本文選題：蛛網(wǎng)膜下腔 + 移植��； 參考：《山東大學(xué)》2017年博士論文

【摘要】：目的:探討bcl-2基因轉(zhuǎn)染雪旺細(xì)胞蛛網(wǎng)膜下腔移植對脊髓損傷大鼠損傷神經(jīng)功能恢復(fù)的影響。方法:體外培養(yǎng)大鼠雪旺細(xì)胞,經(jīng)Ad-EGFP為載體介導(dǎo)端B淋巴細(xì)胞瘤-2基因(bcl-2)基因轉(zhuǎn)染雪旺細(xì)胞,分為3組:對照組、陰性轉(zhuǎn)染組及bcl-2轉(zhuǎn)染組。Western blot檢測雪旺細(xì)胞被轉(zhuǎn)染后第3天和第14天的bcl-2蛋白的表達(dá)。選取成年雌性SD大鼠83只,成功造模72只,隨機分為對照組,SCs組及bcl-2-SCs組,每組24只。依據(jù)改良Allen打擊法建造大鼠急性脊髓損傷模型,分別于造模前、造模后1天、3天、1周、2周、3周、4周進(jìn)行BBB、斜板試驗及改良Tarlov方法對各組大鼠進(jìn)行運動功能評定。造模后7天通過RT-PCR及Western blot檢測檢測脊髓損傷區(qū)周圍膠質(zhì)原纖維酸性蛋白(GFAP)、神經(jīng)絲蛋白(NF-200)的表達(dá),采用TUNEL法檢測神經(jīng)細(xì)胞凋亡情況,造模后4周取材行病理切片HE染色及熒光顯微鏡觀測EGFP標(biāo)記的SCs存活及分布情況,HRP逆行示蹤檢測神經(jīng)纖維修復(fù)情況,通過SEP和MEP觀察大鼠神經(jīng)電生理恢復(fù)情況。結(jié)果:Western blot結(jié)果顯示Ad-EGFP介導(dǎo)的bcl-2基因轉(zhuǎn)染大鼠雪旺細(xì)胞體外能穩(wěn)定表達(dá)bcl-2;大鼠下肢運動功能評價bcl-2-SCs組優(yōu)于SCs組,SCs組優(yōu)于對照組。造模完成72h,bcl-2-SCs組細(xì)胞凋亡數(shù)均明顯低于其他兩組(P0.05)。造模后7天,與對照組和SCs組相比,bcl-2-SCs組GFAP、NF-200基因和蛋白的表達(dá)均較顯著升高P0.05)。造模后4周,HE染色對照組可見脊髓組織缺失及脊髓空洞形成,無神經(jīng)軸索通過。SCs組損傷區(qū)可見少量神經(jīng)軸索樣結(jié)構(gòu),脊髓空洞較小,bcl-2-SCs組可見較多神經(jīng)軸索樣結(jié)構(gòu),未見脊髓空洞。EGFP標(biāo)記的陽性細(xì)胞數(shù):bcl-2-SCs組多于SCs組,對照組數(shù)量最少,各組數(shù)據(jù)有統(tǒng)計學(xué)差異(P0.05)。HRP陽性神經(jīng)纖維數(shù):bcl-2-SCs組SCs組對照組,各組之間有顯著性(P0.05)。造模后4周,SEP和MEP的潛伏期:bcl-2-SCs組SCs組對照組,且各組之間差異有顯著性意義(P0.05);波幅:bcl-2-SCs組SCs組對照組,且各組之間差異有顯著性意義(P0.05)。結(jié)論:bcl-2基因修飾雪旺細(xì)胞移植可促進(jìn)脊髓損傷大鼠神經(jīng)突觸的再生,升高脊髓損傷區(qū)GFAP、NF-200基因和蛋白的表達(dá),減少脊髓損傷區(qū)神經(jīng)細(xì)胞凋亡,提升大鼠的肢體運動功能和電生理功能。
[Abstract]:Aim: to investigate the effect of subarachnoid transplantation of Schwann cells transfected with bcl-2 gene on the recovery of nerve function in spinal cord injury rats.Methods: Schwann cells were cultured in vitro. Schwann cells were transfected with Ad-EGFP mediated terminal B lymphocytoma 2 gene (BCL 2) gene. The cells were divided into 3 groups: control group.The expression of bcl-2 protein in Schwann cells on the 3rd and 14th day after transfection was detected by Western blot in negative transfection group and bcl-2 transfection group.Seventy-two adult female Sprague-Dawley rats were selected and randomly divided into two groups: control group (n = 24) and bcl-2-SCs group (n = 24).The model of acute spinal cord injury (sci) in rats was established according to the modified Allen attack method. The motor function of each group was evaluated by slanting plate test and modified Tarlov method before the model was made and 1 day, 2 weeks, 3 weeks and 4 weeks after the establishment of the model.RT-PCR and Western blot were used to detect the expression of glial fibrillary acidic protein (GFAP) and neurofilament protein (NF-200) around the injured area of spinal cord. The apoptosis of neurons was detected by TUNEL method.The survival and distribution of EGFP labeled SCs were observed by HE staining and fluorescence microscope 4 weeks after the model. The nerve fiber repair was detected by retrograde tracing. The electrophysiological recovery of rat nerve was observed by SEP and MEP.Results the expression of bcl-2in Schwann cells transfected with bcl-2 gene mediated by Ad-EGFP was stable in vitro, and the evaluation of motor function of lower extremity in bcl-2-SCs group was superior to that in SCs group.The number of apoptotic cells in 72h Bcl-2-SCs group was significantly lower than that in the other two groups (P 0.05).After 7 days, compared with control group and SCs group, the expression of GFAP-NF-200 gene and protein in Bcl-2-SCs group was significantly higher than that in control group and SCs group.At 4 weeks after modeling, the absence of spinal cord tissue and the formation of syringomyelia were observed in the control group, and a few axonal structures were observed in the injured area without axons passing through .SCs, and more axonal structures were observed in the smaller syringomyelia bcl-2-SCs group.The number of positive cells labeled in the syringomyelia group was higher than that in the SCs group, and the number of the control group was the least. There was significant difference in the number of positive nerve fibers between the two groups (P 0.05). The number of HRP-positive nerve fibers in the SCs group was 10% bcl-2-SCs, and there was a significant difference between the two groups (P 0.05).Four weeks after modeling, the latent period of SCs and MEP in the SCs group was significantly higher than that in the control group (P 0.05), and the amplitude in the SCs group was significantly higher than that in the control group (P 0.05).Conclusion Schwann cell transplantation with the BCL 2 gene can promote the regeneration of nerve synapses in spinal cord injury rats, increase the expression of GFAPN NF-200 gene and protein in spinal cord injury area, and reduce neuronal apoptosis in spinal cord injury area.Enhance the limb motor function and electrophysiological function of rats.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R651.2

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