Semi-automated flow cytometric analysis of CD34-expressing hematopoietic cells in peripheral blood progenitor cell apheresis products

BACKGROUND: The measurement of CD34+ cells is the most important step in the quality control of peripheral blood progenitor cell apheresis products. For this purpose, flow cytometry is applied. Recently, a new test kit has been introduced for the enumeration of CD34-expressing cells, in combination with software support for semi-automation of data acquisition and analysis. STUDY DESIGN AND METHODS: This study evaluated the ProCOUNT kit. Ninety samples obtained from peripheral blood progenitor cell apheresis products from 39 patients with hemato-oncologic diseases were analyzed. For data acquisition and analysis, ProCOUNT software was used. Data comparison was performed with parallel measurements according to the International Society for Hematotherapy and Graft Engineering (ISHAGE) guidelines and the German reference protocol for analysis of CD34-expressing cells. RESULTS: Correlation of the German and ISHAGE techniques was excellent (r2 = 0.99). The initial correlation coefficient of ProCOUNT analysis with the German protocol was r2 = 0.89. In 21 (23.3%) of 90 ProCOUNT analyses, a warning message was encountered from the ProCOUNT software. Following manual reevaluation of these data with CellQUEST software, a correlation of r2 = 0.96 with the German protocol and r2 = 0.97 with the ISHAGE analyses was obtained. ANOVA testing revealed significant differences between ProCOUNT and ISHAGE techniques (p<0.05) and between ProCOUNT and the German protocol (p<0.05). No statistically significant difference between ISHAGE and German protocol was observed (p = 0.19). CONCLUSION: The ProCOUNT kit and software for semi-automated data acquisition and analysis represents a further step toward standardization of CD34 cell quantitation in peripheral blood progenitor cell apheresis products. However, the occurrence of software warnings is high, and analysis or data reevaluation by experienced staff is still mandatory. Therefore, currently there is no definite advantage of the kit and software over the existing guidelines for CD34+ analysis in peripheral blood progenitor cell grafts.     

Hepatic porto-enterostomy or cholecystostomy in the treatment of extrahepatic biliary atresia. A study of 49 cases.

Hepatic porto-enterostomy or cholecystostomy (Kasai's procedure) was successful in restoring bile flow in 31 of 49 patients with "noncorrectable" extrahepatic biliary atresia. However, all but one of the 31 developed acute or chronic complications such as cholangitis, bile peritonitis, or portal hypertension. During a five-year follow-up period, 26 (53%) died while 9 of the 23 survivors continue to manifest chronic or recurrent cholangitis. Thirteen of the 19 survivors who are more than one year of age have developed portal hypertension. These complications limit the prognosis of infants with "noncorrectable" biliary malformations.     

CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer‐related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre‐loxP‐controlled lentiviral vectors expressing CRIPTO in cell line‐derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft‐derived ex vivo tumour slices. CRIPTO‐overexpressing patient‐derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial‐to‐mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2‐CRIPTO cells formed tumours when injected into immune‐compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High‐level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO‐expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

白芍总苷对大鼠心肌缺血再灌注损伤保护作用及对GRP78表达的影响

目的研究白芍总苷(TGP)对心肌缺血再灌注损伤的保护作用及对Grp78蛋白表达的影响。方法采用传统的在体心肌缺血再灌注模型,观察TGP对大鼠心功能的影响。方法将48只大鼠随机分为6组:假手术组(sham组)、模型组(I/R)、TGP低(50mg/kg)组、TGP中(100mg/kg)组、TGP高(200mg/kg)组、葛根素(100mg/kg)组,分别在术前一周灌胃,假手术组和模型组分别给予等量溶媒,葛根素(PUE)于再灌注前5min经颈总静脉注射,分别测定缺血前、缺血后30min、再灌注90min时心率(HR)、左心室压力变化最大速率(±dp/dtmax)、左心室收缩峰压(LVSP)以评价心脏功能。结果缺血再灌注模型组在缺血30min及再灌注90min后LVSP和±dp/dtmax降低,与I/R组相比,不同剂量的TGP组与PUE组相对应时间点LVSP、±dp/dtmax进行性升高。结论TGP对心肌缺血再灌注损伤有保护作用;其机制可能与通过促进GRP78的表达来发挥内源性的保护作用有关。     

淫羊藿甙对动脉粥样硬化家兔血管平滑肌细胞内质网应激的影响

目的:观察淫羊藿甙对动脉粥样硬化(AS)家兔血管平滑肌细胞(VSMC)中葡萄糖调节蛋白78基因表达的影响,探讨淫羊藿甙防治AS的可能机制.方法:复制家兔AS模型,给予淫羊藿甙治疗,组织原位杂交观察该基因在兔斑块组织中的表达水平.结果:组织原位杂交显示该基因片段在AS斑块组织中表达水平增高.结论:葡萄糖调节蛋白78基因在AS细胞质中表达增高,可能是淫羊藿甙作用靶点之一,如上调该基因则可促进平滑肌细胞凋亡,将对AS治疗产生一定作用.     

Overexpression of GRP78 protects glial cells from endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress induces apoptotic cell death by causing the accumulation of structurally abnormal proteins. The 78-kDa glucose-regulated protein (GRP78) is an ER chaperone that regulates protein folding in the ER and has been suggested to contribute to cell survival. Using the rat C6 glioma cell line and flow cytometry, we assessed GRP78 expression following tunicamycin- and glutamate-induced ER stress. The results showed that GRP78 expression is upregulated following ER stress and has protective effects on injured glial cells. Annexin V and propidium iodide labeling revealed cells transiently expressing GRP78 prior to injury were protected against high-concentrations of tunicamycin and glutamate within 72 h. Our findings support the hypothesis that GRP78 inhibits cell death associated with ER stress.     

不同转移潜能肝癌细胞株GRP78的表达及其对肝癌细胞生物学行为的影响

目的: 研究葡萄糖调节蛋白78(GRP78)在不同转移潜能肝癌细胞株内的表达及其对肝癌细胞生物学行为的影响。方法: 应用GRP78反义寡核苷酸(GRP78 ASODN)转染肝癌细胞株HepG2和MHCC97-H。逆转录聚合酶链式反应(RT-PCR)技术检测肝癌细胞内GRP78 mRNA的表达,Transwell小室实验和细胞黏附实验分别检测2种肝癌细胞的侵袭、迁移及黏附能力。结果: 2种细胞内均有GRP78的表达,MHCC97-H细胞表达较HepG2高(P<0.05)。GRP78 ASODN可以有效地抑制2种肝癌细胞株内GRP78的表达。GRP78 ASODN转染的高侵袭潜能的MHCC97-H肝癌细胞的侵袭、迁移和黏附能力较对照组有明显的下降(P<0.05),而低侵袭潜能的HepG2肝癌细胞侵袭、迁移及黏附能力下降不明显(P>0.05)。结论: 抑制肝癌细胞内GRP78的表达可以削弱其侵袭、迁移及黏附能力,GRP78可能成为肝癌治疗上有效的分子靶点。     

绿色荧光蛋白标记的GRP78蛋白表达和功能研究

目的:应用增强型绿色荧光蛋白(EGFP)来标记葡萄糖调节蛋白78(GRP78),从而研究其与免疫细胞结合的情况。方法:通过在原核表达的GRP78的C末端连接一个EGFP来显示跟踪该蛋白的表达,进而通过流式细胞仪检测该蛋白同小鼠脾脏免疫细胞结合的情况。结果:GRP-EGFP融合蛋白分子量为126kD,Western Blot证实该蛋白表达正确,并且表达GRP78-EGFP的BL21菌在紫外光激发下发射出强烈的绿色荧光;与EGFP单体相比较,GRP78-EGFP主要与中性粒细胞相结合,其平均结合率为5.15%。结论:绿色荧光蛋白标记的GRP78蛋白在原核中的表达具有天然构象并能发挥标记目的蛋白的作用。