[Effects of electroacupuncture on miR-34a-5p/SIRT1 signaling in the trigeminal ganglion of rats with migraine].

OBJECTIVE: To investigate the effect of electroacupuncture(EA) on miR-34a-5p, silent mating type information regulation 2 homolog-1 (SIRT1) and nuclear factor-κB subunit p65 (NF-κB p65) in the trigeminal ganglion of rats with migraine, so as to explore the mechanisms of EA underlying prevention of migraine. METHODS: Male SD rats were randomly divi-ded into normal, sham operation, model, EA, and EA plus EX527(a SIRT1 inhibitor) groups, with 10 rats in each group. The rat model of migraine was established by electrical stimulation of the trigeminal ganglion. Before modeling, EA was applied at "Waiguan"(TE5) and "Fengchi"(GB20) for 20 min each time, once a day for 5 consecutive days, and intraperitoneal injection of EX527 (5 mg/kg) every day simultaneously. Serum prostaglandin E2 (PGE2) and calcitonin gene-related peptide (CGRP) concentrations were measured by enzyme-linked immunosorbent assay. The levels of miR-34a-5p, SIRT1 and interleukin-1ß (IL-1ß) mRNA，and protein expression of SIRT1, IL-1ß, NF-κB p65, NF-κB Ac-p65 and cyclooxygenase-2 （COX2） in trigeminal ganglia were detected by real-time quantitative PCR and Western blot, separately. RESULTS: The serum concentrations of PGE2 and CGRP， the expression of miR-34a-5p, IL-1ß mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expression in the trigeminal ganglion were remarkably increased (P<0.05), while the SIRT1 mRNA and protein decreased (P<0.05) in the model group in contrast to the normal group. Following EA intervention， the serum PGE2 and CGRP concentrations, miR-34a-5p expression, IL-1ß mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expression were significantly down-regulated (P<0.05), and SIRT1 mRNA and protein significantly up-regulated (P<0.05). Compared with the EA group, the serum concentrations of PGE2 and CGRP, IL-1ß mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expressions increased (P<0.05), and SIRT1 protein decreased (P<0.05) in the EA plus EX527 group. CONCLUSION: In migraine rats, EA can inhibit miR-34a-5p expression in the trigeminal ganglion, increase SIRT1 expression, down-regulate IL-1ß/COX2 inflammation signals, reduce PGE2 synthesis, and thus redue CGRP released from the peripheral terminals, which may be one of the mechanisms of EA in preventing migraine.

Molecular epidemiological survey of hemoglobinopathies in the Wuxi region of Jiangsu Province, eastern China.

In order to determine the prevalence and molecular characterization of hemoglobinopathies in the Wuxi region of Jiangsu Province in the People's Republic of China (PRC), a total of 10,297 healthy people selected from a regional hospital were screened. Hemoglobin (Hb) electrophoresis, complete blood cell (CBC) count, polymerase chain reaction (PCR), DNA sequencing, reverse dot-blot and multiplex ligation-dependent probe amplification (MLPA) were used to detect Hb variants, thalassemias and hereditary persistence of fetal Hb (HPFH). Two thousand and twenty-one adult subjects were screened for thalassemia, five cases were identified as α-thalassemia (α-thal) carriers including three cases of the -α(3.7) (rightward) deletion, one case of the - -(SEA) deletion and one case of ß-thal [IVS-II-654 (C>T), (HBB: c.316-197C>T)]. The incidence of Hb variants, thalassemia and HPFH/δß-thal were 0.136% (14/10,297), 0.25% (5/2021) and 0.0001% (1/10,297), respectively. Eight genotypes of Hb variants were found, including Hb E [ß26(B8)Glu→Lys, GAG>AAG; HBB: c.79G>A], Hb J-Bangkok [ß56(D7)Gly→Asp (GGC>GAC); HBB; c.170G>A], Hb G-Coushatta [ß22(4)Glu→Ala (GAA>GCA); HBB: c.68A>C], Hb Queens [α34(B15)Leu→Arg (CTG>CGG) (α2 or α1); HBA2: c.104T>G (or HBA1)], Hb I [α16(A14)Lys→Glu, AAG>GAG (α1); HBA1: c.49A>G], Hb Beijing [α16(A14)Lys→Asn (AAG>AAC or AAT) (α2 or α1); HBA2: c.51G>C (or HBA1) or 51G>T (or HBA1)], Hb Ube-2 [α68(E17)Asn→Asp (AAC>GAC) (α2 or α1); HBA2: c.205A>G (or HBA1)] and Hb G-Taipei [ß22(B4)Glu→Gly (GAA>GGA); HBB: c.68A>G]. A Sicilian δß(0)-thal, identified for the first time in Asia, was also found in this survey.

Prevalence and genotype distribution of human papillomavirus infections in women attending hospitals in Chaozhou of Guangdong province.

BACKGROUND: Human papillomavirus (HPV) infection is the main cause of cervical cancer. Limited epidemiologic data of HPV prevalence are available for women attending hospitals in southern China. This study aimed to evaluate the profiles of HPV infection and cytology status in gynecological outpatients in Chaozhou City. METHODS: A total of 2833 eligible women were enrolled. The HPV GenoArray test was used for HPV detection and genotyping. Nearly one half of the HPV positive women received liquid-based cytology test. Logistic regression analysis was performed to assess the predictable effects of age and genotype for categories of abnormal cytology. RESULTS: The prevalence of overall, high-risk, and low-risk HPV infection were 24.5%, 19.5% and 8.4%, respectively. A U-shaped age-specific prevalence curve was observed in overall HPV and high- risk HPV, but not in low-risk HPV, which declined with age increasing. The 6 most common high-risk HPV type in descending order, were types 52, 16, 58, 18, 68, and 33. Age and HPV genotype were both important determinants of abnormal cytology incidence, the older women (>45 years) and those infected with HPV type 16 and/or 18 having the highest risk for abnormal cytology. CONCLUSION: Our findings support the hypothesis that second-generation HPV prophylactic vaccines including HPV-52 and -58 may offer higher protection for women residing in Chaozhou and neighboring cities in Guangdong.

Epidemiologic characterization of human papillomavirus infection in rural Chaozhou, eastern Guangdong Province of China.

BACKGROUND: Human papilloma virus (HPV) infection was the main cause of cervical cancer. There were only a few reports and detailed data about epidemiological research of HPV infection in rural population of China. MATERIALS AND METHODS: The cervical cells of rural Chaozhou women were collected, and multiplex real time PCR was firstly performed to detect high-risk HPV (HR-HPV) infection, which could detect 13 types of HR-HPV (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). Then, HPV-positive samples were typed by HPV GenoArray test. RESULTS: HR-HPV DNA was detected by multiplex real time-PCR in 3830 of 48559 cases (7.89%). There was a peak incidence in age of 55-60 years group, and a lower incidence in who lived in plain group compared with suburban, mountain and seashore group. 3380 cases of HPV positive sample were genotyped, 11.01% (372/3380) cases could not be classified, among the typed 3008 cases, 101 cases were identified without HR-HPV type infection, 2907 cases were infected with one HR-HPV type at least, the 6 most common HR-HPV types in descending order of infection, were type 52 (33.4%, 16 (20.95%), 58 (15.93%), 33 (9.94%), 68 (9.22%) and 18 (8.36%). The combined prevalence of HPV types 16 and 18 accounted for 28.52% of total infection. However, type 52 plus 58 presented 48.23% of total infection. 2209/2907 cases were infected with a single HPV type and 698/2907 cases were infected with multiple types, and multiple infection constituent ratio increased with age, with a peak incidence in age 55-60 years group. CONCLUSIONS: Our findings showed low prevalence of HPV vaccine types (16 and 18) and relatively high prevalence of HPV-52 and -58, support the hypothesis that the second-generation HPV vaccines including HPV-52 and -58 may offer higher protection for women in rural Guangdong Province.

The biological characteristics of glioma stem cells in human glioma cell line SHG44.

Gliomas are the most common tumors of the central nervous system (CNS) and a frequent cause of death. The treatment of malignant gliomas is often palliative due to their high recurrence rate. A growing body of evidence suggests that glioma may arise from cancer stem cells (CSC) correlated with neural stem cells (NSC), with the capacity for self-renewal and multipotency. CSCs have been isolated from human gliomas and numerous other solid tumors. It is assumed that a number of established malignant cell lines also contain a rare subpopulation of stem cells. This study was designed to investigate the proportion of CSCs in the human glioma cell line SHG44 and to study the limitations of CD133 immunophenotyping in glioma stem cell research. SHG44 cells were cultured in both serum-containing and serum-free medium. The similar shape in growth curves (in the exponential growth phase) revealed that most cells participated in the population amplification. Time gradient BrdU labeling and monoclonal assay revealed that almost every single cell participated in the division growth (98.82%) and possessed the ability to form clones (96.19%). No significant difference was found in the proportions of CD133+ cells in the serum-containing and serum-free groups (38.25%/37.92%). In addition, no significant difference was noted in the proportions of CD133+ cells among monoclones selected randomly in the serum-containing group. These results suggested that CD133- cells generate CD133+ cells and have the ability to form clones. Thus, we concluded that most SHG44 cells were CSCs and serum-free medium was not necessary for the generation of CSCs. In this line, CD133- cells also possessed clonogenic, self-renewal capacities and were also CSCs.

Several types of soft tissue sarcomas originate from the malignant transformation of adipose tissue-derived stem cells.

The cellular origin of soft tissue sarcomas (STSs) is not fully understood. The cancer stem cell hypothesis presumes that tumors originate from the malignant transformation of stem cells. As a type of multipotent stem cell, adipose tissue-derived stromal/stem cells (ADSCs), which possess an unexpected degree of plasticity and often reside in other tissues, may represent a potential source of soft tissue sarcoma. To ascertain whether ADSCs are responsible for the formation of STSs, ADSCs from mice were cultured and treated with 3-methycholanthrene to derive transformed cells. These transformed ADSCs were then injected subcutaneously into immunodeficient mice to test their tumorigenic potential. We found that they generated several types of STSs, including synovial sarcoma, malignant fibrous histiocytoma and fibrosarcoma. This is the first study to report that ADSCs may be the potential initiating cells for synovial sarcoma. Our findings indicate that STSs might originate from malignantly transformed ADSCs.

Inhibition of the replication of hepatitis B virus in vitro by oxymatrine.

This study investigated the inhibitory capacity of oxymatrine on in vitro hepatitis B virus (HBV) replication. HepG2.2.15 cells were treated with oxymatrine 50, 100, 200, 400, 800 or 1000 mg/l, or with human interferon-alpha2b (IFN-alpha2b 1000 U/l) as a positive control. Levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and HBV-DNA in cell supernatants were determined by enzyme-linked immunosorbent assay and fluorescent quantitative-polymerase chain reaction, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometric analysis and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labelling were used to evaluate the cytotoxicity of oxymatrine. The inhibitory effects of oxymatrine gradually increased as the concentration increased from 200 to 1000 mg/l for HBsAg and HBeAg, and from 200 to 400 mg/l for HBV-DNA. There was no inhibitory effect of oxymatrine at concentrations < 200 mg/l. No significant difference was seen between human IFN-alpha2b (positive control) and oxymatrine >or= 200 mg/l. It is concluded that oxymatrine can inhibit in vitro HBV replication and antigen expression at concentrations >or= 200 mg/l.

[Human amnion cells express the phenotypes of neural cells and adipocytes in vitro].

OBJECTIVE: To search for culture conditions in which the cells from human amnion could diferentiate into neural cells and hence to explore a new cell source for neural transplantaion. METHODS: Amnion cells from human were cultured with tissue piece method, passaged by trypsin digestion and identified with immunocytochemistry. RESULTS: Amnion cells migrated from explants and primary culture was established; they could multiply and expand steadily in a short time, and they could be passaged by trypsin digestion. When cultured in serum-free neural stem cell media, these cells could form the same spheroid shape as the neurospheres of neural stem cells. They could express alpha smooth muscle actin and differentiate into smooth muscle cells spontaneously, and could express nestin and vimentin, the markers for neural progenitors. Moreover, they could he stained by anti-beta III-tubulin, anti-neurofilament 200 and anti-NSE; the majorities could he stained by antityrosine hydroxylase, the marker for dopaminergic neurons. Lower than 0.1% of the total cells were stained by GFAP, indicating the existence of astrocytes. The amnion cells could also differentiate into adipocytes under specific induction. CONCLUSION: Amnion cells could differentiate into adipocytes and smooth muscle cells; they could express the protein for neural cells; thus may represent an alternative stem cell source for CNS cell transplantation.

[Long-term culture and differentiation of human glioma stem cells ].

OBJECTIVE: To explore the culture and subculture conditions for glioma stem cells(GSCs) and to investigate the differentiation potential of GSCs. METHODS: The cells from human glioma were mechanically dissociated. Cells were cultured in N2 or B27 medium with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), and they were identified by immunocytochemistry. RESULTS: Glioma stem cells from human glioma have been successfully cultured. They formed typical neurospheres in suspension, and they could be cultured and passaged steadily in vitro. The majorities of the cells expressed vimentin and nestin, which were the markers for neural stem cells. They could differentiate into neurons and astrocytes, and express glial fibrillary acidic protein and beta III-tubulin respectively. CONCLUSION: Human glioma stem cells could be cultured from gliomas in vitro, and they could differentiate into neurons and astrocytes, thus providing a basis for further studies.

[Intraventricular transplantation of human neural stem cells into embryonic rats].

OBJECTIVE: To explore the migration and distribution of human neural stem cells (NSCs) after they were transplanted into lateral cerebroventricles of embryonic rats. METHODS: This study was conducted with the proxy consent and the permission from the Hospitals Ethical Review Committee. The cells from the embryonic human cortices were mechanically dissociated. N2 medium was used to culture and expand the cells. And the cells were identified by immunocytochemistry; then after being labelled with BrdU or adenovirus carrying LacZ gene, they were implanted into the lateral cerebroventricles of embryonic rats. The rats were sacrificed after they were born and the brain sections were examined by immunocytochemistry at different time points. RESULTS: NSCs from embryonic humans were successfully cultured; they formed typical neurospheres in suspension, and the majorities of the cells expressed nestin, which was the marker for neural progenitor cells, and the cells could differentiate into neurons, astrocytes and oligodendrocytes. NSCs could steadily expressed exogenous gene in vivo and in vitro; after transplantation into embryonic rat brains, the fetal human NSCs incorporated individually into all major compartments of the brains, generating widespread CNS chimerism and having a wide distribution in the host fore-, mid-, and hindbrain. CONCLUSION: NSCs could express foreign gene steadily and were the ideal cell sources for cell and gene therapy. These cells could incorporate into developing brains and generate widespread chimerism. These chimeras provide a unique model for studying human neural cell migration and differentiation in a functional nervous system.

Differentiation of adult human bone marrow mesenchymal stem cells into Schwann-like cells in vitro.

OBJECTIVE: To investigate the differentiative capability of adult human bone marrow mesenchymal stem cells (BMSCs) into Schwann-like cells. METHODS: Bone marrows were aspirated from healthy donors and mononuclear cells were separated by Percoll lymphocytes separation liquid (1.073 g/ml) with centrifugation, cells were cultured in DMEM/F12 (1:1) medium containing 10% fetal bovine serum (FBS), 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF). Cells of passage 1 were identified with immunocytochemistry. RESULTS: Mononuclear cells separated by Percoll's were passaged 10 times by trypsin/ethylenediaminetetraacetic acid (EDTA) digestion in 40 days, and BMSCs increased about 6x10(7) times in this short period. Immunohistochemistry identified that BMSCs were CD34- and CD31-, but they expressed neuron specific enolase; 0.01%-0.02% of total cells expressed nestin, the marker for neural progenitor cells; 40%-50% cells stained heavily by neurofilament 200; and no glial fibrillary acidic protein (GFAP) positive cells were identified; S100 expression was detected among 0.1%-0.2% cells. CONCLUSIONS: Bone marrow contains the stem cells with the ability of differentiating into Schwann-like cells, which may represent an alternative stem cell sources for neural transplantation.

[Stromal cells from human Wharton's jelly differentiate into neural cells].

OBJECTIVE: To investigate the neural differentiating capability of the umbilical cord tissue-derived stromal cells (UCSCs) in the attempt to find a new cell source for neural transplantation. METHODS: UCSCs from umbilical cord of human were cultured with tissue piece method, passaged by trypsin digestion. And Salvia miltiorrhiza was used to induce the cells to differentiate. Cells were identified with immunocytochemistry. RESULTS: Stromal cells that migrated from explants and primary culture were obtained. These cells could differentiate into smooth muscle cells spontaneously and expressed smooth muscle actin; they could be passaged by trypsin digestion. Salvia miltiorrhiza could induce these cells to differentiate into the neuron-like cells, which displayed typical neuron morphology, expressed nestin, beta III-tubulin and NSE at the early stage of differentiation, and were stained by anti-neurofilament 200 at the late stage of differentiation. With optimal conditions, about 90% of UCSCs expressed neuronal phenotypes, lower than 1% of the differentiated cells expressed GFAP, and no myelin basic protein expression was detected in the differentiated cells, indicating the absence of oligodendrocyte differentiation from stromal cells. CONCLUSION: The data supported the hypothesis that umbilical cord contains the stem cells with the ability of differentiating into neurons, which may provide an alternative stem cell source for CNS cell transplantation.

[Adipose tissue-derived stromal cells as vector for gene therapy in central nervous system].

OBJECTIVE: To investigate exogenous gene expressing ability of adipose tissue-derived stromal cell (ADSCs) and cell distribution after they were transplanted into brains, and to get the genetically modified cells for autografting. METHODS: ADSCs were transfected by Ad5beta gal adenovirus containing a report gene, LacZ gene, then they were transplanted into the adult brain of rats, or ADSCs labeled by Hoechst33258 were transplanted into the adult brain of rats to investigate the migration and distribution of cells. RESULTS: ADSCs showed a good expression of LacZ with X-gal staining after transfecting and transplantation into adult brains, and they could incorporate into the host brain tissues and no disruption was observed. These cells showed good compatibility with the host brains. CONCLUSION: The results indicate that ADSCs could incorporate into host brains and express exogenous gene steadily when they were transplanted into adult brain tissues, no overproliferation and gliosis were identified, and ADSCs may be used as a therapeutic gene delivery vehicle in treating CNS disorders in humans.

Culture of skin-derived precursors and their differentiation into neurons.

OBJECTIVE: To investigate the culture method of skin-derived precursors (SKPs) and to explore a new cell source for cell transplantation of central nervous system. METHODS: Cells from skins of juvenile and adult mice were isolated and cultured in serum-free medium. A mechanical method was chosen to passage these cells and they were identified by the immunocytochemistry assay. RESULTS: SKPs could be isolated from adult and neonatal skins. They could be maintained in vitro for long periods with stable proliferation, and expanded as undifferentiated cells in culture for more than 12 passages. About 50% of SKPs expressed nestin and majority of these cells expressed fibronectin when they were plated on polyornithine and laminin coated plates. About 5% cells showed neuronal differentiation and expressed neurofilament-M (NF-M) and NSE when SKPs were plated in serum-containing medium, and these cells could also differentiate into adipocytes and fibroblast-like cells. CONCLUSIONS: The data support the hypothesis that adult skin contains stem cells capable of differentiating into neurons, adipocytes, and fibroblast-like cells. They may represent an alternative autologous stem cell source for CNS cell transplantation.

The in vitro myelin formation in neurospheres of human neural stem cells.

OBJECTIVE: To explore the culture conditions of human neural stem cells and to investigate the ultrastructure of neurospheres. METHODS: The cells from the embryonic human cortices were mechanically dissociated. N2 medium was adapted to culture and expand the cells. The cells were identified by immunocytochemistry and EM was applied to examine the ultrastructure of neurospheres. RESULTS: The neural stem cells from human embryonic brains were successfully cultured and formed typical neurospheres in suspension, and most of the cells expressed vimentin, which was a marker for neural progenitor cells, and the cells could differentiate into neurons, astrocytes and oligodendrocytes. In vitro myelin formation in neurospheres were observed at an early stage of culture. CONCLUSIONS: Human neural stem cells can be cultured from embryonic brains, can form the typical neurospheres in suspension in vitro and have the ability of myelinating, and may be potential source for transplantation in treating myelin disorders.