Study on the electrophysiological characteristics of ion channels of stem cell derived cardiomyocytes of mouse

AIM: To study the electrophysiological characteristics of ion channels of stem cell derived cardiomyocytes(SCDC) of mouse. METHODS: Embryonic stem cells of D₃ line(ES-D₃) were cultured on the MEF feeder layer with BRL conditioned medium, and fetal mouse heart cells(FMHC)were cultured in vitro. Then ES-D₃ cells were induced to differentiate into many kinds of cells. SCDC were harvested on day 12 after differentiation initiating and identified by electro-microscope and immunocytochemistry. SCDC and FMHC were prepared for the patch-clamp research. Sodium and calcium currents together were elicited and compared between SCDC and FMHC. RESULTS: The current characteristics of sodium and calcium channels of SCDC were very similar to FMHC. CONCLUSION: The functional expression of ion channels occurred during ES-D₃ cells differentiation and the electrophysiological characteristics of sodium and calcium channels of SCDC are very similar to FMHC.     

Amplification and sequence analysis of anti-D variable region gene with leader peptide sequence

AIM:To amplify from leader peptide region and obtain human monoclonal anti-D variable region gene with high specificity and affinity, and analyze the nucleotide and deduced amino acid sequences.METHODS:The total RNA was extracted from an Epstein-Barr-virus-transformed cell line secreting monoclonal anti- (rhesus D) antibody. The leader region primers containing a ribosome recognition site were designed. By using PCR method, the cDNA of human anti-(rhesus D) antibody (IgM κ) variable region gene was amplified. Cloning and subsequent sequence analysis of the variable region gene was performed. The deduced amino acid sequence was also compared and analyzed with previously published sequences.RESULTS:A band of approximate 440 and 410 base pairs were amplified using heavy chain primers and light chain primers, respectively. Sequence analysis indicated that the deduced amino acid sequence was in agreement with the characterization of the amino acid present in the human Ig variable region. CONCLUSION:The cloning and sequencing of a human anti- (Rhesus D) antibody variable region cDNA will make benefits for production of recombinant anti-(Rhesus D) antibody and prevention of Rh haemolytic disease in newborns.     

Expression of MIP-1α and RANTES gene and protein in the bronchus of murine asthma

AIM: To study the role of the gene and protein expression of MIP-1α and RANTES in the bronchus of murine asthma. METHODS: 20 male BALB/C mice were randomly divided into two groups: the control group (A₀ group) and asthma group (B₀ group). In the experiment, the mice model of asthma was established by the ovalbumin (OVA) challenge methods. The protein expression of MIP-1α and RANTES were detected by immunohistochemistry methods. The gene expressions of MIP-1α and RANTES were detected by in situ hybridization methods. RESULTS: Immunohistochemistry showed that the expressions of MIP-1α protein and RANTES protein around the bronchus of group B₀ were significantly higher than those of group A₀ (P<0.01), the epithelial cells were the chief expression cells; (2) In situ hybridization showed that the expressions of MIP-1α gene and RANTES gene around the bronchus of group B₀ were significantly higher compared to those of group A₀ (P<0.01), the epithelial cells were the chief expression cells. CONCLUSION: MIP-1α and RANTES are high expression in the bronchus epithelial cells in experimental murine asthma.     

Development of embryonic stem cells into medulloepithelioma in the eyes of nude mice

AIM: To investigate the intraocular growth characteristics of mice embryonic stem (ES) cells in nude mice.METHODS: Murine embryonic stem cells (D3 cell lines) were cultured and maintained in an undifferentiated state in vitro, then transplanted into the eyes of nude mice. In 6-45 d, the nude mice were executed for Morphological and immunohistochemical examinations.RESULTS: ES cells were developed into masses which enlarged gradually in the anterior chamber and vitreous cavity. Morphological examination showed different component: cysts, sheets and cords of medullary epithelium and rosettes in the eyes of the nude mice. Most of cells were highly stained by NSE, and some cells were moderately stained by GFAP.CONCLUSION: The embryonic stem cells(D3 cell lines) could differentiated into medulloepithelioma-like tissue in the anterior chamber and vitreous cavity of the Balb/c nude mice.     

miR-485-5p inhibits viability and migration and invasion abilities of hepatocellular carcinoma Hep3B cells by targeting SOX5

AIM: To investigate the effects of microRNA-485-5p (miR-485-5p) on the viability, migration and invasion abilities of hepatocellular carcinoma cells and the underlying mechanism. METHODS: The expression levels of sex determining region Y-box 5 (SOX5) mRNA and miR-485-5p in the hepatocellular carcinoma Hep3B cells were detected by RT-qPCR with normal hepatocyte THLE-3 as control. Western blot was used to measure the expression levels of SOX5, proliferating cell nuclear antigen (PCNA), Ki67, cyclin D1 and matrix metalloproteinase-2 (MMP-2). The viability of Hep3B cells was measured by MTT assay. The migration and invasion abilities of the Hep3B cells were detected by Transwell assay. Dual-luciferase reporter assay system was applied to verify the relationship between miR-485-5p and SOX5. RESULTS: Compared with the control cells, the expression level of miR-485-5p was decreased in hepatocellular carcinoma cells Hep3B, Huh7 and HCCLM3 (P<0.05), while the expression of SOX5 at mRNA and protein levels were significantly increased (P<0.05). Over-expression of miR-485-5p inhibited the viability, migration and invasion of Hep3B cells. miR-485-5p targeted the 3′-UTR of SOX5 and negatively regulated the expression of SOX5. Knocking-down of SOX5 expression inhibited the viability, migration and invasion of Hep3B cells. Over-expression of SOX5 partially reversed the inhibitory effect of miR-485-5p over-expression on the viability, migration and invasion of Hep3B cells. CONCLUSION: miR-485-5p inhibits the viability, migration and invasion of Hep3B cells by targeting SOX5 gene. miR-485-5p is a potential molecular target for hepatocellular carcinoma.     

IGF-1R,p-ERK is involved in LDLs-induced mouse peritoneal macrophage survival

AIM: To study the effects of low density lipoproteins (LDLs) on insulin-like growth factor-1 receptor (IGF-1R), phosphorylated extracellular signal-regulated kinase (p-ERK), Bcl-2 and Bax protein expression in mouse peritoneal macrophages (MPMs). METHODS: Using the methods of immunocytochemistry and Western blotting, the expression of IGF-1R, p-ERK, Bcl-2 and Bax protein in MPMs treated with LDLs, anti-IGF-1R antibody (α-IR-3) or ERK inhibitor (PD98059) were detected. RESULTS: oxLDL significantly increased the IGF-1R protein expression in a dose and time-dependent manner. P-ERK protein expression induced by oxLDL peaked at 5 min. Moreover, oxLDL could induced ERK translocation from cytoplasm to nuclear. When given α-IR-3, p-ERK protein expression was significantly inhibited, and ERK translocation disappeared. oxLDL increased Bcl-2 protein expression and reduced Bax expression in a dose and time-dependent manner. When given PD98059, Bcl-2 and Bax protein expression induced by oxLDL altered significantly. Native LDL had no significant effect on the expression of these four proteins. CONCLUSIONS: oxLDL may promote cultured MPMs survival at least by enhancing IGF-1R expression and ERK phosphorylation, and there may be many pathways involved in MPMs survival induced by oxLDL, Whereas native LDL had no effects on culture MPMs.     

应用性信息素缓释剂迷向防治桃树梨小食心虫研究

为明确性信息素缓释剂防治梨小食心虫的持效期及合理使用密度,我们于2008、2009年在河南省郑州市对每根应用0.24 g梨小食心虫性信息素丝迷向防治桃园梨小食心虫进行了试验。结果表明,在梨小食心虫越冬代活动前以350、500、650根·hm-2处理,处理后5个月内均保持了较好的防治效果;350根·hm-2处理在8月份以后防效迅速降低,密度较高处理则保持相对较好防效。结合防治成本,建议中早熟品种可使用350根·hm-2密度进行处理,晚熟品种使用500根·hm-2以上密度处理。     

Effects of sex on chronic pancreatitis induced by L-arginine in Kunming mice

AIM: To explore the effect of sex on the formation of chronic pancreatitis (CP) by comparing the differences in L-arginine-induced CP model between male and female mice.METHODS: Male (n=42) and female (n=42) Kunming mice were randomly divided into 4 groups:female control group, female CP group, male control group and male CP group (n=18 in each control group, n=6 at each time point; n=24 in each CP group, n=8 at each time point). The mice in CP groups were intraperitoneally injected with 20% L-arginine (3 g/kg, twice/d, 1 d/week). After modeling for 2 weeks, 4 weeks and 6 weeks, the mice were anesthetized and sacrificed. The morphological changes and the degree of fibrosis in the pancreas were observed by HE and Masson staining. The positive expression rate of F4/80 in the pancreatic tissues was observed by the method of immunohistochemistry. The mRNA expression of interleukin-6 (IL-6), α-smooth muscle actin (α-SMA) and fibronectin (FN) in the pancreas were detected by real-time PCR. The protein of pancreas was extracted to detect the expression of α-SMA and FN by Western blot.RESULTS: At 2 weeks, 4 weeks and 6 weeks after intraperitoneal injection of L-arginine, the pancreatic tissues were obviously injured and exhibited different degrees of fibrosis in female and male CP groups. At the same time, there were significant differences in the degree of pancreatic injury between male and female mice, and the degree of pancreatic lesion in the male mice was significantly more severe. The positive rate of F4/80 in the pancreas of male CP mice was significantly higher than that in female CP group at the same time point after modeling. At every time point, the mRNA expression of IL-6 in the pancreas was increased in both female and male CP groups, but that in male CP group was higher (P<0.05). The fibrosis indexes, α-SMA and FN, were both highly expressed at mRNA and protein levels after modeling, but compared with the female group, the time of positive expression in male mice was earlier and the expression level was higher (P<0.05).CONCLUSION: The CP model is successfully established by intraperitoneal injection of 20% L-arginine, and a difference in the degree of pathologic alteration in pancreas between male and female mice exists. CP is more effectively induced by L-arginine in male mice, and the degree of fibrosis is more pronounced. The reason may be related to the sensitivity of male mice to L-arginine, causing more serious inflammatory response. Therefore, male mice are more suitable for establishing CP animal model.     

十字花科蔬菜芜菁花叶病毒的RT-PCR快速检测

利用RT PCR技术 ,建立了一种简便、快速、准确地检测十字花科蔬菜芜菁花叶病毒的方法。应用该方法 ,不需复杂的提取、纯化病毒RNA过程 ,直接对田间病样简单处理后进行RT PCR检测。通过获得包含该病毒CP基因的cDNA片段 ,对一些十字花科蔬菜田间病样进行了芜菁花叶病毒的快速检测     

Effects of deoxynivalenol on apoptosis and proliferation of mouse thymocytes in vivo

AIM:To explore the effect of deoxynivalenol on apoptosis and proliferation of mouse thymocytes in vivo.METHODS:Effect of deoxynivalenol at different concentrations on apoptosis and proliferation of mouse thymocytes in vivo were studied with animal experiment, electron microscopic observation, DNA agarose gel electrophoresis and flow cytometric analyses. RESULTS:FCM analysis showed that the apoptosis rates of the thymocytes in DON groups (0.5 mg/kg, 1 mg/kg, 2 mg/kg, 4 mg/kg and 8 mg/kg) were significantly higher than that in control (P＜0.01). In the concentration ranging from 0.5 mg/kg to 8 mg/kg, a significant dose-dependant response correlation could be found between apoptosis rate and DON concentration. The result of DNA agarose gel electrophoresis showed that characteristic DNA ladder was found in groups treated DON at relatively higher concentrations (8 mg/kg and 4 mg/kg). Ultrastructurally, chromatin condensation and nuclear budding phenomenon could be found in the cells treated with DON. In addition to the effects on apoptosis, 4 mg/kg and 8 mg/kg DON treatment could significantly decrease the PIs of the treated thymocytes in vivo as compared with control.CONCLUSION:DON can induce and enhance murine thymocytes apoptosis in a dose-dependant manner and inhibit the proliferation activities of the treated cells.     

2012年园艺植物染色体倍性操作与遗传改良学术研讨会预备通知

自2008年11月在中国农业科学院郑州果树研究所成功召开园艺植物染色体倍性操作与遗传改良研讨会以来,我国科研人员在该领域的研究取得了可喜的成绩。为进一步总结近几年来在该领域的研究进展,促进园艺植物倍性     

小西葫芦黄花叶病毒外壳蛋白基因导入西瓜的遗传转化

研究了西瓜品种ZKM的再生体系和农杆菌介导小西葫芦黄花叶病毒(Zucchini yellowmosaic virus,ZYMV)的外壳蛋白(cp)基因导入西瓜的遗传转化体系。结果表明,质量分数0.6%琼脂是适宜种子萌发的基本培养基,切取5d龄的西瓜无菌苗子叶为外植体,接种在MS+BA2.0mg/L诱导培养基上,诱导不定芽效率最高。选用75mg/L卡那霉素筛选西瓜抗性芽较为合适,外植体经预培养2～3d,菌液浓度以OD600nm值为0.4,共侵染10min有利于提高西瓜转化的效率。经PCR检测,ZYMVcp基因已经导入西瓜植株,转化频率为0.15%。     

A method of inducing mouse embryonic stem cells cultured in vitro to differentiate into cardiomyocytes

AIM:To evaluate the method of inducing mouse embryonic stem (ES) cells in vitro to differentiate into cardiomyocytes without using any chemical reagents.METHODS:BRL conditioned medium was used to promote the growth of ES cells and maintain them in an undifferentiated state before the experiment of differentiation. Then a three-step method including ES cell culture in hanging drops and in suspending was used to induce the differentiation of ES cells. RESULTS:Rhythmically contracting cells were observed among differentiated cells, which were proved to be cardiomyocytes with electron microscope and immunocytochemistry. CONCLUSION:A simple and economical method was established to induce mouse ES cells cultured in vitro to differentiate into cardiomyocytes without using any chemical reagents.     

LC3 protein expression and localization in mouse follicular granulosa cells

AIM: To investigate the expression and localization of autophagy related protein microtublule associated protein 1 light chain 3 (LC3) at various stages of follicular development and atresia in the mice.METHODS: On 0,1,2,3,4 and 5 day after intraperitoneal injection of pregnant mare serum gonadotropin (PMSG), expression and positioning situation of autophagy related protein LC3 and apoptosis related protein cleaved caspase-3 were examined by the me-thod of immunohistochemical staining. The protein levels of cleaved caspase-3 and LC3 were determined by Western blot in cultured mouse granulosa cells after incubation under serum-free conditions in the absence or presence of FSH. LC3 subcellular localization in granulosa cells were studied by the method of immunofluorescence.RESULTS: The LC3 protein expressed in granulosa cells during all developmental stages mainly. Granulosa cells of atretic follicles that showed intense staining of cleaved caspase-3 and LC3. The protein levels of cleaved caspase-3 and LC3-Ⅱ in the granulosa cells significantly decreased at 1 d and 2 d after intraperitoneal injection of PMSG (P<0.05). The protein levels of cleaved caspase-3 and LC3-Ⅱ in the granulosa cells increased in turn on 3, 4 and 5 day after intraperitoneal injection of PMSG. The positive correlation between LC3-Ⅱ and cleaved caspase-3 protein levels was observed (r²=0.8299, P<0.05). The LC3-Ⅱ protein expressed with punctuate structures in granulosa cell cytoplasm cultured under serum-free conditions in the presence of FSH.CONCLUSION: LC3 is expressed in the follicular granulosa cells with cell specificity and regional specificity. Autophagy is induced mainly in granulosa cells during folliculogenesis and shows positive correlation with apoptosis. Ovarian granulosa cell autophagy and apoptosis are gonadotropic hormone dependent.     

Heterogeneity of basal intracellular calcium concentration and its relations to the reactivity in mouse peritoneal macrophages

AIM: To investigate the heterogeneity of basal intracellular free calcium concentration([Ca²⁺]_i) in peritoneal macrophages(PM) and whether it is relative to the reactivity of PM at the single cell level. METHODS:[Ca²⁺]_iimplicated stimulated were measured by fluorescent microscopic imaging system after loading with fluorescent probe fura-2/AM. Superoxide(O₂^-)produced by single PM was determined by modified NBT test. RESULTS: The values of basal[Ca²⁺]_idetermined in 392 PMs of 7 mice showed normal distribution [(54±24) nmol/L, n=392] with wide range(less than 20 nmol/L to more than 100 nmol/L), among which about 50% were in the range of 40-60 nmol/L. When stimulated with PMA or fMLP,[Ca²⁺]_iwas increased, the peak values were positively correlated with the basal[Ca²⁺]_iin one mouse(PMA stimulated cells: r=0.52, P<0.01, n=58; fMLP stimulated cells:r=0.59, P<0.01, n=44. Both of the experiments were repeated in 3 mice, the results in the other 2 mice were similar). The O₂^- in PMA stimulated PMs were also positively correlated with the basal i(r=0.42, P<0.01, n=43, repeated in 4 mice, the results in the other 3 mice were similar). CONCLUSION: Basal[Ca²⁺]_iin murine PM is heterogeneous, and the value of basal[Ca²⁺]_iis tightly correlated to the reactivity of PM stimulated by proinflammatory factors.     

Identification of potential molecular markers for flower sex determination in Vernicia fordii

Vernicia fordii is cultivated as an important woody oil tree, with a long history in China. It is a monoecious and diclinous species. Altering the sex ratio to increase the number of female flowers would lead to better yields. The mechanism of flower development is, however, not yet clear. In this study, the proteins that are differentially expressed between male and female flowers were isolated using a combination of two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). A proteomic analysis led to the identification of 14 proteins, of which two were expressed specifically in male flowers and 10 in female flowers. Among the 10 female-specific proteins, aspartic proteinase, ascorbate peroxidase, and cyclophilin were thought to be involved in stamen abortion and gynoecium formation during the development of female flowers. Further results suggested that these proteins might be the sex-related proteins that are the potential molecular markers for flower sex determination in V. fordii.     

Preliminary study on induction of mouse embryonic stem cells into neural stem cells in vitro

AIM:To explore the feasibility of inducing mouse embryonic stem cells into neural stem cells in vitro. METHODS:Embryonic body induced by retinoic acid and retinal müller cells were selected in neural stem cell-defined medium for 7 days, and the morphological changes were observed. The selected cells were stained immunocytochemically with anti-nestin, anti-BrdU antibodies, and their ability of expansion and differentiation were analyzed. RESULTS:Large amounts of neurospheres were derived from embryonic body in the selected medium on the 7th day, which could be passaged and differentiated, stained positive with nestin and BrdU, and expressed nestin, glutaminase and Brn-3 genes. CONCLUSION:Neural stem cells could be derived from embryonic body induced by RA and müller cells in the selected medium, which would offer an alternative to treat neuropathy such as glaucoma and retinal degeneration in the future.