The potential role of long non-coding RNA Dnm3os in the activation of cardiac fibroblasts / 生物医学工程学杂志

Long non-coding RNA (lncRNA) Dnm3os plays a critical role in peritendinous fibrosis and pulmonary fibrosis, but its role in the process of cardiac fibrosis is still unclear. Therefore, we carried out study by using the myocardial fibrotic tissues obtained by thoracic aortic constriction (TAC) in an early study of our group, and the

Study on visfatin-induced inflammation and necroptosis via LOX-1 in human umbilical vein endothelial cells / 生物医学工程学杂志

The aim of the study is to identify the effects and underlying mechanisms of visfatin on inflammation and necroptosis in vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with visfatin or pretreated with Polyinosinic acid (LOX-1 inhibitor). By using the Western blot, RT-PCR, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), MTT and flow cytometry technique, the occurrence of inflammation and necroptosis in HUVECs were evaluated. Our results showed that 100 ng/mL visfatin significantly increased the mRNA and protein expression of monocyte chemotactic protein 1 (MCP-1) and LOX-1 after 24 hours' treatment in HUVECs. However, pretreatment with Polyinosinic acid could significantly reduce the expression of MCP-1 compared with visfatin group. Additionally, 100 ng/mL visfatin could induce the production of necrotic features and increase the mRNA expression of BMF (one of the markers of necroptosis), while pretreating with Polyinosinic acid markedly downregulated the mRNA expression of BMF gene and promoted the cell proliferation. These results indicate that visfatin might induce inflammation and necroptosis via LOX-1 in HUVECs, suggesting that visfatin plays a central role in the development of atherosclerosis.

Overexpression of miR-130a-3p attenuates cardiomyocyte hypertrophy / 生物医学工程学杂志

This study aimed to explore the role of miR-130a-3p in cardiomyocyte hypertrophy and its underlying mechanisms. Pressure-overload induced myocardial hypertrophy mice model was constructed by thoracic aortic constriction (TAC). , norepinephrine (NE) was used to stimulate neonatal rat cardiomyocytes (NRCMs) and H9c2 rat cardiomyocytes to induce hypertrophic phenotypes. The expression of miR-130a-3p was detected in mice hypertrophic myocardium, hypertrophic NRCMs and H9c2 cells. The mimics and inhibitors of miR-130a-3p were transfected into H9c2 cells to observe the role of miR-130a-3p on the hypertrophic phenotype change of cardiomyocytes separately. Furthermore, whether miR-130a-3p regulated hypertrophic related signaling pathways was explored. The results showed that the expression of miR-130a-3p was significantly decreased in hypertrophic myocardium, hypertrophic NRCMs and H9c2 cells. After transfection of miR-130a-3p mimics, the expression of hypertrophic marker genes, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC), and the cell surface area were notably down-regulated compared with the control group (mimics N.C. + NE group). But after transfection of miR-130a-3p inhibitor, the expression of ANP, BNP and β-MHC in H9c2 cells increased significantly, and the cell area increased further. By Western blot, it was found that the protein phosphorylation level of Akt and mTOR were down-regulated after over-expression of miR-130a-3p. These results suggest that miR-130a-3p mimics may alleviate the degree of cardiomyocyte hypertrophy, meanwhile its inhibitor can further aggravate cardiomyocyte hypertrophy. Over-expression of miR-130a-3p may attenuate cardiomyocytes hypertrophy by affecting the Akt pathway.

The potential role of calnexin in the activation of cardiac fibroblasts / 生物医学工程学杂志

Calnexin is a lectin-like molecular chaperone protein on the endoplasmic reticulum, mediating unfolded protein responses, the endoplasmic reticulum Ca homeostasis, and Ca signals conduction. In recent years, studies have found that calnexin plays a key role in the heart diseases. This study aims to explore the role of calnexin in the activation of cardiac fibroblasts. A transverse aortic constriction (TAC) mouse model was established to observe the activation of cardiac fibroblasts , and the cardiac fibroblasts activation model was established by transforming growth factor β1 (TGFβ1) stimulation. The adenovirus was respectively used to gene overexpression and silencing calnexin in cardiac fibroblasts to elucidate the relationship between calnexin and cardiac fibroblasts activation, as well as the possible underlying mechanism. We confirmed the establishment of TAC model by echocardiography, hematoxylin-eosin, Masson, and Sirius red staining, and detecting the expression of cardiac fibrosis markers in cardiac tissues. After TGFβ1 stimulation, markers of the activation of cardiac fibroblast, and proliferation and migration of cardiac fibroblast were detected by quantitative PCR, Western blot, EdU assay, and wound healing assay respectively. The results showed that the calnexin expression was reduced in both the TAC mice model and the activated cardiac fibroblasts. The overexpression of calnexin relieved cardiac fibroblasts activation, in contrast, the silencing of calnexin promoted cardiac fibroblasts activation. Furthermore, we found that the endoplasmic reticulum stress was activated during cardiac fibroblasts activation, and endoplasmic reticulum stress was relieved after overexpression of calnexin. Conversely, after the silencing of calnexin, endoplasmic reticulum stress was further aggravated, accompanying with the activation of cardiac fibroblasts. Our data suggest that the overexpression of calnexin may prevent cardiac fibroblasts against activation by alleviating endoplasmic reticulum stress.

Preliminary Study of Necroptosis in Cardiac Hypertrophy Induced by Pressure Overload / 生物医学工程学杂志

The aim of this study was to observe whether necroptosis is involved in the process of cardiac hypertrophy induced by pressure overload. SD rats underwent transverse abdominal aortic constriction (TAC) operation for establishing cardiac hypertrophy model. The structure and function of the left ventricle of rats were evaluated via echocardiography, left ventricular mass index, the expression of markers of cardiac hypertrophy and histological detection. Real-time PCR and Western blot were used to measure the gene and protein expression of receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3, the necroptosis markers) respectively. Four weeks after TAC operation, rat model for cardiac hypertrophy was established. The experimental data showed that the gene and protein expressions of RIPK1 and RIPK3 in the rat heart hypertrophic tissues after TAC for 4 weeks were increased significantly compared with those in the sham group. HE staining showed cardiomyocytes injury and hypertrophy in the hearts of TAC rat models. By transmission electron microscope, we observed that mitochondria of cardiomyocytes were damaged seriously in the TAC models. Treatment with losartan used, the selective antagonist of angiotensin II type I receptor could improve the cardiac function of TAC rats. Moreover, losartan treatment decreased the expression of RIPK1 and RIPK3 in heart tissues of TAC rats. The results suggest that necroptosis occurrs in the process of cardiac hypertrophy with pressure overload, and losartan could alleviate the cardiac hypertrophy and inhibit necroptosis.

Expression of Myocardial Specificity Markers MEF-2C and Cx43 in Rat Bone Marrow-derived Mesenchymal Stem Cells Induced by Electrical Stimulation In Vitro / 生物医学工程学杂志

Bone marrow-derived mesenchymal stem cells (BMSCs) for repairing damaged heart tissue are a new kind of important treatment options because of their potential to differentiate into cardiomyocytes. We in this experiment investigated the effect of different electrical stimulation time on the expression of myocardial specificity gene and protein in rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. The rBMSCs of second or third generation were randomly divided into three groups, i.e, electrical stimulation (ES) group, 5-Azacytidine (5-Aza) group and the control group. The rBMSCs in the ES groups with complete medium were exposed to 2 V, 2 Hz, 5 ms electrical stimulation for 0. 5 h, 2 h, 4 h, and 6 h respectively every day for 10 days. Those in the 5-Aza group were induced by 5-Aza (10 μmol/L) for 24 h, and then cultured with complete medium for 10 days. Those in the control group were only cultured with complete medium, without any treatment, for 10 days. The rBMSCs' morphological feature in each group was observed with inverted phase microscope. The mRNA expression of myocyte-specific enhancer factor 2C (MEF-2C) and connexin 43 (Cx43) were examined with Real-Time quantitative PCR and the protein expression of MEF-2C, Cx43 were detected with Western Blot method. The results showed that the mRNA expression level of the MEF-2C, Cx43 and the protein expression level of MEF-2C, Cx43 were significantly higher in the ES group and 5-Aza group than those in the relative control group (P < 0.05). It suggests that electrical stimulation could play a part of role in the induction of the rBMSCs to differentiate into the cariomyocyte-like cells in vitro and the effectiveness of the electrical stimulation with 2 h/d had the best in our experiment. But the mechanism how electrical stimulation promotes the differentiation of rBMSC into cardiomyocyte is still unclear.

Effects of Inhibiting Necroptosis on H9c2 Cardiomyocytes Injury Induced by Hypoxia/Reoxygenation / 生物医学工程学杂志

The aim of this study is to construct specific shRNA expressing plasmids, and to observe their effects on H9c2 cardiomyocytes injury induced by hypoxia/reoxygenation (H/R). RIPK1 and RIPK3 are the key kinases mediating the process of necroptosis. Using recombinant DNA technology, we inserted the synthetic shRNA into pSUPER vector to construct RIPK1-shRNA or RIPK3-shRNA plasmid respectively. We transfected H9c2 cardiomyocytes with the two shRNA plasmids respectively, before we treated them with H/R stimulation. Then, we measured the relevant genes and proteins by real-time PCR and Western blot. Meanwhile,we detected the markers of necroptosis and cardiomyocytes injury. The results showed that inhibition of ripk1 or ripk3 gene expression by its specific shRNA might protect the cardiomyocytes injury induced by H/R stimulation.

Preliminary investigation into the mechanism of cardiomyocyte hypertrophy induced by visfatin / 生物医学工程学杂志

The aim of the current study is to investigate the effect of visfatin on cardiomyocyte hypertrophy. Cultured H9c2 cardiomyocytes were exposed to visfatin at different concentrations for different periods of time, and the markers of cardiomyocyte hypertrophy were detected. Moreover, pravastatin, the inhibitor of endoplasmic reticulum stress (ERS) or thapsigargin, an ERS agonist was used respectively to pre-treat the cells before visfatin stimulation. F-actin staining was performed to measure the cell surface change. The mRNA expressions of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and ERS markers including glucose-regulated protein 78(GRP78), C/EPB homologous protein (CHOP) and activating transcription factor 6 (ATF6) were assessed by real time RT-PCR. The change of protein level of GRP78 and CHOP was detected by Western blot. The experimental data demonstrated that exposure to 100 or 150 ng/mL concentrations of visfatin for 24 h, or 100 ng/mL of visfatin for 24 or 48 h, significantly increased the expression of markers for cardiomyocyte hypertrophy. Visfatin stimulation provoked ERS in H9c2 cells. Furthermore, pre-treatment with pravastatin partially inhibited the visfatin-induced mRNA expression of ANP and BNP in H9c2 cells, whereas thapsigargin promoted the visfatin-induced expression of cardiomyocyte hypertrophy markers. The results suggest that visfatin might induce cardiomyocyte hypertrophy via ERS -dependent pathways.

Differentiation of rat bone marrow-derived mesenchymal stem cells into cardiomyocyte-like cells induced by cyclic stretching strain / 生物医学工程学杂志

Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent stem cells that differentiate into a variety of cell types and widely used in tissue regeneration engineering. The purpose of this study is to investigate whether the cyclic biaxial stretching strain could promote the rat BMSCs (rBMSCs) to differentiate into cardiomyocyte-like cells in vitro. The second or third generation of rBMSCs were randomly divided into the cyclic stretching stain group, the control group and the blank group. Those rBMSCs in the cyclic stretching strain group were seeded on a silicone membrane with complete medium were exposed to biaxial stretching strain of 10% of membrane at a frequency of 1 Hz lasting for 6 h, 12 h and 24 h. Those in the control group were seeded on silicone membrane with complete medium. Those in the blank group were seeded in the 6-wells plates with complete medium. The mRNA expression of GATA4 and myocyte-specific enhancer factor 2C (MEF-2C) were detected by the real-time fluorescent quantification PCR and the protein expression of connexin 43 (Cx43) was detected by using the Western blot method. The results showed that the mRNA expression level of the GATA4 and MEF-2C, and the protein expression level of Cx43 were significantly higher in the cyclic stretching strain groups, compared with those in the relative control groups (P < 0.05). It suggests that cyclic biaxial stretching strain could play a part in the induction of rBMSCs to differentiate into cardiomyocyte-like cells in vitro, but the differentiation mechanism is still unclear.

Effects of electrical stimulation on the differentiation of mesenchymal stem cells into cardiomyocyte-like cells / 生物医学工程学杂志

The aim of this study is to investigate the effects of electrical stimulation ES) on the induction of rat bone marrow mesenchymal stem cells (rBMSCs) to differentiate into cardiomyocyte-like cells in vitro. The third or fourth-generation of the rBMSCs was randomly divided into three groups, i. e. ES group, 5-Azacytidine (5-Aza) group, and control group. Those in the ES group with complete medium were exposed to 1,2,4 and 6V, 2Hz, 5ms ES for 2h everyday,lasting for 10d. Those in the 5-Aza group were induced by 10 micromol/L 5-Aza for 24h, then the medium was changed to complete medium without 5-Aza. Those in the control group were only cultured with complete medium. The growth status and morphological features of rBMSCs were observed by inverted phase microscope. The mRNA expressions of GATA4, a-actin, ACTN2 and TNNT2 were determined by Real-time fluorescent quantification PCR, and the protein expression of TNNT2 was detected with immunofluorescence staining. The results showed that the mRNA expression level of the GATA4, a-actin, ACTN2 and TNNT2 and the protein expression level of the TNNT2 were significantly higher in the ES group and 5-Aza group, compared to those in the control group(P<0. 05). It suggested that ES could induce rBMSCs differentiation into cardiomyocyte-like cells in vitro.

Effect of pulsed electromagnetic field with different frequencies on the proliferation, apoptosis and migration of human ovarian cancer cells / 生物医学工程学杂志

Pulsed electromagnetic field (PEMF), a non-invasive physical treatment modality, is now used clinically to promote bone formation for osteoporosis. The patients after ectomy of ovarian cancer are easily complicated with osteoporosis. However, the safety parameters of PEMF treatment for the osteoporosis patients after resection of ovarian cancer remain unknown. Therefore, this study was designed to examine the effect of different frequency of PEMF on the proliferation, apoptosis and migration of human ovarian cancer cells (SKOV3 cells). Cultured SKOV3 cells were exposed to PEMF stimulation daily with radiation of 8 Hz, 16 Hz, 32 Hz and 64 Hz, respectively. We used sinusoidal waves with strength of 1 mT, twice a day with an interval of 12 hours. An exposure to the waves lasted 30 minutes, for 3 days, with those no PEMF stimulation serving as the control. The proliferation of cells was detected using EdU assay, and the apoptosis of cell was assessed with Annexin V-FITC fluorescence. The migration of cells was measured with the scratch wound assay. The data showed that exposure to PEMF of 1 mT, 8 Hz for 3 days could significantly inhibit the proliferation of SKOV3 cells and induce the apoptosis of the cells. The migrated distance and number were increased by 1 mT, 8 Hz or 32 Hz PEMF stimulation, but decreased by 1 mT, 16 Hz treatment. The results suggested that we should be careful about the safety of PEMF treatment and strictly choose the optical parameters in preventing or treating the osteoporosis of the patients after resection of ovarian cancer.

Low frequence pulsed electromagnetic fields induce chondrocyte-like cells differentiation of rat bone marrow-derived mesenchymal stem cells in vitro / 生物医学工程学杂志

Mesenchymal stem cells (MSCs) are multipotent stem cells that differentiate into a variety of cell types. Low frequency pulsed electromagnetic fields (LFPEMFs) therapy can causes biochemical changes at the cellular level to accelerate tissue repair in mammals. So, we tested the hypothesis that LFPEMFs can promote chondrogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs) in vitro. The rBMSCs were isolated by adherence method and the third-generation of the rBMSCs were randomly divided into LFPEMFs groups, chondrocyte-induced group and control group. LFPEMFs groups with complete medium were exposed to 50Hz, 1mT PEMFs for 30 min every day, lasting for 10, 15 and 20 d, respectively. Chondrocyte-induced group were treated with chondrogenic media, while control groups were only cultured with complete medium. The mRNA expressions of type II-collagen (Col II) and aggrecan were determined by Real-time fluorescent quantitation PCR. The protein expression of Col II and aggrecan were detected with toluidine blue stain or immunocytochemical stain, respectively. The result showed that the mRNA and protein expression level of Col-II and aggrecan were significantly higher in the LFPEMFs group or chondrocyte-induced group, compared to the control group. It suggest that LFPEMFs could contribute to rBMSCs to differentiate into chondrogenic differentiation in vitro.

The effects of PEMF on the activation of human monocytes / 生物医学工程学杂志

The aim of the present study is to investigate the effect of pulsed electromagnetic field (PEMF) on the activation of human monocytes (THP-1). Cultured THP-1 cells were exposed to PEMF stimulation with radiation of 32Hz or 64Hz respectively, using sinusoidal wave, and 1mT, twice a day, 30 minutes each time, with an interval of 8 hours, for 3 days. Those with 0Hz stimulation served as the controls. Monocytes activation was monitored by measuring both the release of monocyte chemoattractant protein-1 (MCP-1) from monocytes and their adhesion to monolayers of human umbilical vein endothelial cells (HUVECs). The adhesion of THP-1 cells to HUVECs was evaluated by cell counting method. The secretion of MCP-1 from THP-1 cells was detected by ELISA and MCP-1 mRNA expression was assessed by real time quantitative RT-PCR. The data showed that exposure to PEMF with above parameters could significantly inhibit the adhesion of THP-1 cells to HUVECs and decrease the MCP-1 mRNA and protein expression. The results demonstrated that exposure to PEMF of 1mT, 32Hz or 64Hz for 3 days could significantly inhibit the activation of THP-1 cells.

The effects of pulsed electromagnetic fields on the induction of rat bone marrow mesenchymal stem cells to differentiate into cardiomyocytes-like cells in vitro / 生物医学工程学杂志

The aim of this study is to investigate the effects of pulsed electromagnetic fields (PEMFs) on the induction of rat bone marrow mesenchymal stem cells (rBMSCs) to differentiate into cardiomyocytes-like cells in vitro. rBMSCs were randomly divided into PEMFs groups, 5-Azacytidine (5-Aza) groups and control groups. PEMFs groups were exposed to 50 Hz, 1 mT PEMFs for 30 min every day, lasting for 10 d, 15 d and 20 d, respectively. 5-Aza groups were induced by 10 micromol/L 5-Aza for 1 day, then the medium was changed to complete medium without 5-Aza. And control groups were only cultured with complete medium, rBMSCs growth status and morphological features were observed by inverted phase microscope every day. The mRNA expressions of cardiac troponin T (TNNT2) and alpha-actinin (ACTN2) were determined by Real-Time PCR. The results showed that rBMSCs were spindle, polygon or fusiform in control groups. The cells gradually got longer and grew close together after being stimulated by PEMFs and 5-Aza, and with the extension of induction time, the tendency became obvious. At 20th day after PEMFs or 5-Aza treatment, rBMSCs gathered like a long chain, got much longer obviously at the high magnification, and some of them even fused with their neighbors. Compared with control groups, the levels of TNNT2 mRNA expression in 5-Aza groups were 19.40 fold (P < 0.01), 21.02 fold (P < 0.01) and 2.38 fold at 10 d, 15 d, 20 d and the levels of ACTN2 mRNA expression in 5-Aza groups were 6.64 fold (P < 0.01), 6.67 fold (P < 0.01) and 0.76 fold at 10 d, 15 d, 20 d. However, the levels of TNNT2 mRNA expression in PEMFs groups were 15.78 fold (P < 0.01), 6.73 fold (P < 0.05) and 2.73 fold (P < 0.01) of control groups at 10 d, 15 d, 20 d and the levels of ACTN2 mRNA expression in PEMFs groups were 4.93 fold (P < 0.01), 1.89 fold and 0.64 fold, respectively. Compared with 5-Aza groups, the levels of TNNT2 mRNA expression in PEMFs groups were 0.81 fold, 0.32 fold (P < 0.01) and 1.15 fold at 10 d, 15 d, 20 d and the levels of ACTN2 mRNA expression in PEMFs groups were 0.74 fold, 0.28 fold (P < 0.01) and 0.83 fold at 10 d, 15 d, 20 d. PEMFs could contribute to the induction of rat marrow rBMSCs to differentiate into cardiomyocytes-like cells in vitro, and the best exposure time might be 10 days, but further investigation is still needed.

Activin A and BMP-4 induce cardiomyocyte-like cells differentiation of human amniotic epithelial cells / 生物医学工程学杂志

Amniotic epithelial cells (AECs) have been expected to be a good cell source for stem cell-based cardiac repair. Activin A signaling is required for cardiac differentiation in human embryonic stem cells (ESCs), and bone morphogenetic protein-4 (BMP-4) is an important regulator that controls stem cell fates. Previous study has established an efficient protocol to generate cardiomyocytes from human ESCs via induction with Activin A and BMP-4. The aim of present study was to test the hypothesis that Activin A and BMP-4 could also induce AECs to differentiate into cardiomyocytes in vitro. Human AECs (hAECs) were isolated from human term placenta by trypsin digestion according to the previous reports. Freshly isolated hAECs were examined to detect the expression of cytokeratin 19 by immunocytochemistry. High-density undifferentiated hAECs at passage 1 were sequential treated with 100 ng/ ml human recombinant Actvin A and 10 ng/ml BMP-4. The expression of cardiac-specific genes was examined before and after in vitro induction of cellular differentiation. Freshly isolated hAEC could express cytokeratin 19, the specific marker of epithelial cells. The data showed that hAECs treated with Activin A and BMP-4 were able to express cardiac-specific genes, including Nkx2.5 and alpha-actinin. Our results demonstrated that Activin A and BMP-4 could induce cardiomyocyte differentiation of hAECs, which might be a novel approach to induce differentiation of AECs into cardiomyocytes-like cells.

Application of rotary biopsy forceps in diagnosis of gastric pre-cancerous lesions / 中华消化内镜杂志

Objective To evaluate the sampling efficiency of rotary biopsy forceps in gastric precancerous lesions. Methods A total of 60 gastric lesions with suspected pre-cancerous characters under narrow band imaging were enrolled,and consecutive samples were taken from same lesion by one endoscopist with routine and rotary forceps,respectively. The most severe pathological diagnosis was regarded as the final diagnosis. Results There was a significant difference between rotary biopsy forceps and routine ones in regarding of sample quality and capability of minimize tissue damage (P＜0. 05). The concordance rate with final pathological diagnosis from sample taken by rotary biopsy forceps was higher than that from routine ones,but without significance (P＞0. 05). Conclusion The rotary biopsy forceps is superior to routine ones in sampling of gastric pre-cancerous lesions.

The changes of hemodynamic parameters, pathology and c-kit mRNA expression in myocardium after acute myocardial infarction in rats / 生物医学工程学杂志

This study was aimed to investigate the changes of hemodynamic parameters, pathology and c kit mRNA expression in myocardium after acute myocardial infarctionin (AMI) in rats, and to elucidate the relationship between these three kinds of changes. Sixty six adult male SD rats were randomly divided into normal group, Sham groups and ligation groups. The rat model of AMI was set up by ligating the left anterior descending artery. Hemodynamic parameters, pathological changes and c kit mRNA expression in myocardiam were examined. The results revealed that there were no statistically significant differences in hemodynamic parameters between normal group and Sham groups. Compared with the normal group, all ligation groups exhibited significantly decreased left ventricular systolic pressure (LVSP) and +/-dp/dtmax (P<0.01), and increased left ventricular end diastolic pressure (LVEDP, P<0.01). In the other ligation groups, compared with 6th hour group after ligation, there appeared striking increase of LVSP, LVEDP and +/-dp/dtmax (P<0.05). HE staining in myocardiam showed that there are necrosis and derangement at 24th hour group after ligation ,and a great number of inflammatory cells infiltration around the infarct zone at 3rd day group after ligation, and granulation tissue infiltrated into the infarct zone at 14th day group after ligation. In all five time points groups after ligation, the levels of c-kit mRNA expression were 0.99 fold, 1.06 fold, 1.46 fold, 1.91 fold and 2.67 fold, respectively, compared with Sham groups. The results suggest that cardiac stem cells in myocardium might contribute to the role of regenerating myocardium via self proliferation after acute myocardial infarction, but further investigation is still needed.

Effects of CTGF gene silencing on the proliferation and myofibroblast differentiation of human lung fibroblasts / 生物医学工程学杂志

Connective tissue growth factor (CTGF) is involved in the differentiation of lung fibroblast into myofibroblast and is considered as an important mediator in the pathogenesis of pulmonary fibrosis. In the present study, a CTGF small interference RNA (siRNA) expressing plasmid (CTGF-siRNA) was constructed and stably transfected into human lung fibroblast cell line, MRC-5. Stable clones with CTGF gene silencing (CTGF-siRNA/MRC-5) were successfully established by G418 screening and further confirmed by real-time quantitative PCR and Western blot. Cell proliferation was investigated by growth curve analysis, and cell doubling time of the CTGF-siRNA/MRC-5 cells was markedly longer than that of the control cells (P < 0.05). Compared with control cells, the expression of alpha-smooth muscle actin (alpha-SMA), the marker of myofibroblast differentiation, was decreased in CTGF-siRNA/MRC-5 cells. Moreover, the deposition of extracellular matrix (ECM) proteins (such as collagen type I and fibronectin) in CTGF-siRNA/MRC-5 cells was also declined. Our data suggest that CTGF may play an important role in the differentiation of lung fibroblast into myofibroblast, and that siRNA targeting CTGF gene might provide a new strategy for gene therapy of pulmonary fibrosis.

Effect of antisense oligonucleotides targeting focal adhesion kinase on the proliferation and activation of hepatic stellate cells / 生物医学工程学杂志

Hepatic stellate cell (HSC) plays a pivotal role in liver fibrosis and isconsidered as one of the therapeutic targets for the treatment of hepatic fibrosis. Focal adhesion kinase (FAK) has been shown to play an important role in the HSC activation. The aim of the study is to explore the role of FAK in the proliferation and activation in culture-activated rat HSCs by using a specific antisense oligonucleotides targeting FAK (FAK-ASON). Rat HSCs were prepared from SD rats by in situ perfusion of pronase and collagenase and single-step density Nycodenze gradient. Culture-activated HSCs were transfected with the FAK-ASON (1 microM) by lipofectamine 2000 for 24, 48 or 72 hours. The proliferation of HSC was detected by MTT assay. The expression of the marker of HSC activation, alpha-smooth muscle actin (alpha-SMA), was assessed by reverse transcription- polymerase chain reaction (RT-PCR) and Western blot. The inhibition rates for HSC proliferation 24, 48 and 72 hours after transfection were 65.5% +/- 5.8%, 46.8% +/- 4.3% and 35.7% +/- 5.2% respectively. Transfection of FAK-ASON could significantly inhibit the proliferation of HSC. Meanwhile, treatment with the FAK-ASON could markedly decrease the mRNA and protein expression of alpha-SMA in rat HSC. The specific FAK-ASON may have an inhibitory effect on the proliferation and activation in rat HSC.

Effects of fluid shear stress on bone resorption in rat osteoclasts / 生物医学工程学杂志

This study was aimed to assess the effects of fluid shear stress on the bone resorption in rat osteoclasts. The osteoclasts were derived from the lumbar vertebrae marrow cells which were isolated from the 6-month-old female Sprague Dawley rats, cultured on the slide at 1 x 10(6) cell/ml, and induced with 1, 25 (OH)2 Dihydroxyvitamin D3. The slide containing osteoclasts was taken out on day 7 after culture and then put into the flow chamber. The loads of fluid shear stress applied to the osteoclasts were 5.97, 11.36, 16.08 and 20.54 dyne/cm2, respectively, for 30 minutes. The osteoclasts unloading fluid shear stress were used as control. The bone resorptive pits were studied by light microscopy and scanning electron microscopy. The tartrate-resistant acid phosphatase (TRAP) secreted by osteoclasts was detected with ultraviolet spectrophotometry. The results showed that fluid shear stress can increase the activity of TRAP and significantly increase the number and area of bone resorptive pits made by osteoclasts,and the effect of fluid shear stress on the bone resorption of osteoclasts is the same as that on the activity of TRAP. The reaction of the osteoclasts to the fluid shear stress in this study also suggested that the bone resorption of osteoclasts be increased in a magnitude of fluid shear stress-dependent manner, and that the changes of TRAP activity be closely related to the changes of the number and area of resorptive pits of the osteoclasts.