【摘要】：目的：缺氧缺血性腦病(hypoxic-ischemic encephalopathy, HIE)是導(dǎo)致新生兒急性死亡及慢性神經(jīng)系統(tǒng)損傷的主要原因之一。研究表明，促紅細(xì)胞生成素(erythropoietin, EPO)有腦保護(hù)作用，本實(shí)驗(yàn)通過(guò)建立新生鼠缺氧缺血性腦損傷(hypoxic-ischemic brain damage, HIBD)模型，腹腔內(nèi)注射外源性重組人促紅細(xì)胞生成素(recombinant human erythropoietin, rhEPO)，觀察EPO對(duì)新生鼠HIBD時(shí)缺氧誘導(dǎo)因子-1α(hypoxia inducible factor-1α, HIF-1α)及存活素(Survivin)表達(dá)的影響，探討其抗凋亡的可能機(jī)制，為臨床應(yīng)用EPO治療新生兒HIE提供新的理論依據(jù)。 方法：將7d Wistar大鼠120只隨機(jī)分為3組：假手術(shù)組(n=8)、HIBD組(n=56)、rhEPO治療組(n=56)，后兩者根據(jù)處死時(shí)間分為：3h組，6h組，12h組，24h組，48h組，3d組，7d組，每組8只。假手術(shù)組新生大鼠行常規(guī)消毒后切開頸部皮膚，分離左側(cè)頸總動(dòng)脈，不予缺氧處理；HIBD組新生大鼠分離左側(cè)頸總動(dòng)脈并給予結(jié)扎，后置于缺氧倉(cāng)中（8%O2和92%N2混合氣體）2h；rhEPO治療組于HIBD模型建立后即刻、24h、48h連續(xù)腹腔注射rhEPO(5 000 IU/kg/d)。各組新生鼠于相應(yīng)時(shí)間點(diǎn)處死取腦組織后觀察腦外觀的變化，采用HE染色鏡下觀察腦組織的病理學(xué)改變，應(yīng)用免疫組化法測(cè)定HIF-1、Survivin的表達(dá)，用脫氧核糖核苷酸末端轉(zhuǎn)移酶介導(dǎo)的缺口末端標(biāo)記法（terminal-deoxynucleotidyl transferasemediated nick end labeling, TUNEL）對(duì)細(xì)胞凋亡指數(shù)（apoptosis index, AI）進(jìn)行檢測(cè)。 結(jié)果：（1）新生大鼠缺氧缺血后可見大腦左側(cè)有不同程度的水腫，rhEPO治療后左側(cè)腦水腫程度較HIBD組減輕。（2）HE染色：假手術(shù)組新生大鼠腦組織結(jié)構(gòu)和細(xì)胞層次清楚，細(xì)胞排列規(guī)整，形態(tài)尚正常，可見少數(shù)細(xì)胞胞漿皺縮，核深染；HIBD組各時(shí)間點(diǎn)新生大鼠左側(cè)腦組織呈現(xiàn)不同程度的水腫，可見壞死，胞核固縮、溶解，神經(jīng)細(xì)胞數(shù)減少；rhEPO治療組各時(shí)間點(diǎn)大鼠腦組織損傷程度與HIBD組相比有所減輕。（3）免疫組化染色：①HIF-1的表達(dá)：HIF-1陽(yáng)性細(xì)胞主要表達(dá)于大腦皮層和海馬區(qū)，陽(yáng)性著色位于神經(jīng)細(xì)胞胞質(zhì)和（或）胞核，呈棕黃色細(xì)顆粒沉積。HIF-1的表達(dá)在假手術(shù)組中呈少量表達(dá)；在HIBD組，缺氧缺血后3h即增強(qiáng)，12h達(dá)高峰，后逐漸降低(P＜0.01)；rhEPO治療組12h、24h、48h、3d的HIF-1表達(dá)水平較HIBD組明顯減少(P＜0.05)。②Survivin的表達(dá)：Survivin陽(yáng)性細(xì)胞主要表達(dá)于大腦皮層和海馬區(qū)，陽(yáng)性著色位于神經(jīng)細(xì)胞胞質(zhì)和（或）胞核，呈棕黃色細(xì)顆粒沉積。Survivin的表達(dá)在假手術(shù)組僅少量表達(dá)；在HIBD組，缺氧缺血后12h開始增高，7d明顯增高，與假手術(shù)組相比，除3h、6h組，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)；rhEPO治療組12h、24h、48h、3d的Survivin表達(dá)較HIBD組明顯增高(P＜0.05)。（4）神經(jīng)細(xì)胞凋亡趨勢(shì)：細(xì)胞AI在假手術(shù)組中較低；在HIBD組缺氧缺血6h即增高，7d明顯增高，與假手術(shù)組相比，除3h組，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)；rhEPO治療組24h、48h、3d、7d的細(xì)胞AI值較HIBD組明顯降低(P＜0.05)。 結(jié)論：(1)通過(guò)鏡下觀察HE染色結(jié)果證實(shí)，rhEPO治療組各時(shí)間點(diǎn)與HIBD組比較，新生鼠腦組織病理?yè)p傷程度有所減輕，從病理學(xué)證實(shí)應(yīng)用EPO治療后可減輕HIBD。(2)腦缺氧缺血可導(dǎo)致腦皮質(zhì)及海馬區(qū)神經(jīng)細(xì)胞凋亡，同時(shí)可提高HIF-1和Survivin的表達(dá)，說(shuō)明HIF-1和Survivin共同參與了新生大鼠HIBD的病理過(guò)程。(3)應(yīng)用EPO治療后，通過(guò)抑制HIF-1的過(guò)表達(dá)及提高Survivin的表達(dá)，進(jìn)而抑制神經(jīng)細(xì)胞凋亡，從而在新生鼠HIBD時(shí)發(fā)揮其腦保護(hù)作用，這可能是EPO腦保護(hù)作用機(jī)制之一。
[Abstract]:Objective: The hypoxic ischemic encephalopathy (HIE) is one of the main causes of acute death and chronic nervous system injury in neonates. It is shown that erythropoietin (EPO) has the protective effect of brain. This experiment is to establish a model of hypoxic-ischemic brain injury (HIBD) in neonatal rats, and inject exogenous recombinant human erythropoietin (rhEPO) into the abdominal cavity. Objective To observe the effect of EPO on the expression of hypoxia-inducible factor-1 gene and survivin in neonatal rat HIBD, and to explore the possible mechanism of anti-apoptosis, and to provide a new theoretical basis for clinical application of EPO in the treatment of neonatal HIE. Methods: 120 Wistar rats were randomly divided into 3 groups: sham operation group (n = 8), HIBD group (n = 56), rhEPO treatment group (n = 56). The two groups were divided into three groups: 3h group, 6h group, 12h group, 24h group, 48h group, 3d group, 7d group, each group 8 The left carotid artery was isolated from the neonatal rats of HIBD group and ligated and placed in anoxic bin (8% O2 and 92% N2 mixed gas) 2h; rhEPO treatment group was established after the HIBD model was established. engraved, 24h, 48h continuous intraperitoneal injection of rhEPO (5,000 IU/ kg/ day The changes of brain appearance were observed after the rats were killed at corresponding time points, and the pathological changes of brain tissues were observed under HE staining. The expressions of survivin and Survivin were determined by immunohistochemistry. The apoptosis index (AI) was examined by TUNEL-mediated nick end labeling (TUNEL). Results: (1) After hypoxic ischemia in neonatal rats, there were different degrees of edema left on the left side of the brain, and the left brain edema after rhEPO treatment was higher than that of HIBD. (2) HE staining: The structure and cell level of brain tissue of neonatal rats in sham operation group are clear, the cell arrangement is regular, the morphology is still normal, few cell cytoplasm shrinkage and nuclear deep staining can be seen, and the left brain tissues of neonatal rats in the HIBD group show different degree of edema, which can See necrosis, cell nucleus contraction, dissolution, number of nerve cells decreased, and the degree of damage of brain tissue in rhEPO treatment group was compared with that of HIBD group. (3) Immunohistochemical staining: The expression of p27-1 was mainly expressed in the cerebral cortex and hippocampus, and the positive staining was located in the cytoplasm and/ or nucleus of the nerve cells. The expression of CD44v6-1 in the sham operation group was expressed in a small amount; in the HIBD group, after the hypoxia-ischemia 3h, the expression level was increased, the peak of 12h reached the peak, and then gradually decreased (P <0.01). The expression level of OPG-1 in rhEPO treatment group was significantly lower than that in HIBD group (P <0. 01). 05). Survivin expression: Survivin positive cells are mainly expressed in the cerebral cortex and hippocampus, and the positive staining is located in the cytoplasm and/ or nucleus of nerve cells. The expression of Survivin was only a small amount in sham operation group; in HIBD group, 12h after hypoxic ischemia increased, and 7d was significantly increased. Compared with sham operation group, the difference was significant (P <0.01); rhEPO treated group 12h, 24h. The expression of Survivin in 48h and 3d was higher than that in HIBD group (P <0.01). 05). (4) Neuronal apoptosis trend: The AI was lower in the sham operation group; in the HIBD group, the hypoxia ischemia 6h was increased, and 7d was significantly increased. Compared with the sham operation group, the difference was statistically significant (P <0.01). The rhEPO treatment group was 24h, 48h. The AI values of 3d and 7d were significantly lower than that in HIBD group (P <0.01). Conclusion: (1) Compared with HIBD group, the pathological damage degree of neonatal rat's brain tissue was decreased, and the pathological findings of rhEPO treated group decreased after EPO therapy. Mild HIBD. (2) Cerebral ischemia can lead to neuronal apoptosis in cerebral cortex and hippocampus, meanwhile, the expression of survivin-1 and Survivin can be improved, and the expression of survivin-1 and Survivin in neonatal rat HIBD is demonstrated. (3) After EPO is applied, the expression of survivin and the expression of Survivin are inhibited, so that the apoptosis of nerve cells can be inhibited, so that the brain protection effect can be exerted when the newborn mouse HIBD is HIBD, which may be EPO brain protection.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R722.1

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