胚胎干细胞来源平滑肌细胞的纯化及功能研究
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摘要
背景:平滑肌细胞(smooth muscle cell, SMC)是构成血管主要的细胞成分之一。无论在生理情况下,还是在动脉粥样硬化、高血压、癌症等病理情况下,SMC都发挥重要的作用。与骨骼肌或心肌细胞这些“终末分化”细胞不同的是,SMC在不同的分化阶段、甚至在成体器官中仍然具有明显的可塑性,并能够发生表型转换。SMC从静止、收缩的分化表型转变为增殖和分泌功能活跃的去分化表型被认为是许多SMC相关疾病重要的发病机制之一。全面理解SMC正常发育、成熟和分化的调控机制不仅对于认识血管疾病发生发展的关键环节、阐明其发病机制并为这些疾病的治疗提供新的靶点至关重要,而且还有助于加深对先天血管发育缺陷性疾病的理解。此外,阐明SMC正常发育机制还可能为SMC相关性疾病的细胞治疗、血管组织工程和血管组织构建的治疗学奠定基础。但是要达到上述目的,首先亟须获得能够再现平滑肌正常发育及成熟过程的SMC。
然而到目前为止,由于缺乏理想的体内及体外模型,导致人们对SMC发育及成熟机制仍知之甚少。例如,从成体组织分离的SMC虽然被广泛应用于表型转换的机制研究,但是这些细胞在体外培养时既不能再现正常的SMC分化,也不能维持收缩这一SMC特性,因此限制了其在分化研究中的应用。在其他模型中,虽然可以通过添加不同的诱导剂诱导10T1/2细胞、神经嵴来源的MONC-1细胞、P19胚胎癌细胞或成体干细胞分化形成表达多种SMC标志物的细胞,但是由于这些细胞并不是SMC真正的前体细胞,且分化形成的SMC没能体现出成熟SMC确切的功能特性(如收缩功能),因此这些细胞向SMC分化的过程是否与在体情况相似仍不确定。
胚胎干细胞(embryonic stem cells, ESC)是从胚胎早期内细胞团分离得到的全能干细胞,具有分化为三个胚层来源细胞的能力。在体外悬浮培养时,ESC能够自发分化形成囊状结构的胚胎小体(embryoid body,EB),EB中包含了三个胚层来源的细胞。有研究表明EB可形成可见的自发收缩的SMC,这表明ESC来源的SMC可以再现SMC的分化和成熟过程。就这点而言,ESC-EB系统似乎是较为理想的研究SMC分化的模型。但值得注意的是,这一模型也有其不足之处。ESC的多能性虽然为多种功能细胞提供了利于其分化的环境因素,但当对某种特定细胞类型(如SMC)进行研究时,其他非兴趣细胞的“污染”反而阻碍了研究的进行。解决这一难题的关键在于将我们所要研究的细胞类型(本研究中为SMC)从ESC分化形成的多种细胞类型中分离纯化出来。
目的:利用平滑肌特异性启动子驱动的嘌呤霉素抗性(puromycin acetyl transferase, pac)基因及增强型绿色荧光蛋白(enhanced green fluorescence protein, EGFP)基因双表达载体,通过抗性筛选纯化ESC来源的SMC,并进一步研究其功能。
方法:
1.平滑肌特异性SM22α启动子调节的pac基因及EGFP双表达载体构建
(1)SM22α启动子克隆:从小鼠基因组中用PCR法扩扩增541 bp的SM22α启动子,引物两端加AseI/NheI酶切位点及保护性碱基。上游引物:AGTTATATTAATTTTGCATAGTGCCTGGTTG ;下游引物: GCGCTAGCTA CAAGGCTTGGTCGTTTG。将SM22α启动子序列克隆到pMD19-T Simple载体扩增,然后用AseI/NheI双酶切获得SM22α启动子片段。AseI/NheI双酶切去掉pIRES2-EGFP中的CMV启动子。将SM22α启动子插入得到中间载体pSM22α-IRES2-EGFP。(2)Pac基因获取:HindIII/ClaI双酶切载体pSM2C得到663 bp的pac基因,将其亚克隆到pSUPER.basic中得到pSUPER-PAC。(3)BglII/AccI双酶切pSUPER-PAC获得pac基因并将其插入到pSM22-IRES2-EGFP中,构建pSM22α-PAC-IRES2-EGFP,经酶切鉴定成功的载体送测序。将测序成功的载体转染小鼠微血管内皮细胞(endothelial cell, EC)系SVEC及SMC,验证该载体是否有效。
2. ESC培养及pSM22α-IRES2-EGFP转基因ESC制备
获取孕12.5-14.5d小鼠胚胎制备原代小鼠胚胎成纤维细胞(mouse embryonic fibroblast, MEF),使用前用丝裂霉素C处理2 h,作为ESC的饲养层细胞。小鼠ESC细胞株R1常规培养于饲养层细胞MEF上。每天换液,隔天进行传代。转染前,将ESC传代到0.1%明胶包被的无饲养层培养皿中。将线性化的pSM22α-PAC-IRES2-EGFP质粒DNA用脂质体转染ESC。5 h后换ESC培养液培养24 h,然后用含有500μg/ml G418的ESC培养液筛选。共筛选约2周,传代1次。挑取阳性克隆进行扩增。制作EB观察有无EGFP表达,对于有EGFP表达的克隆进一步应用RT-PCR扩增pac基因转录子鉴定ESC转染是否成功。鉴定转染成功的ESC克隆被命名为SPIE-ESC。
3. EB培养、诱导分化及SMC纯化
将SPIE-ESC消化成单细胞悬液,接种到细菌培养皿中悬浮培养5 d,形成EB。第6 d将EB接种到明胶包被的组织培养皿中使其贴壁分化,分化培养基中添加10-8 mol/L全反式维甲酸(all-trans retinoic acid,ATRA)诱导分化5 d,每天换液。第11 d时用10μg/ml嘌呤霉素筛选诱导分化细胞3 d。筛选后存活的细胞重新接种到新的培养皿中。筛选到的细胞被命名为SM22α-SMC。流式细胞仪分析筛选所得细胞的纯度。
4.流式细胞仪测定SM22α-SMC纯度
将未筛选的SPIE-ESC和SM22α-SMC消化成单细胞并用70μm尼龙网过滤。用流式细胞仪获取10 000个细胞,结果用CellQuest软件分析。以正常ESC作为阴性对照。
5.免疫细胞化学
将SM22α-SMC接种到0.1%明胶包被的盖玻片上。4%多聚甲醛固定,0.2% TritonX-100通透,5% BSA封闭。一抗为小鼠抗SMα-actin,兔抗平滑肌肌球蛋白重链(smooth muscle myosin heavy chain, SM-MHC)及兔抗SM22α。二抗为相应的四甲基异硫氰酸罗丹明(tetramethyl rhodamine iso-thiocyanate, TRITC)标记的IgG。DAPI染核后封片并在荧光显微镜下观察染色情况。
6. Western blot分析
搜集大鼠原代主动脉SMC、未经筛选的SPIE-ESC分化细胞及SM22α-SMC并裂解。离心后取上清。BCA法测定蛋白浓度。等量上样后经10% SDS-PAGE胶电泳分离。湿法转膜至PVDF膜。5%脱脂奶粉封闭非特异性结合位点。一抗为小鼠抗SMα-actin,兔抗平滑肌肌球蛋白重链(smooth muscle myosin heavy chain, SM-MHC)及兔抗SM22α。二抗为辣根过氧化物酶标记的相应IgG。用增强化学发光试剂盒显影。
7. SMC收缩功能测定
将纯化的SM22α-SMC在0.1 %明胶包被的盖玻片上培养24h。更换新鲜培养液,并向培养液中加入卡巴可,使其终浓度为含10μmol/L。从加入卡巴可时开始采集图像,每隔30 s采集一张,总观察时间30 min。计算收缩面积百分比并与阴性对照SVEC比较。
8.利用Matrigel测定SMC募集功能
用Matrigel包被盖玻片并将其置于24孔板中,将等量小鼠微血管内皮细胞SVEC及SM22α-SMC(各1×104个)混合培养于Matrigel上。12 h或60 h荧光显微镜下观察SMC的募集情况。1×104个SVEC单独培养用于观察管腔样结构形成。
9.统计学分析
所有实验均重复至少3次。用SPSS 11.1软件包进行统计学分析。计量资料以均数±标准差表示。两组间比较采用Student’s t检验。p< 0.05被认为差异有显著性。
结果:
1. pSM22α-PAC-IRES2-EGFP载体构建
测序结果证实载体构建成功。该载体在SMC中能驱动EGFP表达,而在SVEC中不能驱动EGFP表达,表明该载体有效且特异。
2. SPIE-ESC建立及ESC来源SM22α-SMC的纯化
G418筛选2周后,挑取并扩增仍然存活的ESC克隆。RT-PCR法检测pac基因转录鉴定转染是否成功。共有4株克隆被证明成功转染pSM22α-PAC- IRES2-EGFP。ATRA诱导分化后荧光显微镜下可见EGFP表达的SM22α-SMC。SM22α-SMC位于贴壁分化EB的周边部位。在没有进行嘌呤霉素筛选之前,可以看见典型的呈未分化形态的细胞集落。而经过嘌呤霉素筛选3 d后,仅剩下EGFP阳性的SM22α-SMC。这些细胞呈梭形,与大鼠主动脉SMC相似。
3.筛选后的SM22α-SMC纯度达到99%
未经筛选的ATRA诱导分化10 d的SPIE-ESC经流式细胞仪测定,EGFP表达阳性的SM22α-SMC为40.05%;经ATRA诱导分化10 d及嘌呤霉素筛选3 d的SPIE-ESC,EGFP表达阳性的SM22α-SMC为98.99%。
4. SM22α-SMC表达平滑肌特异性标志物
免疫荧光染色结果显示SM22α-SMC表达平滑肌特异性标志物SMα-actin和SM-MHC。且所有细胞SM22α染色强阳性。细胞呈SMC样形态,具有发育良好的肌动蛋白应力纤维网。免疫印迹分析显示,纯化的SM22α-SMC组SM22α蛋白表达水平与大鼠主动脉SMC组相当,并高于未纯化组;纯化的SM22α-SMC组SM-MHC的表达水平高于未纯化组,但低于大鼠主动脉SMC组;三组SMα-actin表达水平相当。
5.卡巴可刺激SM22α-SMC收缩
卡巴可处理后,SM22α-SMC收缩明显,细胞面积减少28±6.4%。而SVEC对卡巴可无反应。
6. SM22α-SMC能够整合到Matrigel中形成的管腔样结构中
SVEC单独培养于Matrigel上在12 h及60 h可以形成管腔样结构。SM22α-SMC/SVEC共培养在同一时间点形成相似结构。荧光显微镜下可见EGFP表达阳性SM22α-SMC募集到SVEC形成的管腔样结构边缘。
结论:首次成功构建了SM22α启动子驱动的pac基因及EGFP双表达载体。利用该载体成功筛选出高纯度、ESC来源的有收缩及募集功能的SMC。
Background: As a major cellular component of blood vessels, smooth muscle cells (SMC) play an important role under physiological conditions as well as in a large number of human diseases, including atherosclerosis, hypertension and cancer. Unlike either skeletal or cardiac muscle those are terminally differentiated, SMC possess remarkable plasticity and undergo phenotypic switch at different stages of development, even in adult organisms. The phenotypic switch from a quiescent, contractile differentiation state to a highly proliferative, synthetic dedifferentiation state of SMC is believed to contribute to pathogenesis of many SMC-related diseases. An full understanding of the normal regulation of SMC development, maturation and differentiation will not only provide the foundation for elucidating how these processes may be disrupted in vascular disease, accelerating appreciation of pathogenesis and providing novel therapeutic targets, but will also be critical to understanding congenital defects in vascular development. In addition, an appreciation of normal SMC developmental mechanisms may also contribute to novel cell-based therapies for SMC-related diseases as well as for tissue engineering and reconstruction. To achieve the goals mentioned above, SMC recapitulating normal development and maturation are badly needed.
Unfortunately however, the mechanisms underlying SMC development and maturation are poorly understood partly due to the lack of good in vitro and in vivo models. For example, SMC separated from adult tissues, which are widely used in mechanism study for phenotype switch, neither recapitulate normal SMC differentiation nor maintain contractile function when cultured in vitro, so their usefulness in differentiation study is limited. Other models like induction of 10T1/2 cells, neural crest-derived MONC-1 cells, P19 embryonal carcinoma cells and even adult stem cells by different agents produce cells expressing several SMC markers, but whether the cells undergo differentiation similar to in vivo process are questionable due to their controversial origin.
Embryonic stem cell (ESC) lines have been previously established from the inner cell mass of blastocysts and have been shown to have the potential to generate all embryonic cell lineages when they undergo differentiation. When cultured suspendedly, ESC spontaneously differentiates into cyst-like structures, termed embryoid bodies (EB), which contain derivatives of the three primitive germ layers. EB have been shown to form regions of visibly spontaneously contractile SMC which indicates SMC derived from ESC recapitulate SMC differentiation and maturation, so it seems that ESC-EB system is an ideal model for differentiation study of SMC. But this model also has a defect, pluripotency of ESC not only provide environmental clues favoring development of various functional cell types, but also make study of specific cell type (like SMC) difficult due to“contamination”of other non-interested cells derived from ESC. To overcome all these disadvantages, purification of functional SMC from background of all cell type derived from ESC is needed.
Objective: To develop a novel method to obtain purified population of SMC derived from ESC by using puromycin resistance gene expressing vector driven by smooth muscle specific promoter, and then to assess the biological function of these cells.
Methods:
1. Construction of plasmid encoding puromycin resistance gene (also known as puromycin acetyl transferase gene or pac gene) and enhanced green fluorescence protein (EGFP) under the control of SM22αpromoter
(1) Clone of SM22αpromoter: The 541bp SM22αpromoter that works specifically in smooth muscle lineage cells was amplified from mouse genomic DNA using PCR with forward primer: AGTTATATTAATTTTGCATAGTGCCTGGTTG; and reverse primer: GCGCTAGCTACAAGGCTTGGTCGTTTG. The PCR product was cloned into the pMD18-T Simple vector, and was subsequently excised with AseI/NheI and subcloned into the same sites of the pIRES2-EGFP vector, removing the CMV promoter. The intermediate vector was termed pSM22α-IRES2-EGFP. (2) Clone of pac gene: The 663 bp pac gene was excised from pSM2C with HindIII/ClaI and subcloned into pSUPER.basic to produce pSUPER-PAC. (3)By using enzyme BglII/AccI, pac gene was cut from pSUPER-PAC and subsequently inserted into pSM22α-IRES2-EGFP to construct pSM22α-PAC-IRES2-EGFP, which encodes pac gene and EGFP under the control of SM22αpromoter. The constructed vectors were identified by DNA sequencing and further verified in both murine microvascular endothelial cell (EC) line SVEC and SMC to see whether the promoter can work.
2. Culture of ESC and generation of transgenic ESC
Feeder cells, mouse embryonic fibroblasts (MEF), were prepared from mouse fetuses harvested between day 12.5-14.5d of gestation. Before use, MEF were treated with Mitomycin C for 2 hours. Mouse ESC R1 were routinely cultured on treated MEF. Cells were fed every day and replated every second day. Before transfection, ESC trypsinized to single cell suspension were cultured on plate coated by 0.1% gelatin without feeder layer. Then ESC were transfected with linearized pSM22α-PAC- IRES2-EGFP plasmid DNA using lipofectamine for 5 hours. Transgenic ESC clones were selected by culture medium with 500 ug/mL G418 24 hours after transfection. Colonies derived from single cells under G418 selection were picked and amplified and screened for the presence of the pac gene by reverse transcription polymerase chain reaction (RT-PCR). Transgenic ESC were termed as SPIE-ESC.
3. EB Culture, induced differentiation and SMC purification
To generate EB, SPIE-ESC were typsinized to single cells and cultured in suspension in a petri dish with differentiation medium without LIF. On day 6, suspended EB were plated onto a surface coated with 0.1% gelatin in differentiation medium supplemented with 10 nmol/L all-trans retinoic acid (ATRA). The culture was continued for 5 days with a daily change of fresh ATRA-containing differentiation medium. On day 11, SMC were selected by differentiation medium containing 10μg/mL puromycin for 3 days. The dead cells were removed by a PBS washing every day after selection. The sorted cells were replated in a new plate after selection. The purified SMC were termed as SM22α-SMC. The homogeneity of the population was determined by flow cytometry.
4. Flow cytometry
Unsorted SPIE-ESC and sorted SM22α-SMC were trypsinized and passed through a 70μm filter to obtain single cell suspension. Acquisition of 10 000 events was made with FACScan and data analysis was done with CellQuest software. The cells derived from normal ESC were used as a negative control.
5. Immunocytochemistry
SM22α-SMC were plated on 0.1% gelatin-coated cover slips and cultured in differentiation medium. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 5% BSA. Primary antibodies were mouse anti-SMα-actin and rabbit anti-SM myosin heavy chain (SM-MHC) and rabbit anti-SM22α. After cells incubated with corresponding tetramethyl rhodamine iso-thiocyanate (TRITC) conjugated secondary antibodies and counterstained with DAPI, cover slips were sealed and examined by fluorescence microscopy.
6. Western blot analysis
Rat primary aortic SMC, unsorted cells derived from ESC and sorted SM22α-SMC were disrupted in lysis buffer. Samples were centrifuged and clear supernatants were collected. Protein concentration was determined using BCA protein assay kit. Equal amounts of proteins were run on a 10% SDS-PAGE gel and transferred to a PVDF membrane. Nonspecific binding sites were blocked by incubation with 5% nonfat dry milk in TBS-T. The primary antibodies used for Western analysis were as follows: mouse anti-SMα-actin, rabbit anti-SM22α, and rabbit anti-SM MHC. The specific binding was detected with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagents.
7. Contractile assay
Purified SM22α-SMC were plated onto 0.1 % gelatin coated cover slips with differentiation medium. After 24 hours, fresh medium was replenished and added with Carbachol at a final concentration of 10μmol/L. Pictures were acquired at 30 second intervals for 15 min. Average cell area reduction of 10 SM22α-SMC were calculated and compared to negative control SVEC.
8. Matrigel assay of SMC recruitment
Equal amount of 1×104/mL murine microvascular endothelial cell line SVEC and sorted SM22α-SMC were mixed and plated on cover slips coated by Matrigel (BD) with differentiation medium. SMC localization was examined by fluorescence microscope at 12h or 60 h. SVEC cultured alone on Matrigel were used to observe the tube like structure formation.
9. Statistical analysis
All experiments were performed in duplicates or triplicates and were repeated at least three times. Data analyses were performed using SPSS 11.1 software. Quantitative data were presented as means±standard deviation (SD). Differences between two groups were analyzed by two-tailed Student’s t test and were considered significant when p< 0.05.
Results:
1. Construction of plasmid vector pSM22α-PAC-IRES2-EGFP
Results of DNA sequencing showed that the plasmid vector was successfully constructed. Further verification of the vector in EC and SMC showed that it works in SMC but not in SVEC.
2. Generation of SPIE-ESC and isolation of ESC Derived SM22α-SMC
After 2 weeks selection with G418, positive ESC colonies were picked and amplified. Detection of pac gene transcription by RT-PCR showed that four colonies were successfully transfected with pSM22α-PAC-IRES2-EGFP. ATRA induced differentiation of transgenic ESC to EGFP expressing SM22α-SMC detected by fluorescence microscope. SM22α-SMC located peripherally in the EB outgrowth. In the absence of puromycin, cell colonies with the typical cell morphology of undifferentiated cells remained. In contrast, when cells were treated with puromycin for an additional 3 days, only EGFP expressing SM22α-SMC were observed. These SMC showed a spindle like shape similar to rat aortic SMC.
3. Selected SM22α-SMC has a high purity of 99%
The amount of EGFP expressing SM22α-SMC in the 10-day-old differentiated SPIE-ESC in the presence of ATRA, or in the presence of both ATRA (for 10 days) and puromycin (for 3 days) as quantified by fluorescence-activated cell sorter analysis was 40.05% and 98.99% respectively.
4. Expression of SM22α-SMC with smooth muscle specific markers
Immunofluorescence studies of the selected cells showed that SM22α-SMC stained for the SMC selective markers, SMα-actin and SM-MHC. All the SM22α-SMC stained strongly for SM22α. The cells displayed SMC-like morphology and appearance with a well-developed actin stress fiber network. Immunoblotting analyses revealed that the levels of SM22αproteins in purified SM22α-SMC group were higher than unpurified group, while comparable to rat aortic SMC group. The highly selective SMC protein, SM-MHC, expressed in the purified cell population after puromycin selection were higher than unpurified group and a little lower than rat aortic SMC group. The levels of SMα-actin are similar in three groups.
5. SM22α-SMC contracted in response to carbachol
SM22α-SMC showed significant contraction after carbachol treatment, with an average of 28±6.4% cell area reduction. No obvious response to carbachol was observed on SVEC.
6. SM22α-SMC integrated into tube like structure in a Matrigel assay
SVEC seeded alone on Matrigel formed tube like structures after either 12 h or 60 h culture. SM22α-SMC/SVECs formed similar structures at the same time points observed under phase contrast microscope. Under fluorescence microscope, EGFP expressing SM22α-SMC were seen to integrated into the tube like structures formed by SVEC.
Conclusions: By using a novel vector expressing pac gene and EGFP under control of smooth muscle specific SM22αpromoter, we successfully obtained highly purified and functional SMC derived from ESC.
然而到目前为止,由于缺乏理想的体内及体外模型,导致人们对SMC发育及成熟机制仍知之甚少。例如,从成体组织分离的SMC虽然被广泛应用于表型转换的机制研究,但是这些细胞在体外培养时既不能再现正常的SMC分化,也不能维持收缩这一SMC特性,因此限制了其在分化研究中的应用。在其他模型中,虽然可以通过添加不同的诱导剂诱导10T1/2细胞、神经嵴来源的MONC-1细胞、P19胚胎癌细胞或成体干细胞分化形成表达多种SMC标志物的细胞,但是由于这些细胞并不是SMC真正的前体细胞,且分化形成的SMC没能体现出成熟SMC确切的功能特性(如收缩功能),因此这些细胞向SMC分化的过程是否与在体情况相似仍不确定。
胚胎干细胞(embryonic stem cells, ESC)是从胚胎早期内细胞团分离得到的全能干细胞,具有分化为三个胚层来源细胞的能力。在体外悬浮培养时,ESC能够自发分化形成囊状结构的胚胎小体(embryoid body,EB),EB中包含了三个胚层来源的细胞。有研究表明EB可形成可见的自发收缩的SMC,这表明ESC来源的SMC可以再现SMC的分化和成熟过程。就这点而言,ESC-EB系统似乎是较为理想的研究SMC分化的模型。但值得注意的是,这一模型也有其不足之处。ESC的多能性虽然为多种功能细胞提供了利于其分化的环境因素,但当对某种特定细胞类型(如SMC)进行研究时,其他非兴趣细胞的“污染”反而阻碍了研究的进行。解决这一难题的关键在于将我们所要研究的细胞类型(本研究中为SMC)从ESC分化形成的多种细胞类型中分离纯化出来。
目的:利用平滑肌特异性启动子驱动的嘌呤霉素抗性(puromycin acetyl transferase, pac)基因及增强型绿色荧光蛋白(enhanced green fluorescence protein, EGFP)基因双表达载体,通过抗性筛选纯化ESC来源的SMC,并进一步研究其功能。
方法:
1.平滑肌特异性SM22α启动子调节的pac基因及EGFP双表达载体构建
(1)SM22α启动子克隆:从小鼠基因组中用PCR法扩扩增541 bp的SM22α启动子,引物两端加AseI/NheI酶切位点及保护性碱基。上游引物:AGTTATATTAATTTTGCATAGTGCCTGGTTG ;下游引物: GCGCTAGCTA CAAGGCTTGGTCGTTTG。将SM22α启动子序列克隆到pMD19-T Simple载体扩增,然后用AseI/NheI双酶切获得SM22α启动子片段。AseI/NheI双酶切去掉pIRES2-EGFP中的CMV启动子。将SM22α启动子插入得到中间载体pSM22α-IRES2-EGFP。(2)Pac基因获取:HindIII/ClaI双酶切载体pSM2C得到663 bp的pac基因,将其亚克隆到pSUPER.basic中得到pSUPER-PAC。(3)BglII/AccI双酶切pSUPER-PAC获得pac基因并将其插入到pSM22-IRES2-EGFP中,构建pSM22α-PAC-IRES2-EGFP,经酶切鉴定成功的载体送测序。将测序成功的载体转染小鼠微血管内皮细胞(endothelial cell, EC)系SVEC及SMC,验证该载体是否有效。
2. ESC培养及pSM22α-IRES2-EGFP转基因ESC制备
获取孕12.5-14.5d小鼠胚胎制备原代小鼠胚胎成纤维细胞(mouse embryonic fibroblast, MEF),使用前用丝裂霉素C处理2 h,作为ESC的饲养层细胞。小鼠ESC细胞株R1常规培养于饲养层细胞MEF上。每天换液,隔天进行传代。转染前,将ESC传代到0.1%明胶包被的无饲养层培养皿中。将线性化的pSM22α-PAC-IRES2-EGFP质粒DNA用脂质体转染ESC。5 h后换ESC培养液培养24 h,然后用含有500μg/ml G418的ESC培养液筛选。共筛选约2周,传代1次。挑取阳性克隆进行扩增。制作EB观察有无EGFP表达,对于有EGFP表达的克隆进一步应用RT-PCR扩增pac基因转录子鉴定ESC转染是否成功。鉴定转染成功的ESC克隆被命名为SPIE-ESC。
3. EB培养、诱导分化及SMC纯化
将SPIE-ESC消化成单细胞悬液,接种到细菌培养皿中悬浮培养5 d,形成EB。第6 d将EB接种到明胶包被的组织培养皿中使其贴壁分化,分化培养基中添加10-8 mol/L全反式维甲酸(all-trans retinoic acid,ATRA)诱导分化5 d,每天换液。第11 d时用10μg/ml嘌呤霉素筛选诱导分化细胞3 d。筛选后存活的细胞重新接种到新的培养皿中。筛选到的细胞被命名为SM22α-SMC。流式细胞仪分析筛选所得细胞的纯度。
4.流式细胞仪测定SM22α-SMC纯度
将未筛选的SPIE-ESC和SM22α-SMC消化成单细胞并用70μm尼龙网过滤。用流式细胞仪获取10 000个细胞,结果用CellQuest软件分析。以正常ESC作为阴性对照。
5.免疫细胞化学
将SM22α-SMC接种到0.1%明胶包被的盖玻片上。4%多聚甲醛固定,0.2% TritonX-100通透,5% BSA封闭。一抗为小鼠抗SMα-actin,兔抗平滑肌肌球蛋白重链(smooth muscle myosin heavy chain, SM-MHC)及兔抗SM22α。二抗为相应的四甲基异硫氰酸罗丹明(tetramethyl rhodamine iso-thiocyanate, TRITC)标记的IgG。DAPI染核后封片并在荧光显微镜下观察染色情况。
6. Western blot分析
搜集大鼠原代主动脉SMC、未经筛选的SPIE-ESC分化细胞及SM22α-SMC并裂解。离心后取上清。BCA法测定蛋白浓度。等量上样后经10% SDS-PAGE胶电泳分离。湿法转膜至PVDF膜。5%脱脂奶粉封闭非特异性结合位点。一抗为小鼠抗SMα-actin,兔抗平滑肌肌球蛋白重链(smooth muscle myosin heavy chain, SM-MHC)及兔抗SM22α。二抗为辣根过氧化物酶标记的相应IgG。用增强化学发光试剂盒显影。
7. SMC收缩功能测定
将纯化的SM22α-SMC在0.1 %明胶包被的盖玻片上培养24h。更换新鲜培养液,并向培养液中加入卡巴可,使其终浓度为含10μmol/L。从加入卡巴可时开始采集图像,每隔30 s采集一张,总观察时间30 min。计算收缩面积百分比并与阴性对照SVEC比较。
8.利用Matrigel测定SMC募集功能
用Matrigel包被盖玻片并将其置于24孔板中,将等量小鼠微血管内皮细胞SVEC及SM22α-SMC(各1×104个)混合培养于Matrigel上。12 h或60 h荧光显微镜下观察SMC的募集情况。1×104个SVEC单独培养用于观察管腔样结构形成。
9.统计学分析
所有实验均重复至少3次。用SPSS 11.1软件包进行统计学分析。计量资料以均数±标准差表示。两组间比较采用Student’s t检验。p< 0.05被认为差异有显著性。
结果:
1. pSM22α-PAC-IRES2-EGFP载体构建
测序结果证实载体构建成功。该载体在SMC中能驱动EGFP表达,而在SVEC中不能驱动EGFP表达,表明该载体有效且特异。
2. SPIE-ESC建立及ESC来源SM22α-SMC的纯化
G418筛选2周后,挑取并扩增仍然存活的ESC克隆。RT-PCR法检测pac基因转录鉴定转染是否成功。共有4株克隆被证明成功转染pSM22α-PAC- IRES2-EGFP。ATRA诱导分化后荧光显微镜下可见EGFP表达的SM22α-SMC。SM22α-SMC位于贴壁分化EB的周边部位。在没有进行嘌呤霉素筛选之前,可以看见典型的呈未分化形态的细胞集落。而经过嘌呤霉素筛选3 d后,仅剩下EGFP阳性的SM22α-SMC。这些细胞呈梭形,与大鼠主动脉SMC相似。
3.筛选后的SM22α-SMC纯度达到99%
未经筛选的ATRA诱导分化10 d的SPIE-ESC经流式细胞仪测定,EGFP表达阳性的SM22α-SMC为40.05%;经ATRA诱导分化10 d及嘌呤霉素筛选3 d的SPIE-ESC,EGFP表达阳性的SM22α-SMC为98.99%。
4. SM22α-SMC表达平滑肌特异性标志物
免疫荧光染色结果显示SM22α-SMC表达平滑肌特异性标志物SMα-actin和SM-MHC。且所有细胞SM22α染色强阳性。细胞呈SMC样形态,具有发育良好的肌动蛋白应力纤维网。免疫印迹分析显示,纯化的SM22α-SMC组SM22α蛋白表达水平与大鼠主动脉SMC组相当,并高于未纯化组;纯化的SM22α-SMC组SM-MHC的表达水平高于未纯化组,但低于大鼠主动脉SMC组;三组SMα-actin表达水平相当。
5.卡巴可刺激SM22α-SMC收缩
卡巴可处理后,SM22α-SMC收缩明显,细胞面积减少28±6.4%。而SVEC对卡巴可无反应。
6. SM22α-SMC能够整合到Matrigel中形成的管腔样结构中
SVEC单独培养于Matrigel上在12 h及60 h可以形成管腔样结构。SM22α-SMC/SVEC共培养在同一时间点形成相似结构。荧光显微镜下可见EGFP表达阳性SM22α-SMC募集到SVEC形成的管腔样结构边缘。
结论:首次成功构建了SM22α启动子驱动的pac基因及EGFP双表达载体。利用该载体成功筛选出高纯度、ESC来源的有收缩及募集功能的SMC。
Background: As a major cellular component of blood vessels, smooth muscle cells (SMC) play an important role under physiological conditions as well as in a large number of human diseases, including atherosclerosis, hypertension and cancer. Unlike either skeletal or cardiac muscle those are terminally differentiated, SMC possess remarkable plasticity and undergo phenotypic switch at different stages of development, even in adult organisms. The phenotypic switch from a quiescent, contractile differentiation state to a highly proliferative, synthetic dedifferentiation state of SMC is believed to contribute to pathogenesis of many SMC-related diseases. An full understanding of the normal regulation of SMC development, maturation and differentiation will not only provide the foundation for elucidating how these processes may be disrupted in vascular disease, accelerating appreciation of pathogenesis and providing novel therapeutic targets, but will also be critical to understanding congenital defects in vascular development. In addition, an appreciation of normal SMC developmental mechanisms may also contribute to novel cell-based therapies for SMC-related diseases as well as for tissue engineering and reconstruction. To achieve the goals mentioned above, SMC recapitulating normal development and maturation are badly needed.
Unfortunately however, the mechanisms underlying SMC development and maturation are poorly understood partly due to the lack of good in vitro and in vivo models. For example, SMC separated from adult tissues, which are widely used in mechanism study for phenotype switch, neither recapitulate normal SMC differentiation nor maintain contractile function when cultured in vitro, so their usefulness in differentiation study is limited. Other models like induction of 10T1/2 cells, neural crest-derived MONC-1 cells, P19 embryonal carcinoma cells and even adult stem cells by different agents produce cells expressing several SMC markers, but whether the cells undergo differentiation similar to in vivo process are questionable due to their controversial origin.
Embryonic stem cell (ESC) lines have been previously established from the inner cell mass of blastocysts and have been shown to have the potential to generate all embryonic cell lineages when they undergo differentiation. When cultured suspendedly, ESC spontaneously differentiates into cyst-like structures, termed embryoid bodies (EB), which contain derivatives of the three primitive germ layers. EB have been shown to form regions of visibly spontaneously contractile SMC which indicates SMC derived from ESC recapitulate SMC differentiation and maturation, so it seems that ESC-EB system is an ideal model for differentiation study of SMC. But this model also has a defect, pluripotency of ESC not only provide environmental clues favoring development of various functional cell types, but also make study of specific cell type (like SMC) difficult due to“contamination”of other non-interested cells derived from ESC. To overcome all these disadvantages, purification of functional SMC from background of all cell type derived from ESC is needed.
Objective: To develop a novel method to obtain purified population of SMC derived from ESC by using puromycin resistance gene expressing vector driven by smooth muscle specific promoter, and then to assess the biological function of these cells.
Methods:
1. Construction of plasmid encoding puromycin resistance gene (also known as puromycin acetyl transferase gene or pac gene) and enhanced green fluorescence protein (EGFP) under the control of SM22αpromoter
(1) Clone of SM22αpromoter: The 541bp SM22αpromoter that works specifically in smooth muscle lineage cells was amplified from mouse genomic DNA using PCR with forward primer: AGTTATATTAATTTTGCATAGTGCCTGGTTG; and reverse primer: GCGCTAGCTACAAGGCTTGGTCGTTTG. The PCR product was cloned into the pMD18-T Simple vector, and was subsequently excised with AseI/NheI and subcloned into the same sites of the pIRES2-EGFP vector, removing the CMV promoter. The intermediate vector was termed pSM22α-IRES2-EGFP. (2) Clone of pac gene: The 663 bp pac gene was excised from pSM2C with HindIII/ClaI and subcloned into pSUPER.basic to produce pSUPER-PAC. (3)By using enzyme BglII/AccI, pac gene was cut from pSUPER-PAC and subsequently inserted into pSM22α-IRES2-EGFP to construct pSM22α-PAC-IRES2-EGFP, which encodes pac gene and EGFP under the control of SM22αpromoter. The constructed vectors were identified by DNA sequencing and further verified in both murine microvascular endothelial cell (EC) line SVEC and SMC to see whether the promoter can work.
2. Culture of ESC and generation of transgenic ESC
Feeder cells, mouse embryonic fibroblasts (MEF), were prepared from mouse fetuses harvested between day 12.5-14.5d of gestation. Before use, MEF were treated with Mitomycin C for 2 hours. Mouse ESC R1 were routinely cultured on treated MEF. Cells were fed every day and replated every second day. Before transfection, ESC trypsinized to single cell suspension were cultured on plate coated by 0.1% gelatin without feeder layer. Then ESC were transfected with linearized pSM22α-PAC- IRES2-EGFP plasmid DNA using lipofectamine for 5 hours. Transgenic ESC clones were selected by culture medium with 500 ug/mL G418 24 hours after transfection. Colonies derived from single cells under G418 selection were picked and amplified and screened for the presence of the pac gene by reverse transcription polymerase chain reaction (RT-PCR). Transgenic ESC were termed as SPIE-ESC.
3. EB Culture, induced differentiation and SMC purification
To generate EB, SPIE-ESC were typsinized to single cells and cultured in suspension in a petri dish with differentiation medium without LIF. On day 6, suspended EB were plated onto a surface coated with 0.1% gelatin in differentiation medium supplemented with 10 nmol/L all-trans retinoic acid (ATRA). The culture was continued for 5 days with a daily change of fresh ATRA-containing differentiation medium. On day 11, SMC were selected by differentiation medium containing 10μg/mL puromycin for 3 days. The dead cells were removed by a PBS washing every day after selection. The sorted cells were replated in a new plate after selection. The purified SMC were termed as SM22α-SMC. The homogeneity of the population was determined by flow cytometry.
4. Flow cytometry
Unsorted SPIE-ESC and sorted SM22α-SMC were trypsinized and passed through a 70μm filter to obtain single cell suspension. Acquisition of 10 000 events was made with FACScan and data analysis was done with CellQuest software. The cells derived from normal ESC were used as a negative control.
5. Immunocytochemistry
SM22α-SMC were plated on 0.1% gelatin-coated cover slips and cultured in differentiation medium. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 5% BSA. Primary antibodies were mouse anti-SMα-actin and rabbit anti-SM myosin heavy chain (SM-MHC) and rabbit anti-SM22α. After cells incubated with corresponding tetramethyl rhodamine iso-thiocyanate (TRITC) conjugated secondary antibodies and counterstained with DAPI, cover slips were sealed and examined by fluorescence microscopy.
6. Western blot analysis
Rat primary aortic SMC, unsorted cells derived from ESC and sorted SM22α-SMC were disrupted in lysis buffer. Samples were centrifuged and clear supernatants were collected. Protein concentration was determined using BCA protein assay kit. Equal amounts of proteins were run on a 10% SDS-PAGE gel and transferred to a PVDF membrane. Nonspecific binding sites were blocked by incubation with 5% nonfat dry milk in TBS-T. The primary antibodies used for Western analysis were as follows: mouse anti-SMα-actin, rabbit anti-SM22α, and rabbit anti-SM MHC. The specific binding was detected with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagents.
7. Contractile assay
Purified SM22α-SMC were plated onto 0.1 % gelatin coated cover slips with differentiation medium. After 24 hours, fresh medium was replenished and added with Carbachol at a final concentration of 10μmol/L. Pictures were acquired at 30 second intervals for 15 min. Average cell area reduction of 10 SM22α-SMC were calculated and compared to negative control SVEC.
8. Matrigel assay of SMC recruitment
Equal amount of 1×104/mL murine microvascular endothelial cell line SVEC and sorted SM22α-SMC were mixed and plated on cover slips coated by Matrigel (BD) with differentiation medium. SMC localization was examined by fluorescence microscope at 12h or 60 h. SVEC cultured alone on Matrigel were used to observe the tube like structure formation.
9. Statistical analysis
All experiments were performed in duplicates or triplicates and were repeated at least three times. Data analyses were performed using SPSS 11.1 software. Quantitative data were presented as means±standard deviation (SD). Differences between two groups were analyzed by two-tailed Student’s t test and were considered significant when p< 0.05.
Results:
1. Construction of plasmid vector pSM22α-PAC-IRES2-EGFP
Results of DNA sequencing showed that the plasmid vector was successfully constructed. Further verification of the vector in EC and SMC showed that it works in SMC but not in SVEC.
2. Generation of SPIE-ESC and isolation of ESC Derived SM22α-SMC
After 2 weeks selection with G418, positive ESC colonies were picked and amplified. Detection of pac gene transcription by RT-PCR showed that four colonies were successfully transfected with pSM22α-PAC-IRES2-EGFP. ATRA induced differentiation of transgenic ESC to EGFP expressing SM22α-SMC detected by fluorescence microscope. SM22α-SMC located peripherally in the EB outgrowth. In the absence of puromycin, cell colonies with the typical cell morphology of undifferentiated cells remained. In contrast, when cells were treated with puromycin for an additional 3 days, only EGFP expressing SM22α-SMC were observed. These SMC showed a spindle like shape similar to rat aortic SMC.
3. Selected SM22α-SMC has a high purity of 99%
The amount of EGFP expressing SM22α-SMC in the 10-day-old differentiated SPIE-ESC in the presence of ATRA, or in the presence of both ATRA (for 10 days) and puromycin (for 3 days) as quantified by fluorescence-activated cell sorter analysis was 40.05% and 98.99% respectively.
4. Expression of SM22α-SMC with smooth muscle specific markers
Immunofluorescence studies of the selected cells showed that SM22α-SMC stained for the SMC selective markers, SMα-actin and SM-MHC. All the SM22α-SMC stained strongly for SM22α. The cells displayed SMC-like morphology and appearance with a well-developed actin stress fiber network. Immunoblotting analyses revealed that the levels of SM22αproteins in purified SM22α-SMC group were higher than unpurified group, while comparable to rat aortic SMC group. The highly selective SMC protein, SM-MHC, expressed in the purified cell population after puromycin selection were higher than unpurified group and a little lower than rat aortic SMC group. The levels of SMα-actin are similar in three groups.
5. SM22α-SMC contracted in response to carbachol
SM22α-SMC showed significant contraction after carbachol treatment, with an average of 28±6.4% cell area reduction. No obvious response to carbachol was observed on SVEC.
6. SM22α-SMC integrated into tube like structure in a Matrigel assay
SVEC seeded alone on Matrigel formed tube like structures after either 12 h or 60 h culture. SM22α-SMC/SVECs formed similar structures at the same time points observed under phase contrast microscope. Under fluorescence microscope, EGFP expressing SM22α-SMC were seen to integrated into the tube like structures formed by SVEC.
Conclusions: By using a novel vector expressing pac gene and EGFP under control of smooth muscle specific SM22αpromoter, we successfully obtained highly purified and functional SMC derived from ESC.
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