Metastasis of primary tumors remains a challenge for early diagnosis and prevention. The cellular properties and molecular drivers of metastatically competent clones within primary tumors remain unclear. Here, we generated 10-16 single cell-derived lines from each of three colorectal cancer (CRC) tumors to identify and characterize metastatic seeds. We found that intrinsic factors conferred clones with distinct metastatic potential and cellular communication capabilities, determining organ-specific metastasis. Poorly differentiated or highly metastatic clones, rather than drug-resistant clones, exhibited poor clinical prognostic impact. Personalized genetic alterations, instead of mutation burden, determined the occurrence of metastatic potential during clonal evolution. Additionally, we developed a gene signature for capturing metastatic potential of primary CRC tumors and demonstrated a strategy for identifying metastatic drivers using isogenic clones with distinct metastatic potential in primary tumors. This study provides insight into the origin and mechanisms of metastasis and will help develop potential anti-metastatic therapeutic targets for CRC patients.

The effect of compound probiotics on the caecum of broilers under heat stress was assessed in this study. A total of 400 twenty-eight-day-old AA male broilers were randomly divided into 4 treatment groups, where each group had 5 replicates of 20 broilers. The 4 treatment groups were a heat stress control group (broilers receiving a normal diet) and groups HP I, HP II, and HP Ⅲ, consisting of broilers receiving 1, 5, and 10 g of compound probiotics added to each kilogram of feed, respectively. Compound probiotics (L. casei, L. acidophilus, and B. lactis at a ratio of 1:1:2) were used to formulate a compound probiotic powder, with 1 × 1010 CFU/g of effective viable bacteria. Heat stress treatment was performed at 32 ± 1°C from 9:00 to 17:00 every day from 28 d to 42 d. In d 28 to 42, compared with the HC group, the ADG of broilers in the HP II and III groups was significantly increased (P < 0.05); the ADFI difference between groups was not significant (P > 0.05); the FCR of HP II and III broilers was significantly decreased (P < 0.05); and the FCR of the HP I group increased, but the difference was not significant (P > 0.05). Transcriptome results demonstrate that 665 differential genes were screened (DEGs; upregulated: 366, downregulated: 299). The DEGs were enriched in the B cell receptor signaling pathway, the intestinal immune network for IgA synthesis, the Fc epsilon RI signaling pathway, and other signaling pathways, according to KEGG enrichment analysis. Metabolome analysis identified 92 differential metabolites (DAMs; upregulated: 48, downregulated: 44). KEGG enrichment analysis indicated significant enrichment of Pantothenate and CoA biosynthesis and beta-Alanine metabolism. The combined transcriptome and metabolome analysis revealed that the DAMs and DEGs were mostly involved in beta-alanine metabolism, arginine biosynthesis, amino sugar and nucleotide sugar, and alanine, aspartate, and glutamate metabolism. The results of this study suggest that the addition of compound probiotics has a positive effect on intestinal metabolites, improving the growth performance and contributing to the overall health of broilers under heat stress.

Chronic obstructive pulmonary disease (COPD) embraces a number of pathological processes including chronic bronchitis, chronic bronchiolitis and emphysema. The chronic and progressive course of COPD is often aggravated by short periods of increasing symptoms. Respiratory tract infections (RTIs) are the most common causes of COPD exacerbations. Detection and enumeration of respiratory bacteria are important techniques in diagnosing RTIs and in the validation of new treatment methods. We describe here the development and evaluation of real-time PCR assays for the simultaneous direct detection and quantification of a range of respiratory bacteria in individuals with COPD during stable periods and during acute exacerbations of the disease. Sputum samples from 30 subjects in a COPD study were analysed, and results compared with the current gold standard of culture. Real-time PCR assays proved highly sensitive, with no cross-reactivity with other species. The prevalence of bacteria detected by real-time PCR compared with that by culture was substantially higher for Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus spp. and Moraxella catarrhalis. Multiple pathogens were also found with real-time PCR but were not detected by culture. This study demonstrates the potential of such methods in the detection and enumeration of respiratory bacteria.

Pre‐existing drug resistance and tumorigenicity of cancer cells are highly correlated with therapeutic failure and tumor growth. However, current cancer models are limited in their application to the study of intratumor functional heterogeneity in personalized oncology. Here, an innovative two‐dimensional (2D) and three‐dimensional (3D) model for patient‐derived cancer cells (PDCCs) and air–liquid interface (ALI) organotypic culture is established from colorectal cancer (CRC). The PDCCs recapitulate the genomic landscape of their parental tumors with high efficiency, high proliferation rate, and long‐term stability, while corresponding ALI organotypic cultures retain histological architecture of their original tumors. Interestingly, both 2D and 3D models maintain the transcriptomic profile of the corresponding primary tumors and display the same trend in response to 5‐Fluoruracil, regardless of their difference in gene expression profiles. Furthermore, single‐cell‐derived clones() are efficiently established and pre‐existing drug‐resistant clones and highly tumorigenic clones within individual CRC tumors are identified. It is found that tumorigenic cancer cells do not necessarily possess the stem cells characteristics in gene expression. This study provides valuable platform and resource for exploring the molecular mechanisms underlying the pre‐existing drug resistance and tumorigenicity in cancer cells, as well as for developing therapeutic targets specifically for pre‐existing drug‐resistant or highly tumorigenic clones.

旨在通过WGCNA技术解析转录组和代谢组数据,探究驴肉嫩度调控机制.试验动物采用24～36月龄的健康雌性广灵驴(平均体重236.10 kg),将驴肉剪切力和肌内脂肪含量作为表型数据,以每个样品3个重复进行表型数据测定.本试验基于前期研究的具有显著剪切力和肌内脂肪含量差异的14个广灵驴背最长肌样本的转录组和代谢组测序数据,运用WGCNA技术筛选与驴肉嫩度相关的基因及代谢物并进行转录组与代谢组联合分析,解析嫩度相关基因与代谢物.结果表明,利用WGCNA技术通过|r|≥0.5以及P≤0.05筛选标准得到3个与嫩度相关的关键基因模块Greenyellow、Darkgrey、Darkgreen以及2个关键代谢物模块Brown、Yellow.对关键基因模块进行GO富集分析发现,模块内基因主要在甘油磷脂的生物合成、脂质氧化、脂肪酸β-氧化、细胞大分子分解代谢过程、肌肉器官发育、钙离子结合等GO功能上富集.存在于关键模块内的基因及代谢物经KEGG功能富集分析发现,其大多集中在精氨酸和脯氨酸代谢、Wnt信号通路、蛋白质消化吸收、脂肪酸代谢、TCA循环、胰高血糖素信号通路、甘油磷脂代谢、嘌呤代谢、β-丙氨酸代谢等通路上.联合分析表明,丙氨酸、天冬氨酸和谷氨酸代谢、精氨酸和脯氨酸代谢、β-丙氨酸代谢以及PPAR信号通路可能调控驴肉嫩度.WGCNA及联合KEGG共富集分析筛选到的丙氨酸、天冬氨酸和谷氨酸代谢、精氨酸和脯氨酸代谢、β-丙氨酸代谢以及PPAR信号通路可能对调控驴肉嫩度有重要作用;而GAD1、PPAT、NIT2、AGMAT、CARNS1、ACOXL以及腺苷酸基琥珀酸、L-脯氨酸、L-谷氨酸、肌酸、高肌肽、肌肽、泛酸、(9S)-羟基十八碳二烯酸则可能是影响驴肉嫩度的候选基因及代谢物.本试验可为今后广灵驴肉质嫩度的分子调控与改良育种提供一定的理论基础.

旨在检测驴肉挥发性物质成分,探究与广灵驴嫩度相关的关键差异风味物质并解析关键挥发性物质与嫩度基因之间的功能与联系.本研究选用30头生长环境和饲养条件相同、年龄相近的雌性广灵驴为研究对象,进行剪切力和肌内脂肪的测定并依据含量差异将其分为高嫩度组(HT,n=4)和低嫩度组(LT,n=4).通过HS-SPME-GC-MS技术检测广灵驴背最长肌挥发性物质成分,利用香气活性值(odor activity value,OAV)筛选驴肉关键风味物质,并结合多元统计分析方法获得变量重要性投影(variable importance in the projection,VIP)筛选与嫩度相关的关键差异风味物质,后基于Pearson系数与转录组学进行联合分析,寻找出驴肉嫩度关键风味物质与差异基因之间的联系.结果,在广灵驴背最长肌中共鉴定出41种挥发性物质,包括醇类、醛类、烃类、酮类、酯类以及2种其他物质.通过OAV筛选出13种关键风味物质,发现影响驴肉的主要挥发性物质和贡献者是醛类.通过VIP和OAV值筛选出1-辛烯-3-醇、1-辛醇以及月桂醛既是嫩度的差异物质又是对驴肉风味有贡献的关键风味物质.利用Pearson系数筛选与关键风味物质相关的差异基因并对其进行KEGG功能分析,发现1-辛烯-3-醇的相关基因主要富集在果糖和甘露糖代谢、糖酵解/糖异生、胰高血糖素信号通路等;1-辛醇相关基因主要富集在胰岛素信号通路、丁酸代谢、Ⅱ型糖尿病、多种脂肪细胞因子信号通路等;月桂醛相关基因则多富集在次生代谢物的生物合成、胰岛素信号通路、糖酵解/糖异生、戊糖磷酸途径等.这些通路可能参与了驴肉关键风味物质1-辛烯-3-醇、1-辛醇以及月桂醛的形成.本研究利用SPME-GC-MS技术检测了驴肉的风味挥发性物质并分析了驴肉不同嫩度风味差异,筛选出1-辛烯-3-醇、1-辛醇和月桂醛3种嫩度差异物质和关键风味物质.KEGG富集结果分析发现,酵解/糖异生、胰高血糖素信号通路、PPAR信号通路、果糖和甘露糖代谢、MAPK信号通路等可能参与了 1-辛烯-3-醇、1-辛醇和月桂醛3的形成,这些都为广灵驴肉质嫩度和风味的分子改良与育种提供新的方向.

Background Donkey meat has low fat and high protein contents and is rich in various unsaturated fatty acids and trace elements that are beneficial to human digestion and absorption. IMF (intramuscular fat), also known as marbling, is an important indicator of the lean meat to fat ratio, which directly affects the tenderness and juiciness of the meat. At present, the underlying molecular variations affecting IMF content among donkey breeds are unclear. The Guangling donkey is an indigenous species in China. This study explored candidate regulatory genes that affect IMF content in Guangling donkeys. The IMF content of the longissimus dorsi muscle in 30 Guangling donkeys was measured. Six donkeys of similar age were selected according to age factors and divided into two groups, the high (H) and low (L) fat groups, according to their IMF content. Results RNA-seq technology was used to compare the muscle transcriptome between the two groups. More than 75.0% of alternative splicing (AS) events were of the skipped exon (SE) type. A total of 887 novel genes were identified; only 386 novel genes were aligned to the annotation information of various databases. Transcriptomics analysis revealed 167 differentially expressed genes (DEGs), of which 64 were upregulated and 103 were downregulated between the H and L groups. Gene ontology analysis showed that the DEGs were enriched in multiple biological processes and pathways that are related to adipocyte differentiation, lipid synthesis, and neutral lipid metabolism. KEGG pathway analysis suggested that arachidonic acid metabolism, the HIF-1 signalling pathway, fructose and mannose metabolism, glycerophospholipid metabolism, and the AMPK signalling pathway were involved in lipid deposition. In addition, a gene–gene interaction network was constructed that revealed that the DEGs, including SCD , LEPR , CIDEA , DLK1 , DGAT2 , ITGAL , HMOX1 , WNT10B , and DGKA, had significant roles in adipocyte differentiation and adipogenesis. The selected DEGs were further validated by qRT–PCR. Conclusion This study improves the in-depth understanding of gene regulation and protein expression regarding IMF deposition and lays a basis for subsequent molecular breeding studies in Guangling donkeys.

旨在基于转录组与代谢组联合分析,探究驴肉嫩度的分子调控机制.本研究以30头生长环境和饲养条件相同、33～36月龄的雌性广灵驴为研究对象,进行剪切力和肌内脂肪含量的测定.依据剪切力和肌内脂肪含量选择出8头驴并将其分为高嫩度组(HT,n=4)与低嫩度组(LT,n=4),通过转录组和代谢组分析筛选差异表达基因和差异代谢物,之后联合KEGG富集分析,构建相关网络互作图.转录组结果表明,在HT组和LT组中共发现有1 253个差异表达基因,其中832个基因上调,421个基因下调.KEGG分析表明,差异表达基因主要富集在碳水化合物代谢、脂质代谢、内分泌系统、信号转导以及细胞过程等多种途径;代谢组结果表明,在HT组和LT组中鉴定出225个差异代谢物,其中上调的有154个,下调的有71个.KEGG通路分析表明,差异代谢物主要富集到碳水化合物代谢、脂质代谢、氨基酸代谢、核苷酸代谢以及信号转导等多个途径;联合分析表明,差异表达基因与差异代谢物显著富集在甘油磷脂代谢,磷酸戊糖途径,丙氨酸、天冬氨酸和谷氨酸代谢,精氨酸和脯氨酸代谢以及胰高血糖素信号通路中.显著差异表达基因和代谢物在上述代谢通路中起到了关键调控作用,可作为驴肉嫩度的潜在候选基因及代谢物,为今后探究驴肉嫩度的分子调控机制和驴肉分子育种提供理论基础.

[目的]对广灵驴脂肪和肥胖相关基因(fat mass and obesity-associated gene,FTO)进行克隆及序列分析,同时检测其在广灵驴各组织中的表达差异,以探究广灵驴FTO基因结构对其生理代谢功能的影响.[方法]运用RT-PCR技术扩增并克隆广灵驴FTO基因CDS序列,进行基因及蛋白质功能分析,同时运用实时荧光定量PCR方法检测FTO基因在广灵驴7种组织(心脏、肝脏、脾脏、肺脏、肾脏、背最长肌及皮下脂肪)中的差异表达.[结果]广灵驴FTO基因CDS区序列长1 518 bp,编码505个氨基酸,序列提交至NCBI,登录号:MZ169553.广灵驴FTO基因与马、猪、牛、人、羊驼、绵羊和山羊的相似性为99.3％、90.3％、89.5％、90.8％、90.7％、89.2％和89.2％;系统进化树分析发现,广灵驴与马的亲缘关系最近.FTO蛋白分子质量为58.35 ku,理论等电点为5.07,脂肪系数为80.36,不稳定系数为48.82,平均疏水指数为-0.550,为不稳定的酸性亲水蛋白.FTO蛋白无信号肽和跨膜区,主要定位于细胞质中,具有34个磷酸化位点与5个糖基化位点.FTO蛋白二级结构主要以α-螺旋(43.96％)和无规则卷曲(37.82％)为主.实时荧光定量PCR分析发现,FTO基因在广灵驴7个组织中均有表达,其中在肺脏和皮下脂肪中的表达量极显著高于其他组织(P＜().01),在背最长肌中表达量最低.[结论]本试验结果可为下一步基因表达与调控脂肪沉积机制及改善驴肉品质的研究奠定基础.

以广灵驴作为试验材料,使用RT-PCR方法对广灵驴PPARγ基因CDS编码区进行克隆然后对序列进行分析,并利用实时荧光定量PCR技术检测PPARγ基因在广灵驴的心、肝、脾、肺、肾、背最长肌和皮下脂肪7种组织中的表达特征.结果表明,广灵驴PPARγ基因含有1个1 425 bp的开放阅读框,可编码474个氨基酸残基,其核苷酸序列与马的同源性最高;PPARγ蛋白是一种酸性不稳定的亲水性蛋白,二级结构由α-螺旋、延伸链、β-转角和无规则卷曲4种形式组成,蛋白序列中具有3个结构域和48个磷酸化位点,无信号肽剪切位点以及跨膜区域,主要定位在细胞质和细胞核中;PPARγ基因在广灵驴的7种不同组织中均有表达但存在一定差异,在皮下脂肪中的表达水平最为丰富.本试验成功克隆了广灵驴PPARγ基因,并发现其在皮下脂肪中有较高的表达水平,说明PPARγ基因可能与广灵驴脂肪沉积有关.

本研究旨在对广灵驴腺苷琥珀酸裂解酶(adenylo succinatelyase,ADSL)基因进行克隆及生物信息学分析,并检测其在不同组织中的表达情况,为探究A DSL基因在广灵驴肌苷酸合成及风味形成中的作用机制提供理论参考.根据GenBank中公布的马(登录号:XM 001917207.5)、牛(登录号:NM 001102377.2)、猪(登录号:GU249574.1)等物种的A DSL基因mRNA序列,通过Primer Premier 3.0在线工具设计同源引物,RT-PCR法扩增并克隆A DSL基因序列,对编码序列进行结构与功能分析,最后用实时荧光定量PCR检测A DSL基因在广灵驴心脏、肝脏、脾脏、肺脏、肾脏、背最长肌组织中的表达水平.结果 显示,广灵驴ADSL基因CDS长1473 bp,编码490个氨基酸,提交至NCBI,登录号:MW037837,其核苷酸序列与马、牛、双峰驼、猪、绵羊、人、小鼠的相似性分别为99.5％、90.8％、92.3％、90.4％、90.7％、90.5％和86.0％.进化树分析结果表明,广灵驴与马的种属关系最近,与小鼠的亲缘关系最远.ADSL蛋白分子质量为55.44 ku,等电点为6.52,平均疏水指数为-0.243,是一种不稳定的酸性亲水蛋白.ADSL蛋白有41个磷酸化修饰位点,6个糖基化修饰位点,没有信号肽和跨膜结构,有1个卷曲螺旋.ADSL蛋白主要定位在细胞质,α-螺旋(68.98％)是主要的二级结构.ADSL基因在广灵驴6个组织中都有表达,其中肺脏中的表达量最高,显著高于其他组织(P＜0.05),其次是心脏和肝脏,脾脏、肾脏和背最长肌中的表达量最低.本研究结果为今后探究A DSL基因在广灵驴肌苷酸合成及风味形成的分子机制奠定基础.

本研究旨在对广灵驴二酰基甘油酰基转移酶2(DGAT2)基因进行克隆,生物信息学分析和检测其在不同组织中的表达情况,为探究DGAT2基因在广灵驴脂肪沉积和提高乳脂率等方面的作用机制提供理论参考.根据GenBank上公布的马(登录号:XM_023645689.1)、双峰驼(登录号:XM_010973154.1)、绵羊(登录号:XM_027979550.1)等物种的DGAT2基因mRNA序列,利用Primer Premier 3.0在线工具设计同源引物,应用RT-PCR法扩增DGAT2基因序列,用生物信息学方法分析DGAT2基因编码序列,用实时荧光定量PCR技术检测DGAT2基因在广灵驴心脏、肝脏、脾脏、肺脏、肾脏、背最长肌、肌间脂肪、皮下脂肪组织中的表达.结果显示,广灵驴DGAT2基因CDS序列1 086 bp,编码361个氨基酸,提交到GenBank,获得登录号:MT993643,其编码序列与马、牛、双峰驼、猪、绵羊、人、小鼠的同源性分别为99.0％、92.0％、93.5％、92.0％、92.7％、85.3％、84.1％.系统进化树分析表明,驴与马的亲缘关系最近,和小鼠的关系最远.DGAT2蛋白分子质量40.96 ku,脂肪系数92.85,等电点9.16,是一种具有跨膜区的稳定碱性疏水蛋白.DGAT2蛋白有28个磷酸化修饰位点,2个糖基化修饰位点,没有信号肽,主要定位在内质网,α-螺旋(39.89％)和无规则卷曲(36.01％)是主要的二级结构.DGAT2基因在检测的8个组织中都有表达,其中皮下脂肪中的表达量显著高于其余组织(P＜0.05),其次是心脏、肝脏和肾脏,背最长肌中的表达量最低.本试验结果为探究DGAT2基因在广灵驴脂肪沉积和提高乳品质性状的作用奠定基础.

旨在基于转录组(RNA-Seq)和代谢组(UPLC-MS/MS)关联分析,筛选驴肌内脂肪沉积的关键调控因子.本研究选用饲养条件相同、平均体重为236.10 kg的雌性广灵驴30头,对其背最长肌进行肌内脂肪(IMF)含量的测定,选择年龄一致的驴,并根据其IMF含量的高低分为两组:低肌内脂肪组(L组,每组3头)与高肌内脂肪组(H组,每组3头),进行转录组学和代谢组学测序分析,对差异表达基因(DEGs)和差异代谢物(DAMs)进行KEGG关联分析和相关性分析,并构建相关性网络图.结果表明,在L组和H组之间共鉴定出72种差异代谢物,其中27种代谢物上调,45种代谢物下调;关联分析表明,在差异表达基因和差异代谢物之间共有35条共富集通路,其中甘油脂代谢、甘油磷脂代谢、花生四烯酸代谢、亚油酸代谢和不饱和脂肪酸的生物合成5条通路与IMF沉积相关,有8个差异表达基因和15种差异代谢物在这5条通路中富集,包括DGKA、DGAT2、PLA2G3、GPCPD1和SCD等差异表达基因以及甘油-3-磷酸(glycerol-3-phosphate,G3P)、溶血卵磷脂酸16:0(lysophosphatidic acid,LPA)、溶血卵磷脂酰胆碱16:0(lysophosphatidylcholine,LPC)、二十二碳六烯酸(docosahexaenoic acid,DHA)和花生四烯酸(arachidonic acid,AA)等差异代谢物.另外,通过网络图分析发现,甘油脂代谢通路和甘油磷脂代谢通路中的DGA T2、PLA2G3、GPCPD1和DGKA基因与它们相应的代谢产物具有高度相关性.本研究所鉴定差异表达基因可作为IMF沉积相关的潜在候选基因,为今后进一步探究驴IMF沉积的分子调控机制和驴的肉品质分子育种提供理论依据.

The intestinal epithelium is one of the most rapidly renewing tissues, which is fueled by stem cells at the base of the crypts. Strategies of genetic lineage tracing and organoids, which capture major features of original tissues, are powerful avenues for exploring the biology of intestinal stem cells in vivo and in vitro, respectively. The combination of intestinal organoid–culturing system and genetic modification approaches provides an attractive platform to uncover the mechanism of colorectal cancer and genetic disorders in the human minigut. Here, we will provide a comprehensive overview of studies on intestinal epithelium and intestinal stem cells. We will also review the applications of organoids and genetic markers in intestinal research studies. Furthermore, we will discuss the advantages and drawbacks of organoids as disease models compared with mice models and cell lines.

[目的]本试验旨在对广灵驴SCD基因CDS区进行生物信息学分析,并检测其在广灵驴组织中的表达,为探究SCD基因在广灵驴脂肪沉积中的作用提供理论参考。[方法]设计SCD基因同源引物,RT-PCR法克隆CDS序列,对SCD基因编码产物进行生物信息学预测,同时使用qRT-PCR法检测广灵驴7个组织中SCD基因的表达量。[结果]广灵驴SCD基因CDS长1 080 bp,可编码359个氨基酸,提交到GenBank,获得登录号:MW132164。广灵驴编码序列与马、牛、双峰驼、猪、绵羊、人、小鼠、大鼠的同源性分别为99.4%、89.2%、90.7%、90.1%、89.5%、88.2%、81.2%、82.4%。SCD蛋白分子质量为41.55 ku,等电点为9.29,不稳定指数为45.54,平均疏水指数为-0.146,属于不稳定的碱性亲水蛋白。SCD蛋白既没有信号肽也没有卷曲螺旋,有4个跨膜区,主要定位在内质网。二级、三级结构显示,SCD蛋白主要由α-螺旋（40.67%）和无规则卷曲（40.11%）构成。SCD基因在广灵驴7个组织内都表达,在肝脏、肺脏和皮下脂肪中的表达量最高,心脏和背最长肌最低。[结论]本研究结果可为今后探究SCD基因在广灵驴脂肪沉积中的功能,以及提高广灵驴乳肉品质奠定基础。

The intestinal epithelium is one of the most rapidly renewing tissues, which is fueled by stem cells at the base of the crypts. Strategies of genetic lineage tracing and organoids, which capture major features of original tissues, are powerful avenues for exploring the biology of intestinal stem cells in vivo and in vitro, respectively. The combination of intestinal organoid-culturing system and genetic modification approaches provides an attractive platform to uncover the mechanism of colorectal cancer and genetic disorders in the human minigut. Here, we will provide a comprehensive overview of studies on intestinal epithelium and intestinal stem cells. We will also review the applications of organoids and genetic markers in intestinal research studies. Furthermore, we will discuss the advantages and drawbacks of organoids as disease models compared with mice models and cell lines. Copyright (C) 2020, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.

试验旨在对广灵驴的激素敏感脂酶(hormone sensitive lipase,HSL)基因进行克隆和序列分析,并对HSL基因在广灵驴不同组织中的差异表达水平进行分析.使用RT-PCR法扩增并克隆广灵驴HSL基因CDS区部分序列,将序列拼接后得到HSL基因完整的CDS区全长序列,并对序列进行一系列生物信息学分析,通过实时荧光定量PCR检测HSL基因mRNA在广灵驴的心脏、肝脏、脾脏、肺脏、肾脏、背最长肌和皮下脂肪7个组织中的表达情况.结果 显示,广灵驴HSL基因完整的CDS区全长为2 286 bp,共编码761个氨基酸,序列已提交到NCBI,登录号:MN231003.广灵驴HSL基因的核苷酸序列与马、羊驼、骆驼、猪、牛、山羊、小鼠、绵羊相应序列的同源性分别为99.6％、88.9％、88.6％、88.1％、86.9％、85.6％、80.8％、79.1％.系统进化树预测表明,广灵驴HSL基因与马的亲缘关系最近,与小鼠的亲缘关系最远.生物信息学分析发现,HSL蛋白的理论等电点为6.51,不稳定指数为56.83,亲水性的总平均值为-0.048,说明HSL是酸性不稳定的水溶性蛋白.蛋白保守域中存在N-末端结构域、α/β水解酶折叠结构域以及调节结构域.蛋白序列中共存在88个磷酸化修饰位点、25个糖基化修饰位点.蛋白中存在较强的疏水性区域,没有信号肽及跨膜区域.二级结构显示此蛋白是由α-螺旋、延伸链、β-转角和无规则卷曲构成的,分别占45.33％、11.70％、5.65％、37.32％.实时荧光定量PCR检测结果显示,HSL基因mRNA在广灵驴的7种组织中均有表达但存在差异,在皮下脂肪中表达量最高,在心脏中表达量最低,说明广灵驴HSL基因可能在体内脂肪沉积中发挥着非常重要的作用.该试验为进一步研究HSL蛋白功能及其在广灵驴脂肪沉积中代谢调控机制提供了理论基础.

PTEN基因是一个具有双重特异磷酸酶活性的抑癌基因,可影响细胞周期调控和细胞增殖.异常PTEN可导致PI3K通路失调,细胞异常增殖.多种肿瘤中可检测到异常的PTEN突变.最近的研究显示,除了在PI3K通路中的作用外,PTEN还具有DNA损伤修复等其它生物学功能,PTEN缺陷肿瘤细胞对DNA损伤药物敏感.因此,针对PTEN基因异常肿瘤可采取PI3K抑制剂、DNA损伤化疗药物等治疗方法联合使用的个体化治疗策略,本文对人PTEN基因异常肿瘤的个体化治疗进行初步分析.

Curcumin is a natural compound isolated from the rhizome of Curcuma longa. It possesses anti-tumor activity through arresting cell cycles and promoting cell apoptosis. However, the effect of curcumin on DNA damage is not well defined. In this study, we investigated the effect of curcumin on inducing DNA damage and on sensitizing lymphoma cells to anti-tumoral DNA damage drugs. Western blot showed curcumin induced γ-H2AX foci in CH12F3 lymphoma cells, which suggests curcumin induces DNA breaks. In addition, curcumin decreased the expression of Rad51, which suggests curcumin induces DNA damage through regulating Rad51-dependant homologous recombination. Rad51-dependant homologous recombination is a vital DNA repair pathway for cancer cells to resist anti-tumoral DNA damage drugs, therefore, we studied the effect of curcumin on the sensitizing lymphoma cells to various chemotherapeutic drugs. We found low level of curcumin (5μM) sensitized lymphoma cells to anti-tumoral DNA damage agents including cisplatin, methyl methanesulfonate, hydroxyurea and camptothecin. We also found curcumin sensitized CH12F3 lymphoma cells to DNA-PK and PARP inhibitors. Flow cytometry analysis showed curcumin promoted apoptosis and western blot analysis confirmed curcumin activated caspase3-dependent apoptosis. Taken together, these results demonstrate that curcumin induces DNA damage through regulating Rad51-dependant homologous recombination and triggers caspase3-dependent apoptosis, more importantly, curcumin sensitizes lymphoma cells to various DNA damage drugs. Consequently, curcumin would be a potent agent for sensitizing lymphoma cells to anti-tumoral chemotherapeutic agents.

PTEN is a tumor suppressor gene well characterized as a phosphatase. However, more evidences demonstrate PTEN functions in DNA repair independent of its phosphatase activity, which affects the efficacy of DNA damage anti-tumoral drugs in treating cancer cells with PTEN variations. Using BT549 breast cancer cells, we studied the roles of PTEN in DNA repair and in sensitization of breast cancer cells to olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor. Comet assay showed PTEN promoted DNA repair. PTEN-deficient BT549 cells are sensitive to olaparib, which shows the synthetic lethality between PTEN and PARP1. We expressed PTEN in BT549 cells and found PTEN-proficient BT549 cells resist to olaparib. Western blot showed that PTEN up-regulated Rad51 expression, suggesting PTEN promotes DNA repair through Rad51-dependnent homologous recombination. We used 5μM olaparib or 5μM RI-1, a Rad51 inhibitor, to treat PTEN-proficient BT549 cells respectively. The immunofluorescent analysis showed the combination of olaparib and RI-1 induced more than 4-fold of γH2AX foci than either of them. MTT assay showed 5μM RI-1 did not change the survival of PTEN-proficient BT549 cells, however, this dose of RI-1 sensitized PTEN-proficient BT549 cells to olaparib. Consequently, these results demonstrate that inhibition of Rad51 can sensitize BT549 cells with wild type PTEN to olaparib, which would contribute to using PARP inhibitors in individual treatment of breast cancer patients with PTEN variations.