本文選題：腦室周圍白質(zhì)損傷 + 促紅細(xì)胞生成素��； 參考：《東南大學(xué)》2015年博士論文

【摘要】：目的：腦室周圍白質(zhì)損傷(periventricular white matter damage,PWMD)是早產(chǎn)兒腦損傷的主要神經(jīng)病理學(xué)形式,已成為早產(chǎn)兒腦癱、智力障礙等神經(jīng)系統(tǒng)后遺癥的主要原因之一,給家庭和社會(huì)帶來(lái)沉重負(fù)擔(dān)。目前,早產(chǎn)兒腦白質(zhì)損傷的發(fā)病機(jī)制尚未完全明確,缺氧缺血是早產(chǎn)兒PWMD發(fā)生的兩個(gè)主要病因之一,而腦室周圍血管發(fā)育不完善是早產(chǎn)兒發(fā)生PWMD的重要解剖因素,目前針對(duì)早產(chǎn)兒PWMD尚無(wú)有效的干預(yù)措施。所以尋找治療或預(yù)防早產(chǎn)兒腦白質(zhì)損傷的方法、改善早產(chǎn)兒生存質(zhì)量已是目前迫切需要解決的問(wèn)題之一。重組人促紅細(xì)胞生成素(recombinant human erythropoietin,rh-EPO)多年來(lái)廣泛用于早產(chǎn)兒貧血的治療且取得較好的效果,隨著深入的研究發(fā)現(xiàn)EPO不僅具有造血功能,而且具有良好的神經(jīng)保護(hù)作用。研究表明外周靜脈或腹腔內(nèi)注射rh-EPO能通過(guò)血腦屏障直接在腦內(nèi)發(fā)揮神經(jīng)保護(hù)作用。目前國(guó)內(nèi)外對(duì)rh-EPO與腦損傷的關(guān)系及機(jī)制研究主要集中于rh-EPO對(duì)腦卒中及腦外傷等的神經(jīng)保護(hù)作用,與早產(chǎn)兒PWMD的關(guān)系的研究仍少,尤其是其中的機(jī)制目前不明確。因此,本研究通過(guò)建立缺氧缺血性PWMD新生鼠模型,并給缺氧缺血后的新生鼠腹腔內(nèi)注射一定劑量的rh-EPO,觀察rh-EPO對(duì)缺氧缺血的PWMD新生大鼠腦保護(hù)功能,并探討可能機(jī)制。該研究將為臨床治療早產(chǎn)兒PWMD提供一定的實(shí)驗(yàn)信息及依據(jù),為今后進(jìn)一步研究早產(chǎn)兒PWMD的防治提供新的思路和途徑。方法：本實(shí)驗(yàn)采用生后3日齡的新生大鼠,除假手術(shù)(Sham)組外,其余各組大鼠乙醚麻醉后取頸部正中切口,游離右側(cè)頸總動(dòng)脈并結(jié)扎,術(shù)畢放回原飼養(yǎng)環(huán)境中恢復(fù)2-3小時(shí)后置于恒溫密閉容器中,以2L·min-1的速度輸入6%02、94%N2混合氣體,持續(xù)4小時(shí)。Sham組大鼠手術(shù)僅游離右側(cè)頸總動(dòng)脈但不結(jié)扎,不進(jìn)行缺氧通氣。藥物干預(yù)組(HI-EPO)大鼠于缺氧后1小時(shí)內(nèi)給予腹腔注射(5U/g)的rh-EPO。缺氧缺血(hypoxia-ischemia, HI)組和Sham組大鼠給予腹腔注射相同劑量的無(wú)菌生理鹽水。第一部分：整個(gè)實(shí)驗(yàn)過(guò)程中觀察大鼠的生長(zhǎng)發(fā)育狀況,術(shù)后11天(P14)天斷頭取腦行HE染色觀察腦組織病理改變,生后(postnatal day,P)30天動(dòng)物行為檢測(cè)(懸吊實(shí)驗(yàn)、曠場(chǎng)實(shí)驗(yàn)、拒俘反應(yīng)實(shí)驗(yàn)、圓筒實(shí)驗(yàn))和P90天學(xué)習(xí)記憶檢測(cè)(三臂迷宮實(shí)驗(yàn))。每小組完成實(shí)驗(yàn)保證6只。第二部分：分別于術(shù)后2天(P5)、術(shù)后5天(P7)、術(shù)后7天(P10)和術(shù)后11天(P14)斷頭取腦,一部分標(biāo)本用于免疫組化檢測(cè)EPO受體(Eythropoietin receptor, EPOR)和攜氧蛋白神經(jīng)珠蛋白(Neuroglobin,Ngb),一部分標(biāo)本用于Western Blotting方法檢測(cè)EPOR蛋白,另一部分用于Real-time PCR方法檢測(cè)Ngb。每小組完成實(shí)驗(yàn)保證6只。第三部分：分別于術(shù)后2天(P5)、術(shù)后5天(P7)、術(shù)后7天(P10)和術(shù)后11天(P14)斷頭取腦,一部分標(biāo)本用于免疫組化檢測(cè)血管生成細(xì)胞內(nèi)皮祖細(xì)胞(Endothelial progenitor cells,EPCs),一部分標(biāo)本用于Western Blotting法檢測(cè)EPCs,另一部分用Real-time PCR方法檢測(cè)血管內(nèi)皮生長(zhǎng)因子(Vascular endothelial growth factor,VEGF)、血管生成素-1(Angiopoietin-1, Ang-1)、新生微動(dòng)脈標(biāo)志EphrinB2和微靜脈標(biāo)志EphB4。每小組完成實(shí)驗(yàn)保證6只。結(jié)果：1.Sham組幼鼠死亡率為2.50%；HI組幼鼠死亡率為16.12%：HI-EPO組幼鼠死亡率為8.23%；三組間死亡率差異存在統(tǒng)計(jì)學(xué)意義(χ2=8.32,P0.05)。術(shù)前,各組大鼠體重差別無(wú)統(tǒng)計(jì)學(xué)意義(F=0.448,P=0.647),自術(shù)后2天(P5)起HI組大鼠體重增長(zhǎng)幅度低于Sham組,兩組間差異存在統(tǒng)計(jì)學(xué)意義(P0.05)：HI-EPO組大鼠在手術(shù)后初期體重增長(zhǎng)情況不良,從術(shù)后7天始體重增長(zhǎng)加快,體重高于HI組但略低于Sham組,而且與Sham組和HI組間體重差異均存在統(tǒng)計(jì)學(xué)差異(P0.05)2. Sham組鼠雙側(cè)腦室大小相同,細(xì)胞排列整齊,形態(tài)大小正常。HI組鼠右側(cè)腦室較對(duì)側(cè)明顯增大,腦室形狀不規(guī)則,腦室周圍組織疏松,細(xì)胞變性、水腫,部分可見(jiàn)內(nèi)囊部位空腔形成：HI-EPO組鼠腦室周圍白質(zhì)病變減輕。3.懸吊實(shí)驗(yàn)、曠場(chǎng)實(shí)驗(yàn)、拒俘實(shí)驗(yàn)及三臂迷宮評(píng)分結(jié)果均顯示三組間存在統(tǒng)計(jì)學(xué)差異,進(jìn)一步兩兩分析,HI組評(píng)分明顯低于Sham組(P0.05),HI-EPO組評(píng)分改善高于HI組(P0.05),且與Sham組間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。圓筒實(shí)驗(yàn)評(píng)分結(jié)果顯示HI組偏好右前足的頻率與Sham組間存在明顯統(tǒng)計(jì)學(xué)差異(P0.01),HI-EPO組則有改善,但未恢復(fù)到正常,與前兩組間均存在統(tǒng)計(jì)學(xué)差異(P0.05)。4. Sham組大鼠EPOR表達(dá)在一較低水平；大鼠缺氧缺血后腦內(nèi)EPOR在術(shù)后2天(P5)即明顯增多,術(shù)后4天(P7)則逐漸開(kāi)始下降,但一直到術(shù)后11天(P14)仍保持在一較高水平,術(shù)后各時(shí)間點(diǎn)與Sham組比較有統(tǒng)計(jì)學(xué)意義(P0.05)；HI-EPO組大鼠EPOR在術(shù)后2天(P5)較HI組及Sham組均增高且達(dá)到一高峰,與HI組及Sham比較增高有統(tǒng)計(jì)學(xué)意義(P0.05),但很快開(kāi)始下降,在術(shù)后4天(P7)就降至HI組水平,術(shù)后7天(PI0)和術(shù)后11天(P14)進(jìn)一步下降,與Sham組比較無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),與HI組比較存在統(tǒng)計(jì)學(xué)意義(P0.05)。5.Sham組大鼠Ngb隨著日齡的增大而略微增加；大鼠HI后腦內(nèi)Ngb在術(shù)后2天(P5)即開(kāi)始增加,術(shù)后7天(PI0)及11天(P14)未表現(xiàn)出進(jìn)一步增多,術(shù)后各時(shí)間點(diǎn)與Sham組比較有統(tǒng)計(jì)學(xué)意義(P0.05)；HI-EPO組大鼠Ngb在術(shù)后2天(P5)至術(shù)后7天(PI0)較HI組增高,且兩組之間比較有統(tǒng)計(jì)學(xué)意義(P0.05),術(shù)后11天(P14)Ngb則下降,與HI組比較無(wú)統(tǒng)計(jì)學(xué)意義,但與Sham組比較仍有統(tǒng)計(jì)學(xué)意義(P0.05)6. Sham組CD34+細(xì)胞隨著日齡的增加而增加；HI后CD34+細(xì)胞較相同日齡Sham組大鼠初始增多(P5和P7),且兩組間差異存在統(tǒng)計(jì)學(xué)意義(P0.05),但術(shù)后7天起(PI0)開(kāi)始下降,并少于相同日齡Sham組大鼠,兩組間差異存在統(tǒng)計(jì)學(xué)意義(P0.05)；HI-EPO組大鼠CD34+細(xì)胞變化趨勢(shì)同缺氧缺血組,且較相同日齡缺氧缺血組鼠增多,各時(shí)間點(diǎn)兩組間差異存在統(tǒng)計(jì)學(xué)意義(P0.05),同時(shí)與Sham組間差異也存在統(tǒng)計(jì)學(xué)意義(P0.05)。7.Sham組大鼠VEGF、Ang-1表達(dá)隨著日齡的增加而增加：大鼠HI后腦內(nèi)VEGF水平在術(shù)后2天(P5)即開(kāi)始增加,至術(shù)后7天(PI0)達(dá)到高峰,然后下降,各時(shí)間點(diǎn)VEGF表達(dá)量均較Sham組增多；HI-EPO組大鼠術(shù)后2天(P5)VEGF、Ang-1表達(dá)量即較缺氧缺血組大鼠開(kāi)始增加,術(shù)后7天(PI0)達(dá)高峰,術(shù)后11天(P14)又開(kāi)始下降但仍高于缺血缺氧組,各個(gè)時(shí)間點(diǎn)與缺血缺氧組間差異存在統(tǒng)計(jì)學(xué)意義(P0.05)8.Sham組大鼠EphrinB2及EphB4表達(dá)均隨著日齡的增加而增加,各時(shí)間點(diǎn)EphrinB2/EphB4比例接近0.5；大鼠缺氧缺血后腦內(nèi)EphrinB2及EphB4在術(shù)后2天(P5)未見(jiàn)增加,EphrinB2/EphB4為0.49,與Sham組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),但自術(shù)后4天(P7)EphrinB2及EphB4迅速增加,與Sham組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),術(shù)后7天(PI0)開(kāi)始則下降,但EphrinB2表達(dá)較EphB4多,EphrinB2/EphB4為0.56;HI-EPO組大鼠術(shù)后2天(P5)EphrinB2及EphB4表達(dá)較HI組大鼠明顯增加(P0.05),術(shù)后4天(P7)達(dá)高峰然后開(kāi)始下降,但仍高于缺血缺氧組,且EphrinB2表達(dá)較EphB4多。結(jié)論：1.缺氧缺血后腦組織有內(nèi)源性修復(fù)反應(yīng),表現(xiàn)為腦室周圍白質(zhì)EPOR表達(dá)增加,攜氧蛋白Ngb呈現(xiàn)持續(xù)上升表達(dá),并有血管新生反應(yīng)。2. Rh-EPO對(duì)PWMD起到了保護(hù)作用,rh-EPO干預(yù)缺氧缺血腦白質(zhì)損傷鼠可以明顯減輕腦室周圍白質(zhì)區(qū)域的損傷,可以改善大鼠的遠(yuǎn)期神經(jīng)行為學(xué)能力,促進(jìn)腦白質(zhì)內(nèi)EPOR表達(dá)進(jìn)一步上調(diào),給EPO提供受體發(fā)揮效應(yīng)。3. Rh-EPO與EPOR結(jié)合后,一方面可以通過(guò)促進(jìn)Ngb為腦組織提供更多的氧,另一方面可以通過(guò)促進(jìn)血管新生反應(yīng)為腦組織提供更多腦血流發(fā)揮干預(yù)效應(yīng)。
[Abstract]:Objective: periventricular white matter damage (PWMD) is the main neuropathological form of brain injury in preterm infants. It has become one of the main causes of neurological sequelae in premature infants, such as cerebral palsy and mental retardation, and brings heavy burden to the family and society. At present, the pathogenesis of brain white matter injury in premature infants has not yet been found. It is clear that hypoxic ischemia is one of the two main causes of PWMD in preterm infants, and the poor development of peripheral blood vessels around the ventricle is an important anatomical factor for the occurrence of PWMD in preterm infants. There is no effective intervention for premature infant PWMD, so it is necessary to find a method to treat or prevent the brain white matter injury in preterm infants and improve the quality of survival in premature infants. It is one of the urgent problems that need to be solved. Recombinant human erythropoietin (recombinant human erythropoietin, rh-EPO) has been widely used for the treatment of anemia in preterm infants for many years and has achieved good results. With further research, it has been found that EPO not only has hematopoiesis, but also has good neuroprotective effect. Intravenous or intraperitoneal injection of rh-EPO can play a neuroprotective role through the blood-brain barrier directly in the brain. Research on the relationship and mechanism of rh-EPO and brain injury at home and abroad is mainly focused on the neuroprotective effect of rh-EPO on stroke and brain injury, and the study of the relationship with premature infant PWMD is still less, especially the mechanism of which is unclear. Therefore, in this study, a hypoxic ischemic PWMD rat model was established and a dose of rh-EPO was injected into the abdominal cavity of neonatal rats after hypoxic ischemia. The protective function of rh-EPO on hypoxic-ischemic PWMD neonatal rats was observed and the possible mechanism was discussed. This study will provide some experimental information and basis for the clinical treatment of PWMD in the treatment of premature infants. In order to further study the prevention and control of PWMD in preterm infants in the future, a new way of thinking and method is provided. In this experiment, the newborn rats of 3 days of age after birth, except for the sham operation (Sham) group, were taken from the middle incision of the neck after ether anesthesia, free right common carotid artery and ligated, and then returned to the original feeding environment for 2-3 hours after the operation. In a warm closed vessel, 6%02,94%N2 mixed gas was input at the speed of 2L min-1. For 4 hours, the right common carotid artery was free from the right common carotid artery in group.Sham rats, but no anoxic ventilation was carried out. The drug intervention group (HI-EPO) rats were given the rh-EPO. hypoxic-ischemic (hypoxia-ischemia, HI) group and Sham group within 1 hours after hypoxia. Rats were given the same dose of aseptic saline in the abdominal cavity. Part 1: the growth and development of rats were observed during the whole experiment. The brain tissue was observed by HE staining on the 11 day (P14) days (P14) days, and the 30 days after birth (postnatal day, P), the animal behavior test (suspension test, open field experiment, refusing reaction experiment, cylinder experiment) And P90 day learning and memory test (three arm labyrinth test). Each group completed the experiment to guarantee 6. Second parts: 2 days after operation (P5), 5 days after operation (P7), 7 days after operation (P10) and 11 days after operation (P14), and some specimens were used for immunohistochemical detection of EPO receptor (Eythropoietin receptor, EPOR) and oxygen carrying protein Neuroglobin (Neuroglo). Bin, Ngb), some specimens were used to detect EPOR protein by Western Blotting method. The other part was used for the Real-time PCR method to detect Ngb. in each group. 6. Third parts: 2 days after operation (P5), 5 days after operation (P7), 7 days after operation (P10) and 11 days after operation (P14), some specimens were used for immunohistochemistry to detect angiogenesis. Endothelial progenitor cells (EPCs), some specimens are used to detect EPCs in Western Blotting method, and the other part uses Real-time PCR method to detect vascular endothelial growth factor (Vascular endothelial growth), angiopoietin, and microarterioles and venules. The result: the mortality of young rats in group 1.Sham was 2.50%, and the mortality of young rats in group HI was 8.23% in group HI and in group 16.12%:HI-EPO, the mortality difference between groups was statistically significant (F=0.448, P=0.647) before operation (F=0.448, P=0.647), 2 days after operation (P). 5) the weight increase of the HI group was lower than that of the Sham group, and the difference between the two groups was statistically significant (P0.05) the body weight growth in the group:HI-EPO rats was poor after the operation, and the weight increased from 7 days after the operation, and the weight was higher than that of the HI group, but it was slightly lower than the Sham group, and there was a statistical difference between the group of Sham and the HI group (P0.05) 2. Sh. The size of the bilateral ventricle of the AM rats was the same, the cells arranged neatly, the right ventricle of the right ventricle increased obviously in the right ventricle of the normal.HI group, the shape of the ventricle was irregular, the periventricular tissue was loose, the cell degeneration, the edema and the formation of the cavity in the inner capsule: the peripheral white matter in the HI-EPO group was reduced by the.3. suspension experiment, the open field experiment, and the refusal of the capture. The results of the three arm labyrinth score showed that there was a statistical difference between the three groups. The score of group HI was significantly lower than that of group Sham (P0.05), and the improvement of group HI-EPO was higher than that of group HI (P0.05), and there was no statistical difference between the group of Sham (P0.05). The results of the test score of the cylinder experiment showed that the frequency of the HI group preference right front foot was clear between the Sham groups. The significant difference (P0.01), HI-EPO group improved, but did not recover to normal, and there were statistical differences between the first two groups (P0.05).4. Sham group EPOR expression at a lower level, EPOR in the brain after hypoxia ischemia in rats after 2 days (P5), the 4 days (P7) gradually began to decline, but until the 11 day after the operation (P14) still guaranteed. At a high level, there was a significant difference between the Sham group and the Sham group after the operation. The EPOR in group HI-EPO increased and reached a peak at 2 days after the operation (P5), which was higher than that in the HI group and the Sham group. It was significantly higher than the HI group and Sham (P0.05). But it began to descend quickly and decreased to the level of the HI group at the 4 day (P7) after the operation, and 7 days after the operation. After 11 days (P14), there was no significant difference from group Sham (P0.05), and there was a statistically significant difference between group HI and group HI (P0.05) in group.5.Sham, Ngb was slightly increased with the increase of day age, and Ngb in the brain after HI began to increase at the beginning of the operation (P5), and the 7 days (PI0) and 11 days (P14) after the operation did not increase further, and all the time after the operation. The point was statistically significant compared with that in the Sham group (P0.05), and the Ngb in group HI-EPO was higher than that in the HI group at 2 days after the operation (P5) and 7 days after the operation (PI0), and there was a significant difference between the two groups (P0.05), and the 11 days (P14) Ngb decreased, but there was no statistical significance compared with the HI group, but there was still a significant difference between the 6. group and the 6. group. The increase of day age increased; after HI, CD34+ cells increased initially in group Sham (P5 and P7), and there was a significant difference between the two groups (P0.05), but it began to decline (PI0) from the 7 day after the operation, and was less than that of the same age group of Sham rats. The difference between the two groups was significant (P0.05); the CD34+ cell trend of the HI-EPO group was deficient. Oxygen ischemia group, and more than the same age of hypoxia ischemia group increased, there was a statistically significant difference between the two groups at each time point (P0.05), and there was a statistically significant difference between the Sham group (P0.05) and the VEGF in group.7.Sham, Ang-1 expression increased with the increase of day age: the level of VEGF in the brain of the rat HI began to increase at the 2 day after the operation (P5), to the operation. The following 7 days (PI0) reached the peak, then decreased, and the expression of VEGF in each time point increased more than that in the Sham group; the expression of Ang-1 in the HI-EPO group was increased at 2 days (P5), and the expression of Ang-1 was increased in the hypoxic ischemia group, the peak of the 7 day (PI0) after the operation, and the 11 day after the operation (P14), but still higher than the ischemic anoxia group, and the difference between each time point and the ischemic anoxia group. There was statistical significance (P0.05) in group 8.Sham, the expression of EphrinB2 and EphB4 increased with the increase of day age, and the proportion of EphrinB2/EphB4 in each time point was close to 0.5. The EphrinB2 and EphB4 in the brain after hypoxic ischemia in rats did not increase at 2 days (P5), EphrinB2/EphB4 was 0.49, but there was no statistical difference from the Sham group (P0.05), but 4 after the operation. P7 EphrinB2 and EphB4 increased rapidly, compared with the Sham group, the difference was statistically significant (P0.05), and the 7 days after the operation (PI0) began to decrease, but the expression of EphrinB2 was more than EphB4 and EphrinB2/EphB4 was 0.56, and the 2 days after operation (P5) was significantly increased in the HI-EPO group. The expression of EphrinB2 was higher than that of ischemic hypoxia group, and the expression of EphrinB2 was more than that of EphB4. Conclusion: after hypoxic ischemia, the brain tissue has endogenous repair response, which shows an increase in the expression of white matter EPOR around the ventricle, the elevated expression of oxygen carrying protein Ngb, and a neovascularization reaction.2. Rh-EPO to the protection of PWMD, rh-EPO interfered with the white matter injury of hypoxic and ischemic brain. Rats can significantly reduce the damage of the white matter area around the ventricle, improve the long-term neurobehavioral ability of the rat, promote the further up of EPOR expression in the white matter, and provide EPO with the receptor effect of.3. Rh-EPO and EPOR, on one hand, by promoting Ngb to provide more oxygen to the brain tissue, on the other hand it can promote blood by promoting blood. The tube regeneration response provided more cerebral blood flow to the brain tissue and played an intervention role.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R-332;R722.6

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