OBJECTIVE:In this study, we investigated the mechanism of action of LIMK1 in cervical cancer progression. METHODS:The biological role of LIMK1 in regulating the growth, invasion, and metastasis of cervical cancer was studied in SiHa, CaSki cells and nude mice tumor models. The role of LIMK1 in the growth of cervical cancer was evaluated by HE staining. The role of LIMK1 in the invasion, metastasis, and proliferation of cervical cancer was evaluated by cell scratch, Transwell, and monoclonal experiments. The interaction among LIMK1, ROS, and Src was evaluated by Western blotting. The effects of regulating ROS and p-Src expression on LIMK1 in the migration/invasion and proliferation of cervical cancer cells were evaluated through cellular functional assays. RESULTS:Overexpression of LIMK1 promoted tumor growth in nude mice. Cell scratch, Transwell, and monoclonal experiments suggested that LIMK1 promoted the invasion, metastasis, and proliferation of cervical cancer cells. Western blotting suggested that LIMK1 can promote the expression of ROS-related proteins NOX2, NOX4, p-Src, and downstream proteins p-FAK, p-ROCK1/2, p-Cofilin-1, F-actin and inhibit the expression of p-SHP2 protein. Correction experiments showed that LIMK1 regulated the expression of p-FAK and p-Cofilin-1 proteins by regulating ROS and p-Src. Through the detection of cervical cancer cell functions, it was found that the activation of ROS and p-Src induced by LIMK1 is an early event that promotes the migration, proliferation, and invasion of cervical cancer cells. CONCLUSIONS:LIMK1 promotes the expression of F-actin and promotes the development of cervical cancer by regulating the oxidative stress/Src-mediated p-FAK/p-ROCK1/2/p-Cofilin-1 pathway.
Pancreatic cancer is a worldwide medical and health problem. Due to its high heterogeneity and malignant phenotype, the 5-year survival rate of pancreatic cancer is only 7.2%, which is a serious threat to human health. The biological mechanism and drug development research of pancreatic cancer often rely on the traditional two-dimensional culture models (cell lines) and patient-derived xenograft models. However, cell lines lack the three-dimensional environment and heterogeneity of the tumor, while xenograft models have the disadvantages of long culture time, low success rate and difficulty in carrying out high-throughput drug screening. There is an urgent need to develop three-dimensional culture models that can highly reflect the characteristics and molecular variation of pancreatic cancer. As a three-dimensional culture model developed in recent years, human-derived pancreatic cancer organoids are multicellular units extracted from tissue samples and embedded in extracellular matrix gels after mechanical and enzymatic digestion, which can reproduce the histological characteristics and organ characteristics of the original patient, and even have the functions of the original organ. With the continuous development and improvement of pancreatic cancer organoids culture system, its simple, economical and stable culture technology has been gradually established, which promotes the application of human pancreatic cancer organoids to high-throughput drug screening, individualized precision treatment and deeper pathogenesis and targeted drug development research. At the same time, human-derived pancreatic cancer organoids are also a novel semi-in vivo model for the study of clinical molecular typing of pancreatic cancer, especially for the study of etiology, molecular characteristics, histomorphology and somatic mutation burden of patients with high mutation rate and high heterogeneity. The construction of human pancreatic cancer organoid biobank with large sample size is a new platform for biology, basic medicine and clinical science oncology research, which provides a reliable resource for the in-depth study of the pathogenesis of pancreatic cancer and the development of treatment strategies. In summary, the research progress of human pancreatic cancer organoids promotes the application of laboratory models that recapitulate the characteristics of clinical patients, improves the connection between basic laboratory research and clinical patients, and provides clinical sources for drug screening, pathogenesis research, individualized treatment strategies and the construction of biobanks. Therefore, this article reviewed the latest research progress of human pancreatic cancer organoids, in order to provide reference for researchers engaged in the research of human pancreatic cancer organoids.
Objective The present study aimed to observe the changes in Aβ deposition and estrogen receptor levels in the hippocampal tissue of female APP/PS1 mice with Alzheimer's disease(AD)after receiving an ovariectomy at different ages.Methods 3-month-old APP/PS1 female mice were divided into three groups,with 20 mice in each group.Ten mice were treated with bilateral ovarian resection via abdominal surgery as the model(ovx)group.The 10 mice in the control(sham)group underwent surgery in which an equivalent volume fat was removed from the corresponding area.After 3,4,and 5 months,APP/PS1 mice were assigned to 6-,7-,and 8-month age groups.The cognitive function of the APP/PS1 mice of different ages was evaluated using the Morris water maze.Morphological changes to intracellular organelles in the CA1 area of the hippocampus of the two groups of mice were observed using transmission electron microscopy.Immunofluorescence staining was used to detect the deposition of Aβ in the CA1 area of the hippocampus of the two groups of mice.ELISA was used to detect the levels and contents of serum estrogen,ROS,SOD,and MDA,and Western blot was used to determine the expression levels of ERα and ERβ.Results As the ovx group mice increased in age,their escape latency in the water maze test gradually lengthened,the deposition of Aβ in the hippocampal CA1 area increased,the mitochondrial swelling of hippocampal neurons increased,and larger amounts of lipofuscin and amyloid deposition were observed in the cytoplasm.Their serum E2 levels and SOD activity decreased,while their ROS levels and MDA contents increased,and the expression of ERα and ERβ in the hippocampal tissue decreased.Conclusions Ovariectomy in mice with low estrogen status may exacerbate hippocampal Aβ deposition and age-dependent cognitive decline.
Objective To investigate the virulence, drug resistance genes, and whole genome information of Aeromonas dhakensis from shrimp and preliminarily evaluate the pathogenicity of Aeromonas dhakensis. Methods Molecular identification and MLST typing of shrimp isolates were performed. The pathogenicity of this strain was examined by β-hemolysis, extracellular protease, liquid toxicity test, cytotoxicity, and mouse tests, and virulence gene detection. The characteristics of drug resistance were analyzed by a drug sensitivity test and drug resistance gene detection. Eggnog and Prophage Hunter tools were used to analyze the whole genome sequence. Results The 18FX22 strain was identified as Aeromonas dhakensis. The pathogenicity of this strain was characterized by β-hemolysis and extracellular protease activity. It was pathogenic to Caenorhabditis elegans, mouse fibroblasts, and mice. The strain was resistant to amoxicillin/clavulanic acid, cefazolin, and polymyxin, which carried 10 virulence genes and 12 drug resistance genes. Whole genome sequencing showed that the whole genome size of 18FX22 was 4 811 079 bp and GC content was 61.33%. COG annotation indicated that 18FX22 had 3850 genes with 20 classes of functions. Prophage prediction revealed that the 18FX22 genome contained two prophage sequences. Cluster analysis of Aeromonas dhakensis worldwide showed that this strain was more closely related to GCA016729585. Conclusions Aeromonas dhakensis was isolated from commercial shrimp and found to be highly toxic, indicating that the control of seafood food safety should be strengthened. This study provides a reference to prevent and control Aeromonas dhakensis infection.
目的 探讨低氧诱导剂二氯化钴(CoCl2)调控 3T3-L1 脂肪细胞自噬活性从而改善脂肪细胞炎症反应和胰岛素抵抗的机制.方法 常规培养和诱导分化 3T3-L1 脂肪细胞成为成熟的脂肪细胞,CoCl2 作为低氧诱导剂在不同时间、不同浓度条件下干预成熟的脂肪细胞,确认CoCl2 干预脂肪细胞自噬活性的最佳时间和浓度,随后根据此时间和浓度分组,分为 0 h(对照组)、12 h、24 h、48 h CoCl2 处理组,收集细胞样本进行相关指标测定.MTT法评价各组细胞存活情况;Western blot分析各组细胞HIF-1α及其下游蛋白葡萄糖转运蛋白Glut-1、自噬相关蛋白LC3-Ⅱ和Beclin-1的表达情况;免疫荧光法检测各组细胞的自噬水平;ELISA法检测各组细胞上清液中炎症因子TNF-α和IL-6分泌情况.结果 150 μmol/L CoCl2 是调控 3T3-L1 脂肪细胞自噬水平的最佳干预浓度;150 μmol/L CoCl2 干预成熟的脂肪细胞 24 h时,自噬活性水平增高,细胞存活率无显著的减低,脂肪细胞中HIF-1α、LC3-Ⅱ、Beclin-1、Glut-1蛋白的表达水平也显著升高,但炎症因子TNF-α和IL-6 分泌水平无明显增加;48 h时自噬水平减低,细胞存活率出现显著减低,脂肪细胞中HIF-1α、LC3-Ⅱ、Beclin-1、Glut-1 蛋白的表达水平减低,炎症因子TNF-α和IL-6分泌水平出现增加趋势.结论 150 μmol/L CoCl2 可以调控脂肪细胞自噬水平增加,自噬水平的增加以依赖HIF-1α的方式活化,从而使自噬发挥保护脂肪细胞的作用使其免受炎症损伤和改善脂肪细胞胰岛素抵抗.
目的 探讨大蒜素改善高糖诱导的人腹膜间皮细胞-间充质转化的相关机制.方法 培养人腹膜间皮细胞(human peritoneal mesothelial cells,HPMCs)后将其进行两次分组,分组 1:①对照组;②8.5 mmol/L D-葡萄糖诱导组(8.5 mmol/L DG组);③17 mmol/L D-葡萄糖诱导组(17 mmol/L DG组);④34 mmol/L D-葡萄糖诱导组(34 mmol/L DG组);⑤68 mmol/L D-葡萄糖诱导组(68 mmol/L DG组).其中除对照组外,其余组分别用 8.5、17、34、68 mmol/L的D-葡萄糖诱导 48 h.分组 2:①对照组;②34 mmol/L D-葡萄糖诱导组(HG组);③34 mmol/L D-葡萄糖+低剂量大蒜素诱导组(AL-L组);④34 mmol/L D-葡萄糖+中剂量大蒜素诱导组(AL-M组);⑤34 mmol/L-葡萄糖+高剂量大蒜素诱导组(AL-H组);⑥34 mmol/L D-葡萄糖+JAK2 抑制剂诱导组(JAK2 组).其中HG组用 34 mmol/L的D-葡萄糖诱导 48 h,AL-L组、AL-M组、AL-H组用 34 mmol/L的D-葡萄糖预处理 6 h后分别用10、20 和 40 ng/mL大蒜素诱导 48 h,JAK2 组加入 1 μmol/L AG490 预处理 6 h后用 34 mmol/L的D-葡萄糖诱导48 h.ELISA检测HPMCs上清的IL-6、TNF-α和IL-1β的含量;CCK-8检测细胞增殖并观察形态;Western blot检测JAK2、p-JAK2、STAT3、p-STAT3、N-cadherin、E-cadherin、Vimentin、α-SMA、MCP-1、p65、p-p65 蛋白的表达情况.结果 与对照组相比,高糖诱导组HPMCs的相对存活率明显降低(P＜0.01),细胞形态表现异常,促进上皮细胞-间充质转分化(epithelial-mesenchymal transition,EMT)发生的α-SMA、N-cadherin和Vimentin表达明显上调,抑制EMT发生的E-cadherin蛋白表达明显下调,JAK2/STAT3信号通路被激活从而导致EMT的发生(P＜0.01);而大蒜素能明显促进高糖诱导后的HPMCs增殖,恢复异常的细胞形态,调节与EMT发生的相关蛋白水平从而改善HPMCs的上皮间质转分化;与高糖诱导组相比,大蒜素处理组HPMCs的促炎症因子IL-1β、IL-6和TNF-α明显降低,促炎症蛋白p-p50和MCP1表达明显下调,表明大蒜素能改善EMT引起的炎症.结论 大蒜素可通过抑制JAK2/STAT3 信号通路调节EMT发生的标志蛋白、炎症信号蛋白及炎症因子水平从而改善高糖诱导的EMT及炎症.
目的 探讨中等有氧运动和高强度间歇运动对非酒精性脂肪肝小鼠心肌线粒体自噬的影响及可能机制.方法 将 40 只 3 周龄雄性C57BL/6J小鼠随机分为普通饲养组(Chow组,n=10)和高脂饲养组(HFD组,n=30).第 18 周,将HFD组体重超过普通饮食组 20%～30%(包含 20%)者判定为肥胖小鼠(n=26),通过随机抽选 2 只小鼠做肝油红O染色,确定NAFLD小鼠模型建立成功.随机筛选出 16 只NAFLD小鼠分成中等强度有氧运动组(MICT组)和高强度间歇有氧运动组(HIIT组),每组 8 只,分别对两组进行 8 周的运动训练,干预结束后称重取材.Masson染色观察心肌纤维化情况;透射电镜观察心肌细胞超微结构;Western blot检测心肌中线粒体自噬和线粒体生物发生等相关蛋白表达.结果 (1)与Control组相比,Model组体重显著增加、心指数明显下降;与Model组相比,MICT组和HIIT组体重明显增加、MICT组心指数上升.(2)Masson结果显示,与 Control组相比,Model组心肌内胶原纤维含量显著增加,且电镜下心肌肌纤维排列杂乱、断裂,心肌形态杂乱不堪,线粒体肿胀、嵴发生断裂且模糊不清并夹杂多个脂滴;与Model组相比,MICT组和HIIT组心肌内胶原纤维含量显著性减少,透射电镜下心肌肌纤维排列略有恢复,z线清晰可见,线粒体变性程度略有改善,且脂滴也略有减少,其中,MICT组对心脏组织结构改善效果优于HIIT组.(3)Western blot结果显示,与Control组相比,Model组心肌组织中PINK1 和Beclin1 表达水平无明显变化,Parkin、LAMP1 和PGC-1α表达显著下降(P＜0.01),蛋白表达和LC3-Ⅱ/LC3-Ⅰ比值显著升高(P＜0.05,P＜0.01);与Model组相比,MICT组和HIIT组PINK1 和Beclin1 表达水平无明显变化,PGC-1α表达呈上调趋势,其中MICT组Parkin、LAMP1 表达显著升高(P＜0.05),p62 蛋白表达和LC3-Ⅱ/LC3-Ⅰ比值明显降低(P＜0.05,P＜0.01),HIIT组Parkin、LAMP1 表达水平具有上调趋势,LC3-Ⅱ/LC3-Ⅰ比值和p62 蛋白表达明显降低(P＜0.05,P＜0.01).结论 不同方式有氧运动均能有效改善NAFLD小鼠心肌结构和功能损伤,其作用途径可能是通过刺激心肌细胞内线粒体自噬通量,激活自噬,恢复细胞正常自噬功能,从而改善心肌细胞损害,且中等连续有氧运动改善效果更佳.
目的 探究金丝桃苷(Hyp)对肾病综合征(nephrotic syndrome,NS)大鼠肾自噬及AMPK/mTOR/ULK1 通路的影响.方法 32 只 6 周龄SD大鼠分为正常组(N组)、肾病综合征组(NS组)、金丝桃苷组(Hyp组,60 mg/kg Hyp)、金丝桃苷+AMPK抑制剂组(Hyp+CC组,60 mg/kg Hyp+0.2 mg/kg CC),每组 8 只.除N组外,其余各组大鼠采用一次性尾静脉注射阿霉素(6.5 mg/kg)建立NS模型,模型成功率为 75.0%.Hyp组大鼠灌胃给予60 mg/kg的Hyp,Hyp+CC组给予 60 mg/kg的Hyp灌胃和 0.2 mg/kg CC腹腔注射,N组和NS组给予等量溶剂,每天 1 次,连续14 d.给药结束后,全自动分析仪检测24 h尿液总蛋白(UTP)、血尿素氮(BUN)、血清肌酐(Scr)和白蛋白(ALB)水平;HE染色观察肾组织病理形态;透射电子显微镜(TEM)观察肾组织超微结构变化;Western blot检测肾中自噬、足细胞及 AMPK/mTOR/ULK1 通路蛋白表达;免疫荧光染色观察自噬体和足细胞的定位.结果 相比于N组,NS组肾小球体积变大、肾小管萎缩或部分消失、基底膜增厚;UTP、BUN、Scr、基底膜厚度、足突宽度及p-AMPK/AMPK比值显著增加(P＜0.05),ALB、LC3-Ⅱ/Ⅰ、Beclin-1、Atg5、Atg7、NPHS2 蛋白水平、NPHS2、Beclin-1相对荧光强度及p-AMPK/AMPK、p-ULK1/ULK1 比值显著降低(P＜0.05).相比于NS组,Hyp治疗可改善肾小球形态,降低 UTP、BUN、Scr、基底膜厚度、足突宽度及 p-AMPK/AMPK 比值(P＜0.05),增加 ALB、LC3-Ⅱ/Ⅰ、Beclin-1、Atg5、Atg7、NPHS2 蛋白水平、NPHS2、Beclin-1 相对荧光强度及 p-AMPK/AMPK、p-ULK1/ULK1 比值(P＜0.05).相比于Hyp组,Hyp+CC组肾小球体积变大、肾小管萎缩或部分消失、基底膜增厚;UTP、BUN、Scr、基底膜厚度、足突宽度及p-AMPK/AMPK比值显著增加(P＜0.05),ALB、LC3-Ⅱ/Ⅰ、Beclin-1、Atg5、Atg7、NPHS2 蛋白水平、NPHS2、Beclin-1相对荧光强度及p-AMPK/AMPK、p-ULK1/ULK1 比值显著降低(P＜0.05).结论 Hyp可能通过激活AMPK/mTOR/ULK1 通路促进肾细胞自噬活性,减轻NS大鼠的足细胞损伤等肾病变.
阿尔茨海默病(Alzheimer's disease,AD)是一种侵袭性神经退行性疾病,其发病原因迄今未清.神经炎症是中枢神经系统(central nervous system,CNS)中由小胶质细胞和星形胶质细胞激活的一种慢性炎症反应,与多种炎症因子的释放和血脑屏障(blood brain barrier,BBB)的破坏密切相关.研究表明,神经炎症是继β-淀粉样蛋白(amyloid-β protein,Aβ)沉积和神经原纤维缠结(neurofibrillary tangles,NFTs)后AD的第三大病理改变.本文总结了小胶质细胞-星形胶质细胞的交互作用,并对其在神经炎症和AD中的作用进行整理和讨论,以期为AD的发病机制及防治研究提供理论支持和实验参考.
目的 探索六君子汤乙酸乙酯提取物(EAELD)对癌相关成纤维细胞(CAFs)条件培养基下食管癌EC9706 细胞能量代谢影响的分子机制.方法 噻唑蓝(MTT)法检测EAELD对EC9706 增殖活性的影响;比色法检测EAELD对CAFs条件培养基(CAFM)下EC9706 细胞上清中乳酸及葡萄糖含量的影响;Seahorse能量代谢分析系统检测EAELD 对 CAFM 下 EC9706 细胞能量代谢的影响;实时荧光定量 PCR(RT-qPCR)、蛋白免疫印迹(Western blot)法检测EAELD对能量代谢相关分子mRNA及蛋白表达的影响.结果 与DMEM相比,除 10 μg/mL组外,EAELD对EC9706 细胞增殖活性均有明显抑制作用(P＜0.05),选取抑制浓度(IC30)25 μg/mL,半抑制浓度(IC50)40 μg/mL,作为低、高剂量组进行后续实验.在CAFM培养的EC9706细胞各组中,EAELD低、高剂量组都能显著降低非线粒体耗氧、基础呼吸值、最大呼吸值、合成ATP耗氧量、备用呼吸能力、基础糖酵解、补偿糖酵解、糖酵解潜能(P＜0.01),减少 EC9706 细胞上清乳酸含量(P＜0.01),下调 GLUT1 的 mRNA 表达(P＜0.05,P＜0.01),下调p-PKM2、HK2、PKM2、MCT1蛋白表达(P＜0.01);EAELD高剂量组能够下调EC9706 细胞的线粒体耗氧与基础糖酵解比值(P＜0.05),减少EC9706细胞葡萄糖摄取(P＜0.05),下调p-PKM2、GLUT1的蛋白表达(P＜0.01,P＜0.05);EAELD低剂量组能够下调MCT1的mRNA表达(P＜0.05).结论 六君子汤乙酸乙酯提取物能够干预CAFs条件培养基下EC9706细胞的能量代谢,其机制可能与EAELD调控HK2、PKM2、GLUT1、MCT1、MCT4的mRNA和蛋白表达相关.
目的 通过旷场和Morris水迷宫实验,探究黄芩苷调控注意缺陷多动障碍动物模型核心临床症状的疗效及其潜在机制.方法 将 30 只SHR大鼠随机分为模型组、盐酸哌甲酯(MPH)组、黄芩苷组、黄芩苷+丁苯那嗪组和MPH+丁苯那嗪组,每组 6 只,另设 6 只WKY大鼠为对照组.MPH组(1.5 mg/kg)、黄芩苷组(150 mg/kg)按体重(1 mL/100 g)灌胃给药,空白组、模型组给等量生理盐水,MPH+丁苯那嗪组、黄芩苷+丁苯那嗪组在灌胃给药同时,按体重(0.5 mL/100 g)腹腔注射丁苯那嗪(3 mg/kg).连续给药 4 周.在规定时间进行旷场和水迷宫实验,记录并分析实结果.结果 MPH组和黄芩苷组在旷场实验中运动总距离和平均速度较模型组显著减小(P＜0.05),在水迷宫实验中潜伏期较模型组显著缩短(P＜0.05)、在目标象限运动距离和停留时间占比及穿越平台次数较模型组显著增加(P＜0.05),且这两组间无明显差异;黄芩苷+丁苯那嗪组在旷场实验中运动总距离和平均速度较模型组显著减小(P＜0.05),较黄芩苷组明显增大,在水迷宫实验中潜伏期较模型组显著缩短(P＜0.05),较黄芩苷组显著延长(P＜0.05)、在目标象限运动距离和停留时间占比较模型组明显增加,较黄芩苷组显著减少(P＜0.05).结论 黄芩苷能够改善SHR大鼠多动、冲动、注意力不集中等核心症状,其疗效的发挥可能与调控DA囊泡转运相关.
目的 建立有效稳定的小鼠慢性酒精中毒性脑损伤模型.方法 将 40 只C57BL/6J小鼠随机分成对照组和模型组,模型组小鼠除基础饮用水中添加 5%(v/v)酒精外,同时灌胃 28%(v/v)酒精,灌胃剂量前两周呈梯度增长(从 0 逐渐增加至 6 g/kg体重),随后 4 周维持在 6 g/kg体重,对照组饮用水中不添加酒精同时灌胃等量生理盐水.实验结束后,通过行为学实验测定小鼠的认知功能和运动能力,通过组织病理染色检测小鼠脑组织的形态学变化.结果 与对照组比较,模型组小鼠在行为学实验中表现出认知功能和运动功能障碍,模型组小鼠脑组织病理染色显示海马区形态损伤和细胞坏死.结论 实验有效建立了小鼠慢性酒精中毒性脑损伤模型,该模型可应用于慢性酒精中毒性脑病的机制和药物研究.
缺血性脑卒中是一种具有高残疾率和死亡率,严重者可危及生命的脑血管疾病,在全球范围内具有沉重的社会经济负担.缺血性脑卒中的病因及病理过程是由多种分子过程介导,这些分子机制部分转录后被动态调节.越来越多证据表明,微小RNA(micro-ribonucleic-acid,miRNA)作为转录后基因沉默的重要介质在基因表达和缺血性脑卒中病理过程中起着至关重要的作用.在本综述中,我们介绍了miRNA在缺血性脑卒中不同机制中的神经保护作用.由于通过特定的药物和非药物治疗促进或抑制miRNA的表达可能有益于缺血性脑卒中的恢复,本文还详细论述了关于miRNA在缺血性脑卒中的临床诊断和治疗相关作用,旨在对本领域临床和基础研究提供一定的参考.
目的 总结骨肉瘤(Osteosarcoma,OS)动物模型造模要点,为完善其造模方法和评价指标提供参考与建议.方法 通过检索中国知网、万方数据库、PubMed中OS动物模型相关文献,总结其实验动物种类、性别、造模方法、癌细胞株种类、检测指标等,建立数据库进行统计分析.结果 共纳入 284 篇文献,统计分析发现OS模型动物首选BALB/c-nu/nu裸鼠(227 例,75.17%),其次是SD大鼠(20 例,6.62%).造模方式多选用背部皮下细胞液移植法(66 例,21.85%)、腋部皮下细胞液移植法(55 例,18.21%)或原位细胞液移植法(51 例,16.89%)等;癌细胞株种类以人源MG-63 细胞(100 例,33.11%)、鼠源UMR-106 细胞(39 例,12.91%)为主;检测指标主要选择肿瘤组织表观指标(238 例,83.80%)、肿瘤组织HE染色(129 例,45.42%)、动物表观指标(94 例,33.10%)、肿瘤组织免疫组化(89 例,31.34%).结论 目前OS模型多选用 4～6 周龄BALB/c-nu/nu裸鼠作为实验动物,采用人源MG-63 细胞异位移植法(背部、腋下移植)建立OS动物模型,模型检测指标以动物表观指标、肿瘤表观指标、肿瘤组织病理进行整体评价.但目前依旧缺少与临床吻合度高的动物模型制备及评价标准,本文通过文献挖掘、数据分析总结其优缺点,以期为建立良好的OS模型提供参考,更好地应用于OS机制研究及新药开发.
目的 分析长链非编码RNA SNHG16(long non-coding RNA SNHG16,lncRNA SNHG16)通过调控微小RNA-570(miR-570)对肝癌细胞索拉非尼耐药的机制研究.方法 采用实时荧光RT-PCR检测人体正常肝组织、肝癌细胞组织中HepG2、HepG2-R细胞的lncRNA SNHG16、miR-570 表达,并对HepG2-R细胞做转染,后分别记为HepG2-R+pcDNA组、HepG2-R+pcDNA SNHG16 组、HepG2-R+anti-miR-NC组、HepG2-R+anti-miR-570 组、HepG2-R+pcDNA SNHG16+miR-NC组、HepG2-R+pcDNA SNHG16+miR-570 组,用于后续试验,且将miR-NC、miR-570、si-NC、si-SNHG16 用相同方式转染至HepG2 细胞,分别记为miRNC组、miR-570 组、si-NC组、si-SNHG16 组.用MTT法、流式细胞仪、Transwell试验检测细胞增殖、凋亡及侵袭,Western blot法测定细胞CyclinD1、P21、MMP-9、MMP-2表达变化.结果 与人体正常肝组织组相比,肝癌细胞组织组的lncRNA SNHG16 表达升高,miR-570 表达下降(P＜0.05).与正常细胞HepG2-P组相比,HepG2-R组的lncRNA SNHG16 及IC50 值提高,miR-570、HepG2-R细胞在索拉非尼浓度为 1、2、4、8、16 μmol/L中的抑制水平下降(P＜0.05).HepG2-R+pcDNA SNHG16 作为过表达组,其lncRNA SNHG16 表达显著升高(P＜0.05),与HepG2-R+pcDNA组对比,HepG2-R+pcDNA SNHG16 组的迁移的细胞个数及CyclinD1、P21、MMP-9、MMP-2 的表达水平降低,抑制率、凋亡率及 P21 的表达水平上升(P＜0.05).与HepG2-R+anti-miR-NC组相比,HepG2-R+anti-miR-570 组miR-570 表达水平降低(P＜0.05),与HepG2-R+anti-miR-NC组比较,HepG2-R+anti-miR-570 组CyclinD1、MMP-9、MMP-2 表达水平降低,抑制率、凋亡率及P21 的表达水平升高(P＜0.05).双荧光素酶报告实验显示,与miR-NC组相比,miR-570 使WT-SNHG16 荧光素酶活性降低(P＜0.05),而对MUT-SNHG16 荧光素酶活性影响较小(P＞0.05).过表达lncRNA SNHG16 可使HepG2-R细胞中miR-570 表达下降(P＜0.05),与HepG2-R+pcDNA SNHG16+miR-NC组相比,HepG2-R+pcDNA SNHG16+miR-570 组迁移的细胞个数及CyclinD1、MMP-9、MMP-2的表达水平升高,抑制率、凋亡率及P21 的表达水平降低(P＜0.05).结论 lncRNA SNHG16 可调控HepG2-R肝癌细胞的耐药性,其机制与lncRNA SNHG16 靶向调控miR-570 有关,为临床治疗肝癌细胞提供了新的靶点.
目的 探讨长链非编码RNA(LncRNA)FGD5-AS1 对口腔鳞状细胞癌(OSCC)细胞增殖、凋亡、迁移和侵袭的影响与机制.方法 利用在线数据库分析FGD5-AS1 在OSCC中的表达.以在滕州市中心人民医院口腔科收集的 30 例OSCC患者的肿瘤组织、正常组织和体外培养的人口腔黏膜细胞(HOK)和OSCC细胞(SCC-9、HSC-4、SCC-25、CAL-27)为研究对象,采用qRT-PCR法检测FGD5-AS1 和miR-129-5p表达.将FGD5-AS1 表达最高的CAL-27 细胞分成 Control 组、si-NC 组、si-FGD5-AS1 组、si-FGD5-AS1+NC inhibitor 组和 si-FGD5-AS1+miR-129-5p inhibitor组,CCK-8法和克隆形成实验检测细胞增殖能力;流式细胞术检测细胞凋亡水平;划痕愈合实验检测细胞迁移能力;Transwell小室检测细胞侵袭能力;双荧光素酶报告实验验证 FGD5-AS1 与 miR-129-5p 的靶向关系;Western blot检测高迁移率族蛋白B1(HMGB1)蛋白表达.构建体内异种移植瘤模型,并分为sh-NC组、sh-FGD5-AS1 组、miR-129-5p inhibitor组和sh-FGD5-AS1+miR-129-5p inhibitor组,检测肿瘤体积和肿瘤;qRT-PCR检测移植瘤组织FGD5-AS1、miR-129-5p表达;免疫组化检测移植瘤组织HMGB1、Ki67 表达.结果 数据库分析显示,OSCC肿瘤组织中FGD5-AS1 的表达水平是正常组织的4 倍,且FGD5-AS1 表达与OSCC患者分级较差相关.与正常组织或人口腔黏膜细胞相比,肿瘤组织和 OSCC 细胞系中 FGD5-AS1 表达明显升高,miR-129-5p 表达明显降低(P＜0.05),选择FGD5-AS1 表达水平最高的CAL-27 细胞进行转染实验.沉默FGD5-AS1 可升高细胞凋亡率,降低细胞活力、划痕愈合率及侵袭细胞数,并增强miR-129-5p表达,下调HMGB1 表达(P＜0.05).miR-129-5p是FGD5-AS1的靶基因,抑制miR-129-5p表达可逆转沉默FGD5-AS1 对OSCC细胞增殖、凋亡、迁移和侵袭的影响.体内实验显示,沉默FGD5-AS1 明显抑制移植瘤生长和HMGB1、Ki67 表达(P＜0.05),抑制miR-129-5p则相反;抑制miR-129-5p可逆转沉默FGD5-AS1 对肿瘤生长和HMGB1、Ki67 表达的抑制作用(P＜0.05).结论 FGD5-AS1 在OSCC细胞中上调,干扰FGD5-AS1 可通过靶向调控miR-129-5p/HMGB1 轴,抑制OSCC细胞增殖、迁移、侵袭,促进凋亡.
肝是最常见的肿瘤远处转移脏器,miRNAs的表达对肿瘤肝转移的过程非常重要.本文整理分析了相关miRNAs调节消化系统中恶性肿瘤发生肝转移起调节作用的研究进展.查阅相关文献介绍miRNAs在结直肠癌、胃癌、胰腺癌、胆囊癌等几类消化系统肿瘤肝转移过程中发挥的调节作用,为肿瘤肝转移诊断、治疗及研究提供帮助.
猴痘是猴痘病毒(monkeypox virus,MPXV)感染引起的传染性疾病.猴痘病毒的宿主依然没有完全明确,啮齿类与非人灵长类动物被认为是潜在的宿主.猴痘正在全世界范围内逐渐扩散,但我国一直以来并未开展猴痘的动物模型的研究.作为一种严重危害人类健康的病原体,猴痘病毒有多种感染类型;其在人群中的传播呈现新的特点.因此本文论述了猴痘病毒发现的经过与早期疫情、不同的感染类型和共感染.此外,本文还介绍了啮齿类和非人灵长类动物的实验性感染与猴痘动物模型.
阿尔茨海默病(Alzheimer's disease,AD)是一种发病机制复杂,不可逆转的神经退行性疾病.AD患者表现为记忆缺失,突触可塑性受损.本文关注与突触可塑性密切相关的环腺苷酸反应元件结合蛋白(cAMP responsive element-binding protein,CREB),总结CREB的结构、激活途径、下游基因及对记忆的调控作用,关注CREB与AD的联系,为基于神经可塑性的AD的研究提供参考.
帕金森病(Parkinson's disease,PD)是一种神经退行性疾病,目前发病机制不明,患者会出现与疾病相关的运动和非运动症状.啮齿类动物模型只能部分精准模拟患者的症状,导致相关的临床前研究存在局限性,非人灵长类动物模型能够较好的弥补这种缺陷,对非人灵长类PD模型的运动和非运动症状的量化有利于发病机制和治疗的研究,本综述中总结了不同行为学量化方式,同时对比了不同方法之间的优劣性,为进行PD猴模型研究提供了行为学测试的参考.
