【作者】 吴晓娟；

【导师】 魏明发；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 先天性巨结肠(Hirschsprung’s disease，HD)是小儿消化道常见畸形，发病率为1／2000～1／5000，其疾病的发生目前一致认为是由于多种原因使胚胎期神经嵴细胞在消化道移行受阻，直肠、结肠甚至小肠肠壁神经节细胞缺如，病变肠管痉挛收缩，肠内容物排出受阻，近端肠管继发性扩张形成巨结肠。迄今，手术仍然是最有效的治疗手段，虽然，手术方式的不断改进，微创手术的日益进步，但术后并发症仍困扰着患者和医护工作者。而近年来研究的热点之一细胞移植有望成为治疗HD又一有效并且安全的方法，本课题将研究如何获得具有功能的细胞进行移植。骨髓间充质干细胞(bone mesenchymal stem cells，BMSC)在体外容易分离、培养及纯化，多向分化潜能是其最主要的生物学特征之一，现在关于BMSC向神经方向分化的研究报道较多，许多研究表明BMSC在体外一定条件下可分化为神经细胞，移植于损伤脑或脊髓治疗神经系统疾病，这为BMSC治疗肠神经系统疾病提供了理论依据，并避免了神经干细胞、胚胎干细胞移植来源不足及伦理问题。目前BMSC向神经细胞分化的具体机制尚不清楚，可能与诱导剂的共同作用启动了向神经细胞定向分化有关的基因表达或使其表达增强有关。较常见的是用β-巯基乙醇(β-mercaptoethanol，BME)、二甲亚砜(dimethyl sulfoxide，DMSO)及维甲酸等化学物质进行诱导，也有用某些生长因子进行诱导的。神经营养因子的作用也已受到重视，一方面，神经营养因子可在一定培养条件下诱导BMSC向神经细胞分化；另一方面，神经营养因子具有神经保护和促进神经突起生长的作用，缺乏神经营养因子的支持可能导致神经细胞的凋亡发生。本实验先从探讨BMSC向神经细胞分化的条件，以及有关神经营养因子表达的变化，如胶质细胞源神经营养因子(glial cell line-derived neurotrophic factor，GDNF，RET(rearranged during transfection)等开始，初步探讨了其分化的机制，其结果发现碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)及表皮生长因子(epidermal growth factor，EGF)可促进BMSC向神经细胞分化，在向神经细胞分化的过程中，GDNF表达增强，并且RET基因也有表达，GDNF可能在BMSC向神经细胞分化过程中起到重要作用。在BMSC向肠神经元分化的可行性实验研究中，将GDNF作为诱导因子与胎肠培养基(fetal gut condition medium，FGCM)共同作用，诱导BMSC分化为肠神经元，诱导后的细胞，不仅表达神经元标志物神经特异性烯醇化酶(neural specific enolase，NSE)以及肠神经标志物一氧化氮合酶(nitric oxide synthase，nNOS)，而且还能分泌肠神经递质肠道血管活性肽(vasoactive intestinal peptide，VIP)，说明体外分化的肠神经元不仅具备形态学上的特征，而且具有一定程度的功能。为了提高BMSC向肠神经元的分化率，本实验还进行了GDNF基因转入BMSC的研究，结果显示，体外可成功构建表达GDNF的转基因BMSC，在FGCM的诱导下可分化为具有肠神经元表型的细胞，并且诱导分化率高于外源性GDNF组。但关于诱导后的肠神经元是否具备正常肠神经元的功能，移植于无神经节段后能否改善肠段的功能，还有待下一步的在体实验进一步研究。第一部分大鼠骨髓间充质干细胞的分离、培养、纯化和鉴定目的：体外分离培养大鼠骨髓间充质干细胞(bone mesenchymal stem cells，BMSC)，并进行纯化和鉴定。方法：收集Sprague-Dawley(SD)大鼠的股骨骨髓，用含10％胎牛血清的DMEM培养基直接种植于培养瓶，反复传代贴壁法纯化细胞，流式细胞仪检测CD90及CD45。结果：BMSC能在体外贴壁生长，经过反复贴壁，BMSC可融合生长，高表达BMSC标志物CD90(93.4％)，不表达造血干细胞标志物CD45。结论：体外可成功培养BMSC，经过反复贴壁法可纯化BMSC，可获得高纯度的细胞。第二部分骨髓间充质干细胞体外向神经细胞的诱导分化目的：探讨大鼠骨髓间充质干细胞(bone mesenchymal stem cells，BMSC)向神经分化的条件，了解神经细胞标志物的表达情况及胶质细胞源神经营养因子(glialcell line-derived neurotrophic factor，GDNF)的表达变化。方法：体外培养大鼠BMSC，至少传至第4代，进行诱导分化。以10ng／ml碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)或／和10ng／ml表皮生长因子(epidermal growth factor，EGF)在含10％胎牛血清的DMEM中诱导，7d后观察细胞形态的变化，免疫组化检测胶质纤维酸性蛋白(glial fibrillary acidic protein，GFAP)及神经特异性烯醇化酶(neural specific enolase，NSE)的情况。RT-PCR检测GDNF及其受体RET mRNA的变化。结果：诱导7天后，实验组均可见GFAP及NSE的表达，而对照组为阴性。各实验组比较，bFGF组及bFGF+EGF组表达的NSE高于EGF组，而EGF组表达GFAP更高。RT-PCR检测示，BMSC向神经细胞诱导后，GDNF表达显著增强，RET基因也开始表达，以bFGF+EGF组最明显。结论：bFGF及EGF可促进BMSC向神经细胞分化。在向神经细胞分化的过程中，能促进GDNF表达的增强，以及RET基因的表达，GDNF可能在BMSC向神经细胞分化过程中起到重要作用。第三部分骨髓间充质干细胞体外两种方法向肠神经元诱导分化及比较目的：探讨大鼠骨髓间充质干细胞(bone mesenchymal stem cells，BMSC)向肠神经分化的条件及可行性，并比较两种方法的分化效率。方法：体外培养大鼠BMSC，传至第4代后，进行诱导分化。第一种方法：以10ng／ml胶质细胞源神经营养因子(glial cell line-derived neurotrophic factor,GDNF)及10％胎牛血清(fetal bovine serum，FBS)的稀释胎肠培养基(fetal gutcondition medium，FGCM)诱导7天，免疫组化的方法进行鉴定诱导细胞的神经特异性烯醇化酶(neural specific enolase，NSE)、血管活性肠肽(vasoactive intestinalpeptide，VIP)及神经元一氧化氮合酶(neuronal nitric oxide synthase，nNOS)的表达。第二种方法：提取新生大鼠肠组织mRNA，用逆转录PCR方法扩增GDNF全长cDNA，构建其真核表达载体PIRES2-EGFP-GDNF，酶切及测序鉴定，然后转染原代培养的大鼠BMSC，荧光免疫细胞化学及细胞形态学检测GDNF的表达，转染后的细胞在稀释的FGCM条件下诱导分化。同样的方法检测VIP，nNOS。结果：BMSC诱导7天后，两组实验组均可见NSE、VIP及nNOS的表达，而对照组为阴性。表达GDNF的BMSC被诱导分化的肠神经元NSE阳性，VIP及nNOS阳性率高于外源性GDNF诱导的细胞。结论：GDNF联合FGCM可诱导BMSC分化为肠神经元。表达GDNF的BMSC分化为肠神经元的效率更高。第四部分骨髓间充质干细胞向肠神经元分化后神经营养因子表达的变化及其机制的初步探讨目的：探讨大鼠骨髓间充质干细胞(bone mesenchymal stem cells，BMSC)向肠神经分化过程中胶质细胞源神经营养因子(glial cell line-derived neurotrophicfactor，GDNF)及其受体RET以及c-kit基因表达的变化，并对其机制进行初步探讨。方法：同样的方法诱导BMSC为肠神经元，免疫组化的方法进行鉴定诱导细胞的神经特异性烯醇化酶(neural specific enolase，NSE)、血管活性肠肽(vasoactive intestinal peptide，VIP)及一氧化氮合酶(nitric oxide synthase，nNOS)的表达。RT-PCR检测GDNF、RET及c-kit mRNA的变化，Western Blot方法检测GDNF蛋白的表达情况。结果：BMSC向肠神经元诱导后，免疫组化染色NSE，VIP及nNOS阳性，RT-PCR结果显示，诱导前低表达GDNF以及不表达RET、C-kit，诱导后GDNF表达显著增强，RET、c-kit基因表达，而且GDNF在蛋白水平上也有表达。结论：GDNF联合FGCM可诱导BMSC分化为肠神经元，诱导后的细胞表达肠神经元标志物，其GDNF、RET及c-kit基因表达增强；GDNF-RET信号通路在诱导分化过程中起重要作用，c-kit在BMSC向肠神经元分化过程中的某个阶段发挥其作用。更多还原

【Abstract】 Hirschsprung’s disease (HD) is common digestive tract malformation in childrenwith an incidence rate of 1/2000～1/5000. The pathogenesis of HD is currently agreedthat embryonic neural crest cells are blocked in the digestive tract transition becauseof a number of reasons, so rectal and colon wall is absent of ganglion cells. HDmanifestes that spastic contraction of aganglionic segment and the secondarymegacolon due to expansion of the proximal intestine. To date, surgery is still themost effective treatment. Although surgical methods improve continuously andminimally invasive surgery makes great progress, postoperative complications are stillplagued patients and doctors. Cell transplantation, one of the top researches in recentyears, is expected to become another effective and safe treatment manner for HD.Bone mesenchymal stem cells (BMSC) can be easily isolated, cultured andpurified in vitro and multi-directional differentiation potential is one of the mostimportant biological characteristics. Now there are more and more reports aboutneural differentiation of BMSC. Many studies indicate that BMSC can differentiateinto nerve cells under certain conditions in vitro and transplant into the brain or spinalcord to treat the injury of the nervous system, which makes it possible that BMSCtreat intestinal nervous diseases and avoids the absence of neural stem cell and ethicalproblem. However, the mechanism of differentiation of BMSC into nerve cells is notclear, which may be concerned with the combined effect of agents that activated thedifferentiation-related gene expression. More common withβ-mercaptoethanol(BME), dimethyl sulfoxide (DMSO), retinoic and other chemical substances are oftenused as inducers. In addition, some growth factors and neurotrophic factors havegradually be taken seriously. On the one hand, neurotrophic factors may be used asinduced factors under a certain culture condition to induce BMSC to the nerve cell;On the other hand, neurotrophic factors can protect the nerve and promote neuritegrowth. Neural cell apoptosis may occur without the support of neurotrophic factors. In this experiment, we investigated the conditions of differentiation from BMSCto nerve cells, as well as neurotrophic factor expression changes, such as glial cellline-derived neurotrophic factor (GDNF) and rearranged during transfection (RET).The results showed that bFGF and EGF can not only induce neural differentiation ofBMSC, but also increase the expression of GDNF and RET significantly. GDNF mayplay an important role in the process of differentiation of BMSC into nerve cells. Inthe following experiment, GDNF and fetal gut condition medium (FGCM) inducedifferentiation of BMSC, and the induced cells expressed not only neuronal markerNSE and the enteric nervous marker nNOS, but also intestinal neurotransmitters VIP.This showed that differentiated enteric neurons in vitro not only have themorphological characteristics, but also a certain degree of function and it’s possiblethat the induced cells could be transplanted to aganglionic segment in vivo. In order toimprove the differentiation rate of BMSC into enteric neurons, the experiment aboutBMSC expressing GDNF was carried out. The results showed that recombinantgenetic engineered BMSC can successfully express GDNF in vitro. The geneticengineered BMSC in FGCM can differentiate into enteric neurons and induction ofdifferentiation is higher than that of exogenous GDNF group.However, we have no idea whether the induced cells have the function of normalganglion and whether cell transplantation can improve the function of aganglionsegment. It still needs further study in vivo. PartⅠIsolation, cultivation, purification and identification ofrat bone mesenchymal stem cells in vitroObjective: To isolate and observe bone mesenchymal stem cells (BMSC) fromSprague-Dawley (SD) rats in vitro and purify the cells.Methods: Femur bone marrow of rats were harvested and all the cells wereplanted and were cultured in DMEM with 10% FBS. BMSC were purified afterseveral passages. They were identified by flow cytometry of CD90 and CD45.Results: BMSC grew clones after adherence. The cells became fusion afterseveral passages. BMSC at passage 4 were BMSC marker CD90 positive (93.4%) andhematopoietic marker CD45 negative on flow cytometry.Conclusions: BMSC can be successfully cultured in vitro. The cells can bepurified only through repeated adherence.PartⅡDifferentiation from bone mesenchymal stem cells intonerve cells in vitroObjective: To investigate neural differentiation of rat bone mesenchymal stemcells (BMSC) and expression change of neural markers.Methods: At passage 4, BMSC were induced by basic fibroblast growth factor(bFGF, 10ng/ml), epidermal growth factor (EGF, 10ng/ml) and both of them inDMEM with 10% FBS for 7 days, respectively. The expression of neural specificenolase (NSE) and glial fibrillary acidic protein (GFAP) were detected byimmunocytochemistry. The expression of glial cell line-derived neurotrophic factor(GDNF) and rearranged during transfection (RET) mRNA was detected by RT-PCR.Results: After 7 days of induction, a certain number of cells showed expression of GFAP and NSE by immunocytochemistry method in experimental group, while thecells showed no expression of GFAP and NSE in control group. In bFGF group andbFGF+ EGF group, the positive rate of NSE was significantly higher than EGF group,while expression of GFAP in EGF group was obviously higher. BMSC expressed lowlevel GDNF mRNA and no RET mRNA, however, after induction, GDNF mRNAhighly increased and RET mRNA expressed, especially in bFGF+ EGF group.Conclusions: bFGF and EGF can not only induce neural differentiation of BMSC, butalso increase the expression of GDNF and RET significantly. GDNF may play animportant role in the process of differentiation of BMSC into nerve cells. The cellsinduced by bFGF are mainly neurons, while the induced cells by EGF are gliacytes.PartⅢDifferentiation from BMSC into enteric neurons in vitroand comparison of two different methodsObjective: To investigate the conditions and feasibility of differentiation frombone mesenchymal stem cells (BMSC) into enteric neurons in vitro and compare theefficiency of two methods.Methods: BMSC were cultured in DMEM supplemented with 10% fetal bovineserum (FBS) and induced by two methods after passage 4. The first one: BMSC wereinduced by 10ng/ml glial cell line-derived neurotrophic factor (GDNF) and fetal gutcondition medium (FGCM) for 7 days. The second one: The total mRNA wasextracted from newborn intestine by one-step method. GDNF cDNA was acquired byRT-PCR. The vector of PIRES2-EGFP-GDNF was constructed and identified bydouble enzyme digestion. Its sequence was analyzed by sequencing. The transgeneticexpression results were identified with immunofluorescence detection andimmunocytochemical stain. The transgentic cells were induced with diluted FGCM. The expression of neural specific enolase (NSE), vasoactive intestinal peptide (VIP)and nitric oxide synthase (nNOS) was detected by immunohistochemistry.Results: After 7 days of induction, a certain number of cells showed expressionof NSE, VIP and nNOS by immunohistochemistry method in both experimental group,while the cells showed no expression of VIP and NSE in control group. The inducedneurons form BMSC expressing GDNF were VIP and nNOS positive higher thanexogenous GDNF-induced cells.Conclusions: GDNF and FGCM can induce differentiation of BMSC intoenteric neurons. The efficiency of differentiation of GDNF-modified-BMSC intoenteric neurons is higher than that of exogenous GDNF-induced cells.PartⅣNeurotrophic factor expression changes of BMSC afterdifferentiation into enteric neurons and the study of mechanismObjective: To investigate the expression changes of glial cell line-derivedneurotrophic factor (GDNF), the receptor RET and C-kit of bone mesenchymal stemcells (BMSC) after differentiation into enteric neurons.Methods: At passage 4, BMSC were induced by 10ng/mlGDNF and fetal gutcondition medium (FGCM) for 7 days. The expression of neural specific enolase(NSE), vasoactive intestinal peptide (VIP) and nitric oxide synthase (nNOS) wasdetected by immunocytochemistry. The expression change of GDNF, RET and c-kitmRNA was detected by RT-PCR and protein was detected by western blot.Results: After 7 days of induction, a certain number of cells showed expressionof NSE, VIP and nNOS by immunohistochemistry method in experimental group,while the cells showed no expression of VIP and NSE in control group. BMSCexpressed low GDNF and no RET or c-kit mRNA, while after induction, the expression of GDNF mRNA was highly increased RET and C-kit mRNA expressed.GDNF protein was expressed.Conclusions: GDNF and FGCM can not only induce differentiation of BMSCinto enteric neurons, but also increase the expression of GDNF, RET and c-kit mRNA.GDNF-RET singaling pathway may play an important role in enteric neuraldifferentiation. C-kit may play a role at a certain stage of development of entericnervous system and be blocked after receiving a signal of mature enteric neurons.更多还原