[摘 要] 目的：探讨核仁小RNA（snoRNA）71A通过TLR3/PD-L1通路对食管鳞状细胞癌（ESCC）细胞增殖、迁移及侵袭能力的影响。方法：qPCR检测52例ESCC患者的配对肿瘤组织、癌旁组织，以及人ESCC细胞中SNORA71A的表达情况。将反义寡核苷酸（SNORA71A-ASO-29、SNORA71A-ASO-102和NC-ASO）或小干扰RNA（siTLR3、siTLR3-NC）分别转染至人ESCC TE-1细胞，分别记为SNORA71A-ASO1组、SNORA71A-ASO2组、NC-ASO组及siTLR3组、siNC组。另外将过表达质粒pcDNA3.1-SNORA71A和pcDNA3.1空载体质粒分别转染至TE-1细胞，分别记为SNORA71A组和Vector组。细胞功能学实验（MTS实验、划痕愈合实验，以及Transwell侵袭实验）评估各组细胞在敲低或过表达SNORA71A后增殖、迁移和侵袭能力的变化。高通量转录组测序筛选SNORA71A下游作用靶基因，基因本体论（GO）和京都基因与基因组百科全书（KEGG）功能富集分析预测SNORA71A下游作用靶基因可能参与的生物学过程和信号通路。qPCR检测测序筛选出的下游作用靶基因TLR3在ESCC组织以及细胞中的表达。同时，qPCR和WB检测TE-1细胞在敲低或过表达TLR3后PD-L1 mRNA和蛋白质表达变化。细胞功能学实验检测TLR3敲低对SNORA71A所促进的TE-1细胞恶性生物行为（增殖、迁移、侵袭）的影响。结果：在52例ESCC患者的肿瘤组织以及ESCC细胞中SNORA71A表达均呈高水平（P < 0.01或P < 0.05）。敲低SNORA71A抑制TE-1细胞增殖、迁移和侵袭（P < 0.01或P < 0.05），过表达SNORA71A则促进TE-1细胞增殖、迁移和侵袭（P < 0.01或P < 0.05）。高通量转录组测序筛选到TLR3为SNORA71A下游作用靶基因。TLR3在ESCC组织以及TE-1细胞中呈低表达（P < 0.01或P < 0.05）。TLR3正向调控PD-L1 mRNA和蛋白质表达（P < 0.01或P < 0.05）。细胞功能学实验中，TLR3可以部分削弱SNORA71A对PD-L1表达的调控（P < 0.01）。结论：SNORA71A通过调控TLR3/PD-L1通路调节TE-1细胞增殖、迁移和侵袭。

Background/Aims: Systemic Inflammation (SI) is considered a key mechanism in disease progression and development of complications in decompensated liver cirrhosis. SI is mainly driven by portal hypertension and bacterial translocation. Transjugular intrahepatic portosystemic shunt (TIPS) insertion represents an effective treatment for portal hypertension. This study aims to investigate the impact of TIPS insertion on SI and bacterial translocation. Methods: We prospectively included 59 cirrhotic patients undergoing TIPS insertion. Blood samples were collected at TIPS insertion and follow-up (FU) 1, 3, 6, and 12 months thereafter. At all time points, we performed a comprehensive analysis of SI including 43 soluble inflammatory markers (SIMs), and surrogates of bacterial translocation (sCD14, sCD163). To investigate long-term kinetics of SI, C-reactive protein (CRP) and white blood cells (WBC) were retrospectively analyzed in a cohort of 177 patients up to 3 years after TIPS insertion. Results: At TIPS insertion, 30/43 SIMs, sCD14, and sCD163 measured significantly higher in cirrhotic patients compared to healthy controls. By FU6 25 SIMs and sCD14 measured at significantly lower levels compared to baseline. Interestingly, in patients with TIPS indication of refractory ascites, IL-6 decreased to levels documented in earlier stages of cirrhosis. In long-term follow-up, CRP levels significantly decreased after TIPS insertion, which translated into lower mortality in Cox regression analysis (HR 0.968, p=0.042). Notably, patients with residual ascites post-TIPS showed significantly higher CRP and IL-6 levels across all follow-ups compared to patients with resolved ascites. Conclusions Decreasing portal hypertension via TIPS insertion leads to a significant attenuation of SI and bacterial translocation over time.

Background/Aims: Systemic Inflammation (SI) is considered a key mechanism in disease progression and development of complications in decompensated liver cirrhosis. SI is mainly driven by portal hypertension and bacterial translocation. Transjugular intrahepatic portosystemic shunt (TIPS) insertion represents an effective treatment for portal hypertension. This study aims to investigate the impact of TIPS insertion on SI and bacterial translocation. Methods: We prospectively included 59 cirrhotic patients undergoing TIPS insertion. Blood samples were collected at TIPS insertion and follow-up (FU) 1, 3, 6, and 12 months thereafter. At all time points, we performed a comprehensive analysis of SI including 43 soluble inflammatory markers (SIMs), and surrogates of bacterial translocation (sCD14, sCD163). To investigate long-term kinetics of SI, C-reactive protein (CRP) and white blood cells (WBC) were retrospectively analyzed in a cohort of 177 patients up to 3 years after TIPS insertion. Results: At TIPS insertion, 30/43 SIMs, sCD14, and sCD163 measured significantly higher in cirrhotic patients compared to healthy controls. By FU6 25 SIMs and sCD14 measured at significantly lower levels compared to baseline. Interestingly, in patients with TIPS indication of refractory ascites, IL-6 decreased to levels documented in earlier stages of cirrhosis. In long-term follow-up, CRP levels significantly decreased after TIPS insertion, which translated into lower mortality in Cox regression analysis (HR 0.968, p=0.042). Notably, patients with residual ascites post-TIPS showed significantly higher CRP and IL-6 levels across all follow-ups compared to patients with resolved ascites. Conclusions Decreasing portal hypertension via TIPS insertion leads to a significant attenuation of SI and bacterial translocation over time.

Background/Aims: Systemic Inflammation (SI) is considered a key mechanism in disease progression and development of complications in decompensated liver cirrhosis. SI is mainly driven by portal hypertension and bacterial translocation. Transjugular intrahepatic portosystemic shunt (TIPS) insertion represents an effective treatment for portal hypertension. This study aims to investigate the impact of TIPS insertion on SI and bacterial translocation. Methods: We prospectively included 59 cirrhotic patients undergoing TIPS insertion. Blood samples were collected at TIPS insertion and follow-up (FU) 1, 3, 6, and 12 months thereafter. At all time points, we performed a comprehensive analysis of SI including 43 soluble inflammatory markers (SIMs), and surrogates of bacterial translocation (sCD14, sCD163). To investigate long-term kinetics of SI, C-reactive protein (CRP) and white blood cells (WBC) were retrospectively analyzed in a cohort of 177 patients up to 3 years after TIPS insertion. Results: At TIPS insertion, 30/43 SIMs, sCD14, and sCD163 measured significantly higher in cirrhotic patients compared to healthy controls. By FU6 25 SIMs and sCD14 measured at significantly lower levels compared to baseline. Interestingly, in patients with TIPS indication of refractory ascites, IL-6 decreased to levels documented in earlier stages of cirrhosis. In long-term follow-up, CRP levels significantly decreased after TIPS insertion, which translated into lower mortality in Cox regression analysis (HR 0.968, p=0.042). Notably, patients with residual ascites post-TIPS showed significantly higher CRP and IL-6 levels across all follow-ups compared to patients with resolved ascites. Conclusions Decreasing portal hypertension via TIPS insertion leads to a significant attenuation of SI and bacterial translocation over time.

Thermal analysis technology has emerged as a pivotal tool for the identification and quality control of traditional Chinese medicine （TCM） owing to its advantages of high sensitivity and capability for simultaneous multi-parameter detection. The application progress on thermogravimetric analysis （TGA）, differential thermal analysis （DTA）, and differential scanning calorimetry （DSC） in four key areas: authenticity identification of herbal medicines, optimization of processing techniques, evaluation of extract thermal stability, and construction of quality evaluation systems were summarized. Thermal analysis technology enables rapid authentication of medicinal materials by establishing a thermal fingerprint. When integrated with hyphenated techniques （e.g., FTIR and GC-MS）, it facilitates in-depth analysis of compositional differences in complex matrices. In Future, the development of thermal analysis databases and multi-technology integration will be expected to further promote the standardization of TCM quality control.

[摘 要] 目的：基于生物信息学分析和体内、体外实验研究长链非编码RNA00511（LINC00511）敲减对非小细胞肺癌细胞增殖、凋亡、侵袭等恶性生物学行为的影响，并初步探究其作用机制。方法：通过基因表达谱交互分析（GEPIA）数据库分析LINC00511在非小细胞肺癌的表达水平，及其与患者肿瘤分期、生存期等临床特征、肿瘤细胞恶性生物学行为有关基因的相关性；利用shRNA慢病毒载体构建LINC00511敲减的H1299肺癌细胞株，克隆形成实验、划痕愈合实验和流式细胞术分别检测对H1299细胞增殖、迁移、细胞周期和凋亡能力的影响，qRT-PCR检测相关调控基因表达，WB法检测肿瘤相关蛋白的表达；构建裸鼠皮下移植瘤模型，取瘤组织进行免疫组织化学实验检测Ki67表达情况。结果：GEPIA数据库分析表明LINC00511在非小细胞肺癌组织中表达水平升高，且与该病的临床分期情况相关（P < 0.05），LINC00511与肺癌中CASP3、CCNB1、CDK4等多种基因表达均有相关性（P < 0.01）；LINC00511敲减可抑制细胞的克隆形成和迁移能力、促进肺癌细胞凋亡并影响细胞周期进展（P < 0.05，P < 0.01）；LINC00511敲减可下调肺癌细胞CCNB、CDK4、TGF-β1基因的表达（P < 0.01），对CCND1、VEGFA基因表达无明显影响，LINC00511敲减可抑制细胞内MMP9、CTNNB1表达，上调CASP3的表达（P < 0.05，P < 0.01）；裸鼠体内实验证实，LINC00511敲减可抑制移植瘤体组织内Ki67的表达（P < 0.01）。结论：LINC00511在非小细胞肺癌组织中呈高表达，与肺癌临床分期和多种基因表达具有相关性，LINC00511敲减可能通过影响相关基因、蛋白的表达，抑制肺癌H1299细胞的恶性生物学行为。

[摘 要] 目的：为解决野生型B细胞成熟抗原（BCMA）被γ分泌酶切割导致表达不稳定的问题，构建抵抗γ分泌酶切割的BCMA突变体并构建靶细胞，用于评价BCMA CAR-T细胞的杀伤功能。方法：将野生型BCMA的穿膜域替换为人CD8α穿膜域，构建抵抗γ分泌酶切割的BCMA突变体（BCMA-CD8α TM），构建过表达该突变体的U266（U266BCMA Mut）、K562（K562BCMA Mut）、SKOV3（SKOV3BCMA Mut）和CHO（CHOBCMA Mut）细胞；构建装载NFAT-EGFP报告基因的BCMA CAR Jurkat细胞（BCMA-CAR-Jurkat-Reporter）与U266BCMA Mut细胞共培养，采用FCM检测该细胞中EGFP表达水平以指示NFAT激活水平，荧光素酶法检测BCMA CAR-T细胞对Luciferase标记的K562BCMA Mut细胞的杀伤作用，实时无标记动态细胞分析技术（RTCA）检测BCMA CAR-T细胞对SKOV3BCMA Mut和CHOBCMA Mut细胞的杀伤作用。结果：应用γ分泌酶抑制剂LY411575抑制γ分泌酶活性，显著增强野生型U266细胞表面BCMA表达水平，平均荧光强度上调10倍以上；但撤除抑制剂后BCMA表达水平逐渐降低（P<0.01）；BCMA-CD8α TM突变体可抵抗γ分泌酶的切割作用，在U266细胞表面稳定表达（P>0.05）；U266细胞及过表达BCMA-CD8α TM的U266细胞与BCMA-CAR-Jurkat-Reporter细胞共培养后都可激活Reporter系统、增强EGFP表达，但该效应在BCMA-CD8α TM过表达的U266细胞中更显著（P<0.01）；BCMA-CD8α TM在BCMA表达阴性的K562、SKOV3和CHO 3种靶细胞中成功过表达，且在LY411575处理下该突变体的表达水平仅有小幅度升高；荧光素酶法检测结果显示，不同效靶比下，BCMA CAR-T细胞均可特异、高效杀伤过表达BCMA-CD8α TM的K562细胞；RTCA结果显示，不同效靶比下，BCMA CAR-T细胞均可有效识别、杀伤过表达BCMA-CD8α TM的SKOV3和CHO细胞，但同等效靶比下的Mock-T细胞无此效应。结论：本实验构建的BCMA-CD8α TM突变体能够抵抗γ分泌酶的切割，在多种靶细胞表面稳定表达，为评价BCMA CAR-T细胞体外杀伤的有效性和特异性提供多种检测手段。

[摘 要] 目的：利用昆虫杆状病毒表达系统（昆虫BEVS）表达糖酵解酶α-烯醇化酶（ENO1）及其3种酶活性位点缺失突变的ENO1蛋白ENO1-M1、ENO1-M2和ENO1-M3，为后续宫颈癌的代谢治疗研究奠定基础。方法：利用分子克隆技术将优化后ENO1序列插入pFastBacTM1载体，获得含有目的基因的重组质粒pFastBac-ENO1。分别缺失ENO1发挥糖酵解酶功能的3个活性位点，进行优化后将其插入pFastBacTM1载体，获得3个活性位点缺失的重组质粒pFastBac-M1、pFastBac-M2和pFastBac-M3。通过转座、转染后获得重组杆状病毒rBV-ENO1、rBV-M1、rBV-M2和rBV-M3，利用WB法对目的蛋白的表达及特异性进行检测。结果：成功扩增重组杆粒rBacmid-ENO1、rBacmid-M1、rBacmid-M2和rBacmid-M3，获得大小约2 000 bp的基因片段，与预期大小相符。昆虫BEVS可表达ENO1蛋白及其3个酶活位点缺失的重组蛋白ENO1-M1、ENO1-M2和ENO1-M3，其分子量约为52 000，与预期相符。WB法鉴定这些蛋白能与特异性标签His-tag发生反应。结论：通过昆虫BEVS成功表达目的蛋白ENO1及其酶活性位点缺失蛋白ENO1-M1、ENO1-M2和ENO1-M3，这些蛋白具有反应原性，为后续测定这些蛋白与ENO1单抗亲和力创造了条件。

[摘 要] 目的：探讨高良姜素（Gal）是否通过调节cGAS/STING信号通路影响骨肉瘤MG63细胞增殖、迁移、侵袭和凋亡。方法：体外培养人骨肉瘤MG63细胞，分别使用0、5、15、25、50、100、200 μmol/L的Gal培养48 h后，CCK-8法检测Gal对细胞活力的影响。将MG63细胞分为对照组（未处理细胞）、Gal低浓度组（Gal-L组，50 μmol/L Gal处理）、Gal高浓度组（Gal-H组，100 μmol/L Gal处理）和Gal-H+STING抑制剂组（Gal-H+H-151组，100 μmol/L Gal+8 μmol/L H-151处理）。采用EdU染色法、划痕愈合实验、Transwell小室法、流式细胞术检测各组细胞增殖活力、迁移、侵袭和凋亡能力，WB法检测各组细胞中cGAS、STING蛋白的表达水平。结果：与对照组比较，Gal-L组、Gal-H组细胞增殖活力、迁移、侵袭能力均显著降低（均P<0.05），细胞凋亡率和cGAS、STING蛋白表达水平均显著升高（均P<0.05）；与Gal-H组比较，Gal-H+H-151组细胞增殖活力、迁移和侵袭能力均显著升高（均P<0.05），细胞凋亡率和cGAS、STING蛋白表达水平均显著降低（均P<0.05）。结论：Gal可能通过激活cGAS/STING信号通路抑制骨肉瘤MG63细胞增殖、迁移、侵袭并促进细胞凋亡。

[摘 要] 目的：探讨解偶联蛋白2（UCP2）抑制剂京尼平（GEN）对人下咽癌FaDu细胞增殖及线粒体功能的影响。方法：使用不同浓度的GEN作用于FaDu细胞24 h，实验分为GEN 0（对照）、50、100、200和400 μmol/L组。采用CCK-8法检测各组细胞增殖能力，DCFH-DA探针及JC-1染色联合流式细胞术检测GEN对FaDu细胞活性氧（ROS）含量及线粒体膜电位的影响，激光共聚焦显微镜观察GEN对FaDu细胞线粒体膜通透性转换孔的影响，可见分光光度法检测细胞中乳酸的含量，WB法检测细胞中UCP2蛋白的表达变化。结果：与对照组相比，GEN可显著抑制FaDu细胞的增殖活力（P<0.05或P<0.01）、细胞中UCP2蛋白的表达（P<0.05），降低线粒体膜电位（P<0.05或P<0.01）、乳酸含量（P<0.000 1），改变细胞线粒体膜孔道通透性，提高细胞中ROS水平（P<0.05或P<0.01）。结论：GEN通过调节细胞中UCP2的表达水平进而影响细胞的氧化还原能力及线粒体功能，从而发挥抑制人下咽癌FaDu细胞增殖并诱导细胞凋亡的作用。