ECSOD基因转染MSCs移植治疗缺血性脑卒中大鼠的实验研究
发布时间：2018-09-19 20:47
【摘要】：第一部分ECSOD基因表达载体的构建并转染至骨髓间充质干细胞的实验研究 实验目的：构建胞外超氧化物歧化酶(extracellular supero-xide dismutase, ECSOD)基因表达载体,经慢病毒包装后转染至成年Sprague-Dawley(SD)雄性大鼠的骨髓间充质干细胞(bone mar-row mesenchymal stem cell, MSCs),观察和检测ECSOD基因的转染效率及转染后对细胞活力的影响与ECSOD蛋白表达情况。 实验方法：利用基因工程技术从MSCs中提取总的RNA,经PCR扩增、载体双酶切后,获得ECSOD基因片段,将其基因片段连接到GV230-EGFP载体构建GV230-EGFP-ECSOD表达载体,经菌落PCR及测序,鉴定载体构建是否成功,采用慢病毒包装后将GV230-EGFP-ECSOD导入MSCs中,荧光显微镜检测转染效率,实时荧光定量多聚酶链反应(Quantitati-ve Polymerase Chain Reaction, QPCR)对转染后对MSCs中mRNA定量分析,MTT及EDU检测转染后ECSOD基因对细胞活力与增殖影响,Western Blot检测ECSOD基因表达情况。 实验结果：提取RNA产物经PCR扩增、双酶切成功将目的基因连接至真核表达载体GV230-EGFP中,菌落PCR获得目的基因大小和阳性对照一致,菌落PCR阳性转化子经测序分析与目的基因序列完全一致；慢病毒包装的GV230-EGFP-ECSOD成功转染至MSCs,培养48h后在荧光显微镜下观察并计算得MSCs转染率为(75±3.6)%,进一步采用QPCR定量测定转染后mRNA含量明显高于对照组。EDU与MTT检测发现ECSOD基因转染MSCs后对细胞活力无影响。Western Blot检测证实ECSOD基因转染MSCs后,MSCs中ECSOD蛋白呈阳性表达。 实验结论：成功构建GV230-EGFP-ECSOD真核表达载体,通过慢病毒包装转染技术将ECSOD基因转染到MSCs中且表达ECSOD蛋白,进而为下一步研究ECSOD基因转染MSCs治疗脑梗塞动物模型奠定实验基础。 第二部分ECSOD基因转染MSCs移植治疗缺血性脑卒中大鼠神经保护作用的实验研究 实验目的：观察ECSOD基因转染的MSCs移植治疗缺血性脑卒中大鼠的神经保护作用。 实验方法：将80只Sprague DaWley (SD)成年雄性大鼠,随机分为两组6h组(n=40)、12h组(n=40),分别将6h、12h组分为四个亚组：Model组(n=10)、PBS组(n=10)、MSCs组(n=10)、ECSOD-MSCs组(n=10)；根据改良Zea Longa线栓法制作左侧大脑中动脉缺血模型,分别在造模成功后6h、12h脑缺血/再灌注,同时经颅立体定位对后三组脑缺血大鼠分别注射10μLPBS液、MSCs液、ECSOD-MSCs液,Model组不做处理。脑缺血/再灌注后Id、3d、7d、2w、4w进行改良神经功能评分(Modified Neurological Severity Score, mNSS)评估预后,缺血/再灌注后1天、28天分别行MRI检查测定大鼠脑梗塞体积变化情况。4周后取脑组织采用免疫组织化学方法测定脑组织梗塞周围ECSOD、Bax、Bcl-2的表达情况。 实验结果：移植后2w、4w, ECSOD-MSCs组和MSCs组的mNSS评分较PBS组、Model组低(P0.05),其中ECSOD-MSCs组mNSS评分更低。移植后脑梗塞体积变化的相对值比较ECSOD-MSCs组、MSCs组明显大于PBS组、Model组,差异有统计学意义(P0.05),其中ECSOD-MSCs组更大。在脑梗塞移植后28d脑梗塞及周围ECSOD、Bax、Bcl-2的表达情况：ECSOD-MSCs组较MSCs组、PBS组、Model组ECSOD表达高,差异有统计学差异(P0.05); ECSOD-MSCs组、MSCs组较PBS组、Model组Bax阳性细胞数明显少(P0.05), ECSOD组更少；而bcl-2阳性细胞数ECSOD-MSCs组、MSCs组高于Model组、PBS组(P0.05),其中ECSOD组更高。 实验结论：ECSOD基因转染MSCs细胞能预防脑缺血后再灌注损伤,进一步改善大鼠神经功能缺损,挽救梗死边缘的脑组织。
[Abstract]:Part one: Construction and transfection of ECSOD gene expression vector into bone marrow mesenchymal stem cells
OBJECTIVE: To construct an extracellular supero-oxide dismutase (ECSOD) gene expression vector and transfect it into adult Sprague-Dawley (SD) male rat bone Mar row mesenchymal stem cells (MSCs) after packaging with lentivirus. The effect on cell viability and ECSOD protein expression.
METHODS: Total RNA was extracted from MSCs by genetic engineering technology, amplified by PCR, and digested by double enzyme. The ECSOD gene fragment was obtained. The gene fragment was linked to GV230-EGFP vector to construct GV230-EGFP-ECSOD expression vector. The vector was successfully constructed by colony PCR and sequencing. The vector was packaged with lentivirus and then guided by GV230-EGFP-ECSOD. In MSCs, the transfection efficiency was examined by fluorescence microscope, the mRNA in MSCs was quantitatively analyzed by real-time fluorescence quantitative polymerase chain reaction (QPCR), the effect of ECSOD gene on cell viability and proliferation was detected by MTT and EDU, and the expression of ECSOD gene was detected by Western Blot.
The results showed that the RNA product was amplified by PCR, and the target gene was successfully linked to the eukaryotic expression vector GV230-EGFP by double digestion. The size of the target gene obtained by colony PCR was identical with that of the positive control. The positive transformant of colony PCR was completely identical with the target gene sequence by sequencing analysis. The lentivirus-packaged GV230-EGFP-ECSOD was successfully transfected into MSCs. After 48 hours of culture, the transfection rate of MSCs was (75 Da.
Conclusion: Eukaryotic expression vector of GV230-EGFP-ECSOD was successfully constructed, and ECSOD gene was transfected into MSCs by lentiviral packaging transfection technology to express ECSOD protein. This will lay an experimental foundation for further study of ECSOD gene transfected MSCs to treat cerebral infarction animal model.
Part 2 Experimental study on neuroprotective effect of ECSOD gene transfected MSCs transplantation on ischemic stroke rats
Objective: To observe the neuroprotective effect of MSCs transfected with ECSOD gene on ischemic stroke rats.
Methods: 80 Sprague DaWley (SD) adult male rats were randomly divided into two groups: 6 h group (n = 40) and 12 h group (n = 40). The rats were divided into four subgroups: Model group (n = 10), PBS group (n = 10), MSCs group (n = 10), ECSOD-MSCs group (n = 10), and left middle cerebral artery ischemia model was established by modified Zea Longa occlusion method. Six hours and twelve hours after cerebral ischemia/reperfusion, and three groups of rats were injected with 10 mu LPBS, MSCs, ECSOD-MSCs respectively, without treatment in the Model group. Modified Neurological Severity Score (mNSS) was used to evaluate the prognosis of the rats at the 1st and 28th days after cerebral ischemia/reperfusion. The changes of infarct volume were detected by MRI. The expression of ECSOD, Bax and Bcl-2 were detected by immunohistochemistry in the brain tissues around infarction 4 weeks later.
The results showed that the mNSS scores of ECSOD-MSCs and MSCs groups were lower than those of PBS and Model groups (P 0.05), and the mNSS scores of ECSOD-MSCs group were lower than those of PBS group and Model group (P 0.05). The expression of ECSOD, Bax and Bcl-2 was significantly higher in ECSOD-MSCs group than in MSCs group, PBS group and Model group at 28 days after transplantation (P 0.05); the number of Bax-positive cells in ECSOD-MSCs group was significantly lower than that in PBS group, MSCs group, Model group and ECSOD-MSCs group (P 0.05), while the number of bcl-2-positive cells in ECSOD-MSCs group was higher than that in Model group. Group PBS and group P0.05 (ECSOD) were higher.
CONCLUSION: ECSOD gene transfection into MSCs cells can prevent reperfusion injury after cerebral ischemia, further improve neurological deficits in rats, and save the brain tissue at the edge of infarction.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3
[Abstract]:Part one: Construction and transfection of ECSOD gene expression vector into bone marrow mesenchymal stem cells
OBJECTIVE: To construct an extracellular supero-oxide dismutase (ECSOD) gene expression vector and transfect it into adult Sprague-Dawley (SD) male rat bone Mar row mesenchymal stem cells (MSCs) after packaging with lentivirus. The effect on cell viability and ECSOD protein expression.
METHODS: Total RNA was extracted from MSCs by genetic engineering technology, amplified by PCR, and digested by double enzyme. The ECSOD gene fragment was obtained. The gene fragment was linked to GV230-EGFP vector to construct GV230-EGFP-ECSOD expression vector. The vector was successfully constructed by colony PCR and sequencing. The vector was packaged with lentivirus and then guided by GV230-EGFP-ECSOD. In MSCs, the transfection efficiency was examined by fluorescence microscope, the mRNA in MSCs was quantitatively analyzed by real-time fluorescence quantitative polymerase chain reaction (QPCR), the effect of ECSOD gene on cell viability and proliferation was detected by MTT and EDU, and the expression of ECSOD gene was detected by Western Blot.
The results showed that the RNA product was amplified by PCR, and the target gene was successfully linked to the eukaryotic expression vector GV230-EGFP by double digestion. The size of the target gene obtained by colony PCR was identical with that of the positive control. The positive transformant of colony PCR was completely identical with the target gene sequence by sequencing analysis. The lentivirus-packaged GV230-EGFP-ECSOD was successfully transfected into MSCs. After 48 hours of culture, the transfection rate of MSCs was (75 Da.
Conclusion: Eukaryotic expression vector of GV230-EGFP-ECSOD was successfully constructed, and ECSOD gene was transfected into MSCs by lentiviral packaging transfection technology to express ECSOD protein. This will lay an experimental foundation for further study of ECSOD gene transfected MSCs to treat cerebral infarction animal model.
Part 2 Experimental study on neuroprotective effect of ECSOD gene transfected MSCs transplantation on ischemic stroke rats
Objective: To observe the neuroprotective effect of MSCs transfected with ECSOD gene on ischemic stroke rats.
Methods: 80 Sprague DaWley (SD) adult male rats were randomly divided into two groups: 6 h group (n = 40) and 12 h group (n = 40). The rats were divided into four subgroups: Model group (n = 10), PBS group (n = 10), MSCs group (n = 10), ECSOD-MSCs group (n = 10), and left middle cerebral artery ischemia model was established by modified Zea Longa occlusion method. Six hours and twelve hours after cerebral ischemia/reperfusion, and three groups of rats were injected with 10 mu LPBS, MSCs, ECSOD-MSCs respectively, without treatment in the Model group. Modified Neurological Severity Score (mNSS) was used to evaluate the prognosis of the rats at the 1st and 28th days after cerebral ischemia/reperfusion. The changes of infarct volume were detected by MRI. The expression of ECSOD, Bax and Bcl-2 were detected by immunohistochemistry in the brain tissues around infarction 4 weeks later.
The results showed that the mNSS scores of ECSOD-MSCs and MSCs groups were lower than those of PBS and Model groups (P 0.05), and the mNSS scores of ECSOD-MSCs group were lower than those of PBS group and Model group (P 0.05). The expression of ECSOD, Bax and Bcl-2 was significantly higher in ECSOD-MSCs group than in MSCs group, PBS group and Model group at 28 days after transplantation (P 0.05); the number of Bax-positive cells in ECSOD-MSCs group was significantly lower than that in PBS group, MSCs group, Model group and ECSOD-MSCs group (P 0.05), while the number of bcl-2-positive cells in ECSOD-MSCs group was higher than that in Model group. Group PBS and group P0.05 (ECSOD) were higher.
CONCLUSION: ECSOD gene transfection into MSCs cells can prevent reperfusion injury after cerebral ischemia, further improve neurological deficits in rats, and save the brain tissue at the edge of infarction.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3
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