【作者】 王瑞娟；

【导师】 彭瑞云；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病理学与病理生理学， 2009， 博士

【摘要】 目的和意义中子辐射肠损伤重、难恢复,且目前尚无防治良策。本课题组前期研究发现,IL-11可减轻中子辐射对肠道损伤,促进肠上皮再生,其防治机制与IL-11刺激肠上皮细胞IL-11受体表达上调并激活Jak/STAT信号转导通路有关。但有关IL-11诱导的ERK及PI3K通路在中子辐射肠上皮细胞损伤发生发展中的变化以及通路之间的相互作用、IL-11对Bax和Bcl-2的调节研究尚属空白。在中子辐射诱发的损伤中,多种基因表达的变化可能发挥重要作用,基因芯片技术可高通量地筛选损伤后差异表达基因谱,并为深入探讨IL-11信号转导机制提供线索。为此,本研究对上述问题进行探讨,为阐明中子辐射肠道损伤及IL-11调控的分子机制,并为寻找新的防治措施奠定理论基础。材料与方法168只BALB/c二级雄性小鼠,分别经3Gy中子和10Gyγ射线照射,中子照后腹腔注射500μg/kg的rhIL-11,每日1次,连用5 d,于照后6 h、1 d、2 d、3 d和5 d活杀取材。同时采用4Gy中子和10Gyγ射线照射IEC-6细胞,并于照射前12 h或照射后即刻给予100ng/ml的rhIL-11,于rhIL-11处理前12 h分别加入10μM U0126或LY294002。采用基因芯片、RT-PCR、HE染色、MTT、流式细胞术、Hoechst染色、免疫组化、免疫印迹和图像分析等技术,研究中子照射后小鼠空肠组织差异表达基因及肠道病理形态的变化;探讨IL-11及U0126和LY294002对小鼠肠道病理形态、IEC-6细胞增殖活力及凋亡和坏死率的影响以及IEC-6细胞Raf-1、MEK1/2、ERK1/2、PI3K、PDK1、PTEN和Akt表达及活化的影响。实验结果1.小鼠死亡情况及小肠病理改变:(1)3Gy中子照射后小鼠于3~5 d内全部死亡,并出现腹泻症状,体重进行性下降;照射后6 h~3 d,肠黏膜大面积坏死脱落,绒毛上皮细胞稀疏、排列紊乱,细胞肿胀;隐窝细胞数量急剧减少,伤后2 d时隐窝中细胞少见、结构破坏,绒毛上皮及基部细胞浆出现嗜酸性变,核浓缩及核碎片形成,照后3 d偶见隐窝细胞再生;IL-11治疗组照后3~5 d小鼠存活曲线右移,腹泻率降低,治疗后3 d隐窝再生较明显,肠绒毛上皮细胞数目较多。2.小鼠小肠差异表达基因改变:3Gy中子照射后6 h,小鼠空肠组织中存在608个差异表达的基因,其中上调的有277个,下调的有331个,差异基因涉及细胞的增殖与凋亡、MAPK和PI3K/Akt等多条信号通路等。3. IEC-6细胞生物学行为的改变:(1)10Gyγ射线照射后6 h,IEC-6细胞凋亡和坏死率增加、增殖活力下降、细胞周期阻滞;IL-11处理组凋亡和坏死率下降。(2)4Gy中子照射后6 h,IEC-6细胞肿胀变圆、凋亡和坏死率增加、增殖活力下降;照射后24 h,IEC-6细胞凋亡和坏死率无明显变化,增殖活力下降。IL-11处理组,其形态较好、增殖活力升高、凋亡和坏死率下降。4. IEC-6细胞ERK信号通路的改变及U0126对其生物学行为影响:(1)10Gyγ射线照射后6~24h,IEC-6细胞Raf-1表达和活化及MEK1/2活化升高,ERK1/2活化减弱;与照射组比较,IL-11处理组于照射后5~15min可增加Raf-1和MEK1/2活性,其余时间点改变不明显,照射后6 h抑制ERK1/2活化。(2)4Gy中子照后6~24 h,Raf-1表达和活化及MEK1/2活化无明显变化,ERK1/2表达和活化升高;与照射组比较,IL-11处理组照后6 h,Raf-1表达变化不明显,照后6~24 h,MEK1/2活化升高,IL-11在照后6 h抑制ERK1/2表达及活化,照后24 h增加ERK1/2表达及活化。(3)IL-11处理前应用U0126处理IEC-6细胞,4Gy中子照射后6~24 h,其增殖活力较IL-11处理组下降,凋亡和坏死率变化不明显。5. IEC-6细胞PI3K/Akt信号通路的改变及LY294002对其生物学行为影响:(1)10Gyγ射线照射后6 h,IEC-6细胞PI3K表达和Akt活化减弱,Akt表达、PDK1及PTEN活化增加;IL-11处理组PI3K表达和Akt活化增加,Akt表达、PDK1及PTEN活化减弱。(2)4Gy中子照后6 h,IEC-6细胞PI3K表达减少,24 h恢复;照后6~24 h,PDK1及PTEN活化增加,Akt活化减弱;IL-11处理组照后6~24 h,PI3K表达和Akt活化增强,PDK1和PTEN活化减弱。(3)IL-11处理前应用LY294002处理IEC-6细胞,4Gy中子照射后6~24 h,其增殖活力较IL-11处理组下降,凋亡和坏死率变化增加。6. ERK和PI3K/Akt信号通路间的相互调控:(1)U0126处理组IEC-6细胞在照后6~24 h,ERK1/2表达和活化较IL-11处理组无明显变化,但PDK1及Akt活化均增加。(2)LY294002处理组IEC-6细胞与IL-11处理组比较,照后6~24 h,Akt活化及ERK1/2的表达和活化均下降。7.小鼠小肠Bax和Bcl-2表达的改变:(1)Bax于正常小鼠小肠绒毛上皮细胞呈颗粒状阳性,于隐窝细胞呈弱阳性;3Gy中子和10Gyγ线照射后6 h~3 d,Bax表达显著增强;3Gy中子照射后应用IL-11治疗,Bax表达于照后3 d减弱。(2)Bcl-2于正常小鼠肠绒毛和隐窝细胞浆呈弱阳性表达;3Gy中子照射后6 h~3d,Bcl-2表达无明显变化;10Gyγ线照射后6 h~3 d,Bcl-2表达升高;3Gy中子照射后应用IL-11治疗,Bcl-2表达于照后3 d升高。结论1.中子照射可致小鼠空肠多靶点的损伤,涉及包括PI3K/Akt和MAPK在内的多条通路。2.中子照射后肠上皮细胞内ERK1/2持续激活,IL-11对其活化呈先抑制后激活的波动趋势,提示ERK在中子辐射肠上皮细胞损伤与修复的不同阶段发挥不同的作用。3.中子照射时,PI3K/Akt通路活化受抑制,IL-11可通过促进其激活发挥细胞保护作用。4.中子和γ射线对肠上皮细胞ERK和PI3K/Akt的影响不同,可能是二者致伤差异的分子机制。5. IL-11调控中子辐射肠上皮细胞时,ERK和PI3K/Akt通路在IEC-6细胞内可能通过一个反馈环路联系:ERK1/2的持续激活反馈性抑制了PI3K/Akt的活性,IL-11对ERK的抑制作用,则协同IL-11促进了PI3K/Akt途径活性的上调;ERK1/2的活性受PI3K的调节而不依赖于MEK1/2。6.在中子辐射时,LY294002阻断IL-11对肠上皮细胞的保护作用较U0126更显著,提示ERK和PI3K/Akt通路在介导IL-11保护作用中地位不同,ERK主要与细胞的增殖有关,而PI3K/Akt则与细胞的增殖与死亡反应均有关。7.中子辐射肠道损伤时,IL-11可通过减小Bax/Bcl-2比值发挥保护作用。更多还原

【Abstract】 AIMS AND SIGNIFICANCENeutron radiation can cause severe damages to the intestine which is hard to recover. Unfortunately, there is still no good cure so far. Our previous study showed that IL-11 could attenuate the intestinal injury and promote recovery of intestinal epithelium. And the protection mechanism of IL-11 was related to the up-regulations of the IL-11 receptors and Jak/STAT activation. But it’s still unknown whether ERK and PI3K/Akt signaling pathways induced by IL-11 are involved in the intestinal injury and recovery after neutron irradiation. How do the ERK and PI3K/Akt signaling pathways interact in these processes? Are Bax and Bcl-2 attributed to the cytoprotection of IL-11? All the three questions remain to be answered. In addition, various genes may play a role in the regulation of radiation response of the intestine. Gene chip makes it come true to screening out differentially expressed genomics in a high-flux way and the results maybe offer helpful clues to the next exploration of IL-11 signaling. Therefore, in this study we investigated the above topics and sought to elucidate the molecular mechanism of IL-11 regulation in neutron irradiation-induced intestinal injury, which might help to find new potential therapies.MATERIAS AND METHODS168 BALB/c mice were irradiated by 3 Gy neutron or 10 Gyγirradiation and injected with rhIL-11 at a dose of 500μg/kg body weight once per day for 5 days after irradiation. The mice were sacrificed at 6 h, 1d, 2d, 3d and 5d after irradiation. IEC-6 cells were exposed to 4 Gy neutron or 10 Gyγirradiation and treated with 100ng/ml rhIL-11 12 h prior to or immediately after irradiation. 10μM U0126 or LY294002 was added 12 h prior to IL-11 treatment. Gene chip, RT-PCR, HE staining, MTT, flow cytometry, Hoechst staining, immunohistochemistry, Western Blot and image analysis were used to detect the differentially expressed genes, histopathological changes in the mouse jejunum and determine the effect of IL-11, Uo126 and LY294002 on the intestinal pathology, IEC-6 cellular biological behaviors and expressions and activities of Raf-1, MEK1/2, ERK1/2, PI3K, PDK1, PTEN and Akt.RESULTS1. Survival and intestinal histological changes in the mice: (1) All of the mice received 3 Gy neutron irradiation died in 3~5 d after irradiation; and diarrhea occurred in every irradiated mice and the body weight lost rapidly and progressively. The small intestinal mucosa of the neutron irradiated mice showed marked destruction with villus epithelium decrease, disorder and swelling; cryptal cell number decreased sharply and the crypts were destructed with rare cell number at the 2nd d. The epithelia cells showed acidophily, pyknosis and karuorrhexis. Regeneration was rare at 3d. The mice with IL-11 administration showed significant increase in survival fraction, decrease in diarrhea, more crypt regeneration and villous cells.2. Differentially expressed genes in the murine small intestine: After 6 h of 3 Gy neutron irradiation, there were 608 genes differentially expressed in the small intestine, of which 277 genes were up-regulated and 331 genes were down-regulated. The differentially expressed genes were related to cellular proliferation and apoptosis, MAPK, PI3K/Akt signaling and so on.3. Changes of IEC-6 cellular biological behaviors: (1) At 6 h after 10 Gyγirradiation, IEC-6 cells showed higher rates of apoptosis and necrosis, lower proliferating capability and cell cycle arrest. IL-11 treatment down-regulated the rate of apoptosis and necrosis. (2) At 6 h after 4 Gy neutron irradiation, IEC-6 cells became round in shape and showed higher rates of apoptosis and necrosis and lower proliferating capability; at 24 h after neutron irradiation, the rates of apoptosis and necrosis showed no marked changes but the proliferating capability was still lower than normal. IL-11-treated IEC-6 cells showed better in morphology, lower apoptosis rate and stronger proliferating capability.4. Changes of ERK pathway in IEC-6 cells and effect of U0126 on IEC-6 cellular biological behaviors: (1) The expression and activity of Raf-1 and MEK1/2 increased but ERK1/2 activation decreased in 6~24 h after 10 Gyγirradiation. IL-11 increased the activities of Raf-1 and MEK1/2 in 5~15min and inhibited ERK1/2 activity at 6 h after radiation. (2) The expression and activity of Raf-1 and MEK1/2 showed no significant changes in 6~24 h after 4 Gy neutron irradiation while the expression and activity of ERK1/2 increased. IL-11 treatment didn’t change the expression of Raf-1 at 6 h and increased the activity of MEK1/2 in 6~24 h. As well as the expression and activity of ERK1/2 was concerned, IL-11 functioned as inhition at 6 h and promotion at 24 h. (3) Compared with IL-11 treated IEC-6 cells, cells with U0126 added before IL-11 showed lower proliferating capability but no obvious changed in cell death at 6 and 24 h after 4 Gy neutron irradiation.5. Changes of PI3K/Akt pathway in IEC-6 cells and effect of LY294002 on IEC-6 cellular biological behaviors: (1) At 6 h after 10 Gyγirradiation, the expression of PI3K and activation of Akt in IEC-6 cells decreased while the expression of Akt, activities of PDK1 and PTEN increased. IL-11 treatment increased the expression of PI3K and activation of Akt and decreased the expression of Akt, activities of PDK1 and PTEN. (2) After 4 Gy neutron irradiation, the expression of PI3K decreased at 6 h and recovered at 24 h. The activation of PDK1 and PTEN increased, meanwhile the activation of Akt decreased in 6~24 h. IL-11 up-regulated the expression of PI3K and activation of Akt and down-regualted the activation of PDK1 and PTEN. (3) Compared with IL-11 treated IEC-6 cells, cells with LY294002 added before IL-11 showed lower proliferating capability and higher apoptosis and necrosis rate at 6 and 24 h after 4 Gy neutron irradiation.6. Interactions between ERK and PI3K/Akt pathway: (1) Compared with IL-11 treated IEC-6 cells, U0126 brought no significant changes to the expression and activities of ERK1/2 but increased the activations of PDK1and Akt in 6~24 h after neutron irradiation. (2) Compared with IL-11 treated IEC-6 cells, LY294002 decreased the activations of both Akt and ERK1/2 in 6~24 h after neutron irradiation.8. Expressions of Bax and Bcl-2 in the murine small intestine: (1) Immunoreactivity of Bax was granulo-positive in the normal small intestinal villous cytoplasm and weakly positive in the cryptal cytoplasm. After 3 Gy neutron and 10Gyγirradiation, Bax immunoreactivity increased greatly in 6 h~3 d. IL-11 adminstration decreased the expression of Bax at 3 d after 3 Gy neutron irradiation. (2) Immunoreactivity of Bax was weakly positive in the normal small intestinal villous and cryptal cytoplasm. 3 Gy neutron brought no changes to the expression of Bcl-2 in 6 h~3 d. 10Gyγirradiation increased the expression of Bcl-2 in 6 h~3 d.CONCLUSIONS1. Neutron irradiation resulted in injuries to many molecular targets, involved multi pathway like PI3K/Akt and MAPK in the jejunum.2. In IEC-6, ERK1/2 was continuously activated by neutron irradiation. The effect of IL-11 on ERK1/2 activation fluctuated from inhibition to promotion. This suggests ERK played dual roles in different stages of neutron-induced IEC injury.3. PI3K/Akt pathway was inhibited by neutron irradiation and triggered by IL-11 to protect IEC from neutron injury.4. Neutron andγirradiation had different impacts on ERK and PI3K/Akt pathway, which might underlie their different injury response.5. There maybe a feedback loop to connect ERK and PI3K/Akt pathway when IL-11 regulated neutron induced intestinal epithelial injury: (1) Continuously activated ERK1/2 inhibited PI3K/Akt activity, while ERK inhibition synergized with IL-11 to promote PI3K/Akt activation; (2) The activity of ERK1/2 was regulated by PI3K and independent on MEK1/2.6. LY294002 was more effective than U0126 to block IL-11 protection the intestinal epithelium from neutron irradiation. This suggests that ERK and PI3K/Akt signaling played different roles in mediating the cytoprotection by IL-11: ERK was mainly associated to cellular proliferation and PI3K/Akt was involved both cellular proliferation and death.7. IL-11 decreased the ratio of Bax/Bcl-2 to confer protection on the intestine exposed to neutron irradiation.更多还原