RNA干扰技术抑制HLX1基因表达对滋养细胞生物学性能的影响

目的:研究小分子干扰RNA(siRNA)技术抑制人类同源异型盒基因HLX1的表达对胎盘滋养细胞株HTR-8/SVneo细胞生物学性能的影响。方法:常规体外培养HTR-8/SVneo细胞,设实验组(转染HLX1基因的特异性siRNA)、阴性对照组(转染阴性对照siRNA)及空白对照组(除不含siRNA片段,余试剂与另两组相同)3组分别进行瞬时转染。用流式细胞术测定siRNA转染效率,应用实时定量PCR技术和蛋白印迹法测定转染后各组HTR-8/SVneo细胞中HLX1 mRNA和蛋白的表达;应用MTT比色实验、Matrigel侵袭实验分别检测转染后各组细胞的增殖能力与侵袭能力的变化。结果:(1)转染24h后测得siRNA转染效率达(86.3±2.6)%。(2)转染48h后HLX1 mRNA的表达水平下调(77.0±1.2)%(P<0.01),转染72h后HLX1蛋白的表达水平下调(82.6±1.2)%(P<0.01),与HLX1 mRNA的表达水平下调相一致。(3)转染HLX1 siRNA 24h后,HTR-8/SVneo细胞的增殖活性已受到抑制,转染72h后细胞增殖抑制最显著,抑制率达到(58.1±4.4)%(P<0.01)。(4)转染HLX1 siRNA后,HTR-8/SVneo细胞侵袭Matrigel基质胶的能力受到显著抑制,其穿膜细胞数为(29±3)个,与空白对照组(穿膜细胞数为[53±8]个)相比,差异有统计学意义(P<0.01)。结论:HLX1基因表达水平的下降可以显著抑制滋养细胞的增殖活性与侵袭能力;HLX1基因表达异常可能通过影响滋养细胞增殖、侵袭等过程参与IFGR、子痫前期等疾病的发生。     

HLX基因在AML中的表达及预后分析

目的 分析H2.0样同源盒基因（H2.0-like homeobox gene,HLX）在急性髓系白血病（acute myelogenous leukemia,AML）患者中的临床表达情况,探究HLX基因与AML疾病预后的关系。方法 收集56例AML患者的骨髓单个核细胞,实时荧光定量PCR （quantitative real-time polymerase chain reaction,qRT-PCR）检测HLX的表达水平,并结合预后基因进行分析。结果 在临床初发的AML患者中,HLX的表达水平高于正常人（P<0.01）,HLX表达水平与白血病组患者年龄、骨髓原始细胞数显著相关（P<0.05）,HLX表达水平与染色体核型代表的预后分层无明显关联（P>0.05）。结论 HLX基因在AML患者中的表达水平是上调的,可能拥有独立的预后信息,是AML中潜在的干预靶点。     

siRNA沉默HLX基因对AML细胞生长的影响

目的 筛选高表达H2.0样同源盒基因（H2.0-like homeobox gene,HLX）的白血病细胞株,沉默HLX后观察急性髓系白血病（acute myelogenous leukemia,AML）细胞株的生长及分化影响,为HLX在AML中的深入研究提供理论基础。方法 实时荧光定量PCR（quantitative real-time polymerase chain reaction,qRT-PCR）筛选高表达HLX基因的白血病细胞株,并检测p21活化激酶1（p21-activated kinase 1,PAK1）的基因表达;设计siRNA-HLX沉默HLX基因表达最高的AML细胞株中的该基因,运用相差显微镜观察细胞生长情况,MTS/PMS比色分析法检测增殖抑制率。结果 AML细胞株中HLX基因的表达水平显著较高（P< 0.01）,干扰HLX基因后,可见AML细胞株生长速度减慢,增殖被抑制,并且PAK1mRNA表达下调（P< 0.01）。结论 HLX基因可抑制AML细胞株的生长、增殖及分化。     

肝细胞生长因子对滋养细胞HLX1基因的表达及侵袭能力的影响

目的 探讨肝细胞生长因子(HGF)对胎盘滋养细胞株HTR-8/SVneo细胞HLX1基因的表达及侵袭能力的影响.方法 常规体外培养HTR-8/SVneo细胞,用不同浓度HGF(0、10、20、50、100ng/mL)处理细胞48h.设0浓度组为对照组;用HLX1 siRNA转染细胞24h后继续以20 ng/mL HGF刺激48h,分空白对照组(MC)、siRNA+HGF组、MC+HGF 3个实验组;应用实时荧光定量RT-PCR和蛋白印迹法测定各组细胞中HLX1 mRNA和蛋白的表达;明胶酶谱测定上清中MMP-2的表达;Matrigel侵袭实验检测各组细胞的侵袭能力.结果 ① HGF处理组细胞HLX1 mRNA表达水平与对照组比较上调了90%～475%(P<0.01),且与HGF呈剂量依赖性;20ng/mL HGF使HLX1蛋白表达水平上调(156.7±6.4)%,P<0.01;② siRNA+HGF组与MC组比较.细胞HLX1 mRNA和蛋白的表达水平分别下降(54.57±0.31)%及(68.44±2.48)%,P<0.01;③ 20ng/mL HGF处理组细胞其上清MMP-2表达水平与对照组比较上调(394.6±2.9)%,P<0.01;siRNA+HGF组与MC组比较MMP-2表达水平下降(81.5±0.6)%,P<0.01;④ 20ng/mL HGF处理的HTR-8/SVneo细胞穿过Matrigel的细胞数为(71±5)个,与对照组(50±3)个比较侵袭能力明显增强(P<0.01);siRNA+HGF组穿膜细胞数明显减少为(20±4)个,MC组为(43±3)个,两组比较差异有统计学意义(P<0.01).结论 HGF可提高HTR-8/SVneo细胞HLX1基因的表达及细胞的侵袭力,且侵袭力的增加可能与HLX1表达水平升高有关.     

Sequence variants in the HLX gene at chromosome 1q41-1q42 in patients with diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a common birth defect for which few causative genes have been identified. Several candidate regions containing genes necessary for normal diaphragm development have been identified, including a 4–5 Mb deleted region at chromosome 1q41-1q42 from which the causative gene(s) has/have not been cloned. We selected the HLX gene from this interval as a candidate gene for CDH, as the Hlx homozygous null mouse has been reported to have diaphragmatic defects and the gene was described as being expressed in the murine diaphragm. We re-sequenced HLX in 119 CDH patients and identified four novel single nucleotide substitutions that predict amino acid changes: p.S12F, p.S18L, p.D173Y and p.A235V. These sequence alterations were all present in patients with isolated CDH, although patients with both isolated CHD and CDH with additional anomalies were studied. The single-nucleotide substitutions were absent in more than 186 control chromosomes. In-situ hybridization studies confirmed expression of Hlx in the developing murine diaphragm at the site of the junction of the diaphragm and the liver. Although functional studies to determine if these novel sequence variants altered the inductive activity of Hlx on the α-smooth muscle actin and SM22α promoters showed no significant differences between the variants and wild-type Hlx , sequence variants in HLX may still be relevant in the pathogenesis of CDH in combination with additional genetic and environmental factors.     

Efficacy and safety of intravitreal HLX04-O, an anti-VEGF monoclonal antibody, for the treatment of wet age-related macular degeneration

AIM: To evaluate the efficacy and safety of HLX04-O, an investigational ophthalmic formulation of HLX04 (bevacizumab biosimilar) for intravitreal injection, as a treatment for wet age-related macular degeneration (wAMD) in a phase 1/2 clinical trial (NCT04993352). METHODS: Eligible patients with wAMD were enrolled to receive HLX04-O intravitreal injections at a dose of 1.25 mg/0.05 mL every four weeks. Efficacy and adverse events were evaluated every month during study visits. RESULTS: A 76-year-old male with wAMD in his left eye participated in the trial and completed six cycles of HLX04-O intravitreal injections. Changes were observed in macular center point thickness (baseline vs last study visit, 437 vs 255 μm) and best-corrected visual acuity letter score (baseline vs last study visit, 36 vs 77) of the affected eye, which indicated an improvement in wAMD over treatment. No adverse events were reported by the data cutoff date. CONCLUSION: HLX04-O at 1.25 mg/0.05 mL every four weeks is well tolerated in this patient, demonstrating promising safety and efficacy in wAMD treatment. Large-scale studies are required to confirm the outcomes.     

HLX基因的表达与急性髓系白血病相关性

目的 研究HLX表达水平与AML的临床变量及AML不同危险度分层的相关性。方法 应用实时荧光定量PCR方法检测32例初治AML患者骨髓单个核细胞中HLX表达量,分析其与患者FAB分型、血象、骨髓原始细胞、分化程度及AML不同危险度分层关系。结果 初治AML患者HLX表达量在AML-M₃中明显降低（P<0.01）,AML-M₅中明显升高（P<0.05）;HLX表达水明与骨髓原始细胞数呈负相关（P<0.01）,与外周血血小板水平呈正相关（P<0.05）,且AML-M₃ HLX表达量与外周血白细胞水平呈正相关（P<0.05）,AML-M₅ HLX表达量与外周血白细胞水平呈负相关（P<0.05）;AML-M₅组中分化不良组HLX表达水平明显高于分化良好组,（P<0.05）,病例组及非M₃组初治AML患者中,低危组与中危组HLX表达水平差异无统计学意义（P>0.05）。结论 HLX表达水平在部分亚型（如AML-M₅）中表达升高,且在不同的FAB分型中有差异。该基因与白血病细胞的增殖、分化和成熟相关。本研究暂无证据表明HLX在不同AML危险度分层中有差异,仍需进一步实验研究。