Aim To investigate the effect of Sonic Hedgehog on normal hearts and hypoxic-ischemic myo-cardial cells. Methods A method for left anterior de-scending artery ( LAD ) ligation was employed to con-struct the myocardial infarction model, and ultrasonic cardiogram was used for identification. Western blot and immunofluorescence staining were used to detect expressions of Shh, Ptch-1, Smo and Gli-1 in H9C2 cells and H2 O2-induced H9C2 cells, and that in 12 ca-ses of myocardial infarction tissues and 9 cases of nor-mal myocardium, respectively. Agonist and antagonist of Shh pathway were adminstered in the hypoxic-ische-mic myocardial H2 O2-induced H9C2 cell model, and once again expressions and strength of Shh, Ptch-1, Smo, Gli-1 were detected. Results Shh and Gli-1 were not expressed in normal hearts, but expressed in hearts with myocardial infarction;Ptch-1 and Smo were expressed in both normal hearts and hearts with myo-cardial infarction. Under the action of agonist, expres-sions of Shh and Gli-1 increased in the hypoxic-ische-mic H9C2 cell model. Similarly, Shh and Gli-1 were not expressed in normal H9C2 cells, but in H2 O2-in-duced H9C2 cells, and Ptch-1 and Smo were expressed in both normal H9C2 and in H2 O2-induced H9C2 cells. Conclusion Shh signaling pathway can be acti-vated in the condition of ischemia and oxidative stress, and then it promotes the repairing of myocardial cell damage.

It was previously revealed that noncoding RNAs, especially microRNAs, control cardiac genes and regulate heart function.Recently, growing evidence from high-throughput genomic platforms has confirmed that long non-coding RNAs ( lncRNAs) serve as new and enigmatic regulators in cardiac development and homeostasis.Nevertheless, lit-tle is known about their characteristics compared to microRNAs.Here, we review the latest progress on lncRNAs in cardiac biology and diseases, summarizing detailed knowledge of their functions and novel cardiac-related gene regulatory mecha-nisms in epigenetic processes.Finally, we highlight that lncRNAs could be promising therapeutic targets and diagnostic bi-omarkers in cardiac pathophysiology.

AIM:To construct recombinant retroviral vector of short interfering RNAs (siRNA) specific for macrophage migration inhibitory factor (MIF) and to establish the stable knockdown of MIF cell line of mammalian cells by transfecting the recombinant retroviral vectors. METHODS: We synthesized oligo-nucleotides for MIF in vitro, and cloned them into retroviral vector pSuper.retro. Subsequently the plasmids were sequenced and digested to identify the construction of the recombinant retroviral vectors. The vectors RNAi were transfected into packing cell line PHOENIX, which was selected by puromycin later. HeLa cell line was infected by the virus supernatant of stable PHOENIX cell lines, and the stable HeLa cell line showed significantly to silence MIF was established by selecting with puromycin. We also compare the characters of HeLa-pSuper-mock to HeLa-pSuper-MIF cells by using migration assay, adhesion assay, soft agar assay and FACS analysis of the cell-cycle progression. RESULTS: The recombinant retroviral vectors were constructed successfully. The HeLa cell line infected by the supernatant containing the retrovirus of package PHOENIX cells was persistent knockdown of MIF confirmed by Western blotting. Knockdown of MIF in HeLa cells inhibited the migration and adhesion, and decreased the clone formation. FACS analysis revealed that knockdown of MIF arrested HeLa cells in G0/G1 phase. CONCLUSION: We establish the stable HeLa cell line with a persistent knockdown of MIF. Our current studies reveal that MIF is necessary for HeLa cell migration and anchorage-independent growth.

【Ojective】To clone,express and detect activity of VEGF165.【Methods】Using human heart cDNA library as template,amplified the VEGF gene by PCR.The PCR product was ligated to PUC19 plasmid and sequenced.A eukaryotic expression plasmid AdtrackCMV harbouring VEGF165 was constructed.,then transformed into 293 cells.RNA dot blot and Western blotting were performed to demonstrated whether the tranfermants expressed VEGF165 at mRNA and protein level respectively.Furthermore the bio-activity of VEGF165 was preliminarily detected with Miles test.【Results】Sequence of 582bp VEGF165 cDNA was proved correct by sequencer analysis.The expression of VEGF165 mRNA was identified by RNA dot blot,Western blotting indicated that the molecular weight of VEGF165 protein was 22ku.VEGF165 also is of vascular permeability.【Conclusion】VEGF165 gene has been successfully cloned and expressed,which makes a basis for the further study in vivo.

Aim To measure the methylation rate of bone marrow mesenchymal stem cells (BMMSCs) using ultra-performance liquid chromatography-tandem mass spectrometry, and determine the methylation rate in the process of senescence.Methods The DNA extracted from BMMSCs would be digested into individual deoxynucleosides using enzymatic hydrolysis.To quantify the global genomic DNA methylation rate,we developed a method using ultra-performance liquid chromatography-tandem mass spectrometry with multiple reaction monitoring to simultaneously measure the levels of 5-methyl deoxycytidine and deoxyguanosine in digested genomic DNA.Results The DNA methylation rate could be analyzed within two minutes after hydrolyzing 1μg DNA for an hour.The detection limit of DNA methylation rate was 5.00×10-4.The coefficient of variance of the intra-day and inter-day precision was within 7.12×10-2 and 0.119, respectively.With the subculture of BMMSCs, the methylation rate gradually decreased.The methylation rate of stem cells was the lowest in 4~6 generation, and gradually increased with the subculture.Conclusions Using this method, the preliminary data on DNA methylation of BMMSCs in the process of senescence are obtained.The method is simple and convenient for sample preprocessing, and the method is fast and accurate with good sensitivity and repeatability.

AIM: To construct a subtracted cDNA library of differentially expressed genes in human unstable angina lymphocytes. METHODS: Suppression subtractive hybridizations (SSH) were performed between the patients with unstable angina pectoris and stable angina pectoris. Lymphocyte RNA, the obtained forward and reverse cDNA fragments were directly inserted into T/A cloning vector and transformed into E.coli JM 109 to construct a subtractive cDNA library. The inserting fragments were screened by blue and while blot screening and bacterium liqulid PCR. RESULTS: Each subtractive cDNA library contained more than 2000 positive bacteria clones. Most of them distributed between 200-600 bp inserts. CONCLUSION: The library is efficient and lays solid foundation for screening and cloning new and specific expressed genes in unstable angina lymphocyte RNA.

AIM: To examine expression of macrophage migration inhibitroy factor (MIF) gene and protein in macrophages induced by oxidized low density lipoprotein (ox-LDL). METHODS: Macrophages were incubated with ox-LDL at the concentration of 150 mg/L for time course (0-36 h) and with ox-LDL at the different concentrations (0-300 mg/L) for 24 h, expression of MIF mRNA and protein were detected by RT-PCR and ELISA. RESULTS: The results showed that ox-LDL increased MIF gene and protein expression in macrophages in a dose and time-dependent manner. After the exposure of macrophage to ox-LDL, the expression of MIF mRNA level increased consistently with protein. CONCLUSION: MIF may play an important role in atherosclerosis. [

Aim To investigate changes of MAP2 expression level in rat hippocampal pyramidal cells induced by chronic stress, and to explore effects of tianeptine on them. Methods 25 rats were divided randomly into three groups:Control group,Stress group and Stree-tianeptine group. The forced-swimming was performed to rats in stress group and stress-tianeptine. Using the immunohistochemistry and the computerized image technique, expression levels of phosphorated MAP2 and the number the Positive cells were assayed quantitatively in each group. Results Compared with control group (149.34?1.81), the phosphorated MAP2 average gray degree in pyramidal cells of stress group (144.99?4.40) was significantly lower, that of the stress-tianeptine group (148.84?2.73) was significantly higher than that of stress group; The number of phosphorated MAP2 positive cells in stress group (40.36?1.35) was significantly less compared withthat of control group (42.73?1.56); that of stress-tianeptine group (42.14?1.62) was significantly more than that of stress group. Conclusion It is suggested that tianeptine could inhibit the enhancement of phosphorated MAP2 expression in hippocampal pyramidal cells induced by chronic stress.

AIM: To investigate the effects of nitric oxide (NO) inhalation on nitric oxide synthase (NOS) and endothelin-1 (ET-l) of patients with hypoxic pulmonary hypertension. METHODS: Examined 13 pulmonic blood samples to determine the concentration of NOS in leukocyte and ET - 1 in plasma before NO inhalation, 30 minutes after inhalation, 2 and 12 hours after stopping of inhalation respectiviy. RESULTS: The values taken before inhalation was NOS (0.70 ? 0.21 )mol/min?mg-l, ET-1 (78.89 ? 46.59) Pmol/L; 30 minutes after inhalation (0.74?0.14)mol/min.mg-l, ET - 1 (88 .27 ? 45 .41 )pmol/L; 2 hours after stopping of inhalation NOS (0.64 ? 0.22)mol/min.mg-1, ET - 1 (80.76?42.66)pmol/L; and 12 hours after stopping of inhalation NOS (0. 63? 0. 17)mol/min.mg-1, ET-1(61.07?29.44)pmol/L. NO significant difference was found in the values of NOS and ET- 1 before and after inhalation, P＞ 0. 05. CONCLUSION: The effects of NO inhalation on NOS and ET-l in patients with hypoxic pulmonary hypertension are not significant according to the above investigation.

To study the effects of tetrandrine (Tet) on hypertrophied myocardial hydroxyproline content and myosin ATPase activity, left ventricular hypertrophy(LVH) was induced by renovascular hypertension (two-kidney, one-clip) in rats. Eight weeks after operation Tet 50 mg*kg-1*d-1 and enalapril(Ena) 6 mg*kg-1*d-1 were given by gavage for 8 weeks. The results showed that hydroxyproline content in LVH group was much higher than that of sham-operated one 〔(5.9±0.3) vs (3.6±0.4) mg*g-1 dry weight〕, and was decreased by 28.2% and 39.0% in Tet and Ena groups, respectively. Myosin ATPase activity in LVH group was much lower than that of sham-operated group 〔(0.43±0.09) vs (0.97±0.06) mmol Pi*min-1*g-1 protein, P<0.01〕. In Tet and Ena groups they were 60.5% and 118.6% higher than that of LVH group, respectively. The results suggest that Tet or Ena partially reduce the hydroxyproline content and elevate myosin ATPase activity of hypertrophied myocardium in renovascular hypertensive rats.