Novel Therapeutic Potential of Retinoid-Related Orphan Receptor α in Cardiovascular Diseases.

The retinoid-related orphan receptor α (RORα) is one subfamily of nuclear hormone receptors (NRs). This review summarizes the understanding and potential effects of RORα in the cardiovascular system and then analyzes current advances, limitations and challenges, and further strategy for RORα-related drugs in cardiovascular diseases. Besides regulating circadian rhythm, RORα also influences a wide range of physiological and pathological processes in the cardiovascular system, including atherosclerosis, hypoxia or ischemia, myocardial ischemia/reperfusion injury, diabetic cardiomyopathy, hypertension, and myocardial hypertrophy. In terms of mechanism, RORα was involved in the regulation of inflammation, apoptosis, autophagy, oxidative stress, endoplasmic reticulum (ER) stress, and mitochondrial function. Besides natural ligands for RORα, several synthetic RORα agonists or antagonists have been developed. This review mainly summarizes protective roles and possible mechanisms of RORα against cardiovascular diseases. However, there are also several limitations and challenges of current research on RORα, especially the difficulties on the transformability from the bench to the bedside. By the aid of multidisciplinary research, breakthrough progress on RORα-related drugs to combat cardiovascular disorder may appear.

Sirtuin 3 deficiency exacerbates diabetic cardiomyopathy via necroptosis enhancement and NLRP3 activation.

Sirtuin 3 (SIRT3) is a potential therapeutic target for cardiovascular, metabolic, and other aging-related diseases. In this study, we investigated the role of SIRT3 in diabetic cardiomyopathy (DCM). Mice were injected with streptozotocin (STZ, 60 mg/kg, ip) to induce diabetes mellitus. Our proteomics analysis revealed that SIRT3 expression in the myocardium of diabetic mice was lower than that of control mice, as subsequently confirmed by real-time PCR and Western blotting. To explore the role of SIRT3 in DCM, SIRT3-knockout mice and 129S1/SvImJ wild-type mice were injected with STZ. We found that diabetic mice with SIRT3 deficiency exhibited aggravated cardiac dysfunction, increased lactate dehydrogenase (LDH) level in the serum, decreased adenosine triphosphate (ATP) level in the myocardium, exacerbated myocardial injury, and promoted myocardial reactive oxygen species (ROS) accumulation. Neonatal rat cardiomyocytes were transfected with SIRT3 siRNA, then exposed to high glucose (HG, 25.5 mM). We found that downregulation of SIRT3 further increased LDH release, decreased ATP level, suppressed the mitochondrial membrane potential, and elevated oxidative stress in HG-treated cardiomyocytes. SIRT3 deficiency further raised expression of necroptosis-related proteins including receptor-interacting protein kinase 1 (RIPK1), RIPK3, and cleaved caspase 3, and upregulated the expression of inflammation-related proteins including NLR family pyrin domain-containing protein 3 (NLRP3), caspase 1 p20, and interleukin-1ß both in vitro and in vivo. Collectively, SIRT3 deficiency aggravated hyperglycemia-induced mitochondrial damage, increased ROS accumulation, promoted necroptosis, possibly activated the NLRP3 inflammasome, and ultimately exacerbated DCM in the mice. These results suggest that SIRT3 can be a molecular intervention target for the prevention and treatment of DCM.

Dihydromyricetin Improves Endothelial Dysfunction in Diabetic Mice via Oxidative Stress Inhibition in a SIRT3-Dependent Manner.

Dihydromyricetin (DHY), a flavonoid component isolated from Ampelopsis grossedentata, exerts versatile pharmacological activities. However, the possible effects of DHY on diabetic vascular endothelial dysfunction have not yet been fully elucidated. In the present study, male C57BL/6 mice, wild type (WT) 129S1/SvImJ mice and sirtuin 3 (SIRT3) knockout (SIRT3-/-) mice were injected with streptozotocin (STZ, 60 mg/kg/day) for 5 consecutive days. Two weeks later, DHY were given at the doses of 250 mg/kg by gavage once daily for 12 weeks. Fasting blood glucose (FBG) and glycosylated hemoglobin (HbA1c) level, endothelium-dependent relaxation of thoracic aorta, reactive oxygen species (ROS) production, SIRT3, and superoxide dismutase 2 (SOD2) protein expressions, as well as mitochondrial Deoxyribonucleic Acid (mtDNA) copy number, in thoracic aorta were detected. Our study found that DHY treatment decreased FBG and HbA1c level, improved endothelium-dependent relaxation of thoracic aorta, inhibited oxidative stress and ROS production, and enhanced SIRT3 and SOD2 protein expression, as well as mtDNA copy number, in thoracic aorta of diabetic mice. However, above protective effects of DHY were unavailable in SIRT3-/- mice. The study suggested DHY improved endothelial dysfunction in diabetic mice via oxidative stress inhibition in a SIRT3-dependent manner.

Mitochondrial genome and diverse inheritance patterns in Pleurotus pulmonarius.

Pleurotus pulmonarius, a member of the Pleurotaceae family in Basidiomycota, is an edible, economically important mushroom in most Asian countries. In this study, the complete mitochondrial genomes (mtDNA) of three P. pulmonarius strains - two monokaryotic commercial (J1-13 and ZA3) and one wild (X1-15) - were sequenced and analyzed. In ZA3 and X1-15, the mtDNA molecule was found to be a single circle of 68,305 bp and 73,435 bp, respectively. Both strains contain 14 core protein-coding genes and two ribosomal RNA (rRNA) subunit genes. The ZA3 strain has 22 transfer RNA (tRNA) genes and nine introns: eight in cytochrome c oxidase subunit 1 (coxl), and one in the rRNA large subunit (rnl). Monokaryotic J1-13 and ZA3 mtDNAs were found to be similar in their structure. However, the wild strain X1-15 contains 25 tRNA genes and only seven introns in coxl. Open reading frames (ORFs) of ZA3/J1-13 and X1-15 encode LAGLIDADG, ribosomal protein S3, and DNA polymerase II. In addition, mtDNA inheritance in J1-13, ZA3, and X1-15 was also studied. Results showed that the mtDNA inheritance pattern was uniparental and closely related to dikaryotic hyphal location with respect to the parent. Results also show that mtDNA inheritance is influenced by both the parental nuclear genome and mitogenome in the zone of contact between two compatible parents. In summary, this analysis provides valuable information and a basis for further studies to improve our understanding of the inheritance of fungal mtDNA.

Mogrol attenuates lipopolysaccharide (LPS)-induced memory impairment and neuroinflammatory responses in mice.

This study aimed to evaluate whether mogrol, a main bioactive ingredient of Siraitia grosvenorii, could attenuate LPS-induced memory impairment in mice. The behavioral tests and immunohistochemical analysis and Western blot were performed. The present results showed that oral administration of mogrol (20, 40, 80 mg/kg) significantly improved LPS-induced memory impairment in mice. The results also indicated that mogrol treatment significantly reduced the number of Iba1-positive cells, the nuclear NF-κB p65 and levels of TNF-α, IL-1ß and IL-6 both in the hippocampus and frontal cortex of LPS-challenged mice. [Formula: see text].

Dentate nNOS accounts for stress-induced 5-HT1A receptor deficiency: Implication in anxiety behaviors.

BACKGROUND: Anxiety is a common disorder with high social burden worldwide. Dysfunction of serotonin-1A receptor (5-HT1A receptor) in the dentate gyrus (DG) of the hippocampus has been predominantly implicated in the anxiety behavior. However, the molecular mechanism underlying the deficiency of postsynaptic 5-HT1A receptor in regulating anxiety behavior remains unclear. METHODS: Using pharmacological and genetic methods, we investigated the role of detate nNOS in 5-HT1A receptor decline and anxiety behavior induced by chronic mild stress (CMS) in mice. RESULTS: Here we showed that local elevation of glucocorticoids in the DG accounted for chronic stress-induced anxiety behavior. Neuronal nitric oxide synthase (nNOS) mediated chronic stress-induced downregulation of 5-HT1A receptor in the DG through peroxynitrite anion (ONOO•) pathway but not cyclic guanosine monophosphate (cGMP) pathway. By using pharmacological tool drugs and nNOS knockout mice, we found that nNOS in the DG played a key role in chronic stress-induced anxiety behavior. CONCLUSIONS: These findings uncovered an important role of nNOS-5-HT1A receptor pathway in the DG of the hippocampus in chronic stress-induced anxiety. Accordingly, we developed a "dentate nNOS-5-HT1A receptor closed-loop" theory (stress-glucocorticoids-nNOS-Nitric oxide-ONOO•-5-HT1A receptor -nNOS) of stress-related anxiety.

Hippocampal Salt-Inducible Kinase 2 Plays a Role in Depression via the CREB-Regulated Transcription Coactivator 1-cAMP Response Element Binding-Brain-Derived Neurotrophic Factor Pathway.

BACKGROUND: Developing novel pharmacological targets beyond monoaminergic systems is now a popular strategy for finding new ways to treat depression. Salt-inducible kinase (SIK) is a kinase that regulates the nuclear translocation of cyclic adenosine monophosphate response element binding protein (CREB)-regulated transcription coactivator (CRTC) by phosphorylation. Here, we hypothesize that dysfunction of the central SIK-CRTC system may contribute to the pathogenesis of depression. METHODS: Chronic social defeat stress (CSDS) and chronic unpredictable mild stress (CUMS) models of depression, various behavioral tests, viral-mediated gene transfer, Western blotting, coimmunoprecipitation, quantitative real-time reverse transcription polymerase chain reaction, and immunohistochemistry were used in this study (for in vivo studies, n = 10; for in vitro studies, n = 5). RESULTS: Both CSDS and CUMS markedly increased the expression of hippocampal SIK2, which reduced CRTC1 nuclear translocation and binding of CRTC1 and CREB in the hippocampus. Genetic overexpression of hippocampal SIK2 in naïve mice simulated chronic stress, inducing depressive-like behaviors in the forced swim test, tail suspension test, sucrose preference test, and social interaction test, as well as decreasing the brain-derived neurotrophic factor signaling cascade and neurogenesis in the hippocampus. In contrast, genetic knockdown and knockout of hippocampal SIK2 protected against CSDS and CUMS, exerting significant antidepressant-like effects that were mediated via the downstream CRTC1-CREB-brain-derived neurotrophic factor pathway. Moreover, fluoxetine, venlafaxine, and mirtazapine all significantly restored the effects of CSDS and CUMS on the hippocampal SIK2-CRTC1 pathway, which was necessary for their antidepressant actions. CONCLUSIONS: The hippocampal SIK2-CRTC1 pathway is involved in the pathogenesis of depression, and hippocampal SIK2 could be a novel target for the development of antidepressants.

Dihydromyricetin Attenuates Myocardial Hypertrophy Induced by Transverse Aortic Constriction via Oxidative Stress Inhibition and SIRT3 Pathway Enhancement.

Dihydromyricetin (DMY), one of the flavonoids in vine tea, exerts several pharmacological actions. However, it is not clear whether DMY has a protective effect on pressure overload-induced myocardial hypertrophy. In the present study, male C57BL/6 mice aging 8⁻10 weeks were subjected to transverse aortic constriction (TAC) surgery after 2 weeks of DMY (250 mg/kg/day) intragastric administration. DMY was given for another 2 weeks after surgery. Blood pressure, myocardial structure, cardiomyocyte cross-sectional area, cardiac function, and cardiac index were observed. The level of oxidative stress in the myocardium was assessed with dihydroethidium staining. Our results showed that DMY had no significant effect on the blood pressure. DMY decreased inter ventricular septum and left ventricular posterior wall thickness, relative wall thickness, cardiomyocyte cross-sectional areas, as well as cardiac index after TAC. DMY pretreatment also significantly reduced arterial natriuretic peptide (ANP), brain natriuretic peptide (BNP) mRNA and protein expressions, decreased reactive oxygen species production and malondialdehyde (MDA) level, while increased total antioxidant capacity (T-AOC), activity of superoxide dismutase (SOD), expression of sirtuin 3 (SIRT3), forkhead-box-protein 3a (FOXO3a) and SOD2, and SIRT3 activity in the myocardium of mice after TAC. Taken together, DMY ameliorated TAC induced myocardial hypertrophy in mice related to oxidative stress inhibition and SIRT3 pathway enhancement.

[Research Advances in Hydrogen Sulfide and Diabetes Mellitus as Well as Diabetic Complications].

Diabetes and its complications continue to be an increasing global health problem. Hydrogen sulfide (H2S)has emerged as the third gasotransmitter following the nitric oxide and carbon monoxide in the body, and plays an important role in both physiology and pathophysiology. Recent work has suggested important roles for H2S in the regulation of pancreatic ß-cell function, as well as development of insulin resistance and diabetic complications. Targeting H2S represents a novel therapeutic approach in diabetes and its associated complications. This review article summarizes the current body of evidence regarding the role of H2S in diabetes mellitus, and especially in its associated vascular complications, as well as the underlying signaling mechanisms.

Telmisartan attenuates hydrogen peroxide-induced apoptosis in differentiated PC12 cells.

The present study investigated the protective actions of telmisartan, an angiotensin II type 1 receptor blocker (ARBs), against the cell apoptosis induced by exposure to hydrogen peroxide (H2O2) in differentiated PC12 cells. Preincubation of PC12 cells with telmisartan prevented H2O2-induced cytotoxicity as indicated by increased MTT (3,(4,5-dimethylthiazole-2-yl)2,5-diphenyl-tetrazolium bromide) reduction, decreased lactate dehydrogenase (LDH) release, and improved morphological changes. Hoechst 33,258 staining showed that telmisartan markedly reduced shrunken nuclei of the cells, and Western blot analysis indicated that telmisartan significantly attenuated caspase-3 activity, as indicated by decreased ratio of cleaved Caspase-3 to its precursor and increased ratio of Bcl-2/Bax. The present findings showed that telmisartan protected against cellular oxidative damages by inhibiting apoptotic response.

Characterization of pumpkin polysaccharides and protective effects on streptozotocin-damaged islet cells.

The polysaccharides from pumpkin fruit (PP) were obtained and purified by hot-water extraction, anion-exchange chromatography, and gel column chromatography. The physicochemical properties of PP were determined by gel filtration chromatography, gas chromatography, fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. Results indicated that the molecular weight of PP was about 23 kDa and PP was composed of D-Arabinose, D-Mannose, D-Glucose, and D-Galactose with a molar ratio of 1 : 7.79 : 70.32 : 7.05. FTIR and NMR spectra indicated that PP was the polysaccharide containing pyranose ring. Additionally, PP protected islets cells from streptozotocin (STZ) injury in vitro via increasing the levels of super-oxide dismutase (SOD) and malondialdehyde (MDA) and reducing the production of NO. The experiment of reverse transcriptase-polymerase chain reaction further proved that PP inhibited apoptosis via modulating the expression of Bax/Bcl-2 in STZ-damaged islet cells. In conclusion, PP could be explored as a novel agent for the treatment of diabetes mellitus.

[Effect of Ganoderma lucidum polysaccharides on oxidative stress of hyperlipidemic fatty liver in rats].

OBJECTIVE: To study the effect of Ganoderma lucidum polysaccharides on oxidative stress of hyperlipidemic fatty liver in rats. METHOD: Seventy-two SD rats were randomly divided into six groups, namely the normal control group (NG), the model group (MG), the G. lucidum polysaccharides groups of low, middle and high dose (GLPs-LG, GLPs-MG, GLPs-HG) and the Simvastatin group (SV). The rats were fed with high fat diet to establish the model of hyperlipidemic fatty liver in rats. After administration for 12 weeks, rats in each group were tested with the following indexes: total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) in serum as well as the contents of SOD, MDA, GSH-Px and T-AOC in hepatic tissues. Histopathological changes of hepatic tissues were observed under light glass. RESULTS: The contents of TC, TG and LDL-C were significantly increased in the model group (P < 0.01). Compared with the model group, both the GLPs-M group and the GLPs-H group showed significant decreases in TC, TG and LDL-C (P < 0.05 or P < 0.01), while the GLPs-H group showed a notable increase in HDL-C (P < 0.05). Compared with the model group, both the GLPs-M group and the GLPs-H group showed significant decreases in MDA (P < 0.05 or P < 0.01) and notable increases in SOD, GSH-Px, T-AOC (P < 0.05 or P < 0.01). The GLPs-M group and the GLPs-H group proved a remarkable alleviation in fatty degeneration of hepatic cells. CONCLUSION: G. lucidum polysaccharides can significantly reduce the blood fat level of hyperlipidemic fatty liver in rats and effectively inhibit oxidant stress, showing the effect on preventing and treating hyperlipidemic fatty liver in rats to some extent.

Stable suppression of gene expression in murine embryonic stem cells by RNAi directed from DNA vector-based short hairpin RNA.

Murine embryonic stem (ES) cells are an ideal system for the research of directed differentiation in vitro. Long double-stranded RNA, which can induce RNA interference (RNAi) effectively in many organisms, has been shown to suppress target gene expression efficiently and specifically in undifferentiated ES cells. However, it cannot be used in differentiated ES cells due to unspecific inhibition of gene expression resulting from the activation of interferon pathway following differentiation. Using green fluorescent protein (GFP) as a reporter system, we show here that a short hairpin RNA (shRNA) expression vector driven by the murine U6 small nuclear RNA promoter can specifically induce potent gene knockdown effect (i.e., inhibit GFP expression specifically) when transfected transiently into ES cells. Furthermore, when the expression vector is stably integrated into the genome of the cell, it can still show specific RNAi effect, which can be maintained at least for 10 days. These transfected ES cells showed no obvious differences in the morphology or growth rate in culture compared with untransfected cells, suggesting that the activation of shRNA-directed RNAi did not affect the properties of ES cells and that the RNAi effect in ES cells is specific and persistent. Our results prove the feasibility of the U6 promoter-driven shRNA expression technique to be used to study the function of genes expressed in ES cells. These ES cells, after integration of the U6-based RNAi vector into their genome, could be used to generate gene knockdown mice.

Labeling of three different mouse ES cell lines with the green fluorescent protein.

The linearized plasmid pEGFP-N3 was electroporated into three different mouse ES cell lines MESPU-13, MESPU-35 and MESPU-62 derived from 129/ter, C57BL/6J and BALB/c mouse strains respectively. Resistant clones were selected in the presence of G418 and then were identified under the fluorescence microscope through blue exciting light. Positive green clones were primarily expanded and further sorted using FACS(fluorescence activated cell sorter). Finally five EGFP stable integrated cell strains were obtained and were expanded (2 strains from 129/ter, 1 strain from C57BL/6J and 2 strains from BALB/c). Each of the five cell strains presents high proliferation growth rate and typical morphology characters of ES cells and their colonies. More than 85% cells of each cell strain contain normal diploid karyotype. Then some analysis such as the AP (alkaline phosphatase) staining, oct4 gene expression assay, embryonic body formation and differentiated test in vivo and in vitro were made. The results indicated that the stable labeled ES cell strains had the normal karyotypes and maintained the ES cell typical characteristics.

Comparison of the method establishing embryonic stem cell lines from five different mouse strains.

We compared the characteristics of the method establishing embryonic stem cell lines from five different mouse strains using the medium containing 70% rat heart cell-conditioned medium (RH-CM) as ES cell culture medium, using the primary murine embryo fibroblast as feeder cells, and using the digestive enzyme buffer containing 1% chicken serum and "the series digestive method". We first reported new ES cell lines established from the outbred strain mice KM and ICR using the improved method in our lab and the ratio of establishment of ES cell lines from KM and ICR strain mice is up to 12% and 42.1% respectively. Compared with routine method of establishing ES cell lines, the improved method made distinct differences, increasing the ratio of ES cell line's establishment of 129/ter mouse from 11.8% to 33.3%, that of C57BL/6J mouse from 3.7% to 13.3%, that of BALB/c mouse from 2.9% to 19.4%. We tested the appropriate dispersing occasion, that is proliferating period of the ICM, affected the formation of ES clones and the ratios of ES cell lines established. It was shown that the most appropriate dispersed occasion for the ICM of 129/ter, C57BL/6J, BALB/c, KM and ICR mice was 4-6 d, 3-3.5 d, 4 d, 4-5 d, 4-5 d after ICM proliferation respectively. At the same time, the effects of the concentration of digestive enzyme buffer were discussed. It was found that the ES cells from BALB/c mice were sensitive to the high concentration of digestive enzyme buffer and the 0.05% Trypsin-0.008% EDTA is an ideal concentration for their establishment and maintenance. It was shown that 'the series dispersed method' was much better than 'the once dispersed method' on the aspect of dispersing the proliferating ICM and formation of ES clones. Compared with the routine ES cell culture medium containing mLIF, the RH-CM not only remarkably inhibited the differentiation of murine ES cells and maintained their diploid karyotype, but also promoted the attachment and growth of ES cells. This improved method of establishment and culture of ES cell lines effectively maintained a series of their characteristics of pluripotent embryonic stem cells.

[RNA interference directed by small hairpin RNA expressed in COS-7 cells].

RNA interference is a phenomenon of gene silencing directed by double-stranded RNA. It can specifically inhibit gene expression by degrading mRNA efficiently and has been widely used to knockdown gene expression in Caenorhabditis elegans, Drosophila melanogaster, etc. For mammalian cells, dsRNA directed RNAi was detected only in murine undifferentiated ES or embryonic carcinoma (EC) cells. Our previous work proved the existence of RNAi effect for reporter gene GFP and endogenous gene Oct4 in undifferentiated murine ES cells. Yet in other kinds of mammalian cells, because of the existence of interferon pathway, long dsRNA will induce the cells to shutdown global protein translation and go to apoptosis. Therefore, dsRNA longer than 30 bp cannot be used to induce specific gene knockdown effect in these cells. Elbashir et al found that in vitro synthesized small interfering RNA (siRNA) (19-23 nt) could induce potent RNAi as effective as long dsRNA without showing unspecific effect, so that the interferon pathway could be bypassed. It was shown that during RNAi process, long dsRNA was first degraded into 19-23 nt siRNA and then recruited into RISC (RNA induced silencing complex) to degrade corresponding mRNA. However, the synthesis of siRNA is expensive and the effect is transient because the knockdown effect can only be maintained for about a week. Recently, it has been shown that U6 promoter directed small hairpin RNA (shRNA) can induce potent gene knockdown effect in murine P19 Embryonic Carcinoma cell. The RNAi effect of U6 promoter-driven shRNA corresponding to Green Fluorescence Protein (GFP) in COS-7 cells was checked. And it was found that the U6 promoter-driven shRNA for GFP can specifically and potently knockdown the GFP's expression in COS-7 cells. The result established the feasibility of using RNAi technique directed by U6 promoter-driven shRNA to study genes' function in COS-7 cell line.

[RNA interference in three ES cell lines from different mouse strains].

RNA interference phenomenon in three different murine ES cell lines (MESPU13, B3, and R1) is reported. A vector(pdsGFP) was used that transcribed hairpin double-stranded RNA of GFP gene to transfect ES cells by using lipofectin. The transient transcription of dsRNA induced RNAi (RNA interference) in the ES cells. That is, the double-stranded RNA of GFP gene potently turned down the expression of the GFP gene. On the hand, the linearized plasmid pdsGFP-puro was electroporated into MESPU13 ES cells, and the expression level of GFP after puromycin screening was turned down obviously in about 30% ES cell clones; and in a few clones, the expression level of GFP was not observed under the fluorescence microscope and GFP mRNA was not detectable by RT-PCR. Further more, another vector (pdsOCT4) was constructed that transcribed double-stranded RNA of OCT-4 gene which is specifically expressed in ES cells. ES cell clones that stably integrated the vector were screened after the electrotransfection of the cells with the above construct. 51 random-selected clones were amplified and 48 of them were checked by semi-quantitative RT-PCR. In 11 of them the mRNA of OCT-4 was undetectable by RT-PCR. This means that RNAi can be used to study mammal and human gene's function in ES cell lines from different strain mice.

[Current state of establishing and maintaining human embryonic stem cell lines and key problems in these studies].

Derivation of human embryonic stem cell and embryonic germ cell lines has widespread and far-reaching significance on human basic research and transplantation therapies. Human pluripotential stem cells provide an exciting new model for studying early human embryogenesis, understanding normal human development and abnormal development, provide a powerful system for discovering human novel genes and testing their function, offer new strategies for discovering of novel growth factors and medicines and promise a renewable source of cells for tissue transplantation, cell replacement and gene therapies. Research history of establishment of human ES and EG cell lines is reviewed. Several methods of establishment of these cell lines involving in the protocol, route, significance and possibility are discussed. Selection of the feeder layer, medium, and supplemental cytokines and their roles in establishing and maintaining human ES and EG cell lines at present are illustrated in detail systematically. Effects and used methods of several kinds of digestive en-zyme in propagations are prepared. Several methods for identifying human ES and EG cells are summarized. At the end, some key problems which are urgent to resolve in these studies at present are put forward and analyzed.

[Researches on the methodology of establishment of BALB/c murine embryonic stem cell lines].

Several methods and processes of establishment and culture of BALB/c mouse ES cell lines were discussed detailedly. A new method to establish and culture ES cell lines derived from BALB/c mouse was set up successfully using mouse embryonic fibroblast feeder layer and rat-heart-cell-conditioned medium (RH-CM). These culture conditions not only maintain the undifferentiated state and normal diploid karyotypes of BALB/c mouse ES cells effectively, but also maintain a series of their characteristics of murine stem cells. two different kinds of digestive methods and Two kinds of digestive juice with different concentrations were designed to dissociate proliferous inner cell mass (ICM) and ES cell colonies derived from dissociative ICM. two different kinds of digestive methods are "single time dissociation method" and "several times dissociation method", two kinds of digestive juice are 0.25% Trypsin-0.04% EDTA and 0.05% Trypsin-0.008% EDTA. At the same time, appropriate dissociated occasion of ICM and the effect of RH-CM on establishment and culture of BALB/c mouse ES cell lines were discussed. The results suggested that it is a reasonable method to establish BALB/c mouse ES cell lines using low concentration digestive juice and "several times dissociation method" to dissociate ICM after 4 days' proliferation. Judged by the form of ES cells and its colonies, proliferous capability, karyotypes examine, alkaline phosphatase activity assay and differentiation capability in vitro and in vivo, the 9 ES cell lines that we established satisfied the all traits of murine ES cell line.