【作者】 李成林；

【导师】 余佩武；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 人类对外层空间的探索以及太空资源的开发和利用带来航天活动的蓬勃发展。近年来,我国的航天事业取得了长足的进步。但航天事业的发展离不开航天医学的支持。航天器围绕地球旋转时,处于失重状态。但由于受到残余大气阻力、航天器自旋等因素影响,其内部表现为微重力环境。微重力环境对已适应地球重力影响的哺乳动物生物机能的影响,会阻碍人类的空间探索,因此已成为空间生命科学与空间细胞生物科学的研究热点。失重对生物机体的研究,最好将生物体置于宇宙空间飞行真实失重环境中进行。但由于航天飞行的载荷量低,空间狭小,成本高,大量的相关研究工作基本为地面模拟失重状态下进行。模拟失重的大鼠动物模型一般采用尾悬吊法。实验大鼠尾部悬于笼顶30°,前肢踏于笼底,可自由活动。此模型可以较理想地模拟失重条件下体液移动和制动的效果,是一种较好的失重模拟方法,几十年的航天实践及模拟失重研究证实,空间飞行会导致心血管功能障碍、骨质丢失、肌肉萎缩、免疫能下降、内分泌功能紊乱及空间运动病等多种生理和病理变化,研究也取得了很大的成果。但是对消化系统失重生理病理学的研究却十分局限,迄今国内外尚未见有关失重和模拟失重环境下肠道黏膜屏障的研究报道。胃肠道除作为消化吸收的器官,完成食物的消化、营养物质的吸收及废物的排泄外,由于肠道内存在众多的化学物质和大量的微生物,为避免这些外来物质对机体的损害,胃肠道还有一套完整的防御体系,即胃肠道的粘膜屏障功能。该屏障由粘膜机械屏障、免疫屏障、生物屏障和化学屏障四个层次构成。肠黏膜上皮细胞及其紧密连接是粘膜机械屏障的重要组成部分,它的完整性是黏膜屏障的基础。在肠道免疫中sIgA扮演着重要角色,它主要由肠粘膜固有层中的IgA浆细胞产生,当发生肠粘膜上皮受损时,势必影响到sIgA的分泌,削弱机体的免疫屏障功能。肠道淋巴样系统由具有分化功能并能产生局部免疫的淋巴细胞亚群所组成,参与免疫调节。任何原因造成免疫功能受损,均可引起肠粘膜免疫屏障功能减弱。本研究依据目前航天医学的现状而设立课题,进行模拟失重动物实验,观察微重力环境对肠黏膜机械及免疫屏障的的影响。研究方法:(1)选用清洁级成年健康雄性Wistar大鼠,适应饲养1周后进行实验。依照陈杰等设计的尾悬吊法(tail-suspension)建立模拟失重大鼠模型。各时相点实验结束时,腹腔注射戊巴比妥钠(45 mg/kg)将实验及对照动物麻醉,无菌操作,剖腹取材。(2)实验一:Wistar大鼠,随机分为5组:模拟失重1w组、模拟失重2w组、模拟失重3w组、模拟失重4w组以及地面对照组。酶法检测血中D-乳酸的变化水平,鲎试剂法测定门静脉内毒素水平,透射电镜观察回肠黏膜的超微结构变化。(3)实验二:Wistar大鼠,随机分为模拟失重组和对照组,分别设1 d、2 d、4 d、及7 d共4个时相点,用双抗体夹心ABC-ELISA法测定TNF-α的水平,免疫组织化学法检测肠道黏膜NF-κB表达,TUNEL法检测肠黏膜上皮细胞凋亡。(4)实验三:Wistar大鼠,随机分为模拟失重组和对照组,1 d、2 d、4 d、及7 d不同的时相点,免疫组织化学法检测肠黏膜sIgA的表达,TUNEL法原位检测肠黏膜下淋巴细胞的凋亡,免疫组织化学法检测肠黏膜CD4细胞数量的变化。(5)实验四:在模拟失重下,分6h,12h,24h,48h,96h不同时相点,应用免疫组化检测大鼠黏膜上皮Hsp70的表达。结果:(1)模拟失重对大鼠的肠壁超微结构破坏明显,表现为微绒毛排列紊乱脱落,外观极不平整,细胞间隙明显增宽,细胞桥粒连接模糊。线粒体内质网肿胀、空泡变性。以1W组明显。模拟失重1w组大鼠的血乳酸水平明显增高,然后逐渐下降。模拟失重4w组的血乳酸水平接近对照组水平。模拟失重1w组大鼠的门静脉血内毒素的水平明显增高,然后略有下降。3w、4w组的内毒素水平仍处高水平。(2)模拟失重状态下,血浆TNF-α的水平从第2d开始升高,4d最高,7d恢复到正常水平。1d组肠道黏膜NF-κB表达阳性率较对照组开始升高,2d、4d、7d阳性率明显高于对照组,7d模拟失重组的阳性表达仍在高水平。黏膜上皮细胞的凋亡1d明显增加,以后开始减慢,但7d后仍高于对照组。(3)模拟失重1d、2d组大鼠肠道黏膜sIgA分泌明显下降,4d开始明显上调、7d组sIgA的分泌量增多,但仍未恢复正常水平。模拟失重1d、2d组肠道黏膜的淋巴细胞凋亡明显,模拟失重4d、7d组凋亡率明显下降,逐渐恢复正常水平。模拟失重1d组肠道黏膜CD4细胞的数量明显下降,模拟失重2d、4d开始逐渐上升、7d组CD4细胞的数量接近正常水平。(4)模拟失重大鼠从6h开始肠黏膜高度表达Hsp70,24h达到高峰,48h略有下降,96h仍处在高表达的水平。结论:(1)血D-乳酸和门静脉内毒素浓度的增高,提示模拟失重造成了大鼠肠道黏膜通透性的增加。(2)模拟失重造成了肠黏膜超微结构的改变和黏膜上皮细胞的凋亡增加,这可能是黏膜通透性改变的基础。(3)模拟失重引起大鼠肠道黏膜NF-κB应激活化和血浆TNF-α的增高,提示炎性反应在失重状态下的肠道黏膜损害中起到一定作用。(4)模拟失重造成了大鼠肠道黏膜sIgA的分泌减少,淋巴细胞的凋亡增加,CD4细胞数量的减少,显示肠道黏膜的免疫屏障功能降低。(5)模拟失重是一种能诱导大鼠肠道黏膜Hsp70快速表达的典型应激原。更多还原

【Abstract】 The spaceflight has gotten a great achievement along with man’s exploring to outspace. Our country has also made a greate progress in space program. The ambitious exploration rises challenges to space medical researchers.Gravity has been a constant physical factor during the evolution and development of life on Earth. But the spaceflight activity is under the weightleness or macrogravity. The physiological changes caused by weightlessness will harm the astronauts. More medical researches should be done to meet the need.Ground-based models play a significant role in space exploration because the real spaceflight researchs have too many limits.Although the absence of gravity can not accurately be simulated on ground, several kinds of models could mimic some responses observed after exposure to microgravity. Tail-suspension or hindlimb unloading in rodents is one of the most commonly used models which can mimics many of the physiological alterations in various organ systems caused by actual spaceflight. The hindlimb unloading technique has been approved by the NASA Ames Research Center Animal Care and Use Committee as a rat model for simulating spaceflight.Many serious adverse physiological changes occur during spaceflight. Some of these include fluid redistribution, increased kidney filtration, sensory input changes, cardiovascular deconditioning, bone deterioration, muscle loss, and impaired immune system function. However, there are scanty research reports on the pathophysiological alterations of digestive system under microgravity. To our knowledge ,the effects of the simulated weightlessness on intestinal barrier have not been elucidated. Accordingly, the present study was designed to document the intestinal epithelial barrier and immune changes in rat under simulated weightlessness and to determine whether there is associated stress response with alteration that microgravity imposed upon intestine.Methods:(1) The experiments were performed after approval by the local Ethics Committee and conducted on Male Wistar rats (Laboratory Animals Center, China Agriculture University). The procedure of tail-suspension described by Chen et al was adopted in the present study. At the end of the experiment, animals were anesthetized with pentobarbital (45 mg/kg) and laparotomy via middle line was made. Heparinized blood sample were drawn from the portal vein or vena cava. A small section of terminal ileum samples obtained from control and suspended animals were processed for transmission electron microscopy evaluation.The other terminal ileums were preserved in zinc-buffered formalin.Subsequently,tissue were processed for routine hematoxylin and eosin staining to assess general architecture and injury. (2) Part one:Male Wistar rats weighted 300±20 g were randomly assigned to 5 experimental groups: suspended for 1w, 2w, 3w, 4w and 0w (control). The ultrastructural alterations of intestinal epithelial cells were assessd under transmission electron microscopy; Endotoxin values were measured by use of kinetic test with ET ELISA Kit; D-lactic acid was measured by using D-lactic acid UV-method.(3) Part two:64 male Wistar rats weighted 280-310 g were randomly divided into simulated weightlessness group and control group, each with 4 subgroups :1-day group;2-day group ;4-day group and 7-day group.The expressions of NF-κB p65 were detected by using immunohistochemistry PV-6001. The apoptosis of intestinal epithelial cells was detected by terminal deoxy-nucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Measurement of tumor necrosis factor-α(TNF-α) was performed in blood samples using the immunoassay kit. (4) Part three:The animals and experimental groups were same as in part two.Lymphocytes apoptosis beneath the intestinal epithelium was measured using TUNEL method. The sIgA expression in intestinal mucosa was detected using immunohistochemistry PV-6001 and the number of CD4 in intestinal mucosa was calculated using immunohistochemistry stain.(5) Part four: 48 male Wistar rats weighted 300±20 g were randomly assigned to 6 experimental groups: suspended for 6 h, 12 h, 24 h, 48 h, 96 h and 0 h (control). Hsp70 expression in intestinal mucosa was detected in the first 96h also using immunohistochemistry method.Results:(1) Ultrastructural alterations in the intestine epthilial cells were observed under transmission electron microscopy. They consisted of dilatation of the endoplasmic reticulum, mitochondrial swelling and dilatations within the tight junctions.Endotoxin values in 1w-group were singnificantly higher than control group. Though it slightly declined then, the values in 2w-, 3w-,4w- groups were still in high level. The density of D-lactic in 1w-group was higher then control group, then declined gradually, the level in 4w-group approach the control group’s. (2)Under the simulated macrogravity ,the plasma TNF-αlevel rised in 2-day subgroup, reached its peak in 4-day subgroup then declined to level of control groups in 7-day. The tail-suspension significantly increased intestine NF-κB expression in the rats, compared with the control(P<0.01), with peak expression in 2-day subgroups, followed by the slight declining, but the expression in 7-day subgroup was still higher. By TUNEL method, an increase in the percentages of apoptosis index was noticed in the intestine epithelial cells of all experimental rats under simulated microgravity compared with controls. The maximum percentage increase of apoptotic index was observed in 1-day group of suspension.(3) The sIgA expressions were reduced in experimental groups especially in 1-day, 2-day groups compared with control groups. It gradually elevated in 4-day,7-day groups. The percentage of apoptosis index of lymphocyte increased in all experimental groups, the maximum was in 1-day group. The number of CD4 in intestinal mucusa decreased markedly in 1-day group, then gradully increase in 2-day,4-day group.It near normal in 7-day goup.(4) The tail-suspension significantly increased Hsp70 expression levels in intestine, moreover it mainteined at high level in 96h-group.Conclusions: These findings suggest that: (1) Simulated microgravity by tail-suspension increased the intestine permeability which can be inferred from increased endotoxin value and D-lactic level; (2) The ultrastructural alteration and apoptosis of intestinal epithelial cells promoted by simulated microgravity may be the fundamental reason for increased intestinal permeability. (3) The tail-suspension has the effects of inducing activation of NF-κB and elevation of TNF-αin intestine especially in the early stage which suggests that NF-κB plays important roles in the inflammatory reactions of intestine to the weightlessness stress. (4) The simulated weightlessness promotes lymphocyte apoptosis and reduces sIgA secretion and CD4 cells which lead to lower the intestinal immune function.(5) The simulated weightlessness acts as a typical cellular stressor to induce high Hsp70 expression in intestine.更多还原