以Matrigel作為神經(jīng)干細胞移植支架治療急性脊髓損傷的實驗研究
發(fā)布時間：2018-07-26 09:03
【摘要】：目的本研究旨在探討Matrigel作為急性脊髓損傷后細胞移植支架材料的可行性。主要方法1.取新生24 h的SD大鼠大腦海馬組織,在體外用無血清的神經(jīng)干細胞培養(yǎng)液[成分為DMEM/F12(1∶1)、20 ng/ml EGF、20 ng/ml b FGF、1%PS]進行擴增培養(yǎng)。加入胎牛血清使其自然分化,應用細胞免疫熒光技術(shù)對細胞進行鑒定。體外分別采用Matrigel、可吸收明膠海綿(absorbale sponge)、TCP/POC作為支架材料培養(yǎng)感染Ad-G FP腺病毒的NSCs,于培養(yǎng)后2 h、1 d、3 d、7 d、14 d在熒光顯微鏡下觀察細胞在不同支架材料中的存活情況。將Matrigel/NSCs混合物接種至裸鼠皮下成瘤,對瘤體進行組織學分析觀察移植的NSCs在M atrigel中的生長和分化情況。2.制備SD大鼠脊髓的Allen’s打擊損傷模型。將動物分為三組:PBS對照組(A組,于損傷位點注射PBS),單純Matrigel組(B組,于損傷位點單純注射Matrigel),Matrigel/NSCs組(C組,于損傷位點植入Matrigel與NSCs的復合物)。暴露T10節(jié)段脊髓后予10×2.5g·cm的能量打擊。各組在SCI后7 d進行移植處理,C組將Matrigel與用PKH67綠色熒光染料標記的第三代NSCs(細胞密度為5×107/ml)在體外混勻后植入大鼠SCI后7 d的脊髓損傷位點,分別于移植后1d,7d,14d,28d在激光共聚焦掃描顯微鏡下觀察脊髓損傷部位移植NSCs的存活情況。術(shù)后通過BBB評分評估大鼠后肢運動功能,觀察8周;移植處理后4,8周取損傷段脊髓行HE染色和免疫組織學檢測,觀察和評估Matrigel對損傷脊髓以及移植細胞的作用。結(jié)果1.新生鼠海馬組織中分離出的NSCs具有較強的克隆能力,可形成細胞團,呈不規(guī)則球形,免疫熒光結(jié)果提示早期形成的神經(jīng)球Nestin抗原呈陽性;加入血清促使其分化2周,細胞免疫熒光結(jié)果提示分化后的細胞GFAP抗原、MAP2抗原呈陽性。2.與absorbale sponge和TCP/POC兩種支架材料相比較,NSCs在Matrigel中的存活率以及存活時間優(yōu)于另外兩組,NSCs可在Matrigel中存活較長時間;免疫熒光結(jié)果提示NSCs可在Matrigel中分化為β-tubulin 3陽性的神經(jīng)元;裸鼠皮下成瘤瘤體病理學檢測結(jié)果提示Matrigel中神經(jīng)細胞生長良好,并可見少許新生血管;組織免疫學檢測結(jié)果提示移植的細胞可分化為β-tubulin 3和MAP2陽性的神經(jīng)元。3.PKH67示蹤NSCs結(jié)果示NSCs在脊髓損傷部位可存活較長時間。4.C組各時相點BBB評分最高,與其他兩組比較差異有統(tǒng)計學意義(p0.05);A組與B組各時相點無統(tǒng)計學差異(p0.05)。植入處理4周時,脊髓組織HE染色結(jié)果顯示A組損傷部位神經(jīng)組織液化壞死形成體積不等的空洞;B組損傷部位空洞被植入的Matrigel填補,Matrigel中可見細胞生長;C組損傷部位空洞被植入的Matrigel填補,可見大量神經(jīng)細胞在植入的Matrigel中生長,且有少許血管生成。免疫組織學結(jié)果顯示:A組損傷部位無neun、NF-200、MAP2陽性的神經(jīng)元,損傷部位邊緣可見較多Nestin陽性的神經(jīng)干細胞和GFAP陽性的星形膠質(zhì)細胞,損傷中心則無Nestin表達;在B、C組損傷部位可見Nestin、neun、NF-200、MAP2、GFAP陽性的神經(jīng)細胞,C組表達多于B組。植入處理8周時,支架材料有一定程度降解,上述神經(jīng)細胞標記物表達降低。結(jié)論神經(jīng)干細胞在Matrigel中可以存活和分化;以Matrigel作為急性脊髓損傷后神經(jīng)干細胞移植支架材料,具有使用方便,不易造成二次損傷的特點。
[Abstract]:Objective the purpose of this study was to explore the feasibility of Matrigel as a scaffold for transplantation of cells after acute spinal cord injury. 1. the main method was to take the brain hippocampus of SD rats with new 24 h, and to expand the culture in vitro using serum free neural stem cell culture medium (DMEM/F12 (1: 1), 20 ng/ml EGF, 20 ng/ml B FGF, 1%PS], and to add fetal bovine blood. The cells were identified by cell immunofluorescence technology. Matrigel was used in vitro to absorb gelatin sponge (absorbale sponge) and TCP/POC as scaffold material to cultivate NSCs of Ad-G FP adenovirus. After culture, 2 h, 1 D, 3 D, 7 d, and 14 d in different scaffold materials under fluorescence microscope. The Matrigel/NSCs mixture was inoculated into the subcutaneous tumor of nude mice, and the tumor body was histologically analyzed to observe the growth and differentiation of the transplanted NSCs in M atrigel.2. to prepare the Allen 's damage model of the spinal cord of SD rats. The animals were divided into three groups: the PBS control group (A group, PBS injecting site injection), and the simple Matrigel group. The injury site was simply injected with Matrigel), group Matrigel/NSCs (group C, the complex of Matrigel and NSCs at the site of damage). After exposure to the T10 segment spinal cord, the energy shock was given to 10 * 2.5G. Cm. Each group was transplanted after SCI 7 d. The C group was in vitro (the cell density of 5 x) marked with the green fluorescent dye. The spinal cord injury site of 7 d after SCI was implanted, and the survival of NSCs was observed in 1D, 7d, 14d and 28d after the laser confocal scanning microscope after the transplantation. The hind limb movement function was evaluated by BBB score for 8 weeks, and the spinal cord of the injured segment of the transplanted spinal cord was stained with HE and immuno tissue after the transplantation. The effects of Matrigel on the injured spinal cord and the transplanted cells were observed and evaluated. Results the isolated NSCs in the hippocampus of 1. neonatal rats had a strong clone ability, which could form a cell group, which showed irregular sphere. The immunofluorescence results suggested that the early formation of the neural ball Nestin was positive, and the serum was added to promote its differentiation for 2 weeks. The immunofluorescence results suggest that the differentiated cell GFAP antigen, MAP2 antigen positive.2. and absorbale sponge and TCP/POC two scaffolds are compared, the survival rate and survival time of NSCs in Matrigel are better than the other two groups, NSCs can survive for a long time in Matrigel, and the immunofluorescence results suggest that NSCs can be divided into beta in Matrigel. Ulin 3 positive neurons; the pathological examination results of subcutaneous tumor tumor in nude mice suggest that the nerve cells in Matrigel grow well, and a few neovascularization is visible; the tissue immunological detection results suggest that the transplanted cells can differentiate into the.3.PKH67 NSCs of the neurons of beta -tubulin 3 and MAP2 positive neurons indicating that NSCs can survive in the injured part of the spinal cord. The BBB score of each time point in the.4.C group was the highest for a long time, and there was significant difference with the other two groups (P0.05). There was no statistical difference between the A group and the B group (P0.05). At the time of implantation, the spinal tissue HE staining showed that the nerve tissue of the injured part of the group A was liquefied and necrotic to form a void in the group A, and the cavity in the lesion site was planted in the group B. In the Matrigel filling, the cell growth was seen in Matrigel, and the injured site in group C was filled with the implanted Matrigel, and a large number of nerve cells were grown in the implanted Matrigel, and a little angiogenesis was found. The immunohistochemical results showed that there were no NeuN, NF-200, MAP2 positive neurons in the A group and more Nestin in the edge of the injury site. Positive neural stem cells and GFAP positive astrocytes had no Nestin expression in the damage center; in B, Nestin, NeuN, NF-200, MAP2, GFAP positive neurons were seen in group C, and the expression of C group was more than that in B group. The scaffold material was degraded to a certain extent and the expression of nerve cell markers decreased at 8 weeks. Conclusion nerve stem was found. Conclusion nerve stem Cells can survive and differentiate in Matrigel, and Matrigel is used as a scaffold for neural stem cell transplantation after acute spinal cord injury. It is easy to use and is not easy to cause two damage.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R651.2
本文編號：2145519
[Abstract]:Objective the purpose of this study was to explore the feasibility of Matrigel as a scaffold for transplantation of cells after acute spinal cord injury. 1. the main method was to take the brain hippocampus of SD rats with new 24 h, and to expand the culture in vitro using serum free neural stem cell culture medium (DMEM/F12 (1: 1), 20 ng/ml EGF, 20 ng/ml B FGF, 1%PS], and to add fetal bovine blood. The cells were identified by cell immunofluorescence technology. Matrigel was used in vitro to absorb gelatin sponge (absorbale sponge) and TCP/POC as scaffold material to cultivate NSCs of Ad-G FP adenovirus. After culture, 2 h, 1 D, 3 D, 7 d, and 14 d in different scaffold materials under fluorescence microscope. The Matrigel/NSCs mixture was inoculated into the subcutaneous tumor of nude mice, and the tumor body was histologically analyzed to observe the growth and differentiation of the transplanted NSCs in M atrigel.2. to prepare the Allen 's damage model of the spinal cord of SD rats. The animals were divided into three groups: the PBS control group (A group, PBS injecting site injection), and the simple Matrigel group. The injury site was simply injected with Matrigel), group Matrigel/NSCs (group C, the complex of Matrigel and NSCs at the site of damage). After exposure to the T10 segment spinal cord, the energy shock was given to 10 * 2.5G. Cm. Each group was transplanted after SCI 7 d. The C group was in vitro (the cell density of 5 x) marked with the green fluorescent dye. The spinal cord injury site of 7 d after SCI was implanted, and the survival of NSCs was observed in 1D, 7d, 14d and 28d after the laser confocal scanning microscope after the transplantation. The hind limb movement function was evaluated by BBB score for 8 weeks, and the spinal cord of the injured segment of the transplanted spinal cord was stained with HE and immuno tissue after the transplantation. The effects of Matrigel on the injured spinal cord and the transplanted cells were observed and evaluated. Results the isolated NSCs in the hippocampus of 1. neonatal rats had a strong clone ability, which could form a cell group, which showed irregular sphere. The immunofluorescence results suggested that the early formation of the neural ball Nestin was positive, and the serum was added to promote its differentiation for 2 weeks. The immunofluorescence results suggest that the differentiated cell GFAP antigen, MAP2 antigen positive.2. and absorbale sponge and TCP/POC two scaffolds are compared, the survival rate and survival time of NSCs in Matrigel are better than the other two groups, NSCs can survive for a long time in Matrigel, and the immunofluorescence results suggest that NSCs can be divided into beta in Matrigel. Ulin 3 positive neurons; the pathological examination results of subcutaneous tumor tumor in nude mice suggest that the nerve cells in Matrigel grow well, and a few neovascularization is visible; the tissue immunological detection results suggest that the transplanted cells can differentiate into the.3.PKH67 NSCs of the neurons of beta -tubulin 3 and MAP2 positive neurons indicating that NSCs can survive in the injured part of the spinal cord. The BBB score of each time point in the.4.C group was the highest for a long time, and there was significant difference with the other two groups (P0.05). There was no statistical difference between the A group and the B group (P0.05). At the time of implantation, the spinal tissue HE staining showed that the nerve tissue of the injured part of the group A was liquefied and necrotic to form a void in the group A, and the cavity in the lesion site was planted in the group B. In the Matrigel filling, the cell growth was seen in Matrigel, and the injured site in group C was filled with the implanted Matrigel, and a large number of nerve cells were grown in the implanted Matrigel, and a little angiogenesis was found. The immunohistochemical results showed that there were no NeuN, NF-200, MAP2 positive neurons in the A group and more Nestin in the edge of the injury site. Positive neural stem cells and GFAP positive astrocytes had no Nestin expression in the damage center; in B, Nestin, NeuN, NF-200, MAP2, GFAP positive neurons were seen in group C, and the expression of C group was more than that in B group. The scaffold material was degraded to a certain extent and the expression of nerve cell markers decreased at 8 weeks. Conclusion nerve stem was found. Conclusion nerve stem Cells can survive and differentiate in Matrigel, and Matrigel is used as a scaffold for neural stem cell transplantation after acute spinal cord injury. It is easy to use and is not easy to cause two damage.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R651.2
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