Comparison of rumen archaeal diversity in adult and elderly yaks(Bos grunniens)using 16S rRNA gene high-throughput sequencing

This study was conducted to investigate the phylogenetic diversity of archaea in the rumen of adult and elderly yaks. Six domesticated female yaks, 3 adult yaks （（5.3±0.6） years old）, and 3 elderly yaks （（10.7±0.6） years old）, were used for the rumen contents collection. Illumina MiSeq high-throughput sequencing technology was applied to examine the archaeal composition of rumen contents. A total of 92 901 high-quality archaeal sequences were analyzed, and these were assigned to 2 033 operational taxonomic units （OTUs）. Among these, 974 OTUs were unique to adult yaks while 846 OTUs were unique to elderly yaks; 213 OTUs were shared by both groups. At the phylum level, more than 99% of the obtained OTUs belonged to the Euryarchaeota phylum. At the genus level, the archaea could be divided into 7 archaeal genera. The 7 genera （i.e., Methanobrevibacter, Methanobacterium, Methanosphaera, Thermogymnomonas, Methanomicrobiu, Meth- animicrococcus and the unclassified genus） were shared by all yaks, and their total abundance accounted for 99% of the rumen archaea. The most abundant archaea in elderly and adult yaks were Methanobrevibacterand Thermogymnomonas, respectively. The abundance of Methanobacteria （class）, Methanobacteriales （order）, Methanobacteriaceae （family）, and Methanobrevibacter （genus） in elderly yaks was significantly higher than in adult yaks. In contrast, the abundance of Ther-mogymnomonas in elderly yaks was 34% lower than in adult yaks, though the difference was not statistically significant. The difference in abundance of other archaea was not significant between the two groups. These results suggested that the structure of archaea in the rumen of yaks changed with age. This is the first study to compare the phytogenetic differences of rumen archaeal structure and composition using the yak model.

High-throughput sequencing analysis of differentially expressed mi RNAs and target genes in ischemia/reperfusion injury and apelin-13 neuroprotection

miRNAs regulate a variety of biological processes through pairing-based regulation of gene expression at the 3＇ end of the noncoding region of the target miRNA, miRNAs were found to be abnormally expressed in ischemia/reperfusion injury models. High-throughput sequencing is a recently developed method for sequencing miRNAs and has been widely used in the analysis of miRNAs. In this study, ischemia/reperfusion injury models were intracerebroventricularly injected with 50 pg/kg apelin-13. High-throughput sequencing showed that 357 known miRNAs were differentially expressed among rat models, among which 78 changed to 〉 2-fold or 〈 0.5-fold. Quantita- tive real-time polymerase chain reaction was selected to confirm the expression levels of four miRNAs that were differentially expressed, the results of which were consistent with the results of high-throughput sequencing. Gene Ontology analysis revealed that the predicted targets of the different miRNAs are particularly associated with cellular process, metabolic process, single-organism process, cell, and binding. Kyoto Encyclopedia of Gene and Genome analysis showed that the target genes are involved in metabolic pathways, mitogen-ac- tivated protein kinase signaling pathway, calcium signaling pathway, and nuclear factor-KB signaling pathway. Our findings suggest that differentially expressed miRNAs and their target genes play an important role in ischemia/reperfusion injury and neuroprotection by apelin-13.

High throughput RNA sequencing utility for diagnosis and prognosis in colon diseases

RNA sequencing is the use of hight hroughput next generation sequencing technology to survey, characterize, and quantify the transcriptome of a genome. RNA sequencing has been used to analyze the pathogenesis of several malignancies such melanoma, lung cancer, and colorectal cancer. RNA sequencing can identify differential expression of genes(DEG's), mutated genes, fusion genes, and gene isoforms in disease states. RNA sequencing has been used in the investigation of several colorectal diseases such as colorectal cancer, inflammatory bowel disease(ulcerative colitis and Crohn's disease), and irritable bowel syndrome.

High Throughput Sequencing of circRNAs in Tomato Leaves Responding to Multiple Stresses of Drought and Heat

Our aim is to study the roles of a new emerging group of non-coding RNAs, circRNAs, in tomato(Solanum lycopersicum L.) plants grown at the combination of drought and heat, two of the most usual stress conditions known to frequently happen in field. Tomato seedlings from cultivar‘Jinling Meiyu’ were treated without stresses(control), at water shortage, high temperature and subjected the multiple stresses. In total, 467 circRNAs were identified with 87.82% from exon using high throughput sequencing technology. Among the circRNAs, 70 were from chr1 with the range from 23 to 49 from the other chromosomes. In detail, 156 circRNAs were shared in the four libraries, while 21, 17 and 36 circRNAs were only shown in drought, heat and multiple stresses libraries, respectively. Through a differential expression analysis, four, seven and nine circRNAs were differentially regulated in tomato at drought, heat and multiple stresses as compared with control. These circRNAs played roles on photosynthesis, starch and sucrose metabolism, RNA transport, RNA degradation, spliceosome, ribosome, etc. Our study underlined the potential role of circRNAs involved in the abiotic stress response in tomato, which might pave the way for studying biological roles of circRNAs responding to multiple stresses in plants.

Microbial Diversity in Rhizosphere Soil of Cotinus coggygria Based on High Throughput Sequencing

In order to reveal the influence of different plant configurations on the microbial community structure and diversity in rhizosphere soil of Cotinus coggygria in Fragrant Hills park,the ITS+5.8S rDNA gene and 16S rDNA gene V3-V4 region sequencing analysis for fungi and bacteria,respectively,were conducted by high throughput sequencing(Illumina MiSeq).The results showed that the fungal diversity in the rhizosphere soil samples of C.coggygria in Fragrant Hills park in 2018 was significantly higher than that in 2016,and it was higher in the rhizosphere soil of healthy C.coggygria in Xunlupo than that in diseased ones in 2018.Verticillium dahliae,which is the causal agent of C.coggygria wilt,was detected in five soil samples.In 2018,the bacterial diversity in the rhizosphere soil of diseased C.coggygria in Xunlupo was the lowest,while it was the highest in the rhizosphere soil of healthy C.coggygria under Platycladus orientalis in Langfengting.

High-throughput Sequencing Technology and Its Application

Gene sequencing is a great way to interpret life, and high-throughput sequencing technology is a revolutionary technological innovation in gene sequencing researches. This technology is characterized by low cost and high-throughput data. Currently, high-throughput sequencing technology has been widely applied in multi-level researches on genomics, transcriptomics and epigenomics. And it has fundamentally changed the way we approach problems in basic and translational researches and created many new possibilities. This paper presented a general description of high-throughput sequencing technology and a comprehensive review of its application with plain, concisely and precisely. In order to help researchers finish their work faster and better, promote science amateurs and understand it easier and better.

Genome-wide identification of RNA editing in seven porcine tissues by matched DNA and RNA high-throughput sequencing

Background: RNA editing is a co/posttranscriptional modification mechanism that increases the diversity of transcripts, with potential functional consequences. The advent of next-generation sequencing technologies has enabled the identification of RNA edits at unprecedented throughput and resolution. However, our knowledge of RNA editing in swine is still limited.Results: Here, we utilized RES-Scanner to identify RNA editing sites in the brain, subcutaneous fat, heart, liver,muscle, lung and ovary in three 180-day-old Large White gilts based on matched strand-specific RNA sequencing and whole-genome resequencing datasets. In total, we identified 74863 editing sites, and 92.1% of these sites caused adenosine-to-guanosine(A-to-G) conversion. Most A-to-G sites were located in noncoding regions and generally had low editing levels. In total, 151 A-to-G sites were detected in coding regions(CDS), including 94 sites that could lead to nonsynonymous amino acid changes. We provide further evidence supporting a previous observation that pig transcriptomes are highly editable at PRE-1 elements. The number of A-to-G editing sites ranged from 4155(muscle) to 25001(brain) across the seven tissues. The expression levels of the ADAR enzymes could explain some but not all of this variation across tissues. The functional analysis of the genes with tissuespecific editing sites in each tissue revealed that RNA editing might play important roles in tissue function.Specifically, more pathways showed significant enrichment in the fat and liver than in other tissues, while no pathway was enriched in the muscle.Conclusions: This study identified a total of 74863 nonredundant RNA editing sites in seven tissues and revealed the potential importance of RNA editing in tissue function. Our findings largely extend the porcine editome and enhance our understanding of RNA editing in swine.

Microbial diversity in Huguangyan Maar Lake of China revealed by high-throughput sequencing

Huguangyan Maar Lake is a typical maar lake in the southeast of China. It is well preserved and not disturbed by anthropogenic activities. In this study, microbial community structures in sediment and water samples from Huguangyan Maar Lake were investigated using a high-throughput sequencing method. We found significant differences between the microbial community compositions of the water and the sediment. The sediment samples contained more diverse Bacteria and Archaea than did the water samples. Actinobacteria, Betaproteobacteria, Cyanobacteria, and Deltaproteobacteria predominated in the water samples while Deltaproteobacteria, Anaerolineae, Nitrospira, and Dehalococcoidia were the major bacterial groups in the sediment. As for Archaea, Woesearchaeota (DHVEG-6), unclassified Archaea, and Deep Sea Euryarchaeotic Group were detected at higher abundances in the water, whereas the Miscellaneous Crenarchaeotic Group, Thermoplasmata, and Methanomicrobia were significantly more abundant in the sediment. Interactions between Bacteria and Archaea were common in both the water column and the sediment. The concentrations of major nutrients (NO^3-, PO4^3-, SiO3^2- and NH4^+) shaped the microbial population structures in the water. At the higher phylogenetic levels including phylum and class, many of the dominant groups were those that were also abundant in other lakes;however, novel microbial populations (unclassified) were often seen at the lower phylogenetic levels. Our study lays a foundation for examining microbial biogeochemical cycling in sequestered lakes or reservoirs.

NON-SYNDROMIC HEARING LOSS AND HIGH- THROUGHPUT STRATEGIES TO DECIPHER ITS GENETIC HETEROGENEITY

Hearing loss (HL) is the most common sensory disorder, affecting all age groups, ethnicities, and gen-ders. According to World Health Organization (WHO) estimates in 2005, 278 million people worldwide have moderate to profound HL in both ears. Results of the 2002 National Health Interview Survey indicate that nearly 31 million of all non-institutionalized adults (aged 18 and over) in the United States have trouble hearing. Epidemiological studies have estimated that approximately 50%of profound HL can be attributed to genetic causes. With over 60 genes implicated in nonsyndromic hearing loss, it is also an extremely het-erogeneous trait. Recent progress in identifying genes responsible for hearing loss enables otolaryngologists and other clinicians to apply molecular diagnosis by genetic testing. The advent of the $1000 genome has the potential to revolutionize the identification of genes and their mutations underlying genetic disorders. This is especially true for extremely heterogeneous Mendelian conditions such as deafness, where the muta-tion, and indeed the gene, may be private. The recent technological advances in target-enrichment methods and next generation sequencing offer a unique opportunity to break through the barriers of limitations im-posed by gene arrays. These approaches now allow for the complete analysis of all known deafness-causing genes and will result in a new wave of discoveries of the remaining genes for Mendelian disorders. This re-view focuses on describing genotype-phenotype correlations of the most frequent genes including GJB2, which is responsible for more than half of cases, followed by other common genes and on discussing the im-pact of genomic advances for comprehensive genetic testing and gene discovery in hereditary hearing loss.

Differential mRNA expression profiling of oral squamous cell carcinoma by high-throughput RNA sequencing

Differentially expressed genes are thought to regulate the development and progression of oral squamous cell carcinomas （OSCC）. The purpose of this study was to screen differentially expressed mRNAs in OSCC and matched paraneoplastic normal tissues, and to explore the intrinsic mechanism of OSCC development and progres- sion. We obtained the differentially expressed mRNA expression profiles in 10 pairs of fresh-frozen OSCC tissue specimens and matched paraneoplastic normal tissue specimens by high-throughput RNA sequencing. By using Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analyses, the functional significance of the differentially expressed genes were analyzed. We identified 1,120 sig- nificantly up-regulated mRNAs and 178 significantly down-regulated mRNAs in OSCC, compared to normal tissue. The differentially expressed mRNAs were involved in 20 biological processes and 68 signal pathways. Compared to adjacent normal tissue, the expression of MAGEAll was up-regulated; TCHH was down-regulated. These find- ings were verified by real-time PCR. These differentially expressed mRNAs may function as oncogenes or tumor suppressors in the development and progression of OSCC. This study provides novel insights into OSCC. However, further work is needed to determine if these differentially expressed mRNAs have potential roles as diagnostic bio- markers and candidate therapeutic targets for OSCC.

Characterization of Bacterial Community Associated with Four Organs of the Yesso Scallop (Patinopecten yessoensis) by High-Throughput Sequencing

We used Illumina high-throughput sequencing of PCR-amplified V3-V4 16 S rRNA gene regions to characterize bacterial communities associated with the adductor muscles, gills, gonads and intestines of the Yesso scallop(Patinopecten yessoensis) from waters around Zhangzidao, Dalian, China. Overall, 421,276 optimized reads were classified as 25 described bacterial phyla and 308 genera. Firmicutes, Proteobacteria, Tenericutes, Bacteroidetes, Chlamydiae and Spirochaetae accounted for > 97% of the total reads in the four organs. The bacterial 16 S rDNA sequences assigned to Firmicutes and Proteobacteria were abundant in the adductor muscles, gills and gonads; while reads from Tenericutes were dominant in the intestines, followed by those from Firmicutes, Chlamydiae, Proteobacteria and Bacteroidetes. At the genus level, the dominant genera in the adductor muscles, gills and gonads appeared to be Bacillus, Enterococcus and Lactococcus, whereas Mycoplasma was dominant in the intestines. The relative abundances of Bacillus, Enterococcus, Lactococcus, Alkaliphilus, Raoultella, Paenibacillus and Oceanobacillus were significantly lower in the intestine than in the other three organs. Cluster analysis and principal coordinates analysis of the operational taxonomy units profile revealed significant differences in the bacterial community structure between the intestine and the other three organs. Taken together, these results suggest that scallops have intestine-specific bacterial communities and the adductor muscles, gills and gonads harbor similar communities. The difference in the bacterial community between organs may relate to unique habitats, surroundings, diet and their respective physiological functions.

A New High-throughput Real-time PCR Assay for the Screening of Multiple Antimicrobial Resistance Genes in Broiler Fecal Samples from China

Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology,and screened multiple AMR genes in broiler fecal samples.Methods A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection.A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.Results The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction.The sensitivity rate,specificity rate,positive predictive value,negative predictive value and correct indices were 99.30%,98.08%,95.31%,99.79%,and 0.9755,respectively.Utilizing this assay,we demonstrate that AMR genes are widely spread,with positive detection rates ranging from 0 to 97.07%in 273 broiler fecal samples.bla CTX-M,bla TEM,mcr-1,fex A,cfr,optr A,and int I1 showed over 80%prevalence.The dissemination of AMR genes was distinct between the two farms.Conclusions We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples.The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.

Gelatin filter capture-based high-throughput sequencing analysis of microbial diversity in haze particulate matter

Airborne particulate matter(PM),especially PM2.5,can be easily adsorbed by human respiratory system.Their roles in carrying pathogens for spreading epidemic diseases has attracted great concern.Herein,we developed a novel gelatin filter-based and culture-independent method for investigation of the microbial diversity in PM samples during a haze episode in Tianjin,China.This method involves particle capture by gelatin filters,filter dissolution for DNA extraction,and high-throughput sequencing for analysis of the microbial diversity.A total of 584 operational taxonomic units(OTUs)of bacteria and 370 OTUs of fungi at the genus level were identified during hazy days.The results showed that both bacterial and fungal diversities could be evaluated by this method.This study provides a convenient strategy for investigation of microbial biodiversity in haze,facilitating accurate evaluation of airborne epidemic diseases.

RNA-Seq and Bulked Segregant Analysis of Genes Related to High Growth in <i>Ginkgo biloba</i>Half-Sibling Families

The lifetime of G. biloba is very long, and its growth is relatively slow. However, little is known about growth-related genes in this species. We combined mRNA sequencing (RNA-Seq) with bulked segregant analysis (BSA) to fine map significant agronomic trait genes by developing polymorphism molecular markers at the transcriptome level. In this study, transcriptome sequencing of high growth (GD) and low growth (BD) samples of G. biloba half-sib families was performed. After assembling the clean reads, 601 differential expression genes were detected and 513 of them were assigned functional annotations. Single nucleotide polymorphism (SNP) analysis identified SNPs associated with 119 genes in the GD and BD groups;58 of these genes were annotated. Two Homeobox-leucine zipper protein genes were up-regulated in the GD group compared with the BD group;therefore, these are very likely related to high growth of G. biloba. This study provides molecular level data that could be used for seed selection of high growth G. biloba half-sib families for future breeding programs.

鸭绿江口海域浮游植物粒级结构的环境差异响应

海洋浮游植物面临着与气候及环境变化相关的新挑战,种种迹象表明,海水营养盐浓度下降和失衡,以及海水升温、酸化会导致海洋浮游植物粒级结构趋向小型化,但在自然海域这一趋势缺乏直接的验证与深入研究。本文在黄海北部的鸭绿江口设置了16个采样点,2020年3−12月期间,每月采集一次样品(包括浮游植物环境DNA样品和海水样品),对鸭绿江口海域浮游植物粒级结构和环境指标〔包括氮、磷、硅营养盐,水温、溶解氧(DO)、pH以及化学需氧量(COD)等〕进行了系统分析,利用非度量多维尺度分析(NMDS),筛选出各环境因子不同浓度梯度站位,结合高通量测序和实验室镜检手段对浮游植物粒级分级和定量,以验证鸭绿江口海域浮游植物粒级结构的环境差异响应。结果表明:鸭绿江口海域含氮营养盐平均浓度呈现出东西两侧河口区域较高、中部海域相对较低的分布特征,磷、硅营养盐的分布趋势与氮营养盐基本一致。同时,pH和DO浓度在西侧河口区域相对较低,而COD浓度在该区域相对较高。水温在3−12月期间呈倒U型变化趋势,7月达到最高值。验证结果表明,海水中氮、磷、硅营养盐浓度与小粒级浮游植物生物量均呈负相关,即营养盐浓度越低,小粒级浮游植物生物量占比越高。同时,水温的上升也促进了小粒级浮游植物的生长,使其生物量占比增加。此外,发现小粒级浮游植物对有机物的利用效率高于大粒级浮游植物,且对低氧环境具有一定的耐受性。这表现为小粒级浮游植物生物量占比随着COD浓度的升高而升高,随DO浓度的升高而下降。研究显示,近岸海域氮、磷、硅营养盐浓度下降以及水温上升、缺氧加剧,均可导致浮游植物粒级结构趋于小型化,这可能会对生态系统的稳定和海水贝类养殖业的可持续发展构成潜在威胁。因此在全球气候变化的背景下,建议对河口区域陆源氮、磷等营养盐的调控政策进行实时调整,以利于海洋生态系统的健康和海洋经济可持续发展。

基于高通量测序的青花菜KASP标记开发与应用

为提高青花菜分子标记辅助育种水平,本研究基于高通量测序数据开发了70组KASP标记,并采用KASP检测平台对43份青花菜试验材料进行分型,根据分型结果筛选出分型效果好、多态性高的标记,用于指纹图谱构建、遗传关系确定和纯度鉴定。结果表明,70组KASP标记中,未分型成功有3个,未分型材料>5份有13个,多态性差的有6个,分型成功48个(成功率68.6%)。48个标记中,多态性信息含量(PIC)值≥0.30的标记有37个(占比77.08%),其中PIC值为0.37的标记数量最多,为11个,0.38的标记有3个。将48个标记的分型结果转化为二元编码数据,获得了43份青花菜育种材料的SNP-DNA指纹图谱。聚类分析结果发现,Br05与其他材料的遗传关系较远;Br12和Br19、Br33和Br34之间遗传系数均为100,Br14和Br32的遗传系数为99,表明两两间为同一株系的可能性大,这与在田间的表型观察结果基本一致。10组标记对30株Br19的一致性进行鉴定,发现纯度值区间为83.3%~96.7%,说明Br19株系间存在差异;采用10组标记对亲本Br19、Br35和杂交种F1(Br19×Br35)进行基因分型,结果有5个标记(SNP07、SNP09、SNP10、SNP11和SNP19)在三者间的分型结果不同,可用于杂交制种田F1(Br19×Br35)种子纯度鉴定。本研究结果对青花菜种质资源鉴定、分子标记辅助育种与新品种保护具有重要的应用价值。

福建养殖皱纹盘鲍线粒体基因组全测序及分析

为有效鉴别鲍的种类,探讨其来源,通过高通量测序获得福建养殖皱纹盘鲍线粒体基因组全序列,分析比较其序列与GenBank中收录的3个皱纹盘鲍线粒体基因组全序列或近全序列。试验结果显示,皱纹盘鲍线粒体基因组全长17037 bp,具有37个编码基因,包括13个蛋白质编码基因、22个tRNA基因和2个rRNA基因。除控制区存在碱基差异外,当前4个皱纹盘鲍线粒体全序列基本一致。基于鲍属线粒体基因组全序列构建系统发育树,4个皱纹盘鲍聚为一支,再与红鲍、黑鲍及黑足鲍聚为一支,其中福建养殖皱纹盘鲍连江群体与韩国巨济群体关系较近。笔者分析了不同皱纹盘鲍群体间的线粒体碱基组成和鲍属贝类系统进化关系,试验结果丰富了皱纹盘鲍遗传信息库,可为东南沿海鲍种的鉴定和良种选育提供参考。

辣椒胞质雄性不育恢复基因CAPS标记的PARMS转换及其应用

Co1Mod1-CAPS是一个可广泛应用于筛选辣椒胞质雄性不育恢复源的CAPS分子标记。为了将其转化为可高通量检测的分子标记以提高选择效率,本研究以Co1Mod1-CAPS标记扩增10份不同类型的辣椒三系材料,分析其扩增产物在不育系、保持系和恢复系材料间的序列差异,从而基于序列差异将其转化为PARMS高通量检测标记并对321份辣椒种质材料进行恢复源筛选。结果表明,在Co1Mod1-CAPS标记扩增产物第601个碱基处存在一个T→C碱基的变异,即不育系和保持系材料在该处的碱基均为T,而恢复系材料在该处的碱基为C,后基于该碱基变异成功将Co1Mod1-CAPS转化为高通量标记Co1Mod1-PARMS,并在321份种质材料中筛选出146份资源携带纯合恢复基因RfRf,其中小果形且辣味重的辣椒类型携带RfRf基因型的个体占比高,指形椒的占比最高,为59.5%,樱桃椒占比次之,为56.5%,甜椒类型辣椒(灯笼椒)携带RfRf基因型的个体占比最低,为8.3%。该研究结果与前人研究较为一致,表明该标记有效。因此,Co1Mod1-PARMS高通量检测标记可应用于辣椒胞质雄性不育恢复系的分子标记辅助选育,从而促使胞质雄性不育恢复系选育效率的提升。

基于PacBio测序解析浓香型白酒酒醅发酵过程中细菌群落及其代谢特性变化

利用16S r RNA基因全长测序技术PacBio解析浓香型白酒酒醅发酵过程中细菌群落结构及其代谢特性变化,并对细菌菌群与挥发性风味物质间的相关性进行分析。结果表明,根据细菌群落多样性的变化,浓香型白酒酿造过程可分为变化期(0~14 d)和稳定期(14~80 d)。在种水平上注释到12种优势细菌(相对丰度≥1.00%),其中,金山醋酸乳杆菌(Acetilactobacillus jinshanensis)从酿造14 d开始相对丰度>90%。白酒酿造过程中的微生物与挥发性风味物质之间具有一定的相关性,其中,与未命名的罗尔斯通氏菌(Ralstonia sp.)、丝状菌(Thiothrix eikelboomii)相关的物质数量较多,2,4-二叔丁基苯酚和戊酸乙酯几乎与所有优势细菌种存在显著相关性(P<0.05)。功能预测结果表明,与白酒酿造相关的代谢通路中甘氨酸的生物合成和代谢、脂质代谢、核苷酸代谢的相对丰度在发酵过程中提高,但氨基酸代谢、其他次生代谢产物生物合成、辅因子和维生素代谢的相对丰度呈下降趋势。

氧化苦参碱对牙周膜干细胞干性标志物表达和成骨分化的作用

背景:人牙周膜干细胞是牙周再生组织工程潜在的功能细胞,然而长期体外培养会导致牙周膜干细胞干性减低并发生复制性衰老,从而影响其治疗效果。目的:探讨氧化苦参碱在体外对牙周膜干细胞干性维持和骨向分化的影响,并寻找潜在的影响机制。方法:采用组织块酶消化法从人牙周膜组织中分离、培养得到牙周膜干细胞,并使用流式细胞仪进行间充质细胞表面标志物鉴定。用0,2.5,5,10μg/mL氧化苦参碱孵育牙周膜干细胞,通过CCK8实验检测氧化苦参碱对牙周膜干细胞增殖活性的影响,筛选后续实验合适的药物质量浓度,采用Western blot检测牙周膜干细胞中干细胞非特异性蛋白SOX2和OCT4的表达,采用qRT-PCR、Western blot检测牙周膜干细胞中成骨相关基因和蛋白表达水平。结果与结论:①CCK8实验结果显示2.5μg/mL氧化苦参碱对牙周膜干细胞增殖活性有显著增强作用,后续实验选用2.5μg/mL氧化苦参碱进行干预;②与空白对照组相比,氧化苦参碱组牙周膜干细胞的干性标志物SOX2蛋白表达水平变化不明显(P>0.05),OCT4蛋白表达明显上调(P<0.05);③与成骨诱导组相比,氧化苦参碱+成骨诱导组牙周膜干细胞成骨相关基因ALP、RUNX2 mRNA表达及成骨相关蛋白ALP蛋白表达明显下调(P<0.05);④氧化苦参碱上调牙周膜干细胞干性标志物表达,抑制牙周膜干细胞骨向分化,高通量测序结果表明可能与WNT2、WNT16、COMP、BMP6有关。