Single nucleus/cell RNA-seq of the chicken hypothalamicpituitaryovarian axis offers new insights into the molecular regulatory mechanisms of ovarian development

The hypothalamic-pituitary-ovarian(HPO)axis represents a central neuroendocrine network essential for reproductive function.Despite its critical role,the intrinsic heterogeneity within the HPO axis across vertebrates and the complex intercellular interactions remain poorly defined.This study provides the first comprehensive,unbiased,cell type-specific molecular profiling of all three components of the HPO axis in adult Lohmann layers and Liangshan Yanying chickens.Within the hypothalamus,pituitary,and ovary,seven,12,and 13 distinct cell types were identified,respectively.Results indicated that the pituitary adenylate cyclase activating polypeptide(PACAP),follicle-stimulating hormone(FSH),and prolactin(PRL)signaling pathways may modulate the synthesis and secretion of gonadotropin-releasing hormone(GnRH),FSH,and luteinizing hormone(LH)within the hypothalamus and pituitary.In the ovary,interactions between granulosa cells and oocytes involved the KIT,CD99,LIFR,FN1,and ANGPTL signaling pathways,which collectively regulate follicular maturation.The SEMA4 signaling pathway emerged as a critical mediator across all three tissues of the HPO axis.Additionally,gene expression analysis revealed that relaxin 3(RLN3),gastrin-releasing peptide(GRP),and cocaine-and amphetamine regulated transcripts(CART,also known as CARTPT)may function as novel endocrine hormones,influencing the HPO axis through autocrine,paracrine,and endocrine pathways.Comparative analyses between Lohmann layers and Liangshan Yanying chickens demonstrated higher expression levels of GRP,RLN3,CARTPT,LHCGR,FSHR,and GRPR in the ovaries of Lohmann layers,potentially contributing to their superior reproductive performance.In conclusion,this study provides a detailed molecular characterization of the HPO axis,offering novel insights into the regulatory mechanisms underlying reproductive biology.

Quercetin ameliorates oxidative stress-induced senescence in rat nucleus pulposus-derived mesenchymal stem cells via the miR-34a-5p/SIRT1 axis

BACKGROUND Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Oxidative stress,which is highly associated with the progression of IDD,increases senescence of nucleus pulposus-derived mesenchymal stem cells(NPMSCs)and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs(IVDs).Quercetin(Que)has been demonstrated to reduce oxidative stress in diverse degenerative diseases.AIM To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism.METHODS In vitro,NPMSCs were isolated from rat tails.Senescence-associatedβ-galactosidase(SA-β-Gal)staining,cell cycle,reactive oxygen species(ROS),realtime quantitative polymerase chain reaction(RT-qPCR),immunofluorescence,and western blot analyses were used to evaluated the protective effects of Que.Meanwhile the relationship between miR-34a-5p and Sirtuins 1(SIRT1)was evaluated by dual-luciferase reporter assay.To explore whether Que modulates tert-butyl hydroperoxide(TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway,we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression.In vivo,a puncture-induced rat IDD model was constructed,and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo.RESULTS We found that TBHP can cause NPMSCs senescence changes,such as reduced cell proliferation ability,increased SA-β-Gal activity,cell cycle arrest,the accumulation of ROS,and increased expression of senescence-related proteins.While abovementioned senescence indicators were significantly alleviated by Que treatment.Que decreased the expression levels of senescence-related proteins(p16,p21,and p53)and senescence-associated secreted phenotype(SASP),including IL-1β,IL-6,and MMP-13,and it increased the expression of SIRT1.In addition,the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown.In vivo,X-ray,and histological analyses indicated that Que alleviated IDD in a punctureinduced rat model.CONCLUSION In summary,the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway,suggesting that Que may be a potential agent for the treatment of IDD.

Nucleus Pulposus Cells from Calcified Discs Promote the Degradation of the Extracellular Matrix through Upregulation of the GATA3 Expression

Objective Disc calcification is strongly associated with disc degeneration;however,the underlying mechanisms driving its pathogenesis are poorly understood.This study aimed to provide a gene expression profile of nucleus pulposus cells(NPCs)from calcified discs,and clarify the potential mechanism in disc degeneration.Methods Primary NPCs were isolated from calcified and control discs(CAL-NPC and CON-NPC),respectively.The proliferation and extracellular matrix(ECM)metabolism capacities of the cells were evaluated using MTT and Western blotting,respectively.RNA sequencing was used to identify differentially expressed genes(DEGs)in the CAL-NPCs.The biological functions of the DEGs were analyzed using the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases.The transcription factor database and Cytoscape software were used to construct the transcription factor-DEGs regulatory network.The role of the verified transcription factor in NPC proliferation and ECM metabolism was also investigated.Results The CAL-NPCs exhibited a lower proliferation rate and higher ECM degradation capacity than the CON-NPCs.In total,375 DEGs were identified in the CAL-NPCs.The GO and KEGG analyses showed that the DEGs were primarily involved in the regulation of ribonuclease activity and NF-kappa B and p53 signaling pathways.GATA-binding protein 3(GATA3)with the highest verified levels was selected for further studies.Overexpression of GATA3 in the CON-NPCs significantly inhibited their proliferation and promoted their ECM degradation function,while the knockdown of GATA3 in the CAL-NPCs resulted in the opposite phenotypes.Conclusion This study provided a comprehensive gene expression profile of the NPCs from the calcified discs and supported that GATA3 could be a potential target for reversing calcification-associated disc degeneration.

Ultrastructural Features of Nucleus Degradation During Programmed Cell Death of Starchy Endosperm Cells in Rice

Ultrastructural features of nucleus degradation during programmed cell death (PCD) of starchy endosperm cells in rice ( Oryza sativa L.) were observed using transmission electron microscopy. Several distinct morphological features of PCD have been found in the developing starchy endosperm cells, e.g. nucleus deformation, chromatin condensation, nuclear envelope disruption, and nuclear matrix leakage. DNA ladder displayed a smear of large DNA fragments from nucleus and evident bands of small DNA fragments (140-180 bp) from both nucleus and cytoplasm. In contrast with the rapid nucleus degradation, cell organelles in cytoplasm, such as rough endoplasmic reticulum, amyloplast, and mitochondrion, maintained their metabolic functions for a longer time. Seed reserves were continually synthesized and accumulated in the starchy endosperm cells despite the nucleus degradation during the PCD process. These results suggest that starchy endosperm cells remain active during reserve material synthesis and accumulation in the PCD process. The specific relationships between nucleus and cytoplasm in the developing endosperm cells and the morphological changes of nucleus in the endosperm PCD process were also discussed.

Differentiation of Mesenchymal Stem Cells into Nucleus Pulposus Cells In Vitro

To find a new source of seed cells for constructing tissue-engineered intervertebral disc, nucleus pulposus （NP） cells and mesenchymal stem cells （MSCs） were isolated from New Zealand white rabbits. The nucleus pulposus cells population was fluorescence-ladled and co-cultured with MSCs with or without direct contact. Morphological changes were observed every 12 h. Semi-quantitaive reverse transcriptase-polymerase chain reaction was performed to assess the expression levels of Sox-9, aggreacan and type Ⅱ collagen every 24 h after the co-culture. MSCs treated with direct contact rounded up and presented a ring-like appearance. The expression of marker genes was significantly increased when cells were co-cultured with direct contact for 24 h. No significant change was found after coculture without direct contact. Co-culture of NP cells and MSCs with direct contact is a reliable method for generating large amount of NP cells used for cell-based tissue engineering therapy.

6-gingerol protects nucleus pulposus-derived mesenchymal stem cells from oxidative injury by activating autophagy

BACKGROUND To date,there has been no effective treatment for intervertebral disc degeneration(IDD).Nucleus pulposus-derived mesenchymal stem cells(NPMSCs)showed encouraging results in IDD treatment,but the overexpression of reactive oxygen species(ROS)impaired the endogenous repair abilities of NPMSCs.6-gingerol(6-GIN)is an antioxidant and anti-inflammatory reagent that might protect NPMSCs from injury.AIM To investigate the effect of 6-GIN on NPMSCs under oxidative conditions and the potential mechanism.METHODS The cholecystokinin-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of 6-GIN.ROS levels were measured by 2´7´-dichlorofluorescin diacetate analysis.Matrix metalloproteinase(MMP)was detected by the tetraethylbenzimidazolylcarbocyanine iodide assay.TUNEL assay and Annexin V/PI double-staining were used to determine the apoptosis rate.Additionally,autophagy-related proteins(Beclin-1,LC-3,and p62),apoptosisassociated proteins(Bcl-2,Bax,and caspase-3),and PI3K/Akt signaling pathwayrelated proteins(PI3K and Akt)were evaluated by Western blot analysis.Autophagosomes were detected by transmission electron microscopy in NPMSCs.LC-3 was also detected by immunofluorescence.The mRNA expression of collagen II and aggrecan was evaluated by real-time polymerase chain reaction(RT-PCR),and the changes in collagen II and MMP-13 expression were verified through an immunofluorescence assay.RESULTS 6-GIN exhibited protective effects against hydrogen peroxide-induced injury in NPMSCs,decreased hydrogen peroxide-induced intracellular ROS levels,and inhibited cell apoptosis.6-GIN could increase Bcl-2 expression and decrease Bax and caspase-3 expression.The MMP,Annexin V-FITC/PI flow cytometry and TUNEL assay results further confirmed that 6-GIN treatment significantly inhibited NPMSC apoptosis induced by hydrogen peroxide.6-GIN treatment promoted extracellular matrix(ECM)expression by reducing the oxidative stress injury-induced increase in MMP-13 expression.6-GIN activated autophagy by increasing the expression of autophagy-related markers(Beclin-1 and LC-3)and decreasing the expression of p62.Autophagosomes were visualized by transmission electron microscopy.Pretreatment with 3-MA and BAF further confirmed that 6-GIN-mediated stimulation of autophagy did not reduce autophagosome turnover but increased autophagic flux.The PI3K/Akt pathway was also found to be activated by 6-GIN.6-GIN inhibited NPMSC apoptosis and ECM degeneration,in which autophagy and the PI3K/Akt pathway were involved.CONCLUSION 6-GIN efficiently decreases ROS levels,attenuates hydrogen peroxide-induced NPMSCs apoptosis,and protects the ECM from degeneration.6-GIN is a promising candidate for treating IDD.

Nucleus Transfer Efficiency of Ear Fibroblast Cells Isolated from Bama Miniature Pigs at Various Ages

Summary： Somatic cell nucleus transfer （SCNT） has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a pan- city of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer （NT）. The present study examined the NT efficiency of ear fibroblasts from fetal, new- born, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells ini- tially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were ＂S-shaped＂ in different age groups. The cell proliferation of postnatal ear fi- broblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts （P〈0.05 or P〈0.01）. Two-month- and 4-month-old ear fibroblasts had a sig- nificantly higher proportion of G1 stage cells （85% to 91%） than those at 6 and 12 months （66% to 74%, P〈0.01）. The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group （8.06%）. It was concluded that 〈4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.

Stromal cell-derived factor-1α promotes recruitment and differentiation of nucleus pulposus-derived stem cells

BACKGROUND Intervertebral disc(IVD) degeneration is a condition characterized by a reduction in the water and extracellular matrix content of the nucleus pulposus(NP) and is considered as one of the dominating contributing factors to low back pain. Recent evidence suggests that stromal cell-derived factor 1α(SDF-1α) and its receptor CX-C chemokine receptor type 4(CXCR4) direct the migration of stem cells associated with injury repair in different musculoskeletal tissues.AIM To investigate the effects of SDF-1α on recruitment and chondrogenic differentiation of nucleus pulposus-derived stem cells(NPSCs).METHODS We performed real-time RT-PCR and enzyme-linked immunosorbent assay to examine the expression of SDF-1α in nucleus pulposus cells after treatment with pro-inflammatory cytokines in vitro. An animal model of IVD degeneration was established using annular fibrosus puncture in rat coccygeal discs. Tissue samples were collected from normal control and degeneration groups.Differences in the expression of SDF-1α between the normal and degenerative IVDs were analyzed by immunohistochemistry. The migration capacity of NPSCs induced by SDF-1α was evaluated using wound healing and transwell migration assays. To determine the effect of SDF-1α on chondrogenic differentiation of NPSCs, we conducted cell micromass culture and examined the expression levels of Sox-9, aggrecan, and collagen II. Moreover, the roles of SDF-1/CXCR4 axis in the migration and chondrogenesis differentiation of NPSCs were analyzed by immunofluorescence, immunoblotting, and real-time RT-PCR.RESULTS SDF-1α was significantly upregulated in the native IVD cells cultured in vitro with pro-inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, mimicking the degenerative settings. Immunohistochemical staining showed that the level of SDF-1α was also significantly higher in the degenerative group than in the normal group. SDF-1α enhanced the migration capacity of NPSCs in a dose-dependent manner. In addition, SDF-1α induced chondrogenic differentiation of NPSCs, as evidenced by the increased expression of chondrogenic markers using histological and immunoblotting analyses. Realtime RT-PCR, immunoblotting, and immunofluorescence showed that SDF-1αnot only increased CXCR4 expression but also stimulated translocation of CXCR4 from the cytoplasm to membrane, accompanied by cytoskeletal rearrangement.Furthermore, blocking CXCR4 with AMD3100 effectively suppressed the SDF-1α-induced migration and differentiation capacities of NPSCs.CONCLUSION These findings demonstrate that SDF-1α has the potential to enhance recruitment and chondrogenic differentiation of NPSCs via SDF-1/CXCR4 chemotaxis signals that contribute to IVD regeneration.

Urolithin a alleviates oxidative stress-induced senescence in nucleus pulposus-derived mesenchymal stem cells through SIRT1/PGC-1α pathway

BACKGROUND In degenerative intervertebral disc(IVD),an unfavorable IVD environment leads to increased senescence of nucleus pulposus(NP)-derived mesenchymal stem cells(NPMSCs)and the inability to complete the differentiation from NPMSCs to NP cells,leading to further aggravation of IVD degeneration(IDD).Urolithin A(UA)has been proven to have obvious effects in delaying cell senescence and resisting oxidative stress.AIM To explore whether UA can alleviate NPMSCs senescence and to elucidate the underlying mechanism.METHODS In vitro,we harvested NPMSCs from rat tails,and divided NPMSCs into four groups:the control group,H2O2 group,H2O2+UA group,and H2O2+UA+SR-18292 group.Senescence-associatedβ-Galactosidase(SA-β-Gal)activity,cell cycle,cell proliferation ability,and the expression of senescence-related and silent information regulator of transcription 1/PPAR gamma coactivator-1α(SIRT1/PGC-1α)pathway-related proteins and mRNA were used to evaluate the protective effects of UA.In vivo,an animal model of IDD was constructed,and Xrays,magnetic resonance imaging,and histological analysis were used to assess whether UA could alleviate IDD in vivo.RESULTS We found that H2O2 can cause NPMSCs senescence changes,such as cell cycle arrest,reduced cell proliferation ability,increased SA-β-Gal activity,and increased expression of senescence-related proteins and mRNA.After UA pretreatment,the abovementioned senescence indicators were significantly alleviated.To further demonstrate the mechanism of UA,we evaluated the mitochondrial membrane potential and the SIRT1/PGC-1αpathway that regulates mitochondrial function.UA protected mitochondrial function and delayed NPMSCs senescence by activating the SIRT1/PGC-1αpathway.In vivo,we found that UA treatment alleviated an animal model of IDD by assessing the disc height index,Pfirrmann grade and the histological score.CONCLUSION In summary,UA could activate the SIRT1/PGC-1αsignaling pathway to protect mitochondrial function and alleviate cell senescence and IDD in vivo and vitro.

Normal and Degenerated Rabbit Nucleus Pulposus Cells in in vitro Cultures: A Biological Comparison

This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus （NP） cells in vitro in order to provide seed cells for intervertebral disc （IVD） tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models ofintervertebral disc degeneration （IDD）. Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix （ECM）-related genes （aggrecan and type II col- lagen） were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding （P〈0.05）. The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance （.4） value at passages 4-7 was obviously decreased as compared with that at passage 1 （P〈0.05）. Cell cycle analysis showed that the proportion of normal NP cells at G1 phase was 65.4%-3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase With the value being 77.6%-4.8%. The degen- erated NP cells were predominantly arrested at Gt phase and failed to enter S phase. The expression of type II collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.

How to enhance the ability of mesenchymal stem cells to alleviate intervertebral disc degeneration

Intervertebral disc(ID)degeneration(IDD)is one of the main causes of chronic low back pain,and degenerative lesions are usually caused by an imbalance between catabolic and anabolic processes in the ID.The environment in which the ID is located is harsh,with almost no vascular distribution within the disc,and the nutrient supply relies mainly on the diffusion of oxygen and nutrients from the blood vessels located under the endplate.The stability of its internal environment also plays an important role in preventing IDD.The main feature of disc degeneration is a decrease in the number of cells.Mesenchymal stem cells have been used in the treatment of disc lesions due to their ability to differentiate into nucleus pulposus cells in a nonspecific anti-inflammatory manner.The main purpose is to promote their regeneration.The current aim of stem cell therapy is to replace the aged and metamorphosed cells in the ID and to increase the content of the extracellular matrix.The treatment of disc degeneration with stem cells has achieved good efficacy,and the current challenge is how to improve this efficacy.Here,we reviewed current treatments for disc degeneration and summarize studies on stem cell vesicles,enhancement of therapeutic effects when stem cells are mixed with related substances,and improvements in the efficacy of stem cell therapy by adjuvants under adverse conditions.We reviewed the new approaches and ideas for stem cell treatment of disc degeneration in order to contribute to the development of new therapeutic approaches to meet current challenges.

The Relationship between the Morphology of Nucleus of Liver Cells and the Liver Functions and Prognosis of Portal Hypertension due to Hepatic Cirrhosis

The morphology of nucleus of liver cells from 30 patients with portal hypertension due to hepatic cirrhosis and 5 normal persons were measured using an image analyzer coupled with a computer. It was found that the diameters, perimeters, areas and form factor (FF) of the nucleus of liver cirrhosis porial hypertension patients were significantly increased as compared with those of the normal subjects (P<0. 05 or P<0. 01). There was a very significant difference in this parameters between the normal persons and patients with Child-Pugh A liver funetion or patients with Child-Pugh C liver function (P<0. 01 for both). Significant difference in these parameters existed between the normal persons or patients with Child-Pugh A liver function and patients with liver functian of Child-Pugh B (P<0. 05). No significant difference in the parameter of optic density (OD) were found between the normal persons and patients with impairment of liver function of varying degrees (Child-Pugh Classification) (P>0. 05). Our results suggest that the hepatocytes of patients with portal hypertension due to hepatic cirrhosis became juvenile and the morphology of the hepatocytes of patients with impairment of liver function of Child-Pugh C changed obviously. The enlargement and sparsity of nucleus of hepatocytes as revealed by pathological examination is a sign of severe impairment of liver function.

Ultrastructural Pathology of Nerve Cells in Mouse Brain Infected with Epidemic B Encephalitis Virus

An animal model of epidemic(Japanese)B encephalitis was estabilisged by injecting theJin Wei Yah 1 stain of B encephalitis virus into the peritoneal cavity of mice.The ultrastructuralchanges of the nerve cells in their brains were studied,special attention being paid to some types ofnerve cell in the cerebellar cortex.The infectet Purkinje cells and especially the granular cells showedspecial and inter,sting pathological features.These were compared with the changes found in the in-fected nerve cells in the cerebral cortex,diencephalon and mesencephalon.A radiating structure consisting of a microveside-microtubule aggregation body at the centerand endoplastic reticulum or virus replication multivesicular structures around it was often found in thein fected nerve cells.Its morphological features were described in detail,and its significance and the se-quenoe of events of its development discussed.In the late stage of infection,virus particles were found in the nuclei of part of the necroticcells.It is considered that they entered the nuclei from the cytoplasm during or after the death of theinfected cells.The observation smade in this study have comfimed in the granular cell of the cerebellum theidea of Chert et al.that the encephalitis B virus particle can he formed in the perinudear cistem ofthe infected nerve cell,and have brought forth further information in this respect.The way of releaseof the virus particles from the infected nerve cells observed in this study is fundamentally consistentwith that observed by Chen et al.but most of the virus particles left the nerve cell via the cell pro-

The prognostic molecular markers in hepatocellular carcinoma

The prognosis of hepatocellular carcinoma (HCC) still remains dismal, although many advances in its clinical study have been made. It is important for tumor control to identify the factors that predispose patients to death. With new discoveries in cancer biology, the pathological and biological prognostic factors of HCC have been studied quite extensively. Analyzing molecular markers (biomarkers) with prognostic significance is a complementary method. A large number of molecular factors have been shown to associate with the invasiveness of HCC, and have potential prognostic significance. One important aspect is the analysis of molecular markers for the cellular malignancy phenotype. These include alterations in DNA ploidy, cellular proliferation markers (PCNA, Ki-67, Mcm2, MIB1, MIA, and CSE1L/CAS protein), nuclear morphology, the p53 gene and its related molecule MD M2, other cell cycle regulators (cyclin A, cyclin D, cyclin E, cdc2, p27, p73), oncogenes and their receptors (such as ras, c-myc, c-fms, HGF, c-met, and erb-B receptor family members), apoptosis related factors (Fas and FasL), as well as telomerase activity. Another important aspect is the analysis of molecular markers involved in the process of cancer invasion and metastasis. Adhesion molecules (E-cadherin, catenins, serum intercellular adhesion molecule-1, CD44 variants), proteinases involved in the degradation of extracellular matrix (MMP-2, MMP-9, uPA, uPAR, PAI), as well as other molecules have been regarded as biomarkers for the malignant phenotype of HCC, and are related to prognosis and therapeutic outcomes. Tumor angiogenesis is critical to both the growth and metastasis of cancers including HCC, and has drawn much attention in recent years. Many angiogenesis-related markers, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF), thrombospondin (TSP), angiogenin, pleiotrophin, and endostatin (ES) levels, as well as intratumor microvessel density (MVD) have been evaluated and found to be of prognostic significance. Body fluid (particularly blood and urinary) testing for biomarkers is easily accessible and useful in clinical patients. The prognostic significance of circulating DNA in plasma or serum, and its genetic alterations in HCC are other important trends. More attention should be paid to these two areas in future. As the progress of the human genome project advances, so does a clearer understanding of tumor biology, and more and more new prognostic markers with high sensitivity and specificity will be found and used in clinical assays. However, the combination of some items, i.e., the pathological features and some biomarkers mentioned above, seems to be more practical for now.

Epigenetics and chromatin plasticity in embryonic stem cells

The study of embryonic stem cells is in the spotlight in many laboratories that study the structure and function of chromatin and epigenetic processes. The key properties of embryonic stem cells are their capacity for selfrenewal and their pluripotency. Pluripotent stem cells are able to differentiate into the cells of all three germ layers, and because of this property they represent a promising therapeutic tool in the treatment of diseases such as Parkinson's disease and diabetes, or in the healing of lesions after heart attack. As the basic nuclear unit, chromatin is responsible for the regulation of the functional status of cells, including pluripotency and differentiation. Therefore, in this review we discuss the functional changes in chromatin during differentiation and the correlation between epigenetics events and the differentiation potential of embryonic stem cells. In particular we focus on post-translational histone modification, DNA methylation and the heterochromatin protein HP1 and its unique function in mouse and human embryonic stem cells.

Specific Inhibitory Protein Dkk-1 Blocking Wnt/β-catenin Signaling Pathway Improve Protectives Effect on the Extracellular Matrix

The present study examined the role of Wnt/β-catenin signaling pathway in the degeneration of nucleus pulposus cells and the protective effect of DKK1 on nucleus pulposus cells. The model of nucleus pulposus cell degeneration was induced by intra-disc injection of TNF-α, and the expression of β-catenin protein was detected by Western blotting. The cultured rabbit nucleus pulposus cells were divided into 4 groups. In group A, the cells were cultured with normal medium and served as control group. In group B, the cells were cultured with TNF-α and acted as degeneration group. In group C, the cells were cultured with TNF-α and transfected with Adv-eGFP and was used as fluorescence control group. In group D, the cells were cultured with TNF-α and transfected with Adv-hDKK1-eGFP, serving as intervention group. The expression of typeⅡcollagen, proteoglycan, β-catenin, and MMP-13 in each group was detected by immunocytochemistry and RT-PCR. The result showed that TNF-α increased the expression of β-catenin and MMP-13, and significantly inhibited the synthesis of type Ⅱ collagen and proteoglycan, which resulted in the degeneration of nucleus pulposus cells. This effect could be obviously reversed by DKK1. We are led to concluded that TNF-α could activate the Wnt/β-catenin signaling pathway, and increase the expression of MMP-13, thereby resulting in disc degeneration. Specifically blocking Wnt/β-catenin signaling pathway by DKK-1 could protect the normal metabolism of intervertebral disc tissue. The Wnt pathway plays an important role in the progression of the intervertebral disc degeneration.

Olfactory ensheathing cells promote nerve regeneration and functional recovery after facial nerve defects

Olfactory ensheathing cells from the olfactory bulb and olfactory mucosa have been tbund to increase axonal sprouting and pathfinding and promote the recovery of vibrissae motor performance in facial nerve transection injured rats. However, it is not yet clear whether olfactory ensheathing cells promote the reparation of facial nerve defects in rats. In this study, a collagen sponge and silicone tube neural conduit was implanted into the 6-mm defect of the buccal branch of the facial nerve in adult rats. Olfactory ensheathing cells isolated from the olfactory bulb of newborn Sprague-Dawley rats were injected into the neural conduits connecting the ends of tile broken nerves, the morphology and function of the regenerated nerves were compared between the rats implanted with olfactory ensheathing cells with the rats injected with saline. Facial paralysis was assessed. Nerve electrography was used to measure facial nerve-induced action potentials. Visual inspection, anatomical microscopy and hematoxylin-eosin staining were used to assess the histomorphology around the trans planted neural conduit and the morphology of the regenerated nerve. Using fluorogold retrograde tracing, toluidine blue staining and lead uranyl acetate staining, we also measured the number of neurons in the anterior exterior lateral f：acial nerve motor nucleus, the number of myelinated nerve fibers, and nerve fiber diameter and myelin sheath thickness, respectively. After surgery, olfactory ensheathing cells de- creased facial paralysis and the latency of the facial nerve-induced action potentials. There were no differences in the general morphology of the regenerating nerves between the rats implanted with olfactory ensheathing cells and the rats injected with saline. Between-group results showed that olfactory ensheathing cell treatment increased the number of regenerated neurons, improved nerve fiber morphology, and increased the number of myelinated nerve fibers, nerve fiber diameter, and myelin sheath thickness. In conclusion, implantation of olfactory ensheathing cells can promote regeneration and functional recovery after facial nerve damage in rats.

Nuclear and mitochondrial DNA microsatellite instability in hepatocellular carcinoma in Chinese

AIM：To study the nuclear microsatellite instability (nMSI) at BAT26 and mitochondral microsalellite instability (mtMSI) in the occurrence and development of hepatocellular carcinoma and the relationship between nMSI and mtMSI.METHODS: nMSI was observed with PCR and mtMSI with PCR-SSCP in 52 cases of hepatocellular carcinoma.RESULTS:mtMSI was detected in 11 out of the 52 cases of hepatocellular carcinoma (21.2%). Among the 11 cases of hepatocellular carcinoma with mtMSI, 7 occured in one locus and 4 in 2 loci. The frequency of mtMSI in the 52 cases of hepatocellular carcinoma showed no correlation to sex, age,infection of hepatitis B, liver cirrhosis as well as positive AFP of the patients (P>0.05). In addition, nMSI was detected in 3 out of 52 cases of hepatocellular carcinoma (5.8%) and there was no correlation of the incidence of mtMSI to that of nMSI (P>0.05).CONCLUSION:mtMSI may be involved in the coccurrence and development of hepatocellular carcinoma and it is independent of nMSI.

Morphometric study on Leydig cells in capsulotomized testis of rats

AIM: To further clarify the changes occurred in the testicular capsulotomized rats. METHODS: In testicular capsulotomized and sham-operated rats, the cross sectional area, the nucleus diameter and the number of Leydig cells were morphologically analyzed by the Vidas Image Processing System connected to a microscope. RESULTS: In the capsulotomized animals, the cross sectional area of Leydig cells was gradually increased from 30 days onwards. There was no obvious change in the nucleus diameter of Leydig cells. However, The Leydig cell number was significantly increased from day 30 onwards. CONCLUSION: In rats, testicular capsulotomy may induce hyperplasia/hypertrophy of Leydig cells in the testis.