Antisense drug targeting with VEGF mRNA designed by computer aid and inhibitory effects on K562 cell line in vitro

AIM:The effective antisense sequences targeted VEGF mRNA with computer software would be screened and designed, and effect of them on growth K562 cells and protein expression of VEGF were studied with experiments.METHODS:Seven antisense sequences were selected and synthesized, which consisted of 18-20 deoxynucleotide acid and were modified with phosphorothioate, according to principle of low free energy of overall △G₃₇ Overall. Cell growth was assayed by trypan blue dye exclusion assay and level of VEGF protien in the media was determined by ELISA.RESULTS:Six of seven sequences were capable of inhibing growth of K562 cells and downregulating the VEGF protein expression significantly, compared with Scrambed control group. It was found that there was a close correlation between low level of overall △G₃₇ and antisense effectiveness (r=0.887,P<0.01).CONCLUSION:VEGF mRNA antisense oliogdeoxynucleotides, which were designed by computer software of RNAstructure, were able to inhibit growth of K562 and its protein expression. The VEGF mRNA may be new target attached by drugs. At same time, the computer aided design is useful methods to obtain the effective antisense.     

and laminin on adhesion and proliferation of human hepatocellular carcinoma cell

AIM:To explore the effects of PMA(phorbol-12-myristate-13-acetate, a tumor promoter, mimicking the action of diacylglycerol on PKC)and laminin on the adhesion and the proliferation of human hepatocellular carcinoma cells, and provide a new clue to liver cancer treatment.METHODS:Human hepatocellular carcinoma cell line(BEL-7402)was used to identify the endogenous laminin and protein kinase C-α(PKC-α) expression, and the effects of laminin and PMA on the adhesion and the proliferation were also investigatedin vitro.RESULTS:By the effect of exogenous laminin, human hepatocellular carcinoma cell (BEL-7402) possessed endogenous laminin expression and increased the adhesion and the proliferation, which was showed the synergistic action by the effect of PMA in combination. By the action of PMA alone, the proliferation and the PKC-α expression increased by exogenous laminin were decreased, and the adhesion and the endogenous laminin expression were increased.CONCLUSIONS:The finding suggested that the adhesion and the proliferation of human hepatocellular carcinoma cell were closely related to the effects of endogenous or exogenous laminin, which were associated with cPKC-α activity. Therefore, the application of anti-laminin antibody in combination with PKC antagonist might be a new clue to find out the therapy for liver cancer.     

Quick detection of sex determining region protein gene in mouse fetuses

AIM: To detect quickly the Y-chromosome specific sex determining region protein (Sry) gene in mouse fetuses on embryonic day 14.5 with a PCR method. METHODS: We designed specific primers with the OLIGO 5. 0 software. Templates were prepared in 30 minutes by the following way. About 1 mg embryonic tissue but not fetal liver was suspended, and treated with 200μL of lysis buffer, consisting of PCR buffer containing 20 mg/L proteinase K, 0. 5% NP-40, and 0.05% Tween 40, at 60°C for 15 minutes, heated for 5 minutes at 100 °C, 10μL was used as template. The PCR react ion was performed in 50μL, using two sets of primers specific for Sry gene (chromosome Y) and IL-3 gene (chromosome 11) . PCR conditions and cycle numbers were optimized. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The specificity of the method was conf irmed by fluorescent in situ hybridization (FISH) using a specific male probe on embryonic tissue cells. RESULTS: Electrophoresis showed that PCR product of male control DNA consisted of a 649 bp product representing the IL-3 gene and a 444 bp product representing the Y-specific Sry gene, female control DNA only one 649 bp product. Fetuses with two bands matching those as seen inmale control DNA are the presumpt ive male fetuses. Fetuses, only the IL-3-associated 649 bp band, are the presumptive female fetuses. These were confirmed by FISH. The ent ire procedure took <3. 5 h. CONCLUSION: The established PCR assay offers a quick, simple, accurate, and sensitive detection of sex determining region protein gene in mouse fetuses. This method allowed the preparation and culture of pure male and female hematopoietic stem cells from fetal tissue.     

‘三棱榄''橄榄果实香气成分分析

1 材料与方法选取广东优良鲜食橄榄品种‘三棱榄’,2001年12月6日采样,采用固相微萃取法(SPME)富集香气成分(鲜橄榄果肉于15℃下捣碎后取样1.0 g放入4 mL聚四氟乙烯硅橡胶垫密封螺口玻璃瓶中,插入100μm聚二甲基硅氧烷纤维头于室温25-30℃顶空取样2 h),用美国Finnigan TRACE GC-MS气相色谱-质谱联用仪进行分析。气相色谱柱为DB-1弹性毛细管柱30 m×0.25 mm,载气为He(99.99％),流速1.0mL/min。程序升温从40℃开始先保持10 min,后以2℃/min的升温速率升至150℃保持10 min。质谱条件:电子能量70 eV,离子源温度250℃,质量范围35-450 aum,不分流进样。2002年12月18日采样重复分析。     

The application of atomic force microscope in clinic diagnosis of erythrocytes

AIM: To investigate the application of atomic force microscope (AFM) in clinic diagnosis. METHODS: Topographic images and some parameters of large range field and microstructures of erythrocytes in the blood of normal subjects, lung cancer and myelodisplastic syndrome (MDS) patients were examined by atomic force microscope. RESULTS: Many clear topographic images of many erythrocytes, single erythrocyte, and microstructure of erythrocyte membrane surface were obtained. Many erythrocytes in lung cancer patients were found to change into echinocytes. One erythrocyte had 10-20 protuberances, most of which, with a mean width of 589. 0 nm and a length of 646. 7 nm, were on the edge of cells. The protuberances on the center of echinocytes are lodged and embedded. The erythrocytes of MDS patients were biconcave in shape. Many apertures with different diameters of tens to hundreds nanometer appeared on the surface of cell membrane. CONCLUSIONS: AFM can be widely applied in clinic pathological inspection, including quantification of cells, obtainment and comparison of many parameters (such as diameter, thickness, volume, surface, surface area/volume ratio), observation of topograph of single cell, and observation and comparison of membrane surface microstructure of cells, and so on.     

Inhibitory effects of LDL-ACM complex on subcutaneous implanted tumors in nude mice()

AIM: In order to evaluate the applicable value of LDL as a targeted vehicle for chemotherapeutic agents, we investigated and compared the inhibitory effects of LDL-ACM complex and free ACM on nude mice's subcutaneous implanted tumors derived from gastric cancer cell lines, SGC-7901 and NKM-45. METHODS: LDL-ACM complex was prepared and the tumor model of nude mice was established by subcutaneous implantation of SGC-7901 and NKM-45. Then, the groups of nude mice developed subcutaneous implanted tumors were received either LDL-ACM complex or free ACM. Subsequently, the tumor size, weight and leukemia cell counts were measured and the rates of tumor-inhibition and the survival were compared among the groups. RESULTS: The inhibitory effects of LDL-ACM complex on the tumors, especially on SGC-7901 implanted tumors were much more obvious than that of free ACM. It was also indicated that the action of LDL-ACM complex was mediated by LDL receptor. CONCLUSION: These results showed that LDL-ACM complex had significant inhibitory effects on the implanted tumors and the effect might be mediated by LDL receptor.     

Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of HL60 and K562 cells to daunorubicin

AIM:To investigate whether the bcl-2 antisense oligonucleotide increases the sensitivity of HL60 and K562 cell lines to daunorubicin.METHODS:IC50 for HL60 and K562 was determined with MTT method, the expression levels of Bcl-2 protein were assayed by immunofluorescence using fluoresce isothiocyanate labeling. In addition, apoptosis was detected by morphological observation and flow cytometric analysis of DNA fragmentation.RESULTS:It was found that the two oligonucleotides directed against the coding region and the translation initiation of bcl-2 mRNA, combined respectively with daunorubicin, inhibited expression of bcl-2 protein, increased apoptosis in HL60 and K562 cells, and decreased IC50 of daunorubicin significantly (P＜0.05). Compared to the antisense oligonucleotide directed against the translation initiation of bcl-2 mRNA, the antisense oligonucleotide directed against the coding region showed stronger effects in the aspects of increasing the sensitivity of HL60 cells to daunorubicin (P＜0.05).CONCLUSIONS:These two antisense sequences in the translation initiation and the coding region of bcl-2 mRNA increased the sensitivity of HL60 and K562 cell lines to daunorubicin in a sequence-specific manner.     

Sca-1+ cells from mouse fetal liver can differentiate into neurons in vitro

AIM: To study whether Sca-1⁺ cells from fetal liver can be induced to differentiate into neuronal cells in vitro. METHODS:Sca-1⁺cells from 14 5-days-old murine fetal liver were isolated with a magnetic cell sorting kit, and were cultured in Dulbecco s modif ied Eagle s medium(DMEM)/F12 supplemented with 10%fetal bovine serum(FBS), and passaged at a rat io of 1 3 when cells reached more than 80%confluence.The 5 passage cells were induced by 10^-3mol/Lβ-mercaptoethanol(β-ME)and 5×10^-7 mol/L all-trans-retinoic acid(RA)for 24 hours, and then incubated in serum-free medium for 5 hours to 5 days.The characteristics of treated cel s were assayed by immunocytochemistry staining analysis at 5 hours, or 5 days.RESULTS: Cells treated with β-ME and RA exhibited neuronal phenotype and expressed neuron-specific protein such as neuron-specific nuclear protein (NeuN), neuronfilament-M, and neuron-specific tubulin-1 (TuJ-1) but not tau, MAP-2, or the astrocyte-specific marker glial fibrillary acidic protein (GFAP).CONCLUSION: Sca-1⁺ cells from fetal liver, of which most are regarded as hematopoietic stem cells, could differentiate into early immature neuronal cells in vitro. These findings suggest that Sca-1⁺ cells from fetal liver may be an alternative source in cell therapy and gene therapy of neural dysfunction.     

Effect of Yigu capsule drug-containing serum on apoptosis and tartrate resistant acid phosphatase secretion in rat osteoclasts

AIM:The effects of Yigu capsule on tartrate resistant acid phosphatase (TRAP) secretion and apoptosis in rat osteoclasts were investigated in order to further explore its mechanism of preventing and treating osteoporosis.METHODS:(1) Twenty-month-old Sprague-daweley rats were randomly divided into two groups(Yigu capsule group and saline group), and the drug-containing serum and control serum were prepared. (2) The newborn Sprague-daweley rat osteoclasts were cultured with different concentrations of Yigu capsule drug-containing serum. At different time point, TRAP activity was measured and the survival osteoclast was counted under reverse microscope.The percentage of osteoclast apoptosis was observed under fluorescence microscope after acridine orange staining.RESULTS:TRAP activity was lower and the percentage of osteoclast apoptosis was higher in drug-containing serum group than in control group at 24, 48 and 72 h(P<0.01), respectively, and the survival osteoclasts were less in drug-containing serum group than in control groups at 24, 48 and 72 h(P<0.01).CONCLUSIONS:These data suggest that Yigu capsule drug-containing serum induces apoptosis and inhibits TRAP activity in osteoclasts, which may be one of the mechanisms of Yigu capsule preventing and treating osteoporosis.     

The expression of IFN-γ and IL-4 on T lymphocytes that infiltrate in nasal polyps

AIM:To investigate the expression of Th1-typed cytokine IFN-γ and Th2-typed cytokine IL-4 on T lymphocytes that infiltrate in nasal polyps for searching the pathogenesis of nasal polyps. METHODS:Nasal polyps tissue samples and peripheral blood were obtained from 21 patients. Normal human inferior turbinate mucosa and peripheral blood were obtained as well. Flow cytometry was adopted to detect the expression of IFN-γ and IL-4 of T lymphocytes. RESULTS: Th cytokines were rarely detected in inferior turbinate from normal human. Nasal polyps tissue consisted of abundant T lymphocytes. The expression of IL-4 and IFN-γ increased in peripheral blood from patients compared with normal human (P<0.05). The expression of IL-4 increased but the expression of IFN-γ decreased in nasal polyps compared with that of peripheral blood from the same patient (P＜0.05). CONCLUSION:There were generous of T lymphocytes infiltrating in nasal polyps. There was abnormal immune status in the local nasal mucosa from the patients, and the predomination of Th cytokine secretion changed compared with peripheral blood from the same patients, which resulted in the change of microenvironment of nasal mucosa and possibly close related to the formation of nasal polyps.