Efficient differentiation of vascular smooth muscle cells from Wharton's Jelly mesenchymal stromal cells using human platelet lysate: A potential cell source for small blood vessel engineering

BACKGROUND The development of fully functional small diameter vascular grafts requires both a properly defined vessel conduit and tissue-specific cellular populations.Mesenchymal stromal cells(MSCs) derived from the Wharton's Jelly(WJ) tissue can be used as a source for obtaining vascular smooth muscle cells(VSMCs),while the human umbilical arteries(h UAs) can serve as a scaffold for blood vessel engineering.AIM To develop VSMCs from WJ-MSCs utilizing umbilical cord blood platelet lysate.METHODS WJ-MSCs were isolated and expanded until passage(P) 4. WJ-MSCs were properly defined according to the criteria of the International Society for Cell and Gene Therapy. Then, these cells were differentiated into VSMCs with the use of platelet lysate from umbilical cord blood in combination with ascorbic acid,followed by evaluation at the gene and protein levels. Specifically, gene expression profile analysis of VSMCs for ACTA2, MYH11, TGLN, MYOCD, SOX9,NANOG homeobox, OCT4 and GAPDH, was performed. In addition,immunofluorescence against ACTA2 and MYH11 in combination with DAPI staining was also performed in VSMCs. HUAs were decellularized and served as scaffolds for possible repopulation by VSMCs. Histological and biochemical analyses were performed in repopulated h UAs.RESULTS WJ-MSCs exhibited fibroblastic morphology, successfully differentiating into"osteocytes", "adipocytes" and "chondrocytes", and were characterized by positive expression(> 90%) of CD90, CD73 and CD105. In addition, WJ-MSCs were successfully differentiated into VSMCs with the proposed differentiation protocol. VSMCs successfully expressed ACTA2, MYH11, MYOCD, TGLN and SOX9. Immunofluorescence results indicated the expression of ACTA2 and MYH11 in VSMCs. In order to determine the functionality of VSMCs, h UAs were isolated and decellularized. Based on histological analysis, decellularized h UAs were free of any cellular or nuclear materials, while their extracellular matrix retained intact. Then, repopulation of decellularized h UAs with VSMCs was performed for 3 wk. Decellularized h UAs were repopulated efficiently by the VSMCs. Biochemical analysis revealed the increase of total hydroyproline and s GAG contents in repopulated h UAs with VSMCs. Specifically, total hydroxyproline and s GAG content after the 1 st, 2 nd and 3 rd wk was 71 ± 10, 74 ± 9 and 86 ± 8 μg hydroxyproline/mg of dry tissue weight and 2 ± 1, 3 ± 1 and 3 ± 1μg s GAG/mg of dry tissue weight, respectively. Statistically significant differences were observed between all study groups(P<0.05).CONCLUSION VSMCs were successfully obtained from WJ-MSCs with the proposed differentiation protocol. Furthermore, h UAs were efficiently repopulated by VSMCs. Differentiated VSMCs from WJ-MSCs could provide an alternative source of cells for vascular tissue engineering.

心肌素基因在结直肠癌中的抑癌作用

目的研究心肌素(myocardin, MYOCD)基因对结直肠癌(colorectal cancer, CRC)细胞增殖、迁移侵袭能力的影响及机制。方法 TCGA数据库、荧光实时定量PCR与Western blot检测结直肠癌组织与正常组织中的MYOCD基因表达差异;构建高表达MYOCD结直肠癌细胞系,CCK-8实验检测细胞增殖能力,Transwell迁移实验、Transwell侵袭实验、划痕实验检测CRC细胞迁移、侵袭能力,运用生物信息学方法研究MYOCD对CRC组织干细胞特性的影响,并用克隆形成实验进一步验证。结果 CRC组织中MYOCD与正常组织相比呈低表达状态,MYOCD的过表达可抑制CRC细胞的增殖,但对CRC细胞的迁移与侵袭无明显影响,生物信息学分析提示MYOCD可抑制CRC细胞的干细胞特性,细胞实验进一步证明过表达MYOCD可抑制CRC细胞的CD133表达,并明显抑制CRC细胞克隆形成能力。结论MYOCD基因可抑制CRC细胞的增殖但对细胞迁移、侵袭能力无明显影响,该过程可能是通过抑制CRC的干细胞特性实现。

基于miRNA-mRNA网络筛查并验证胃癌诊断和预后评估相关的标志物及其潜在分子机制

目的:通过生物信息学方法探索并实验验证胃癌相关标志物miR-1-3p对胃癌细胞增殖的作用及其分子机制。方法:收集TCGA数据库中胃癌(n=375)及癌旁组织(n=45)的转录组数据,构建胃癌特异性mRNA-miRNA网络,筛选潜在的miRNA类标志物,利用TargetScan预测标志物的下游靶基因且分析它们的功能。选取人正常胃上皮细胞GES-1及胃癌细胞AGS、MKN45、NCI-N87,用q PCR法检测细胞中miR-1-3p和心肌蛋白(MYOCD)的表达,用lipofectamine 2000将miR-1-3p模拟物转染至胃癌细胞中,CCK-8法测定轨染后细胞的增殖能力,WB法测定MYOCD的表达量,双荧光素酶报告基因实验验证miR-1-3p与MYOCD之间的靶向结合关系。结果:通过数据库数据分析得到差异表达的259个miRNA和7 545个mRNA,构建胃癌特异性mRNAmiRNA调节网络,分析网络中脆弱结构后确定miR-1-3p为潜在的胃癌标志物,ROC曲线和Kaplan-Meier分析显示其对胃癌的诊断和预后评估有重要意义。细胞实验显示miR-1-3p在胃癌细胞中呈低表达(P<0.05),过表达miR-1-3p可抑制胃癌细胞AGS和MKN-45的增殖能力(P<0.05或P<0.01),且可抑制MYOCD的表达(P<0.01)。TargetScan数据库预测到MYOCD的3’UTR区域中有两个与miR-1-3p结合的位点,双荧光素酶报告基因实验证实miR-1-3p与MYOCD靶向结合且负调控MYOCD的表达(P<0.01)。结论:miR-1-3p可能是胃癌诊断和预后相关潜在的标志物,且miR-1-3p可能是通过靶向MYOCD来影响胃癌细胞的增殖。