn)行一元方差分析Excel軟件作圖;vs對(duì)照組,*示P0.05,**示P0.01;vs照射組,#示P0.05,##示P0.01。 實(shí)驗(yàn)結(jié)果 一、小鼠整體行為、空腸組織學(xué)及超微結(jié)構(gòu)改變 (一)小鼠整體行為的改變:3Gy中子照射及應(yīng)用姜黃素后,小鼠精神狀態(tài)逐漸變差,不進(jìn)食水,體重進(jìn)行性下降,并出現(xiàn)嚴(yán)重腹瀉癥狀;于照射后3~5d內(nèi)全部死亡,出現(xiàn)了腸型放射病典型的“三天半效應(yīng)”。 (二)小鼠空腸組織學(xué)改變:3Gy中子照射后,光鏡下可見(jiàn)腸黏膜大面積壞死脫落,絨毛上皮細(xì)胞稀疏、排列較亂,細(xì)胞腫脹,隱窩細(xì)胞見(jiàn)核固縮、碎裂,其數(shù)量進(jìn)行性減少;照射后3~5d可見(jiàn)隱窩細(xì)胞再生,但隱窩中細(xì)胞數(shù)目較少;姜黃素組在治療3d和5d時(shí)隱窩細(xì)胞再生較明顯,且可見(jiàn)豐富的絨毛深入腸腔。 (三)小鼠空腸超微結(jié)構(gòu)改變:3Gy中子照射后3d,電鏡下,小鼠空腸隱窩細(xì)胞減少,可見(jiàn)胞核碎裂,核染色質(zhì)濃縮邊移,隱窩細(xì)胞呈空泡狀,出現(xiàn)壞死和凋亡的細(xì)胞,以壞死為主,照射后5d可見(jiàn)小腸絨毛有輕度增生;Cur組于3Gy中子照射后3~5d,空腸組織中可見(jiàn)有新生微絨毛及隱窩,隱窩內(nèi)細(xì)胞數(shù)目較照射組略有增加。 二、小鼠空腸上皮細(xì)胞嗜銀蛋白及凋亡改變 (一)小鼠空腸上皮細(xì)胞嗜銀蛋白和DNA含量:3Gy中子照射照射后6h~5d,N組和Cur組小鼠空腸上皮細(xì)胞嗜銀蛋白和DNA含量進(jìn)行性下降(P0.01),照射后3~5d,Cur組小鼠空腸上皮細(xì)胞嗜銀蛋白和DNA含量均較照射組增加(P0.05)。 (二)小鼠空腸上皮細(xì)胞凋亡TUNEL檢測(cè)結(jié)果:3Gy中子照射照射后6h~1d,N組小鼠空腸上皮可見(jiàn)大量凋亡細(xì)胞;照射后3d,N組和Cur組小鼠空腸上皮細(xì)胞偶見(jiàn)凋亡細(xì)胞。 三、小鼠空腸上皮細(xì)胞NF-κB及通路中信號(hào)分子的變化 (一)小鼠空腸上皮細(xì)胞NF-κB的表達(dá)變化:NF-κB在正常腸絨毛及隱窩上皮細(xì)胞胞漿內(nèi)呈弱陽(yáng)性,3Gy中子照射后6h~5d,NF-κB于上皮細(xì)胞核呈陽(yáng)性,并于照射后5d達(dá)到高峰(P0.01),Cur組在照射后3d和5d,NF-κB在絨毛及隱窩上皮細(xì)胞核及漿內(nèi)表達(dá)呈陽(yáng)性,強(qiáng)度弱于照射組(P0.05)。 (二)小鼠空腸組織NF-κB的DNA結(jié)合活性變化:3Gy中子照射后6h~3d,N組小鼠空腸組織中NF-κB與DNA結(jié)合活性明顯增強(qiáng);Cur組在照射后3d,小鼠空腸組織中NF-κB與DNA結(jié)合活性較N組明顯降低。 (三)小鼠空腸組織NF-κB通路中信號(hào)分子的變化:(1)小鼠空腸組織NF-κB和IKKβ蛋白于3Gy中子照射后6h和1d表達(dá)上調(diào)(P0.05),照射后3d和5d表達(dá)明顯上調(diào)(P0.01);Cur組在照射后3d和5d,NF-κB和IKKβ表達(dá)下調(diào)(P0.05)。(2)小鼠空腸組織IκBα蛋白于3Gy中子照射后6h和1d表達(dá)下調(diào)(P0.05),照射后3d和5d表達(dá)明顯下調(diào)(P0.01);Cur組在照射后3d和5d,IκBα表達(dá)上調(diào)(P0.05)。(3)3Gy中子照射后6h小鼠空腸組織IKKβmRNA表達(dá)增加(P0.05),至照射后24h表達(dá)明顯增加(P0.01);Cur組在照射后6h和24h,IKKβmRNA表達(dá)水平較N組明顯降低(P0.05)。 四、小鼠空腸上皮細(xì)胞PI3K、Akt的表達(dá)及Akt和IKKβ相互作用變化 (一)小鼠空腸組織PI3K和Akt的表達(dá)變化:小鼠空腸組織PI3K和Akt蛋白于3Gy中子照射后6h和1d表達(dá)上調(diào)(P0.05),照射后3d和5d表達(dá)明顯上調(diào)(P0.01);Cur組在照射后3d和5d,PI3K和Akt表達(dá)下調(diào)(P0.05)。 (二)小鼠空腸組織Akt和IKKβ的相互作用:3Gy中子照射后6h和1d,小鼠空腸組織中Akt和IKKβ相互作用明顯增強(qiáng)(P0.01),照射后3d和5d,Akt和IKKβ的相互作用減弱(P0.05);Cur組在照射后3d和5d,Akt和IKKβ相互作用較照射組減弱(P0.05)。 五、IEC-6細(xì)胞形態(tài)、增殖活力、凋亡及壞死率改變 (一)IEC-6細(xì)胞形態(tài)的變化:4Gy中子和10Gyγ射線(xiàn)照射后6h和24h,細(xì)胞腫脹,形態(tài)變圓,折光性增強(qiáng);應(yīng)用LY294002處理后,培養(yǎng)液漂浮大量死細(xì)胞。 (二)IEC-6細(xì)胞增殖活力、凋亡及壞死率改變:4Gy中子和10Gyγ射線(xiàn)照射后,細(xì)胞增殖活力較對(duì)照組明顯下降(P0.01),細(xì)胞凋亡與壞死率均顯著增加(P0.01),輻射后6h凋亡達(dá)高峰,24h以壞死為主;應(yīng)用LY294002處理后,細(xì)胞的增殖活力較照射組明顯降低(P0.05),凋亡和壞死率增加(P0.05)。 六、IEC-6細(xì)胞NF-κB通路中信號(hào)分子變化及蛋白磷酸化改變 (一)IEC-6細(xì)胞NF-κB通路中信號(hào)分子變化:(1)IEC-6細(xì)胞內(nèi)NF-κB、IKKα和IKKβ蛋白于4Gy中子照射后6h表達(dá)上調(diào)(P0.05),24h表達(dá)明顯上調(diào)(P0.01);應(yīng)用LY294002處理組,于照射后6h和24h,上述蛋白表達(dá)均下調(diào)(P0.05)。(2)IκBα于4Gy中子照射后6h表達(dá)下調(diào)(P0.05),24h表達(dá)明顯下調(diào)(P0.01);應(yīng)用LY294002處理組,于照射后6h和24h,IκBα表達(dá)上調(diào)(P0.05)。 (二)IEC-6細(xì)胞NF-κB信號(hào)通路中蛋白磷酸化改變:(1)10Gyγ射線(xiàn)照射后15~30min,IEC-6細(xì)胞內(nèi)磷酸化NF-κB和磷酸化IKKα/β蛋白表達(dá)上調(diào)(P0.05),照射后1h表達(dá)明顯上調(diào)(P0.01);應(yīng)用LY294002處理組,三種磷酸化蛋白的表達(dá)均下調(diào)(P0.05)。(2)IEC-6細(xì)胞內(nèi)磷酸化IκBα蛋白在10Gyγ射線(xiàn)照射后15~30min未見(jiàn)表達(dá),1h見(jiàn)明顯表達(dá)(P0.01);應(yīng)用LY294002處理組,于照射后1h磷酸化IκBα的表達(dá)較照射組明顯上調(diào)(P0.01)。 結(jié)論 一、3Gy中子照射可引起小鼠輕度腸型放射病,腸上皮嚴(yán)重?fù)p傷,上皮細(xì)胞凋亡與壞死并存,細(xì)胞增殖能力降低;表明3Gy中子照射成功建立了中子輻射小鼠空腸損傷的動(dòng)物模型。 二、姜黃素治療可減輕中子輻射腸道損傷的程度,促進(jìn)空腸粘膜上皮再生修復(fù),對(duì)中子輻射腸上皮損傷具有保護(hù)作用。 三、3Gy中子照射可使小鼠空腸NF-κB信號(hào)通路活化,通路中關(guān)鍵信號(hào)分子IKKβ表達(dá)上調(diào),NF-κB表達(dá)上調(diào)且發(fā)生核轉(zhuǎn)位,進(jìn)而可能通過(guò)調(diào)控多種參與炎癥反應(yīng)的靶基因,促進(jìn)炎癥因子的表達(dá),導(dǎo)致中子照射后腸道炎癥發(fā)生。 四、姜黃素可抑制中子照射后小鼠空腸NF-κB信號(hào)通路活化,下調(diào)NF-κB和IKKβ的表達(dá)水平,這可能是其發(fā)揮保護(hù)作用的機(jī)制之一。 五、3Gy中子照射后,小鼠空腸Akt和NF-κB信號(hào)通路中關(guān)鍵激酶IKKβ存在相互作用,表明中子照射后NF-κB信號(hào)通路受到PI3K/Akt信號(hào)通路的調(diào)控。 六、4Gy中子和10Gyγ射線(xiàn)照射均可使IEC-6細(xì)胞增殖活力降低,凋亡與壞死率增加,應(yīng)用LY294002可以進(jìn)一步降低照射后IEC-6細(xì)胞增殖活力,增加凋亡和壞死率。 七、4Gy中子和10Gyγ射線(xiàn)照射均可使IEC-6細(xì)胞NF-κB信號(hào)通路活化,應(yīng)用PI3K的抑制劑LY294002可相應(yīng)抑制NF-κB信號(hào)通路的活化,表明NF-κB信號(hào)通路活化可對(duì)IEC-6細(xì)胞損傷發(fā)揮保護(hù)作用。
[Abstract]:The radiation of neutron radiation is heavier than that of gamma ray, and the intestinal tract is a highly sensitive target organ of neutron radiation. The intestinal damage of neutron radiation is heavy and difficult to recover. The pathological characteristics of the intestinal damage of neutron radiation are basically clear, but the damage mechanism is still not fully elucidated, and the effective treatment measures are still lacking. Therefore, this subject has been replicated on the basis of previous research. The effects of NF- kappa B signaling pathway on the intestinal damage of neutron radiation are the breakthrough point, and the regulation of the NF- kappa B signal pathway in the intestinal epithelial cell damage caused by neutron radiation is studied. The molecular mechanism of the neutron radiation of intestinal epithelial cell damage is clarified to find new preventive measures. And the discovery of new therapeutic targets provides a basis.
Materials and methods
First, group and model making method of experimental animals
120 male BALB/c mice were randomly divided into control group (group C, n=30), 3Gy neutron irradiation group (N group, n=60) and 3Gy neutron irradiation + curcumin group (Cur group, n=30), N group and Cur group were irradiated with 3Gy neutrons all over the whole body. After irradiation, the intraperitoneal injection of curcumin suspension was given 1 times and the dose was 200. The mice in N and C groups were intraperitoneally injected with the same volume of normal saline for 5 days (once a day).
2. Observation of whole behavior and jejunum structure in mice by light and electron microscopy
The overall behavior, weight change, diarrhea and death of the animals were observed and recorded every day after irradiation. After irradiation, 6h, 1D, 3D and 5D were killed in C and N groups. The Cur group killed the 3D and 5D after irradiation and left the jejunum tissues of each time point in each group. The tissue structure of the jejunum in the mice after the 3Gy neutron irradiation and the Cur after the use of Cur was observed by light and electron microscopy. Changes in the ultrastructure.
3. Detection of proliferation and death of mouse jejunal epithelial cells
AgNOR and Feulgen staining were used to detect the changes of eosinophils and DNA in the jejunum epithelial cells after neutron irradiation and the use of curcumin. The apoptosis of mouse jejunum epithelial cells was detected by TUNEL method.
4. Detection of signal molecules in NF-kappa B pathway in mouse jejunum
The expression and activity changes of NF- kappa B in jejunum epithelial cells after neutron irradiation and curcumin were detected by IHC and EMSA methods. The expression of NF- kappa B, IKK beta, I kappa B alpha, PI3K and Akt were detected by WB. Changes in IKK beta mRNA.
Five, IEC-6 cell culture, grouping, irradiation and treatment
After subculture, IEC-6 cells were randomly divided into 5 groups: the control group (C), the 4Gy neutron irradiation group (N), the 4Gy+LY294002 group (LY), the 10Gy gamma ray irradiation group (R) and the 10Gy gamma ray +LY294002 group (LY2). The culture solution containing LY294002 at the final concentration of 10 micromol/L was replaced.
Six, IEC-6 cell proliferation and death detection
After irradiation of neutron and gamma rays 6h and 24h, the morphological changes of IEC-6 cells were observed by IPCM. After the neutron irradiation, 6h and 24h collected the cells to collect the samples. After gamma ray irradiation, the samples were collected by 15min, 30min, 1H, 6h and 24h, and the proliferation activity and the apoptosis and necrosis rate of the cells were detected by MTT methods.
Seven, detection of signal molecules in the NF- kappa B pathway of IEC-6 cells
WB method was used to detect the expression of NF- kappa B, IKK alpha / beta and I kappa B alpha and their phosphorylation levels in IEC-6 cells after neutron and gamma irradiation.
Eight, image analysis and quantitative method
(I) the AgNOR, DNA content and the immunohistochemical results of NF- kappa B in mouse jejunum cells, using CMIAS- II Series multi-function true color pathological image analysis system. Under the 10 x 40 fold visual field of light microscope, 10 fields of vision were collected from each group, and their MOD and IOD. were detected.
(two) WB results were analyzed by Image Pro 5 software, and the IOD of the electrophoresis strip was measured. The ratio of each target protein strip to the IOD ratio of the corresponding internal reference GAPDH was quantitatively analyzed.
(three) IKK beta and corresponding GAPDH mRNA were quantitatively analyzed by 7300 system SDS software.
(four) Co-IP results were analyzed by IOD and quantitative analysis using CMIAS- II image analysis system.
Nine, statistical treatment
The data in this paper are represented by mean and standard deviation (? X + s); Excel software is made by using SPSS 13 statistical software for the analysis of one variance analysis software; vs control group, * P0.05, vs irradiation group, vs exposure group, P0.01. P0.01., P0.01. P0.01..
experimental result
(1) the whole behavior of mice, the histological and ultrastructural changes of jejunum.
(1) changes in the overall behavior of mice: after 3Gy neutron irradiation and curcumin application, the mental state of the mice gradually became worse, no water was eaten, the weight of the mice decreased and the symptoms of severe diarrhea occurred. After the irradiation, all the mice died in 3~5d, and the typical "three half day and half effect" of intestinal type radiation disease was found.
(two) histological changes in the jejunum of mice: after 3Gy neutron irradiation, large area necrosis and shedding of intestinal mucosa were observed under light microscope, and the villous epithelial cells were sparsely scattered, the cells were swollen, the recess cells were reformed, the number of the recess cells were reduced, and the recess cells regenerated after irradiation, but the number of the recess cells was less; the curcumin group was in the treatment of 3D. The regeneration of crypt cells was more obvious when compared with 5D, and abundant villus were seen in the intestinal cavity.
(three) the ultrastructural changes in the jejunum of mice: 3Gy neutron irradiated 3D, under electron microscope, the cells of the jejunum recess decreased, the nuclei of the nucleus were broken, the nucleus chromatin was concentrated, the recess cells were vacuoled, necrotic and apoptotic cells appeared, the necrotic cells were necrotic, and the 5D of the small intestine had mild hyperplasia after irradiation; group Cur was irradiated with 3Gy neutrons, 3~5d, jejunum after irradiation. Microvilli and crypt were seen in the tissue. The number of cells in the crypt increased slightly compared with that in the irradiated group.
Two, the changes of Ag protein and apoptosis in mouse jejunal epithelial cells.
(1) the content of Ag and DNA in the mouse jejunum epithelial cells: after irradiation of 3Gy neutron irradiation, 6h~5d, N and Cur mice were decreased (P0.01) in the jejunum epithelial cells (P0.01). The contents of eosinophils and DNA in the jejunum epithelial cells of the mice were increased after irradiation (P0.05).
(two) TUNEL detection results of apoptosis in mouse jejunum epithelial cells: a large number of apoptotic cells were found in the jejunum epithelium of group N after irradiation of 3Gy neutron irradiation, and apoptotic cells were found in 3D, N group and Cur group of jejunum epithelial cells.
Three, the changes of NF- kappa B and signal molecules in the pathway of mouse jejunal epithelial cells.
(1) the expression of NF- kappa B in the mouse jejunum epithelial cells: NF- kappa B was weakly positive in the cytoplasm of normal intestinal villi and recess epithelial cells, 6h~5d after 3Gy neutron irradiation, NF- kappa B in epithelial cell nuclei and 5D reached its peak after irradiation (P0.01). Cur group was expressed in the nucleus and pulp of the villus and recess epithelium after irradiation. The intensity is weaker than that of the irradiated group (P0.05).
(two) the DNA binding activity of NF- kappa B in the jejunum of mice: 3Gy neutron irradiated 6h~3d, and the binding activity of NF- kappa B and DNA in the jejunum tissue of group N mice increased obviously; the Cur group was 3D after irradiation, and the binding activity of NF- kappa and kappa was significantly lower in the jejunum tissue of mice than that in the mice.
(three) the changes in the signal molecules in the NF- kappa B pathway in the jejunum tissue of mice: (1) the expression of 6h and 1D in the jejunum tissue of mice was up to up (P0.05) after 3Gy neutron irradiation, and the expression of 3D and 5D after irradiation was obviously up (P0.01). After irradiation, the expression of 6h and 1D was down (P0.05), and the expression of 3D and 5D was obviously down (P0.01). The expression of 3D and 5D, I kappa B alpha was up (P0.05) in Cur group after irradiation. (3) after irradiation, the expression of the mouse jejunum tissue was increased, and the expression was obviously increased after irradiation. Decrease (P0.05).
Four, the expression of PI3K, Akt and the interaction of Akt and IKK beta in mouse jejunal epithelial cells.
(1) the expression of PI3K and Akt in the jejunum tissue of mice: the expression of PI3K and Akt protein in the jejunum tissue of mice was up regulated by 6h and 1D after 3Gy neutron irradiation (P0.05), and the expression of 3D and 5D increased obviously after irradiation (P0.01).
(two) the interaction of Akt and IKK beta in the jejunum tissue of mice: 6h and 1D after 3Gy neutron irradiation, the interaction of Akt and IKK beta in the jejunum tissue of mice increased significantly (P0.01). The interaction between 3D and 5D, Akt and IKK beta was weakened after irradiation.
Five, IEC-6 cell morphology, proliferation activity, apoptosis and necrosis rate change.
(1) changes in IEC-6 cell morphology: 6h and 24h after irradiation of 4Gy neutrons and 10Gy gamma rays, the cells swelled, the morphology became round, and the refraction was enhanced; after the LY294002 treatment, the culture fluid was floating a large number of dead cells.
(two) the proliferation activity, apoptosis and necrosis rate of IEC-6 cells were changed: after 4Gy neutron and 10Gy gamma ray irradiation, the cell proliferation activity was significantly lower than that of the control group (P0.01), the apoptosis and necrosis rate increased significantly (P0.01), the apoptosis reached the peak of 6h after radiation, and the 24h was mainly necrotic, and the proliferation activity of the cells was significantly lower than that of the irradiated group after the use of LY294002. Low (P0.05), the increase of apoptosis and necrosis (P0.05).
Six, changes in signal molecules and protein phosphorylation in NF- kappa B pathway in IEC-6 cells.
(1) the change of signal molecules in the NF- kappa B pathway of IEC-6 cells: (1) NF- kappa B in IEC-6 cells, IKK A and IKK beta protein expression up up (P0.05) after 4Gy neutron irradiation, and the expression of 24h expression was obviously up-regulated. The expression of h was significantly down regulated (P0.01), and the expression of 6h and 24h, I kappa B was increased (P0.05) in LY294002 treated group.
(two) the changes in protein phosphorylation in the NF- kappa B signaling pathway in IEC-6 cells: (1) the expression of phosphorylated NF- kappa B and phosphorylated IKK alpha / beta protein expression in IEC-6 cells increased after 10Gy gamma ray irradiation (P0.05), and the 1H expression was obviously up (P0.01) after irradiation. The expression of three phosphorylated proteins decreased (2) phosphoric acid within the cells. The expression of I kappa B alpha protein was not expressed in 15~30min after 10Gy gamma ray irradiation, and the expression of 1H was obviously expressed (P0.01). The expression of I kappa B alpha in 1H phosphorylated after irradiation was significantly up (P0.01) in the LY294002 treatment group than in the irradiated group.
conclusion
First, 3Gy neutron irradiation can cause mild intestinal radiation disease in mice, the intestinal epithelium is seriously damaged, the apoptosis and necrosis of epithelial cells coexist, and the cell proliferation ability is reduced, which indicates that 3Gy neutron irradiation has successfully established the animal model of the jejunum damage in mice irradiated by neutron radiation.
Two, curcumin treatment can reduce the degree of intestinal injury induced by neutron irradiation, promote the regeneration and repair of jejunal epithelium, and protect the intestinal epithelium from neutron irradiation.
Three, 3Gy neutron irradiation can activate the NF- kappa B signal pathway of jejunum in mice. The expression of key signal molecule IKK beta in the pathway is up-regulated and the expression of NF- kappa B is up-regulated and has nuclear transposition, which may promote the expression of inflammatory factors by regulating various target genes involved in the inflammatory response, leading to the occurrence of intestinal inflammation after the irradiation of the middle children.
Four, curcumin can inhibit the activation of NF- kappa B signal pathway in jejunum of mice after neutron irradiation, and down regulate the expression level of NF- kappa B and IKK beta, which may be one of its protective mechanisms.
Five, after 3Gy neutron irradiation, the key kinase IKK beta in the Akt and NF- kappa B signaling pathway in the mouse jejunum was interacted, indicating that the NF- kappa B signal pathway was regulated by the PI3K/Akt signaling pathway after the neutron irradiation.
Six, 4Gy neutrons and 10Gy gamma rays can reduce the proliferation of IEC-6 cells and increase the apoptosis and necrosis rate. LY294002 can further reduce the proliferation activity of IEC-6 cells after irradiation and increase the rate of apoptosis and necrosis.
Seven, both 4Gy neutrons and 10Gy gamma rays can activate the NF- kappa B signaling pathway in IEC-6 cells. The activation of NF- kappa B signaling pathway can be inhibited by PI3K inhibitor LY294002, indicating that NF- kappa B signaling pathway can be activated for IEC-6 cells.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R363

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