Cell sheet technology: a promising strategy in regenerative medicine

Regenerative medicine is a burgeoning field that is important to combat challenging diseases and functional impairments. Compared with traditional cell therapies with evident shortcomings (e.g., cell suspension injection or tissue engineering with scaffolds), scaffold-free cell sheet technology enables transplanted cells to be grafted and fully maintain their viability on target sites. Clinical and experimental studies have advanced the application of cell sheet technology to numerous tissues and organs (e.g., liver, cornea and bone). However, previous reviews have failed to discuss vital aspects of this rapidly developing technology, and many new challenges are gradually emerging. This review aims to provide a comprehensive introduction to cell sheet technology from cell selection to the ultimate applications of cell sheets, and challenges and future visions are also described.     

Molecular cloning and sequencing of the upstream region of the major Bacillus subtilis autolysin gene: a modifier protein exhibiting sequence homology to the major autolysin and the spoIID product.

The upstream region of the N-acetylmuramoyl-L-alanine amidase gene (cwlB; a major Bacillus subtilis autolysin) was cloned into Escherichia coli by chromosome walking. Sequencing of the region showed the presence of two open reading frames, one (designated as cwbA) which starts at a UUG codon and encodes a polypeptide of 705 amino acids with an M(r) of 76,725, and the other (designated as lppX), upstream of cwbA, comprising 102 amino acids and having a signal sequence characteristic of a lipoprotein. Purification of the CwbA protein and determination of its N-terminal amino acid sequence revealed that it contains a presumed signal peptide which is processed after Ala at position 25 from the N-terminal, and that the M(r) of the mature form is 75,000. The amino acid sequences of the N-terminal and C-terminal regions of CwbA were found to be highly homologous with those of the cell wall binding domain of CwlB and the spoIID gene product, respectively. CwbA stimulated the major autolysin activity approximately threefold in vitro. These data indicate that CwbA is the modifier protein of the major autolysin reported by Herbold, D. R. & Glaser, L. (1975; Journal of Biological Chemistry 250, 1676-1682). In-frame fusion between the lppX and lacZ genes demonstrated that lppX is translated in vivo and expressed during the exponential growth phase.     

QTL Mapping of Agronomic Waterlogging Tolerance Using Recombinant Inbred Lines Derived from Tropical Maize (Zea mays L) Germplasm

Waterlogging is an important abiotic stress constraint that causes significant yield losses in maize grown throughout south and south-east Asia due to erratic rainfall patterns. The most economic option to offset the damage caused by waterlogging is to genetically incorporate tolerance in cultivars that are grown widely in the target agro-ecologies. We assessed the genetic variation in a population of recombinant inbred lines (RILs) derived from crossing a waterlogging tolerant line (CAWL-46-3-1) to an elite but sensitive line (CML311-2-1-3) and observed significant range of variation for grain yield (GY) under waterlogging stress along with a number of other secondary traits such as brace roots (BR), chlorophyll content (SPAD), % stem and root lodging (S&RL) among the RILs. Significant positive correlation of GY with BR and SPAD and negative correlation with S&RL indicated the potential use of these secondary traits in selection indices under waterlogged conditions. RILs were genotyped with 331 polymorphic single nucleotide polymorphism (SNP) markers using KASP (Kompetitive Allele Specific PCR) Platform. QTL mapping revealed five QTL on chromosomes 1, 3, 5, 7 and 10, which together explained approximately 30% of phenotypic variance for GY based on evaluation of RIL families under waterlogged conditions, with effects ranging from 520 to 640 kg/ha for individual genomic regions. 13 QTL were identified for various secondary traits associated with waterlogging tolerance, each individually explaining from 3 to 14% of phenotypic variance. Of the 22 candidate genes with known functional domains identified within the physical intervals delimited by the flanking markers of the QTL influencing GY and other secondary traits, six have previously been demonstrated to be associated with anaerobic responses in either maize or other model species. A pair of flanking SNP markers has been identified for each of the QTL and high throughput marker assays were developed to facilitate rapid introgression of waterlogging tolerance in tropical maize breeding programs.     

阿拉善高原2种荒漠植物根系构型及生态适应性特征

根系构型决定了植物对资源的吸收方式，根系构型的变化是植物对环境的生态适应和有效生存策略。在阿拉善高原西南缘红砂（Reaumuria soongarica）-珍珠猪毛菜（Salsola passerina）混生群落采用传统挖掘法收集两种植物根系，基于量化的根系形态指标，利用几何拓扑学及分形理论分析了根系构型特征，探讨了该地区2种植物对干旱生境的生态适应策略。结果表明：红砂和珍珠猪毛菜根系均以水平分布占优，根系浅层化分布明显，混生的两种植物占据不同的生态位；2种荒漠植物均具有较大的比根长（SRL）和比表面积（SRA），红砂SRL=21.3 cm/g，SRA=7.6 cm²/g，珍珠SRL=22.4 cm/g，SRA=6.5 cm²/g，有利于水分和养分的获取；红砂根系拓扑指数（TI）、修正拓扑参数（q_a和q_b）分别为0.86、0.52、0.49，珍珠猪毛菜对应参数分别为0.93、0.76、0.73，表明2种植物根系均趋向于鱼尾形分支结构；根系分形维数值（FD_红=1.488、FD_珍=1.422）较小，而分形丰度值（lgK_红=1.855、lgK_珍=1.774）较大，表明2种植物分支相对简单，但空间拓展能力强，有利于对营养空间的占有。上述特征可能是阿拉善西南缘红砂-珍珠猪毛菜群落2种荒漠植物植物对干旱贫瘠生境的重要生态适应策略。     

肿瘤特异性蛋白B7-H4纳米抗体的筛选和鉴定

羊驼体内存在天然缺少轻链的重链抗体,克隆重链抗体可变区(VHH),即可构建单域抗体(single-domain antibodies,sdAbs),又称纳米抗体(nanobody,Nb)。利用非免疫羊驼噬菌体文库筛选肿瘤特异性蛋白B7-H4的纳米抗体,经过4轮淘选,ELASE鉴定阳性克隆噬菌体,测序获得其DNA序列后体外转录为mRNA,将修饰纯化后的mRNA转染到肿瘤细胞,利用细胞免疫荧光检测mRNA在肿瘤细胞内是否表达,Western印迹进一步验证其表达情况;通过CCK-8法鉴定其对肿瘤细胞的增殖抑制能力;划痕实验鉴定其对肿瘤细胞迁移能力的影响;Transwell法鉴定其对肿瘤细胞的侵袭能力的影响;裸鼠荷瘤模型瘤旁注射mRNA,鉴定其在体内实验对肿瘤组织的作用。结果显示,通过淘选获得1个高亲和性的噬菌体株菌H6;DNA测序并导出的氨基酸序列符合羊驼纳米抗体结构;将其mRNA导入肿瘤细胞,能有效表达出纳米抗体H6;转染H6-mRNA的肿瘤细胞(0.84±0.08)与未转染H6-mRNA的对照组(1.83±0.04)相比,其增殖能力明显受到抑制,P<0.01,其迁移和侵袭能力(78.60±5.36)明显低于空白对照组(197.80±21.04),效果优于B7-H4 mRNA的siRNA(95.40±16.56);在裸鼠乳腺癌模型中能有效抑制肿瘤生长,效果优于紫杉醇和B7-H4 mRNA的siRNA。这说明筛选所得抗B7-H4纳米抗体H6能特异结合B7-H4蛋白并封闭其功能,导致肿瘤细胞的增殖、迁移和侵袭受到抑制。该结果为利用B7-H4作为治疗癌症的靶点提供了实验基础。