面神经损伤大鼠脑内移植SPIO标记的GFP转基因胎鼠神经干细胞后的MRI示踪研究

【作者】 李宗芳；

【导师】 刘流； 田伟； 赵德萍； 李逸松； 代晓明；

【作者基本信息】 昆明医学院 ， 外科学， 2009， 博士

【摘要】 通过将SPIO标记的GFP转基因胎鼠NSCs移植入面神经损伤大鼠脑内,并利用MRI对移植的NSCs进行活体示踪,探讨了移植的NSCs的在脑内的迁移情况及用MRI对移植NSCs进行活体示踪的可行性。结果显示移植的NSCs可在脑内存活并向损伤侧面神经核区域迁移,MRI可以显示移植细胞的迁移。因此,本课题为面神经损伤后的修复提供了一种新的思路（中枢神经元的修复）和建立了一种活体检测方法（MRI活体示踪移植细胞）,为进一步深入研究奠定了良好的实验基础。详细摘要如下:实验一面神经损伤大鼠模型的建立目的动物实验是研究面神经疾病的基础,无论是研究面神经损伤后的病理生理变化、神经再生过程,还是研究面神经疾病的病因和治疗,都需要建立理想的动物模型。本实验旨在建立面神经高位损伤模型,为面神经损伤后脑内移植NSCs研究奠定模型基础。方法SD大鼠12只,随机分为术后1周和3周组,然后将右侧面神经主干在茎乳孔根部牵拉出约3 mm切断。按规定时间处死大鼠并取材、制备石蜡切片,行HE、TB和TUNEL染色,观察双侧面神经元的凋亡情况。结果健侧和术后1周组损伤侧面神经元均未见明显凋亡,术后3周组损伤侧见大量凋亡面神经元。结论采用将面神经主干在茎乳孔根部牵拉出约3 mm切断的方法建立的面神经高位损伤模型成功诱导了面神经元的凋亡,且重复性好,为进一步研究奠定了良好的模型基础。实验二GFP转基因胎鼠NSCs的分离、培养和鉴定目的建立GFP转基因胎鼠NSCs的分离、培养和鉴定的方法,为面神经损伤后脑内移植NSCs研究准备供体细胞。方法①分离GFP转基因胎鼠的海马组织,用吸管吹打机械方法制成单细胞悬液。采用含B27、碱性成纤维细胞生长因子（bFGF）和表皮生长因子（EGF）的无血清培养基培养。②利用Nestin抗体对原代和传代细胞进行特异性鉴定。③用胎牛血清诱导分化后,荧光显微镜下观察分化细胞的形态和GFP表达情况。利用NeuN、GFAP和O₄抗体分别对分化后的神经元、星形胶质细胞和少枝胶质细胞进行特异性鉴定。结果①从GFP转基因胎鼠海马组织分离的细胞以悬浮方式生长,可形成典型的神经球,能在体外传代培养和连续形成克隆,且原代和传代细胞GFP表达均呈阳性。②免疫细胞化学染色示原代和传代细胞均为Nestin阳性。③血清诱导分化后的细胞可分别表达神经元、星形胶质细胞和少枝胶质细胞特异性抗原NeuN、GFAP和O₄,且分化后的细胞GFP表达呈阳性。结论从GFP转基因胎鼠海马组织成功分离培养出了NSCs。培养出的细胞Nestin阳性且具有增殖、自我更新能力及向神经元及神经胶质细胞分化的潜力,且均可稳定表达GFP,因此可作为NSCs脑内移植实验研究的供体细胞。实验三GFP转基因胎鼠NSCs的SPIo标记及体外磁共振成像目的探索用SPIO体外标记NSCs的可行性及1.5 T MR仪对磁标记干细胞的显示能力,为进一步利用MRI活体示踪移植于面神经损伤脑内的NSCs奠定基础。方法①将SPIO和PLL混悬制备SPIO-PLL复合物,加入无血清NSCs条件培养液,使其中铁的终浓度为25μg/ml,PLL的终浓度为0.75μg/ml,然后与NSCs在37℃、5%CO₂培养箱中共同孵育48小时,以便对NSCs进行SPIO标记。②用倒置相差显微镜和荧光显微镜观察标记细胞的形态和GFP表达。③于标记后24小时和诱导分化后5天行普鲁士蓝染色观察细胞内铁颗粒。④于标记后2、4、6天行台盼蓝拒染试验观察细胞活性。⑤于标记后2、4、6、8天用MTT法检测标记细胞的增殖。⑥用流式细胞仪检测标记后2天细胞的凋亡和坏死及细胞周期并用TUNEL方法检测细胞凋亡。⑦用NeuN、GFAP和O₄抗体分别对标记细胞分化后的神经元、星形胶质细胞和少枝胶质细胞进行特异性鉴定。⑧用1.5 T MR仪对不同数量级的标记细胞行体外MRI。结果①SPIO标记和未标记的NSCs在光镜下形态无明显差别,但标记细胞颜色呈棕黄色;荧光显微镜下两组细胞绿色荧光强弱无明显差别。②普鲁士蓝染色显示SPIO标记NSCs和分化后的细胞胞质内均见大量蓝色颗粒,而未标记NSCs和分化后的细胞胞质内未见蓝色颗粒。③SPIO标记NSCs 2、4、6天组和未标记NSCs的台盼蓝拒染率比较均无统计学差异。④SPIO标记NSCs 2、4、6、8天组利未标记NSCs的OD值比较均无统计学差异。⑤SPIO标记NSCs 2天和未标记NSCs的凋亡和坏死及细胞周期比较无统计学差异,TUNEL法亦未检测到细胞凋亡。⑥SPIO标记NSCs诱导分化后的细胞可分别表达神经元、星形胶质细胞和少枝胶质细胞特异性抗原NeuN、GFAP和O₄。⑦GRE T₂^*WI中,1×10⁶管和1×10⁵管均可见明显低信号改变,1×10⁴管、1×10³管和琼脂糖管未见明显低信号改变;FSE T₂WI中,1×10⁶管可见明显低信号改变,余4管未见明显低信号改变;T₁WI中,5管的信号未见有明显差别。结论①SPIO-PLL复合物可用于体外标记NSCs,应用SPIO-PLL复合物标记NSCs安全、有效。②在1.5 TMR仪上,SPIO标记NSCs可引起明显信号降低,且信号降低程度与标记细胞的数量和扫描序列有关,其中GRE T₂^*WI对信号改变最敏感。实验四面神经损伤大鼠脑内移植SPIO标记的GFP转基因胎鼠NSCs后的MRI示踪研究目的探讨大鼠面神经损伤后脑内移植SPIO标记的GFP转基因胎鼠的NSCs的迁移情况及用MRI对移植NSCs进行活体示踪的可行性。方法24只SD大鼠随机分为4组:A组为面神经切断后移植SPIO标记的GFP转基因胎鼠NSCs悬液;B组为面神经切断后移植未用SPIO标记的GFP转基因胎鼠NSCs悬液;C组为面神经切断后移植含SPIO的NSCs培养液;D组为正常大鼠移植SPIO标记的GFP转基因胎鼠NSCs悬液。造模后1周,行立体定向移植,注射点为前囟后方11.30 mm、背侧9.00 mm、正中线位置。分别于移植后第3天及第2、3、4周行MRI,并于第4周处死大鼠,取脑组织冰冻切片后用荧光显微镜和普鲁士蓝染色观察GFP和SPIO双标的NSCs的迁移,并将该结果与MRI相比较。结果①移植部位:MRI:A、C、D各组动物移植后第3天至第4周均呈类圆形低信号,且无明显改变,B组动物移植后第3天至第4周均无明显低信号;普鲁士蓝染色:A、C、D组对应于MRI低信号区呈蓝染,B组未见蓝染区域;GFP荧光检测:A、B、D组见多少不等的GFP阳性细胞,其中A、D组与普鲁士蓝蓝染区对应,C组未见GFP阳性细胞;②损伤侧面神经核区域（桥脑右份腹外侧区）:MRI:移植后第3天及第2周所有大鼠均未见低信号改变,移植后第3周及第4周A组动物中有3只（3/6）可见条形或点状低信号,A组动物中另外3只（3/6）及B、C、D组均未见低信号;普鲁士蓝染色:只有A组见多少不等的蓝染,与MRI低信号区相符,B、C、D组均未见蓝染;GFP荧光检测示:A、B组均见多少不等的GFP阳性细胞,其中A组与普鲁士蓝蓝染区对应,C、D组未见GFP阳性细胞。③健侧面神经核区域（桥脑左份腹外侧区）:MRI:4组均未见低信号改变;普鲁士蓝染色示:4组均未见蓝染;GFP荧光检测示:4组均未见GFP阳性细胞。④血管内:在移植部位附近和远离移植部位均见GFP阳性细胞位于血管内;⑤移植处与面神经核团区域之间:未见GFP阳性细胞或蓝色相连。结论SPIO和GFP双标记的NSCs移植入面神经损伤大鼠脑内后,可在脑内存活并向损伤侧面神经核区域迁移,利用MRI技术可以对移植后脑内的标记细胞进行初步活体示踪。更多还原

【Abstract】 Experiment one:Establishment of the model of facial nerve injury on ratObjective Animal experiment is the basis of the study on facial nerve diseases.It all needs to construct an ideal model for the research on changes of pathology and physiology after facial nerve injury,on process of nerve regeneration and on etiological factor and treatment of facial nerve diseases.This experiment aims to establish a model of facial nerve injury on rat in order to set up the foundation for the study of transplanting NSCs into rat brain after facial nerve injury.Methods 12 SD rats were randomly divided into 2 groups:1 week and 3 week after model induction.Then the right facial nerve was softly drug out about 3 mm from stylomastoid foramen before cut off at the root of stylomastoid foramen.1 week and 3 week following model induction,rats were killed respectively.Then paraffin sections of brain tissues were prepared.HE,TB and TUNEL staining were made to observe the neuronal apoptosis of facial nucleus on both sides.Results There were no obvious neuronal apoptosis of facial nucleus on the healthy side and injured side of 1 week group.But on the injured side of 3 week group there was remarkable neuronal apoptosis of facial nucleus.Conclusion The model made in this experiment induces remarkable neuronal apoptosis of facial nucleus successfully and have good reproducibility.So it settles good foundation for the study of transplanting NSCs into rat brain after facial nerve injury.Experiment two:Isolation,culture and identification of NSCs derived from GFP transgenic embryonic miceObjective To prepare donor cells for the study of transplanting NSCs into rat brain after facial nerve injury by isolating,culturing and identifying NSCs derived from GFP transgenic embryonic mice.Methods①Hippocampus isolated from GFP transgenic embryonic mice was blown mechanically and collected in centrifuge tube.Serum-free medium containing 1% B27,20 ng/ ml bFGF and 20 ng/ ml EGF was added into the tube after centrifugalization to suspend the cells.After the cells were dissociated into single cell, the density was adjusted to 2×10⁵ cells/ml.Then cells were inoculated into culture plate and cultured in 37℃,5%CO₂ incubator.About 5 to 7 days the cells proliferated and formed neurospheres.Then cells were passage by isolating mechanically.After that,cells were passage every 5 to 7 days.Cells growth and GFP expression were observed with invert phase-contrast light microscope and fluorescence microscope respectively.②Primary and passage cells were identified by anti-nestin immunocytochemical staining.③Cells morph and GFP expression were observed with fluorescence microscope after differentiation induced by fetal bovine serum. Identification of neuron,astrocyte and oligodendrocytes were performed by NeuN, GFAP and O₄ immunocytochemical staining respectively.Results①Cells isolated from GFP transgenic embryonic mice were grown in the matter of suspensions and formed typical neurospheres after culturing 48 hours.They were able to serial passage and form clones in vitro.Meanwhile the primary and passage cells both showed GFP positive.②Immunocytochemical method showed the primary and passage cells were nestin positive.③Cells induced by FBS differentiated and immunocytochemical method showed they were NeuN,GFAP and O₄ positive respectively.The differentiated cells also showed GFP positive.Conclusion The cells isolated from GFP transgenic embryonic mice are nestin positive and possess the abilities of proliferation,self-renew and multipotency of differentiating into neuron,astrocyte and oligodendrocyte which are believed to be the main characteristics of NSCs.They also stably express GFP.So the cells isolated from GFP transgenic embryonic mice could be considered as one of the ideal donor cells in experimental research on NSCs transplantation therapy in future. Experiment three:SPIO labeling and in vitro MRI of NSCs derived from GFP transgenic embryonic miceObjective To explore the feasibility of magnetic labeling of NSCs with SPIO and to investigate the possibility of in vitro detection of magnetically labeled NSCs with a clinical 1.5 T MR.Methods①SPIO was mixed with PLL to obtain a complex of SPIO-PLL,in which the ultimate concentration of Fe and PLL was 25ug/ml and 0.75ug/ml respectively. Then NSCs were cultured in the culture media containing SPIO-PLL in 37℃,5%CO₂ incubator for about 48 hours to label NSCs with SPIO.②Morph and expression of GFP of labeled NSCs were observed with invert phase-contrast light microscope and fluorescence microscope respectively.③Prussian blue staining was performed for demonstrating intracytoplastic nanoparticles at 24 hours and 5 days after cells induced differentiation respectively.④Trypan blue exclusion test for cell viability were performed at 2,4,6 days after labeling respectively.⑤MTT test for cell proliferation were performed at 2,4,6,8 days after labeling respectively.⑥Cell cycle,cellular apoptosis and necrosis of labeled NSCs were studied by flow cytometry.Meanwhile cellular apoptosis were also detected by TUNEL staining.⑦After differentiation induced by FBS,identification of neuron,astrocyte and oligodendrocytes were performed by NeuN,GFAP and O₄ immunocytochemical staining respectively.⑧1×10⁶,1×10⁵,1×10⁴ and 1×10³ cells were respectively suspended in 0.5 ml gelatin （1%） for in vitro MRI at 1.5 T MR imaging system.A tube contain gelatin used as negative control.Results①There were no significantly difference in cell morph and GFP expression between labeled and unlabeled cells.But the colour of labeled cells was buffy under light microscope.②Numerous intracytoplastic iron particles were stained with Prussian blue staining in the SPIO-PLL labeled NSCs and differentiated cells.But there were no iron particles in unlabeled cells.③There were no significantly difference in Trypan blue exclusion test for cell viability between labeled and unlabeled cells at 2,4,6 days after labeling.④There were no significantly difference in MTT test for cell proliferation between labeled and unlabeled cells at 2,4,6,8 days after labeling.⑤There were no significantly difference in cell cycle,cellular apoptosis and necrosis between labeled and unlabeled cells at 2 days after labeling. Cellular apoptosis were not detected by TUNEL staining.⑥Immunocytochemical method showed differentiated NSCs induced by FBS were NeuN,GFAP and O₄ positive respectively.⑦On GRE T₂^*WI only tubes of 1×10⁶ and 1×10⁵ cells presented obvious low signal intensity.On FSE T₂WI only tubes of 1×10⁶ cells presented obvious low signal intensity.On T₁WI there was no obvious difference among all five tubes.Conclusion①It is feasible,efficient and safe to label NSCs with SPIO-PLL complex.②NSCs labeled with SPIO-PLL complex can result in a negative enhancement on GRE T₂^*WI and FSE T₂WI,which can be detected by a clinical 1.5 T MR imaging system.The degree of negative enhancement is correlated with the cell number.GRE T₂^*WI is the most sensitive sequence to detect negative enhancement of labeled cells.Experiment four:Magnetic resonance tracking of transplanted NSCs derived from GFP transgenic embryonic mice labeled with SPIO in rat brain after facial nerve injuryObjective To explore the migration of transplanted NSCs derived from GFP transgenic embryonic mice labeled with SPIO in rat brain after facial nerve injury and the feasibility of magnetic resonance tracking of this migration.Methods NSCs were derived from the brain of GFP transgenic embryonic mice and labeled with SPIO.24 adult SD rats were randomly divided into 4 groups:A group, transplanted SPIO labeled NSCs in rat brain after facial nerve injury;B group, transplanted unlabeled NSCs in rat brain after facial nerve injury;C group, transplanted nutrient solution contained SPIO in rat brain after facial nerve injury;D transplanted SPIO labeled NSCs in normal rat brain.1 week following model induction,NSCs stereotaxis transplantation was performed,and the injection point was 11.30 mm to pone of the anterior fontanelle,9.00 mm to dorsal side,at the median line.MR scanning was performed to monitor the transplanted cells after implantation in 3 day and 2,3,4 weeks respectively.After MRI on the fourth week all rats were killed and frozen sections of brain tissues were prepared.Fluorescence microscope and Prussian blue staining of the histological sections were employed to observe the migration of GFP and SPIO co-labeled NSCs and the results were compared with MRI.Results①At grafting region:MRI:there was a round area of low signal intensity in groups A,C and D except B from the third day to the fouth week after transplantation. Prussian blue staining:there were blue staining area in groups A,C and D except B. And this area was matched with the low signal intensity area on MRI;Fluorescence microscope:GFP-positive cells were observed in groups A,B and D except C.In groups A and D GFP-positive cells were matched with blue staining area on Prussian blue staining.②In the area around facial nucleus of injured side facial nerve（ventral side of right part of pons）:MRI:From the third day to the second week after transplantation there was no area of low signal intensity in all groups.From the third week to the fourth week after transplantation there was low signal intensity presented on 3 animals（3/6） of group A.No low signal intensity was observed on the other animals.Prussian blue staining:Only group A had blue staining area which was matched with the low signal intensity area on MRI.Fluorescence microscope:In groups A and B GFP-positive cells were observed migration to this area.And in groups A GFP-positive cells were matched with blue staining area on Prussian blue staining.③In the area around facial nucleus of healthy side facial nerve（ventral side of left part of pons）:MRI:There was no area of low signal intensity in all groups;Prussian blue staining:There was no area of blue staining in all groups;Fluorescence microscope:No GFP-positive cell was observed migration to this area in all groups.④In blood vessel:Some GFP-positive cells were observed in blood vessel near or away from the injection point.⑤The area between grafting region and facial nucleus: No GFP-positive cell or blue staining was observed connecting the two points.Conclusion NSCs co-labeled with SPIO and GFP could migrate into area around nucleus of injured side facial nerve after transplanted into rat brain and 1.5T MRI is feasible to tracking the migration of the labeled neural stem cells.更多还原