骨髓间充质干细胞向心肌细胞和内皮细胞分化的研究
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摘要
背景:由于人们生活水平的提高,冠心病成为现代社会发病率和死亡率不断上升的疾病,而急性心肌梗死是重要的死亡原因。心肌梗死后,心肌细胞坏死、凋亡造成心肌细胞数目减少,由于心肌细胞不能再生,只能由成纤维细胞填充,因此在心肌缺血发生后坏死的心肌由瘢痕组织取代,并逐步发生心室重构导致心脏收缩功能下降和心力衰竭。目前心肌梗死后的治疗手段主要包括药物治疗、介入治疗和手术治疗。药物治疗通过减少心肌氧耗,在一定程度上缓解了病人的症状。冠脉介入治疗及心脏搭桥手术治疗只能恢复局部血液供应,挽救梗死区内存活的心肌细胞,延缓左室重构,改善病人预后,但无法使已梗死的心肌细胞再生,没能从根本上修复梗死区,因此仍有部分病人不可避免地发展为心功能不全甚至导致心源性死亡。
近年来发展的细胞移植技术为心肌梗死的治疗提供了新的方向并受到了人们的广泛关注。通过细胞心肌移植术增加有功能的心肌细胞数目,可能是心肌梗死后心衰治疗的最有效方法之一。此外近年来提出的治疗性血管新生也已发展成为治疗缺血性疾病的新方法。骨髓间充质干细胞(mesenchymal stem cells,MSCs)是骨髓中不同于造血干细胞的一类细胞,它在体内能够自我更新,保持未分化状态,在体外合适的培养条件下,易于扩增,具有向多种细胞类型分化的潜能。1999年,Makino等首次在体外成功地诱导MSCs定向分化为心肌细胞。同时MSCs具有分化为内皮细胞的理论基础和实验基础。2004年,Oswald等人发现在体外MSCs与VEGF_(165)和2%胎牛血清共培养可诱导分化为内皮样细胞。这些实验为MSCs诱导分化为心肌细胞和内皮细胞进行细胞移植治疗缺血性心脏病提供了良好的实验基础。
Notch信号通路在脊椎动物和无脊椎动物中高度保守,它在决定细胞分化上起重要作用。它对正常心血管系统的发育有重要作用,Notch信号表达不足或过量都会造成动物因心血管异常而死亡。此外,Notch信号系统在血管再生方面也有重要作用。Notch信号通路在MSCs向内皮细胞分化的过程中可能也起着重要的调节作用。本实验主要研究了大鼠MSCs体外向心肌细胞及内皮细胞诱导分化的可行性,并研究了Notch信号通路对大鼠MSCs向内皮细胞诱导分化产生的影响,以期为缺血性心脏病的细胞移植治疗提供良好的种子细胞。
研究目的
1.体外分离培养大鼠骨髓MSCs,研究大鼠骨髓MSCs的生物学特性。用5-氮胞苷(5-Azacytidine,5-aza)定向诱导MSCs向心肌细胞分化,研究MSCs体外向心肌细胞分化的可行性。
2.定向诱导大鼠骨髓MSCs向内皮细胞分化,研究MSCs体外向内皮细胞分化的可行性。研究诱导前后细胞增殖能力、迁移能力和形成毛细血管样结构能力的变化。
3.采用RT-PCR方法检测大鼠骨髓MSCs上Notch信号受体和配体mRNA的表达情况,研究MSCs向内皮细胞诱导分化前后细胞上Notch信号受体和配体mRNA的表达变化。向诱导后内皮细胞中导入VEGF_(165)基因来促进细胞的增殖和迁移能力。检测转染VEGF_(165)基因前后细胞上Notch信号受体和配体mRNA的表达变化,检测细胞增殖能力、迁移能力和形成毛细血管样结构能力的变化,研究Notch信号对诱导后内皮细胞的增殖能力、迁移能力和形成毛细血管样结构能力的影响,以期更好的诱导MSCs向内皮细胞分化,促进诱导后内皮细胞的增殖、迁移和形成毛细血管样结构的能力,为缺血性心脏病的细胞移植治疗提供良好的种子细胞。
方法
1.采用密度梯度离心法和贴壁细胞分离培养法体外分离培养大鼠骨髓单个核细胞,显微镜下观察细胞的形态,采用流式细胞仪检测法鉴定细胞表面抗原的表达情况;采用5-aza诱导MSCs向心肌细胞分化,显微镜下观察诱导前后细胞形态的变化,采用免疫荧光法检测心肌细胞特异性蛋白Connexin 43和TroponinⅠ在诱导后细胞上的表达情况。
2.采用密度梯度离心法和贴壁细胞分离培养法体外分离培养骨髓单个核细胞,采用VEGF_(165)和bFGF诱导MSCs向内皮细胞分化,显微镜下观察诱导前后细胞形态的变化;采用免疫荧光法检测内皮细胞特异性蛋白Flk1和CD31在诱导后细胞上的表达情况;将细胞接种在半固体凝胶上观察细胞形成毛细血管样结构的能力;采用~3H-TdR掺入法实验和细胞划痕实验检测诱导前后细胞增殖和迁移能力的变化。
3.采用RT-PCR方法检测大鼠骨髓MSCs上Notch信号受体和配体mRNA的表达情况;后面的实验共分4组,A组:正常大鼠MSCs;B组:诱导后内皮细胞;C组:导入了VEGF_(165)基因的诱导后内皮细胞;D组:阻断了Notch信号的C组细胞。采用RT-PCR方法检测四组细胞上Notch信号受体和配体mRNA的表达差异;采用~3H-TdR掺入法实验和细胞划痕实验检测细胞增殖和迁移能力的变化;将细胞接种在半固体培养基上检测细胞形成毛细血管样结构的能力。
结果
1.大鼠骨髓MSCs的形态主要表现为长梭形和纺锤形,细胞表达整合素家族成员CD29、粘附分子CD44,但不表达造血前体细胞标志抗原CD34、泛白细胞标志抗原CD45;经5-aza诱导后,细胞形态变化不大,细胞表达心肌细胞特异性蛋白Connexin 43和TroponinⅠ。
2.大鼠MSCs经VEGF_(165)和bFGF诱导后,细胞形态由原来的长梭形、纺锤形变成圆形、椭圆形,细胞具备了内皮细胞的形态特征;细胞表达内皮细胞特异性标志物Flk1和CD31;诱导后细胞能够在半固体培养基上形成毛细血管样结构,且其增殖能力较大鼠MSCs有所降低(P＜0.01),迁移能力有所增强(P＜0.01)。
3.大鼠MSCs上表达有Notch信号受体Notch1和配体Jagged1的mRNA;大鼠MSCs向内皮细胞诱导后,细胞上Notch1和配体Jagged1的mRNA的表达无明显变化,诱导后细胞的增殖能力下降(P＜0.01),迁移能力增强(P＜0.01);给诱导后的内皮细胞导入VEGF_(165)基因后,细胞上Notch信号配体Jagged1的mRNA的表达增强(P＜0.01),细胞的增殖能力、迁移能力和形成毛细血管样结构的能力增强(P＜0.05,P＜0.01,P＜0.05);当阻断了诱导后内皮细胞的Notch信号后,细胞的增殖能力、迁移能力和形成毛细血管样结构的能力更进一步增强(P＜0.05,P＜0.05,P＜0.05)。
结论
1.大鼠MSCs体外向心肌细胞诱导分化是可行的,诱导后的细胞获得了部分心肌细胞的表型。
2.大鼠MSCs体外向内皮细胞诱导分化是可行的,诱导后的细胞具备部分内皮细胞的表型,并获得成熟内皮细胞的功能。
3.大鼠MSCs上表达有Notch信号受体Notch1和配体Jagged1的mRNA。当向诱导后内皮细胞中导入VEGF_(165)基因后,细胞上Jagged1 mRNA的表达水平上调。大鼠MSCs向内皮细胞诱导后,细胞的增殖能力有所下降,迁移能力有所增强。VEGF可以促进诱导后内皮细胞的增殖能力、迁移能力和形成毛细血管样结构的能力,而Notch信号对细胞的增殖能力、迁移能力以及形成毛细血管样结构的能力有抑制作用。
Baekgroud The incidence and the fatality of coronary artery disease are ascending with the development of the living standard of the people, acute myocardial infarction is one of the main causes leading to the death to the people who have the cornary artery disease.After myocardial infarcation,the necrosis and the apoptosis of the cardiomyocytes resulted in the decrease of the cells' number,the necrosis cardiomyocyte substitute by the scar tissue,and ventricular remodeling appeared step by step and at last,cardiac systolic function decreased and even congestive heart failure was happened.Now the thearpy of the cardial infarcion include drugs therapy,interventional therapy and surgical theatment.By decrease the consumption myocardium oxygen,the drugs can release the symptom of the patients to a certain degree.Interventional therapy and surgical treatment can improve the blood supply to the heart and retrieve the survival cells,deferred the ventricular remodeling and improved the prognosis,but they cannot regenerate the death cells,so there are some patients finally developed in the cardiac insufficiency and even resulted in cardiac death.
Cell transplant therapy providing a new method to the cardial infraction and received wide public concern.By increase the cell's number,cell transplant therapy will be a effective therapy to cardial infraction.In addition,angiogenes therapy have been developed into a new method for the ischemic disease at the moment,but the key problem is the resouce of the seed cells.Mesenchymal stem cells(MSCs) are defined as cells that can differentiate into multiple mesenchymal lineage cells such as adipocyte,muscle cells,cartilage cells,nerve cells, endothelial cells,osteoblasts and so on.Because of the multilineage potential of MSCs for differentiation and relatively easy isolation,there has been a continuing interest in therapeutic applications of MSCs from the bone.Makino first sussessfully induced MSCs differentiate into cardiomyocyte by 5-azacytidine(5-aza) in vitro.At the same time,MSCs have basic theory and experiment background to differentiate into endothelial cells.Oswald has discovered that VEGF can induced MSCs differentiate into endothlial cells in vitro.These studies provide some experiment bases for the cell transplant therapy for coronary cardial diease.
The notch pathway is an evlutionary highly conserved signaling machinery with roles in invertebrates as well as in almost erery veterbrate organ and tissue.The notch pathway is a versatile regulator of cell fate specification,growh,differentiation,and patterning processes in metazoan organisms.It has a important role in the development of the cardiovascular,its dificient or overdose will result in death because of the abnormal in cardiovascular.What's more,the notch pathway has a important role in the vascular regeneration.Notch signaling mybe has a significant role in the MSCs differentiation into endothelial cells.In this experiment,in order to provide perfect seed cells for the cell transplant therapy for coronary cardial disease,we studied the feasibility of MSCs differentiation into cardiomyocyte and endothelial cells in vitro,explored the effect of notch signaling on the differentiation of MSCs into endothelial cells.
Aim
1.Isolate and cultivate rat bone MSCs in vitro,study the biological characteristics of MSCs.Induced MSCs to differentiate into cardiomyocytes in vitro.Research the feasility of MSCs differentiation into cardiomyocytes.
2.Induced MSCs to differentiate into endothelial cells by VEGF and bFGF in vitro.Research the feasility of MSCs differentiation into endothelial cells.Detecte the change of the differentiated cells' proliferation ability,migration ability and capillary-like structure' s informing ability.
3.The mRNA expressions of notch signaling receptors and ligands on rat bone MSCs were detected by RT-PCR.Research the change of mRNA expression of Notch receptor and its ligand on the cells treated by VEGF and bFGF.the gene of VEGF was imported into cells which were treated by VEGF and bFGF to promote the proliferation and migration ability of the cells,mRNA expression change of the Notch receptor and its ligand on these cells also detected.The proliferation ability,migration ability and capillary-like structure's informing ability of these cells were mesured.Explore the effect of notch signaling on the differentiation of MSCs into endothelial cells.
Moths
1.Isolate the rat bone mononuclearcells by density gradient centrifugation,through change the culture fluid,non-adherent cells were removed.The form of the cells were observed under the microscope,surface antigen of MSCs were detected by flow cytometry;MSCs were induced to differentiate into cardiomyocyte by 5-aza,the form of the differentiated cells were observed under the microscope;in order to identificate the differentiated cells' nature, cardiomocyte specific markers of connexin 43 and troponin I were detected by immunofluorescence.
2.Isolate the rat bone mononuclearcells by density gradient centrifugation,through change the culture fluid,non-adherent cells were removed.MSCs were induced to differentiate into endothelial cells by VEGF and bFGF,the form of the differentiated cells were observed under the microscope;in order to identificate the differentiated cells' nature,endothlial cells specific markers of Flk1 and CD31 were detected by immunofluorescence;inoculate MSCs and the differentiated cells on the semisolid gel to study the cells' ability of informing the capillary-like structure.The proliferation and migration ability of the differentiated cells were mesured by ~3H-thymidine incorporation and by scarification test.
3.The receptors and the ligands of the Notch signaling were detected by RT-PCR;There are four groups in the following experiment:group A: rat bone MSCs;group B:MSCs which treated by VEGF and bFGF; group C:the cells in group B were imported in the gene of VEGF_(165); group D:the cells in group C which were inhibited the notch signaling.Notch signaling receptor and its ligand on these cells were detected by RT-PCR;the proliferation and migration ability of these cells were mesured by ~3H-thymidine incorporation and by scarification test.Inoculate these cells on the semisolid gel to study theirs ability of informing the capillary-like structure.
Results
1.The shape of rat bone MSCs were mainly appeared as long fusiform and fusiform,it expressed antigens of CD29 and CD44,do not expressed antigens of CD34 and CD45,after treated by 5-aza,the cells form didn't has significant change and they expressed the cardiomyocyte' specific markers of connexin 43 and torponin I.
2.The experiment showed that after induced by VEGF and bFGF,the cells' form mainly appeared as round and ellipse,they have the morphological characteristic of the endothlial cells and they expressed the endothelial cells' specific markers of Flk1 and CD31,at the same time,they also gained the ability of mature endothelial cells to inform the capillary-like structure on the semisolid gel.Compared to rat bone MSCs,the proliferation ability of the differentiated cells was decreased(P＜0.01 ) and theirs migration ability was increased(P＜0.01 ).
3.The results RT-PCR experiment showed that there are Notch 1 and Jaggedl's mRNA expressed on rat bone MSCs;the mRNA expression of the receptor Notch1 and its ligand Jagged 1 have no significant change on the differentiated cells;After treated by VEGF and bFGF, the cells' proliferation ability were decreased(P＜0.01),and its migration ability were increased(P＜0.01),at the same time,they gained the ability to inform the capillary-like structure on semisolid gel.When imported the gene of VEGF_(165) into the cells which have treated by VEGF and bFGF,the mRNA expression of notch signaling ligand Jaggedl was strenghened(P＜0.01) and the cells' proliferation ability,migration ability and informing capillary-like structure' ability were all increased(P＜0.05,P＜0.05,P＜0.05);when notch signaling were inhibited,these abilities of these cells were further increased(P＜0.05,P＜0.05,P＜0.05 ).
Conclusions:
1.The differentiation of rat bone MSCs into cardiomyocytes after treated by 5-aza in vitro is feasible,the defferentiated cells gained part of the phenotypes of the cardiomyocytes.
2.The differentiation of rat bone MSCs into endothelial cells after treated by VEGF and bFGF in vitro is feasible,the defferentiated cells have part of the phenotypes of the endothelial cells and at the same time,the differentiated cells also gained the function of the mature endothelial cells.
3.There are Notch signaling receptor Notch1 and its ligand Jaggedl's mRNA expressed on rat bone MSCs.After imported the gene of VEGF into the differentiated cells,the mRNA expression level of the Jagged 1 was upgrade.When rat bone MSCs were induced to differentiate into endothelial cells,its proliferation ability was decreased and its migration ability was increased,and they also gained the ability to inform the capillary-like structure on semisolid gel.VEGF can promote the differentiated cells' proliferation ablility, migration ablility and informing capillary-like structure' ability but notch signaling has the reversed effect.
近年来发展的细胞移植技术为心肌梗死的治疗提供了新的方向并受到了人们的广泛关注。通过细胞心肌移植术增加有功能的心肌细胞数目,可能是心肌梗死后心衰治疗的最有效方法之一。此外近年来提出的治疗性血管新生也已发展成为治疗缺血性疾病的新方法。骨髓间充质干细胞(mesenchymal stem cells,MSCs)是骨髓中不同于造血干细胞的一类细胞,它在体内能够自我更新,保持未分化状态,在体外合适的培养条件下,易于扩增,具有向多种细胞类型分化的潜能。1999年,Makino等首次在体外成功地诱导MSCs定向分化为心肌细胞。同时MSCs具有分化为内皮细胞的理论基础和实验基础。2004年,Oswald等人发现在体外MSCs与VEGF_(165)和2%胎牛血清共培养可诱导分化为内皮样细胞。这些实验为MSCs诱导分化为心肌细胞和内皮细胞进行细胞移植治疗缺血性心脏病提供了良好的实验基础。
Notch信号通路在脊椎动物和无脊椎动物中高度保守,它在决定细胞分化上起重要作用。它对正常心血管系统的发育有重要作用,Notch信号表达不足或过量都会造成动物因心血管异常而死亡。此外,Notch信号系统在血管再生方面也有重要作用。Notch信号通路在MSCs向内皮细胞分化的过程中可能也起着重要的调节作用。本实验主要研究了大鼠MSCs体外向心肌细胞及内皮细胞诱导分化的可行性,并研究了Notch信号通路对大鼠MSCs向内皮细胞诱导分化产生的影响,以期为缺血性心脏病的细胞移植治疗提供良好的种子细胞。
研究目的
1.体外分离培养大鼠骨髓MSCs,研究大鼠骨髓MSCs的生物学特性。用5-氮胞苷(5-Azacytidine,5-aza)定向诱导MSCs向心肌细胞分化,研究MSCs体外向心肌细胞分化的可行性。
2.定向诱导大鼠骨髓MSCs向内皮细胞分化,研究MSCs体外向内皮细胞分化的可行性。研究诱导前后细胞增殖能力、迁移能力和形成毛细血管样结构能力的变化。
3.采用RT-PCR方法检测大鼠骨髓MSCs上Notch信号受体和配体mRNA的表达情况,研究MSCs向内皮细胞诱导分化前后细胞上Notch信号受体和配体mRNA的表达变化。向诱导后内皮细胞中导入VEGF_(165)基因来促进细胞的增殖和迁移能力。检测转染VEGF_(165)基因前后细胞上Notch信号受体和配体mRNA的表达变化,检测细胞增殖能力、迁移能力和形成毛细血管样结构能力的变化,研究Notch信号对诱导后内皮细胞的增殖能力、迁移能力和形成毛细血管样结构能力的影响,以期更好的诱导MSCs向内皮细胞分化,促进诱导后内皮细胞的增殖、迁移和形成毛细血管样结构的能力,为缺血性心脏病的细胞移植治疗提供良好的种子细胞。
方法
1.采用密度梯度离心法和贴壁细胞分离培养法体外分离培养大鼠骨髓单个核细胞,显微镜下观察细胞的形态,采用流式细胞仪检测法鉴定细胞表面抗原的表达情况;采用5-aza诱导MSCs向心肌细胞分化,显微镜下观察诱导前后细胞形态的变化,采用免疫荧光法检测心肌细胞特异性蛋白Connexin 43和TroponinⅠ在诱导后细胞上的表达情况。
2.采用密度梯度离心法和贴壁细胞分离培养法体外分离培养骨髓单个核细胞,采用VEGF_(165)和bFGF诱导MSCs向内皮细胞分化,显微镜下观察诱导前后细胞形态的变化;采用免疫荧光法检测内皮细胞特异性蛋白Flk1和CD31在诱导后细胞上的表达情况;将细胞接种在半固体凝胶上观察细胞形成毛细血管样结构的能力;采用~3H-TdR掺入法实验和细胞划痕实验检测诱导前后细胞增殖和迁移能力的变化。
3.采用RT-PCR方法检测大鼠骨髓MSCs上Notch信号受体和配体mRNA的表达情况;后面的实验共分4组,A组:正常大鼠MSCs;B组:诱导后内皮细胞;C组:导入了VEGF_(165)基因的诱导后内皮细胞;D组:阻断了Notch信号的C组细胞。采用RT-PCR方法检测四组细胞上Notch信号受体和配体mRNA的表达差异;采用~3H-TdR掺入法实验和细胞划痕实验检测细胞增殖和迁移能力的变化;将细胞接种在半固体培养基上检测细胞形成毛细血管样结构的能力。
结果
1.大鼠骨髓MSCs的形态主要表现为长梭形和纺锤形,细胞表达整合素家族成员CD29、粘附分子CD44,但不表达造血前体细胞标志抗原CD34、泛白细胞标志抗原CD45;经5-aza诱导后,细胞形态变化不大,细胞表达心肌细胞特异性蛋白Connexin 43和TroponinⅠ。
2.大鼠MSCs经VEGF_(165)和bFGF诱导后,细胞形态由原来的长梭形、纺锤形变成圆形、椭圆形,细胞具备了内皮细胞的形态特征;细胞表达内皮细胞特异性标志物Flk1和CD31;诱导后细胞能够在半固体培养基上形成毛细血管样结构,且其增殖能力较大鼠MSCs有所降低(P＜0.01),迁移能力有所增强(P＜0.01)。
3.大鼠MSCs上表达有Notch信号受体Notch1和配体Jagged1的mRNA;大鼠MSCs向内皮细胞诱导后,细胞上Notch1和配体Jagged1的mRNA的表达无明显变化,诱导后细胞的增殖能力下降(P＜0.01),迁移能力增强(P＜0.01);给诱导后的内皮细胞导入VEGF_(165)基因后,细胞上Notch信号配体Jagged1的mRNA的表达增强(P＜0.01),细胞的增殖能力、迁移能力和形成毛细血管样结构的能力增强(P＜0.05,P＜0.01,P＜0.05);当阻断了诱导后内皮细胞的Notch信号后,细胞的增殖能力、迁移能力和形成毛细血管样结构的能力更进一步增强(P＜0.05,P＜0.05,P＜0.05)。
结论
1.大鼠MSCs体外向心肌细胞诱导分化是可行的,诱导后的细胞获得了部分心肌细胞的表型。
2.大鼠MSCs体外向内皮细胞诱导分化是可行的,诱导后的细胞具备部分内皮细胞的表型,并获得成熟内皮细胞的功能。
3.大鼠MSCs上表达有Notch信号受体Notch1和配体Jagged1的mRNA。当向诱导后内皮细胞中导入VEGF_(165)基因后,细胞上Jagged1 mRNA的表达水平上调。大鼠MSCs向内皮细胞诱导后,细胞的增殖能力有所下降,迁移能力有所增强。VEGF可以促进诱导后内皮细胞的增殖能力、迁移能力和形成毛细血管样结构的能力,而Notch信号对细胞的增殖能力、迁移能力以及形成毛细血管样结构的能力有抑制作用。
Baekgroud The incidence and the fatality of coronary artery disease are ascending with the development of the living standard of the people, acute myocardial infarction is one of the main causes leading to the death to the people who have the cornary artery disease.After myocardial infarcation,the necrosis and the apoptosis of the cardiomyocytes resulted in the decrease of the cells' number,the necrosis cardiomyocyte substitute by the scar tissue,and ventricular remodeling appeared step by step and at last,cardiac systolic function decreased and even congestive heart failure was happened.Now the thearpy of the cardial infarcion include drugs therapy,interventional therapy and surgical theatment.By decrease the consumption myocardium oxygen,the drugs can release the symptom of the patients to a certain degree.Interventional therapy and surgical treatment can improve the blood supply to the heart and retrieve the survival cells,deferred the ventricular remodeling and improved the prognosis,but they cannot regenerate the death cells,so there are some patients finally developed in the cardiac insufficiency and even resulted in cardiac death.
Cell transplant therapy providing a new method to the cardial infraction and received wide public concern.By increase the cell's number,cell transplant therapy will be a effective therapy to cardial infraction.In addition,angiogenes therapy have been developed into a new method for the ischemic disease at the moment,but the key problem is the resouce of the seed cells.Mesenchymal stem cells(MSCs) are defined as cells that can differentiate into multiple mesenchymal lineage cells such as adipocyte,muscle cells,cartilage cells,nerve cells, endothelial cells,osteoblasts and so on.Because of the multilineage potential of MSCs for differentiation and relatively easy isolation,there has been a continuing interest in therapeutic applications of MSCs from the bone.Makino first sussessfully induced MSCs differentiate into cardiomyocyte by 5-azacytidine(5-aza) in vitro.At the same time,MSCs have basic theory and experiment background to differentiate into endothelial cells.Oswald has discovered that VEGF can induced MSCs differentiate into endothlial cells in vitro.These studies provide some experiment bases for the cell transplant therapy for coronary cardial diease.
The notch pathway is an evlutionary highly conserved signaling machinery with roles in invertebrates as well as in almost erery veterbrate organ and tissue.The notch pathway is a versatile regulator of cell fate specification,growh,differentiation,and patterning processes in metazoan organisms.It has a important role in the development of the cardiovascular,its dificient or overdose will result in death because of the abnormal in cardiovascular.What's more,the notch pathway has a important role in the vascular regeneration.Notch signaling mybe has a significant role in the MSCs differentiation into endothelial cells.In this experiment,in order to provide perfect seed cells for the cell transplant therapy for coronary cardial disease,we studied the feasibility of MSCs differentiation into cardiomyocyte and endothelial cells in vitro,explored the effect of notch signaling on the differentiation of MSCs into endothelial cells.
Aim
1.Isolate and cultivate rat bone MSCs in vitro,study the biological characteristics of MSCs.Induced MSCs to differentiate into cardiomyocytes in vitro.Research the feasility of MSCs differentiation into cardiomyocytes.
2.Induced MSCs to differentiate into endothelial cells by VEGF and bFGF in vitro.Research the feasility of MSCs differentiation into endothelial cells.Detecte the change of the differentiated cells' proliferation ability,migration ability and capillary-like structure' s informing ability.
3.The mRNA expressions of notch signaling receptors and ligands on rat bone MSCs were detected by RT-PCR.Research the change of mRNA expression of Notch receptor and its ligand on the cells treated by VEGF and bFGF.the gene of VEGF was imported into cells which were treated by VEGF and bFGF to promote the proliferation and migration ability of the cells,mRNA expression change of the Notch receptor and its ligand on these cells also detected.The proliferation ability,migration ability and capillary-like structure's informing ability of these cells were mesured.Explore the effect of notch signaling on the differentiation of MSCs into endothelial cells.
Moths
1.Isolate the rat bone mononuclearcells by density gradient centrifugation,through change the culture fluid,non-adherent cells were removed.The form of the cells were observed under the microscope,surface antigen of MSCs were detected by flow cytometry;MSCs were induced to differentiate into cardiomyocyte by 5-aza,the form of the differentiated cells were observed under the microscope;in order to identificate the differentiated cells' nature, cardiomocyte specific markers of connexin 43 and troponin I were detected by immunofluorescence.
2.Isolate the rat bone mononuclearcells by density gradient centrifugation,through change the culture fluid,non-adherent cells were removed.MSCs were induced to differentiate into endothelial cells by VEGF and bFGF,the form of the differentiated cells were observed under the microscope;in order to identificate the differentiated cells' nature,endothlial cells specific markers of Flk1 and CD31 were detected by immunofluorescence;inoculate MSCs and the differentiated cells on the semisolid gel to study the cells' ability of informing the capillary-like structure.The proliferation and migration ability of the differentiated cells were mesured by ~3H-thymidine incorporation and by scarification test.
3.The receptors and the ligands of the Notch signaling were detected by RT-PCR;There are four groups in the following experiment:group A: rat bone MSCs;group B:MSCs which treated by VEGF and bFGF; group C:the cells in group B were imported in the gene of VEGF_(165); group D:the cells in group C which were inhibited the notch signaling.Notch signaling receptor and its ligand on these cells were detected by RT-PCR;the proliferation and migration ability of these cells were mesured by ~3H-thymidine incorporation and by scarification test.Inoculate these cells on the semisolid gel to study theirs ability of informing the capillary-like structure.
Results
1.The shape of rat bone MSCs were mainly appeared as long fusiform and fusiform,it expressed antigens of CD29 and CD44,do not expressed antigens of CD34 and CD45,after treated by 5-aza,the cells form didn't has significant change and they expressed the cardiomyocyte' specific markers of connexin 43 and torponin I.
2.The experiment showed that after induced by VEGF and bFGF,the cells' form mainly appeared as round and ellipse,they have the morphological characteristic of the endothlial cells and they expressed the endothelial cells' specific markers of Flk1 and CD31,at the same time,they also gained the ability of mature endothelial cells to inform the capillary-like structure on the semisolid gel.Compared to rat bone MSCs,the proliferation ability of the differentiated cells was decreased(P＜0.01 ) and theirs migration ability was increased(P＜0.01 ).
3.The results RT-PCR experiment showed that there are Notch 1 and Jaggedl's mRNA expressed on rat bone MSCs;the mRNA expression of the receptor Notch1 and its ligand Jagged 1 have no significant change on the differentiated cells;After treated by VEGF and bFGF, the cells' proliferation ability were decreased(P＜0.01),and its migration ability were increased(P＜0.01),at the same time,they gained the ability to inform the capillary-like structure on semisolid gel.When imported the gene of VEGF_(165) into the cells which have treated by VEGF and bFGF,the mRNA expression of notch signaling ligand Jaggedl was strenghened(P＜0.01) and the cells' proliferation ability,migration ability and informing capillary-like structure' ability were all increased(P＜0.05,P＜0.05,P＜0.05);when notch signaling were inhibited,these abilities of these cells were further increased(P＜0.05,P＜0.05,P＜0.05 ).
Conclusions:
1.The differentiation of rat bone MSCs into cardiomyocytes after treated by 5-aza in vitro is feasible,the defferentiated cells gained part of the phenotypes of the cardiomyocytes.
2.The differentiation of rat bone MSCs into endothelial cells after treated by VEGF and bFGF in vitro is feasible,the defferentiated cells have part of the phenotypes of the endothelial cells and at the same time,the differentiated cells also gained the function of the mature endothelial cells.
3.There are Notch signaling receptor Notch1 and its ligand Jaggedl's mRNA expressed on rat bone MSCs.After imported the gene of VEGF into the differentiated cells,the mRNA expression level of the Jagged 1 was upgrade.When rat bone MSCs were induced to differentiate into endothelial cells,its proliferation ability was decreased and its migration ability was increased,and they also gained the ability to inform the capillary-like structure on semisolid gel.VEGF can promote the differentiated cells' proliferation ablility, migration ablility and informing capillary-like structure' ability but notch signaling has the reversed effect.
引文
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