淫羊藿苷诱导骨髓间充质干细胞分化心肌样细胞的实验研究
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摘要
心肌梗塞后，存活的心肌细胞数量减少，纤维疤痕组织增生，心功能下降，最终发展为缺血性心力衰竭甚至死亡。目前临床所用的治疗手段不能逆转已坏死的心肌，心脏移植由于费用和排斥反应等问题不能得到推广应用。因此，寻找一种新的、更好的治疗手段已成为当务之急。大量的研究人员把研究重点放到了干细胞的移植治疗上，而干细胞分为胚胎干细胞和成体干细胞两大类。其中胚胎干细胞由于取材困难等因素的限制，成体干细胞逐渐成为人们研究的重点。骨髓间充质干细胞(MSCs)是成体干细胞的一种，来源于中胚层的间充质。在一定的环境和刺激因子的作用下可分化为多种组织细胞，在组织工程中，MSCs作为一种重要的种子细胞越来越引起人们的关注，将MSCs诱导分化为心肌细胞移植治疗心肌梗塞，为中药与干细胞联合治疗心血管疾病提供新的理论依据。
MSCs属于非造血细胞，其特点是贴壁生长，呈纺锤型的纤维细胞样，国内外研究认为MSCs具有独特的表征和巨大的培养增殖能力，并认为其是多向分化潜能的干细胞，在一定条件下可以分化为骨骼、软骨、肌肉、神经和造血微环境组织等，具有很高的可塑性。国内外学者在体外培养中将MSCs经传代后用5-氮胞苷(5-aza)对其进行诱导分化，可见有心肌细胞样超微结构，有心肌特异性基因表达，并有持续的动作电位，表明他们成功地诱导出心肌细胞。另有研究人员将MSCs直接注射入自体心肌组织内，发现有心肌样细胞形成，具有肌钙蛋白T和肌球蛋白重链染色阳性的心肌细胞特异性标志，表明MSCs在体内微环境条件下可以诱导分化为心肌细胞。MSCs作为一种重要的种子细胞越来越引起人们的关注，研究者把研究重点放在如何对其进行诱导分化为心肌细胞，以期为移植治疗心梗提供新的理论依据。
用于MSCs体外诱导分化为心肌细胞的诱导剂主要是化学物质5-Aza，但5-Aza有一定的毒性，关于中药诱导的报道较少，这可能与干细胞诱导分化研究涉及的技术问题较多，中药介入的条件还不太成熟有关。寻求一种高效安全的诱导剂是干细胞诱导分化研究的关键。
淫羊藿是补肾壮阳类中药，常用于治疗阳虚证，淫羊藿苷(ICA)是其主要活性成分，ICA药理活性主要表现在改善心血管系统功能、增强机体免疫力、促进DNA合成及诱导肿瘤细胞分化等方面。在改善心脑血管系统功能方面，其主要功效为补肾强心、宁心安神、养血除烦。对改善冠心病患者心绞痛等症状及缺血型心电图有较好疗效，并有镇静和降血脂作用。在对该成分体外定向诱导ES细胞分化成心肌细胞的研究中发现：ICA与ES细胞的定向分化呈一定的量效、时效关系。我们考虑ICA是否也与MSCs的定向分化呈一定的量效、时效关系。该诱导分化是否也能促进MSCs在体内对梗死心肌的修复作用。
本课题研究希望通过检测ICA诱导MSCs后的α-MHC、β-MHC等基因的表达，以及Desmin、cTnT、VEGF和BFGF的表达，来探讨ICA是否能诱导MSCs分化为心肌细胞，其最佳的诱导浓度和诱导时间是多少；在体内实验研究中，诱导后的细胞是否能有效改善心梗大鼠的受损心肌，为研究心梗的治疗提供新的研究思路。
目的探讨淫羊藿苷体外诱导MSCs分化为心肌样细胞的最佳时效和量效关系，并以此为基础，进一步探讨诱导后的MSCs移植治疗心梗大鼠的心肌修复。
方法本课题分为四个实验进行
实验一、按照不同的药物浓度将ICA分为10~(-6)M、10~(-7)M、10~(-8)M、10~(-9)M、10~(-10)M五个浓度梯度，利用MTT法检测不同浓度组对MSCs的增殖影响。同时通过形态学和免疫组化对分离所得的细胞进行表型分子鉴定。
实验二、利用RT-PCR对ICA各浓度组(10~(-6)M、10~(-7)M、10~(-8)M、10~(-9)M)诱导MSCs后，心肌细胞特有的α-MHC、β-MHC和GATA-4的表达情况，探讨ICA诱导MSCs表达心肌细胞特有的α-MHC、β-MHC和GATA-4的时效、量效关系。同时以5-Aza为阳性对照。
实验三、在实验二的基础上进一步探讨诱导后的MSCs分泌VEGF和BFGF的表达情况，同样以5-Aza为阳性对照。
实验四、在实验二和实验三的基础上，把ICA体外诱导后的MSCs经尾静脉注射入大鼠体内2W和4W，通过免疫组化检测心梗部位心肌Desmin、cTnT、VEGF和BFGF的表达情况，同时通过HE染色和Masson染色观察该治疗方法对受损心肌的修复情况。
结果
实验一、采用无菌分离大鼠胫、股骨骨髓腔细胞，并用全贴壁培养法所获得的细胞经传代培养，去掉其中的造血细胞，得到的是骨髓间充质干细胞。与空白对照组比较，10~(-7)M、10~(-8)M的ICA与MSCs共培养48h后，能明显促进MSCs的增殖，P<0．01。
实验二、ICA体外促进MSCs分泌α-MHC、β-MHC的最佳浓度是10~(-7)M，最佳的诱导时间是共培养24h后，换成完全培养基培养1W。
实验三、再次确认ICA体外促进MSCs分泌VEGF和BFGF的最佳浓度是10~(-7)M，最佳的诱导时间是共培养24h后，换成完全培养基培养1W。
实验四、把经ICA最佳浓度和时间诱导后的MSCs移植入心梗大鼠体内后，与移植治疗2W组比较，4W组能明显促进Desmin、VEGF和BFGF的表达，梗塞部位心肌纤维化程度减轻，有一定数量的新生血管形成，对梗塞心肌的修复作用明显。
结论
由实验一至实验三可知：①10~(-7)M和10~(-8)M的ICA在体外能促进MSCs增殖；②10~(-7)M和10~(-8)M的ICA能有效促进MSCs心肌发育依赖的α-MHC、β-MHCmRNA的表达，而诱导24h后再换液培养1周是其最佳的诱导时间。③10~(-6)M和10~(-7)M的ICA能显著促进细胞分泌VEGF、bFGF表达。综合以上实验结果，我们认为：ICA体外诱导MSCs分化为心肌样细胞的最佳浓度是10~(-7)M，最佳诱导时间是诱导24h后再换液培养1周。
在体外实验结果的基础上对MSCs进行诱导，再把诱导后的细胞通过尾静脉注射输入心梗大鼠体内，实验发现：把经10~(-7)M的ICA诱导24h再换液培养1周的MSCs经尾静脉移植，能有效地修复受损心肌，促进该部位Desmin、cTnT、VEGF和BFGF的表达。
已有大量的实验研究针对骨髓间充质干细胞(MSCs)移植治疗心梗，较多的研究人员提出：将MSCs通过一定的途径移植到心梗大鼠体内能有效地修复受损心肌。而在本实验中，我们却发现：MSCs没有预先经过一定的处理而直接进行移植治疗，Desmin、cTnT、VEGF和BFGF的表达相对较弱，仅比不做任何治疗的模型组稍好；而经过与ICA共培养后再进行移植，其修复效果理想，Desmin、cTnT、VEGF和BFGF的表达强，效果满意。
综上所述，ICA能促进体外分离的MSCs增殖；还能促进MSCs的α-MHC、β-MHCmRNA的表达；对VEGF和BFGF的分泌同样有促进作用；通过尾静脉注射移植诱导后的MSCs细胞，能修复受损心肌，促进心肌修复所需的Desmin、cTnT、VEGF和BFGF的表达。因此，由本课题研究我们认为：10~(-7)M的ICA与MSCs共培养24h后，在更换为完全培养基培养1周，能诱导MSCs分化为心肌样细胞，经其诱导后的细胞能有效改善心梗后的受损心肌。
The decrease of cardiac muscle cells、hyperplasia of scar tissue andventricle reconstructions after myocardial infraction.It will be evolved intoheart failure,arrhythmia and even death.Nowadays,in clinic,those treatmentscould not replace the nectrotic cardiac muscle cells because of the impossibleregeneration of cardiomyocytes.Heart transplantation could not be spreadbeacause of its rejection and expensive expenditure.So it is important to finda effective treatment.
The focus of the study has been put on the treatment of transplantationof mesenchyma stem cells.The mesenchyma stem cells is divided into twocategories:embryonic stem cells and adult stem cells.The focus of the studyhas been put on adult stem cells beacause of the rich sources and the easycultivation.Bone marrow mesenchyma stem cells(MSCs)is a kind of the AdultStem Cells.It develop from the mesenchymal layer of mesoderm.In certaincondition and actions of stimulation factors,it can be differentiated intomulti-histiocytes.As a important seed cell,people pay more and more attentionto MSCs in tissue engineering.To use the cardiac cells induced anddifferentiated by MSCs to cure myocardial infraction give some new theoreticbasis for the treatment of cardiovascular diseases by the combination of bothTCM and stem cells.
MSCs belongs to nonhematopoietic cell and its basic character is adherentgrowth and looks like spindle fibrocyte in shape.It is considered that MSCsis a kind of stem cell which has potency of pluripotential progenitor and canbe differentiated into bones,cartilage,muscle,fat,tendon and nerve,etc.It has high plasticity.Scholars both here and abroad used 5-aza into induceMSCs after passage in culture in vitro.And the cardiac cell like ultrastructure,specific gene expression of cardiac cell and persistent actionpotential showed that they induced cardiac cells successfully.Some scholarsmedication experience of ten years,which has the function of promoting blood circulation directly injected MSCs into self cardiac muscle cells tissue and cardiac musclelike cytomorphosis was found.It had specific symbols,for example,thechromatin reconstitution of cardiac troponin I and myosin heavy chain,ofcardiac muscle cells.It showed that MSCs could be induced and differentiatedinto cardiac muscle cells in microenvironment in vitro.
Icariin (ICA)is one of the constituents of epimedium,a traditionalChinese herbal medicine.It possesses many kinds of biological actions,particularly in cardiovascular function improvement,hormone regulation,immunological function modulation,anti-tumor and anti-virus activity.It hasbeen used for the treatment of heart disease.
It could be fund that ES cells were remarkably induced into therhythmically beating EBs with ICA in a concentration-and time-dependent manner.So it will be considered that if the concentration-and time-dependent mannercan be fund with MSCs induced by ICA.
This study was designed by the detection of gene and protein of cardiacmuscle such asα-MHC、β-MHC、Desmin、cTnT、VEGF and bFGF etc.This study can be divided into four parts.We hope that we can explore the relationshipbetween ICA and MSCs.Objective
Explore the best concentration-and time-dependent manner of MSCs beeninduced by ICA,and explore the improvement of myocardial infraction by thetransplantation in the treatment of MSCs induced by ICA.Method This study can be divided into four parts.
Firstly:ICA was divided into 5 concentration gradient(10~(-6)M、10~(-7)M、10~(-8)M、10~(-9)M,10~(-10)M).It can be studied the effects of MSCs induced by ICA withmethod of MTT.At the same time,MSCs can be identified withimmunohistochemistry method and Morphology.
Secondly:It can be explore the concentration-and time-dependent mannerof the specific gene ofα-MHC、β-MHC and GATA-4 in myocardial cells whichdetected by reverse transcription-poly-merase chain reaction(RT-PCR)analysis.
Thirdly:On the basis of the second part,It can be explore the expressionof VEGF and bFGF with Western Blot method compared with 5-aza further.
Fourthly:On the basis of above experiments,the MSCs induced by ICA for2 weeks and 4 weeks was injected into MI rats through intravenous of tail. The specific Protein Desmin、cTnT、VEGF and BFGF can be detectived withimmunohistochemistry method.And we can observed the improvement of myocardialinfraction by the transplantation in the treatment of MSCs induced by ICA withstaining of HE and Masson.Results
Firstly:MSCs were isolated from adult rat's gradient centrifugationand anchoring culture,the MSCs were purified by culture-expanded.Comparedwith control group,10~(-7)M and 10~(-8)M of ICA could promoted MSCs Proliferationobviously,P<0.01.
Secondly:It showed that RT-PCR analysis of the expression ofcardiomyocyte-specific genes in differentiated rat MSCs cells.The expressionof the 10~(-7)M of ICA group forα-MHC andβ-MHC were more strongly than othergroups,and that 10~(-7)M of ICA could promoted MSCs proliferation obvirsly for24h and a 7-days culture in primary culture.
Thirdly:It showed that Western Blot analysis of the expression of VEGFand bFGF in differentiated rat MSCs cells.the 5-aza group were used as apositive control.The expression of the 10~(-7)M of ICA group for specificprotein---VEGF and bFGF were more strongly than other groups,that 10~(-7)M of ICA could promoted MSCs proliferation obviously for 24h and a 7-days culturein primary culture.
Fourthly:It was obviously that the expression of Desmin、cTnT、VEGF andbFGF in Myocardial infraction of 4-weeks of ICA group was strongly comparedwith 2-weeks of ICA group、model group and simpel MSCs group.
Conclusion
It can be inferred that 10~(-7)M of ICA could promoted MSCs differentiatedinto cardiomyocyto-like cells for 24h and a 7-days culture in primary culture.It can be concluded that the expression of the 10~(-7)M of ICA of groupcardiomyocyte-specific genes and protein forα-MHC、β-MHC、Desmin、cTnT、VEGF and bFGF were more strongly than other groups than other groups.Thereare a lot of experimental research for bone marrow-derived mesenchymal stemcells (MSCs)transplantation in the treatment of myocardial infraction,moreresearchers think that MSCs through certain channels to myocardial infractionin rats in vivo transplantation can be effective in repairing damagedmyocardium.But,it was found that MSCs which had not been treated could notexpress Desmin、cTnT、VEGF and bFGF obviously.The most ideally treatment is that 10~(-7)M of ICA could promoted MSCs differentiated into cardiomyocyto-likecells for 24h and a 7-days culture in primary culture.The method can inducedMSCs differentiated into cardiomyocyto-like cells.
MSCs属于非造血细胞，其特点是贴壁生长，呈纺锤型的纤维细胞样，国内外研究认为MSCs具有独特的表征和巨大的培养增殖能力，并认为其是多向分化潜能的干细胞，在一定条件下可以分化为骨骼、软骨、肌肉、神经和造血微环境组织等，具有很高的可塑性。国内外学者在体外培养中将MSCs经传代后用5-氮胞苷(5-aza)对其进行诱导分化，可见有心肌细胞样超微结构，有心肌特异性基因表达，并有持续的动作电位，表明他们成功地诱导出心肌细胞。另有研究人员将MSCs直接注射入自体心肌组织内，发现有心肌样细胞形成，具有肌钙蛋白T和肌球蛋白重链染色阳性的心肌细胞特异性标志，表明MSCs在体内微环境条件下可以诱导分化为心肌细胞。MSCs作为一种重要的种子细胞越来越引起人们的关注，研究者把研究重点放在如何对其进行诱导分化为心肌细胞，以期为移植治疗心梗提供新的理论依据。
用于MSCs体外诱导分化为心肌细胞的诱导剂主要是化学物质5-Aza，但5-Aza有一定的毒性，关于中药诱导的报道较少，这可能与干细胞诱导分化研究涉及的技术问题较多，中药介入的条件还不太成熟有关。寻求一种高效安全的诱导剂是干细胞诱导分化研究的关键。
淫羊藿是补肾壮阳类中药，常用于治疗阳虚证，淫羊藿苷(ICA)是其主要活性成分，ICA药理活性主要表现在改善心血管系统功能、增强机体免疫力、促进DNA合成及诱导肿瘤细胞分化等方面。在改善心脑血管系统功能方面，其主要功效为补肾强心、宁心安神、养血除烦。对改善冠心病患者心绞痛等症状及缺血型心电图有较好疗效，并有镇静和降血脂作用。在对该成分体外定向诱导ES细胞分化成心肌细胞的研究中发现：ICA与ES细胞的定向分化呈一定的量效、时效关系。我们考虑ICA是否也与MSCs的定向分化呈一定的量效、时效关系。该诱导分化是否也能促进MSCs在体内对梗死心肌的修复作用。
本课题研究希望通过检测ICA诱导MSCs后的α-MHC、β-MHC等基因的表达，以及Desmin、cTnT、VEGF和BFGF的表达，来探讨ICA是否能诱导MSCs分化为心肌细胞，其最佳的诱导浓度和诱导时间是多少；在体内实验研究中，诱导后的细胞是否能有效改善心梗大鼠的受损心肌，为研究心梗的治疗提供新的研究思路。
目的探讨淫羊藿苷体外诱导MSCs分化为心肌样细胞的最佳时效和量效关系，并以此为基础，进一步探讨诱导后的MSCs移植治疗心梗大鼠的心肌修复。
方法本课题分为四个实验进行
实验一、按照不同的药物浓度将ICA分为10~(-6)M、10~(-7)M、10~(-8)M、10~(-9)M、10~(-10)M五个浓度梯度，利用MTT法检测不同浓度组对MSCs的增殖影响。同时通过形态学和免疫组化对分离所得的细胞进行表型分子鉴定。
实验二、利用RT-PCR对ICA各浓度组(10~(-6)M、10~(-7)M、10~(-8)M、10~(-9)M)诱导MSCs后，心肌细胞特有的α-MHC、β-MHC和GATA-4的表达情况，探讨ICA诱导MSCs表达心肌细胞特有的α-MHC、β-MHC和GATA-4的时效、量效关系。同时以5-Aza为阳性对照。
实验三、在实验二的基础上进一步探讨诱导后的MSCs分泌VEGF和BFGF的表达情况，同样以5-Aza为阳性对照。
实验四、在实验二和实验三的基础上，把ICA体外诱导后的MSCs经尾静脉注射入大鼠体内2W和4W，通过免疫组化检测心梗部位心肌Desmin、cTnT、VEGF和BFGF的表达情况，同时通过HE染色和Masson染色观察该治疗方法对受损心肌的修复情况。
结果
实验一、采用无菌分离大鼠胫、股骨骨髓腔细胞，并用全贴壁培养法所获得的细胞经传代培养，去掉其中的造血细胞，得到的是骨髓间充质干细胞。与空白对照组比较，10~(-7)M、10~(-8)M的ICA与MSCs共培养48h后，能明显促进MSCs的增殖，P<0．01。
实验二、ICA体外促进MSCs分泌α-MHC、β-MHC的最佳浓度是10~(-7)M，最佳的诱导时间是共培养24h后，换成完全培养基培养1W。
实验三、再次确认ICA体外促进MSCs分泌VEGF和BFGF的最佳浓度是10~(-7)M，最佳的诱导时间是共培养24h后，换成完全培养基培养1W。
实验四、把经ICA最佳浓度和时间诱导后的MSCs移植入心梗大鼠体内后，与移植治疗2W组比较，4W组能明显促进Desmin、VEGF和BFGF的表达，梗塞部位心肌纤维化程度减轻，有一定数量的新生血管形成，对梗塞心肌的修复作用明显。
结论
由实验一至实验三可知：①10~(-7)M和10~(-8)M的ICA在体外能促进MSCs增殖；②10~(-7)M和10~(-8)M的ICA能有效促进MSCs心肌发育依赖的α-MHC、β-MHCmRNA的表达，而诱导24h后再换液培养1周是其最佳的诱导时间。③10~(-6)M和10~(-7)M的ICA能显著促进细胞分泌VEGF、bFGF表达。综合以上实验结果，我们认为：ICA体外诱导MSCs分化为心肌样细胞的最佳浓度是10~(-7)M，最佳诱导时间是诱导24h后再换液培养1周。
在体外实验结果的基础上对MSCs进行诱导，再把诱导后的细胞通过尾静脉注射输入心梗大鼠体内，实验发现：把经10~(-7)M的ICA诱导24h再换液培养1周的MSCs经尾静脉移植，能有效地修复受损心肌，促进该部位Desmin、cTnT、VEGF和BFGF的表达。
已有大量的实验研究针对骨髓间充质干细胞(MSCs)移植治疗心梗，较多的研究人员提出：将MSCs通过一定的途径移植到心梗大鼠体内能有效地修复受损心肌。而在本实验中，我们却发现：MSCs没有预先经过一定的处理而直接进行移植治疗，Desmin、cTnT、VEGF和BFGF的表达相对较弱，仅比不做任何治疗的模型组稍好；而经过与ICA共培养后再进行移植，其修复效果理想，Desmin、cTnT、VEGF和BFGF的表达强，效果满意。
综上所述，ICA能促进体外分离的MSCs增殖；还能促进MSCs的α-MHC、β-MHCmRNA的表达；对VEGF和BFGF的分泌同样有促进作用；通过尾静脉注射移植诱导后的MSCs细胞，能修复受损心肌，促进心肌修复所需的Desmin、cTnT、VEGF和BFGF的表达。因此，由本课题研究我们认为：10~(-7)M的ICA与MSCs共培养24h后，在更换为完全培养基培养1周，能诱导MSCs分化为心肌样细胞，经其诱导后的细胞能有效改善心梗后的受损心肌。
The decrease of cardiac muscle cells、hyperplasia of scar tissue andventricle reconstructions after myocardial infraction.It will be evolved intoheart failure,arrhythmia and even death.Nowadays,in clinic,those treatmentscould not replace the nectrotic cardiac muscle cells because of the impossibleregeneration of cardiomyocytes.Heart transplantation could not be spreadbeacause of its rejection and expensive expenditure.So it is important to finda effective treatment.
The focus of the study has been put on the treatment of transplantationof mesenchyma stem cells.The mesenchyma stem cells is divided into twocategories:embryonic stem cells and adult stem cells.The focus of the studyhas been put on adult stem cells beacause of the rich sources and the easycultivation.Bone marrow mesenchyma stem cells(MSCs)is a kind of the AdultStem Cells.It develop from the mesenchymal layer of mesoderm.In certaincondition and actions of stimulation factors,it can be differentiated intomulti-histiocytes.As a important seed cell,people pay more and more attentionto MSCs in tissue engineering.To use the cardiac cells induced anddifferentiated by MSCs to cure myocardial infraction give some new theoreticbasis for the treatment of cardiovascular diseases by the combination of bothTCM and stem cells.
MSCs belongs to nonhematopoietic cell and its basic character is adherentgrowth and looks like spindle fibrocyte in shape.It is considered that MSCsis a kind of stem cell which has potency of pluripotential progenitor and canbe differentiated into bones,cartilage,muscle,fat,tendon and nerve,etc.It has high plasticity.Scholars both here and abroad used 5-aza into induceMSCs after passage in culture in vitro.And the cardiac cell like ultrastructure,specific gene expression of cardiac cell and persistent actionpotential showed that they induced cardiac cells successfully.Some scholarsmedication experience of ten years,which has the function of promoting blood circulation directly injected MSCs into self cardiac muscle cells tissue and cardiac musclelike cytomorphosis was found.It had specific symbols,for example,thechromatin reconstitution of cardiac troponin I and myosin heavy chain,ofcardiac muscle cells.It showed that MSCs could be induced and differentiatedinto cardiac muscle cells in microenvironment in vitro.
Icariin (ICA)is one of the constituents of epimedium,a traditionalChinese herbal medicine.It possesses many kinds of biological actions,particularly in cardiovascular function improvement,hormone regulation,immunological function modulation,anti-tumor and anti-virus activity.It hasbeen used for the treatment of heart disease.
It could be fund that ES cells were remarkably induced into therhythmically beating EBs with ICA in a concentration-and time-dependent manner.So it will be considered that if the concentration-and time-dependent mannercan be fund with MSCs induced by ICA.
This study was designed by the detection of gene and protein of cardiacmuscle such asα-MHC、β-MHC、Desmin、cTnT、VEGF and bFGF etc.This study can be divided into four parts.We hope that we can explore the relationshipbetween ICA and MSCs.Objective
Explore the best concentration-and time-dependent manner of MSCs beeninduced by ICA,and explore the improvement of myocardial infraction by thetransplantation in the treatment of MSCs induced by ICA.Method This study can be divided into four parts.
Firstly:ICA was divided into 5 concentration gradient(10~(-6)M、10~(-7)M、10~(-8)M、10~(-9)M,10~(-10)M).It can be studied the effects of MSCs induced by ICA withmethod of MTT.At the same time,MSCs can be identified withimmunohistochemistry method and Morphology.
Secondly:It can be explore the concentration-and time-dependent mannerof the specific gene ofα-MHC、β-MHC and GATA-4 in myocardial cells whichdetected by reverse transcription-poly-merase chain reaction(RT-PCR)analysis.
Thirdly:On the basis of the second part,It can be explore the expressionof VEGF and bFGF with Western Blot method compared with 5-aza further.
Fourthly:On the basis of above experiments,the MSCs induced by ICA for2 weeks and 4 weeks was injected into MI rats through intravenous of tail. The specific Protein Desmin、cTnT、VEGF and BFGF can be detectived withimmunohistochemistry method.And we can observed the improvement of myocardialinfraction by the transplantation in the treatment of MSCs induced by ICA withstaining of HE and Masson.Results
Firstly:MSCs were isolated from adult rat's gradient centrifugationand anchoring culture,the MSCs were purified by culture-expanded.Comparedwith control group,10~(-7)M and 10~(-8)M of ICA could promoted MSCs Proliferationobviously,P<0.01.
Secondly:It showed that RT-PCR analysis of the expression ofcardiomyocyte-specific genes in differentiated rat MSCs cells.The expressionof the 10~(-7)M of ICA group forα-MHC andβ-MHC were more strongly than othergroups,and that 10~(-7)M of ICA could promoted MSCs proliferation obvirsly for24h and a 7-days culture in primary culture.
Thirdly:It showed that Western Blot analysis of the expression of VEGFand bFGF in differentiated rat MSCs cells.the 5-aza group were used as apositive control.The expression of the 10~(-7)M of ICA group for specificprotein---VEGF and bFGF were more strongly than other groups,that 10~(-7)M of ICA could promoted MSCs proliferation obviously for 24h and a 7-days culturein primary culture.
Fourthly:It was obviously that the expression of Desmin、cTnT、VEGF andbFGF in Myocardial infraction of 4-weeks of ICA group was strongly comparedwith 2-weeks of ICA group、model group and simpel MSCs group.
Conclusion
It can be inferred that 10~(-7)M of ICA could promoted MSCs differentiatedinto cardiomyocyto-like cells for 24h and a 7-days culture in primary culture.It can be concluded that the expression of the 10~(-7)M of ICA of groupcardiomyocyte-specific genes and protein forα-MHC、β-MHC、Desmin、cTnT、VEGF and bFGF were more strongly than other groups than other groups.Thereare a lot of experimental research for bone marrow-derived mesenchymal stemcells (MSCs)transplantation in the treatment of myocardial infraction,moreresearchers think that MSCs through certain channels to myocardial infractionin rats in vivo transplantation can be effective in repairing damagedmyocardium.But,it was found that MSCs which had not been treated could notexpress Desmin、cTnT、VEGF and bFGF obviously.The most ideally treatment is that 10~(-7)M of ICA could promoted MSCs differentiated into cardiomyocyto-likecells for 24h and a 7-days culture in primary culture.The method can inducedMSCs differentiated into cardiomyocyto-like cells.
引文
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