Table of Content

05 July 2013, Volume 33 Issue 7

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The role of PcG protein Ezh2 in the process of differentiation of mouse induced pluripotent stem cells introduced by atRA

2013, 33(7):  787-792. 

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Objective To investigate the role of Histone Methyltransferase EZH2 when mouse induced pluripotent stem (iPS) cell line IP14D-1 differentiates into primordial germ cells(PGCs) in vitro. Methods IP14D-1-derived EBS were maintained in culture medium containing 1μM atRA for 2d, 4d and 7d, respectively. RT-PCR、Western blot and immunofluorescence were performed to detect the expression changes of Ezh2 and Stra8 in the atRA processing IP14D-1-EBs around. Results The expression features of genes showed that Ezh2 mRNA rapidly reached peak value in 2d, and with the induction of prolonged expression decreased; In contrast, Stra8 expression increased when the EBs induced with atRA to extend. Immunofluorescence showed positive signals of Ezh2 and Stra8 were located on the membrane and in the cytoplasm, this result was consistent with the RT-PCR. Conclusions The EZH2 appeares earlier in IP14D-1-derived EBS and is due to the induction of the atRA, and the expression of Ezh2 and Stra8 is not coordinated. Low expression of EZH2 may occur earlier in the differentiation of PGCs with atRA introduction, the enhanced expression of Stra8 may start the differentiation of IP14D-1 to the germ cell.

S100A6 upregulate β-catenin and its mechanism in human breast cancer cell MCF-7

2013, 33(7):  793-798. 

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To investigate the impact of S100A6 on β-catenin and its molecular mechanism in human breast cancer cell line MCF-7. Methods MCF-7 cells were infected by recombinant adenoviruses carrying human S100A6 and its siRNA gene, AdS100A6 and AdsiS100A6 respectively. RT-PCR,Western blot and immunocytochemistry were used to detect expression and/or distribution of β-catenin,E-cadherin and p-GSK3β. Results In MCF-7 cells, S100A6 increased the protein level of β-catenin（P<0.01）,but no significant difference in transcriptional level of β-catenin between S100A6 group and control group（GFP group）（P>0.05）; The mRNA,protein level and distribution of E-cadherin had no significant difference between S100A6 group and control group（P>0.05）; S100A6 increased phosphorylation of GSK3β（P<0.01）.Conclusion S100A6 may upregulate β-catenin by promoting phosphorylation of GSK3β，rather than via effect on E-cadherin.

Calpain mediates estrogen-enhanced survival in MCF-7 breast cancer cells

2013, 33(7):  799-803. 

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Objective To investigate the effects of estrogen（E2）on the gene expression of calpain1 and calpain activity, and a possible role of calpain in the E2-enhanced survival in breast cancer cells. Methods Human breast cancer cell line MCF-7 was employed as a model system. RT-PCR was used to analyze the levels of mRNA expression and western blotting was employed to access proteolysis of FAK. Serum starvation was used to induce cell death, and Trypan blue staining and cell counting were used to evaluate cell viability. Results Stimulation of MCF-7 cells with E2 led to increased mRNA expression of calpain1 and calpain activity. E2 also increased cell viability by 14.29 % (P<0.05) during serum starvation, while calpain inhibitors significantly suppressed the E2-increased expression of calpain1 mRNA, calpain activity , and cell viability by 19.17% (P<0.05). Conclusion E2 causes increased expression of calpain1 mRNA and calpain activity, and this may contribute to the E2-stimulated survival during serum starvation in MCF-7 cells.

Glucose controls the fibroblast proliferation through mTORC1 signal pathway

2013, 33(7):  804-807. 

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Object To study the effect of glucose on the mouse skin fibroblast proliferation. Methods The cells cultured in different concentrations of glucose medium including 5.5mmol/L、15mmol/L、25mmol/L, and tested the cells proliferation by MTT. The formation of translation initiation factor 4F complex was analyzed by 7-MGTP pulldown test and the mTORC1 signal pathway was analyzed by Western blot. Result In contrasted with 5.5mmol/L, when the concentration of glucose medium was 15mmol/L, it promoted the cell proliferation and the translation initiation, activated the mTORC1 signal pathway. In contrasted with 5.5mmol/L, 25mmol/L inhibited the cell proliferation and the translation initiation, inactivated the mTORC1 signal pathway (included the 4EBP1 related with cell proliferation and the Akt related with cell survival), but the S6K1 was over activatied. Conclusion Glucose controls the fibroblast proliferation through the mTORC1 signal pathway.

The impact to MDC1 and 53BP1 after silence H2AX in esophageal carcinomas ECA109

2013, 33(7):  808-813. 

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Objective To discover the impact to MDC1 and 53BP1 after silence H2AX and when ionizing radiation in esophageal high grade squamous cell carcinoma ECA109. Methods Construction of lentiviral vector silencing H2AX , then transfecting lentivirus to esophageal cancer ECA109 and detecting the silence effect after transfection. Immunofluorescence detection the nuclear focus of r-H2AX、MDC1 and 53BP1 in ECA109 with ionizing radiation before and after transfection. As well as detection the expression of three proteins by western blot. Results 1) Successfully constructed the silence of H2AX in ECA109 cells. 2) Ionizing radiation can cause r-H2AX expression increases but not MDC1 and 53BP1. Ionizing radiation-induced nuclear focus of r-H2AX, MDC1 and 53BP1 are similar. 3) The nucleus focus of r-H2AX, MDC1 and 53BP1 are significantly reduced in ECA109 after silence H2AX. Protein expression did not change. Conclusion H2AX is one of early ionizing radiation-reaction protein in ECA109 and is located in the damage response upstream and can adjust the position of MDC1 and 53BP1.

A case of 48, XXYY syndrome with diabetes and analysis of the parental origin of X chromosome and skewed X-inactivation

2013, 33(7):  814-818. 

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Objective: To explore the clinical features of 48, XXYY syndrome with diabetic; to analyze parental origin of X-chromosome and skewed X-inactivation. Methods: The clinical data were collected; Genomic DNA was extracted from a whole blood sample. The parental origin of X chromosome and skewed X-inactivation were analyzed using PCR combined Hpa Ⅱ restriction enzyme digestion. Results: The patient was diagnosed as 48, XXYY syndrome with diabetes according to the clinical presentations and laboratory examinations. Plasma glucose level remained stable after metformin plus testosterone treatment. The X chromosome were inherited from the patient’ father and mother. The degree of skew of X-inactivation was 0.52. Conclusion: The pathogenesis of diabetes in 48,XXYY patient may be associated with low testosterone levels and insulin resistance. The extra X chromosome of our case and other had reported eight cases of 48, XXYY syndrome were paternal origin. Not all of 48,XXYY syndrome patients had skew X-inactivation.

The role of calpain in the responsiveness of transformed mammary epithelial cells to estrogen stimulation

2013, 33(7):  819-823. 

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Objective To investigate the sensibility of 17βestradiol(E2)-transformed MCF-10A mammary epithelial cells in response to E2 stimulation and the role of calpain (CANP) in the E2-induced effect, so as to understand the mechanism underlying the E2 signaling. Methods E2-transformed MCF-10A mammary epithelial cells were prepared and used as a model system. Western blotting was employed to observe proteolysis of focal adhesion kinase (FAK), a sensitive calpain substrate, to access calpain activity, and wound healing assay was performed to investigate cell migration. Calpain inhibitor was used to understand a possible role of calpain in the E2-induced effect. Results In non-transformed MCF-10A cells, focal adhesion kinase (FAK) was expressed primarily in the form of wild type, while in E2(50 nmol/L)- transformed cells FAK was significantly proteolyzed, indicating increased activity of calpain. Furthermore, transformed cells showed increased migration as compared with control (P<0.01). Treatment of transformed cells with E2 (10 nmol/L) triggered further truncation of FAK and enhancement of migration (P<0.01), while non-transformed cells were not sensitive to E2 stimulation. Additionally, pre-treatment of transformed cells with calpain inhibitor-1 (ALLN, 10 μmol/L) abrogated E2-enhanced FAK processing and migration (P<0.01). Conclusion Transformed MCF10-A mammary epithelial cells displays increased responsiveness to E2 stimulation, and this effect may be mediated through activation of calpain, indicating an active E2-CANP-FAK signaling in the transformed cells.

Down regulation of the expressions of OCT-4 and GDNF by Cyclophosphamide in rat testis

2013, 33(7):  824-828. 

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Objective To observe the effects of cyclophosphamide on the expressions of GDNF and OCT-4 that related functions of spermatogonial stem cells in rats. Methods Twelve 8 weeks male SD rats were randomly divided into control and experimental group. The rats in experimental group were injected cyclophosphamide (50mg?kg-1?d-1) intraperitoneally for three days. The testis and epididymis were removed after 4 weeks of injection to assess the weight , sperm mobility, sperm and spermatogonia numbers and diameter of seminiferous tubule. The expressions of OCT-4 and GDNF were measured by immunohistochemistry assay. Results Compared with the control group, the weights of testis and epididymis were reduced. Although there was no observed changes of testicular histology, the epididymal sperm mobility, and the numbers of epididymal sperm and spermatogonia were reduced. The expression of GDNF was decreased and that of OCT-4 was not changed. Conclusion The spermatogenesis was damaged by short-time treatment of cyclophosphamide, it might be that cyclophosphamide caused the reduction of spermatogonia through the downregulation of GDNF.

Neuregulin-1 (Nrg1) regulates cell adhesion molecule L1 expression in U87-MG glioblastoma cells and contributes to cell migration

Wei-jiang ZHAO

2013, 33(7):  829-833. 

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Objectives To investigate the regulating role of Neuregulin-1 (Nrg1) on cell adhesion molecule L1 expression and its effects on the migration of U87-MG cells. Methods Cells were administered with recombinant Nrg1α (rNrg1α) for 24 hours (h) and 48 h, and RT-PCR was used to investigate the mRNA levels of L1. Cells were then treated with rNrg1α and rNrg1β at 2.5 nmol/L for 48 h, and Western blot was applied to study the expression of L1 in response to Nrg1. siRNA targeting Nrg1 was also applied to study its effects on L1 expression. In addition, cells treated with Nrg1 siRNA was also subjected to wound healing assay. Immunofluorescence staining was used to study Nrg1α/β and L1 expression in cells on the wound edge. Results Administration of Nrg1α at 2.5 nmol/L can appartently increase the L1 mRNA levels at 24 h and 48 h time points. In addition, both 2.5 nmol/L of Nrg1α and Nrg1β can significantly increase the protein level of L1 at 48 h (p＜0.01 and p＜0.05, respectively). siRNA targeting Nrg1 for 48 h can appartently reduce the expression of Nrg1, accompanied with the reduction of L1 levels. Wound healing assay indicated that downregulating Nrg1 expression not only reduced the expression of L1 in cells on the wound edge, but also reduced cell migration, as was indicated by the wound space with a higher width. Conclusions Nrg1 can regulate L1 expression in human glioblastoma U87-MG cells, which may partially contribute to cell migration.

The inhibition of brain amyloid Aβ burden with cholera toxin B subunit and anti-Aβ monoclonal antibody conjugate in senile dementia mice

2013, 33(7):  834-839. 

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Objective To study the inhibition effect of anti-Aβ monoclonal antibody(IgG) conjugated with cholera toxin B subunit(CB) on brain Aβ burden in senile dementia mice. Methods IgG was conjugated with CB by improved sodium metaperiodate method. CB-IgG amount that accessed into the brain of mice was measured by indirect ELISA, which was also employed to measure IgG amount of brain different regions and blood sample after 3 hours of IgG administration. Transgenic mice were used to study the role of CB–IgG in inhibition of Aβ burden compared with IgG IN group, IgG IV group and wild type mice, after 14 weeks of IgG administration, Aβ level of brain was assessed by sandwich ELISA and Aβ deposits and senile plaques were observed by immunostaining. Results The trace of IgG in brain could be detected after 0.3 hours of CB–IgG intranasal administration and IgG amount achieved to peak at 3 hours when the amount of IgG in hippocampus was 10-fold higher in CB-IgG group than IgG IN group or IgG IV group (P<0.05),and IgG amount in brain was similar between IgG IN and IgG IV group. Compared with positive control group, the Aβ level of CB-IgG IN group was markedly reduced (P<0.05), percent Aβ load in hippocampus and cortex in CB-IgG IN group were both reduced by 80％. Conclusion CB-IgG treatment via intranasal was a promising approach that can effectively accessed into brain, and decrease Aβ deposits and senile plaques, which is more effective than the other way.

Construction of an infectious clone of enterovirus 71

2013, 33(7):  840-844. 

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Objective To construct full length infectious cDNA clone of enterovirus 71. Methods A cDNA fragment with a T7 promoter at the 5’ end and a poly (A) tail at the 3’ end was amplified by RT-PCR from the genome of the EV71/87-2008 Xi’an strain directly. This fragment was then ligated to pBR322 via SalI and BspEI site. Then the in vitro synthesized RNA transcripts were transfected into RD cells to produce the rescued virus. The negative-strand viral RNA was detected from the RD cells lysates by RT-PCR. The one step growth curve of recovered EV71 was measured by plaque assay. Results The full length cDNA clone of EV71/87-2008 Xi’an was constructed and the infectious of this clone was validated successfully. The EV71 recovered virus was proved functionally identical to its template. Conclusion Results suggest that the infectious clone of EV71/87-2008 Xi’an strain was constructed successfully.

Modified gelatin zymography for detecting MMP-2 and -9 activity in plasma of spontaneously hypertensive rats

2013, 33(7):  845-848. 

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Objective To improve the method of zymography for MMP-2 and MMP-9 detection in plasma of spontaneously hypertensive rats (SHR). Methods The effects of changed incubation time and working solution components, different pH values of developing buffer and plasma freeze / thaw cycles on the activity of MMPs were observed based on gelatin-containing SDS-polyacrylamide gel electrophoresis (SDS-PAGE)-dependent zymography. The relative activity of plasma MMPs was assayed by optimized zymography in Wistar and SHR rats. Results Gel incubation time reduced from 42 h to 17 h did not affect the test results of zymography. Rinsing step was omitted and renaturing buffer only containing 2.5% Triton X-100, NaN3 and NaCl were removed from developing buffer and deionized water was replaced for distilled water in steps after electrophoresis did not affect the test results of zymography. The pH value within 7.2~8.8 of the developing buffer can be used for zymography. Repeated freezing and thawing plasma samples up to 6 times did not affect the test results of zymography. The activities of MMP-2 and MMP-9 detected by optimized zymography were significantly increased in SHR compared with Wistar rats. Conclusions The optimized zymography is simpler and more economical and the experiment results are the same compared with conventional method.

Macrophages Enhance the Inhibition of H2O2 on the Proliferation of Human Vascular Endothelial Cells and Rabbit Smooth Muscle Cells

2013, 33(7):  849-853. 

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Objects Investigate the changes of inflammatory factors secreted by macrophages exposed to oxidative stress, and those effect on the proliferation of vascular endothelial cells and smooth muscle cells. Methods The groups described as followings: control, macrophage, 0.5mmol/L H2O2, macrophage+0.5mmol/L H2O2. The growth curves of VECs and VSMCs under different conditions were measured by CCK-8. The IL-6, IL-10, MCP-1, TNF-α and VEGF were detected by ELISA. The level of PCNA, cyclinD1 and eNOS were analysised by Western blot. Results The proliferation of control group was higher than the others in VEC(P<0.05), while, in VSMC, the control group was lower than the macrophage group(P<0.05). And, in both VEC and VSMC, macrophages+0.5mmol/L H2O2 conditioned medium groups were higher than 0.5mmol/L H2O2 groups(P<0.05), while were lower than macrophage groups(P<0.05). The secretions of IL-6, IL-10, and MCP-1 in macrophages were increasing with time and H2O2 concentration(P<0.05), while the amount of TNF-α didn’t change significantly. Only the concentration of H2O2 reached 1.0mmol/L, did the secretion of VEGF increase significantly(P<0.05). Conclusions The oxidative stress treatment can affect the secretions of inflammatory cytokines secreted by macrophages, and then affect the proliferations of vascular endothelial cells and smooth muscle cells.

Inhibition of hepatitis B virus(HBV) replication using antisense LNA targeting to C gene in HBV transgenetic mice

2013, 33(7):  854-858. 

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Objective To investigate the inhibitory effects on hepatitis B virus(HBV) replication of antisense locked nucleic acid (LNA) targeting to C gene in transgenic mice. Methods Antisense LNA were injected into transgenic mice via the tail vein. Serum HBV DNA was tested by real-time PCR. Serum HBeAg was tested by time-resolved fluorescence immune assay.The expression of HBcAg in the liver was detected by immuneohistochemistry. Results Five days after LNA injection,serum HBV DNA levels in the AS-LNA group were reduced by 44.47%,and serum HBeAg levels were decreased by 63.46%.These values were significantly higher than those in the control groups(all P<0.05). The expression levels of HBcAg in the liver were significantly lower than those in the control groups. Conclusion The present study proves that antisense LNA targeting to C gene has strong inhibition effect on HBV replication and expression in transgenic mice, and suggests that C gene being the effected site for gene therapy.

The expression and clinical significance of RIN1 in breast cancer

2013, 33(7):  859-863. 

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Objective To investigate the expression of RIN1 in breast cancer, and analyze its association with the prognosis. Methods To analyze the tissues of 85 breast cancer and 20 benign breast disease patients by immunhistochemical techniques, and followed up the 85 cases of breast cancer; RIN1 in 20 cases of breast cancer and adjacent tissues’ expressions by Real Time PCR and Western blot assay. Kaplan – Meier survival analyze the average survival time.Results A varying degrees of RIN1 were expressed in 85 cases of breast cancer ,high expression in 44 cases,RIN1 was highly expressed in breast cancer than in no-cancer tissues (p﹤0.05);The expression of RIN1 were significantly associated with tumor size、histological grade、family history (p﹤0.05). the average survival time of high expression group was significantly shorter than low expression group(p﹤0.05).The high expression of RIN1 was related to the tumor disease-free survival (p﹤0.05). mRNA and its protein were expressed in 20 cases of breast cancer and its adjacent tissues. The expression of RIN1 mRNA and its protein significantly increased in cancer tissues.Conclusion RIN1 expression played a role in breast cancer pathogenesis and Maybe its high expression could be used as prognostic marker in breast cancer.

Creatine phosphate decreases the apoptosis and autophagy in myocardial ischemia/reperfusion rat heart

2013, 33(7):  864-867. 

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Objective To investigate the role of Creatine phosphate in myocardial ischemia-reperfusion (I/R) injury of rat heart in vivo. Methods 24 male SD rats weighing 200g～250g, were randomly divided into a Sham group; a I/R group and a Creatine phosphate（CP）group. CP group used the intravenous administration of 4 mg/kg Creatine Phosphate Sodium before the reperfusion. TUNEL method was used to detect apoptosis of cardiomyocytes. The formation of autophagosomes was observed by transmission electron microscopy. Expression of LC3-II was measured by the Western blotting. Results Comparing with Sham group, I/R aggravates injury of mitochondria, and increase autophagic vacuoles (AVs) (P<0.01) and apoptosis of cardiomyocytes (P<0.01). However, CP group alleviates injury of mitochondria and reduce autophagic vacuoles (P<0.05) and apoptosis of cardiomyocytes (P<0.05) comparing to I/R group. LC3-II formation, an autophagy marker, was down-regulated in the CP group (P<0.01), which less increased than I/R-injured rats (P<0.05). Conclusion These results suggest that Creatine phosphate inhibits apoptosis and excessive autophagy to diminish the cell death induced by the myocardial I/R injury.

Homer expression in the pancreases of type 2 Diabetes mellitus rats

2013, 33(7):  868-872. 

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Objective To study the expression and correlation of Homer and insulin in rats with Type 2 Diabetes mellitus. Methods 22 Diabetes mellitus rats and 15 normal rats were sacrificed, the pancreases of rats were taken to detect Homer (Homerlc, Homer2 and Homer3) and insulin by using Polymerase Chain Reaction (PCR), Immunohistochemistry and double immunofluorescent labeling methods. Results Homerl, Homer2 and Homer3 were positive in the pancreases. In rats with Diabetes mellitus, leverages of insulin were much lower, but expressions of Homer1c, Homer2 and Homer3 protein were much higher. Conclusion The result shows that there is a significant change in the expression of Homer1c, Homer2,Homer3 and insulin between two groups．Homer may play important roles in insulin secretion，Homer1c, Homer2,Homer3 may act as inhibitors.

FN from pig blood promotes healing of skin Ⅲ-degree burns in mice

2013, 33(7):  873-875. 

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Objective To study the effects and differences of different purities of FN on wound healing of Ⅲ-degree burns in mice under the same concentration. Methods Establishing a model of skin Ⅲ-degree burns in mice, and dividing randomly into five groups: FN high purity group (85%), middle purity group (75%), low purity group (65%), Bei Fuji positive control group and blank control group. Observing the wound healing time,decrustation time and pathological changes in each group. Results All the different purities groups of FN could shorten the wound healing time and decrustation time,and significantly improved pathological morphology of the burn skin,compared with the blank control group. Conclusions FN can significantly improve the healing time of skin Ⅲ-degree burns in mice. The middle purity group might have the clinical value.

Cloning, expression and biological activity of the bile acid sulfate sulfatase gene BSS H1

2013, 33(7):  876-880. 

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Objective To clone a gene encoding BSS H1 from Pseudomonas aeruginosa and express the product. To study its enzymatic activities. Methods A target sequence named BSS H1 was identified in Pseudomonas aeruginosa genome through sequence alignment. The complete sequence of BSS H1 was amplified by PCR. The obtained segment was inserted into pET30b(+) vector. The recombinant expression vector was transformed into Escherichia Coli BL21(DE3) and was induced to express the proteion. A high expression yield was achieved by optimizing the conditions of induction. The BSS-HSD double enzyme-linked method was employed in the study of the enzymatic characters. Results The highest level expression of the enzyme was achieved when the engineered E. coli having grown for 3 hrs was induced by IPTG with a final concentration of 1 mM for 5hrs. The hydrolyzates of bile acid sulfate included both 3α-and 3β-hydroxy bile acid. At pH8.5 the activity of the cloned enzyme was (632±65) U/L for 3α- catalyzing and (52±4) U/L for 3β-catalyzing respectively. Conclusion An engineered strain of high BSS H1 expression was obtained. The unique characteristic of BSS H1 that it can transform the substrate into two types of stereoisomers has not been reported in the study of other BSS. The mechanism is unknown, which is worthy of further research.

The molecular mechanism of regulation of TRAIL-insensitive breast cancer cell migration by miR-146a

2013, 33(7):  881-887. 

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Objective To investigate the effection of miR-146a and its target gene EGFR on the migration of breast cancer cells. Methods Recombinant soluble TRAIL (rsTRAIL) was used at a final concentration of 50, 100, 200, and 500 μg /L to continually stimulate TRAIL-sensitive MDA-MB-231 cells for four weeks, respectively, and a TRAIL-insensitive breast cancer cell line named MDA-MB-231/TR was screened. MiR-146a expression level was evaluated using RT-PCR. Transwell cell invasion assay and wound-healing experiment were performed to examine the migration of breast cancer cells. The relationship between miR-146a level and EGFR expression was identified in MDA-MB-231/TR cells by dual luciferase reporter assay and Western blot. The expression of DR4, DR5, IRAK1, CXCR4, p-IκBα and apoptosis-related proteins were detected by Western blot. Chromatin immunoprecipitation(ChIP) analysis revealed the binding activity of NF-κB p65 subunit to the binding sites of miR-146a promoter element in MDA-MB-231/TR cells. Result MiR-146a expression in a TRAIL-insensitive breast cancer cell line MDA-MB-231/TR was decreased significantly. Down-regulation of miR-146a increased its target EGFR expression, and eventually promoted breast cancer cell migration. Furthermore, we found that NF-κB regulated the low expression of miR-146a in the MDA-MB-231/TR cells. Conclusion miR-146a plays an important regulatory role in the migration of TRAIL-insensitive breast cancer cells.

RNA interference-mediated pirin low expression deduced the erythroid differentiation of K562 cells

2013, 33(7):  888-891. 

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Objective This article is aimed at researching the influence of pirin knocking down on the differentiation and proliferation of K562 cells. Methods Techniques of shRNA interference was applied to generate stable cell strain of pirin low expression. Western blot was used to analyse the interference efficiency of pirin-shRNA. MTT and benzidine staining were applied to detect the cell viability and hemoglobin synthesis of the stable cell line in the presence of inducers Results Western blot showed that the pirin knockdown stable cell strain had a lower expression of pirin compared to the control group, MTT results showed that there was no difference between the pirin knockdown cell strain and the control group, but the hemoglobin synthesis had been reduced as compared to the control group. Conclusions pirin knocking down will not affect the cell viability of K562 cells, but it can inhibit the erythroid differentiation of K562 cells.

Establishment of a serum-free and feeder-free culture system for human embryonic stem cells

2013, 33(7):  892-897. 

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Objective To establish a serum-free and feeder-free culture system for human embryonic stem cells (HESc). Methods The HESX-01 cells were cultured in Knockout medium (the first group), self-made serum-free medium (the second group) and improved serum-free medium with bFGF and heparin (the third group), respectively. The morphology, clone number and clone size were observed after the twenty-fifth generation. And the surface labeling immunocytochemical analysis, Fluorescence-activated cell sorter analysis (FACS) and embryoid bodies formation were utilized to determine the character of HESc. Results After twenty-fifth passages the clones of the second group were gradually different- tiated, and the HESX-01 cells in the first and third group showed typical human embryonic stem cell morphology, they all expressed Nanog, Oct-4，Tra-1-60 and SSEA-4, but did not express SSEA-1, the same results were observed from FACS analysis. The HESc could form embryoid body in vivo. Besides, the significant differences were oberserved in the second group compared with the first and third group in clone nuber and size per view.Conclutions The self-made serum-free and feeder-free culture system for HESX-01 cells has been established, and the culture effect is similar with the publish recognized Knockout medium.

Construction and identification of siRNA expression vector of neuralized

2013, 33(7):  898-903. 

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Objective To construct Neuralized siRNA vector and to observe the effect of siRNA plasmids on expression of Neuralized in cells. Method Short hairpin oligonucleotide sequence producted by chemical synthesis was first inserted into linearized plasmid. After indentificated by enzymatic digestion and sequencing, Neuralized siRNA plasmids were transfected into HEK293 cells, the expression level and change of location of Neuralized were analysis by Western-Blot and Immunofluorescence. Result Sequencing result from Neuralized siRNA vector showed that the target nucleotide sequences were successfully inserted into the expected sites of vectors and sequences were correct. After the plasmids were transfected HEK293 cells, Western-Blot displayed that neuralized expression was decreased in cells. Immunofluorescence displayed that the fluorescence of neuralized lacted on the cell membrane was dimmed when the cells were transfected the four kinds of neuralized siRNA plasmids. Conclusion The neuralized siRNA plasmids were successfully constructed and could decrease expression level of neuralized in cells.

IgA Nephropathy associated to HIV infection: a study of 4 patients

2013, 33(7):  904-907. 

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Objective To study IgAN patients associated to HIV infection, clinical, pathological and treatment. Method The patients came from PUMC hospital, who hospitalized between 2002 and 2012, diagnosed as HIV associated with IgAN by renal biopsy. Results 4 HIV patients had proteinuria, SCr level were normal. Renal biopsy showed IgAN. All 4 patients received HAART，3 patients receieved ACEI/ARB treatment， 1 patient proteinuria decreased, then 2 patients were given prednisone treatment，HIV virus copies were not increased. Conclusion HIV infection could be associated with many types of renal diseases including IgAN. Renal biopsy could be necessary if diagnosis was not confirmed.

BRL37344 enhanced 3T3-L1 preadipocyte pERK1/2 expression

2013, 33(7):  908-909. 

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CDK4 and cycinD1 suppresses proliferation of breast cancer MCF-7 cells interfering

2013, 33(7):  910-911. 

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Inducing Newborn Rabbit Bone Marrow Stromal Cells To Differentiate Into Osteogenic Cells

2013, 33(7):  912-914. 

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Progress on type II transmembrane serine protease

2013, 33(7):  915-918. 

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The type II transmembrane serine protease (TTSP) family includes a group of trypsin-like enzymes that are anchored on the cell surface via an integral transmembrane domain near the N-terminus. The activated TTSPs cleave their specific substrates to participate in a variety of physiological and pathological processes. Dysregulation of these TTSPs may lead to serious diseases such as cancer, hearing loss, anemia and cardiovascular disease.

Zebrafish: a novel animal model for demyelination and remyelination

2013, 33(7):  919-922. 

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Multiple sclerosis is an immune-mediated disorder in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms. Zebrafish is an ideal model for investigating vertebrate demyelination and remyelination, due to the rapid external development and transparency of the embryos. Transgenic zebrafish allows visualization of cells in living zebrafish and provides a rapid, real-time in vivo system for the analysis of gene expression patterns.

The study for relationship of long noncoding RNA, smaller non-coding RNA and virus

2013, 33(7):  923-926. 

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Noncoding RNA,including miRNA,siRNAand piRNA as the representative of the short RNA and long chain non coding RNA.long non-coding RNAs (lncRNAs) are hot spots which are different from other smaller non-coding RNAs. Through researches, lncRNAs have been found to affect species evolution, embryonic development, metabolism and tumorigenesis. MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation.

Review of study on Dengue virus-like particles vaccine

2013, 33(7):  927-930. 

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The disease caused by Dengue virus is an acute life-threatening mosquito-borne infectious disease. So far, an effective and safe vaccine for dengue is not yet available. Recently a new virus-like particles vaccine can be capable of inducing the vaccinated mice to produce neutralizing antibody, and long-lasting virus-specific memory T lymphocytes. What more, it also can be widely explored by using yeast expression system. Its special priorities make it become a hopeful ideal dengue vaccine.

Internship training of medical students at community health care centers

2013, 33(7):  931-934. 

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Objective To learn the participation of the internship at community health care centers for the preclinical medical students in PUMC, the course outcomes and suggestions for the course improvement. Methods The survey was conducted among medical students admitted in the year from 2007 to 2009. A self-filled questionnaire for all students of 3 years and two focus-group interviews for students of class 2008 were used for data collection. A total of 198 students completed the questionnaire. Results The students who had participated in the internship showed more awareness on the community health care such as predominant population served, major diseases managed, routines activities, and advantages of them. Most respondents thought that it was suitable to arrange the internship in the second semester of the fourth year with a length of 18 hours. But nearly one half of them suggested visiting community centers in the mornings should be re-considered. They also suggested the internship should focus more on the activities specific for community health centers and they hope arrange training of knowledge and skills for the students before the internship. Conclusion The internship improved medical students’ awareness about the community health care, and it is still facing the challenges to guarantee the quality of different centers and achieve aims of the internship in limited course-hours.