OBJECTIVE:Macrophage polarization and function are regulated by epigenetic mechanisms and microRNAs transported by exosomes; however, the specific mechanisms are not clear yet. The study aims to investigate whether exosomes regulate miR-155-5p maturation through methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification, thereby driving M1 macrophage polarization and providing potential targets for inflammation and tumor immunotherapy. METHOD:THP-1 cells were differentiated into M0 macrophages by PMA and subsequently polarized to the M1 phenotype with LPS and IFN-γ. Exosomes were extracted from culture supernatants using ultracentrifugation, and identified by dynamic light scattering (DLS), transmission electron microscopy (TEM), and western blotting (WB). Genes for overexpression or knockdown of METTL3/miR-155-5p were loaded into M0 exosomes, respectively, and M0-exos were then used to deliver them to recipient macrophages or injected into mice. RESULTS:Dot blot analysis showed an increase in m6A modification levels in the OE-METTL3 group. Flow cytometry (FCM), Enzyme-linked immunosorbent assay (ELISA), RT-qPCR, and WB results indicated that the OE-METTL3 group exhibited significantly elevated expression of M1 macrophage markers CD86, iNOS and HLA-DR, cytokines IL-1β, IL-6, TNF-α, and IL-18, signaling molecules NLRP3, TLR4, and caspase-1, whereas the expression of M2 macrophage markers CD206, CD163, Arg-1, IL-10 and TGF-β was decreased. The SI-METTL3 group showed the opposite trends. Further results demonstrated that miR-155-5p was correspondingly upregulated or downregulated. Additional intervention with miR-155-5p confirmed its regulatory effect on m6A modification and M1 polarization (p < 0.05). In vivo experiments yielded consistent results. CONCLUSION:Exosomes promote miR-155-5p maturation through METTL3-mediated m6A modification, activate the NLRP3/TLR4/caspase-1 signaling pathway, drive M1 macrophage polarization, and exacerbate inflammatory responses. This research may provide novel potential targets for inflammation and tumor immunotherapy.

Background: The roles of small extracellular vesicles (sEVs) and mRNA modifications in regulating hepatitis B virus (HBV) transmission, replication, and related disease progression have received considerable attention. However, the mechanisms through which methyltransferase-like 3 (METTL3) and insulin-like growth factor 2 (IGF2BP2), key genes that mediate m6A modifications, regulate HBV replication in sEVs remain poorly understood. Therefore, this study investigated the molecular mechanisms through which the key molecules (METTL3 and IGF2BP2) in sEVs mediate m6A epigenetic modification to regulate HBV replication. Methods: Small extracellular vesicles were extracted from the supernatants of HepG2.2.15 and HepG2 cells via ultracentrifugation, followed by purification with hepatitis B virus surface antigen (HepBsAg) immunomagnetic beads. The sEVs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and Western blotting (WB). Methylation enrichment in the two types of sEVs was analyzed by dot blotting and quantitative reverse transcription-PCR (RT-qPCR). The cells were treated with HepG2.2.15–sEVs transfected with either the METTL3 plasmid, METTL3 siRNA, the IGF2BP2 plasmid, or the IGF2BP2 siRNA. After 48 h, the expression of METTL3, IGF2BP2, and HBV DNA expressions were assessed via dot blotting, quantitative-PCR (qPCR), RT-qPCR, and WB. Co-immunoprecipitation (co-IP) was performed to investigate the interactions between METTL3 and IGF2BP2. Results: By conducting TEM, DLS, and WB analyses, we confirmed that the isolated sEVs exhibited typical characteristics. HepG2.2.15-derived sEVs presented elevated levels of m6A modifications, with increased METTL3 and IGF2BP2 mRNA and protein expression levels, respectively (p < 0.05). In the overexpression (OE)-METTL3 group, the expression levels of HBV pregenomic RNA (HBV pgRNA), HBV DNA, HBV relaxed circular DNA (HBV rcDNA), HBV covalently closed circular DNA (HBV cccDNA), HBsAg, hepatitis B virus core antigen (HBcAg), and hepatitis B virus e antigen (HBeAg) were significantly elevated compared to those in the control group (p < 0.01). In contrast, results for the small interfering (SI)-METTL3 group were the opposite. Similarly, in the OE-IGF2BP2 group, HBV pgRNA, HBV DNA, HBV rcDNA, HBV cccDNA, HBsAg, HBcAg, and HBeAg expression were greater than in the control group (p < 0.05), whereas the opposite results were recorded in the SI-IGF2BP2 group. Co-immunoprecipitation confirmed that METTL3 and IGF2BP2 interact synergistically. Conclusion: Small extracellular vesicles increase METTL3 and IGF2BP2 expression, synergistically promoting HBV replication by regulating m6A modification levels.

MicroRNAs regulate neural stem cell function. Argonaute 2 protein, constituent of the RNA-induced silencing complex, plays an important role in regulating microRNA function for post-transcriptional gene silencing. Although Argonaute 2 and microRNAs are recognized as central regulators of RNA-induced silencing complex, their precise role in adult neural stem cell function has remained unclear. In particular, it was not known whether Argonaute 2 is required for sustaining neural stem cell proliferation, neurogenesis, and oligodendrogenesis in the adult brain, or how its loss might influence recovery after ischemic injury. The present study examined the effect of Argonaute 2 deletion in adult neural stem cells on neurogenesis and oligodendrogenesis. Adult transgenic mice with conditional and inducible ablation of Argonaute 2 in Ascl1-lineage neural stem cells exhibited the reduction of neurogenesis in the ventricular-subventricular zone of the lateral ventricle and in the subgranular zone of the dentate gyrus, as evidenced by a decrease in neural stem cell proliferation and neuroblast numbers. Argonaute 2 deletion also reduced oligodendrogenesis in the corpus callosum, as indicated by the reduction of oligodendrocyte progenitor cell proliferation and the number of mature oligodendrocytes. Additionally, deleting Argonaute 2 in adult neural stem cells of ischemic mice exacerbated impairments of sensorimotor and cognitive functions. Mechanistically, Argonaute 2 ablation in neural stem cells reduced the stability of mature microRNAs and downregulated genes involved in the Shh (Sonic Hedgehog), Notch, and TGF beta (transforming growth factor beta) signaling pathways, which regulate the functions of neural stem cells. Collectively, our study demonstrates that Argonaute 2 is essential for adult neural stem cell-mediated neurogenesis and oligodendrogenesis, with its deletion worsening recovery after ischemia. By revealing that Argonaute 2 stabilizes mature microRNAs, our work uncovers a novel mechanism of neural stem cell regulation and highlights Argonaute 2 as a potential therapeutic target for neurodegenerative and ischemic brain diseases.

Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent and aggressive form of pancreatic cancer. Gemcitabine (GEM), the first‑line treatment for PDAC, which alleviates symptoms and enhances the quality of life of patients. However, it is prone to lead to the development of drug resistance during treatment. Interferon (IFN)‑γ exhibits antitumor and immunomodulatory properties. The present study aimed to explore the impact of IFN‑γ on the viability, migration and apoptosis of GEM‑resistant pancreatic cancer cells. Firstly, a GEM‑resistant pancreatic cancer cell line, named PANC‑1/GEM, was constructed. Hematoxylin and eosin staining analyzed the cell morphology, whereas reverse transcription‑quantitative PCR (RT‑qPCR) assessed the expression levels of the drug‑resistance genes multidrug resistance‑associated protein (MRP) and breast cancer resistance protein (BCRP). The MTT assay and cell counting techniques were used to determine the appropriate concentration of IFN‑y and its effects on cell viability. The IFN‑γ‑induced apoptosis of PANC‑1/GEM cells was assessed using an Apoptosis Detection Kit, whereas the impact of IFN‑γ on the migration of these cells was evaluated using a wound‑healing assay. The MTT assay revealed a resistance index of 22.4 in the PANC‑1/GEM cell line. RT‑qPCR indicated that, compared with in wild‑type cells, the PANC‑1/GEM resistant strain exhibited lower MRP and higher BCRP mRNA expression levels. The optimal concentration of IFN‑γ for affecting PANC‑1/GEM cells was determined to be 0.3 µg/ml. At this concentration, IFN‑γ induced PANC‑1/GEM cell apoptosis, along with a notable reduction in migration. Following treatment of PANC‑1/GEM cells with IFN‑γ, MRP expression increased whereas BCRP mRNA expression decreased, indicating a reversal in their drug‑resistance gene expression. In conclusion, IFN‑γ exhibited antitumor immune properties by upregulating MRP and downregulating BCRP expression, reversing drug‑resistance gene expression, and reducing cell viability and migration, while promoting apoptosis in PANC‑1/GEM cells. IFN‑γ could potentially serve as a treatment option for patients with GEM‑resistant pancreatic cancer.

Small extracellular vesicles (sEVs) have the ability to transfer genetic material between cells, but their role in mediating HBV infection and regulating M1 macrophages to promote immune evasion remains unclear. In this study, we utilized PMA + LPS + IFN-γ to induce THP-1 into M1 macrophages. We then extracted sEVs from HepG2.2.15 cell and treated the M1 macrophages with these sEVs. QPCR detection revealed the presence of HBV-DNA in the M1 macrophages. Additionally, RT-qPCR and WB analysis demonstrated a significantly decreased in the expression of TLR4, NLRP3, pro-caspase-1, caspase-1p20, IL-1β and IL-18 in the M1 macrophages (P < 0.05). Furthermore, RT-qPCR results displayed high expression levels of that miR-146a and FEN-1 in the sEVs derived from HepG2.2.15 cells (P < 0.01). RT -qPCR and WB analysis showed that these sEVs enhanced the expression of FEN-1 or miR-146a in the M1 macrophages through miR-146a or FEN-1 (P < 0.05), while simultaneously reducing the expression of TLR4, NLRP3, caspase-1p20, IL-1β and IL-18 in the M1 macrophages (P < 0.05). In summary, our findings indicate that sEVs loaded with HBV inhibit the inflammatory function of M1 macrophages and promote immune escape. Additionally, miR-146a and FEN-1 present in the sEVs play a crucial role in this process.

Objective To investigate the molecular mechanism by which methytransferase like3 (METTL3) cooperates with insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) to regulate HBV replication through N6-methyladenosine (m6A) modifications. Methods HBV stably replicating cell line HepG2.2.15 and its source cells HepG2 were used as models. Spot hybridization was used to analyze m6A modification level, and RT-qPCR and Western blotting were utilized to detect the expression of METTL3 and IGF2BP2 at mRNA and protein levels. Bioinformatics analysis and co-immunoprecipitation assay were applied to analyze METTL3 and m6A reading proteins and their interactions. After METTL3 plasmid, METTL3 siRNA and/or IGF2BP2 siRNA were transfected into HepG2.2.15 cells, respectively, the cells were assigned into OE-METTL3 group, si-METTL3 group, si-IGF2BP2 group, OE-METTL3+si-IGF2BP2 group, si-METTL3+si-IGF2BP2 group, and the cells untreated were subjected as control group. Copy numbers of HBV DNA, HBV rcDNA, and HBV cccDNA were detected by qPCR, and the expression of HBV pgRNA, METTL3, and IGF2BP2 were measured with RT-qPCR or Western blotting. Results Compared with HepG2 cells, m6A modification was enriched, and the expression levels of METTL3 and IGF2BP2 were elevated in HepG2.2.15 cells (P < 0.05). Compared with the control group, m6A modification enrichment was enhanced and IGF2BP2 expression level was elevated in the OE-METTL3 group (P < 0.05), and HBV replication-related indexes (HBV DNA, HBV rcDNA, HBV cccDNA, and HBV pgRNA) were increased (P < 0.01); and opposite phenomena were observed in the si-METTL3 group. Bioinformatics analysis and co-immunoprecipitation assay showed that METTL3 and IGF2BP2 were interacting proteins. Compared with the control group, m6A modification enrichment was attenuated and METTL3 expression level was reduced (P < 0.01), and HBV replication-related indexes were decreased in the si-IGF2BP2 group (P < 0.01). In the OE-METTL3+si-IGF2BP2 group, HBV replication-related indexes were reduced when compared with the OE-METTL3 group (P < 0.01). In the si-METTL3+si-IGF2BP2 group, HBV replication-related indexes were reduced when compared with the si-METTL3 group and si-IGF2BP2 group (P < 0.05). Conclusion METTL3 enriches m6A by depending on IGF2BP2, replicates the virus by enhancing the conversion of HBV rcDNA to cccDNA, and in turn enhances pgRNA reverse transcription.

目的:探究多聚甲醛固定外泌体对外泌体本身、细胞活性和功能的影响,为研究外泌体相关策略选择提供基础.方法:采用透射电镜和Western blot鉴定外泌体,通过外泌体转染HepG2、Panc-1、Hela细胞后的形态和功能变化,鉴定多聚甲醛固定和未固定DIOC18(3)(3,3'-二十八烷氧碳菁高氯酸盐)染色对外泌体生物学特性的影响.采用CCK-8、MTT试验、细胞凋亡试验、细胞分泌外泌体的功能和细胞本身的功能来确定多聚甲醛固定和未固定DIO染色外泌体对细胞活力和功能的影响.结果:多聚甲醛固定DIO染色外泌体转染细胞阳性率比多聚甲醛未固定组高(20.21±0.05)％(P<0.05),前者的凋亡率比后者高(12.65±0.01)％(P<0.05).多聚甲醛未固定DIO染色外泌体转染的细胞存活率比多聚甲醛固定组高(22.84±0.16)％(P<0.05),前者外泌体分泌率比后者高(28.10±5.65)％(P<0.05).多聚甲醛未固定DIO染色HepG2.2.15细胞上清来源的外泌体转染HepG2细胞后,HBV DNA拷贝数比多聚甲醛固定DIO染色外泌体组高3.33倍(P<0.05).结论:多聚甲醛固定DIO染色外泌体对外泌体本身特性及示踪定位研究具有良好效果;多聚甲醛未固定DIO染色外泌体亦能达到一定的示踪目的,但更能准确评价外泌体对细胞功能调控的作用.

This study examined exosomal hepatitis B virus (HBV)-DNA levels in chronic HBV infection (CHB). Patients were grouped according to the European Association for the Study of the Liver classification (1: HBV-DNA-positive CHB, normal alanine aminotransferase [ALT]; 2: HBV-DNA-positive CHB, elevated ALT; 3: HBV-DNA-negative HBeAb-positive CHB, normal ALT; 4: HBV-DNA-positive HBeAg-negative HBeAb-positive CHB, elevated ALT; 5: HBV-DNA-negative, HBcAb-positive; 6: HBV-negative, normal ALT). Exosomes were isolated, comparative analysis of exosomes and serum HBV-DNA. The HBV-DNA content was lower in exosomes than in serum for groups 1, 2, and 4 (all P < 0.05). In the groups negative for serum HBV-DNA (groups 3 and 5), the exosomal HBV-DNA levels were higher than the serum HBV-DNA levels (all P < 0.05). The exosomal and serum HBV-DNA levels were correlated in groups 2 (R (2) = 0.84) and 4 (R (2) = 0.98). The exosomal HBV-DNA levels were correlated with total bilirubin (R (2) = 0.94), direct bilirubin (R (2) = 0.82), and indirect bilirubin (R (2) = 0.81) in group 5 (all P < 0.05). In patients with CHB and negative for serum HBV-DNA, exosomal HBV-DNA was detectable and could be used to monitor the treatment effects. Exosomal HBV-DNA could be used in patients with a high suspicion of HBV infection but negative for serum HBV-DNA.

Background: Somatostatin (SST) lineage neurons, the second largest family of GABAergic interneurons in the neocortex, play a critical role in cortical activity and neuroplasticity after stroke. microRNAs (miRNAs) play an important role in mediating ischemic brain repair. Elucidating changes of miRNAs in SST neurons after stroke may provide molecular insight into SST neuron function. Methods: To systematically determine miRNA expression in SST neurons, we employed a miRNA tagging and affinity-purification (miRAP) method to select specific SST neurons via the Cre-loxp binary system in male SST-tAgo2 (tAgo2: a GFP-MYC-Ago2 fusion protein) mice subjected to 7 day middle cerebral artery occlusion (MCAO) (N=3/group). Results: Using miRNA sequencing, we found a total of 523 known and novel miRNAs with distinct profiles that were expressed in SST neurons isolated from ischemic hemispheres compared to miRNAs from sham operated mice. Among them, 70 and 7 miRNAs showed significantly increased and decreased expression, respectively, with a fold change above 2.0 and over 100 copy numbers. Top 3 upregulated miRNAs included miR-381-3p, miR-543-3p and miR-433-3p, while the top 3 downregulated ones were miR-9-5p, miR-423-5p and miR-486b-5p. Furthermore, using miRAP method in combination with RNA-sequencing, we uncovered over 1000 differential gene expression with at least 1.5-fold enrichment in ischemic SST neurons. Integrative analysis of miR-mRNA network by IPA algorithm revealed that 23 miRNAs were predicted to target deregulated mRNAs. Enriched pathways targeted by miRNAs were involved in NTRK3 (neurotrophic receptor tyrosine kinase 3), insulin, PI3K, EGFR1, mTOR signaling, etc, which are associated with neurodevelopment, neuronal growth and survival. These data highlight the importance of these gene regulatory mechanisms to SST neuron function after stroke. Summary/Conclusion: Taken together, our study provides comprehensive miRNA and transcriptional signatures for SST neurons after stroke. These data will facilitate a deeper understanding of the neuroplasticity after brain injury, potentially leading to new therapeutic targets against ischemic stroke. Funding: NIDDK Diacomp 21AU4165 and AHA/ASA 18IPA34170331 (X.S.L).

莨菪是一类主要生活在我国东北、华北、西北和西南的茄科、天仙子属草本植物,其主要成分含莨菪碱(0.02％～0.17％)、阿托品(0.018％)、东莨菪碱(0.01％～0.08％)、山莨菪碱(0.007％)等生物碱.目前,有关莨菪的化学组成研究较少,特别是其中的抗肿瘤活性物质结构还不清楚,但有一些试验结果表明莨菪提取物中的主要成份能够显著抑制某些癌症癌细胞生长.文章总结了有关莨菪提取物中生物碱抗肿瘤作用的研究方法及结果,以期为进一步研究的构效机制和药物研究方法提供理论依据.

Abstract Background Breast cancer (BC) is one of the most common cancers among women. Since diverse features can be collected, how to stably select the powerful ones for accurate BC diagnosis remains challenging. Methods A hybrid framework is designed for successively investigating both feature ranking (FR) stability and cancer diagnosis effectiveness. Specifically, on 4 BC datasets (BCDR-F03, WDBC, GSE10810 and GSE15852), the stability of 23 FR algorithms is evaluated via an advanced estimator (S), and the predictive power of the stable feature ranks is further tested by using different machine learning classifiers. Results Experimental results identify 3 algorithms achieving good stability ( $$S \ge 0.55$$ S ≥ 0.55 ) on the four datasets and generalized Fisher score (GFS) leading to state-of-the-art performance. Moreover, GFS ranks suggest that shape features are crucial in BC image analysis (BCDR-F03 and WDBC) and that using a few genes can well differentiate benign and malignant tumor cases (GSE10810 and GSE15852). Conclusions The proposed framework recognizes a stable FR algorithm for accurate BC diagnosis. Stable and effective features could deepen the understanding of BC diagnosis and related decision-making applications.

目的:比较煮沸法与苯酚-氯仿法提取血清及外泌体内HBV-DNA,Taqman法与SYBR法qPCR检测HBV-DNA表达的优缺点,并寻找更为准确敏感的诊疗参考指标.方法:收集高病毒载量组与低病毒载量组的血清,提取血清外泌体并鉴定,分别提取血清与外泌体内HBV-DNA,比较分析煮沸法与苯酚-氯仿法提取及qPCR Taqman法与SYBR法检测之间的表达差异.结果:高病毒载量组(HBV-DNA>5E+02 U/ml)中煮沸法提取血清HBV-DNA,Taqman法检测值高于SYBR法(P<0.05);Taqman测定血清HBV-DNA、SYBR测定外泌体HBV-DNA,煮沸法提取效率均高于苯酚-氯仿法(P<0.05);血清HBV-DNA表达高于外泌体(P<0.05).低病毒载量组(HBV-DNA<5E+02 U/ml)中煮沸法与苯酚-氯仿法提取、Taqman法与SYBR法qPCR检测血清及外泌体HBV-DNA差异均无统计学意义.外泌体HBV-DNA表达高于血清(P<0.05).结论:两种提取及检测方法均具有一致的灵敏度与特异度.外泌体包裹的HBV-DNA水平可能是临床诊断和治疗慢性乙肝中不可或缺的关键因素.

目的:探究赶黄草活性成分槲皮素和没食子酸对CCl4所致小鼠急性肝损伤的保护作用及机制.方法:将小鼠随机分为4组(每组10只):正常组、模型组、槲皮素组(100 mg/kg)和没食子酸组(100 mg/kg).正常组和模型组小鼠灌胃等量生理盐水,其余各组按分组给予相应药物灌胃7 d,末次灌胃后2 h,腹腔注射0.2％CCl4植物油溶液(15 ml/kg),并16 h后收集小鼠血清和肝脏组织.HE染色观察小鼠肝脏病理变化;ELISA检测小鼠血清谷氨酸氨基转移酶(ALT)、肿瘤坏死因子-α(TNF-α)和肝组织还原型谷胱甘肽(GSH)水平;Western blot和免疫荧光检测肝脏中血红素加氧酶-1(HO-1)水平.结果:相比于模型组小鼠,槲皮素和没食子酸干预后,可降低其肝脏指数(P<0.05),降低血清ALT和TNF-α水平(P<0.01),增加肝组织中GSH水平(P<0.01),增加肝组织中HO-1表达.结论:赶黄草活性物质槲皮素和没食子酸对于肝脏损伤具有保护作用,其机制可能与增强抗氧化损伤能力,降低炎症因子水平有关.

Introduction: Dendritic cells (DC) are involved in immune recognition, response and immunomodulation mechanisms related to the onset of cancer.Objective: To explore DCs mechanism in the inhibition of autophagy in hepatoma cells.Methods: Human peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation and induced into DCs, which were co-cultured with HepG2 cells by Transwell migration assay.HepG2 cell activity was determined using the CCK8 assay.LC3 autophagy index expression was measured with Western blot analysis, and the expression and secretion of cytokines, with qRT-PCR and ELISA.Results: In the co-culture system, DCs were able to reduce HepG2 cells viability; IL-2, IL-12, IL-10 and IFN-g expression in DCs was also significantly inhibited, although IL-2 and IFN-g were still expressed 0.6 and 0.53 more than in the control group.Conclusion: DCs can regulate autophagy in hepatocellular carcinoma cells.The mechanism may be related to the synthesis and release of cytokines such as IL-2, IL-12 and IFN-g by DCs.

目的:探究赶黄草活性成分槲皮素和没食子酸对CCl 4 所致小鼠急性肝损伤的保护作用及机制。方法:将小鼠随机分为4组（每组10只）:正常组、模型组、槲皮素组（100 mg/kg）和没食子酸组（100 mg/kg）。正常组和模型组小鼠灌胃等量生理盐水，其余各组按分组给予相应药物灌胃7 d,末次灌胃后2 h,腹腔注射0.2%CCl 4 植物油溶液（15 ml/kg）,并16 h后收集小鼠血清和肝脏组织。HE染色观察小鼠肝脏病理变化；ELISA检测小鼠血清谷氨酸氨基转移酶（ALT）、肿瘤坏死因子-α（TNF-α）和肝组织还原型谷胱甘肽（GSH）水平；Western blot和免疫荧光检测肝脏中血红素加氧酶-1（HO-1）水平。结果:相比于模型组小鼠，槲皮素和没食子酸干预后，可降低其肝脏指数（P＜0.05）,降低血清ALT和TNF-α水平（P＜0.01）,增加肝组织中GSH水平（P＜0.01）,增加肝组织中HO-1表达。结论:赶黄草活性物质槲皮素和没食子酸对于肝脏损伤具有保护作用，其机制可能与增强抗氧化损伤能力，降低炎症因子水平有关。

Introduction: Dendritic cells (DC) are involved in immune recognition, response and immunomodulation mechanisms related to the onset of cancer. Objective: To explore DCs mechanism in the inhibition of autophagy in hepatoma cells. Methods: Human peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation and induced into DCs, which were co-cultured with HepG2 cells by Transwell migration assay. HepG2 cell activity was determined using the CCK8 assay. LC3 autophagy index expression was measured with Western blot analysis, and the expression and secretion of cytokines, with qRT-PCR and ELISA. Results: In the co-culture system, DCs were able to reduce HepG2 cells viability; IL-2, IL-12, IL-10 and IFN-γ expression in DCs was also significantly inhibited, although IL-2 and IFN-γ were still expressed 0.6 and 0.53 more than in the control group. Conclusion: DCs can regulate autophagy in hepatocellular carcinoma cells. The mechanism may be related to the synthesis and release of cytokines such as IL-2, IL-12 and IFN-γ by DCs.

现代教育离不开网络,网络是教育的延伸和补充,是必不可少的元素.过程性评价亦然,现代教育离不开过程性评价.现代医学教育更需要培养学生的自主学习能力,因此,在网络信息技术架构下的新型过程性评价体系建立和完善是提高教学效果的必经之路.

Neurogenesis contributes to poststroke recovery. Long noncoding RNAs (lncRNAs) participate in the regulation of stem cell self-renewal and differentiation. However, the role of lncRNAs in stroke-induced neurogenesis remains unknown. In this study, we found that H19 was the most highly upregulated lncRNA in neural stem cells (NSCs) of the subventricular zone (SVZ) of rats subjected to focal cerebral ischemia. Deletion of H19 suppressed cell proliferation, promoted cell death, and blocked NSC differentiation. RNA sequencing analysis revealed that genes deregulated by H19 knockdown were those that are involved in transcription, apoptosis, proliferation, cell cycle, and response to hypoxia. H19 knockdown significantly increased the transcription of cell cycle-related genes including p27, whereas overexpression of H19 substantially reduced expression of these genes through the interaction with chromatin remodeling proteins EZH2 and SUZ12. Moreover, H19 regulated neurogenesis-related miRNAs. Inactivation of H19 in NSCs of ischemic rats attenuated spontaneous functional recovery after stroke. Collectively, our data provide novel insights into the epigenetic regulation of lncRNAs in stroke-induced neurogenesis.

Background and Purpose: Adult neurogenesis contributes to functional recovery after stroke. Long non-coding RNAs (lncRNAs) regulate stem cell self-renewal and differentiation. However, the role of lncRNAs in stroke-induced neurogenesis remains unknown. Methods and Results: Using lncRNA array and in situ hybridization, we analyzed lncRNA profiles of adult neural stem cells (NSCs) isolated from the subventricular zone neurogenic region in rats subjected to middle cerebral artery occlusion. We found that H19 was the most highly upregulated lncRNA (19 fold) in ischemic NSCs compared with non-ischemic NSCs. Reduction of endogenous H19 in NSCs by CRISPR-Cas9 genome editing significantly decreased the proliferation and increased the apoptosis of ischemic NSCs, as assayed by the number of BrdU + cells (56±5% vs 22±3%, p<0.01, n=3) and Caspase-3/7 activity compared to NSCs transfected with scrambled small guide RNA (sgRNA). Knockdown of H19 significantly decreased the number of Tuj1 + neuroblasts (8±2% vs 5±0.4%, p<0.01, n=3) and NG 2 + oliogodendrocyte progenitor cells (10±1% vs 5±0.3%, p<0.01, n=3), suggesting that deletion of H19 suppresses the proliferation and survival and blocks the differentiation of NSCs into neurons and oligodendrocytes. Additional RNA-sequencing and bioinformatics analyses revealed that genes deregulated by H19 knockdown were involved in transcription, apoptosis, proliferation, cell cycle and response to hypoxia. Western blot analysis validated that loss-of-function and gain-of-function of H19 significantly increased and reduced, respectively, the transcription of cell cycle-related genes including p27. Using ChIRP assay, we found that upregulated H19 in NSCs was physically associated with EZH2 which catalyzes the repressive H3K27me3 histone marker. Knockdown of H19 significantly reduced the enrichment of H3K27me3 at the promoter of p27, leading to the upregulation of p27 expression and consequently inhibition of NSC proliferation. Conclusions: H19 mediates stroke-induced neurogenesis by regulating genes involved in cell cycle and survival through the interaction with chromatin remodeling proteins. Our data provide novel insights into epigenetic regulation of gene expression by lncRNA in neurogenesis.

新冠肺炎疫情影响下,"停课不停学、停课不停教",促使教学团队对新形势下实验课程网络教学模式进行深入的探索和研究.这不仅是形势所趋,也是当代教育教学发展的需要.利用现代网络技术建构的实验教学模式应在尽可能符合学习认知规律的前提下丰富和多样化,通过声音、图形、影像等感官多维度刺激达到甚至超越传统教学模式的教育教学效果.教研组以医学免疫学实验网络教学为例对实验网络在线教学模式的各环节加以探索和研究,以期为完善实验网络在线教学模式奠定基础.