本文選題：膽紅素腦損傷　切入點(diǎn)：皮層星形膠質(zhì)細(xì)胞　出處：《重慶醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的：觀察反式激活蛋白-NEMO結(jié)合域(TAT-NBD)對(duì)膽紅素誘導(dǎo)的大鼠皮層星形膠質(zhì)細(xì)胞核因子-κB(NF-κB)活化及炎癥反應(yīng)的作用。初步探究腹腔內(nèi)注射TAT-NBD治療膽紅素腦病新生鼠對(duì)星形膠質(zhì)細(xì)胞核因子-κB (NF-κB)活化的影響,能否改善運(yùn)動(dòng)、學(xué)習(xí)記憶能力。方法：分離、培養(yǎng)、鑒定SD新生鼠大腦皮層2代星形膠質(zhì)細(xì)胞,隨機(jī)分為三組：對(duì)照組,膽紅素組,TAT-NBD干預(yù)組。免疫熒光染色法觀察細(xì)胞形態(tài),改良MTT法檢測(cè)細(xì)胞相對(duì)存活率,AnnexinV-FITC/PI雙染法觀察凋亡情況,Western blot檢測(cè)NF-KBp65表達(dá),EMSA檢測(cè)NF-κB的入核、活化。ELISA檢測(cè)上清中炎癥因子IL-1β、TNF-α、IL-6的水平。用5日齡SD新生鼠隨機(jī)分組后造模成三組：對(duì)照組,膽紅素組,TAT-NBD干預(yù)組。細(xì)胞免疫熒光觀測(cè)建模后24h各組腦組織切片中細(xì)胞形態(tài)和NF-κBp65表達(dá)；建模時(shí)及建模后3d記錄新生鼠每天體重變化,記錄建模后各組新生鼠30d以內(nèi)存活狀態(tài)；取28日齡各組SD大鼠進(jìn)行轉(zhuǎn)棒測(cè)試和避暗實(shí)驗(yàn)檢測(cè)平衡運(yùn)動(dòng)、學(xué)習(xí)記憶能力。結(jié)果：膽紅素組的NF-KBp65蛋白灰度比值顯著高于對(duì)照組(p0.01),2h、12h達(dá)高峰(p0.05); TAT-NBD干預(yù)使NF-κBp65蛋白表達(dá)明顯低于膽紅素組(p0.01)。IL-1β、TNF-α、IL-6的釋放與膽紅素誘導(dǎo)的NF-KBp65表達(dá)有時(shí)間相關(guān)性,于12h達(dá)高峰,且TAT-NBD干預(yù)2h、12h的IL-1β、TNF-α、IL-6濃度均低于膽紅素組(p0.01)。星形膠質(zhì)細(xì)胞的存活率與膽紅素作用時(shí)間負(fù)相關(guān),TAT-NBD干預(yù)2h、12h的相對(duì)存活率均高于膽紅素組(p0.05)。TAT-NBD能維持細(xì)胞正常形態(tài),減輕細(xì)胞凋亡和損傷。建模后24h,膽紅素模型鼠腦組織中GFAP、NF-KBp65的表達(dá)及NF-κB入核、活化較對(duì)照組明顯增多,TAT-NBD干預(yù)可減少膽紅素組腦組織中GFAP、NF-KBp65的表達(dá)和NF-κB的入核、活化。TAT-NBD干預(yù)組SD新生鼠建模后體重增長(zhǎng)趨勢(shì)連續(xù)3d高于膽紅素組但無(wú)明顯差異(p0.05),顯著低于對(duì)照組(p0.01)；近期死亡率顯著低于膽紅素組(p0.01)；遠(yuǎn)期生存狀態(tài)較膽紅素組好轉(zhuǎn)差異無(wú)統(tǒng)計(jì)學(xué)意義。平衡運(yùn)動(dòng)和避暗逃避實(shí)驗(yàn)中,TAT-NBD干預(yù)組在轉(zhuǎn)棒上停留時(shí)間及進(jìn)入黑箱的潛伏期均顯著短于對(duì)照組(p0.01),長(zhǎng)于膽紅素組(p0.05)。結(jié)論：膽紅素可誘導(dǎo)原代培養(yǎng)的大鼠皮層星形膠質(zhì)細(xì)胞NF-κB激活、過(guò)表達(dá),TAT-NBD抑制NF-κB的活化高峰、減少炎癥因子釋放發(fā)揮抗炎保護(hù)作用,可用于防治膽紅素腦損傷。腹腔注射TAT-NBD在急性期可顯著減輕膽紅素腦病新生鼠腦組織內(nèi)GFAP、NF-κB的激活和過(guò)表達(dá),減少NF-κB的入核、活化；顯著提升膽紅素腦病新生鼠覓食能力,明顯降低其近期死亡率；遠(yuǎn)期可適當(dāng)改善其生存狀態(tài),顯著提升其平衡運(yùn)動(dòng)、學(xué)習(xí)記憶能力。
[Abstract]:Objective: to observe the effects of transactivator protein Nemo binding domain (TAT-NBDD) on activation and inflammatory response of rat cortical astrocyte nuclear factor- 魏 B (NF- 魏 B) induced by bilirubin, and to explore the effect of intraperitoneal injection of TAT-NBD in the treatment of bilirubin encephalopathy in neonatal rats with bilirubin encephalopathy. Effects of glial nuclear factor-魏 B on the activation of NF- 魏 B, Methods: two generations of astrocytes were isolated, cultured and identified in the cerebral cortex of neonatal SD rats. They were randomly divided into three groups: control group, TAT-NBD intervention group, and immunofluorescence staining method. The relative survival rate of cells was detected by modified MTT method and the apoptosis was observed by Annexin V-FITC / Pi double staining. The expression of NF-KBp65 was detected by Western blot and NF- 魏 B was detected by EMSA. Activation. Elisa was used to detect the level of IL-1 尾 TNF- 偽 and IL-6 in the supernatant. Five day old SD newborn rats were randomly divided into three groups: the control group, the control group, and the control group. The cell morphology and NF- 魏 Bp65 expression in brain slices of each group were observed 24 hours after modeling, the changes of body weight of newborn rats were recorded during modeling and 3 days after modeling, and the survival status of newborn rats within 30 days after modeling was recorded. SD rats of 28 days old were tested by rotation test and dark avoidance test to detect the balance movement. Results: the gray level ratio of NF-KBp65 protein in bilirubin group was significantly higher than that in control group (P 0.01 / 12 h), and the expression of NF- 魏 Bp65 protein in bilirubin group was significantly lower than that in bilirubin group (P 0.01). IL-1 尾 TNF- 偽 IL-6 release was correlated with NF-KBp65 expression induced by bilirubin. The concentration of IL-1 尾 TNF- 偽 IL-6 was significantly lower than that of bilirubin group (P 0.01). The survival rate of astrocytes was negatively correlated with the time of bilirubin action. The relative survival rate of TAT-NBD for 2 h was higher than that of bilirubin group for 2 h. TAT-NBD could maintain the normal morphology of the cells. The expression of NF-KBp65 and the entry of NF- 魏 B into the brain tissue of bilirubin model were significantly increased compared with the control group. TAT-NBD intervention decreased the expression of GFAPNF-KBp65 and the entry of NF- 魏 B in the brain tissue of bilirubin group. The trend of body weight gain in the activated. TAT-NBD intervention group after modeling was higher than that in the bilirubin group for 3 days, but there was no significant difference between the two groups, which was significantly lower than that in the control group (p0.01), the short-term mortality was significantly lower than that in the bilirubin group (P 0.01), and the long-term survival status was better than that in the bilirubin group. There was no significant difference between the two groups. In the balanced exercise and avoidance experiments, the time of staying on the rotating rod and the latency of entering the black box in TAT-NBD intervention group were significantly shorter than those in the control group (p0.01) and longer than that in the bilirubin group. Conclusion: bilirubin can induce primary culture. NF- 魏 B was activated in cultured rat cortical astrocytes. Overexpression of TAT-NBD inhibits the peak of NF- 魏 B activation, reduces the release of inflammatory factors, plays an anti-inflammatory and protective role, and can be used to prevent and treat bilirubin brain injury. Intraperitoneal injection of TAT-NBD can significantly reduce the activation and overexpression of GFAP- 魏 B in the brain tissue of newborn rats with bilirubin encephalopathy at acute stage. Reduce NF- 魏 B entry into the nucleus, activation; significantly improve the feeding ability of neonatal rats with bilirubin encephalopathy, significantly reduce their short-term mortality; in the long run, can improve their survival status, significantly improve their balanced movement, learning and memory ability.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R722.1

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