Protocol for Robust In Vivo Measurements of Erythrocyte Aggregation Using Ultrasound Spectroscopy

Erythrocyte aggregation is a non-specific marker of acute and chronic inflammation. Although it is usual to evaluate this phenomenon from blood samples analyzed in laboratory instruments, in vivo real-time assessment of aggregation is possible with spectral ultrasound techniques. However, variable blood flow can affect the interpretation of acoustic measures. Therefore, flow standardization is required. Two techniques of flow standardization were evaluated with porcine and equine blood samples in Couette flow. These techniques consisted in either stopping the flow or reducing it. Then, the sensibility and repeatability of the retained method were evaluated in 11 human volunteers. We observed that stopping the flow compromised interpretation and repeatability. Conversely, maintaining a low flow provided repeatable measures and could distinguish between normal and high extents of erythrocyte aggregation. Agreement was observed between in vivo and ex vivo measures of the phenomenon (R² = 82.7%, p value < 0.0001). These results support the feasibility of assessing in vivo erythrocyte aggregation in humans by quantitative ultrasound means.     

CRISPR/Cas9系统及其在血液病基因治疗中的研究进展

近年来随着基因编辑技术的兴起,可以实现对基因组中特定的基因进行编辑,使基因突变性疾病的治愈成为可能。在众多基因编辑技术中,CRISPR/Cas9系统因其操作简便性、靶向特异性、经济性等特点备受关注,已广泛应用于分子生物学以及生命科学的领域研究中。本文就此系统结构、作用机制以及在血友病、β-地中海贫血、慢性髓系白血病等血液病中基因治疗的最新研究进展作一综述。     

Non-Invasive Liver Ablation Using Histotripsy: Preclinical Safety Study in an In Vivo Porcine Model

This study investigates the safety profile for use of histotripsy, a non-invasive ultrasonic ablation method currently being developed for the treatment of liver cancer, for liver ablation in an in vivo porcine model. Histotripsy treatments were applied to the liver and hepatic veins of 22 porcine subjects, with half of the subjects receiving systemic heparinization. Vital signs (heart rate, blood pressure, temperature, electrocardiogram and SpO₂) were monitored throughout the procedure and for 1 h post-treatment. Blood was drawn at six points during the experiment to analyze blood gases, liver function and free hemoglobin levels. All treatments were guided and monitored by real-time ultrasound imaging. After treatment, the tissue was harvested for histological analysis. Results indicated that histotripsy generated well-defined lesions inside the liver and around the treated hepatic veins of all subjects in both treatment groups. Vital signs and blood analysis revealed that animals responded well to histotripsy, with all animals surviving the treatment. One animal in the non-heparinized group had a transient increase in pH and decreases in blood pressure, heart rate and PCO₂ during the 15-min vessel treatment, with these changes returning to baseline levels soon after the treatment. Overall, the results indicate that histotripsy can safely be performed on the liver without the need for systemic heparinization, even in regions containing large hepatic vessels, supporting its future use for the treatment of liver cancer.     

In Vivo Gene Delivery by Nonviral Vectors: Overcoming Hurdles?

The promise of cancer gene therapeutics is hampered by difficulties in the in vivo delivery to the targeted tumor cells, and systemic delivery remains to be the biggest challenge to be overcome. Here, we concentrate on systemic in vivo gene delivery for cancer therapy using nonviral vectors. In this review, we summarize the existing delivery barriers together with the requirements and strategies to overcome these problems. We will also introduce the current progress in the design of nonviral vectors, and briefly discuss their safety issues.     

基于CRISPR/Cas9技术构建人CLCN7基因编辑载体

目的为修复石骨症致病基因(CLCN7)进行前期探索,采用CRISPR/Cas9技术构建CLCN7基因编辑表达载体。方法根据sgRNA设计原则,在CLCN7外显子区设计一对sgRNA,合成四条单链DNA寡核苷酸,退火后连接于CRISPR/Cas9质粒的BspQ I酶切位点处,然后转染感受态大肠杆菌DH5α,并通过氨苄青霉素抗性筛选阳性克隆,扩大培养。结果最后经过PCR和Sanger测序鉴定获得了两个基因编辑表达载体CLCN7-sg1RNA-CRISPR-Cas9和CLCN7-sg2RNACRISPR-Cas9。结论基因编辑载体的成功构建为CLCN7突变修复提供了工作基础。     

利用CRISPR/Cas9技术建立SOST荧光报告基因i PSCs细胞系

目的利用CRISPR/Cas9技术构建硬骨素(SOST)基因荧光报告诱导性多能干细胞(i PSCs)细胞株。方法使用PCR检测i PSCs分化过程不同阶段成骨基因的表达。利用CRISPR/Cas9基因编辑技术将p2A-td TomatoNeo质粒转染至i PSCs细胞。使用PCR凝胶电泳检测转染的效果,而后分别在光镜下观察成骨不同阶段i PSCs的形态变化、茜素红染色及SOST-td Tomato荧光表达情况,并利用茜素红染色及PCR评估转染细胞的成骨效果及成骨基因表达情况。结果 PCR检测结果发现,SOST只在骨细胞中稳定表达,可作为骨细胞的标志性基因。利用CRISPR/Cas9技术将td Tomato插入到SOST终止密码子处,转染后的i PSCs细胞株不影响其成骨效果及SOST基因的表达。结论利用CRISPR/Cas9技术能够成功构建td Tomato-SOST荧光报告基因的i PSCs细胞株。     

应用CRISPR/Cas9技术构建Raji-Luc CD19 KO淋巴瘤细胞系

目的 应用CRISPR/cas9技术构建敲除CD19的Raji-Luc淋巴瘤细胞,并对其免疫逃逸能力进行初步验证。方法构建PB-CRISPR-CD19小向导RNA(small guide RNA, sgRNA)质粒,筛选最优的sgRNA序列,用pCAG-PBase转座酶、PB-CD19 sgRNA及PB-CRISPR-Cas9共同电转染Raji-Luc细胞,采用流式分选和极限稀释法筛选得到稳定敲除的单克隆细胞株。经过基因序列检测对敲除效果进行验证;酶标仪检测细胞系表面荧光素酶的表达情况;以实验室前期构建的CD19 CAR-T和CD38 CAR-T作为效应细胞,用荧光素酶化学发光法验证Raji-Luc CD19 KO细胞株的免疫逃逸能力。结果 电转染制备的Raji-Luc CD19 KO细胞转染效率较高,筛选所得两组单克隆细胞敲除效率均达到99%以上,与原始Raji-Luc细胞的荧光素酶表达无显著差异,且不能激活CD19 CAR-T细胞对其进行杀伤。结论 成功构建了Raji-Luc CD19 KO淋巴瘤细胞系。     

In Vivo time harmonic elastography of the human heart

Time harmonic elastography is introduced as a modality for assessing myocardial elasticity changes during the cardiac cycle. It is based on external stimulation and real-time analysis of 30-Hz harmonic shear waves in axial direction of a parasternal line of sight through the lateral heart wall. In 20 healthy volunteers, the externally induced waves showed smaller amplitudes during systole (76.0 ± 30.8 μm) and higher amplitudes during diastole (126.7 ± 52.1 μm). This periodic wave amplitude alteration preceded ventricular contraction and dilation by about 100 ms. The amplitude ratio of 1.75 ± 0.49 indicates a relative change in myocardial shear elasticity on the order of 14 ± 11. These results well agree with observations made by cardiac magnetic resonance elastography for a similar displacement component and region of the heart. The proposed method provides reproducible elastodynamic information on the heart in real-time and may help in diagnosing myocardial relaxation abnormalities in the future.     

基于CRISPR/Cas系统诊断平台的研究进展

目前核酸检测技术已被广泛应用于临床实验室诊断,常规检测技术如实时荧光定量PCR技术耗时长且依赖特定的仪器设备。成簇规律间隔短回文重复序列(CRISPR)/CRISPR相关蛋白(Cas)系统是细菌和古细菌在与病毒斗争过程中获得的适应性免疫防御机制,已被发展成强大的基因组编辑技术。最近CRISPR领域的先驱团队基于Cas13a、Cas12a和新发现的Cas14蛋白开发出SHERLOCK、DETECTR等新型核酸检测工具,在传染性疾病的快速诊断、癌症中基因突变的检测和基因分型等方面意义重大,其灵敏度高、特异性强且快速经济,在临床分子诊断领域具有巨大潜力。本文综述了CRISPR/Cas系统的作用机制及新型诊断平台的原理和应用进展,总结了新型检测技术的优缺点并对其发展前景进行展望。     

利用CRISPR/Cas9技术敲除K562细胞中的SMIM1基因

目的利用慢病毒介导的CRISPR/Cas9基因编辑技术,在K562细胞系中敲除编码Vel血型抗原的SMIM1基因。方法设计5条sgRNA序列,构建Cas9-sgRNA共表达质粒,在293T细胞中初步筛选切割效率较高的sgRNA序列。将筛选后的sgRNA通过第二代慢病毒包装系统包装慢病毒并感染K562细胞,提取细胞基因组DNA,进行Sanger测序和TA克隆检测。结果使用易转染的293T细胞建立sgRNA的初步筛选平台,筛选出切割效率较高的sgRNA4序列。CRISPR/Cas9系统成功在K562细胞中SMIM1基因的sgRNA4识别位点发挥基因编辑活性。结论证实了利用CRISPR/Cas9技术在K562细胞中敲除SMIM1基因的可行性,为构建Vel抗原表型阴性的细胞模型奠定基础,也为后续编辑其他血型抗原的基因提供实验依据。     

CRISPR/Cas技术在病原体检测中的应用

1987 年在大肠埃希菌基因组中首次发现了成簇间隔短回文重复序列(CRISPR),具有切割外来入侵病毒核酸的获得性免疫功能[1].CRISPR及其相关蛋白(CRISPR/Cas)系统是由编码 Cas 蛋白基因和由启动子加上重复序列与入侵核酸同源的间隔序列交替而构成[2].该系统的获得性免疫机制可以分为 3 个阶段:获得...     

CRISPR/Cas系统在感染性疾病诊断中的应用

成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）系统,作为原核生物的适应性免疫防御系统,近年来已经被开发成为一种高效的分子诊断工具,其特异、灵敏、快速、便捷等优势使得其在分子诊断领域,尤其是在感染性疾病的诊断上有着极佳的发展前景。本文旨在对CRISPR/Cas系统在感染性疾病诊断的应用现状、优...     

CRISPR/Cas系统在分子检测中的应用

近年来,成簇规律间隔短回文重复序列/成簇规律短回文重复序列相关蛋白(CRISPR/Cas)系统凭借其简单、高效的基因编辑能力,已被广泛应用于生物、医学等多个研究领域。随着CRISPR技术的快速发展,CRISPR/Cas系统已被开发为一种快速、便携、低成本、高灵敏度的分子检测工具,在病原体检测、耐药性分析、单核苷酸多态性(SNP)分型、肿瘤基因突变检测等方面取得重大突破。文章就不同Cas蛋白在分子检测中的最新研究进展进行综述,并对其应用前景进行展望,以期为从事相关领域的科研工作者提供参考与帮助。