Biological characteristics of JAK2 transduced CD34+ cells from cord blood during ex vivo expansion

AIM: To explore the feasibility and biological characterization of long-term regulated expansion of JAK2 transduced human CD34⁺ cord blood cells in vitro.METHODS: A retrovirus (RV) vector which contains JAK2 catalytic domain and two binding sites for a chemical inducer,dimerization (AP20187),was cloned (designated MGI-F₂JAK2).CD34⁺cells were enriched from cord blood with a MiniMACS system.The purified CD34⁺ cells were transfected with supernatant from the retrovirus packaging cell line that expressed JAK2.Following transduction,cells were expanded into four groups: AP20187 alone,FL alone,TPO,alone,AP20187+FL+TPO,respectively.The expanded cells were monitored by GFP expression,immunophenotyping,progenitor colony assay,karyotype analysis as well as tumorigenesis in nude mice.RESULTS: The purity of selected CD34⁺ cells was over 91% and gene transfer rate was 49.32%±6.21%.Only the group of AP20187 +FL+ TPO was obtained a significant sustained outgrowth of the transduced CD34⁺ cord blood cells.The percentage of GFP+ cells consistently produced a rise to the 90% peak level by the end of 8th week of culture.Flow cytometry analysis showed that the phenotype of the expanded cells was CD33⁺,CD61⁺ and Gly-A⁺ partial positive;CD38⁺ and HLA-DR⁺ strong positive,while CD2,CD7 and CD19 were almost negative.Colony assays performed in methycelluos,which can give rise to BFU-E,CFU-GM and CFU-Mix,the CFU-GM was predominantly in all colonies.The tumor was not observed in nude mice and the karyotype analysis was normal from expanded cells.CONCLUSION: The results demonstrate that AP20187-mediated activation of JAK2 signaling is capable of stimulating expansion JAK2 transduced CB CD34⁺ cells in combination with FL and TPO.This system may have applications for studies in signaling transduction,hematopoiesis,and for gene and cell therapy.     

Experimental study on how brain-dead state affects the heart structure and function of Ba-Ma mini pigs and the mechanism

AIM:To investigate how brain-dead state affects the heart structure and function and the effect of PKC-α in BA-Ma mini pigs.METHODS:Ten Ba-Ma mini pigs were randomized into 2 groups: brain-dead group (n=5),and control group (n=5). The brain-dead model was made by increasing intracranial pressure,while the control group was maintained anesthesia for 24 h. The concentrations of cTnT,TNF-α,IL-1β and IL-6 in serum were determined at 6,12 and 24 h after brain death. At 24 h,heart tissues were observed by HE staining and electron microscope. The expression of PKC-α was detected by immunohistochemistry and RT-PCR.RESULTS:(1) Histological changes of myocardium: flaky bleeding under endocardium and dissolution of myocardium were found in optical microscope. In electron microscope dropsical mitochondria and confluent muscle fiber were found. (2) Changes of serum cTnT: serum cTnT for brain-dead group began to increase gradually since 6 h,and were significantly higher at each time point than those in control group (P<0.05). (3) Changes of inflammatory factors: IL-1β,IL-6,and TNF-α in brain-dead group began to increase gradually since 6 h,and were significantly higher at each time point than those in control group (P<0.05). (4) Changes of PKC-α expression: PKC-α mRNA and protein expressions in brain-dead group increased significantly at 24 h (P<0.05).CONCLUSION:Brain death may evoke heart structure and functional injury,and increase the levels of inflammatory factors and PKC-α. The activation of PKC-α may participate in the process of heart injury.     

Effects of STK15 overexpression on growth of human esophageal carcinoma cell line KYSE150

AIM:To investigate the effects of serine/threonine kinase 15 (STK15) overexpression on the growth of human esophageal squamous-cell carcinoma (ESCC) cell line KYSE150. METHODS:Recombinant pEGFP-C1-STK15 expression vector was transfected into KYSE150 cells using LipofectamineTM 2000 and the expression of STK15 was detected by fluorescence microscopy and Western blotting. The proliferation of the cells in vitro was measured by MTT assay. The cell cycle distribution and apoptosis were detected by flow cytometry. The proliferation of the cells in vivo was measured by tumorigenicity experiment in nude mice. RESULTS:After recombinant pEGFP-C1-STK15 expression vector was stably transfected into KYSE150 cells, GFP-STK15 fusion protein localized to centrosome and spindle. The STK15-overexpressing colonies were further confirmed by Western blotting. MTT assay showed that the proliferation of the cells in STK15 overexpression group was increased compared with control group (P<0.01). Flow cytometry analysis showed that the percentage of the cells in G0/G1 phase and the cell apoptosis in STK15 overexpression group were decreased compared with control group (P<0.01). The tumorigenicity experiment in nude mice showed that the proliferation of the cells in STK15 overexpression group was increased compared with control group (P<0.01). CONCLUSION: Overexpression of STK15 in human ESCC KYSE150 cells promotes the cell growth in vitro and in vivo, indicating that STK15 may serve as a novel therapeutic target for esophageal carcinoma.     

日本血吸虫GSK3蛋白编码cDNA的克隆、表达及其重组蛋白的免疫保护研究

本研究利用生物信息学分析了日本血吸虫(Schistosoma japonicum,sj)Glycogen synthase kinase 3(GSK3)蛋白,并对其中一个GSK3蛋白的编码cDNA进行了克隆和原核表达,制备了特异性的多克隆抗体.同时,还初步评估了重组蛋白的免疫保护效果.生物信息学分析表明在日本血吸虫数据库存在两种GSK3蛋白,且其中一个SjGSK3在日本血吸虫不同发育时期均有转录.Western blot结果表明本研究制备的抗体能特异性识别日本血吸虫SjGSK3重组蛋白,表明该重组蛋白具有良好的免疫原性.动物实验表明免疫SjGSK3重组蛋白的动物与佐剂对照组比较分别获得了平均10.6％减虫率和40.5％肝脏减卵率.     

Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.     

Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

In vivo, resumption of oocyte meiosis occurs in large ovarian follicles after the preovulatory surge of luteinizing hormone (LH). The LH surge leads to the activation of a broad signaling network in mural granulosa cells equipped with LH receptors. The signals generated in the mural granulosa cells are further augmented by locally produced peptides or steroids and transferred to the cumulus cell compartment and the oocyte itself. Over the last decade, essential progress has been made in the identification of molecular events associated with the final maturation and ovulation of mammalian oocytes. All new evidence argues for a multiple roles of mitogen-activated protein kinase 3/1 (MAPK3/1) in the gonadotropin-induced ovulation processes. However, the knowledge of gonadotropin-induced signaling pathways leading to MAPK3/1 activation in follicular cells seems limited. To date, only the LH-induced transactivation of the epidermal growth factor receptor/MAPK3/1 pathway has been described in granulosa/cumulus cells even though other mechanisms of MAPK3/1 activation have been detected in other types of cells. In this review, we aimed to summarize recent advances in the elucidation of gonadotropin-induced mechanisms leading to the activation of MAPK3/1 in preovulatory follicles and cultured cumulus-oocyte complexes and to point out a specific role of this kinase in the processes accompanying final maturation of the mammalian oocyte.     

Properties of cod metallothionein,its presence in different tissues and effects of Cd and Zn treatment

One isoform of the low-molecular-weight metal-binding protein metallothionein (MT) has been isolated from the liver of Atlantic cod by size-exclusion and ion-exchange chromatography. Cod MT contained 33% cysteine, no aromatic amino acids or arginine. As is the case for other piscine MTs, the N-terminus of cod MT lacked the asparagine in position 4 which is present in mammalian MTs. In addition, cod MT differed from all other vertebrate MTs described in that the N-terminal methionine was not acetylated. Antibodies were raised in rabbits against hepatic MT from cod by repeated injections of native protein mixed with adjuvant. Anti-cod MT antisera cross reacted with similarly-sized proteins in liver, brain, anterior kidney, posterior kidney, spleen, intestine, gills and ovaries. The putative MT in cod brain migrated differently to that of the other tissues in native gel electrophoresis. Intraperitoneally injected Cd (1 mg/kg) was nearly entirely associated with the MT-peak in hepatic and renal cytosols, whereas a single injection of Zn (10 mg/kg) resulted in increases in all cytosolic Zn pools of the liver and no apparent change in cytosolic Zn, Cu, Ni or Cd in kidney.     

Role of ILK siRNA on expression of GSK-3β and β-catenin in human tubular epithelial cells stimulated by high glucose

AIM：To investigate the effects of siRNA targeting integrin-linked kinase (ILK) on the expression of glycogen synthase kinase 3β (GSK-3β) and β-catenin during epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial cell line HKC induced by high glucose. METHODS：HKC cells were divided into 4 groups:normal glucose (NG) group, high glucose (HG) group, HG+HK (a vector containing the non-specific siRNA designed as negative control) group and HG＋ILK siRNA group. The inverted fluorescence microscope was used to examine the expression of green fluorescent protein (GFP). The expression of ILK at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of p-GSK-3β and β-catenin was observed by immunocytochemical staining. The protein expression of total GSK-3β, p-GSK-3β, nuclear β-catenin, total β-catenin, E-cadherin and α-smooth muscle actin (α-SMA) was measured by Western blotting. RESULTS：GFP was observed in HKC cells, indicating that the transfection was successful. Both the protein and mRNA of ILK were down-regulated in HG＋ILK siRNA group compared with HG group and HG+HK group, but still higher than those in NG group. Silencing of ILK down-regulated the expression of p-GSK-3β and nuclear β-catenin. No difference of total GSK-3β or total β-catenin was observed among the 4 groups. CONCLUSION:These data support a functional role of ILK, GSK-3β and β-catenin in tubular EMT induced by high glucose. ILK may promote tubular EMT by regulating the activity of GSK-3β and β-catenin, the downstream effectors of the Wnt/β-catenin pathway.     

Effect of Raptor on invasion ability of glioma cells

AIM：To study the influence of Raptor on the invasion ability of glioma cells. METHODS：The technique of RNA interference was used. U87 cells were transfected with Raptor restricted siRNA plasmid, and the expression level of Raptor in the transfected cells was detected by Western blotting. The invasive ability of the cancer cells in vitro was determined. The phosphorylation level of ARK5 and the expression of MMP-2 and MMP-9 were detected by Western blotting. The expression levels of Raptor in the tumor samples of low-grade gliomas (WTO grade I and grade II) and high-grade gliomas (WTO grade III and grade IV) were also analyzed by immunohistochemical staining. RESULTS：Raptor siRNA was transfected into U87 cells and the cells were named siRaptor/U87 cells. The cells transfected with the control plasmid was named Scr/U87 cells. The expression level of Raptor in siRaptor/U87 cells was lower than that in Scr/U87 cells. The results of in vitro invasion assay showed that the number of siRaptor/U87 cells penetrating the Matrivgel matrix membrane was less than that of Scr/U87 cells (P＜0.01). The protein expression of MMP-2 and MMP-9, and phosphorylation of ARK5 protein in the cells in the experimental group were lower than those in control group. The correlation between the expression of Raptor in gliomas and the degree of deterioration was also observed (P＜0.01). CONCLUSION：The expression of Raptor may contribute to the invasion ability of glioma cells by phosphorylation of ARK5 and increase in the levels of MMP-2 and MMP-9.