Objective To establish a Hr mutant knockout mouse model to study the function of Hr gene.Methods Transcription activator-like effector nucleases ( TALENs) technique was used to disrupt the mouse Hr locus,creating heritable mutations that eliminate Hr function to explore the effects of Hr on hair development and provide a good model to study the function of Hr gene.The phenotype of Hr -/- mice was observed after birth and skin histology of the transgenic mice was studied by light microscopy.Results It was shown that a F0 mouse with the 2 -bp deletion in Hr gene ranging from 86 to 87 base pairs was obtained.The male mice with clear deletion of the Hr fragment and with obvious frame shifting were mated with wild-type female mice, and F1 mice were achieved.The heterozygous males mated with females to generate the F2 homozygous mice.The first hair coat of Hr-/- mice developed normally.Beginning from 14 days after birth, however, there was a rapid hair loss.The mices were completely hairless except for a few vibrissae at 30 days. Histologically, two characteristic structures appeared, the utriculus and dermal cyst.Conclusions The results suggest that Hr -/- mice are successfully created using TALENs, and Hr is important for regulating hair development, which could explain at least in part the hair loss and be applied to study the mechanism of hair growth and development disorder.

Ranpirnase (onconase, ONC) is a new drug, with weak RNase activity and strong cytotoxicity to various tumor cells in vitro and in vivo. This study is to obtain recombination onconase (rONC) with high bioactivity. Based on the codon preference of Pichia pastoris, we designed and synthesized the gene according to cDNA sequences of ONC and the α mating factor's prepeptide. We screened positive clones after transforming the recombination plasmids into P. pastoris X-33, GSS115 and SMD1168. We screened the best combination of seven different vectors and host strains. Moreover, we optimized culture condition in shake flasks and 10 L bioreactor, and purified rONC from the supernatant after inducing it with 0.25% methanol by aqueous two-phase extraction coupling G50 molecular exclusion method. The highest rONC production was 13 mg/L in pPICZα-A/X-33/ONC combination under the condition of pH 5.5 and 23 degrees C in shake flasks for 7 d; and that the highest rONC production was 180 mg/L when the induction is performed in the lower basic salt medium with pH 5.5 in the 10 L bioreactor for 7 d. The yield of rONC is more than 90% at a purity of above 95%. rONC can kill various tumor cells in vitro. The expression and purification of rONC would be useful for further investigation of this new drug.
Antineoplastic Agents ; metabolism ; Bioreactors ; Cell Line, Tumor ; Codon ; DNA, Complementary ; Genetic Vectors ; Humans ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; Ribonucleases ; biosynthesis

Objective_To study the effect of human insulin on cell cycle progression and apoptosis of rat liver cell line BRL-3A in vitro.Methods_MTT method was used to observe the effect of insulin on cell activity, and flow cytometry was used to detect cell apoptosis and cell cycle.qRT-PCR was used to evaluate the expression of related genes.Results_Human insulin induced the proliferation of BRL-3A cells in a dose-dependent manner ( P<0.05 or P<0.01);After 3 days treated by human insulin (500 nmol/L), the proportion of cells in G0/G1 phases re-markably decreased (P<0.05).Moreover, pro-apoptotic BAX was down-regulated (P<0.05), while cell prolif-eration-related gene CCNA2 was up-regulated (P<0.05).Conclusions_Human insulin may inhibit the apoptosis of BRL-3 A cell line and induce proliferation due to the down-regulated expression of BAX and up-regulated expres-sion of CCNA2 .

Hepatic oval cells (OCs) are the stem cells of the liver. They can differentiate into liver cells, bile duct epithelial cells and other cells, and also are closely related to liver regeneration and liver diseases. In this article, we give a brief summary focused on the source, location, proliferation, differentiation, apoptosis, transplantation of hepatic oval cells, and its role in liver regeneration and liver diseases.

Pit cells (PCs) are hepatic natural killer cells(HNKCs), and play an important role in resisting tumor, resisting virus, regulating hepatocyte growth and differentiation, inhibiting liver fibrosis and so on. In the paper, research progress in liver pit cells is briefly summarized.

Hepatic stellate cells (HSCs) are a group of nonparenchymal cells of liver, and play an important role in lipid, especially vitamin A-storing, extracellular matrix synthesis, microcirculation regulation of hepatic sinusoid, etc. They are also closely related to lots of liver diseases, e. g. hepatofibrosis. In this review, the latest research advance on HSCs including their isolation, identification, proliferation and regulation, and role in liver regeneration, hepatofibrosis, and so on is summarized.

ObjectiveTo study the transcript atlas of cell immunity-associated genes in 8 liver cell types including hepatocytes, bile duct epithelial cells, oval cells, hepatic stellate cells, sinus endothelial cells, Kupffer cells, pit cells and dendritic cells from the rat regenerating liver. Methods Isolation of rat 8 liver cell types, detection of their genes expression change and the prediction physiological activities were accorded to cluster program, and the methods of bioinformation and systematic biology, and their gene expression patterns were analyzed by Microsoft Excel software. Results A total of 40 blood coagulation-associated genes yielded the meaningful expression changes in liver regeneration. Serpine1, a2m in eight liver cell types, vwf, klkb1 in seven liver cell types other than Kupffer cells, and other genes in the two or more liver cell types yielded the meaningful expression changes. It suggested that the synthesis of kallikrein and prothrombin、the formation of thrombin were enhanced at the priming and progressing phases of liver regeneration. The biological activities, such as fibrin monomer aggregated into fibrin polymers, were elevated at termination phase.Conclusion The rat liver regeneration is closely associated with the blood coagulation.

ObjectiveTo explore the correlation of unknown gene AI408225 and humoral immunity involved in rat regenerating liver. MethodsIsolation of rat 8 liver cell types, detection of their genes expression change were accorded to Percoll density gradient centrifugation combined with immune magnetic beads separation method, and their genes sequence homoeology and coexpression relation were analyzed by Microsoft Excel, BLAST software, and the prediction physiological activities were analyzed by bioinformatics and systematic biology. Results A total of 93 humoral immunity-ass ociated genes yielded the meaningful expression changes in liver regeneration, the corresponding gene numbers in 8 liver cell types included hepatocytes, bile duct epithelial cells, oval cells, hepatic stellate cells, sinus endothelial cells, Kupffer cells, pit cells and dendritic cells were 33, 46, 17, 59, 48, 35, 53, 68. Among that AI408225 and cd4 are homology and coexpression.Conclusion The rat liver regeneration is closely associated with humoral immunity, and AI408225 participates in the formation of compound of MHC II-antigen peptide-CD4-TCR.

ObjectiveTo study the transcript atlas of cell immunity-associated genes in 8 liver cell types including hepatocytes, bile duct epithelial cells, oval cells, hepatic stellate cells, sinus endothelial cells, Kupffer cells, pit cells and dendritic cells from the rat regenerating liver. Methods The 8 liver cell types were isolated respectively by Percoll density gradient centrifugation combined with immune magnetic beads separation method. Their expression changes in the regenerating livers were detected by Rat Genome 230 2.0 Array, the expression patterns and the predicted physiological activities of cell immunity-associated genes were analyzed by Cluster program, and the methods of bioinformation and systematic biology. Results A total of 40 cell immunity-associated genes yielded the meaningful expression changes in liver regeneration, the corresponding gene numbers in 8 liver cell types were 19, 19, 9, 19, 19, 21, 22 and 21, respectively. It suggested that the formation of antigen peptide-MHC complexes, the NF-κB kinase activity and the production of cytokines like IL-2 were enhanced at the priming and progressing phases of liver regeneration. The biological activities, such as NF-κB-promoting cell differentiation and caspase-induced T cell apoptosis, were elevated at termination phase.Conclusion The rat liver regeneration is associated with cell immunity.

Kupffer cell is a member of the liver nonparenchymal cells, it participates in a variety of physiological activities through phagocytosis, secreting cytokines, antigen-presenting pathways, and is closely related to a variety of diseases such as liver cancer, liver fibrosis, liver injury. This paper presents the progress of research on physiological functions of kupffer cells and its relations with liver.