本文選題：氙氣 + CLIC4　； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的腹腔內(nèi)注射脂多糖聯(lián)合缺氧缺血制作3日齡新生大鼠腦白質(zhì)損傷模型,并對(duì)腦白質(zhì)損傷新生大鼠給予氙氣干預(yù)。通過(guò)檢測(cè)其腦組織中CLIC4 m RNA、Bcl-2蛋白的表達(dá),探討氙氣對(duì)新生大鼠腦白質(zhì)損傷的神經(jīng)保護(hù)作用機(jī)制。方法將3日齡SD新生大鼠120只隨機(jī)分為空白對(duì)照組(n=24),腦損傷組(n=24)和氙氣干預(yù)組(n=72)。空白對(duì)照組腹腔注射生理鹽水(0.05mg/kg),僅游離右側(cè)頸總動(dòng)脈不做低氧處理;腦損傷組腹腔注射脂多糖(lipopolysaccharide,LPS,0.05mg/kg),游離并結(jié)扎右側(cè)頸總動(dòng)脈并進(jìn)行低氧處理,但不進(jìn)行氙氣處理;氙氣干預(yù)組腹腔注射LPS(0.05mg/kg),游離并結(jié)扎右側(cè)頸總動(dòng)脈并進(jìn)行低氧處理及氙氣吸入處理(50%氙氣+30%氧氣+20%氮?dú)獾某錆M(mǎn)20L恒溫密閉箱,0.5L/min緩慢維持,持續(xù)處理3小時(shí))。根據(jù)腦損傷后氙氣延遲干預(yù)的時(shí)間(0小時(shí)、2小時(shí)和5小時(shí)),將氙氣干預(yù)組分為C1、C2、C3三個(gè)亞組(n=24)。各組大鼠分別于0h、24h、48h、72h各取6只行多聚甲醛灌注后斷頭取腦組織,進(jìn)行腦組織病理檢測(cè)、蛋白質(zhì)印跡法(Western Blot,WB)檢測(cè)B淋巴細(xì)胞瘤-2(B-cell lymphoma-2,Bcl-2)蛋白含量、逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(reverse transcriotion-polymerase chain reaction,RT-PCR)檢測(cè)氯離子通道蛋白4(Chloride Intracellular Channel 4,CLIC4)m RNA的表達(dá)。采用獨(dú)立樣本t檢驗(yàn)、單因素方差分析和Bonferroni法進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果(1)實(shí)驗(yàn)新生鼠行為學(xué)監(jiān)測(cè):腹腔注射脂多糖及缺氧缺血處理后,腦損傷組和氙氣干預(yù)組的新生鼠出現(xiàn)肢體抖動(dòng)、煩躁、紫紺、呼吸急促等行為學(xué)異常;在氙氣處理之后,C1組新生鼠的癥狀在約2小時(shí)后消失;而腦損傷組、C2組和C3組的癥狀約在5小時(shí)后消失;空白對(duì)照組新生鼠未出現(xiàn)明顯異常反應(yīng)。(2)HE染色觀察腦組織病理變化:腦損傷組新生鼠可見(jiàn)腦白質(zhì)蒼白,結(jié)構(gòu)疏松,細(xì)胞腫脹,核膜不清楚,細(xì)胞核固縮,可檢測(cè)到細(xì)胞變性和壞死,神經(jīng)纖維無(wú)序,膠質(zhì)細(xì)胞數(shù)量增加;氙氣干預(yù)組腦白質(zhì)染色淡染,結(jié)構(gòu)相對(duì)較完整,較少細(xì)胞變性壞死,各亞組之間無(wú)明顯差異;而空白對(duì)照組腦組織結(jié)構(gòu)正常,染色清晰,細(xì)胞排列整齊。(3)WB檢測(cè)Bcl-2蛋白含量:在相同時(shí)間點(diǎn),各組Bcl-2蛋白的表達(dá)量相比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05);與腦損傷組相比,C1組和C2組中Bcl-2蛋白的表達(dá)在各時(shí)間點(diǎn)均顯著增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05),但在C3中組的Bcl-2蛋白的表達(dá)在48h和72h與腦損傷組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);與C1組相比,C2組和C3組Bcl-2蛋白表達(dá)在48h和72h顯著降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。(4)RT-PCR檢測(cè)CLIC4 m RNA含量:與空白對(duì)照組相比較,在各時(shí)間點(diǎn)腦損傷組中新生鼠腦室周?chē)踪|(zhì)CLIC4 m RNA的表達(dá)水平增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05);與腦損傷組相比較,氙氣干預(yù)各亞組中CLIC4 m RNA表達(dá)均明顯減少,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論(1)腹腔內(nèi)注射LPS聯(lián)合缺氧缺血可成功制作新生兒大鼠腦白質(zhì)損傷模型;(2)氙氣吸入干預(yù)可減輕新生兒大鼠腦白質(zhì)損傷,減少細(xì)胞壞死;(3)新生大鼠腦白質(zhì)損傷后Bcl-2蛋白表達(dá)水平升高,氙氣可使其表達(dá)可下降,表明氙氣可能通過(guò)抑制神經(jīng)元凋亡進(jìn)而減輕白質(zhì)損傷作用;(4)氙氣干預(yù)可下調(diào)CLIC4基因的表達(dá),表明氙氣可能通過(guò)抑制CLIC4基因的表達(dá)作用于Bcl-2蛋白相關(guān)途徑抑制神經(jīng)元凋亡而發(fā)揮神經(jīng)保護(hù)作用;(5)氙氣治療腦白質(zhì)損傷的時(shí)間窗至少有5小時(shí),且越早干預(yù)效果越好。
[Abstract]:Objective to make intraperitoneal injection of lipopolysaccharide (lipopolysaccharide) and hypoxic ischemia to make a white matter injury model of 3 day old neonatal rats, and to give xenon intervention to neonatal rats with brain white matter injury. By detecting the expression of CLIC4 m RNA and Bcl-2 protein in the brain tissue, the mechanism of neuroprotective effect of xenon on brain white matter injury in newborn rats was investigated. The method was a new method of 3 days of age SD. 120 rats were randomly divided into blank control group (n=24), brain injury group (n=24) and xenon intervention group (n=72). The blank control group was intraperitoneally injected with normal saline (0.05mg/kg), and only the right common carotid artery was free from hypoxic treatment. The brain injury group was injected with lipopolysaccharide (lipopolysaccharide, LPS, 0.05mg/kg), free and ligated the right common carotid artery and carried out. Hypoxia treatment, but no xenon treatment; xenon intervention group intraperitoneal injection of LPS (0.05mg/kg), free and ligated right common carotid artery and conducting hypoxia treatment and xenon inhalation treatment (50% xenon +30% oxygen +20% nitrogen filled 20L constant temperature closed tank, 0.5L/min slow maintenance, sustained treatment for 3 hours). Xenon gas delayed intervention after brain injury, after the delayed intervention of xenon gas (0 hours, 2 hours and 5 hours), the xenon intervention group was divided into C1, C2, and C3 three subgroups (n=24). The rats in each group were respectively taken from 0h, 24h, 48h, and 72h to take the brain tissue after paraformaldehyde perfusion, and the brain tissue was detected, and the protein content was detected by the Western blot (Western Blot, WB), and the inverse of the protein content. Transcription polymerase chain reaction (reverse transcriotion-polymerase chain reaction, RT-PCR) was used to detect the expression of chloride channel protein 4 (Chloride Intracellular Channel 4, CLIC4) m RNA. Independent sample t test, single factor analysis of variance and statistical analysis were used. Results (1) behavioral monitoring of neonatal rats: intraperitoneal injection After the treatment of lipopolysaccharide and hypoxic ischemia, the newborn rats in the brain injury group and the xenon group appeared limb jitter, irritability, cyanosis, and respiratory shortness. After xenon treatment, the symptoms of newborn rats in group C1 disappeared after about 2 hours, while the symptoms of brain injury group, group C2 and C3 disappeared after 5 hours; the blank control group of newborn rats did not. There was an obvious abnormal reaction. (2) the pathological changes of brain tissue were observed by HE staining: the white matter was pale, the structure was loose, the cells were swollen, the nuclear membrane was not clear, the nuclear condensation was not clear, the cell degeneration and necrosis were detected, the nerve fiber disorder, the number of glial cells increased, the white matter in the xenon intervention group was pale staining, and the structure was relatively complete. Whole, less cell degeneration and necrosis, there was no significant difference between the subgroups, while the blank control group had normal brain structure, clear staining and orderly cells. (3) WB detection of Bcl-2 protein content: at the same time point, the expression of Bcl-2 protein in each group was statistically significant (P0.05); compared with the brain injury group, the C1 and C2 group Bcl-2 eggs The expression of white was increased at all time points, and the difference was statistically significant (P0.05), but there was no significant difference in the expression of Bcl-2 protein in C3 group in 48h and 72h with brain injury group (P0.05). Compared with the C1 group, the expression of Bcl-2 protein in C2 group and C3 group decreased significantly in 48h and 72h. (4) M RNA content: compared with the blank control group, the expression level of white matter CLIC4 m RNA around the ventricle of newborn rats in each time point brain injury group increased, the difference was statistically significant (P0.05). Compared with the brain injury group, the RNA expression of CLIC4 m in the xenon group was significantly decreased, the difference was statistically significant (P0.05). Conclusion (1) intraperitoneal injection (P0.05). LPS combined with hypoxia ischemia can successfully produce white matter injury model of neonatal rat brain; (2) xenon inhalation intervention can reduce brain white matter injury and decrease cell necrosis in neonatal rats. (3) the expression level of Bcl-2 protein in newborn rats after white matter injury increases, xenon can decrease its surface, indicating that xenon may inhibit neuronal apoptosis by inhibiting apoptosis. (4) xenon intervention can down regulate the expression of CLIC4 gene, indicating that xenon may play a neuroprotective role by inhibiting the expression of CLIC4 gene to inhibit neuronal apoptosis by Bcl-2 protein related pathway; (5) xenon treatment of brain white matter injury by xenon at at least 5 hours, and the earlier intervention the better.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R742

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本文編號(hào)：1885304