【作者】 李浩明；

【导师】 金国华；

【作者基本信息】 苏州大学 ， 人体解剖与组织胚胎学， 2012， 博士

【摘要】 LIM同源盒基因8（LIM homeobox8, Lhx8）与胚胎基底前脑胆碱能神经元的发育有关，但Lhx8在中枢胆碱能神经再生及海马齿状回胆碱能神经再生过程中所起的作用尚未见到报道，本研究主要探讨Lhx8在海马胆碱能神经再生中所起的作用。一．目的：体外培养大鼠海马源性放射状胶质细胞（Radial glia cells, RGCs），观察并检测海马提取液在RGCs增殖和向胆碱能神经元分化过程中所发挥的效用；构建Lhx8过表达载体并经慢病毒包装，转染至体外培养的RGCs，同时注入大鼠海马齿状回中，对Lhx8在海马胆碱能神经再生过程中所起的作用进行初步的探讨。二．方法：（1）切割大鼠右侧穹窿海马伞并且制备正常侧及切割侧海马提取液；（2）体外培养海马源性RGCs，在培养液中加入正常侧及切割侧海马提取液，观察海马提取液对RGCs增殖和向神经元分化方面的作用；（3）联合应用海马提取液及NGF共同促进海马RGCs向胆碱能神经元分化，观察Lhx8在ChAT阳性的神经元中的表达和定位；（4）克隆Lhx8cDNA序列，构建pLenti6.3-Lhx8-IRES2-EGFP表达载体并经慢病毒包装，将收集的慢病毒Lenti6.3-Lhx8转染RGCs，观察RGCs向胆碱能神经元分化的情况，检测Lhx8和ChAT的基因及蛋白的表达变化；（5）将慢病毒Lenti6.3-Lhx8注入正常侧及切割穹窿海马伞侧大鼠海马齿状回中，观察正常侧及切割侧海马齿状回中ChAT阳性的胆碱能神经元的表达和定位，检测Lhx8和ChAT的基因及蛋白的表达变化。三．结果：（1） RGCs具有典型的双突起形态学特征，同时可表达胶质相关标记物BLBP、GFAP、Vimentin，并且这些胶质相关标记物能与干细胞相关标记物Nestin进行双标，此外RGCs还能够分化为神经元、星形胶质细胞以及少突胶质细胞；（2）切割穹窿海马伞侧的海马提取液能够明显促进RGCs的增殖和向神经元分化；切割侧海马提取液联合NGF能够促进RGCs向ChAT阳性的胆碱能神经元分化，这些ChAT阳性的胆碱能神经元能够表达Lhx8；（3） RGCs转染慢病毒Lenti6.3-Lhx8后，分化为MAP2阳性神经元的总体数目没有明显增加，但是能够提高ChAT阳性细胞在MAP2阳性神经元中所占的比例，Lhx8和ChAT的基因及蛋白的表达量也明显增高，说明转染慢病毒Lenti6.3-Lhx8后，能够促进RGCs向胆碱能神经元分化；（4）在正常海马齿状回中注入慢病毒Lenti6.3-Lhx8后，能够明显提高齿状回内ChAT/Lhx8阳性的胆碱能神经元数量；切割穹窿海马伞并联合慢病毒注射后，发现齿状回内ChAT/Lhx8阳性的胆碱能神经元的数量增加更为明显，Lhx8和ChAT的基因及蛋白的表达量也明显增高，说明切割穹窿海马伞并且联合应用Lenti6.3-Lhx8慢病毒可以明显地促进海马内的胆碱能神经再生水平。四．结论：（1）体外培养的海马源性RGCs具有典型的双突起形态学特征，这些细胞能够同时表达胶质细胞相关标记物和干细胞相关标记物。体外应用切割穹窿海马伞侧海马提取液能够较好的模拟切割穹窿海马伞后的海马再生微环境。在RGCs扩增培养阶段切割侧海马提取液能明显地促进RGCs增殖，而在RGCs分化阶段切割侧海马提取液也能够促进RGCs更多地向神经元分化；（2）慢病毒Lenti6.3-Lhx8能够促进体外培养的海马源性RGCs向胆碱能神经元分化，但对RGCs分化产生的神经元总体数量没有影响。Lhx8作为LIM同源盒基因家族成员，仅在神经元胆碱能表型确立方面起到作用，而不能促进体外培养的海马源性RGCs更多地向神经元分化；（3）慢病毒Lenti6.3-Lhx8注入海马齿状回后，齿状回ChAT阳性胆碱能神经元的数量与正常海马比较明显增多。在切割穹窿海马伞侧的海马齿状回中注入慢病毒Lenti6.3-Lhx8后，发现ChAT阳性的神经元数量增加更为明显，说明LIM同源盒基因家族成员Lhx8在切割穹窿海马伞后的海马胆碱能神经再生过程中发挥了重要作用。更多还原

【Abstract】 Lhx8（LIM homeobox8）is associated with the development of embryo basalcholinergic neurons, however the function of Lhx8on cholinergic neural regeneration incentral nervous system and hippocampal dentate gyrus is still unclear, this research willstudy the function of Lhx8on hippocampal cholinergic neural regeneration.Objective:Radial glia cells （RGCs） derived from rat hippocampus was cultured in vitro, weinvestigated and detected the effects of normal and transected hippocampal extracts in theproliferation and differentiation of RGCs, and then we studied the function of Lhx8onhippocampal cholinergic neural regeneration.Methods:（1） The rats’ hippocampal right fimbria-fornix was transected and the normal andtransected hippocampal extracts were prepared successfully;（2） The RGCs was cultured in vitro, normal and transected hippocampal extractswas added into the culture mediums, then investigated the function of hippocampalextracts in the proliferation and differentiation of RGCs;（3） Using hippocapal extracts and NGF to promote the cholinergic differentiationof RGCs, then investigate the expression and location of Lhx8in ChAT positive cells;（4） Cloning of Lhx8cDNA sequence, the expression recombinant plasmidpLenti6.3-Lhx8-IRES2-EGFP was constructed successfully and packaged by lentivirus, thecollected lentivirus Lenti6.3-Lhx8were transfected into RGCs, then investigated thecholinergic differentiation of RGCs and the dynamic gene and protein expression of Lhx8and ChAT;（5） The lentivirus Lenti6.3-Lhx8were injected into the normal and fimbria-fornixtransected hippocampal dentate gyrus, then investigated the expression and location of theChAT positive cholinergic neurons in normal and transected hippocampal dentate gyrusand the dynamic gene and protein expression of Lhx8and ChAT. Results:（1）RGCs presented typical double processes morphological feature, they expressedastroglial markers BLBP, GFAP, Vimentin, and these astroglial markers co-expressed withstem cells marker Nestin, and these RGCs could differentiate into neurons, astrocytes andoligodendrocytes;（2） The transected hippocampal extracts could promote the proliferation andneuronal differentiation of RGCs; the transected hippocampal extracts combined with NGFcould promote the RGCs differentiation into cholinergic neurons, and these cholinergicneurons could co-express with Lhx8;（3） Lenti6.3-Lhx8was transfected into RGCs, the number of differentiated MAP2positive neurons was not increased, but the proportion of ChAT positive cells to MAP2cells was increased, and the gene and protein expression of Lhx8and ChAT were increasedsignificantly, it indicated that transfection of Lenti6.3-Lhx8could promote the cholinergicneurons differentiation;（4） The Lenti6.3-Lhx8were injected into normal hippocampal dentate gyrus, thenumber of ChAT/Lhx8positive cholinergic neurons were increased; the hippocampalfimbria-fornix transection combined with Lenti6.3-Lhx8injection of dentate gyrus, thenumber of ChAT/Lhx8positive cholinergic neurons were increased significantly, the geneand protein expression of Lhx8and ChAT were also increased, it indicated that thehippocampal fimbria-fornix transection combined with Lenti6.3-Lhx8injection couldpromote the level of cholinergic neural regeneration in hippocampus.Conclusions:（1） The RGCs presented typical double processes morphological feature in vitro,these cells co-expressed astroglial markers with stem cells marker. The fimbria-fornixtransected hippocampal extracts could mimic the hippocampal neural regeneration niche invitro. On the proliferation stage the transected hippocampal extracts could promote theproliferation of RGCs, on the differentiation stage the transected hippocampal extracts alsocould promote the RGCs differentiation into neurons;（2） Lentivirus Lenti6.3-Lhx8could promote the hippocampal RGCs differentiationinto cholinergic neurons, but had no effect on the total number of neurons. As a familymember of LIM homeobox genes, Lhx8was only associated with the establishment of cholinergic phenotype, but Lhx8could not promote hippocampal RGCs differentiation intomore neurons in vitro;（3） Lentivirus Lenti6.3-Lhx8were injected into the hippocampal dentate gyrus, theChAT positive cholinergic neurons was increased, Lenti6.3-Lhx8were injected into thefimbria-fornix transected hippocampal dentate gyrus, the number of ChAT positivecholinergic neurons was significantly increased, it indicated that LIM homeobox geneLhx8played an important role in the hippocampal cholinergic neural regeneration afterfimbria-fornix transection.更多还原