發(fā)布時(shí)間：2018-05-25 10:38

  本文選題：脊髓損傷 + VEGF　； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的探索脊髓損傷后內(nèi)皮祖細(xì)胞的脊髓內(nèi)移植是否為脊髓的恢復(fù)提供神經(jīng)保護(hù)作用,為內(nèi)皮祖細(xì)胞移植治療脊髓損傷的研究提供理論基礎(chǔ)。方法從80-100 g SD大鼠雙后肢提取骨髓經(jīng)梯度密度離心后,經(jīng)誘導(dǎo)培養(yǎng)基培養(yǎng)兩周后用細(xì)胞形態(tài)學(xué)和細(xì)胞功能學(xué)鑒定為內(nèi)皮祖細(xì)胞(Endothelial progenitor cells,EPCs),用CM-Dil染料標(biāo)記后制成濃度為1×105個(gè)/ml細(xì)胞懸液備用。用改良Allen法在成年SD大鼠上制造脊髓損傷模型,并立即在損傷部位硬膜下注射EPCs懸液,同時(shí)注射EPCs培養(yǎng)基作為對照。對所有大鼠脊髓損傷模型分別在損傷后1、2、3、4、5、6w評(píng)估大鼠后肢功能運(yùn)動(dòng)評(píng)分(BBB score)。分別在1w和2w末取出脊髓,直接在熒光顯微鏡下觀察CM-Dil標(biāo)記細(xì)胞位置。用WB測量脊髓損傷部位VEGF蛋白表達(dá)量,免疫組織化學(xué)方法檢測局部血管數(shù)量及血管新生程度、凋亡細(xì)胞比例。脊髓損傷6w末用免疫組化方法檢測局部神經(jīng)元和星形膠質(zhì)細(xì)胞數(shù)量、HE染色橫斷面觀察脊髓損傷情況。結(jié)果(1)脊髓損傷后硬膜下移植EPCs后,EPCs在局部損傷部位可以存活,并且可以遷移到脊髓實(shí)質(zhì)。(2)脊髓損傷1w、2w后EPCs移植組VEGF蛋白表達(dá)量明顯高于對照組(P0.05)。在2w末EPCs移植后脊髓局部內(nèi)皮細(xì)胞數(shù)目比例(38.32±1.49%vs19.02±0.47%,P0.05)和新生血管(16.12±0.79%versus7.61±0.63%,P0.05)較對照組較多,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。(3)手術(shù)2w后實(shí)驗(yàn)組大鼠局部脊髓凋亡細(xì)胞數(shù)量比例明顯少于對照組(38.50±5.01%vs 18.67±1.86%,P0.05)。(4)脊髓損傷6w后EPCs移植大鼠損傷部位神經(jīng)元數(shù)量比例(32.04±3.33%vs 19.34±2.70%,P0.05)明顯多于對照組大鼠,HE染色觀察實(shí)驗(yàn)組組織空洞面積小于對照組(27±4.59%vs59±3.27%,P0.05),差異有統(tǒng)計(jì)學(xué)意義。(5)。隨著手術(shù)后時(shí)間的延長,實(shí)驗(yàn)組和對照組大鼠的運(yùn)動(dòng)功能均有改善,BBB評(píng)分逐漸升高,且在每個(gè)時(shí)間點(diǎn)實(shí)驗(yàn)組大鼠BBB評(píng)分稍高于對照組大鼠,在術(shù)后第6w EPCs移植大鼠評(píng)分達(dá)到7.7±1.34,EPCs培養(yǎng)基移植大鼠評(píng)分為6.1±1.10,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論脊髓損傷后移植內(nèi)皮祖細(xì)胞可以為損傷局部細(xì)胞提供神經(jīng)保護(hù)作用,從而促進(jìn)運(yùn)動(dòng)功能恢復(fù)。
[Abstract]:Objective to investigate whether intraspinal transplantation of endothelial progenitor cells after spinal cord injury can provide neuroprotective effect for spinal cord recovery and provide theoretical basis for the treatment of spinal cord injury by transplantation of endothelial progenitor cells. Methods Bone marrow was extracted from 80-100 g SD rats by gradient density centrifugation. Endothelial progenitor cells were identified as Endothelial progenitor cells (EPCs) by cell morphology and cell function after two weeks of culture on the induction medium, and 1 脳 105 / ml cell suspensions were prepared with CM-Dil dye. The spinal cord injury model was established on adult SD rats by modified Allen method. EPCs suspension was injected into the epidural immediately and EPCs medium was injected as control. All the spinal cord injury models in rats were evaluated for functional motor score of hind limbs and BBB scoreboard at 1: 2, 3, 4, 5 and 5 weeks after injury. The spinal cord was removed at the end of 1 and 2 weeks, and the location of CM-Dil labeled cells was observed directly under fluorescence microscope. The expression of VEGF protein in spinal cord injury was measured by WB. The number of local blood vessels, the degree of angiogenesis and the proportion of apoptotic cells were detected by immunohistochemistry. At the end of 6 weeks after spinal cord injury, the number of neurons and astrocytes were detected by immunohistochemical method. Results 1) EPCs could survive and migrate to spinal parenchyma after subdural transplantation of EPCs after spinal cord injury. The expression of VEGF protein in EPCs transplantation group was significantly higher than that in control group (P 0.05) 2 weeks after spinal cord injury. At the end of 2 weeks, the ratio of the number of spinal cord endothelial cells (38.32 鹵1.49%vs19.02 鹵0.47) and neovascularization (16.12 鹵0.79%versus7.61 鹵0.63) was higher than that in the control group. The number of apoptotic cells in local spinal cord in experimental group was significantly lower than that in control group (38.50 鹵5.01%vs 18.67 鹵1.86P 0.05, P 0.05). After 6 weeks of spinal cord injury, the number of neurons in the injured site of EPCs transplanted rats was 32.04 鹵3.33%vs 19.34 鹵2.70P0.05) significantly higher than that in control group. The tissue cavity area of the experimental group was smaller than that of the control group (27 鹵4.59%vs59 鹵3.27) by HE staining. The difference was statistically significant. With the extension of the time after operation, the motor function of the experimental group and the control group improved gradually, and the BBB score of the experimental group was slightly higher than that of the control group at each time point. At the 6th week after operation, the score of EPCs transplantation rats reached 7.7 鹵1.34.The score of rats transplanted with EPCs medium was 6.1 鹵1.10.The difference was statistically significant (P 0.05). Conclusion Endothelial progenitor cells transplanted after spinal cord injury can provide neuroprotective effect for local injured cells and promote the recovery of motor function.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R651.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 Jin-lan Jiang;Xu-dong Guo;Shu-quan Zhang;Xin-gang Wang;Shi-feng Wu;;Repetitive magnetic stimulation affects the microenvironment of nerve regeneration and evoked potentials after spinal cord injury[J];Neural Regeneration Research;2016年05期

2 程素利;徐家淳;王劍歌;焦召華;周震;孫立明;李巖;;急性脊髓損傷大鼠Allen's造模方法的改良及行為學(xué)評(píng)價(jià)[J];四川中醫(yī);2015年10期

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本文編號(hào)：1933129