【摘要】：[目的]研究POSTN對新生大鼠缺氧缺血(hypoxic-ischemic, HI)后神經(jīng)干細胞增殖分化的影響,為臨床新生兒腦損傷的治療提供新的思路。 [方法]通過7日齡新生大鼠左側(cè)頸總動脈結(jié)扎離斷后8%氧氣缺氧2h制備動物模型,隨機分為HI組和假手術(shù)組。干預(yù)組進行側(cè)腦室注射POSTN蛋白干預(yù)治療,非干預(yù)組注射PBS。采用BrdU標記結(jié)合免疫熒光化學方法觀察各組大鼠不同時間點腦室下區(qū)(subventricular zone, SVZ)和海馬齒狀回(dentate gyrus,DG)神經(jīng)干細胞的增殖及其向神經(jīng)元、星形膠質(zhì)細胞分化情況。采用Morris水迷宮實驗方法觀察各組大鼠學習記憶功能變化。 [結(jié)果]POSTN干預(yù)治療能夠顯著增加SVZ和海馬DG新生細胞(BrdU+)數(shù)量,其中新生神經(jīng)干細胞(BrdU+/Nestin+)在術(shù)后7d達高峰,而新生神經(jīng)元(BrdU+/Map-2+)和星形膠質(zhì)細胞(BrdU+/GFAP+)在14d達高峰,差異有統(tǒng)計學意義。Morris水迷宮實驗結(jié)果顯示,POSTN干預(yù)治療能夠降低大鼠的逃避潛伏期和游泳距離,增加站臺穿越次數(shù)和目標象限停留時間比例,差異有統(tǒng)計學意義。 [結(jié)論]POSTN干預(yù)可促進新生大鼠缺氧缺血性腦損傷后的神經(jīng)干細胞增殖和分化,改善其學習記憶功能,有助于腦損傷后的神經(jīng)修復。 [目的]通過體外培養(yǎng)獲得的Olig2+-GFP+-OPC移植入新生大鼠缺氧缺血性(hypoxia-ischemia, HI)腦損傷模型,觀察Olig2+-GFP+-OPC定向分化成熟為少突膠質(zhì)細胞的能力和植入細胞的存活情況,探討外源性植入神經(jīng)前體細胞OPC治療早產(chǎn)兒腦室周白質(zhì)軟化(periventricular leukomalacia, PVL)的可行性。 [方法]Olig2+-GFP+-mES細胞通過擬胚體(embryonic body, EB)介導的神經(jīng)誘導法,即綜合全反式維甲酸(retinoic acid, RA)誘導法和五步法使Olig2+-GFP+-mES定向分化為OPC。采用免疫細胞化學染色,利用倒置相差顯微鏡和熒光顯微鏡來觀察OPC的分化成熟能力。3日齡新生大鼠左側(cè)頸總動脈結(jié)扎離斷后37℃條件下6%氧氣缺氧2.5h制備動物模型,通過HE(haematoxylin-eosin)染色方法評估模型情況。通過免疫熒光染色方法觀察側(cè)腦室注射植入的Olig2+-GFP+-OPC存活情況。 [結(jié)果]Olig2+-GFP+-mES在胚胎干細胞培養(yǎng)基中形成EB,在含有RA(0.5μM)和SHH(100ng/ml)神經(jīng)分化培養(yǎng)基中,逐漸發(fā)育為神經(jīng)前體細胞球。在EB4d開始出現(xiàn)Olig2+-GFP+-陽性細胞,在EB12d時表達出現(xiàn)高峰。Olig2+-GFP+-陽性細胞具有體外條件下分化為OPC,再定向分化為成熟少突膠質(zhì)細胞并具備成髓鞘的能力。HI組腦室下區(qū)神經(jīng)細胞出現(xiàn)變性、壞死,細胞層次紊亂、排列松散,部分細胞出現(xiàn)核固縮、核溶解,胞漿深染,結(jié)構(gòu)不清。對照組則無明顯病理變化。在側(cè)腦室周邊及附近腦區(qū)有GFP的表達,證實植入OPC成功,并向周圍遷移。 [結(jié)論]體外EB介導的神經(jīng)誘導法能夠高效誘導產(chǎn)生Olig2+-GFP+-OPC,并具有分化為成熟少突膠質(zhì)細胞且有成髓鞘能力。在HI腦損傷模型中側(cè)腦室植入Olig2+-GFP+-OPC細胞能夠存活。 [目的]研究缺氧缺血后亞低溫對新生大鼠海馬部位細胞的存活及星形膠質(zhì)細胞活化增殖的影響,探討亞低溫對缺氧缺血腦損傷保護作用的機制。 [方法]3日齡新生大鼠海馬腦片,培養(yǎng)至第4天進行氧糖剝奪(oxygen-glucose deprivation, OGD)制備模型,分為亞低溫組(33℃,24h)和常溫組(37℃)；對照組不進行OGD處理,37℃培養(yǎng)7d。采用碘化丙啶(propidium iodide, PI)和免疫熒光染色觀察海馬腦片的細胞存活和星形膠質(zhì)細胞的活化增殖。動物實驗：7日齡新生大鼠采用左側(cè)頸總動脈結(jié)扎離斷并在8%02+92%N2中缺氧2h,制備缺氧缺血模型,分為手術(shù)常溫組(肛溫36-37℃,24h)和手術(shù)亞低溫組(肛溫32-33℃,24h)。假手術(shù)組僅分離左側(cè)頸總動脈,不結(jié)扎,也不缺氧,分為假手術(shù)常溫組和假手術(shù)亞低溫組。各組于術(shù)后3d和7d處死取材,采用DAPI (4'-6-diamidino-2-phenylindole)和免疫組織化學方法檢測海馬組織細胞存活和星形膠質(zhì)細胞的活化增殖。 [結(jié)果]體外和體內(nèi)實驗結(jié)果都表明缺氧缺血后3d新生大鼠海馬組織中存活的細胞常溫組較亞低溫組少。海馬腦片培養(yǎng)GFAP (glial fibrillary acidic protein, GFAP)染色顯示,亞低溫組GFAP陽性細胞數(shù)較常溫組明顯減少,兩組比較具有統(tǒng)計學差異(P0.05)。動物實驗海馬組織GFAP染色結(jié)果也顯示,缺氧缺血后3d和7d手術(shù)亞低溫組GFAP陽性細胞較手術(shù)常溫組顯著減少,同一時間點兩組比較具有統(tǒng)計學意義(P0.05)。 [結(jié)論]亞低溫能減輕新生大鼠缺氧缺血后海馬星形膠質(zhì)細胞的活化增殖。
[Abstract]:[Objective] To study the effects of POSTN on the proliferation and differentiation of neural stem cells after hypoxic-ischemic (HI) in neonatal rats. [Methods] The animal model was prepared by ligation of the left common carotid artery of 7-day-old neonatal rats and 8% oxygen hypoxia for 2 h. The model was divided into HI group and sham operation. Group. The intervention group was treated by intraventricular injection of POSTN protein, and the non-intervention group was injected with PB. S. The proliferation of the subventricular zone (SVZ) and the dentate gyrus (DG) neural stem cells in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampal dentate gyrus (DG) and the differentiation of the neurons and astrocytes were observed by means of the BrdU label and the immunofluorescence chemical method. The learning and memory function of each group was observed by the Morris water maze test method. [Results] POSTN intervention could significantly increase the number of the newly-born cells (BrdU +) in the SVZ and the hippocampus, in which the new neural stem cells (BrdU +/ Nestin +) reached the peak at 7 d after the operation, while the new neurons (BrdU +/ Map-2 +) and the astrocytes (BrdU +/ GFAP +) peaked at 14 d and the difference was statistically significant. The results of the Morris water maze show that the POSTN intervention can reduce the escape latency and the swimming distance of the rat, increase the number of crossing times of the platform and the proportion of the residence time of the target quadrant, and the difference is statistics. [Conclusion] POSTN intervention can promote the proliferation and differentiation of neural stem cells after hypoxic-ischemic brain injury in neonatal rats, improve their learning and memory function, and contribute to brain injury Nerve repair.[Objective] To observe the ability and implantation of the Olig2 +-GFP +-OPC (Olig2 +-GFP +-OPC) transplanted into the hypoxic-ischemic (HI) brain injury model of the neonatal rats by in-vitro culture, and observe the ability and the implantation of the oligodendrocytes in the Olig2 +-GFP +-OPC directional differentiation. The survival of the cells was investigated, and the periventricular leukomalacia of the premature infants treated with the exogenous pre-implantation of the neural precursor cells (OPC) was discussed. The feasibility of VL is that the OLlig2 +-GFP +-mES cell is mediated by an embryonic body (EB), i.e., an integrated all-trans-retinoic acid (RA) method and a five-step method to make the Oli2 +-GFP +-mE S-directional differentiation was OPC. The differentiation and maturation of the OPC were observed by using an inverted phase-contrast microscope and a fluorescence microscope. The animal model was prepared by using an inverted phase-contrast microscope and a fluorescence microscope at 37 鈩，

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