GDNF与NT-3双基因修饰的大鼠骨髓基质干细胞移植治疗先天性巨结肠的初步研究
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摘要
第一部分大鼠骨髓间充质干细胞的分离培养
目的：建立一种简便、可靠的Sprague-Dawley(SD)大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)体外分离培养的方法。
方法：单纯贴壁法分离培养SD大鼠骨髓间充质干细胞,倒置显微镜观察细胞生长形态,噻唑蓝(MTT)法测定细胞生长曲线,免疫组化法检测骨髓间充质干细胞标志抗原CD90、CD44和造血干细胞标志抗原CD45的表达情况。
结果：培养的BMSCs为多角形或梭形,大小均一,呈漩涡状排列生长。免疫组化检测显示培养细胞高表达BMSCs于细胞标志抗原CD90、CD44,而不表达造血干细胞标志抗原CD45.
结论：单纯贴壁法可有效分离、培养和纯化大鼠BMSCs,为其组织程的临床研究应用提供了实验基础。
第二部分SD大鼠试验性巨结肠模型的建立和鉴定
目的：建立一种大鼠巨结肠实验动物模型,为临床展开干胞移植提供实验基础。
方法：200±15g重SD大鼠110只,随机分为两组。摸型用5mL/L-1的苯扎氯胺(BAC)经肛处理大鼠结肠45min，对照组用生理盐水。于术后1、2、4、8周行大体观察、结肠测压、取处理段结肠行HE染色、神经元特异性烯醇化酶(neural specific enolase,NSE)和蛋白基因产物9.5(protein gene product 9.5,PGP9.5)免疫荧光染色以及乙酰胆碱酯酶(AchE)组织化学染色观察,RT-PCR检测AchE、胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)神经营养素3(Neurotrophin-3, NT-3)的表达情况。
结果：术后一周实验组大鼠逐渐出现腹胀,排便减少,解剖发现处理段肠管痉挛狭窄,上端肠管肠内容物潴留,反射性收缩消失,组织学检查显示肠神经节细胞消失,且Real-Time PCR也证明AchE、GDNF、NT-3的表达明显下调。对照组则无明显改变。
结论：经肛应用5 mL/L-1BAC的方法成功建立了无神经节细胞肠段的大鼠实验模型,且该模型稳定、可靠、重复性好,为深入研究先天性巨结肠的病理生理提供了一个可靠的模型基础。
第三部分GDNF和FNT-3双基因真核表达载体的构建
目的：构建GDNF和NT-3双基因共表达的真核表达载体。
方法：从新生大鼠脑组织中采用逆转录PCR方法扩增GDNF和NT-3基因序列,将扩增产物分别克隆到pEGFP-N1载体,然后双酶切各pEGFP-N1载体,回收目的基因片段,将目的基因片段克隆至真核表达质粒pEGFP-N1中,构建其真核表达载体pEGFP-EGFP-GDNF-NT-3,并对重组体进行酶切及测序鉴定。
结果：PCR扩增片段与预期结果一致,GDNF-NT-3共表达载体构建成功,双酶切和测序结果正确。
结论：成功构建了pEGFP-EGFP-GDNF-NT-3双基因共表达真核表达质粒载体。
第四部分pEGFP-EGFP-GDNF-NT-3真核表达载体在大鼠骨髓间充质干细胞的表达及成神经诱导
目的：探讨重组体pEGFP-EGFP-GDNF-NT-3双基因表达载体转染大鼠间充质干细胞后的表达以及诱导分化为神经细胞的可行性。
方法：全骨髓法分离培养BMSCs,流式细胞术检测骨髓间充质干细胞标志CD90和造血干细胞标志CD45。转染带荧光的GDNF和NT-3基因,在荧光显微镜下观察绿色荧光蛋白(GFP)的表达及细胞的形态变化；免疫荧光检测神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF)和神经胶质酸性蛋白(GFAP)的表达；Western blot检测细胞GDNF及NT-3蛋白表达。对照组为未转染GDNF和NT-3基因的BMSCs。
结果：BMSCs能在体外成功分离培养,细胞高表达CD90(92.7%),不表达CD45。诱导分化后,BMSCs胞体变圆,伸出明显突起,并可见多数细胞相互交织成网状结构,呈神经细胞样形态。免疫荧光标记检测可见实验组细胞表达NSE和NF,而不表达GFAP。而对照组阴性。Western blot检测可见细胞GDNF及NT-3蛋白表达增强。
结论：研究表明重组质粒pEGFP-EGFP-GDNF-NT-3转染后可在BMSCs中成功表达,转染重组质粒后的BMSCs可分化为神经样细胞并表达神经元标志。该研究为基因治疗神经系统疾病提供了实验基础
第五部分共表达GDNF和NT-3基因的大鼠骨髓间充质干细胞向肠神经样细胞分化的研究
目的：初步研究大鼠骨髓间充质干细胞(BMSCs)体外诱导分化为肠神经样细胞以治疗先大性巨结肠的可行性。
方法：体外分离培养BMSCs,流式细胞术方法检测CD90和CD45的表达,传代至第五代进行诱导分化。实验组采用NanoJuiceTM转染试剂将目的基因胶质细胞源性神经营养因子(GDNF)和神经营养素3(NT-3)共转染至BMSCs内,并联合胎肠培养基(fetal gut culture medium, FGCM)诱导,设阳性对照组(单纯转染pEGFP-GDNF-NT-3)和阴性对照组(未转染质粒的BMSCs),免疫荧光显微镜观察转染及表达情况,免疫荧光鉴定细胞的神经特异性烯醇化酶(neural specific enolase, NSE)、蛋白基因产物9.5 (Protein gene production 9.5,PGP9.5)血管活性肠肽(vasoactive intestinal peptide, VIP)、一氧化氮合酶(nitric oxidesynthase, nNOS)及神经胶质酸性蛋白(glial fibrillary acidic protein,GFAP)的表达,RT-PCR检测目的基因表达情况。
结果：体外成功培养及纯化BMSCs,流式细胞术检测高表达骨髓间充质干细胞标志CD90,而不表达造血干细胞标志CD45。转染后24h即可在免疫荧光显微镜下观察到GFP表达,G418筛选4周后,细胞形态呈神经元样改变,免疫荧光实验组可见NSE、PGP9.5、VIP及nNOS阳性表达,GFAP阴性,阳性对照组亦有NSE阳性表达,而PGP9.5、VIP、nNOS及GFAP阴性,两组的阳性率有统计学差异,阴性对照组未见表达。RT-PCR显示目的基因在BMSCs内成功表达。
结论：GDNF和NT-3双基因修饰联合FGCM诱导的BMSCs可分化为肠神经细胞并表达肠神经标志,为相关肠神经系统疾病如先天性巨结肠的基因治疗提供了实验基础。
第六部分双基因修饰的大鼠骨髓间充质干细胞移植治疗实验性巨结肠的初步研究
目的：研究GDNF和NT-3双基因修饰的大鼠骨髓间充质干细胞移植实验性巨结肠大鼠模型肠壁的存活和基因表达情况,探讨双基因修饰的大鼠骨髓间充质干细胞移植治疗实验性巨结肠的可行性。
方法：体外分离培养大鼠骨髓间充质干细胞,并采用共表达胶质细胞源性神经营养因子和神经营养素3基因的真核表达载体转染修饰骨髓间充质干细胞。将双基因修饰的细胞移植入实验性巨结肠模型鼠去神经支配肠段肠壁。分别于术后1、2、4、8周行大体观察、病理学检测、免疫荧光检测PGP9.5、VIP的表达；real-time PCR检测GDNF、NT-3及RET mRNA的表达情况
结果：1.采用贴壁法成功分离培养和纯化了骨髓间充质干细胞。2.通过基因修饰骨髓间充质干细胞可诱导分化为肠神经样细胞。3.运用0.5%BAC处理大鼠结肠后一周,病理学检查可见神经节细胞缺失。骨髓间充质干细胞移植后1、2、4周后免疫荧光检测可见PGP9.5、VIP阳性的神经节细胞,而PBS移植组未见阳性表达。同时,与PBS移植组相比,干细胞移植组RET, GDNF and NT-3 mRNA的表达逐渐增加。
结论：双基因修饰的骨髓间充质干细胞可以在实验性巨结肠大鼠模型肠壁定植存活并表达相关基因,部分恢复结肠神经肌肉调节功能,为细胞移植治疗先天性巨结肠提供了实验基础。
PartⅠIsolation and culture of rat bone marrow derive Mesenchymals tem cells in vitro
Objective:To establish a simple and reliable method for isolation, culture of rat bone mesenchymal stem cells (BMSC) in vitro.
Methods:MSCs were isolated and culture by the attachment culture method. The morphologic changes of the cells were observed under an inverted lightmicroscope and the growth curve of the cell was determined by MTT assay. The expression of the BMSC marker CD90、CD44 and hematopoietic marker CD45 were detected by immunohistochemisty assay。
Results:The cultured BMSCs were shuttle or polygonal shape, The size of rBMSCs was homogeneous and showing whirlpool-shape. The cell showed positive expression of CD90 and CD44, but no expression of CD45 by immunohistochemisty assay.
Conclusions:BMSC can be successfully isolated、cultured and purified by the attachment culture method in vitro, and to provide the experimental basis for the clinical application of tissue engineering
Part II Identification and establishment of the rat model with experimental congenital megacolon
Objective:To establish a rat model of experimental congenital megacolon on purpose to provide the experimental basis for stem cell transplantation.
Methods:One hundred ten SD rats, weighted about200±15g, were randomly divided into two groups:Test group (n=55) and control group (n=55).In treatment group,5 mL/L-1 benzalkonium chloride (BAC) was applied into the colon for 45 minutes by transanal method and 9mL/L-1 saline was applied in the control group. The models were examined through gross observation , colon manometry , AchE histochemistry , HE staining, neural specific enolase(NSE) and protein gene product 9.5(PGP9.5) immunofluorescence and the expression of AchE,GDNF,NT-3 by RT-PCR at the end of the 1st,2nd,4th and 8th weeks post-operation respectively.
Results:After one week of BAC treatment, the rats in test group began to lose appetite and abdominal distention. Manometry showed the abolition of reflex contraction in the treatment group. Autopsy revealed a narrow segment and marked dilation of the proximal segment. Histologic examination showed lack of of ganglion cells at the site of BAC treatment. The mRNA expression ofAchE, GDNF and NT-3 decreased dramatically after BAC treatment. No visible change in bowel was observed in the control group.
Conclusion:Per anum application of 5 mL/L-1 BAC thus successfully produced a aganglionic segment of bowel in normal rat.. The method has satisfactory stability and repeatability. This model provides the basis for future studies to investigate the pathophysiology of Hirschsprung's disease (HD)
Part III Construction and identification of eukaryotic expression vector for pEGFP-GDNF-NT-3
Objective:To construct the eukaryotic co-expression vector containing both GDNF and NT-3.
Methods:The GDNF and NT-3 cDNA was acquired from neonatal rat cortex of cerebrum by RT-PCR. The products were cloned into an eukaryotic expression plasmid pEGFP-N1, The constructed plasmid pEGFP-EGFP-GDNF-NT-3 was confirmed by double enzyme digestion and DNA sequencing.
Results:The DNA fragment amplified by PCR from the total RNA was identical to GDNF and NT-3 from the gene library. The inserted target fragment was consistent with the target gene GDNF and NT-3 by sequence analysis, The identical DNA fragment was also amplified from the recombinants by double enzyme digestion, and the eukaryotic expression vector pEGFP-EGFP-GDNF-NT-3 was successfully constructed.
Conclusions:The pEGFP-EGFP-GDNF-NT-3 eukaryotic co-expression vector has been successfully constructed.
PartⅣExpression of pEGFP-EGFP-GDNF-NT-3 eukaryotic expression vector in rat bone marrow mesenchymal stem cells and the induction of Neuron
Objective:To investigate the expression of glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3(NT-3) in rat bone marrow mesenchymal stem cells (MSCs) and the feasibility of differentiate into neuron-like cells.
Methods:BMSCs isolated by the whole bone marrow culture, they were characterized by flow cytometry of CD90 and CD45. BMSCs were transfected by GDNF and NT-3 gene, the expression of green fluorescent protein (GFP) and morphological change was measured with fluorescence microscope. The expression of neuronalmarkers NSE (neural specific enolase), NF (neurofilament) and glial cellmarker GFAP ( glial fibrillary acidic protein) were detected by Immunofluorescence assay. The change of GDNF and NT-3 protein expression was quantified with western blot. The control group was BMSCs without GDNF and NT-3 gene.
Results:BMSCs were cultured and purified in vitro. The cultured BMSCs were positive for CD90 (92.7%) and negative for CD45 on flow cytometry. After the induction of differentiation, BMSCs became round or cone-shaped with distinctive outgrowth of protrusions. These cells revealed neuron-like morphological changes, and most of these cells were intertwined into a network structure Immunofluorescence assay showed positive expression of NSE and NF without the expressions of GFAP in experimental group, while the cells showed no expressions of NSE、NF and GFAP in control group. The expression of GDNF and NT-3 protein was highly increased.
Conclusions:The study indicates that recombinant plasmid pEGFP-EGFP-GDNF-NT-3 can express in BMSCs. The BMSCs transfected with GDNF and NT-3 were able to differentiate into neuronal-like cells and express nerve markers. This study provides an experimental basis for gene therapy to treat nervous system-related disorders.
PartⅤDifferentiation of GDNF and NT-3 Dual Gene-modified Rat Bone Marrow Mesenchymal Stem Cells into Enteric Neuron-like Cells
Objective:The purpose of our study was to investigate the feasibility of using in vitro induced enteric neuron-like cells, differentiated from rat BMSCs, for the treatment of Hirschsprung's disease (HD).
Methods:BMSCs isolated by the whole bone marrow culture, They were characterized by flow cytometry of CD90 and CD45. At passage 5, BMSCs were transfected with eukaryotic expression plasmids coexpressing GDNF and NT-3, differentiation induced in fetal gut culture medium (FGCM), The control group was BMSCs without GDNF and NT-3 gene. The expression of green fluorescent protein (GFP) and morphological change was measured with fluorescence microscope; The expression of the neuronal marker NSE(neural specific enolase, NSE), glial cell marker GFAP (glial fibrillary acidic protein,GFAP) and the enteric neuronal markers PGP9.5 (Protein gene production 9.5,PGP9.5)、VIP(vasoactive intestinal peptide, VIP) and nNOS(nitric oxide synthase, nNOS) were detected by Immunofluorescence assay.To detect the expression of mRNA of GDNF and NT-3 by RT-PCR.
Results:BMSCs were cultured and purified in vitro. The cultured BMSCs were positive for CD90 (92.7%) and negative for CD45 on flow cytometry. The transfected cells displayed neuron-like changes after the induction of differentiation, Immunofluorescence assay showed positive expression of the neuronal marker NSE and the enteric neuronal markers PGP9.5, VIP and nNOS, but no expression of GFAP in experimental group, while the cells showed no expressions of NSE、PGP9.5、VIP、nNOS and GFAP in control group. RT-PCR revealed successful expression of GDNF and NT-3 in transfected BMSCs.
Conclusions:Therefore, the present stud y indicates that genetically modified BMSCs coexpressing GDNF and NT-3 were able to differentiate into enteric neuronal cells and express enteric nerve markers when induced with FGCM. This study provides an experimental basis for gene therapy to treat enteric nervous system-related disorders, such as Hirschsprung's disease.
Part VI Preliminary study of GDNF and NT-3 modified BMSCs transplantation for treatment of Hirschsprun's discase in vivo
Objective:To study the survival and gene expression of transplanted GDNF and NT-3 modified BMSCs in rat colonic myenteron with experimental aganglionosis. To investigate the feasibility of the treatment of experimental aganglionosis by GDNF and NT-3 modified BMSCs transplantation in vivo.
Methods:BMSCs isolated by the Adhesive-screening method ,and were transfected with eukaryotic expression plasmids coexpressing GDNF and NT-3. GDNF and NT-3 modified BMSCs were transplantated into the denervated colon by microinjection,. Macroscopic、HE staining、AchE histochemistry、protein gene product 9.5(PGP9.5) and VIP immunofluorescence and the expression of RET ,GDNF ,NT-3 by RT-PCR at the end of the 1st,2nd,4th and 8th weeks post-transplantation respectively.
Results:1.BMSCs were cultured and purified by Adhesive-screening method.2. BMSCs able to differentiate into enteric neuronal-like cells by genetically modified in vitro 3. After one week of 0.5%BAC treatment, Histologic examination showed lack of ganglion cells at the site of BAC treatment. Immunohistofluorescence assay showed that there were positive cells of PGP9.5、VIP at 1w,2w and 4w after BMSCs transplantation. No positive cells were observed after PBS transplantation. The mRNA expression of RET, GDNF and NT-3 increased dramatically in test group as compared to the control group
Conclusions:GDNF and NT-3 modified BMSCs can survive and expression elated genes in the denervated colon of experimental aganglionosis rat, partially recovering the neuromuscular modulation of colonic This study provides an experimental basis for cell transplantation therapy to treat Hirschsprung's disease.
目的：建立一种简便、可靠的Sprague-Dawley(SD)大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)体外分离培养的方法。
方法：单纯贴壁法分离培养SD大鼠骨髓间充质干细胞,倒置显微镜观察细胞生长形态,噻唑蓝(MTT)法测定细胞生长曲线,免疫组化法检测骨髓间充质干细胞标志抗原CD90、CD44和造血干细胞标志抗原CD45的表达情况。
结果：培养的BMSCs为多角形或梭形,大小均一,呈漩涡状排列生长。免疫组化检测显示培养细胞高表达BMSCs于细胞标志抗原CD90、CD44,而不表达造血干细胞标志抗原CD45.
结论：单纯贴壁法可有效分离、培养和纯化大鼠BMSCs,为其组织程的临床研究应用提供了实验基础。
第二部分SD大鼠试验性巨结肠模型的建立和鉴定
目的：建立一种大鼠巨结肠实验动物模型,为临床展开干胞移植提供实验基础。
方法：200±15g重SD大鼠110只,随机分为两组。摸型用5mL/L-1的苯扎氯胺(BAC)经肛处理大鼠结肠45min，对照组用生理盐水。于术后1、2、4、8周行大体观察、结肠测压、取处理段结肠行HE染色、神经元特异性烯醇化酶(neural specific enolase,NSE)和蛋白基因产物9.5(protein gene product 9.5,PGP9.5)免疫荧光染色以及乙酰胆碱酯酶(AchE)组织化学染色观察,RT-PCR检测AchE、胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)神经营养素3(Neurotrophin-3, NT-3)的表达情况。
结果：术后一周实验组大鼠逐渐出现腹胀,排便减少,解剖发现处理段肠管痉挛狭窄,上端肠管肠内容物潴留,反射性收缩消失,组织学检查显示肠神经节细胞消失,且Real-Time PCR也证明AchE、GDNF、NT-3的表达明显下调。对照组则无明显改变。
结论：经肛应用5 mL/L-1BAC的方法成功建立了无神经节细胞肠段的大鼠实验模型,且该模型稳定、可靠、重复性好,为深入研究先天性巨结肠的病理生理提供了一个可靠的模型基础。
第三部分GDNF和FNT-3双基因真核表达载体的构建
目的：构建GDNF和NT-3双基因共表达的真核表达载体。
方法：从新生大鼠脑组织中采用逆转录PCR方法扩增GDNF和NT-3基因序列,将扩增产物分别克隆到pEGFP-N1载体,然后双酶切各pEGFP-N1载体,回收目的基因片段,将目的基因片段克隆至真核表达质粒pEGFP-N1中,构建其真核表达载体pEGFP-EGFP-GDNF-NT-3,并对重组体进行酶切及测序鉴定。
结果：PCR扩增片段与预期结果一致,GDNF-NT-3共表达载体构建成功,双酶切和测序结果正确。
结论：成功构建了pEGFP-EGFP-GDNF-NT-3双基因共表达真核表达质粒载体。
第四部分pEGFP-EGFP-GDNF-NT-3真核表达载体在大鼠骨髓间充质干细胞的表达及成神经诱导
目的：探讨重组体pEGFP-EGFP-GDNF-NT-3双基因表达载体转染大鼠间充质干细胞后的表达以及诱导分化为神经细胞的可行性。
方法：全骨髓法分离培养BMSCs,流式细胞术检测骨髓间充质干细胞标志CD90和造血干细胞标志CD45。转染带荧光的GDNF和NT-3基因,在荧光显微镜下观察绿色荧光蛋白(GFP)的表达及细胞的形态变化；免疫荧光检测神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF)和神经胶质酸性蛋白(GFAP)的表达；Western blot检测细胞GDNF及NT-3蛋白表达。对照组为未转染GDNF和NT-3基因的BMSCs。
结果：BMSCs能在体外成功分离培养,细胞高表达CD90(92.7%),不表达CD45。诱导分化后,BMSCs胞体变圆,伸出明显突起,并可见多数细胞相互交织成网状结构,呈神经细胞样形态。免疫荧光标记检测可见实验组细胞表达NSE和NF,而不表达GFAP。而对照组阴性。Western blot检测可见细胞GDNF及NT-3蛋白表达增强。
结论：研究表明重组质粒pEGFP-EGFP-GDNF-NT-3转染后可在BMSCs中成功表达,转染重组质粒后的BMSCs可分化为神经样细胞并表达神经元标志。该研究为基因治疗神经系统疾病提供了实验基础
第五部分共表达GDNF和NT-3基因的大鼠骨髓间充质干细胞向肠神经样细胞分化的研究
目的：初步研究大鼠骨髓间充质干细胞(BMSCs)体外诱导分化为肠神经样细胞以治疗先大性巨结肠的可行性。
方法：体外分离培养BMSCs,流式细胞术方法检测CD90和CD45的表达,传代至第五代进行诱导分化。实验组采用NanoJuiceTM转染试剂将目的基因胶质细胞源性神经营养因子(GDNF)和神经营养素3(NT-3)共转染至BMSCs内,并联合胎肠培养基(fetal gut culture medium, FGCM)诱导,设阳性对照组(单纯转染pEGFP-GDNF-NT-3)和阴性对照组(未转染质粒的BMSCs),免疫荧光显微镜观察转染及表达情况,免疫荧光鉴定细胞的神经特异性烯醇化酶(neural specific enolase, NSE)、蛋白基因产物9.5 (Protein gene production 9.5,PGP9.5)血管活性肠肽(vasoactive intestinal peptide, VIP)、一氧化氮合酶(nitric oxidesynthase, nNOS)及神经胶质酸性蛋白(glial fibrillary acidic protein,GFAP)的表达,RT-PCR检测目的基因表达情况。
结果：体外成功培养及纯化BMSCs,流式细胞术检测高表达骨髓间充质干细胞标志CD90,而不表达造血干细胞标志CD45。转染后24h即可在免疫荧光显微镜下观察到GFP表达,G418筛选4周后,细胞形态呈神经元样改变,免疫荧光实验组可见NSE、PGP9.5、VIP及nNOS阳性表达,GFAP阴性,阳性对照组亦有NSE阳性表达,而PGP9.5、VIP、nNOS及GFAP阴性,两组的阳性率有统计学差异,阴性对照组未见表达。RT-PCR显示目的基因在BMSCs内成功表达。
结论：GDNF和NT-3双基因修饰联合FGCM诱导的BMSCs可分化为肠神经细胞并表达肠神经标志,为相关肠神经系统疾病如先天性巨结肠的基因治疗提供了实验基础。
第六部分双基因修饰的大鼠骨髓间充质干细胞移植治疗实验性巨结肠的初步研究
目的：研究GDNF和NT-3双基因修饰的大鼠骨髓间充质干细胞移植实验性巨结肠大鼠模型肠壁的存活和基因表达情况,探讨双基因修饰的大鼠骨髓间充质干细胞移植治疗实验性巨结肠的可行性。
方法：体外分离培养大鼠骨髓间充质干细胞,并采用共表达胶质细胞源性神经营养因子和神经营养素3基因的真核表达载体转染修饰骨髓间充质干细胞。将双基因修饰的细胞移植入实验性巨结肠模型鼠去神经支配肠段肠壁。分别于术后1、2、4、8周行大体观察、病理学检测、免疫荧光检测PGP9.5、VIP的表达；real-time PCR检测GDNF、NT-3及RET mRNA的表达情况
结果：1.采用贴壁法成功分离培养和纯化了骨髓间充质干细胞。2.通过基因修饰骨髓间充质干细胞可诱导分化为肠神经样细胞。3.运用0.5%BAC处理大鼠结肠后一周,病理学检查可见神经节细胞缺失。骨髓间充质干细胞移植后1、2、4周后免疫荧光检测可见PGP9.5、VIP阳性的神经节细胞,而PBS移植组未见阳性表达。同时,与PBS移植组相比,干细胞移植组RET, GDNF and NT-3 mRNA的表达逐渐增加。
结论：双基因修饰的骨髓间充质干细胞可以在实验性巨结肠大鼠模型肠壁定植存活并表达相关基因,部分恢复结肠神经肌肉调节功能,为细胞移植治疗先天性巨结肠提供了实验基础。
PartⅠIsolation and culture of rat bone marrow derive Mesenchymals tem cells in vitro
Objective:To establish a simple and reliable method for isolation, culture of rat bone mesenchymal stem cells (BMSC) in vitro.
Methods:MSCs were isolated and culture by the attachment culture method. The morphologic changes of the cells were observed under an inverted lightmicroscope and the growth curve of the cell was determined by MTT assay. The expression of the BMSC marker CD90、CD44 and hematopoietic marker CD45 were detected by immunohistochemisty assay。
Results:The cultured BMSCs were shuttle or polygonal shape, The size of rBMSCs was homogeneous and showing whirlpool-shape. The cell showed positive expression of CD90 and CD44, but no expression of CD45 by immunohistochemisty assay.
Conclusions:BMSC can be successfully isolated、cultured and purified by the attachment culture method in vitro, and to provide the experimental basis for the clinical application of tissue engineering
Part II Identification and establishment of the rat model with experimental congenital megacolon
Objective:To establish a rat model of experimental congenital megacolon on purpose to provide the experimental basis for stem cell transplantation.
Methods:One hundred ten SD rats, weighted about200±15g, were randomly divided into two groups:Test group (n=55) and control group (n=55).In treatment group,5 mL/L-1 benzalkonium chloride (BAC) was applied into the colon for 45 minutes by transanal method and 9mL/L-1 saline was applied in the control group. The models were examined through gross observation , colon manometry , AchE histochemistry , HE staining, neural specific enolase(NSE) and protein gene product 9.5(PGP9.5) immunofluorescence and the expression of AchE,GDNF,NT-3 by RT-PCR at the end of the 1st,2nd,4th and 8th weeks post-operation respectively.
Results:After one week of BAC treatment, the rats in test group began to lose appetite and abdominal distention. Manometry showed the abolition of reflex contraction in the treatment group. Autopsy revealed a narrow segment and marked dilation of the proximal segment. Histologic examination showed lack of of ganglion cells at the site of BAC treatment. The mRNA expression ofAchE, GDNF and NT-3 decreased dramatically after BAC treatment. No visible change in bowel was observed in the control group.
Conclusion:Per anum application of 5 mL/L-1 BAC thus successfully produced a aganglionic segment of bowel in normal rat.. The method has satisfactory stability and repeatability. This model provides the basis for future studies to investigate the pathophysiology of Hirschsprung's disease (HD)
Part III Construction and identification of eukaryotic expression vector for pEGFP-GDNF-NT-3
Objective:To construct the eukaryotic co-expression vector containing both GDNF and NT-3.
Methods:The GDNF and NT-3 cDNA was acquired from neonatal rat cortex of cerebrum by RT-PCR. The products were cloned into an eukaryotic expression plasmid pEGFP-N1, The constructed plasmid pEGFP-EGFP-GDNF-NT-3 was confirmed by double enzyme digestion and DNA sequencing.
Results:The DNA fragment amplified by PCR from the total RNA was identical to GDNF and NT-3 from the gene library. The inserted target fragment was consistent with the target gene GDNF and NT-3 by sequence analysis, The identical DNA fragment was also amplified from the recombinants by double enzyme digestion, and the eukaryotic expression vector pEGFP-EGFP-GDNF-NT-3 was successfully constructed.
Conclusions:The pEGFP-EGFP-GDNF-NT-3 eukaryotic co-expression vector has been successfully constructed.
PartⅣExpression of pEGFP-EGFP-GDNF-NT-3 eukaryotic expression vector in rat bone marrow mesenchymal stem cells and the induction of Neuron
Objective:To investigate the expression of glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3(NT-3) in rat bone marrow mesenchymal stem cells (MSCs) and the feasibility of differentiate into neuron-like cells.
Methods:BMSCs isolated by the whole bone marrow culture, they were characterized by flow cytometry of CD90 and CD45. BMSCs were transfected by GDNF and NT-3 gene, the expression of green fluorescent protein (GFP) and morphological change was measured with fluorescence microscope. The expression of neuronalmarkers NSE (neural specific enolase), NF (neurofilament) and glial cellmarker GFAP ( glial fibrillary acidic protein) were detected by Immunofluorescence assay. The change of GDNF and NT-3 protein expression was quantified with western blot. The control group was BMSCs without GDNF and NT-3 gene.
Results:BMSCs were cultured and purified in vitro. The cultured BMSCs were positive for CD90 (92.7%) and negative for CD45 on flow cytometry. After the induction of differentiation, BMSCs became round or cone-shaped with distinctive outgrowth of protrusions. These cells revealed neuron-like morphological changes, and most of these cells were intertwined into a network structure Immunofluorescence assay showed positive expression of NSE and NF without the expressions of GFAP in experimental group, while the cells showed no expressions of NSE、NF and GFAP in control group. The expression of GDNF and NT-3 protein was highly increased.
Conclusions:The study indicates that recombinant plasmid pEGFP-EGFP-GDNF-NT-3 can express in BMSCs. The BMSCs transfected with GDNF and NT-3 were able to differentiate into neuronal-like cells and express nerve markers. This study provides an experimental basis for gene therapy to treat nervous system-related disorders.
PartⅤDifferentiation of GDNF and NT-3 Dual Gene-modified Rat Bone Marrow Mesenchymal Stem Cells into Enteric Neuron-like Cells
Objective:The purpose of our study was to investigate the feasibility of using in vitro induced enteric neuron-like cells, differentiated from rat BMSCs, for the treatment of Hirschsprung's disease (HD).
Methods:BMSCs isolated by the whole bone marrow culture, They were characterized by flow cytometry of CD90 and CD45. At passage 5, BMSCs were transfected with eukaryotic expression plasmids coexpressing GDNF and NT-3, differentiation induced in fetal gut culture medium (FGCM), The control group was BMSCs without GDNF and NT-3 gene. The expression of green fluorescent protein (GFP) and morphological change was measured with fluorescence microscope; The expression of the neuronal marker NSE(neural specific enolase, NSE), glial cell marker GFAP (glial fibrillary acidic protein,GFAP) and the enteric neuronal markers PGP9.5 (Protein gene production 9.5,PGP9.5)、VIP(vasoactive intestinal peptide, VIP) and nNOS(nitric oxide synthase, nNOS) were detected by Immunofluorescence assay.To detect the expression of mRNA of GDNF and NT-3 by RT-PCR.
Results:BMSCs were cultured and purified in vitro. The cultured BMSCs were positive for CD90 (92.7%) and negative for CD45 on flow cytometry. The transfected cells displayed neuron-like changes after the induction of differentiation, Immunofluorescence assay showed positive expression of the neuronal marker NSE and the enteric neuronal markers PGP9.5, VIP and nNOS, but no expression of GFAP in experimental group, while the cells showed no expressions of NSE、PGP9.5、VIP、nNOS and GFAP in control group. RT-PCR revealed successful expression of GDNF and NT-3 in transfected BMSCs.
Conclusions:Therefore, the present stud y indicates that genetically modified BMSCs coexpressing GDNF and NT-3 were able to differentiate into enteric neuronal cells and express enteric nerve markers when induced with FGCM. This study provides an experimental basis for gene therapy to treat enteric nervous system-related disorders, such as Hirschsprung's disease.
Part VI Preliminary study of GDNF and NT-3 modified BMSCs transplantation for treatment of Hirschsprun's discase in vivo
Objective:To study the survival and gene expression of transplanted GDNF and NT-3 modified BMSCs in rat colonic myenteron with experimental aganglionosis. To investigate the feasibility of the treatment of experimental aganglionosis by GDNF and NT-3 modified BMSCs transplantation in vivo.
Methods:BMSCs isolated by the Adhesive-screening method ,and were transfected with eukaryotic expression plasmids coexpressing GDNF and NT-3. GDNF and NT-3 modified BMSCs were transplantated into the denervated colon by microinjection,. Macroscopic、HE staining、AchE histochemistry、protein gene product 9.5(PGP9.5) and VIP immunofluorescence and the expression of RET ,GDNF ,NT-3 by RT-PCR at the end of the 1st,2nd,4th and 8th weeks post-transplantation respectively.
Results:1.BMSCs were cultured and purified by Adhesive-screening method.2. BMSCs able to differentiate into enteric neuronal-like cells by genetically modified in vitro 3. After one week of 0.5%BAC treatment, Histologic examination showed lack of ganglion cells at the site of BAC treatment. Immunohistofluorescence assay showed that there were positive cells of PGP9.5、VIP at 1w,2w and 4w after BMSCs transplantation. No positive cells were observed after PBS transplantation. The mRNA expression of RET, GDNF and NT-3 increased dramatically in test group as compared to the control group
Conclusions:GDNF and NT-3 modified BMSCs can survive and expression elated genes in the denervated colon of experimental aganglionosis rat, partially recovering the neuromuscular modulation of colonic This study provides an experimental basis for cell transplantation therapy to treat Hirschsprung's disease.
引文
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