pEGFP-N2-BMP2转染P19细胞诱导向心肌分化
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摘要
目的:心肌梗死是一种严重危害人类健康的疾病,心肌梗死后功能性心肌细胞丢失和疤痕组织形成使存活的心肌细胞减少、继发心室重构,是造成心肌梗死患者发生心力衰竭甚至死亡的重要原因。人体内心肌细胞不能再生,损伤的心肌细胞不能修复和分化,故临床药物治疗、介入治疗不能逆转已坏死的心肌,无法促使梗死的心肌再生,使心肌梗死患者的远期预后不能得到显著改善。而心脏移植由于手术复杂、供体来源困难和费用高等原因,在临床很难推广。治疗心肌梗死的关键是增加梗塞区内功能性心肌细胞的数量,目前,细胞移植治疗正成为一种新的治疗方法。因此,寻找可代替死亡细胞的合适的种子细胞来防治心肌梗死后的心衰、心室重构,改善心功能,是近年来研究的主要问题。
骨形态蛋白(bone morphogenetic protein, BMP)家族至少包括40个成员,至今已鉴定了的BMP有15种之多,都属于转化生长因子-β(transforming growth factor-β,TGF-β)家族,以功能域的同源性为基础,它又分成几个BMP超家族,即BMP-2、4超家族,BMP-5、6、7、8超家族,生长分化因子-5(growth and differentiationfactor,GDF-5)、GDF-6(BMP-13)、GDF-7(BMP-12)超家族,以及BMP-3与GDF-10(BMP-3b)超家族。其中,BMP-2、4超家族具有很高的生物活性,有研究表明BMP-2、4参与调控心脏发生和心肌分化过程。对鸡胚的研究证实,在早期鸡胚的心脏发育部位,能够检测到BMP2的表达。另有研究表明外源加入BMP2蛋白可诱导骨髓间充质细胞分化为心肌样细胞,且有资料表明BMP信号途径是心脏早期转录因子GATA-4、NKx2.5上游效应分子。此类研究表明, BMP2在胚胎时期的心脏发育、心肌分化中起着非常重要的作用,但是BMP2在心肌分化中的作用机制,尤其是与心肌细胞转录因子如NKx2.5、GATA-4等之间的相互作用国内外未见报道,另外,外源表达BMP2基因能否高效诱导P19细胞心肌分化亦未见报道。因此本实验选用BMP2基因转染P19细胞,观察其诱导心肌分化的作用。
P19胚胎瘤细胞( P19 embryonal carcinoma cells, P19ECCs)是从C3H/He雄性小鼠畸胎瘤中分离得到,可以在体外迅速大量扩增,多次传代也不丧失其分化能力。P19细胞是多潜能细胞,可分化为内胚层、中胚层、外胚层3个胚层的不同类型的细胞。在体外不同的诱导条件下可被诱导为包括心肌细胞、骨骼肌细胞及神经元在内的多种类型的细胞,因而是一种较为理想的用于研究细胞分化和发育的工具细胞,如果在体外诱导向心肌分化后进行移植,可能成为较理想的替代坏死心肌的种子细胞。因此,P19细胞常作为研究心脏发育和心肌分化的一个良好体外模型。但在P19细胞向心肌细胞分化的过程中细胞聚集体的形成和诱导剂的结合是两个必需条件,目前尚没有明确的资料证明P19细胞在单层培养的情况下可以向心肌细胞分化。因此,缺少这两个条件之一, P19细胞不能向心肌细胞分化。
本课题拟将BMP2基因转染P19细胞,聚集培养后,观察其在没有诱导剂的情况下向心肌分化的情况。并研究在二甲基亚砜(dimethyl sulfoxide DMSO)的作用下,稳定表达BMP2的P19细胞在单层培养时向心肌分化的情况,为P19细胞有效转化为心肌细胞探索一个比较好的诱导方式,为心肌梗死的细胞移植治疗探索一个合适的种子细胞。
方法:
1 pEGFP-N2-BMP2真核表达质粒的构建与鉴定
以pEFSA- BMP2质粒为模板, PCR扩增出BMP2的序列,上游引物:5’- GCCAGATCTAAAGGTCGACCATGGT -3',引入酶切位点BglⅡ;下游引物: 5'- TTAGAATTCGCGACACCCACAACC -3',引入酶切位点EcoRI。反应完毕后,经1%琼脂糖凝胶电泳分离,胶回收纯化靶片断。将条带单一的PCR产物经1%琼脂糖凝胶电泳分离,小剂量胶回收试剂盒回收,并用限制性内切酶BglⅡ和EcoR I双酶切纯化BMP2 DNA。限制性内切酶BglⅡ和EcoRI双酶切pEGFP-N2载体并纯化。将PCR扩增的BMP2 DNA与双酶切后的pEGFP-N2经T4 DNA连接酶进行连接反应,连接产物转化至大肠杆菌DH5α感受态细胞,涂布于固体LB培养基上,37℃倒置培养16-24 h;挑取单个阳性菌落,提取重组质粒,进行菌落PCR鉴定有无目的片段,再经BamH I和HindⅢ双酶切鉴定、测序鉴定。
2 pEGFP-N2-BMP2转染P19细胞后聚集培养诱导向心肌分化
实验分实验组和对照组。实验组为转染pEGFP-N2-BMP2的P19细胞,转染之前通过预实验确定合适的细胞接种密度,转染最佳体系,G418筛选浓度。本实验采用阳离子转染试剂Lipofectamine2000将质粒pEGFP-N2-BMP2转染P19细胞,转染之后24h将细胞按1×105/ml密度传代,G418稳定筛选14天后,将筛出细胞聚集培养4天形成聚集体,聚集体移入培养皿中贴壁生长。在贴壁的4天、8天、12天、16天取细胞,免疫细胞化学检测心肌肌钙蛋白T (cardiac troponin T ,cTnT)和心肌肌动蛋白α(α-cardiac actin)的表达;RT-PCR检测GATA-4、NKx2.5两个基因的表达;Western blot检测α-cardiac actin和cTnT蛋白的表达。并取贴壁16天的细胞透射电镜观察细胞超微结构的变化。对照组将P19细胞直接聚集培养,观察细胞分化的情况,对照组取材时间和检测指标同实验组。
3 DMSO诱导转染pEGFP-N2-BMP2的P19细胞单层培养时向心肌分化
实验分为实验组和对照组,实验组为转染pEGFP-N2-BMP2质粒的P19细胞,DMSO诱导单层细胞培养,对照组为未转染质粒的P19细胞,DMSO诱导单层细胞培养。两组均在诱导的4天、8天、12天、16天取细胞,免疫细胞化学检测α-cardiac actin和cTnT的表达;RT-PCR检测GATA-4、NKx2.5两个基因的表达;Western blot检测α-cardiac actin和cTnT蛋白的表达。并取贴壁16天的细胞做透射电镜观察细胞超微结构的变化。
结果:
1 pEGFP-N2-BMP2真核表达质粒的构建与鉴定
以pEFSA- BMP2质粒为模板, PCR扩增出约1.2 kb的基因片段,与BMP2基因编码框(1191bp)大小一致。经BglⅡ和EcoR I双酶切分别得到带有BglⅡ和EcoR I酶切位点的约1.2 kb的BMP2基因片段和4.7kb的pEGFP-N2载体,然后回收、连接转化后,筛出新生菌落。进行PCR反应,结果显示从重组质粒中可扩增出大小约1.2 kb的基因片段。所提质粒经BamH I和HindⅢ双酶切之后,可获得约1.2 kb和4.7 kb的条带,分别与BMP2基因(1191bp)和载体pEGFP-N2(4.7kb)的大小一致。用启动子CMV引物进行测序,测序结果校对后经BLAST分析,表明此核酸序列与GeneBank中BMP2 mRNA序列(NM-001200)的编码序列完全吻合,酶切位点完整,说明已将BMP2基因编码框正确插入pEGFP-N2载体的多克隆位点(MCS)。确定在本实验中,pEGFP-N2-BMP2重组质粒构建成功。
2 pEGFP-N2-BMP2转染P19细胞后聚集培养诱导向心肌分化通过反复实验确定:细胞传代密度:2×105/ml,转染比率1:2.5,G418稳定筛选浓度:600μg/ml。实验组在转染质粒pEGFP-N2-BMP2之后的18-24h可见部分细胞表达EGFP-BMP2融合蛋白, G418筛选2周后,大多数细胞可观察到绿色荧光,这些细胞为稳定表达BMP2的P19细胞。实验组和对照组的细胞在软琼脂糖培养基内悬浮培养4天形成聚集体,将聚集体接种到培养皿中贴壁培养,细胞由周边爬出,细胞胞体变大变长,伸出突起相连在一起。免疫细胞化学结果:实验组细胞贴壁培养4天时,α-cardiac actin和cTnT都不表达,第8天的时候,两种蛋白均有表达,但是表达量较少,12天和16天的表达量逐渐增多,阳性反应产物分布在细胞质中,呈现棕黄色,随着时间的延长,细胞中出现明显棕黄色束状结构。阳性细胞吸光度值统计结果显示:两种蛋白表达量8天、12天、16天与对照组比有显著差异(P<0.01),其他天数间比较也有显著差异(P<0.01)。对照组没有检测到两种蛋白的阳性表达。RT-PCR结果:实验组GATA-4和Nkx2-5在贴壁培养的4天开始有表达,但表达量较弱,8天、12天和16天表达逐渐增多,统计结果显示:GATA-4和NKx2.5的表达4天、8天、12天、16天与对照组比有显著差异(P<0.01),其他天数间比较也有显著差异(P<0.01)。对照组没有检测到两种基因的阳性表达。Western Blot结果:实验组细胞,α-cardiac actin和cTnT两种蛋白在4天没都有表达,在8天的时候有表达,12天和16天时表达量逐渐增高。统计结果显示:两种蛋白的表达8天、12天、16天与对照组比有统计学差异(P<0.01),其他天数间比较也有统计学差异(P<0.01)。对照组细胞两种蛋白均没有表达。透射电镜观察发现,实验组的细胞经聚集、贴壁培养16天后,细胞胞质中可见较多的高尔基复合体、游离核糖体、粗面内质网、线粒体等细胞器,在一些细胞的胞质中,可见平行排列的肌丝样结构;相邻细胞分化出细胞连接,细胞连接的胞质内出现肌丝样结构,未发现肌节样结构。对照组未发现分化的细胞。
3 DMSO诱导转染pEGFP-N2-BMP2的P19细胞单层培养时向心肌分化在DMSO诱导时,随着诱导时间延长,实验组和对照组细胞均有聚集生长的趋势,生长缓慢。两组细胞在培养过程中都没有发现细胞跳动现象。免疫细胞化学结果:实验组细胞α-cardiac actin和cTnT在4天的时候都没有表达,在8天、12天、16天时均有表达,并随时间延长而逐渐增高,细胞中棕黄色成束丝样结构增多。阳性细胞吸光度值统计结果显示:两种蛋白在8天、12天、16天的表达量与对照组相比有显著差异(P<0.01),其他天数间比较也有显著差异(P<0.01)。对照组细胞在DMSO诱导的4天、8天、12天、16天均没有检测到α-cardiac actin和cTnT的阳性表达。RT-PCR结果:实验组,稳定表达BMP2的P19细胞在DMSO诱导下、单层培养后, GATA-4和NKx2.5在第4天开始有表达,但表达量较弱,8天、12天、16天表达逐渐增多。统计结果表明:GATA-4的表达4天、8天、12天、16天与对照组比有显著差异(P<0.01),其他天数间比较也有显著差异(P<0.01或P<0.05);NKx2.5的表达4天、8天、12天、16天与对照组比有显著差异(P<0.01),其他天数间比较除12天与16天无统计学差异(P>0.05)外也均有显著差异(P<0.01)。在对照组,各时间点均未检测到两种基因的阳性表达。Western Blot结果:实验组,稳定表达BMP2的P19细胞在DMSO诱导下、单层培养后,α-cardiac actin和cTnT在4天都没有表达,在8天的时候开始有表达,12天和16天时表达逐渐增多。统计结果显示:α-cardiac actin在4天、8天、12天、16天与对照组比有统计学差异(P<0.01或P<0.05),其他天数间比较也有统计学差异(P<0.01)。cTnT在8天的时候有表达,但与对照组比没有统计学差异(P>0.05),其他天数间比较有统计学差异(P<0.01)。对照组细胞两种蛋白均没有表达。透射电镜观察发现,实验组细胞胞质中可见较多的高尔基复合体、游离核糖体、粗面内质网、线粒体等细胞器,在一些细胞的胞质中,可见平行排列的肌丝样结构;相邻细胞分化出细胞连接,细胞连接的胞质内出现微丝样结构,未发现肌节样结构。对照组未发现分化的细胞。
结论:
1本实验成功构建了pEGFP-N2-BMP2真核表达质粒,为以后研究BMP2基因的作用奠定了基础。
2本实验将pEGFP-N2-BMP2真核表达质粒转染P19细胞,可见其在P19细胞中表达,因此,pEGFP-N2-BMP2真核表达质粒可以用于真核细胞的转染和稳定筛选。
3转染了质粒pEGFP-N2-BMP2的P19细胞不需要诱导剂的诱导,只聚集培养即可分化为心肌样细胞,表达心脏早期转录因子、心肌特异基因和心肌特异蛋白,在基因和蛋白水平表达心肌源性细胞的特异性标志物。结果表明外源表达BMP2基因可以诱导P19细胞在没有诱导剂诱导时即向心肌分化,为P19细胞有效转化为心肌细胞探索了一个比较好的诱导方式。4转染了质粒pEGFP-N2-BMP2的P19细胞不需要诱导剂的诱导,只聚集培养即可分化为心肌样细胞,表明BMP2基因在P19细胞向心肌细胞分化过程中具有促心肌分化作用,说明BMP信号在P19细胞分化为心肌细胞过程中起着非常重要的作用。
5 DMSO诱导使单层培养的稳定表达BMP2基因的P19细胞向心肌分化,表达心脏早期转录因子、心肌特异基因和心肌特异蛋白,在基因和蛋白水平表达心肌源性细胞的特异性标志物,表明BMP2基因可以促进P19细胞单层培养时向心肌细胞分化,进一步说明BMP信号在P19细胞分化为心肌细胞过程中起着非常重要的作用。
Objective: Myocardial infarction (heart attack) is greatly harmful to human health. In myocardial infarction, the loss of functional myocardial cells and the formation of scar tissue occurs, which makes the living cardiomyocytes become less and less. The loss of functional myocardial cells and the following ventricle reconstruction are the main reasons of heart failure, even death of the patients. Myocardial cells neither be regenerated nor differentiated once the damage is done. Therefore, clinical medicine treatment, interventional therapy and surgery usually do not produce satisfactory outcome. Owing to the complexity of the surgical procedure, rare donors and expensive expenditure, heart transplantation can not be generalized clinically. The key point in treating myocardial infarction is how to increase the number of functional cardiomyocytes in the infarcted area. Currently, cell transplantation is becoming a new trend to treat heard attacks. So the major challenge is to find suitable seed cells, which can substitute for dead cells to prevent heart failure and improve heart function.
The bone morphogenetic protein (BMP) belong to the transforming growth factor-βfamily. There are at least 40 members in BMP family. Up to now, more than 15 of them have been identified. Based on the homology of functional domain, it is divided into several BMP superfamilies, such as BMP 2 and 4 superfamily, BMP 5, 6, 7 and 8 superfamily, growth and differentiation factor (GDF-5), GDF-6(BMP-13) and GDF-7(BMP-12) super family, and BMP-3 and GDF-10(BMP-3b) super family, among which BMP-2 and BMP-4 super family are highly bioactive. Researches show that BMP-2 and BMP-4 are involved in regulating cardiogenesis and myocardial differentiation. Researches show that the BMP2 expression which can be detected in the heart develoment area were improved at early stage in chick embryo. Some evidence show that exogenous BMP2 protein can induce the differentiation of marrow stroma cells to cardiomyocyte. Evidence also show that BMP signal is the upper stream effector molecule of some of heart’s early transcription factor( such as GATA-4 and NKx2.5). These researches show that BMP2 plays an important role in heart development and myocardial differentiation at the early stage of embryo. However, the mechanism of BMP2 in myocardial differentiation, and especially the interaction between BMP2 and myocardial transcription factors such as NKx2.5 and GATA-4 is not unclear after a thorough literature review. Moreover, whether the exogenous expression of BMP2 can induce the differentiation of P19 cell to cardiomyocytes still remains unknow. As such, our experiment chose gene transfection technique to transfect BMP2 into P19 cell and observe the role of BMP2 in myocardial differentiation.
P19 embryonal carcinoma cells (P19ECCs) are amplified rapidly in vitro and can continue to differentiate after many generations. P19 cells can be differentiated into three different types of cells, which contains endoderm, mesoderm and ectoderm. Under different extraorgan induction conditions, it can be induced into many kinds of cells including cardiomyocytes, skeletal muscle cells and neuron. For this reason, it is a relatively ideal instrumental cell that can be used to study cell differentiation. If transplanted after extraorgan induction towards myocardial differentiation, it may become a relatively ideal seed cell to replace the necrotic cardiomyocytes. Experiments show that during the differentiation of P19 cells to cardiomyocytes, the related gene expression about cell growth and electrophysiological feature mainly imitate the myocardial growth of the normal mouse. So P19 cells are considered as a good model in vitro to study heart development and myocardial differentiation. However, during the differentiation of P19 cells to cardiomyocytes, the formation of cell aggregates and the inductor are two important and necessary processes. Up to now, it has not been clearly proved that P19 cells can differentiate to cardiomyocytes in monolayer culture. Without the formation of cell aggregates and the inductor, P19 cells can’t differentiate to cardiomyocytes.
Therefore, there are two main project in the study. First is designed to transfect pEGFP-N2-BMP2 into P19 cells, and observed the differentiation of P19 cells to cardiomyocytes without inductor. Second is to study the differentiation of P19 cells which stable expressing of BMP2 gene in monolayer culture with DMSO induction. We also would be explore a better way of P19 cells differentiate into cardiomyocytes. It would be better, if we can search for a suitable seed cell for cell transplantation in the treatment of myocardial infarction.
Methods:
1 The construction and identification of eukaryotic expression plasmid pEGFP-N2-BMP2
We used plasmid pEFSA- BMP2 as a template and transformed it into competent E. coli DH5αby the conventional CaCl2 method. The BMP2 gene sequence was amplified with PCR. Up stream primer: 5’-GCCAGATCTAAAGGTCGACCATGGT-3', the introduction of endonuclease sites BglⅡ; down stream primer: 5'-TTAGAATTCGCGACACCCACAACC-3', the introduction of endonuclease sites EcoRI. After the reaction completed, PCR products were subjected to agarose gel electrophoresis and target fragment was recovered from the agarose gel. The Coding sequence of BMP2 and vector pEGFP-N2 were digested by BglⅡand EcoRI overnight and recovered respectively. Restriction enzymesBglⅡand EcoRI were used to purify BMP2 DNA and pEGFP-N2 vector. BMP2 DNA was amplified by PCR and both the BMP2 and pEGFP-N2 will have coupled reaction through T4 DNA ligase. The ligation product was transformed into competent E. coli DH5α.cells and coated to the solid LB medium plate containing kanamycin and cultured inverted for 16-24h at 37℃. Single positive colony is chosen to extract recombinant plasmid and detect if there is target fragment through PCR, then by BamH I and HindⅢdouble digestion identification. At the same time, the recombinant plasmid pEGFP-N2-BMP2 was sent to Shanghai Sangon Co. Ltd. for sequencing identification.
2 Cardiomyocyte differentiation from P19 cells with exogenous expression BMP2 gene can be induced by cell aggregate formation.
The experimental subjects were divided into two groups- experimental group and control group. In the experimental group were P19 cells transfected with pEGFP-N2-BMP2. Before transfection, preliminary experiment was used to determine the suitable cell seeding density, the best transfection system, and the appropriate G418 selection concentration. Using the cationic liposome reagent, Lipofectamine 2000, the plasmid pEGFP-N2-BMP2 were transfected into P19 cells. After 14th days of stable selection by G418, the survival cells were suspension cultured to form aggregates for 4th days and the aggregates were transferred to the Petri dish for adherent culture. The cells were collected at 4th day, 8th day, 12th day and 16th day after adherent culture. The expression of cTnT andα-cardiac actin was detected with immunocytochemical staining method. RT-PCR was used to detect the expression of GATA-4 and NKx2.5 genes. Western blot was used to detect the expression ofα-cardiac actin and cTnT proteins. In the experimental group at 16th day after adherent culture, the ultrastructural changes of the cells were observed. In the control group, P19 cells were aggregated and cultured directly.The detection index and sampling time were same as that of experimental group.
3 Dimethylsulfoxide induced monolayer cultured P19 cells which transfected with pEGFP-N2-BMP2 to differentiate toward cardiomyocyte
There were experimental group and control group in the experiment. In the experimental group, DMSO induced monolayer cultured P19 cells which transfected with pEGFP-N2-BMP2. In the control group, DMSO induced monolayer cultured P19 cells. 1% DMSO was used to induce the cells both in experimental group and control group. Cells were collected at 4th day, 8th day, 12th day and 16th day after induction in two groups. The expression of cTnT andα-cardiac actin was detected with immunocytochemical staining method. RT-PCR was used to detect the expression of GATA-4 and NKx2.5 genes. Western blot was used to detect the expression ofα-cardiac actin and cTnT ultrastructural changes of the cells were observed. In the control group, P19 cells were induced culture with 1% DMSO directly.The detection index and sampling time of control group were same as that of experimental group.
Results:
1 Construction and identification of pEGFP-N2-BMP2 eukaryotic expression plasmid
Modeled by pEFSA- BMP2 eukaryotic expression plasmid, a gene fragment of about 1.2 kb was obtained, whose size was as the same as the human BMP2 gene encoding frame (1191bp). Vector pEGFP-N2 and BMP2 gene were digested with restriction enzyme BglⅡand EcoR I respectively. About 1.2 kb BMP2 gene fragment and 4.7kb pEGFP-N2 vector with BglⅡand EcoR I restriction enzyme sites were obtained then they were retrieved and connected. A gene fragment of about 1.2kb was obtained by PCR from the recombinant plamid after transformation and connection. Two gene fragments of about 1.2kb and 4.7 kb were obtained from the recombinant plasmid after double enzyme digesting with BamH I and HindⅢ. With CMV promoter primer for sequencing, analyzed by BLAST after proofread, the sequence result showed that the nucleotide sequence was agreed with the gene encoding sequence of BMP2 mRNA(NM-001200) in GeneBank and the endonuclease sites were complete. There was an intact Kozak sequence in the gene start area. Terminator of gene was mutated and the reading frame was correctly identified. This means BMP2 gene encoding frame has been correctly inserted in MCS of pEGFP-N2 vector. It is confirmed that recombinant plasmid pEGFP-N2-BMP2 was constructed successfully in this experiment. 2 Cardiomyocyte differentiation from P19 cells with exogenous expression BMP2 gene can be induced by cell aggregate formation.
The experiment confirmed that the stable selection concentration of G418 was 600μg/ml and the appropriate cell seeding density for transfection was 2×105/ml. The best transfection system was the plasmid DNA(μg):Lipofectamine2000 (μl)=1:2.5. In the experimental group, at the 24th hour after transfection with plasmid pEGFP-N2-BMP2, some cells expressed the green fluorescence. Most cells expressed green fluorescence after being selected with 600μg/ml G418 two weeks later, which were P19 cells with exogenous expression BMP2 gene. Cells in experimental group and control group were collected and cultured for 4th days to form aggregates. After adhesion of the aggregates, some cells grew out and formed outgrowth around the aggregates. In the outgrowth, the cell body grew bigger and longer, and evections reach out and connected with each other. Immunocytochemical staining results showed that, in experimental group, at 4thday two kinds of antibody,α-cardiac actin and cTnT, were not expressed. At 8th day, they were all expressed in a little amount. At 12th day and 16th day, two kinds of antibody were all expressed, and the expression was gradually increased. Brown positive reaction products were in cytoplasm. With time going by, there was obviously brown filament-like structure in the cytoplasm of the cells at 12th day and 16th day. The statistical results of positive cell absorbance value showed that there was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference(P<0.01)between 8th day, 12th day and 16th day in experimental group. Positive expression of the two antibodies was not detected in control group. RT-PCR analysis showed that, in experimental group, GATA-4 and Nkx2.5 expressed at 4th day after adherent culture, but it is deficient in quantity. At 8th day, 12th day and 16th day, the expression of GATA-4 and Nkx2.5 was gradually increased. The statistical result showed that there was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference(P<0.01) between 8th day, 12th day and 16th day in experimental group. No positive expressions of the two kinds of genes was detected in control group. Western Blot result showed that, in experimental group, at 4thdayα-cardiac actin and cTnT, were not expressed. At 8th day, they were all expressed in a little amount. At 12th day and 16th day they were all expressed, and the expression was gradually increased. The statistical result
showed that there was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference (P<0.01) between 8th day, 12th day and 16th day in experimental group. Positive expression of the two proteins was not detected in control group. The observation of ultrastructure showed that after aggregation and adherent culture for 16th days, some cells became elongated cylindrical and there appeared cell junction between the adjacent cells in experimental group. Some microfilament-like structure appeared beside the cell junction. There was some dense myofilament-like structure arranged in parallel way in cytoplasm of some cells,but sarcomere-like structure was not founed. no differentiated cell was found in control group.
3 Dimethylsulfoxide induced monolayer cultured P19 cells which transfected with pEGFP-N2-BMP2 to differentiate toward cardiomyocyte
Cells both of experimental group and control group induced monolayer culture with 1% DMSO and grew slowly compared with the P19 cells without inducing. And cells accumulated in a state similar to adherent aggregates-like structure as increased in number. The cells in the center of the aggregates were small and dense, while the periphery cells became longer, crawling and radiating out in all directions with cell body elongated to be fusiformis and stretched processes. Beating cells were not observed in either of the two groups during culture. Immunocytochemical staining result showed that, in experimental group, at 4thday after being induced by 1% DMSOα-cardiac actin and cTnT were not expressed. At 8th day, they were all expressed in a little amount. At 12th day and 16th day two kinds of antibody were all expressed, and the expression was gradually increased. Brown positive reaction products were in cytoplasm. With time going by, there was obviously brown filament-like structure in the cytoplasm of the cells at 12th day and 16th day. The statistical result of positive cell absorbance value showed that there was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference (P<0.01) between 8th day, 12th day and 16th day in experimental group. Positive expression of the two kinds of antibody was not detected in control group. RT-PCR analysis showed that, in experimental group, GATA-4 and Nkx2.5 expressed at 4th day after induced by DMSO, but it is deficient in quantity. At 8th day, 12th day and 16th day the expression of two genes was gradually increased. The statistical result showed that the expression of GATA-4 was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference (P<0.01 or P<0.05 ) between 8th day, 12th day and 16th day in experimental group. the expression of Nkx2.5 was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and in experimental group there was also significant difference (P<0.01 ) between 8th day, 12th day and 16th day, but there was no significant difference(P>0.05)between 12th day and 16th day. No positive expressions of the two kinds of genes was detected in control group. Western Blot result showed that, in experimental group, at 4thdayα-cardiac actin and cTnT were not expressed. At 8th day they were all expressed in a little amount. At 12th day and 16th day they were all expressed, and the expression was gradually increased. The statistical result showed that the expression ofα-cardiac actin was distinct difference (P<0.01 or P<0.05) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference (P<0.01) between 8th day, 12th day and 16th day in experimental group. the expression of cTnT was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, but there was no significant difference (P>0.05 ) between 8th day and control group. And there was also significant difference (P<0.01) between 8th day, 12th day and 16th day in experimental group. Positive expression of the two proteins was not detected in control group. The observation of ultrastructure showed that after induced by DMSO culture for 16th days, some cells became elongated cylindrical and there appeared cell junction between the adjacent cells in experimental group. Some microfilament-like structure appeared beside the cell junction. There was some dense myofilament-like
structure arranged in parallel way in cytoplasm of some cells, but sarcomere-like structure was not founed. no differentiated cell was found in control group.
Conclusions:
1 The eukaryotic expression plasmid pEGFP-N2-BMP2 was successfully constructed, which would lay a foundation for the further studies on the role of BMP2 in myocardial differentiation and heart development.
2 Eukaryon expression plasmid pEGFP-N2-BMP2 can be expressed in P19 cells and can be used for the transfection and stable selection of eukaryotic cells.
3 Without inducing agents, exogenous expression of BMP2 gene induced P19 cells to differentiate towards cardiomyocytes only with aggregates formation. P19 cells transfected with pEGFP-N2-BMP2 induced the expression of cardiac-specific markers at the level of gene and protein which increased gradually with prolongation of culture time. It shows that exogenous expression of BMP2 gene can induce myocardial differentiation from P19 cells. This study would explore a reasonable induction way for myocardial differentiation from P19 cells .
4 Without inducing agents, exogenous expression of BMP2 gene induced P19 cells to differentiate towards cardiomyocytes only with aggregates formation. This shows that BMP2 gene can promote myocardial differentiation from P19 cells and indicates that BMP signal path plays a very important role in the process of myocardial differentiation from P19 cells.
5 With monolayer culture and induced by DMSO, P19 cells which transfected with pEGFP-N2-BMP2 can differentiate towards cardiomyo- cytes, which show the expression of cardiac-specific markers at the level of gene and protein. It suggests that BMP2 gene can promote P19 cells to differentiate towards cadiocyte under monolayer culture condition. It is proved further that BMP2 signal path plays a very important role in the process of myocardial differentiation from P19 cells.
骨形态蛋白(bone morphogenetic protein, BMP)家族至少包括40个成员,至今已鉴定了的BMP有15种之多,都属于转化生长因子-β(transforming growth factor-β,TGF-β)家族,以功能域的同源性为基础,它又分成几个BMP超家族,即BMP-2、4超家族,BMP-5、6、7、8超家族,生长分化因子-5(growth and differentiationfactor,GDF-5)、GDF-6(BMP-13)、GDF-7(BMP-12)超家族,以及BMP-3与GDF-10(BMP-3b)超家族。其中,BMP-2、4超家族具有很高的生物活性,有研究表明BMP-2、4参与调控心脏发生和心肌分化过程。对鸡胚的研究证实,在早期鸡胚的心脏发育部位,能够检测到BMP2的表达。另有研究表明外源加入BMP2蛋白可诱导骨髓间充质细胞分化为心肌样细胞,且有资料表明BMP信号途径是心脏早期转录因子GATA-4、NKx2.5上游效应分子。此类研究表明, BMP2在胚胎时期的心脏发育、心肌分化中起着非常重要的作用,但是BMP2在心肌分化中的作用机制,尤其是与心肌细胞转录因子如NKx2.5、GATA-4等之间的相互作用国内外未见报道,另外,外源表达BMP2基因能否高效诱导P19细胞心肌分化亦未见报道。因此本实验选用BMP2基因转染P19细胞,观察其诱导心肌分化的作用。
P19胚胎瘤细胞( P19 embryonal carcinoma cells, P19ECCs)是从C3H/He雄性小鼠畸胎瘤中分离得到,可以在体外迅速大量扩增,多次传代也不丧失其分化能力。P19细胞是多潜能细胞,可分化为内胚层、中胚层、外胚层3个胚层的不同类型的细胞。在体外不同的诱导条件下可被诱导为包括心肌细胞、骨骼肌细胞及神经元在内的多种类型的细胞,因而是一种较为理想的用于研究细胞分化和发育的工具细胞,如果在体外诱导向心肌分化后进行移植,可能成为较理想的替代坏死心肌的种子细胞。因此,P19细胞常作为研究心脏发育和心肌分化的一个良好体外模型。但在P19细胞向心肌细胞分化的过程中细胞聚集体的形成和诱导剂的结合是两个必需条件,目前尚没有明确的资料证明P19细胞在单层培养的情况下可以向心肌细胞分化。因此,缺少这两个条件之一, P19细胞不能向心肌细胞分化。
本课题拟将BMP2基因转染P19细胞,聚集培养后,观察其在没有诱导剂的情况下向心肌分化的情况。并研究在二甲基亚砜(dimethyl sulfoxide DMSO)的作用下,稳定表达BMP2的P19细胞在单层培养时向心肌分化的情况,为P19细胞有效转化为心肌细胞探索一个比较好的诱导方式,为心肌梗死的细胞移植治疗探索一个合适的种子细胞。
方法:
1 pEGFP-N2-BMP2真核表达质粒的构建与鉴定
以pEFSA- BMP2质粒为模板, PCR扩增出BMP2的序列,上游引物:5’- GCCAGATCTAAAGGTCGACCATGGT -3',引入酶切位点BglⅡ;下游引物: 5'- TTAGAATTCGCGACACCCACAACC -3',引入酶切位点EcoRI。反应完毕后,经1%琼脂糖凝胶电泳分离,胶回收纯化靶片断。将条带单一的PCR产物经1%琼脂糖凝胶电泳分离,小剂量胶回收试剂盒回收,并用限制性内切酶BglⅡ和EcoR I双酶切纯化BMP2 DNA。限制性内切酶BglⅡ和EcoRI双酶切pEGFP-N2载体并纯化。将PCR扩增的BMP2 DNA与双酶切后的pEGFP-N2经T4 DNA连接酶进行连接反应,连接产物转化至大肠杆菌DH5α感受态细胞,涂布于固体LB培养基上,37℃倒置培养16-24 h;挑取单个阳性菌落,提取重组质粒,进行菌落PCR鉴定有无目的片段,再经BamH I和HindⅢ双酶切鉴定、测序鉴定。
2 pEGFP-N2-BMP2转染P19细胞后聚集培养诱导向心肌分化
实验分实验组和对照组。实验组为转染pEGFP-N2-BMP2的P19细胞,转染之前通过预实验确定合适的细胞接种密度,转染最佳体系,G418筛选浓度。本实验采用阳离子转染试剂Lipofectamine2000将质粒pEGFP-N2-BMP2转染P19细胞,转染之后24h将细胞按1×105/ml密度传代,G418稳定筛选14天后,将筛出细胞聚集培养4天形成聚集体,聚集体移入培养皿中贴壁生长。在贴壁的4天、8天、12天、16天取细胞,免疫细胞化学检测心肌肌钙蛋白T (cardiac troponin T ,cTnT)和心肌肌动蛋白α(α-cardiac actin)的表达;RT-PCR检测GATA-4、NKx2.5两个基因的表达;Western blot检测α-cardiac actin和cTnT蛋白的表达。并取贴壁16天的细胞透射电镜观察细胞超微结构的变化。对照组将P19细胞直接聚集培养,观察细胞分化的情况,对照组取材时间和检测指标同实验组。
3 DMSO诱导转染pEGFP-N2-BMP2的P19细胞单层培养时向心肌分化
实验分为实验组和对照组,实验组为转染pEGFP-N2-BMP2质粒的P19细胞,DMSO诱导单层细胞培养,对照组为未转染质粒的P19细胞,DMSO诱导单层细胞培养。两组均在诱导的4天、8天、12天、16天取细胞,免疫细胞化学检测α-cardiac actin和cTnT的表达;RT-PCR检测GATA-4、NKx2.5两个基因的表达;Western blot检测α-cardiac actin和cTnT蛋白的表达。并取贴壁16天的细胞做透射电镜观察细胞超微结构的变化。
结果:
1 pEGFP-N2-BMP2真核表达质粒的构建与鉴定
以pEFSA- BMP2质粒为模板, PCR扩增出约1.2 kb的基因片段,与BMP2基因编码框(1191bp)大小一致。经BglⅡ和EcoR I双酶切分别得到带有BglⅡ和EcoR I酶切位点的约1.2 kb的BMP2基因片段和4.7kb的pEGFP-N2载体,然后回收、连接转化后,筛出新生菌落。进行PCR反应,结果显示从重组质粒中可扩增出大小约1.2 kb的基因片段。所提质粒经BamH I和HindⅢ双酶切之后,可获得约1.2 kb和4.7 kb的条带,分别与BMP2基因(1191bp)和载体pEGFP-N2(4.7kb)的大小一致。用启动子CMV引物进行测序,测序结果校对后经BLAST分析,表明此核酸序列与GeneBank中BMP2 mRNA序列(NM-001200)的编码序列完全吻合,酶切位点完整,说明已将BMP2基因编码框正确插入pEGFP-N2载体的多克隆位点(MCS)。确定在本实验中,pEGFP-N2-BMP2重组质粒构建成功。
2 pEGFP-N2-BMP2转染P19细胞后聚集培养诱导向心肌分化通过反复实验确定:细胞传代密度:2×105/ml,转染比率1:2.5,G418稳定筛选浓度:600μg/ml。实验组在转染质粒pEGFP-N2-BMP2之后的18-24h可见部分细胞表达EGFP-BMP2融合蛋白, G418筛选2周后,大多数细胞可观察到绿色荧光,这些细胞为稳定表达BMP2的P19细胞。实验组和对照组的细胞在软琼脂糖培养基内悬浮培养4天形成聚集体,将聚集体接种到培养皿中贴壁培养,细胞由周边爬出,细胞胞体变大变长,伸出突起相连在一起。免疫细胞化学结果:实验组细胞贴壁培养4天时,α-cardiac actin和cTnT都不表达,第8天的时候,两种蛋白均有表达,但是表达量较少,12天和16天的表达量逐渐增多,阳性反应产物分布在细胞质中,呈现棕黄色,随着时间的延长,细胞中出现明显棕黄色束状结构。阳性细胞吸光度值统计结果显示:两种蛋白表达量8天、12天、16天与对照组比有显著差异(P<0.01),其他天数间比较也有显著差异(P<0.01)。对照组没有检测到两种蛋白的阳性表达。RT-PCR结果:实验组GATA-4和Nkx2-5在贴壁培养的4天开始有表达,但表达量较弱,8天、12天和16天表达逐渐增多,统计结果显示:GATA-4和NKx2.5的表达4天、8天、12天、16天与对照组比有显著差异(P<0.01),其他天数间比较也有显著差异(P<0.01)。对照组没有检测到两种基因的阳性表达。Western Blot结果:实验组细胞,α-cardiac actin和cTnT两种蛋白在4天没都有表达,在8天的时候有表达,12天和16天时表达量逐渐增高。统计结果显示:两种蛋白的表达8天、12天、16天与对照组比有统计学差异(P<0.01),其他天数间比较也有统计学差异(P<0.01)。对照组细胞两种蛋白均没有表达。透射电镜观察发现,实验组的细胞经聚集、贴壁培养16天后,细胞胞质中可见较多的高尔基复合体、游离核糖体、粗面内质网、线粒体等细胞器,在一些细胞的胞质中,可见平行排列的肌丝样结构;相邻细胞分化出细胞连接,细胞连接的胞质内出现肌丝样结构,未发现肌节样结构。对照组未发现分化的细胞。
3 DMSO诱导转染pEGFP-N2-BMP2的P19细胞单层培养时向心肌分化在DMSO诱导时,随着诱导时间延长,实验组和对照组细胞均有聚集生长的趋势,生长缓慢。两组细胞在培养过程中都没有发现细胞跳动现象。免疫细胞化学结果:实验组细胞α-cardiac actin和cTnT在4天的时候都没有表达,在8天、12天、16天时均有表达,并随时间延长而逐渐增高,细胞中棕黄色成束丝样结构增多。阳性细胞吸光度值统计结果显示:两种蛋白在8天、12天、16天的表达量与对照组相比有显著差异(P<0.01),其他天数间比较也有显著差异(P<0.01)。对照组细胞在DMSO诱导的4天、8天、12天、16天均没有检测到α-cardiac actin和cTnT的阳性表达。RT-PCR结果:实验组,稳定表达BMP2的P19细胞在DMSO诱导下、单层培养后, GATA-4和NKx2.5在第4天开始有表达,但表达量较弱,8天、12天、16天表达逐渐增多。统计结果表明:GATA-4的表达4天、8天、12天、16天与对照组比有显著差异(P<0.01),其他天数间比较也有显著差异(P<0.01或P<0.05);NKx2.5的表达4天、8天、12天、16天与对照组比有显著差异(P<0.01),其他天数间比较除12天与16天无统计学差异(P>0.05)外也均有显著差异(P<0.01)。在对照组,各时间点均未检测到两种基因的阳性表达。Western Blot结果:实验组,稳定表达BMP2的P19细胞在DMSO诱导下、单层培养后,α-cardiac actin和cTnT在4天都没有表达,在8天的时候开始有表达,12天和16天时表达逐渐增多。统计结果显示:α-cardiac actin在4天、8天、12天、16天与对照组比有统计学差异(P<0.01或P<0.05),其他天数间比较也有统计学差异(P<0.01)。cTnT在8天的时候有表达,但与对照组比没有统计学差异(P>0.05),其他天数间比较有统计学差异(P<0.01)。对照组细胞两种蛋白均没有表达。透射电镜观察发现,实验组细胞胞质中可见较多的高尔基复合体、游离核糖体、粗面内质网、线粒体等细胞器,在一些细胞的胞质中,可见平行排列的肌丝样结构;相邻细胞分化出细胞连接,细胞连接的胞质内出现微丝样结构,未发现肌节样结构。对照组未发现分化的细胞。
结论:
1本实验成功构建了pEGFP-N2-BMP2真核表达质粒,为以后研究BMP2基因的作用奠定了基础。
2本实验将pEGFP-N2-BMP2真核表达质粒转染P19细胞,可见其在P19细胞中表达,因此,pEGFP-N2-BMP2真核表达质粒可以用于真核细胞的转染和稳定筛选。
3转染了质粒pEGFP-N2-BMP2的P19细胞不需要诱导剂的诱导,只聚集培养即可分化为心肌样细胞,表达心脏早期转录因子、心肌特异基因和心肌特异蛋白,在基因和蛋白水平表达心肌源性细胞的特异性标志物。结果表明外源表达BMP2基因可以诱导P19细胞在没有诱导剂诱导时即向心肌分化,为P19细胞有效转化为心肌细胞探索了一个比较好的诱导方式。4转染了质粒pEGFP-N2-BMP2的P19细胞不需要诱导剂的诱导,只聚集培养即可分化为心肌样细胞,表明BMP2基因在P19细胞向心肌细胞分化过程中具有促心肌分化作用,说明BMP信号在P19细胞分化为心肌细胞过程中起着非常重要的作用。
5 DMSO诱导使单层培养的稳定表达BMP2基因的P19细胞向心肌分化,表达心脏早期转录因子、心肌特异基因和心肌特异蛋白,在基因和蛋白水平表达心肌源性细胞的特异性标志物,表明BMP2基因可以促进P19细胞单层培养时向心肌细胞分化,进一步说明BMP信号在P19细胞分化为心肌细胞过程中起着非常重要的作用。
Objective: Myocardial infarction (heart attack) is greatly harmful to human health. In myocardial infarction, the loss of functional myocardial cells and the formation of scar tissue occurs, which makes the living cardiomyocytes become less and less. The loss of functional myocardial cells and the following ventricle reconstruction are the main reasons of heart failure, even death of the patients. Myocardial cells neither be regenerated nor differentiated once the damage is done. Therefore, clinical medicine treatment, interventional therapy and surgery usually do not produce satisfactory outcome. Owing to the complexity of the surgical procedure, rare donors and expensive expenditure, heart transplantation can not be generalized clinically. The key point in treating myocardial infarction is how to increase the number of functional cardiomyocytes in the infarcted area. Currently, cell transplantation is becoming a new trend to treat heard attacks. So the major challenge is to find suitable seed cells, which can substitute for dead cells to prevent heart failure and improve heart function.
The bone morphogenetic protein (BMP) belong to the transforming growth factor-βfamily. There are at least 40 members in BMP family. Up to now, more than 15 of them have been identified. Based on the homology of functional domain, it is divided into several BMP superfamilies, such as BMP 2 and 4 superfamily, BMP 5, 6, 7 and 8 superfamily, growth and differentiation factor (GDF-5), GDF-6(BMP-13) and GDF-7(BMP-12) super family, and BMP-3 and GDF-10(BMP-3b) super family, among which BMP-2 and BMP-4 super family are highly bioactive. Researches show that BMP-2 and BMP-4 are involved in regulating cardiogenesis and myocardial differentiation. Researches show that the BMP2 expression which can be detected in the heart develoment area were improved at early stage in chick embryo. Some evidence show that exogenous BMP2 protein can induce the differentiation of marrow stroma cells to cardiomyocyte. Evidence also show that BMP signal is the upper stream effector molecule of some of heart’s early transcription factor( such as GATA-4 and NKx2.5). These researches show that BMP2 plays an important role in heart development and myocardial differentiation at the early stage of embryo. However, the mechanism of BMP2 in myocardial differentiation, and especially the interaction between BMP2 and myocardial transcription factors such as NKx2.5 and GATA-4 is not unclear after a thorough literature review. Moreover, whether the exogenous expression of BMP2 can induce the differentiation of P19 cell to cardiomyocytes still remains unknow. As such, our experiment chose gene transfection technique to transfect BMP2 into P19 cell and observe the role of BMP2 in myocardial differentiation.
P19 embryonal carcinoma cells (P19ECCs) are amplified rapidly in vitro and can continue to differentiate after many generations. P19 cells can be differentiated into three different types of cells, which contains endoderm, mesoderm and ectoderm. Under different extraorgan induction conditions, it can be induced into many kinds of cells including cardiomyocytes, skeletal muscle cells and neuron. For this reason, it is a relatively ideal instrumental cell that can be used to study cell differentiation. If transplanted after extraorgan induction towards myocardial differentiation, it may become a relatively ideal seed cell to replace the necrotic cardiomyocytes. Experiments show that during the differentiation of P19 cells to cardiomyocytes, the related gene expression about cell growth and electrophysiological feature mainly imitate the myocardial growth of the normal mouse. So P19 cells are considered as a good model in vitro to study heart development and myocardial differentiation. However, during the differentiation of P19 cells to cardiomyocytes, the formation of cell aggregates and the inductor are two important and necessary processes. Up to now, it has not been clearly proved that P19 cells can differentiate to cardiomyocytes in monolayer culture. Without the formation of cell aggregates and the inductor, P19 cells can’t differentiate to cardiomyocytes.
Therefore, there are two main project in the study. First is designed to transfect pEGFP-N2-BMP2 into P19 cells, and observed the differentiation of P19 cells to cardiomyocytes without inductor. Second is to study the differentiation of P19 cells which stable expressing of BMP2 gene in monolayer culture with DMSO induction. We also would be explore a better way of P19 cells differentiate into cardiomyocytes. It would be better, if we can search for a suitable seed cell for cell transplantation in the treatment of myocardial infarction.
Methods:
1 The construction and identification of eukaryotic expression plasmid pEGFP-N2-BMP2
We used plasmid pEFSA- BMP2 as a template and transformed it into competent E. coli DH5αby the conventional CaCl2 method. The BMP2 gene sequence was amplified with PCR. Up stream primer: 5’-GCCAGATCTAAAGGTCGACCATGGT-3', the introduction of endonuclease sites BglⅡ; down stream primer: 5'-TTAGAATTCGCGACACCCACAACC-3', the introduction of endonuclease sites EcoRI. After the reaction completed, PCR products were subjected to agarose gel electrophoresis and target fragment was recovered from the agarose gel. The Coding sequence of BMP2 and vector pEGFP-N2 were digested by BglⅡand EcoRI overnight and recovered respectively. Restriction enzymesBglⅡand EcoRI were used to purify BMP2 DNA and pEGFP-N2 vector. BMP2 DNA was amplified by PCR and both the BMP2 and pEGFP-N2 will have coupled reaction through T4 DNA ligase. The ligation product was transformed into competent E. coli DH5α.cells and coated to the solid LB medium plate containing kanamycin and cultured inverted for 16-24h at 37℃. Single positive colony is chosen to extract recombinant plasmid and detect if there is target fragment through PCR, then by BamH I and HindⅢdouble digestion identification. At the same time, the recombinant plasmid pEGFP-N2-BMP2 was sent to Shanghai Sangon Co. Ltd. for sequencing identification.
2 Cardiomyocyte differentiation from P19 cells with exogenous expression BMP2 gene can be induced by cell aggregate formation.
The experimental subjects were divided into two groups- experimental group and control group. In the experimental group were P19 cells transfected with pEGFP-N2-BMP2. Before transfection, preliminary experiment was used to determine the suitable cell seeding density, the best transfection system, and the appropriate G418 selection concentration. Using the cationic liposome reagent, Lipofectamine 2000, the plasmid pEGFP-N2-BMP2 were transfected into P19 cells. After 14th days of stable selection by G418, the survival cells were suspension cultured to form aggregates for 4th days and the aggregates were transferred to the Petri dish for adherent culture. The cells were collected at 4th day, 8th day, 12th day and 16th day after adherent culture. The expression of cTnT andα-cardiac actin was detected with immunocytochemical staining method. RT-PCR was used to detect the expression of GATA-4 and NKx2.5 genes. Western blot was used to detect the expression ofα-cardiac actin and cTnT proteins. In the experimental group at 16th day after adherent culture, the ultrastructural changes of the cells were observed. In the control group, P19 cells were aggregated and cultured directly.The detection index and sampling time were same as that of experimental group.
3 Dimethylsulfoxide induced monolayer cultured P19 cells which transfected with pEGFP-N2-BMP2 to differentiate toward cardiomyocyte
There were experimental group and control group in the experiment. In the experimental group, DMSO induced monolayer cultured P19 cells which transfected with pEGFP-N2-BMP2. In the control group, DMSO induced monolayer cultured P19 cells. 1% DMSO was used to induce the cells both in experimental group and control group. Cells were collected at 4th day, 8th day, 12th day and 16th day after induction in two groups. The expression of cTnT andα-cardiac actin was detected with immunocytochemical staining method. RT-PCR was used to detect the expression of GATA-4 and NKx2.5 genes. Western blot was used to detect the expression ofα-cardiac actin and cTnT ultrastructural changes of the cells were observed. In the control group, P19 cells were induced culture with 1% DMSO directly.The detection index and sampling time of control group were same as that of experimental group.
Results:
1 Construction and identification of pEGFP-N2-BMP2 eukaryotic expression plasmid
Modeled by pEFSA- BMP2 eukaryotic expression plasmid, a gene fragment of about 1.2 kb was obtained, whose size was as the same as the human BMP2 gene encoding frame (1191bp). Vector pEGFP-N2 and BMP2 gene were digested with restriction enzyme BglⅡand EcoR I respectively. About 1.2 kb BMP2 gene fragment and 4.7kb pEGFP-N2 vector with BglⅡand EcoR I restriction enzyme sites were obtained then they were retrieved and connected. A gene fragment of about 1.2kb was obtained by PCR from the recombinant plamid after transformation and connection. Two gene fragments of about 1.2kb and 4.7 kb were obtained from the recombinant plasmid after double enzyme digesting with BamH I and HindⅢ. With CMV promoter primer for sequencing, analyzed by BLAST after proofread, the sequence result showed that the nucleotide sequence was agreed with the gene encoding sequence of BMP2 mRNA(NM-001200) in GeneBank and the endonuclease sites were complete. There was an intact Kozak sequence in the gene start area. Terminator of gene was mutated and the reading frame was correctly identified. This means BMP2 gene encoding frame has been correctly inserted in MCS of pEGFP-N2 vector. It is confirmed that recombinant plasmid pEGFP-N2-BMP2 was constructed successfully in this experiment. 2 Cardiomyocyte differentiation from P19 cells with exogenous expression BMP2 gene can be induced by cell aggregate formation.
The experiment confirmed that the stable selection concentration of G418 was 600μg/ml and the appropriate cell seeding density for transfection was 2×105/ml. The best transfection system was the plasmid DNA(μg):Lipofectamine2000 (μl)=1:2.5. In the experimental group, at the 24th hour after transfection with plasmid pEGFP-N2-BMP2, some cells expressed the green fluorescence. Most cells expressed green fluorescence after being selected with 600μg/ml G418 two weeks later, which were P19 cells with exogenous expression BMP2 gene. Cells in experimental group and control group were collected and cultured for 4th days to form aggregates. After adhesion of the aggregates, some cells grew out and formed outgrowth around the aggregates. In the outgrowth, the cell body grew bigger and longer, and evections reach out and connected with each other. Immunocytochemical staining results showed that, in experimental group, at 4thday two kinds of antibody,α-cardiac actin and cTnT, were not expressed. At 8th day, they were all expressed in a little amount. At 12th day and 16th day, two kinds of antibody were all expressed, and the expression was gradually increased. Brown positive reaction products were in cytoplasm. With time going by, there was obviously brown filament-like structure in the cytoplasm of the cells at 12th day and 16th day. The statistical results of positive cell absorbance value showed that there was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference(P<0.01)between 8th day, 12th day and 16th day in experimental group. Positive expression of the two antibodies was not detected in control group. RT-PCR analysis showed that, in experimental group, GATA-4 and Nkx2.5 expressed at 4th day after adherent culture, but it is deficient in quantity. At 8th day, 12th day and 16th day, the expression of GATA-4 and Nkx2.5 was gradually increased. The statistical result showed that there was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference(P<0.01) between 8th day, 12th day and 16th day in experimental group. No positive expressions of the two kinds of genes was detected in control group. Western Blot result showed that, in experimental group, at 4thdayα-cardiac actin and cTnT, were not expressed. At 8th day, they were all expressed in a little amount. At 12th day and 16th day they were all expressed, and the expression was gradually increased. The statistical result
showed that there was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference (P<0.01) between 8th day, 12th day and 16th day in experimental group. Positive expression of the two proteins was not detected in control group. The observation of ultrastructure showed that after aggregation and adherent culture for 16th days, some cells became elongated cylindrical and there appeared cell junction between the adjacent cells in experimental group. Some microfilament-like structure appeared beside the cell junction. There was some dense myofilament-like structure arranged in parallel way in cytoplasm of some cells,but sarcomere-like structure was not founed. no differentiated cell was found in control group.
3 Dimethylsulfoxide induced monolayer cultured P19 cells which transfected with pEGFP-N2-BMP2 to differentiate toward cardiomyocyte
Cells both of experimental group and control group induced monolayer culture with 1% DMSO and grew slowly compared with the P19 cells without inducing. And cells accumulated in a state similar to adherent aggregates-like structure as increased in number. The cells in the center of the aggregates were small and dense, while the periphery cells became longer, crawling and radiating out in all directions with cell body elongated to be fusiformis and stretched processes. Beating cells were not observed in either of the two groups during culture. Immunocytochemical staining result showed that, in experimental group, at 4thday after being induced by 1% DMSOα-cardiac actin and cTnT were not expressed. At 8th day, they were all expressed in a little amount. At 12th day and 16th day two kinds of antibody were all expressed, and the expression was gradually increased. Brown positive reaction products were in cytoplasm. With time going by, there was obviously brown filament-like structure in the cytoplasm of the cells at 12th day and 16th day. The statistical result of positive cell absorbance value showed that there was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference (P<0.01) between 8th day, 12th day and 16th day in experimental group. Positive expression of the two kinds of antibody was not detected in control group. RT-PCR analysis showed that, in experimental group, GATA-4 and Nkx2.5 expressed at 4th day after induced by DMSO, but it is deficient in quantity. At 8th day, 12th day and 16th day the expression of two genes was gradually increased. The statistical result showed that the expression of GATA-4 was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference (P<0.01 or P<0.05 ) between 8th day, 12th day and 16th day in experimental group. the expression of Nkx2.5 was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, and in experimental group there was also significant difference (P<0.01 ) between 8th day, 12th day and 16th day, but there was no significant difference(P>0.05)between 12th day and 16th day. No positive expressions of the two kinds of genes was detected in control group. Western Blot result showed that, in experimental group, at 4thdayα-cardiac actin and cTnT were not expressed. At 8th day they were all expressed in a little amount. At 12th day and 16th day they were all expressed, and the expression was gradually increased. The statistical result showed that the expression ofα-cardiac actin was distinct difference (P<0.01 or P<0.05) at 8th day, 12th day and 16th day between the experimental group and control group, and there was also significant difference (P<0.01) between 8th day, 12th day and 16th day in experimental group. the expression of cTnT was distinct difference (P<0.01) at 8th day, 12th day and 16th day between the experimental group and control group, but there was no significant difference (P>0.05 ) between 8th day and control group. And there was also significant difference (P<0.01) between 8th day, 12th day and 16th day in experimental group. Positive expression of the two proteins was not detected in control group. The observation of ultrastructure showed that after induced by DMSO culture for 16th days, some cells became elongated cylindrical and there appeared cell junction between the adjacent cells in experimental group. Some microfilament-like structure appeared beside the cell junction. There was some dense myofilament-like
structure arranged in parallel way in cytoplasm of some cells, but sarcomere-like structure was not founed. no differentiated cell was found in control group.
Conclusions:
1 The eukaryotic expression plasmid pEGFP-N2-BMP2 was successfully constructed, which would lay a foundation for the further studies on the role of BMP2 in myocardial differentiation and heart development.
2 Eukaryon expression plasmid pEGFP-N2-BMP2 can be expressed in P19 cells and can be used for the transfection and stable selection of eukaryotic cells.
3 Without inducing agents, exogenous expression of BMP2 gene induced P19 cells to differentiate towards cardiomyocytes only with aggregates formation. P19 cells transfected with pEGFP-N2-BMP2 induced the expression of cardiac-specific markers at the level of gene and protein which increased gradually with prolongation of culture time. It shows that exogenous expression of BMP2 gene can induce myocardial differentiation from P19 cells. This study would explore a reasonable induction way for myocardial differentiation from P19 cells .
4 Without inducing agents, exogenous expression of BMP2 gene induced P19 cells to differentiate towards cardiomyocytes only with aggregates formation. This shows that BMP2 gene can promote myocardial differentiation from P19 cells and indicates that BMP signal path plays a very important role in the process of myocardial differentiation from P19 cells.
5 With monolayer culture and induced by DMSO, P19 cells which transfected with pEGFP-N2-BMP2 can differentiate towards cardiomyo- cytes, which show the expression of cardiac-specific markers at the level of gene and protein. It suggests that BMP2 gene can promote P19 cells to differentiate towards cadiocyte under monolayer culture condition. It is proved further that BMP2 signal path plays a very important role in the process of myocardial differentiation from P19 cells.
引文
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