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標題: | 後感染排除小鼠腸道上清液刺激人類神經母細胞瘤SH-SY5Y之神經纖維生長 Stimulation of neurite outgrowth in human neuroblastoma SH-SY5Y cells by post-infectious mouse intestinal supernatant |
作者: | Kaun-Yang Hung 洪冠陽 |
指導教授: | 余佳慧 |
關鍵字: | 腸躁症,梨形蟲,內臟高敏感性,神經生長, irritable bowel syndrome(IBS),Giardia lamblia,visceral hypersensitivity,nerve outgrowth, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 背景:IBS是一種常見的功能性腸道異常疾病,症狀包括長期反覆性腹痛、腹脹並伴隨排便習慣改變,但卻無法檢測出病原體或腸道結構型態的改變。目前對腸躁症腹痛敏感的病理機制尚不清楚。本研究目的是利用梨形蟲感染後結合避水壓力雙重因子刺激建立的IBS小鼠模式,探討其腸道組織上的分子變化,並將腸清液與人類神經母細胞培養,觀察神經纖維的生長變化,以瞭解神經細胞型態改變與內臟痛覺高敏感化的相關性。方法:小鼠在第0天時給予灌食梨形蟲(Giardia lamblia)或磷酸鹽緩衝液(PBS),在後排除期的第35-44天進行10天的避水壓力(water avoidance stress,WAS)或無處理(non-handle,NH),共分作四大組(PBS-NH, PBS-WAS, Giardia-NH, Giardia-WAS)。將小鼠於第44天時利用結直腸撐張刺激進行內臟動器反應分析,而後犧牲並收集腸段製備成腸組織上清液。利用人類SH-SY5Y神經母細胞株,加入視黃酸促使分化後,再給予腸組織上清液培養,觀察神經纖維生長變化。結果: 神經母細胞經視黃酸和腦源性生長因子作用後,神經纖維生長為4倍,當作一個正對照組。接著測試四大組小鼠腸組織上清液對神經纖維生長的影響,結果顯示PBS-WAS, Giardia-NH, Giardia-WAS小鼠腸清液相比於PBS-NH組,會分別促使神經細胞纖維長度上升約2,2.5和3倍。透過西方轉漬法分析小鼠腸道組織中的神經營養因子表現量發現,在小腸組織方面,Giardia-WAS 組小鼠的NGF總量有明顯上升;在大腸組織方面,BDNF表現量則有上升的趨勢。在神經細胞實驗中,預先給予中和性anti-NGF或anti-BDNF均能顯著降低Giardia-WAS組小鼠腸清液培養後神經纖維的長度;或預先將CCK receptor antagonists (AIH或BIH)加入神經細胞上,亦能抑制神經細胞纖維的延長。此外,若將腹腔注射AIH或BIH小鼠之腸清液與神經細胞培養則發現,BIH注射小鼠之腸清液能抑制神經細胞纖維的延長,AIH則無法抑制。最後透過西方轉漬法分析發現SH-SY5Y神經細胞會常態性的表現CCK-AR, CCK-BR, BDNF,但不表現CCK和NGF;而在給予小鼠腸清液培養後,NGF總量會增加。結論:本篇研究證明了內臟痛覺敏感化的小鼠腸道中含有CCK、NGF與BDNF的存在,而這些腸源性因子會影響神經細胞纖維的延長而參與導致痛覺異常。 Background: Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by chronic abdominal pain and altered bowel habits, without macroscopic abnormality or presence of pathogen. The mechanism of visceral hypersensitivity in IBS is still unclear. The aim is to investigate the molecular changes in intestinal tissues of a mouse model of IBS with visceral hypersensitivity caused by dual stressors of post-infection and psychological stress, and to evaluate whether gut-derived factors may cause neurite outgrowth by using a human neuroblastoma SH-SY5Y cell line. Method: Mice were inoculated with Giardia lamblia or phosphate-buffered saline (PBS) via orogastric gavage on day 0, and subjected to 10-day water avoidance stress (WAS) or non-handle (NH) from day 35-44 during post-clearance phase. Mice were divided to four groups: PBS-NH, PBS-WAS, Giardia-NH, Giardia-WAS. Visceromotor response to colorectal distension was analyzed in mice on day 44, and then intestinal segments were collected for preparation of gut tissue supernatant. SH-SY5Y cells were treated with retinoic acid to induce the cell differentiation and then cultured with gut tissue supernatant for measurement of nerve fiber length. Result: SH-SY5Y cells treated with retinoic acid and brain-derived neurotrophic factor showed 4-times longer nerve fiber length, which served as a positive control. As for the effect of gut-derived factors on neurite outgrowth, incubation of gut supernatant obtained from PBS-WAS, Giardia-NH, and Giardia-WAS mice caused 1.5-, 2-, and 2.5- times increases in nerve fiber length compared to that from PBS-NH mice, respectively. Western blotting results showed increased expression of NGF in small intestine, and increased expression of BDNF in large intestine. Moreover, addition of neutralizing anti-NGF or anti-BDNF reduced the nerve fiber length incubated with G-WAS gut supernatant; pretreatment with CCK receptor antagonist (AIH or BIH) also inhibited neurite outgrowth. In addition, when culturing SH-SY5Y cells with gut supernatant obtained from mice intraperitoneally injected with AIH or BIH, the BIH-injected gut supernatant decreased the nerve fiber length while the AIH-injected gut supernatant did not. Finally, Western blotting showed that SH-SY5Y cells constitutively expressed CCK-AR, CCK-BR, and BDNF, but not CCK or NGF; increased levels of NGF was seen after culturing with gut supernatant. Conclusion: Presence of CCK, NGF, and BDNF was found in the intestine of mice with visceral hypersensitivity, and these gut-derived factors affect the outgrowth of neurites and may lead to abnormal pain sensation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55633 |
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