This study was aimed to investigate the mechanism of all-trans retinoic acid(ATRA) up-regulating apelin expression in vascular smooth muscle cells(VSMCs). The effect of ATRA on apelin expression in the VSMCs was investigated by RT-PCR, real-time PCR and Western blot analysis. To further define whether retinoic acid receptor α(RARα) mediated the induction of apelin by ATRA, endogenous RARα was down regulated by transfection of si RNA against RARα(si-RARα) or RARα was over-expressed by infection of the adenovirus vector p Ad-GFP-RARα in the VSMCs. The results showed that ATRA significantly induced apelin expression in a time- and dose-dependent manner in the VSMCs. Although RARα expression was increased in a time-dependent manner, the expressions of RARβ and RARγ were little changed by the ATRA treatment. When VSMCs were treated with a RARα antagonist Ro 41-5253 prior to the addition of ATRA, or si-RARα was used to down regulate endogenous RARα expression, the blockade of RARα signaling partially reduced the response of apelin to ATRA. Moreover, RARα over-expression, induced by infection of p Ad-GFP-RARα, further increased the induction of apelin by ATRA. In conclusion, ATRA may up-regulate apelin expression in VSMCs, and the mechanism may be RARα dependent.
낭포성 섬유증 막횡단 전도 조절 유전자(cystic fibrosis transmembrane conductance regulator, CFTR)가 cAMP 의존적인 Cl - 통로 단백질이고 상피액체 분비 과정에서 중요한 작용을 한다. 본 연구팀은 초기 작업에서 3',4',5,5',6,7-hexamethoxyflavone(HMF)와 5-히드록시-6,7,3',4'-테트라메톡시프라본(5-hydroxy-6,7...
The AMP-activated protein kinase(AMPK)widely exists in skeletal muscle,liver,pancreas,adipose tissue and central nervous system.As a cellular energy regulator,activation of AMPK can improve insulin resistance in various mechanisms.To overall understand the importance of AMPK in exercise,the article summarized the research progress on AMPK exercise activation in skeletal muscle,liver and adipose tissue as well as exercise improving cardiovascular insulin resistance by AMPK,and looked forward to the study future of AMPK exercise activation.
Chloride channels belong to a superfamily of ion channels that permit passive passage of anions, mainly chloride, across cell membrane. They play a variety of important physiological roles in regulation of cytosolic pH, cell volume homeostasis, organic solute transport, cell migration, cell proliferation, and differentiation. However, little is known about the functional regulation of these channels. In this study, we generated an integrated transgenic worm strain expressing green fluorescence protein(GFP) fused CLCtype chloride channel 1(CLH-1::GFP), a voltage-gated chloride channel in Caenorhabditis elegans(C. elegans). CLH-1::GFP was expressed in some unidentified head neurons and posterior intestinal cells of C. elegans. Interacting proteins of CLH-1::GFP were purified by GFP-Trap, a novel system for efficient isolation of GFP fusion proteins and their interacting factors. Mass spectrometry(MS) analysis revealed that a total of 27 high probability interacting proteins were co-trapped with CLHp-1::GFP. Biochemical evidence showed that eukaryotic translation elongation factor 1(EEF-1), one of these co-trapped proteins identified by MS, physically interacted with CLH-1, in consistent with GFP-Trap experiments. Further immunostaining data revealed that the protein level of CLH-1 was significantly increased upon co-expression with EEF-1. These results suggest that the combination of GFP-Trap purification with MS is an excellent tool to identify novel interacting proteins of voltage-gated chloride channels in C. elegans. Our data also show that EEF-1 is a regulator of voltage-gated chloride channel CLH-1.
This study was aimed to investigate the mechanisms underlying the modulation effect of Mas-related gene (Mrg) C receptors (MrgC) on morphine tolerance. Saline, morphine (20 μg), morphine plus bovine adrenal medulla 8-22 (BAM8-22, 1 nmol) or (Tyr(6))-2-MSH-6-12 (MSH, 5 nmol) were administered intrathecally in rats for 6 days. Pain-related molecules in the spinal cord and dorsal root ganglion (DRG) were examined using Western blot, immunocytochemistry and RT-PCR techniques. The results showed that intrathecal administration of the selective MrgC receptor agonists (BAM8-22 or MSH) remarkably attenuated or abolished chronic morphine-evoked reduction in glutamate transporters (GLAST, GLT-1 and EAAC1) in the spinal cord and increase in neuronal nitric oxide synthase (nNOS) in the spinal cord as well as DRG. In addition, MrgC receptor-like immunoreactivity (IR) was detected in superficial laminae of the spinal cord. Chronic morphine induced significant increases in MrgC receptor-IR in the spinal cord and MrgC receptor mRNA levels in DRG. These results suggest that the modulation of pro-nociceptive mediators in the spinal cord and DRG underlies the inhibition of morphine tolerance by MrgC receptor activation.
The plateau pika (Ochotona curzoniae) and plateau zokor (Myospalax baileyi) are specialized native species of the Qinghai-Tibetan plateau. The goal of this study was to examine physiological differences in skeletal muscle glycolysis and hepatic lactate metabolism between these two species. The partial sequence of pyruvate carboxylase (PC) gene was cloned and sequenced. The mRNA expression levels of PC and lactate dehydrogenases (LDH-A, LDH-B) were determined by real-time PCR. The enzymatic activity of PC was measured using malic acid coupling method. The concentration of lactic acid (LD) and the specific activities of LDH in liver and skeletal muscle of two species were measured. The different isoenzymes of LDH were determined by native polyacrylamide gel electrophoresis (PAGE). The results showed that, (1) LDH-B mRNA level in skeletal muscle of plateau zokor was significantly higher than that of plateau pika (P < 0.01), but no differences was found at LDH-A mRNA levels between them (P > 0.05); (2) PC, LDH-A and LDH-B mRNA levels in liver of plateau pika were significantly higher than those of plateau zokor (P < 0.01); (3) The LDH activity and concentration of LD in skeletal muscle and liver, as well as the PC activity in liver of plateau pika were significantly higher than those of plateau zokor (P < 0.01); (4) The isoenzymatic spectrum of lactate dehydrogenase showed that the main LDH isoenzymes were LDH-A4, LDH-A3B and LDH-A2B2 in skeletal muscle of plateau pika, while the main LDH isoenzymes were LDH-AB3 and LDH-B4 in skeletal muscle of plateau zokor; the main isoenzymes were LDH-A3B, LDH-A2B2, LDH-AB3 and LDH-B4 in liver of plateau pika, while LDH-A4 was the only isoenzyme in liver of plateau zokor. These results indicate that the plateau pika gets most of its energy for sprint running through enhancing anaerobic glycolysis, producing more lactate in their skeletal muscle, and converting lactate into glucose and glycogen in the liver by enhancing gluconeogenesis. As a result, the plateau pika has a reduced dependence on oxygen in its hypoxic environment. In contrast, plateau zokor derives most of its energy used for digging activity by enhancing aerobic oxidation in their skeletal muscle, although they inhabit hypoxic underground burrows.
The aim of the present study is to investigate whether monocyte chemotactic protein-1(MCP-1)-induced vascular smooth muscle cell(VSMC) proliferation is mediated via monocyte chemotactic protein-1 induced protein-1(MCPIP1).MCPIP1 expressions in cultured VSMC were detected by real-time PCR and Western blot following MCP-1 incubation.After MCPIP1 was silenced by siRNA,cell number was counted by hemocytometer,VSMC activity was analyzed by CCK-8 kit,percentage of DNA synthesis was detected by EdU kit,percentage of S phase cell numbers were measured by flow cytometry,and c-fos mRNA expression induced by MCP-1 or platelet-derived growth factor(PDGF) was detected by real-time PCR.The results showed MCP-1 increased MCPIP1 mRNA and up-regulated MCPIP1 protein expression in dose- and time-dependent manners.Cell counts,cellular activity,the percent- age of DNA synthesis,and the percentage of S phase cell numbers were remarkably decreased in MCPIP1 siRNA group,compared with those in MCP-1 group.The enhancing effect of MCP-1 or PDGF on c-fos mRNA expression was inhibited by MCPIP1 siRNA.These results suggest that MCP-1-induced VSMC proliferation is mediated via MCPIP1,and the underlying mechanism may involve c-fos expression up-regulation.
The aim of the present study was to observe the effects of hypoxia on tensions of aortic rings of pika (Ochotona curzoniae) and Sprague-Dawley (SD) rat. The aortic rings were prepared, and in vitro vascular ring perfusion was used to assay the effects of hypoxia or different drugs on contraction responses of the rings with or without endothelium. The results showed that, there was no difference of the contractions to KCl (80 mmol/L) between the aortic rings of the pikas and SD rats. After pre-contraction with NE (1 μmol/L), the aortic rings with endothelium of the SD rats showed obvious relaxation to ACh (1 μmol/L), whereas the aortic rings of the pikas, no matter with or without endothelium, showed significant and unusual contraction to ACh. The aortic rings of pikas, no matter with or without endothelium, exhibited greater contraction when treated by 1 h of hypoxia, compared with those in SD rats; The similar result was showed under hypoxia in combination with Ca(2+) removal. These results suggest that the contraction response to hypoxia in pika is more sensitive compared to that in SD rat, which is dependent on the release of calcium from intracellular calcium store.
The purpose of this research is to investigate the critical period of voltage-gated Na(+) channel development in hippocampal CA1 neurons. Changes of Na(+) currents in acutely isolated hippocampal CA1 neurons of rats at different ages (0-4 weeks after birth) were recorded using the whole-cell patch-clamp technique. The results indicated that the maximum current density of Na(+) channels was increasing with age, and the amplitudes in 1, 2, 3 and 4 weeks respectively grew by (42.76 ± 4.91)%, (146.80 ± 7.63)%, (208.79 ± 5.28)% and (253.72 ± 5.74)% (n = 10, P < 0.05) compared with that in 0 week. The current density in CA1 neurons of 1-2 weeks after birth increased more significantly than those of other groups. The activation curve of Na(+) channel shifted to the left. The half-activation voltages (mV) in 0-2 weeks were -39.06 ± 0.65, -43.41 ± 0.52, -48.29 ± 0.45 (n = 10, P < 0.05), respectively, showing significant age-dependent decrease, and there were no significant changes in other groups. The slope factors of activation curve for each group did not change significantly. There were no regular changes in inactivation curve and no significant changes in half-inactivation voltage. The slope factors of inactivation curve in 1-2 weeks were: 5.77 ± 0.56, 4.42 ± 0.43 (n = 10, P < 0.05). The inactivation rate of the second week after birth was faster than that of the first week, and there were no significant changes during 0-1 week and 2-4 weeks. The recovery from inactivation curve of Na(+) channel shifted to the left. The recovery time declined in 1-3 weeks. Changes of action potential properties were consistent with Na(+) current. These results suggest that the period of 1-2 weeks after birth may be the critical development period of voltage-gated Na(+) channel in hippocampal CA1 neurons. During this time, the distribution of Na(+) channel increases significantly; the activation curve of Na(+) channel shifts to the left; inactivation rate increases as well as recovery time shortens.
To investigate the time-course changes of myogenic tone in mesenteric small artery (MSA) of spontaneously hypertensive rat (SHR), thirty-two 7-week aged SHR rats were randomly divided into four groups (8, 16, 24, 32 weeks of age), and 32 sexand agematched Wistar-Kyoto (WKY) rats were assigned to control groups (CON). On the day of the study, segments of MSA were isolated and then cannulated to the two pipettes. Vascular diameters in response to the increased intraluminal pressure (from 0 mmHg to 150 mmHg, by 25 mmHg steps) of isolated MSA under no-flow conditions were recorded by a Pressure Myograph System both in physiologic salt solution (PSS) (active diameter, Da) and calcium-free PSS (passive diameter, Dp). The myogenic tone was calculated by (Dp- Da)/Dp × 100%. The tail artery pressure and vascular myogenic tone in SHR rats were significantly higher than those of the CON rats. Before 24 weeks, the vascular myogenic tone of MSA in SHR group increased monotonically, but at the end of 32 weeks, the vascular myogenic tone decreased in comparison with that in 24-week group, but was significantly higher than that in CON group. The tail artery pressure in SHR group slowly increased monotonically with increasing weeks of age, and the tail arterial pressure in 32-week group remained significantly higher than that in 24-week group. Vascular myogenic tone may participate in the whole process of hypertension. Early in the development of hypertension, because of the compensatory role of vascular tone, the vascular function has been partially compensated, thus guaranteeing adequate blood supply to organs. Late in the development of hypertension, because of the decompensation of myogenic tone, the vascular function is damaged, leading to the occurrence of severe vascular disease.
Diabetic peripheral neuropathy (DPN), one of the most common chronic complications of diabetes, is characterized by al- lodynia, hyperalgesia and spontaneous pain. Chinese epidemiological studies have shown that at least 25% diabetic patients suffered from painful DPN, which compromises patients’ daily functioning and becomes a major health care problem. Although the pathogenesis of painful DPN is not fully understood and current treatment options are very limited, research in the field has advanced our understanding on the mechanism of painful DPN in the past Decade of Pain Research and Control. This review will mainly focus on evaluation of current diabetic animal models, possible molecular pathways and available therapies, with an emphasis on roles of purinergic receptor and its signaling transduction pathways. Common therapies address one or two DPN symptoms, while others offer wider symptom control, presumably by targeting pathophysiological mechanisms of DPN. Purinergic receptor signaling transduction path- ways might become potential targets for treatment for painful DPN.
Cyclin D1, as a regulatory factor in cell cycle, is highly expressed in many tumors, such as lung cancer, breast cancer and thyroid cancer. The aim of the present study was to study the role of Cyclin D1 in invasion and metastasis of lung cancer cells. Lung adenocarcinoma cell line A549 and squamous cell line SK-MES-1 were selected as the objects, because A549 expresses Cyclin D1 highly, and SK-MES-1 expresses lowly. Nude mice were injected with A549 or SK-MES-1 via tail vein, and were sacrificed after 4 weeks for cancer tissue isolation. The harvested cancer cells were reinjected into another nude mouse. After one more time of such seeding, highly metastatic lung cancer model was established. After A549 and SK-MES-1 were transfected with Cyclin D1 RNAi and expression vector respectively, transwell migration assay was used to analyze transferring capacity of lung cancer cells. Western blot was used to detect Cyclin D1 and WNT/TCF pathway proteins expressions in parental cell lines and cancer tissue from metastasis model animals. The results showed that, along with the increase of seeding times, lung cancer cells from model animals, no matter A549 or SK-MES-1, exhibited augmented metastasis activity and up-regulated Cyclin D1 expression. The transferring capacity was weakened significantly in A549 cells where the Cyclin D1 was interfered by RNAi, and it was enhanced significantly in SK-MES-1 cells which were transfected with the expression vector of Cyclin D1. The expressions of WNT/TCF pathway proteins, including β-catenin, lymphoid enhancer-binding factor (LEF) and T cell factor (TCF), increased significantly in highly metastatic model animals. The parental cell lines showed lower expressions of WNT/TCF pathway proteins compared with cancer tissue from metastasis model animals. These results suggest that Cyclin D1 is closely related with the invasion and metastasis of lung cancer cells, and the WNT/TCF signal pathway may promote the expression of Cyclin D1.
microRNAs (miRNAs) are non-coding endogenous short RNAs which are involved in regulating gene expression at the post-transcriptional level. miR-15 family is increasingly found to play great roles in important cell processes, such as apoptosis, cell differentiation and stress response. Growing evidence indicates that miR-15 family members are implicated in tumor, cardiovascular disease and neurodegenerative disease. miRNAs have emerged as a new promising subset of therapeutic targets. The present paper reviewed the important function of miR-15 family and new approaches for miRNA-based therapy.
Multiple endocrine neoplasia type 1 (MEN1) is a dominantly inherited tumor syndrome characterized by development of various combinations of tumors in multiple endocrine glands,including the pituitary,parathyroid or pancreas.MEN1 results from mutations in tumor suppressor gene Men1,which encodes nuclear protein menin.Menin has been shown to preferentially repress cell proliferation in endocrine tissues including pancreatic beta cells.Herein,the present study was to explore the potential mechanisms underlying menin in repressing cell proliferation in mice MEN1 insulinoma.In the Gene Set Enrichment Analysis (GSEA),Ccnb2 (encoding cyclin B2) was up-regulated in pancreatic islets of Men1-excised mice after 14-day tamoxifen-feeding.Immunofluorescence with antibody against cyclin B2 revealed that the expression of cyclin B2 was greatly increased in MEN1 insulinoma.In Men1/cells,Men1 ablation leaded to an increase in cyclin B2 expression.Immunofluorescent staining by phospho-H3S10 antibody revealed the increasing number of Men1/cells in mitosis.Cells were seeded at a density of 5×10 4,then counted on day 2,4 and 6,and the cell growth curve revealed Men1 ablation increased the cell proliferation.In contrast,knockdown of cyclin B2 by shRNA diminished the number of cells in mitosis and reduced cell proliferation.Further,chromatin immunoprecipitation (ChIP) assay indicated that menin affected the histone modification of the promoter of Ccnb2 by reducing the level of histone H3 lysine 4 tri-methylation (H3K4me3) and histone H3 acetylation but not affecting the level of histone H3 lysine 9 tri-methylation (H3K9me3) or histone H3 lysine 27 tri-methylation (H3K27me3).Our results suggest that menin may inhibit MEN1 insulinoma by suppressing cyclin B2 expression via histone modification.
The present study investigated the effects of intrathecal(i.t.) application of bovine adrenal medulla 22(BAM22),an endogenous opioid peptide potently activating opioid receptors and sensory neuron-specific receptor(SNSR),on a model of complete Freund's adjuvant(CFA)-induced inflammatory pain.Unilateral,but not bilateral,inflammatory pain was induced by intraplantar(i.pl.) injection of CFA in one side,as indicated by the shortened paw withdrawal latency and the increased edema of paw.Paw withdrawal latency test,paw edema determination and immunohistochemistry were used in CFA-induced in?ammatory pain model after i.t.administration of BAM22 or saline.It was found that administration of BAM22 dose-dependently attenuated CFA-induced hyperalgesia and edema,and resumed antinociceptive effects against thermal stimulation in behavioral test.In 10 nmol BAM22 group,paw withdrawal latency was resumed to 83.2% of normal,and edema increased only by 60% of normal at 48 h.The potency of BAM22 was 33.5% of maximal possible effect(MPE) at 24 h,and the antinociception persisted for at least 1 h.Furthermore,i.t.treatment of 10 nmol BAM22 evidently decreased the expressions of CFA-evoked neuronal nitric oxide synthase(nNOS)-positive cells and calcitonin gene-related peptide(CGRP)-immunoreactivity positive nerve fibers by 25.6%(P 0.01) and 25.2%(P 0.001) compared with saline group,respectively,at L3-L5 segments of the spinal cord.Small and medium CGRP-positive cells were 57.4% and 35.2% in dorsal root ganglion(DRG) in 10 nmol BAM22 group,respectively,which were remarkably lower than those in saline group(P 0.001).The present study suggests that BAM22 relieves CFA-induced thermal hyperalgesia in the early phase and resumes antinociceptive effects through down-regulation of nNOS and CGRP expressions in DRG and spinal cord,which is possibly mediated via SNSR.
The aim of the present study was to assess the ototoxicity of kanamycin sulfate(KM) in adult rats and its underlying mech-anism.Forty male Sprague-Dawley rats(6-7 weeks old) were randomly divided into the experimental group and the control group.The animals in the experimental group were injected subcutaneously with KM(500 mg/kg per day) for two weeks,and the control group received equal volume of normal saline.To assess the ototoxicity of KM,the auditory brainstem response(ABR) was recorded to monitor the changes in hearing thresholds,and the density of spiral ganglion cells(SGCs) and morphology of cochlea were ob-served using surface preparations and frozen sections of cochlea.The results showed that the hearing threshold of rats in the experimental group was elevated by more than 60 dB across all the frequencies two weeks after the first administration of KM.And in the experimental group,the density of SGCs became lower,and organ of Corti suffered loss of hair cells.The loss of outer hair cells(OHCs) was more severe than that of inner hair cells(IHCs),correlated with the density decrease of SGCs.We conclude that the ototoxicity of KM in the adult rats was apparent and the underlying mechanism is associated with the loss of SGCs and hair cells.
To explore the potential of the anti-sense nucleic acid of CyclinD1 in lung cancer therapy,the expression vector containing the anti-sense nucleic acid of CyclinD1 was constructed and named pcDNA3.1-CyclinD1.The A549 cells were transfected with pcDNA3.1-CyclinD1 vectors.After being screened by G418,the stable expression positive clones were obtained.MTT method and flow cytometry technique were used to detect cell proliferation and apoptosis,respectively.The results showed the transfected cells exhibited significantly increased apoptosis and inhibited cell growth,compared with negative control and empty vector groups.To investigate the mechanism for anti-sense nucleic acid of CyclinD1 inducing A549 cells apoptosis,the expression levels of retinoblastoma protein(pRb),adenovirus E2 factor-1(E2F-1),vascular endothelial growth factor(VEGF),matrix metalloproteinase(MMP)-2 and MMP-9 were detected by Western blot,and the results showed the expressions of these proteins were all decreased significantly in anti-sense nucleic acid of CyclinD transfected group,compared with those in negative control and empty vector groups.In a word,anti-sense nucleic acid of CyclinD1 induces the apoptosis of lung adenocarcinoma cancer cells,and the depressions of pRb,E2F-1,VEGF,MMP-2 and MMP-9 expressions may be the possible mechanism.
The purpose of this study was to establish an absolute quantitative method to detect IL-1β and Caspase-3 gene expressions in rat brain after cerebral ischemia-reperfusion(I/R) using real-time PCR.Rats were randomized into the following groups:sham operation group,model group(cerebral I/R group),astragaloside IV(AST IV) group,chuanxiongzine-AST IV group and nimodipine group(n = 10 in each group).The rats in all the groups except sham operation group were subjected to cerebral I/R treatment.Sham operation and model groups were treated by normal saline(5 mL/kg).AST IV,chuanxiongzine-AST IV and nimodipine groups received 20 mg/kg AST IV,10 mg/kg chuanxiongzine plus 20 mg/kg AST IV,and 10 mg/kg nimodipine treatments,respectively.The administrations of drugs were performed with intraperitoneal injections at 0 and 12 h,1 d,2 d,3 d,till to 7 d after I/R.A real-time quantitative PCR assay was developed for absolute quantification of the expressions of IL-1β and Caspase-3 genes.The absolute quantification approach relies on the construction of an accurate standard curve.Thus,two plasmids which contained rat IL-1β and Caspase-3 genes respectively were constructed.The cloned circular plasmids were then quantified using a spectrophotometer and used as standards.Standard curves were generated,and the copy numbers of IL-1β and Caspase-3 mRNA isolated from I/R-damaged brain tissue were also calculated by SYBR Green I dye method using specific primers.The results showed that melting curves exhibited sharp peaks,and PCR product also generated prominent band with expected size in agarose gel electrophoresis,which validated the optimization of the selected primer sets of IL-1β and Caspase-3 genes.The optimal annealing temperatures of IL-1β and Caspase-3 genes were 59 °C and 61.2 °C,respectively.Real-time PCR results showed that the expression of IL-1β and Caspase-3 genes in the model group was significantly elevated compared to that in the sham operation group.However,compared to those in the model group,IL-1β and Caspase-3 gene expressions were obviously decreased in AST IV,chuanxiongzine-AST IV and nimodipine groups.Especially in chuanxiongzine-AST IV group,those two genes showed the most significant expression down-regulation.These results suggest the absolute quantitative method established in the present study is capable of detecting the changes of IL-1β and Caspase-3 gene expressions in rat brain damaged by I/R.
The present study aims to investigate the effects of soluble endoglin (sEng) on invasive ability of cultured cytotrophoblasts of first trimester of pregnancy. Cytotrophoblasts of normal 6 to 8-week pregnancy were cultured by trypsin digestion method, and were incubated with cell culture medium without (control group) and with 10 μg/L sEng (sEng group), respectively for 24 h. The invasive ability was determined by transwell invasion assay, and expressions of MMP-2, MMP-9 mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The results showed that the invasive ability of cytotrophoblasts in sEng group was lower than that in control group (P < 0.05). Compared with control group, the expressions of MMP-2 and MMP-9 mRNA and protein of cytotrophoblasts were significantly lower (P < 0.05). In conclusion, sEng may participate in the genesis of preeclampsia by affecting the invasive ability of cytotrophoblasts through regulation of the expression of MMP-2 and MMP-9.
The aim of this study was to investigate the influence of endothelial like cells differentiated from rat bone marrow mesenchymal stem cells (rBMSC-ECs) on angiogenesis and the effect of Rho kinase (ROCK) inhibitor using an in vitro model of cells co-cultured with rat aorta ring. Cell proliferation capability was detected by MTT method. The rBMSC-ECs were co-cultured with rat aorta ring in rat tail collagen and endothelial medium. A ROCK specific inhibitor, HA-1077 at different concentrations (0, 10, 30 and 60 mmol/L, respectively) was added into the medium of ring-cell co-culture. The protein expression of ROCK I and ROCK II were detected by Western blot. On the third day of cultivation, the branch number of neogenetic microvessels increased by 34.5% in ring-cell co-culture group compared with that in simple aorta ring group (P < 0.01). Compared with that in ring-cell co-culture group, it was significantly decreased by 57.70%, 64.13% and 48.23% respectively in three concentrations of HA-1077 groups (all P < 0.01). However, on the sixth day, rBMSC-ECs proliferated and migrated to the nearby aorta ring, and the growth of microvessels became slow. On the ninth day, some of neogenetic microvessels were retracted, some became thicken, coarsen and lengthen, and some of rBMSC-ECs were sprouting and forming capillary like picture. The protein expression of ROCK I/II was slightly higher in ring-cell co-culture group than that in simple aorta ring group. But, in three concentrations of HA-1077 groups, it was slightly lower than that in ring-cell co-culture group. By using rhodamine-phalloidin staining and laser scanning confocal fluorescence microscope, it showed that there were a lot of the F-actin cytoskeletons in neogenesis microvessels of aorta ring, and there were a lot of thick and long stress fibers in the cells. F-actin-rich surface protrusions at the leading edge of the cell were also shown. Another ROCK inhibitor, Y-27632 (10 μmol/L) induced the actin cytoskeleton reorganization: F-actins appeared to be peripheral fibers at outer area of cell; stress fiber and filopodia disappeared. These results suggest that rBMSC-ECs themselves can be differentiated into new microvessels and facilitate angiogenesis when they are co-cultured with rat aorta ring. The mechanisms involve ROCK activation and F-actin cytoskeleton recombination.
