【摘要】：背景創(chuàng)傷性腦損傷包括原發(fā)性損傷和繼發(fā)性損傷,而繼發(fā)性損傷是目前研究的熱點(diǎn)及難點(diǎn)。繼發(fā)性損傷由多種因素參與:興奮性毒性、氧自由基、鈣超載、水腫、炎癥反應(yīng)等,而水腫、炎癥反應(yīng)在其中發(fā)揮著重要的作用,但具體機(jī)制目前還不十分清楚。研究報(bào)道高遷移率族蛋白B1(high-mobility group box 1,HMGB1)在眾多損傷的急性、慢性炎癥及水腫的調(diào)控中發(fā)揮著關(guān)鍵的作用。那么HMGB1在人腦挫裂傷后的繼發(fā)性損傷中發(fā)揮著怎樣的作用值得深入的研究。目的探討HMGB1在人腦挫裂傷組織中的表達(dá)及作用。方法通過收集32例腦挫裂傷病人手術(shù)切除的腦組織標(biāo)本作為實(shí)驗(yàn)組,根據(jù)傷后時(shí)間不同,分為傷后6 h組(n=7)、12 h組(n=9)、1 d組(n=10)、3 d組(n=6),同時(shí)搜集正常腦組織作為對(duì)照組(n=5)。所有新鮮標(biāo)本用4%多聚甲醛固定,然后用30%蔗糖溶液脫水處理,最后制作冰凍切片。然后進(jìn)行蘇木精-伊紅染色(H.E.染色)、免疫熒光染色。H.E.染色:取實(shí)驗(yàn)組和對(duì)照組各標(biāo)本進(jìn)行染色,觀察每張視野下淋巴細(xì)胞和中性粒細(xì)胞數(shù)目,統(tǒng)計(jì)分析各時(shí)間點(diǎn)炎性細(xì)胞浸潤(rùn)情況。免疫熒光染色:取實(shí)驗(yàn)組和對(duì)照組各標(biāo)本進(jìn)行染色,將HMGB1與DAPI(細(xì)胞核染液)共標(biāo),熒光顯微鏡視野下觀察人腦挫裂傷后組織中各個(gè)時(shí)間點(diǎn)HMGB1在胞核內(nèi)及胞漿中的數(shù)目,進(jìn)一步了解HMGB1從細(xì)胞核內(nèi)釋放到核外的情況;將HMGB1與神經(jīng)元標(biāo)志物NeuN進(jìn)行共標(biāo),觀察統(tǒng)計(jì)人腦挫裂傷后早期神經(jīng)元數(shù)目、神經(jīng)元中HMGB1數(shù)目及神經(jīng)元外的HMGB1數(shù)目,進(jìn)一步了解損傷后神經(jīng)元與HMGB1的轉(zhuǎn)移情況;將水通道蛋白4(AQP4)與星形膠質(zhì)細(xì)胞標(biāo)記物(GFAP)進(jìn)行共標(biāo),計(jì)算各時(shí)間點(diǎn)水通道蛋白4(AQP4)表達(dá)的熒光強(qiáng)度,分析水通道蛋白4在星形膠質(zhì)細(xì)胞表達(dá)的情況。采用SPSS 12.0統(tǒng)計(jì)軟件,計(jì)量資料以x±s表示,多組間比較采用OnewayANOVA分析,以P0.05為差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果人腦挫裂傷后,浸潤(rùn)的淋巴細(xì)胞細(xì)胞于1d開始明顯增加(P0.05);浸潤(rùn)的中性粒細(xì)胞于12h開始明顯增加(P0.05)。隨時(shí)間延長(zhǎng),炎性細(xì)胞數(shù)均逐漸升高。正常人腦組織的HMGB1表達(dá)主要集中在細(xì)胞核內(nèi),人腦挫裂傷組HMGB1在細(xì)胞核內(nèi)的細(xì)胞數(shù)于12 h開始下降(P0.05),并持續(xù)下降至72 h;而HMGB1從核內(nèi)轉(zhuǎn)移至胞漿內(nèi)的數(shù)目從6 h即開始增加(P0.05),隨時(shí)間增加HMGB1也隨之大量轉(zhuǎn)移到胞漿中,在人腦挫裂傷后24 h達(dá)到高峰(P0.05)。人腦挫裂傷后早期,HMGB1主要表達(dá)在神經(jīng)元中(P0.05),腦挫裂傷后神經(jīng)元的數(shù)目逐漸減少(P0.05),而HMGB1從神經(jīng)元核內(nèi)轉(zhuǎn)移至胞漿的比例在6 h明顯增加(P0.05),于24 h-72 h達(dá)到高峰(P0.05);同時(shí)研究發(fā)現(xiàn)AQP4主要表達(dá)在星形膠質(zhì)細(xì)胞,并且隨著損傷時(shí)間的延長(zhǎng),表達(dá)量逐漸增加,于傷后3 d達(dá)到高峰。結(jié)論明確人腦挫裂傷后早期炎癥細(xì)胞浸潤(rùn)情況;HMGB1在人腦挫裂傷后早期迅速從細(xì)胞核內(nèi)釋放,參與炎癥反應(yīng);HMGB1可能參與了人腦挫裂傷后神經(jīng)元的壞死或凋亡;HMGB1可能參與了人腦挫裂傷后腦水腫的病理生理過程。
[Abstract]:Background traumatic brain injury includes primary injury and secondary injury. Secondary injury is involved by many factors: excitatory toxicity, oxygen free radical, calcium overload, edema, inflammatory reaction, etc. Edema and inflammatory reaction play an important role, but the specific mechanism is not very clear. It is reported that high mobility group protein B1 (high-mobility group box 1 HMGB1) plays a key role in the regulation of acute, chronic inflammation and edema. So what role HMGB1 plays in the secondary injury after human brain contusion is worth further study. Objective to investigate the expression and role of HMGB1 in human brain contusion and laceration. Methods 32 brain tissue specimens were collected from 32 patients with cerebral contusion and laceration as experimental group. According to the different time after injury, they were divided into two groups: 6 h group (n = 7), 12 h group (n = 9), 1 d group (n = 10) and 3 d group (n = 6), and normal brain tissue was collected as control group (n 5). All fresh specimens were fixed with 4% paraformaldehyde and then dehydrated with 30% sucrose solution. Then hematoxylin-eosin staining (H. E. Immunofluorescence staining. Staining: the numbers of lymphocytes and neutrophils in each field of vision were observed and the infiltration of inflammatory cells was analyzed at each time point. Immunofluorescence staining: the samples of experimental group and control group were stained with HMGB1 and DAPI. The number of HMGB1 in nucleus and cytoplasm of human brain contusion and laceration was observed under fluorescence microscope. To further understand the release of HMGB1 from the nucleus to the nucleus, the HMGB1 was colabeled with the neuron marker NeuN, and the number of neurons, the number of HMGB1 in neurons and the number of HMGB1 outside of neurons were observed and counted after the contusion and laceration of human brain. To further understand the metastasis between neurons and HMGB1 after injury, we colabeled aquaporin-4 (AQP4) with astrocyte marker (GFAP) to calculate the fluorescence intensity of aquaporin-4 (AQP4) expression at different time points. The expression of aquaporin 4 in astrocytes was analyzed. The statistical software SPSS 12.0 was used, the measurement data was expressed as x 鹵s, and the OnewayANOVA analysis was used to compare the data among groups. The difference was statistically significant with P0.05. Results after contusion and laceration of human brain, the number of infiltrating lymphocytes increased significantly at 1 day (P0.05), and the infiltration of neutrophils increased significantly at 12 hours (P0.05). As time went on, the number of inflammatory cells increased gradually. The expression of HMGB1 in normal human brain tissue is mainly concentrated in the nucleus. In the contusion and laceration group, the number of HMGB1 cells in the nucleus began to decrease at 12 h (P0.05) and continued to decrease to 72 h, while the number of HMGB1 transferred from the nucleus to the cytoplasm began to increase from 6 h (P0.05), and the number of HMGB1 transferred to the cytoplasm increased with time. The peak was reached 24 hours after contusion and laceration of human brain (P0.05). HMGB1 was mainly expressed in neurons at the early stage of contusion and laceration (P0.05), and the number of neurons decreased gradually after contusion (P0.05), while the proportion of HMGB1 transferred from the nucleus of neurons to cytoplasm increased significantly at 6 h (P0.05), and reached its peak at 24h-72 h (P0.05). It was found that AQP4 was mainly expressed in astrocytes. With the extension of injury time, the expression level increased gradually and reached the peak 3 days after injury. Conclusion HMGB1 was rapidly released from the nucleus at the early stage of contusion and laceration of human brain and the infiltration of inflammatory cells in the early stage of contusion and laceration of human brain. HMGB1 may be involved in neuronal necrosis or apoptosis after human brain contusion and laceration. HMGB1 may be involved in the pathophysiological process of brain edema after contusion and laceration.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R651.15

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