摘要
摘要目的：研究大鼠骨髓间充质干细胞(Rat mesenchymal stem cells, rMSCs)以及经诱导分化为神经元样细胞后脑源性神经营养因子(BDNF)、神经生长因子(NGF)、神经营养素-3(NT3)、睫状神经营养因子(CNTF)和胶质细胞源性神经营养因子(GDNF)的表达情况。
方法：取Wistar大鼠骨髓,提取、分离、培养骨髓间充质干细胞,用免疫荧光染色及流式细胞仪行CD44、CD45和CD90鉴定。取传第3代的骨髓间充质干细胞用DMEM、0.1 mmol/L2-巯基乙醇和2%二甲基亚砜,预诱导5小时后更换含10%FBS的DMEM、10μg/L碱性成纤维细胞生长因子(FGF)、10μg/L表皮生长因子(EGF)共1ml,置入37℃,5%CO2培养箱中诱导维持7天和14天,荧光相差显微镜观察细胞形态,行神经元标志物Nestin、NF、MAP-2、β-tubulinⅢ、Chat和GFAP免疫荧光染色和流式细胞仪鉴定。分别在细胞传1、3、5代及诱导7天和14天时采用RT-PCR及ELISA测定骨髓间充质干细胞和神经元样细胞表达BDNF、NGF、NT3、CNTF和GDNF的情况。
结果：骨髓间充质干细胞经诱导后大约有80%的细胞呈神经元样改变,免疫荧光染色显示神经元标志物Nestin阳性率40.17%±2.95%、NF阳性率16.00%±1.23%、MAP-2阳性率36.30%±1.78%、tubulinβⅢ阳性率19.00%±1.58%、Chat阳性率19.30%±1.38%和GFAP阳性率39.17%±1.95%。RT-PCR显示骨髓间充质干细胞能够表达BDNF、NGF、NT3、CNTF和GDNF;而ELISA分析证明在骨髓间充质干细胞传第3代时上述神经营养因子处于高峰,CNTF表达量最高,比NT3高15倍；从传第1代到第5代CNTF表达变化小于1倍,但是BDNF表达变化则多于8倍；分化为神经元样细胞后表达上述神经营养因子的能力下降,特别是CNTF及BDNF下降至一半；在诱导7天时NGF、BDNF、GDNF表达略高于诱导14天,而CNTF、NT3的表达则比诱导14天时稍减少。
结论：1.骨髓间充质干细胞能够表达BDNG, NGF, CNTF, GDNF和NT3,且在细胞传第三代时处于高峰,以CNTF的表达量最高。2.骨髓间充质干细胞诱导分化后为神经元样细胞后表达上述神经营养因子含量下降,以CNTF及BDNF下降最明显。3.在诱导7天和14天时,上述神经营养因子表达量相当。
目的：研究骨髓间充质干细胞移植于切断胫神经远端观察表达上述神经营养因子以及向神经元样细胞分化的情况；进一步观察骨髓间充质干细胞延缓失神经支配的骨骼肌萎缩作用和沉默骨髓间充质干细胞表达CNTF对延缓失神经骨骼肌萎缩的影响。
方法：取成年Wistar雄性大鼠,分为4组：Ⅰ,对照组(n=24)；Ⅱ,CNTF注射组(n=24)；Ⅲ,RNA干扰沉默CNTF表达的骨髓间充质干细胞移植组(n=24)；Ⅳ,骨髓间充质干细胞移植组(n=24)。每组大鼠的远端胫神经分别被注射5ulDMEM, 5ulCNTF,2.5×105RNA干扰沉默CNTF表达的骨髓间充质干细胞和2.5×105骨髓间充质干细胞。分别于术后4、8和12周获取标本行腓肠肌张力、腓肠肌湿重比率、腓肠肌纤维横截面积维持率及三色染色观察肌萎缩情况；同时行在12周时行骨髓基质干细胞标志物CD44、CD90；神经元标志物NF、GFAP及神经营养因子BDNF、NGF、NT3、CNTF和GDNF免疫荧光染色观察骨髓间充质干细胞表达神经营养因子与分化情况以及扫描电镜观察神经肌肉接头处突触后膜变化情况。
结果：在移植后4、8和12周腓肠肌肌张力测试发现,在所有各组肌张力呈持续下降趋势,特别是Ⅰ组；而且腓肠肌湿重比率、腓肠肌纤维横截面积维持率下降趋势与肌张力变化相似；各组间两两比较,结果显示腓肠肌湿重比率、腓肠肌纤维横截面积维持率,Ⅳ组与Ⅰ组、Ⅱ组比较差异有显著性(P<0.05)；Ⅲ组与Ⅰ组、Ⅱ组比较差异有显著性(P＜0.05)；Ⅱ组与Ⅰ组比较差异有显著性(P<0.05)；然而,Ⅲ组与Ⅳ组比较差异无显著性(P＞0.05)。腓肠肌三色染色发现,Ⅰ组肌纤维大部分被胶原纤维组织替代,Ⅱ、Ⅲ、Ⅳ组尚有肌纤维组织得以保持,其中,Ⅳ组优于Ⅲ组优于Ⅱ组。在细胞移植后12周取远侧胫神经标本行免疫荧光染色,发现骨髓基质干细胞标志物CD90呈阳性,同时行神经营养因子BDNF、NGF、NT3、CNTF和GDNF染色呈阳性,而神经元标志物NF、GFAP呈阴性。进行描电镜显示,Ⅳ组神经肌肉接头处突触后膜皱襞深度与密度优于Ⅲ组优于Ⅱ组优于Ⅰ组。
结论：1.骨髓基质干细胞具有延缓失神经骨骼肌肌萎缩的作用；；2.骨髓基质干细胞在缺少某种神经营养因子--CNTF的情况下延缓失神经骨骼肌肌萎缩的作用未发生改变；3.骨髓基质干细胞之所以能够延缓失神经骨骼肌肌萎缩可能因为在周围神经中存活并分泌神经营养因子,而不是分化为神经元样细胞；4.突触后膜的变化与腓肠肌形态与功能的变化一致,我们推猜骨髓基质干细胞表达的神经营养因子是通过神经肌肉接头处的突触后膜发挥延缓失神经骨骼肌肌萎缩。
摘要目的：研究骨髓间充质干细胞对突触后膜AchR表达的影响及引起其变化的细胞信号转导通路情况。
方法：将Sol8肌管细胞培养于含10%FBS的高糖DMEM中待其达到60%融合时,通过构建慢病毒载体(LVTHM)将AchR和ERBB3共转染Sol8肌管细胞,通过RT-PCR验证转染效率；将共转染后的Sol8肌管细胞与传第三代的骨髓基质干细胞共培养48小时；实验分两组：A：Sol8肌管细胞组；B：共培养组；通过Western blot测定Sol8肌管细胞AchR表达情况以及细胞信号转导通路ERBB3(细胞膜)→Ras→Raf→MAPK(细胞质)的变化情况。
结果：经RT-PCR测定发现Erbb3转染率为84.3%,AChRδ转染率为83.3%Western blot结果发现,骨髓间充质干细胞与共转染后的Sol8肌管细胞共培养48hr后观察发现,AChRδ表达增加,细胞膜ErbB3表达上调,同时发现Sol8肌管细胞内Ras、Raf、MAPK细胞信号蛋白表达增加。
结论：1.骨髓间充质干细胞能使突触后膜AchR表达上调；2.其机制是通过ERBB3(细胞膜)→Ras→Raf→MAPK(细胞质)信号转导通路。
Objective:Bone marrow stromal stem cells (MScs), which normally differentiate into mesenchymal derivatives, have recently reported to transdifferentiate into neurons. However, the findings from different groups and interpretations have been challenged.The purpose of this paper is to re-evaluate the phenomenon of neuronal trans-differentiation of MSCs and compare the expression levels of neurotrophins in rMSCs and neuronal-like phenotypes derived from rMSCs.
Method:rMSCs were obtained from both bilateral femurs and tibias. rMSCs were differentiated in pre-induction media and then transferred to neuronal induction media for 7 days and 14days. RT-PCR and ELISA were employed to determine the expression level of Brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT3), ciliary neurotrophic factor (CNTF) and glial derived neurotrophic factor (GDNF) in rMSCs and neuronal-like phenotype derived from rMSCs.
Results:The adherent rMSCs exhibited spindle-shaped or large flattened morphology, and expressed CD90 and CD44 with a positive ratio of 95.37%±2.38% and 94.35%±1.73% respectively. After neuronal induction,, neuronal-like phenotypes accounted for about 80% of the total population. Immunological labelling showed that neuronal-like phenotypes cells expressed mature neuronal markers (GFAP 40.17%±2.95%, Tubulin-Ⅲ19.00%±1.58%, CHAT 19.30%±1.38%), NF 16%±1.23%, MAP-2 36.30%±1.78%) RT-PCR and ELISA analysis confirmed expression of neurotrophins in rMSCs and neuronal-like phenotypes. Moreover, the level of BDNF、NGF、NT3、CNTF and GDNF of rMSCs is remarkably higher in rMSCs than the neuronal-like phenotypes, especially CNTF. The expression level of these neurotrophins did not change significantly after enduring induction.
Conclusion:Under certain conditions, rMSCs can transdifferentiate into neurons. The non-induced rMSCs has a dynamic expression profile of neurotrophins and may serves as a better tool than the trans-differentiated rMSCs for transplant therapy.
Objective:Although there are many therapies, it is still difficulty to reverse post-denervation muscle atrophy. Recently, stem cells transplantation seems to be as a method of delaying muscle atrophy. The findings from different groups and interpretations have shown that MSCs from adult bone marrow have the potential to differentiate into a variety of cell types and express many cytokines and cellular factors. The purpose of this paper is to evaluate whether delivery of neurotrophins to denervated gastronemius muscles improved the function of these muscles when adult rat mesenchymal stem cells (rMSCs) were transplanted into the distal tibial nerve. Further, we observed whether rMSCs by silencing CNTF, the maxlmum in neurotrophins, with RNA interference changed its role in delaying muscular atrophy.
Method:After anesthesia, the right leg of each rat (Wistar, about 120g) was denervated by sectioning the tibial never in 1cm above gastrocnemius muscle. The tibial nerve was tightly ligated with surgical silk in two places, cut between the sutures, and a 0.4cm section of the nerve was removed. Both proximal and distal nerve stumps were implanted in the surrouding tissue as far away from each other as possible. After 1 week of muscle denervation, animals were divided into the following four experimental groups:Ⅰ, control group (n=24);Ⅱ,CNTF injection group(n=24);Ⅲ,rMSCs by silencing CNTF with RNA interference transplantation group (n=24);Ⅳ, rMSCs transplantation group (n=24). They were respectively injected with 5ul of culture media,5ul of CNTF,2.5×105 rMSCs by silencing CNTF with RNA interference and 2.5×105 rMSCs at the site of tibial nerve just proximal to the nerve branch of the medial gastrocnemius. The animals were euthanized again at 4、8and 12 weeks after denervation. Tissue samples of tibial nerve and gastrocnemius muscle were taken. Muscle strength assay、gastrocnemius muscle wet、weight、richrome staining and muscle fiber areas from media gastrocnemius muscle were performed. Meanwhile, Electron microscope examination was done to demonstrate the NMJ. All the data was analyzed by Stata.
Results:Fluorescence immunocytochemistry confirmed that rMSCs expressed the neurotrophins, and scarcely differentiate into neuronal-like phenotypes. Further, tissue examination found that mean±SE wet weight ratio was significantly different in all groups. Mean ofⅢandⅣgroup was about 70% higher than that ofⅠgroup, and 30% higher than mean ofⅡgroup. However, mean betweenⅢandⅣgroup was nearly approximate. Moreover, wet weight ratio in all group declined gradually with lasting denervation, especiallyⅠgroup. The variation of Muscle strength, muscle fiber areas and neuromuscular junctions was nearly consistent with wet weight ratio.
Conclusion:rMSCs could improve the function of denervated gastronemius muscles by delivery of neurotrophins when rMSCs were transplanted into the distal tibial nerve. Furthermore, rMSCs lacking CNTF, the maxlmum in neurotrophins, could not reduce remarkably its role in delaying muscular atrophy.
Objective:Bone marrow mesenchymal stem cells(MSCs) contributes to gene therapy and muscle repair. Neuregulins(NRGs) thought to regulate acetylcholine receptor(AChR) expression, which is a hallmark of the inductive events of synapse formation. This present study investigated effect of rMSCs on Sol 8 cells and which signal transduction pathway involve in it.
Method:Lentivirus (LVTHM) was performed according to instruction manual. Sol 8 cells were transfectioned by AChR and ERBB3 gene through LVTHM. RT-PCR was performed to show the mRNA of AChR and ERBB3. Then, we co-culture rMSCs and Sol 8 cells for 48 hours. Compared with the normal group, Western blot showed protein of AChR raised and protein of AChR is up-regulated. The phosphorylation of Raf、Ras and MAPK were indicated also.
Results:The result was consistent with the precious. After 48 hours, protein of AChR and ERBB3 was up-regulated in experimental group. Meanwhile, the protein of Raf、Ras and MAPK were increased.
Conclusion:rMSCs is able to up-regulate the protein of AChR. We infer that Ras→Raf→MAPK pathway may participate in this process.
引文
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