Construction of a Hr mutant knockout mouse model and phenotypic analysis / 中国比较医学杂志

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.

Restoration of CpG Methylation in The Egf Promoter Region during Rat Liver Regeneration.

Epidermal growth factor (EGF) is an important factor for healing after tissue damage in diverse experimental models. It plays an important role in liver regeneration (LR). The objective of this experiment is to investigate the methylation variation of 10 CpG sites in the Egf promoter region and their relevance to Egf expression during rat liver regenera- tion. As a follow up of our previous study, rat liver tissue was collected after rat 2/3 partial hepatectomy (PH) during the re-organization phase (from days 14 to days 28). Liver DNA was extracted and modified by sodium bisulfate. The methylation status of 10 CpG sites in Egf promoter region was determined using bisulfite sequencing polymerase chain reaction (PCR), as BSP method. The results showed that 3 (sites 3, 4 and 9) out of 10 CpG sites have strikingly methylation changes during the re-organization phase compared to the regeneration phase (from 2 hours to 168 hours, P=0.002, 0.048 and 0.018, respectively). Our results showed that methylation modification of CpGs in the Egf promoter region could be restored to the status before PH operation and changes of methylation didn't affect Egf mRNA expression during the re-organization phase.

Expression, purification and characterization of recombinant onconase expressed in Pichia pastoris / 生物工程学报

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.

Human insulin inhibits apoptosis and promotes proliferation of rat liver cell line BRL-3 A / 基础医学与临床

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 .

Application of hydrodynamics-based transgene to gene transfection in rat fatty liver / 解剖学报

Objective To study the condition and method of hydrodynamics-based transgene(HDT) in rat fatty liver. Methods Inject different dosages and concentrations of green fluorescent protein plasmid pEGFP-C1 at different speeds, then collect 4 rats' liver leaves at different time points after injection and prepare their frozen section, finally observe and quantify the GFP expression with fluo rescence microscope at 488 nm excitation wavelength. Results Plasmid pEGFP-C1 concentration 33mg/L, injection speed 2ml/s, injection volume 8.5%of rat body weight, injected plasmid. After 6 hours of injection, GFP-positive cells rate of pedicel leaf is about 18%, left leaf about 14%, middle leaf about 12.5%;R3ight leaf about 10% and tail leaf about 8%. GFP begin to gradually reduce since 24 hours, until 72 hours almost no GFP-positive cells were checked in all liver leaves. Conclusion Hydrodynamics-based transgene can be applied to rat fatty liver, the appropriate conditions of this method are 33mg/L plasmid concentration, 8.5% rat avoirdupois, 2ml/s injection speed, and the suitable time to observe the proportion of GFP-positive cells is 6-24 hours after gene injection.

Research progress on Kupffer cells / 解剖学报

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.

Transcript atlas of cell immunity-associated gene uncovers physiological activities in 8 liver cell types of rat regenerating liver / 解剖学报

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.

Gene transcript profiles of liver cells predict that AI408225 promotes antigen peptide combining with TCR in rat regenerating liver / 解剖学报

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.

Transcript atlas of blood coagulation-associated gene uncovers physiological activities in 8 liver cell types of rat regenerating liver / 解剖学报

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.

Research progress in hepatic stellate cells / 解剖学报

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.

Research progress in liver pit cells / 解剖学报

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.

Research progress on hepatic oval cells / 解剖学报

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.

Hydrodynamics-based transgene directively into rat regenerating liver in vivo / 解剖学报

Objective To study the conditions and methods of hydrodynamics-based transgene into rat regenerating liver in vivo. Methods The solution with concentration 30mg/L gene-containing plasmid was injected into rat tail veins at a speed of 2ml/s, then partial hepatectomy (PH) was performed at different times before/after injection, finally the rat (g) and regenerating liver (g) were weighed, and the liver coefficient (Lc) was calculated. Out of 15 groups which are Lc±Lc*0%, *5%, *10%, *15%, *20%, *25%, *30%, *35%, the most suitable group was chosen as correction coefficient to calculate the most appropriate volume of plasmid solution which was injected into the regenerating liver at different recovery times, and at the same time, right lobe of liver was gathered to make frozen section, then observe and quantify the positive green fluorescent protein (GFP) rate at 488 nm excitation wavelength. Results Injection of either physiological saline or empty plasmid has no significant difference compared with control (only PH performance). The appropriate time of hydrodynamics-based transgene is more than 12 hours before PH or anytime after PH. The solution volume of hydrodynamics-based transgene into liver regenerating rat after PH is rat weight (g) ×9%×1/3×corresponding correction coefficient (Trc). Both vector and target gene have effect on the time and abundance of gene expression. Conclusion Hydrodynamics-based transgene can effectively be applied to gene transfection in rat regenerating liver.

INHIBITION OF GLUCOCORTICOID ON ORNITHINE DECARBOXYLASE DURING RAT LIVER REGENERATION / 解剖学报

Objective The regulation of ornithine decarboxylase (ODC) gene expression and enzyme activity by corticosterone, the main glucocorticoid in rat, during rat liver regeneration induced by partial hepatectomy (PH) was evaluated.Methods Bilateral adrenaleetomies (ADX) and sham-ADX were performed on ether-anesthetized rats 3 days before PH.Corticosterone in sesame oil was injected subcutaneously to adrenalectomied rats. ODC mRNA, ODC protein and enzyme activity were detected by RT-PCR, Western blotting and high performance liquid chromatography (HPLC), respectively. Results The ODC mRNA levels, protein accumulation and enzyme activity were lower in the intact liver compared to the regenerating liver.After PH, mRNA levels were remarkably enhanced in all groups (n=6 in each group) and peaked at 5 hours post-PH. Till 7 hours, the contents in all groups from high to low were ADX group,control group (Sham-ADX group), ADX treated with 10mg/kg and 40mg/kg body weight corticosterone group, respectively. ODC protein accumulation in ADX rats was higher than that in control rats (n=13, the same below), but it decreasod in corticosterone-treated (10mg/kg) rats until 24 hours post-PH, with a strong decline seen in 40mg/kg corticosterone-treated rats. ODC activity was rapidly promoted, and the highest levels were observed at 6 hours after PH in all groups (n=6 in each group). After corticosterone treatment, the activities declined significantly at 6 hours post-PH, with the lowest value found in the 40mg/kg group. Conclusion Corticosterone treatment results in dose-dependent decreases in ODC mRNA and enzyme protein both in the intact liver and the regenerating liver. The change in ODC activity is partially related to alterations of ODC mRNA and protein accumulation.

PPAR-? coupled signaling pathways involved in rat liver regeneration / 基础医学与临床

Objective To investigate the possible contribution of PPAR-? coupled signaling pathways to rat liver regeneration (LR) at transcription level.Methods The above signaling pathways-related genes were obtained by collecting database data and retrieving pertinent literatures. The gene expression during LR was checked by Rat Genome 230 2.0 array, and LR-associated genes were identified by comparing difference between partial hepatectomy (PH) and operation control (OC) groups.Results Sixty four genes were found to be LR-associated. The number of initially and totally expressed genes occurring in initiation phase of LR, G0/G1 transition, cell proliferation, cell differentiation and structure-function reconstruction was 28, 4, 34, 2 and 72, 41, 247, 90 respectively. Classification of their expression patterns into 11 groups reflected diversity and complexity of gene expression alteration in LR. Conclusion PPAR-? coupled signaling pathways may promote glycogen synthesis in forepart, prophase and anaphase of LR, cell proliferation and migration in the whole LR, while inhibits inflammation during LR.

Study on construction of cDNA library of the treated changliver cell and quality analysis.

The study aims to construct cDNA library of Changliver cell by SMART (switching mechanism at 5' end of RNA transcript) technique and analyze its quality. cDNA of Changliver cell was made with RT-PCR and LD-PCR (long-distance PCR), the cDNA library was constructed with SMART cDNA library construction kit. Through testing, the high quality cDNA library containing whole long cDNA of Changliver cell had been constructed. The titer of the amplified cDNA library was 4.5 × 10(10) pfu/ml and the average exogenous inserts of the recombinants is 1.5 kb. These results showed that the Changliver cell cDNA library had an excellent quality and lay foundation for screening whole long cDNA of related genes.

Identification and characterization of 177 unreported genes associated with liver regeneration / 基因组蛋白质组与生物信息学报·英文版

The mammalian liver has a very strong regeneration capacity after partial hepatectomy (PH). To further learn the genes participating in the liver regeneration (LR), 551 cDNAs selected from subtracted cDNA libraries of the regenerating rat liver were screened by microarray, and their expression profiles were studied by cluster and generalization analyses. Among them, 177 genes were identified unreported and up- or down-regulated more than twofold at one or more time points after PH, of which 62 genes were down-regulated to less than 0.5; 99 genes were up-regulated to 2-10 folds, and 16 genes were either up- or down-regulated at different time points during LR. By using BLAST and GENSCAN, these genes were located on responsible chromosomes with 131 genes on the long arms of the chromosomes. The cluster and generalization analyses showed that the gene expression profiles are similar in 2 and 4, 12 and 16, 96 and 144 h respectively after PH, suggesting that the actions of the genes expressed in the same profiles are similar, and those expressed in different profiles have less similarity. However, the types, characteristics and functions of the 177 genes remain to be further studied.

Construction of cDNA library of the treated Changliver cell and quality analysis / 新乡医学院学报

Objective To construct cDNA library of the treated Changliver cell by switching mechanism at 5′ end of RNA transcript (SMART) technique and analyze its quality.Methods cDNA of Changliver cell was aquired with reverse transcription polymerase chain reaction (RT-PCR) and long-distance PCR (LD-PCR),then the cDNA library was constructed with SMART cDNA library construction kit.Results Through testing,the high quality cDNA library containing full length cDNA of Changliver cell had been constructed.The titer of the amplified cDNA library was 4.5 × 1010 pfu*ml-1 and the average exogenous inserts of the recombinants was 1.5 kb.Conclusion These results suggest that the Changliver cell cDNA library has a high quality and lays a solid foundation for researching on Changliver cell and screening

RELATIONSHIP BETWEEN EXPRESSIONAL PROFILES OF APOPTOSIS RELATED GENES AND APOPTOSIS ACTION OF THE RAT REGENERATING LIVER / 解剖学报

Objective To investigate the actions of apoptotic pathways in rat liver regeneration(LR) at the transcription level.Methods The apoptosis genes were obtained by referring to the putative literatures and databases.Apoptotic-related genes expression profiles in rat LR were checked by Rat Genome 230 2.0 array.Identification of LR-associated genes was made by comparing the discrepancies of genes expression between partial hepatectomied(PH) and sham operatied(SO) rats.Results 252 genes were found to be related to liver regeneration.The initial and total expressing gene numbers occurring in the initiation phase of liver regeneration(0.5-4hours after PH),G_0/G_1 transition(4-6hours after PH),cell proliferation(6-66hours after PH),cell differentiation and structure-function reconstruction(72-168hours after PH) were 81,23,155,16 and 161,100,733,192 respectively.This demonstrated that LR-related genes primarily started their expression in the initiation phase,and worked in the different phases.The total times of their up-and down-regulated expression were 795 and 291,demonstrating that expression of most genes was enhanced in LR.Their expression patterns could be classified into 35 types,indicating the expressions of apoptosis-related genes in LR were diverse and complicated.Conclusion There are fifteen kinds of apoptosis pathways that associate to apoptosis regulation in the regenerating liver.

EXPRESSION PATTERNS AND FUNCTION ANALYSIS OF THE GENES RELATED WITH SEX DETERMINATION AND DIFFERENTIATION DURING RAT LIVER REGENERATION / 解剖学报

Objective To study the function of the genes regulating sex determination and differentiation during liver regeneration at transcriptional level.Methods The genes regulating sex determination and differentiation were obtained by referring to the theses and collecting the data of databases at NCBI,GENMAPP,KEGG,BIOCARTA and RGD,and their function and expression changes in rat liver regeneration were analysized by the Rat Genome 230 2.0 array.Results The initial and total expressed gene numbers in the starting phase of liver regeneration [half to four hours after partial hepatectomy(PH)],G_0/G_1 transition(4 to 6 hours after PH),cell proliferation 6 to 66 hours after PH),cell differentiation and tissue structural function reconstruction(72 to 160 hours after PH) were 41,6,18,3 and 41,25,57,41 respectively,which showed that the related genes were mainly triggered in the starting phase,and worked in different phases.Their expression similarity was classified into 5 groups:only up-,predominantly up-,only down-predominantly down-,up-/down-regulation,involving 22,9,15,9 and 7 genes respectively,and the total frequencies of their up-and down-regulation expressions were 231 and 146 respectively,demonstrating that the expression of the major genes was increased,and the minority decreased.Their expression time relevance was classified into 15 groups,showing that the cellular physiological and biochemical activities were phase related during liver regeneration.The gene expression patterns were classified into 20 types,indicating the diversity and complexity of the cellular physiological and biochemical activity.Conclusion The genes regulating male determination,male and female differentiation are enhanced mainly in the late early phase and prophase of liver regeneration,and the genes regulating female determination are enhanced mainly in the prophase.The function of the genes is closely related to liver regeneration.