Evaluation of serum microRNA-30e-5p expression in systemic lupus erythematosus and its association with clinical manifestations: A cross-sectional study / Evaluación de la expresión del microARN-30e-5p sérico en el lupus eritematoso sistémico y su asociación con las manifestaciones clínicas: un estudio transversal

ABSTRACT Background: MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression post-transcriptionally. Accumulating evidence indicates that the miR-30 family takes part in the development of multiple tissues and organs, and is a potential contributor to various dis eases, including autoimmune disorders such as systemic lupus erythematosus (SLE). The aim of this study was to evaluate the expression of miR-30e-5p, a member of the miR-30 fam ily, and investigate its potential relationship to clinical characteristics and possible disease activity in an Egyptian SLE cohort. Methods: Serum samples from 40 SLE patients and 37 age and gender matched healthy sub jects were tested for miR-30e-5p expression level using the Taqman quantitative reverse transcription-polymerase chain reaction. Analysis was performed using the 2 - AACT method. Results: The mean age of the patients was 28.7 ± 7.9 years, with a mean disease duration of 6.4 ±5.3 years. The median fold change in serum miR-30e-5p among our SLE cohort was significantly higher 1.748 (0.223-20.485) compared to the control group 0.877 (0.058-3.522) (P = 0.02). Receiver operating characteristic curve analysis revealed that miR-30e-5p expres sion level can discriminate SLE patients from controls at a cut-off value >1.06 with the area under the curve (AUC) = 0.676 (95% CI: 0.559-0.794, P = 0.02), with 64.3% sensitivity and 61.5% specificity. There was no correlation between any of the demographic features, clinical manifestations (apart from serositis, P = 0.013) or disease activity and miR-30e-5p levels. Conclusion: Our study demonstrated elevated miR-30e-5p expression levels in serum sam ples of SLE patients. Apart from serositis, it was not associated with any other disease characteristics.

RESUMEN Antecedentes: Los microARN (miRNA) son ARN no codificantes que regulan la expresión de los genes después de la transcripción. Las pruebas acumuladas indican que la familia de miR-30 participa en el desarrollo de múltiples tejidos y órganos, y es un posible contribuyente a diversas enfermedades, incluidos los trastornos autoinmunes como el lupus eritematoso sistémico (LES). El objetivo de este estudio fue evaluar la expresión del miR-30e-5p, un miembro de la familia miR-30, e investigar su posible relación con las características clínicas y la posible actividad de la enfermedad en una cohorte egipcia de LES. Métodos: Se analizaron muestras de suero de 40 pacientes con LES y 37 sujetos sanos de edad y sexo similares para determinar el nivel de expresión de miR-30e-5p, utilizando la reacción en cadena de la polimerasa de transcripción inversa cuantitativa Taqman. El análisis se llevó a cabo empleando el método 2-AACT. Los resultados: La edad media de los pacientes fue de 28,7 ± 7,9 años, mientras que la duración media de la enfermedad fue de 6,4 ± 5,3 años. La mediana del cambio de pliegue del suero miR-30e-5p entre nuestra cohorte de LES fue significativamente mayor, 1,748 (0,223-20,485), en comparación con el grupo de control, 0,877 (0,058-3,522) (p = 0,02). El análisis de la curva característica de funcionamiento del receptor reveló que el nivel de expresión del miR-30e-5p puede discriminar a los pacientes con LES de los controles en un valor de corte > 1,06, con el área bajo la curva (AUC) = 0,676 (IC del 95%: 0,559-0,794; p = 0,02), una sensibilidad del 64,3% y una especificidad del 61,5%. No hubo asociación entre ninguna de las características demográficas, manifestaciones clínicas (aparte de la serositis, p = 0,013) o actividad de la enfermedad y los niveles de miR-30e-5p. Conclusión: Nuestro estudio demostró niveles elevados de expresión de miR-30e-5p en mues tras de suero de pacientes con LES. Aparte de la serositis, no se asoció con ninguna otra característica de la enfermedad.

In situ Hybridization for the Detection and Localization of the Bitter Taste Receptor Tas2r108 in the Murine Submandibular Gland

Mammals have 3 pairs of major salivary glands i.e., the parotid, submandibular, and sublingual glands. Saliva secretion of these glands is modulated by taste perception. Salivary glands are composed mainly of acinar and ductal cells. Primary saliva is secreted by acinar cells and modified during ductal flow. Recently, of the murine 35 bitter taste receptors, Tas2r108 was expressed at highest levels in the submandibular gland by qPCR. Further, Tas2r108-transfected cells respond to a range of bitter compounds, such as denatonium, quinine, colchicine, diphenidol, caffeine and dapson. The objective of the present study was to characterize the expression of Tas2r108 mRNA in acinar and/or ductal cells of the submandibular gland using in situ hybridization (ISH). Male 42-60 days old DBA2 mice were used in the study. Messenger RNAs were extracted from the submandibular gland for generating digoxigenin (DIG) labeled-cRNA probes. These probes were transcribed in anti-sense and sense orientation using T7 RNA polymerase. Dot blot hybridization was performed using DIG labeled-cRNA probes, in order to estimate integrity and optimal diluting concentration of these probes. Subsequently, ISH was performed on murine submandibular gland to detect Tas2r108 mRNA. Dot blot hybridization data demonstrated that Tas2r108 DIG labeled-cRNA anti-sense probes specifically detected Tas2r108 cDNA. ISH results showed that the anti-sense probes labeled acinar and ductal cells in the submandibular gland, whereas no staining was visible in sense controls. Interestingly, the Tas2r108 expression levels were higher in acinar than ductal cells. These results suggested that Tas2r108 might be more associated with primary saliva secretion than with ductal modification of saliva composition.

Maltrato institucional hacia el adulto mayor: percepciones del prestador de servicios de salud y de los ancianos / Institutional abuse toward the elderly: Perceptions of health care providers and older adults

Objetivo. Analizar la percepción que el prestador de servicios de salud y el adulto mayor (AM) tienen sobre el maltrato al AM en los servicios públicos de salud, en ciudades seleccionadas de México. Material y métodos. De 2009 a 2012 se realizó un estudio con diseño cualitativo y estrategia de triangulación de fuentes de datos; se efectuaron entrevistas semiestructuradas a 13 prestadores y a 12 ancianos para recuperar su experiencia en el tema. El análisis utilizó procedimientos de la Teoría Fundamentada. Resultados. El maltrato contra el AM es una práctica naturalizada por el personal y por el anciano, la cual se manifiesta de formas diversas. Conclusiones. La institucionalización, profesionalización histórica y falta de conciencia sobre las necesidades de los AM demandan cambios de planeación, organización y supervisión del Sistema de Salud. El personal requiere intervenciones de formación, capacitación y cambio de actitudes/comportamiento, para otorgar atención integral, digna, humana y de respeto a los Derechos Humanos de los AM.

Objective. To analyze the health care providers (HCP) and elderly patients' perceptions about abuse of the elderly by health personnel of public health services, in selected cities in Mexico. Materials and methods. A qualitative study and a strategy of data triangulation were performed during 2009 and 2012; 13 HCPs and 12 elders were interviewed, in order to obtain their experience regarding elder abuse. Grounded Theory proceedings were used for the analysis. Results. Elder abuse is a naturalized practice, from HCP and elderly people's point of view; these perceptions are showed in different ways. Conclusion. Institutionalization, historical professionalization and lack of consciousness about needs of the elderly (sociocultural and economic), require changes in planning, organization and monitoring process in the Health System; training and educational interventions on staff and exchange attitudes and behavior are necessary in order to offer a health care that is comprehensive, decent, human and with respect for the human rights.

Effects of magnetic c-erbB-2 antisense probe of different concentrations on morphology and expression of SK-Br-3 cancer cell lines in vitro / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the effects of the magnetic c-erbB-2 antisense probe of different concentrations on the morphology and expression of SK-Br-3 cancer cells in vitro.</p><p><b>METHODS</b>Breast cancer SK-Br-3 cells were transfected for 24 h by antisense probe at an iron concentration of 5, 10, 25, 50, and 100 mg/L, respectively. The distribution and content of iron particles in SK-Br-3 cells was determined by Prussian blue staining, electron microscopy, and atomic absorption spectrometry. Cell viability was observed by trypan-blue exclusion and CCK-8 test. The protein expression of c-erbB-2 was assessed by the Western blot analysis. The changes of the signal strength were considered by magnetic resonance imaging (MRI).</p><p><b>RESULTS</b>c-erbB-2 antisense probe was uptake by SK-Br-3 cells in a concentration-dependence manner within a certain range (5, 10, and the Medicine Scientific Research Project of Chongqing Health Bureau (062025)25 mg/L). When the probe concentration was 25 mg/L, iron content in cells was (18.38±0.28) pg, the cell vitality, survival, and c-erbB-2 protein expression were reduced significantly (all P<0.05), and the T2 value was lower significantly (P<0.05). However, the results of 50 mg/L or 100 mg/L group showed no significant difference with the 25 mg/L group (P>0.05).</p><p><b>CONCLUSION</b>The magnetic c-erbB-2 antisense probe can effectively transfect and specifically inhibit the expression of SK-Br-3 cell lines at the iron concentration of 25 mg/L.</p>

Effects of antisense p38 α mitogen-activated protein kinase on myocardial cells exposed to hypoxia and burn serum / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the effects of antisense p38α mitogen-activated protein kinase (hereinafter referred to as p38α) on myocardial cells exposed to hypoxia and burn serum.</p><p><b>METHODS</b>Thirty adult SD rats were inflicted with 40% TBSA full-thickness burn on the back to obtain burn serum. The myocardial cells were isolated from 80 neonatal SD rats and cultured, then they were divided into 4 groups according to the random number table: normal control group (N, ordinary culture without any treatment), hypoxia+burn serum group (HB, exposed to hypoxia after being treated with 10% burn rat serum), hypoxia+burn serum+infection group (HBI, exposed to hypoxia and 10% burn rat serum after being infected with antisense p38α gene-carrying adenovirus), hypoxia+burn serum+empty vector infection group (exposed to hypoxia and 10% burn rat serum after being infected with adenovirus empty vector). At post hypoxia hour (PHH) 1, 3, 6, and 12, mRNA and protein expression levels of p38α in the latter 3 groups were determined by RT-PCR and Western blotting, cell viability was determined by methylthianolyldiphenyl-tetrazolium bromide assay, and lactate dehydrogenase (LDH) activity was assayed at the same time point. At PHH 1, 6, and 12, apoptosis rate of myocardial cells was assessed by annexin V staining method. The indexes of group N were determined with the methods mentioned-above. Three wells were set at each time point in each group. Data were processed with one-way analysis of variance and LSD- t test.</p><p><b>RESULTS</b>(1) At PHH 1, 3, and 6, the p38α mRNA level was higher in group HB than in group N and group HBI (with t values from 2.725 to 4.375, P values all below 0.05). (2) At PHH 1, 3, and 6, the p38α protein level was higher in group HB than those in group N and group HBI (with t values from 5.351 to 7.981, P values all below 0.01). (3) At PHH 3, 6, and 12, the cell viability in group HB (0.115 ± 0.007, 0.104 ± 0.006, 0.094 ± 0.005) was lower than that in group N (0.141 ± 0.014) and group HBI (0.136 ± 0.009, 0.124 ± 0.010, 0.112 ± 0.007, with t values from 2.357 to 6.812, P values all below 0.05). (4) The LDH activity was up-regulated in group HB as compared with that in group N and group HBI at each time point (with t values from 22.753 to 201.273, P values all below 0.01). (5) At PHH 1, 6, and 12, the apoptosis rate of myocardial cells in group HB [(5.4 ± 0.7)%, (8.7 ± 1.1)%, (13.6 ± 1.7)%] was higher than that of group N [(3.1 ± 0.3)%] and group HBI [(4.3 ± 0.5)%, (5.1 ± 0.7)%, (7.2 ± 0.9)%, with t values from 2.345 to 9.700, P < 0.05 or P < 0.01].</p><p><b>CONCLUSIONS</b>Antisense p38α can protect the myocardial cells from the injury of hypoxia and burn serum.</p>

Optimal concentration of c-erbB2 antisense probe labeled with superparamagnetic iron oxide nanoparticles for magnetic resonance imaging in tumor-bearing nude mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To determine the optimal concentration of c-erbB2 antisense probe labeled with superparamagnetic iron oxide (SPIO) nanoparticles for in vivo tumor imaging in mice using magnetic resonance imaging (MRI).</p><p><b>METHODS</b>Thirty BALB/c mice bearing SK-Br-3 tumor were randomized into 5 groups to receive injections of different concentrations of SPIO-labeled c-erbB2 antisense probe (containing 6.0, 9.0, 12.0, 15.0, or 18.0 mg Fe/kg). MRI was performed before and 6 h after the injections, and the signal intensities of the tumor were compared among the groups. The tumor tissues were then dissected for microscopic examination with HE and Prussian blue staining.</p><p><b>RESULTS</b>The tumor-bearing mice all survived after injections of the probe at doses of 6.0, 9.0 and 12.0 mg, but injections at higher doses (15.0 and 18.0 mg) caused death in some mice. Injections of the probe at the doses of 12.0, 15.0 and 18.0 mg resulted in significant signal enhancement of the tumor (P<0.001) to allow visual identification, but the changes showed no significant differences among the 3 groups (P>0.05). Pathological examination revealed irregular structures of the tumor issue containing numerous heterogeneous tumor cells aligned into cancer nests; Prussian blue staining visualized scattered blue iron particles in the tumor issue, which was especially obvious in mice injected with 12.0, 15.0 and 18.0 mg labeled probe.</p><p><b>CONCLUSION</b>Injection of 12.0 mg/kg SPIO-labeled c-erbB2 antisense probe allows optimal tumor imaging in BALB/c mice using MRI.</p>

Cloning and expression of human vascular endothelial growth factor gene and inhibition of its expression by antisense in prokaryotic system

Angiogenesis is an important process in physiology and disease pathogenesis and is controlled in a healthy body by a number of stimulatory and inhibitory factors. The aim of this study was to determine the effect of antisense transcript on the sense transcript of the endothelial growth factor [EGF] gene in bacterial system as an approach for the gene regulation in tumors. The hepatoma cell line [HepG2] was stimulated by PMA. VEGF mRNA was used for RT-PCR. VEGF cDNA was synthesised and cloned into T- vector pTZ57R, then sense fragment of VEGF subcloned into pACYC Duet-1 expression vector and antisense VEGF subcloned into pCDNAS expression vector. Recombinant plasmids were transforemed into BL2 1 bacterial cells. Expression of recombinant plasmid was analysed by western blot technique. The recombinant pCDNA3-VEGF [pYZantiVEGF] was successfully expressed in BL21 cells. Western blot analysis showed that the expression of VEGF decreased significantly in the cells transfected with VEGF antisense RNA compared with the pACYCDUET-1 - VEGF [pYZsenseVEGF] transfected and control. The expression of VEGF in BL21 cells was strong. In vitro, antisense of VEGF inhibited VEGF expression significantly in BL21 cells

Construction and identification of recombinant adenoviral vector for antisense klf4 gene / 中国实验血液学杂志

This study was aimed to construct a recombinant adenovirus vector for antisense klf4 gene through AdEasy system. Human klf4 cDNA was reversely inserted into the multiple cloning sites (MCS)of the pShuttle-CMV by using the backbone plasmid AdEasy-l, the antisense klf4 gene was constructed through homologous recombination in E.coli BJ5183, then the adenoviruses were packaged and amplified in the HEK 293 ce1ls. The adenovirus vector for antisense klf4 gene confirmed by PCR, restriction analysis and DNA sequencing. After being transfected with the adenoviruses at 200 MOI for 48 hours, total RNA and protein were extracted from human umbilical vein endothelial cells (HUVEC). Klf4 mRNA and KLF4 protein expression levels were evaluated by Real-time PCR and Western-blot. The results showed that the recombinant adenovirus vector for antisense klf4 gene was successfully constructed, recombinant adenovirus could suppress the expression of klf4 mRNA and KLF4 protein in HUVECs. It is concluded that the adenovirus vector for antisense klf4 gene has been constructed successfully, which provides the material basis for further studying the biologic function and potential application of klf4.

Combined effects of antisense TBRI eukaryotic expressing plasmid and antisense TIMP-1 eukaryotic expressing plasmid on rat liver fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To test the hypothesis that the introduction of antisense transforming growth factor beta receptor I (TBRI) plasmid and antisense tissue inhibitor of matrix metalloproteinase (TIMP-1) eukaryotic expressing plasmid into a rat liver fibrosis model may influence the progression of liver fibrosis.</p><p><b>METHODS</b>Fragments of TBRI cDNA and TIMP-1 cDNA were obtained by reverse transcription polymerase chain reaction (RT-PCR) and then amplified by nest PCR. pcDNA3.1(+)-antisense TBRI eukaryotic expressing plasmid was constructed by directional and inverted joins with the purified linear pcDNA3.1(+) and the purified fragment of TBRI, as well as, pcDNA3.1(+)-antisense TIMP-1 eukaryotic expressing plasmid. The recombinant was identified by restriction endonuclease digestion and DNA sequence analysis. The recombinant plasmids were encapsulated with Lipofectmine 2000, and then they were injected intraperitoneally into the liver fibrosis model rats. The protein expression of type I collagen was evaluated by immunohistochemistry. VG staining of liver slides of the rats was used for histopathological examination.</p><p><b>RESULTS</b>Compared with the empty plasmid control group and the disease control group, the deposition of type I collagen decreased in the three antisense treatment groups: antisense TBRI group (4.37+/-1.30) x 10(5), P less than 0.05; antisense TIMP-1 group (3.40+/-0.91) x 10(5), probability value less than 0.05; antisense TBRI + antisense TIMP-1 group (0.90+/-0.32) x 10(5), P less than 0.01; treatment control group (6.90+/-1.61) x 10(5); disease control group (7.34+/-1.68) x 10(5); and the normal control group (0.41+/-0.21) x 10(5)]. Significant differences in the pathological grades of fibrosis were found between the normal control group and the other five groups (P less than 0.05) and also between the disease control group and the three antisense treatment groups (antisense TBRI group P less than 0.05; antisense TIMP-1 group P less than 0.05; antisense TBRI + antisense TIMP-1 group P less than 0.01), but no difference was found between the empty plasmid control group and disease control group (P more than 0.05).</p><p><b>CONCLUSION</b>Both antisense TBRI eukaryotic expressing plasmid and antisense TIMP-1 eukaryotic expressing plasmid can inhibit the progress of liver fibrosis. A combined action can inhibit the progress of liver fibrosis more.</p>

Construction of antisense recombinant adenoviral vector for c-myc and its antiproliferative effect on rat lymphocytes / 中国应用生理学杂志

<p><b>AIM</b>To observe the antiproliferative effect of antisense recombinant adenoviral vector for c-myc on rat thymus lymphocytes.</p><p><b>METHODS</b>Antisense and sense bacterial plasmids for c-myc were constructed. Bacterial plasmids and El detected adenoviral plasmid were cotransfected into 293 cells. Recombinant adenoviral vectors were obtained after cotransfection. The antiproliferative effects were assayed by MTS. The expression of c-myc mRNA was detected by RT-PCR.</p><p><b>RESULTS</b>The results showed that antisense recombinant adenoviral vector for c-myc could inhibit rat thymus lymphocytes proliferation. The expression of c-myc mRNA was decreased after antisense recombinant adenoviral vector for c-myc was transfected into cells.</p><p><b>CONCLUSION</b>Recombinant antisense adenoviral vector for c-myc could inhibit rat thymus lymphocytes proliferation.</p>

Effects of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 on liver fibrosis in rats / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>No efficient therapy for liver fibrosis has been available. This study was aimed to provide evidence that the introduction of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 (TIMP-1) into a rat model of immunologically induced liver fibrosis can result in the increased activity of interstitial collagenase, thus enhancing the degradation of collagen.</p><p><b>METHODS</b>Real-time nested polymerase chain reaction (RT-Nested-PCR) and gene recombination techniques were used to construct a rat antisense TIMP-1 recombinant plasmid that can be expressed in eukaryotic cells. Both the recombinant plasmid and an empty vector (pcDNA3) were encapsulated with glycosyl-poly-L-lysine and injected into rats suffering from pig serum-induced liver fibrosis. The expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR, and Western blot. Hepatic interstitial collagenase activity was detected using fluorescinisothiocyanate (FITC)-labeled type I collagen. In addition to hepatic hydroxyproline content, hepatic collagen types I and III were detected by immunohistochemical staining, and the stages of liver fibrosis by Van Gieson staining.</p><p><b>RESULTS</b>Exogenous antisense TIMP-1 was successfully expressed in vivo and could block the gene and protein expression of TIMP-1. Active and latent hepatic interstitial collagenase activities were elevated (P < 0.01), hepatic hydroxyproline content and the accumulation of collagen types I and III were lowered, and liver fibrosis was alleviated in the antisense TIMP-1 group (P < 0.01) as compared with the model group.</p><p><b>CONCLUSION</b>The results demonstrate that antisense TIMP-1 recombinant plasmids have some inhibitory effect on liver fibrosis.</p>

Effects of TGF beta1 autocrine blockage on osteosarcoma cells / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To evaluate the effects of transforming growth factor beta1 (TGFbeta1) autocrine blockage on proliferation activity and drug sensitivity of osteosarcoma. METHODS; Northern blot, MTT determination, and 3H thymidine incorporation were used to investigate the effects of antisense TGF beta1 gene on osteosarcoma.</p><p><b>RESULTS</b>The proliferation of osteosarcoma cells transfected by antisense TGF beta1 gene was suppressed markedly, and adriamycin sensitivity was significantly increased.</p><p><b>CONCLUSION</b>Blockage of osteosarcoma cells TGF beta1 autocrine loop inhibits cell proliferation and enhances chemotherapy sensitivity.</p>

Obstruction of TGF-beta1 signal transduction by anti-Smad4 gene can therapy experimental liver fibrosis in the rat / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the therapeutic effects to block the TGF-beta1 (transforming growth factor beta1) signal transduction by antisense Smad4 gene on experimental fibrotic liver.</p><p><b>METHODS</b>Using the rat model of liver fibrosis induced by Carbon Tetrachloride (CCl4)/ethanol, we transfected antisense Smad4 gene mediated by adenovirus via portal vein infusion into the liver, and observed the expression of Smad4 by Retro-Polymerase Chain Reaction (RT-PCR) and Western Blot. We also investigated the pathologic features and collagen expression.</p><p><b>RESULTS</b>In the non-therapeutic cirrhotic liver, the expression of Smad4 mRNA was significantly increased than normal liver, and so was the collagen I. After antisense Smad4 gene being transfected, the expression of Smad4 mRNA and that of collagen I in the therapeutic liver was significantly decreased, compared with the non-therapeutic cirrhotic liver. The fibrous degree of therapeutic liver was also reduced compared with the non-therapeutic fibrous liver.</p><p><b>CONCLUSION</b>These results indicate that because antisense Smad4 gene could block TGF-beta1 signal transduction by reducing the expression of Smad4, so it could inhibit the production of extracellular matrix (ECM) and improve hepatic fibrosis.</p>

Effects of antisense transforming growth factor beta receptor-II (TGFbetaRII) expressing plasmid on experimental liver fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effects of antisense transforming growth factor beta receptor-II (TGFbetaRII) expressing plasmid on experimental liver fibrosis.</p><p><b>METHODS</b>RT-Nest-PCR and gene recombinant techniques were used to construct the rat antisense TGFbetaRII recombinant plasmid which can be expressed in eukaryotic cells. Thirty-six male SD rats were randomly distributed into five groups: 10 in experimental liver fibrosis model induced by pig-serum as disease control group; 10 in antisense TGFbetaRII transfection as treatment group; 10 in pCDNA3 transfection as treatment control group and 6 in normal control group. The recombinant plasmid and empty vector (pCDNA3) were encapsulated by glycosyl-poly-L-lysine and then transducted into rats of pig serum-induced liver fibrosis model respectively. Expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR and Western blot. We also tested ELISA of serum TGF-beta1, the contents of hepatic hydroxyproline, immunohistochemistry of type I and III collagen, and VG staining for pathological study.</p><p><b>RESULTS</b>The antisense TGFbetaRII expressing plasmid could be well expressed in vivo, and could block the mRNA and protein expression of TGFbetaRII in the fibrotic liver induced by pig serum. Its expression also reduced the level of TGF-beta1 [antisense treatment group (23.16+/-3.13) ng/ml, disease control group (32.96+/-3.79) ng/ml; F=36.73, 0.01]. Compared with the disease control group, the contents of hepatic hydroxyproline [antisense treatment group (0.17+/-0.01) mg/g liver, disease control group (0.30+/-0.03) mg/g liver; F=15.48, 0.01] and the deposition of collagens type I and type III decreased in the antisense group (antisense treatment group collagen type I 650.26+/-51.51, collagen type III 661.58+/-55.28; disease control group type I 1209.44+/-116.60, collagen type III 1175.14+/-121.44; F values are 69.87, 70.46, 0.01). And its expression also improved the pathologic classification of liver fibrosis models (0.01).</p><p><b>CONCLUSION</b>The results demonstrate that TGF-beta plays a key role in liver fibrogenesis and the prevention of liver fibrosis by antisense TGFbetaRII recombinant plasmid intervention may be therapeutically useful.</p>

The Effect of Transient Global Ischemia on the Rat Dentate Gyrus: Apoptosis in the Granular Zone and Neurogenesis in the Subgranular Zone / 대한해부학회지

It has been known that granule neurons of the dentate gyrus (DG) are born in adulthood as well as during development. Apoptotic cell death also occurs normally throughout the life of the rat brain. The present study was designed to determine the effect of transient global ischemia on the apoptosis and/or neurogenesis of granule cells in the dentate gyrus. TUNEL study revealed that the ischemia produced an significant increase in apoptosis mainly in the granular zone (GZ) of the DG. The percentage of TUNEL-positive cells in the DG was maximal (37.3+/-2.5%) 8 hr after ischemia and declined thereafter. However, immunocytochemical studies showed that there was an increase in neurogenesis mainly in the subgranular zone (SGZ) although the induction of neurogenesis took longer than the apoptosis. As a neurogenesis marker, proliferating cell nuclear antigen (PCNA)-positive cells, possibly progenitor cells, were significantly increased by 34.1+/-2.2%(n=3, p<0.05) mainly in the dentate SGZ 4 days after ischemia. In addition, the gradual increase in Bcl-2 expression was only paralleled with the neurogenesis in the SGZ, but not with the apoptosis in the GZ of the DG. The expression level of Bcl-2 in the SGZ was increased significantly (optical density 43.7+/-3.4; n = 3, p<0.05) 4 days after the ischemic insult. Furthermore, the ischemia-induced neurogenesis in the SGZ was also indirectly supported by the observation that the expression of synapsin-alpha was significantly increased (176%; n=3 p<0.05) in the CA3 region 4 days after the ischemia. Taken together, these results strongly suggest that the transient global ischemia induces the apoptosis in the GZ, whereas the cell proliferation in the SCZ of the DG. In situ hybridization using the antisense probes to the NR2A and NR2B subunits of NMDA receptors revealed that the ischemia produced a more profound effect on the mRNA expression of NR2A (61.9% reduction) than NR2B (20.5% reduction). Thus, we also suggest a possibility that ischemia could induce the neurogenesis in the SGZ of the DG through downregulation of the number of functional NMDA receptors.

The expression and possible function of RhoA in human gastric cancer cell lines / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the expression and possible function of RhoA in human gastric cancer cell lines.</p><p><b>METHODS</b>The expression of RhoA in human gastrointestinal cancer cell lines was detected by Western blot. Antisense plasmid of RhoA was constructed by pGEFL and transferred into gastric cancer cell line AGS by lipofectamine. Cell survival was examined by MTT assays, and cell cycle was detected by flow cytometry.</p><p><b>RESULTS</b>The expression of RhoA protein in 10 different kinds of human cancer cell lines was much higher than that in immortalized human intestinal epithelial cell line. After being transfected with antisense RhoA, with the decrease in RhoA protein expression, the growth rate of AGS was inhibited, and the number of cells in S phase was increased by 14%.</p><p><b>CONCLUSION</b>RhoA is overexpressed in many human cancer cell lines. Some of the malignant characteristics of a gastric cancer cell line can be partially reversed by inhibiting RhoA expression.</p>

Technetium-99m labeled antisense oligonucleotide-noninvasive tumor imaging in mice

The 15-mer oligonucleotide sequence was synthesized, aminolinked (sense and antisense phosphodiester) and conjugated with S-Acetyl-NHS-MAG3 by a N-hydroxy-succinimide derivative. The purified MAG3-DNA was radiolabeled with 99mTc by transchelation from sodium tartrate and free 99mTc was separated by gel filtration. The radiolabeled antisense and sense probes were injected intravenously in mammary tumor-bearing KM mice(1×106 cells,6 days post inoculation).Biodistribution was studied and the mice were imaged.Essentially complete conjugation was achieved by reverse-phase Sep-Pak C18 chromatography analysis.The MAG3-DNA was labeled with 99mTc at room temperature and neutral pH, with a mean labeling efficiency of 80.11 percent (s.d=2.96 percent , N=4). After labeling, the stability of the DNA in saline or serum was retained as determined by reverse-phase Sep-Pak C18 chromatography analysis, except a shift at 30 min in serum incubation which might suggest a short time serum protein binding. The labeled antisense DNA still remained the ability to hybridize with its complementary DNA. The pharmacodynamics of 99mTc labeled c-myc probes (antisense and sense) in mammary tumor-bearing KM mice did not change with the time postinjection. The highest accumulation of label was in the liver first, with the kidney and small bowel next. The injected activity localized in the lesion as early as 30min and reached a saturation value at 4hr. The accumulation of radioactivity in the tumor was lower at all time points in animals receiving the blocking oligonucleotides or sense probes. All images obtained with 99mTc-MAG3-c-myc antisense probes showed specific accumulation of radioactivity at the site of tumor. Radiolabel rapidly accumulates at the site of tumor and remains associated with the site even though circulation levels of radioactivity have greatly diminished. The tumor was readily evident since 45min and reached the highest tumor-to-muscle ratio at 4hr. The quite encouraging result was obtained at 20hr to 22hr when the background activity was diminished sufficiently. Positive imaging was not obtained in case of control group (in which non-conjugated, non-labeled antisense oligonucleotides were administered 2hr before the radiolabeled antisense probes were injected) and of sense group. Conclusion The 99mTc labeled antisense probe may provide a sensible and specific tool for noninvasive imaging of c-myc oncogene mRNA for a variety of malignant tumors at an earlier stage

The inhibitory effects of an antisense u-PAR vector on invasion of highly invasive human prostate carcinoma PC-3M cell subclones / 华中科技大学学报（医学）（英德文版）

To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer invasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. It was found that in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PAR was declined, and the ability of anchorage-independent growth of those cell subclones was found decreased sharply, with the inhibiting rate becoming 79% and 60%, respectively. Although the antisense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups was statistically significant (P < 0.01). The antisense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclones in vitro but also restrain the growth of those cell subclones in vivo.

Establishment of a cell line with antisense-blocked POLH and the role of POLH in alkylating agent MNNG induced nontargeted mutagenesis / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the function of POLH(polymerase eta) through establishment of the POLH gene-blocked cell line FL-POLH(-).</p><p><b>METHODS</b>A mammalian expression vector expressing antisense POLH gene fragment pMAMneo-amp-POLHA (-) was constructed by cloning the 1473 - 2131 fragment of POLH gene into the mammalian expression vector pMAMneo-amp(-) in antisense orientation. The FL cells were transfected with this antisense RNA expressing vector and selected by G418. The mutation assay was conducted using the shuttle plasmid pZ189.</p><p><b>RESULT</b>The spontaneous mutation frequency of SupF tRNA gene in the plasmid replicated in the FL-POLH(-) was 13.5 x 10(-4), while it was 4.9x10(-4) and 3.7x10(-4) in the control cells FL and FL-M, respectively. The nontargeted mutation frequency of SupF tRNA gene decreased in the plasmid replicated in these cell lines pretreated with MNNG.</p><p><b>CONCLUSION</b>POLH plays an important role in maintenance of genetic stability and genesis of nontargeted mutation.</p>

Construction of antisense transforming growth factor beta 1 gene and its effect on the proliferation by expression in osteosarcoma cells / 华中科技大学学报（医学）（英德文版）

To construct the antisense transforming growth factor beta 1 (TGF beta 1) gene and investigate the effect of TGF beta 1 autocrine loop blockage on the proliferation of osteosarcoma cells. TGF beta 1 cDNA was cloned by RT-PCR from human osteosarcoma cells (MG-63) and inserted into pcDNA3 to construct an antisense expression vector, which was dubbed pcDNA3-TGF beta 1(-). MTT was used to detect the proliferation of osteosarcoma cells transfected by antisense TGF beta 1 gene. Our results showed that the proliferation of the transfected osteosarcoma cells was suppressed markedly. It is concluded that TGF beta 1 autocrine loop blockage in osteosarcoma cells could inhibit cell proliferation, which might be helpful for gene therapy of osteosarcoma.