Effect of ginsenoside Rh2 on cultured PC-3M cells in vitro

AIM:To explore the influence of ginsenoside Rh₂ on cultured PC-3M cell cycle in vitro. METHODS:DNA content was measured using [³H]-TdR incorporation assay. To analysis the cycle changes of PC-3M cells, flow cytometry was used in the experiment.RESULTS:The results indicated that the rate of [³H]-TdR incorporation was lower in Rh₂-treated cells than the control in a dose-dependent manner. Flow cytometry demonstrated that the pecent of G₁ phase treated with Rh₂ was higher than that of control. CONCLUSION:These results suggested that PC-3M cells cultured with Rh₂ for 24 h were blocked at G₁ phase, and Rh₂ inhibited the growth of PC-3M cells in vitro.     

类番茄茄与番茄属间有性杂交的研究

用类番茄茄与番茄属的 9个种进行有性杂交 ,通过人工离体胚培养获得栽培番茄‘粤农二号’×类番茄茄的F1植株。杂种近于不育。用栽培番茄、类番茄茄、野生番茄及(栽培番茄×野生番茄 )与之回交及杂交 ,获得与栽培番茄的回交子一代及与秘鲁番茄、多腺番茄、潘那利番茄、粤农二号×秘鲁番茄F1的杂种 ,但它们的能育性很低。用栽培番茄、野生番茄与它们再回交及杂交 ,未能得到与栽培番茄的二次回交子一代 ,只获得 (粤农二号×类番茄茄 )× (粤农二号×秘鲁番茄 )的F1与秘鲁番茄、多腺番茄的复合杂种。人工苗期接种黄瓜花叶病毒 (CMV)结果表明 ,类番茄茄及其与番茄属的远缘杂种对CMV有较强的抗性。     

Effects of three kinds of calcium activator on the proliferation of cultured vascular smooth muscle cells in rats

AIM: To investigate the effects of intracellular free calcium ([Ca²⁺]i) from different resources on the proliferation mediated by mitogen activated protein kinase (MAPK) in vascular smooth muscle cells (VSMCs). METHODS: Cultured VSMCs were used in all experiments. Calcium influx was stimulated by angiotension Ⅱ(Ang Ⅱ). The release of intracellular calcium stores was induced by inositol trisphosphate (IP₃) and ryanodine (RY). MAPK activity was measured by [γ-³²P]-ATP incorporation MAPK protein expression by western blot, VSMCs proliferation by [³H]-Leucine ([³H]-Leu) and [³H]-Thymidine ([³H]-TdR) incorporation. RESULTS: Compared to the control VSMCs, Ang Ⅱ, IP₃ and RY significantly increased [Ca²⁺]i concentration activity of MAPK and its protein content in VSMCs. The promotion of [³H]-Leu and [³H]-TdR incorporation in VSMCs was also observed (P<0.01). CONCLUSION: The study indicated that calcium activator-induced increase in the activity and protein content of MAPK was involved in the proliferation of VSMCs, which was closely related to the [Ca²⁺]i concentration but independent to its origin.     

Effects of radiation from 188Re on smooth muscle cell proliferation

AIM: Although endovascular radiotherapy inhibits neointimal hyperplasia, the exact alterations induced by β-particles irradiation remain to be elucidated. The objective of this study was to investigate the ability and the cellular mechanism of local β^-particles emission from ¹⁸⁸Re to inhibit vascular smooth muscle cells (SMCs). METHODS: The SMCs in vitro were irradiated by ¹⁸⁸Re with single doses of 2.6 Gy-25.8 Gy. The effects of β-particles on SMCs, such as effective irradiate doses, the period of inhibition for SMCs proliferation, the changes of cell proliferation rate and DNA synthesis rate, cell cycle progression and related gene expression, were investigated by cell count, [³H]-TdR incorporation, cell cycle progression analysis, cell viability and immunocytochemistry, respectivecy. RESULTS: β-particles irradiation with dose of 5.2 Gy could inhibit significantly SMCs proliferation. At dose of 20.6 Gy DNA synthesis inhibitory rate was 92%, SMCs proliferation rate was only 3%. Renoval of ¹⁸⁸Re did not abolish the inhibitory effects of β-particles on SMCs proliferation. The expression of P53 was up regulation and PCNA was down regulation after irradiation. CONCLUSION: β-particles from ¹⁸⁸ Re was significantly effective and permanent in inhibiting SMCs proliferation, and inhibitory effect was in dose-dependet manner ED50was 5 Gy, the best dose to inhibit SMCs proliferation was 20 Gy. β-particles irradiation induced SMCs to occur G₀/G₁ arrest, damaged the ability of SMCs reproliferation and led to cell clonogenic death. P53 and PCNA had regulatiory effects on SMCs proliferation after β-particles irradiation.     

Effects of human growth arrest-specific homeobox gene delivery on proliferation,migration and cell cycle distribution of vascular smooth muscle cells

AIM: To explore a new gene therapeutic strategy for vein graft restenosis by investigating the effects of adenovirus-mediated human growth arrest-specific homeobox (Ad5-hGax) gene delivery on the proliferation, migration and cell cycle distribution of serum-induced rabbit vascular smooth muscle cells (VSMCs). METHODS: The recombinant adenovirus vector containing hGax gene was constructed and transfected into rabbit VSMCs. The expression of hGax in VSMCs was detected by RT-PCR and immunofluorescent staining. Methyl thiazolyl tetrazolium (MTT) assay was used to assess the effect of hGax over-expression on serum-induced proliferation of VSMCs. Wound healing method was applied to examine the distance of serum-induce VSMCs migration. Flow cytometry was employed to analyze the cell cycle distribution. RESULTS: The recombinant adenovirus vector Ad5-hGax was successfully constructed. The results of RT-PCR and immunofluorescent staining revealed that the hGax -transfected cells contained a 174 bp specific fragment of hGax gene and target protein 48 h after transfection. The proliferation of serum-induced VSMCs was significantly inhibited by overexpression of hGax gene as compared with control group. The migration of serum-induced VSMCs was inhibited after hGax gene delivery. Flow cytometry showed that 72 h after serum induction, the cells in G₀/G₁ phase in Ad5-hGax group were significantly increased, whereas the cells in G₂/M+S phase were significantly decreased. CONCLUSION: Overexpression of hGax gene inhibits the proliferation and migration of serum-induced rabbit VSMCs, and arrests the cells in G₀/G₁ phase. It is likely that hGax gene is a potential target for the gene therapy of vein graft restenosis.     

Taurine attenuated β-glycerophosphate-stimulated calcification in cultured rat vascular smooth muscle cells

AIM: To investigate the effect of taurine on calcification of vascular smooth muscle cells (VSMCs).METHODS: Calcified VSMCs of rat in vitro were induced by β-glycerophosphate. Cellular calcium content, alkaline phosphatase(ALP) activities and [⁴⁵Ca]accumulation were measured. DNA synthesis were evaluated by [³H]-thymidine ( [³H]-TdR) incorporation. RESULTS: Calcium content, ALP activities and [⁴⁵Ca]uptake of calcified VSMCs stimulated by taurine (5-20 mmol/L) were greatly decreased in a concentration-dependent manner as compared with calcified group (P＜0.01). Taurine also inhibited the proliferation of calcified cells in a concentration-dependent manner. Cell countingz, [³H]-TdR incorporation of calcified cells stimulated by taurine were greatly decreased as compared with calcified VSMCs (P＜0.01). CONCLUSION: It was demonstrated that calcification of VSMCs may be alleviated by taurine.     

Effects of p204 expression on expression of p21 and proliferation of vascular smooth muscle cells in rats

AIM: To observe the effect of interferon-inducible protein 204 (p204) on the expression of p21 and proliferation of vascular smooth muscle cells (VSMCs) in rats. METHODS: Interferon alpha (IFN-α) and small interference RNA (siRNA) targeting p204 gene ( Ifi204 ) was used to intervene cultured VSMCs in vitro instantaneously, then the cell vitality was determined by MTT assay to reflect the cell proliferation. The cell cycle was analyzed by flow cytometry. The expression of p204 and p21 at mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blotting. RESULTS: In rat VSMCs, IFN-α induced the increase in the expression of p204 at mRNA and protein levels, reduced the cell vitality and the G₁/S phase transition, and up-regulated the expression of p21 at mRNA and protein levels. Transfection of Ifi204 siRNA restrained the expression of p204 and p21, increased the cell vitality and promoted the G₁/S phase transition. CONCLUSION: The expression of p204 restrains the proliferation of rat VSMCs, probably by activating the expression of p21.     

Effect of fibronectin on proliferation and collagen synthesis in cardiac fibroblasts from spontaneously hypertensive rats

AIM:To investigate the effects of fibronectin (FN) on the proliferation and collagen synthesis in cultured rat cardiac fibroblasts (CFb) derived from SHR (CFb_SHR) and WKY (CFb_WKY). METHODS:CFb derived from 12-week-old spontaneously hypertensive rat (SHR) and WKY was cultured by outgrowth of tissue block. Cell proliferation of CFb was measured by cell number counting and[³H]-TdR incorporation using 24-well plates pre-coated with 5 μg/cm² of FN. Collagen synthesis was determined by [³H]-proline incorporation. RESULTS:As compared with control, the cell number of fibroblasts derived from SHR and WKY were significantly increased to 163.75% and 170.42% respectively after 72 h incubation with FN in the presence of 0.4% FCS from a intial cell density of 1×10⁴ cells/mL. DNA synthesis of CFb was markedly promoted by FN. FN induced an increased in [³H]-proline incorporation in both CFb_SHR and CFb_WKY. CONCLUSION:FN is able to promote cell proliferation and collagen synthesis of CFb derived both from SHR and WKY.     

Effects of human xeroderma pigmentosum group D gene on proliferation of human vascular smooth muscle cells induced by interleukin-6

AIM: To investigate the effects of human xeroderma pigmentosum group D (XPD) gene on the proliferation of human vascular smooth muscle cells (VSMCs) induced by interleukin-6 (IL-6). METHODS: Recombinant plasmid pEGFP-N2/XPD and vacant plasmid pEGFP-N2 were transfected into VSMCs by liposome, and then these cells were incubated with IL-6 at 1×10⁵ U/L for 48 h. The cells were divided into 6 groups: blank control group; pEGFP-N2 group; pEGFP-N2/XPD group; IL-6 group; IL-6 + pEGFP-N2 group; IL-6 + pEGFP-N2/XPD group. The expression of green fluorescent protein was observed under fluorescence microscope. The cell growth was detected by MTT method. The cell cycle and apoptosis rate were examined by flow cytometre. The expression levels of XPD, Bcl-2, Bax and wild type P53 (wt-P53) were detected by RT-PCR and Western blotting.RESULTS: Green fluorescence was observed in the cells transfected with pEGFP-N2/XPD or pEGFP-N2, indicating successful transfection MTT results showed that the transfection of pEGFP-N2/XPD inhibited the cell growth, and reduced the positive effects of IL-6 on VSMCs growth. Flow cytometry results showed that the transfection of pEGFP-N2/XPD increased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, decreased the cell numbers in S phase, and reduced the effects that IL-6 decreased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, and increased the cell numbers in S phase. The results of RT-PCR and Western blotting showed that the transfection of pEGFP-N2/XPD increased the expression of XPD, Bax and wt-P53, decreased the expression of Bcl-2, and reduced the effects that IL-6 decreased the expression of Bax and wt-P53, and increased the expression of Bcl-2. CONCLUSION: XPD gene inhibits VSMCs proliferation, promotes VSMCs apoptosis, and reduces the effects that IL-6 promotes VSMCs proliferation and inhibits VSMCs apoptosis. Therefore, XPD gene is likely to be potential molecular target for treatment of atherosclerosis.     

Inhibitory effect of Salidroside on the proliferation of rabbit pulmonary artery smooth muscle cells under hypoxia

AIM: To investigate the effect of Salidroside on the proliferation, DNA synthesis, intracellular Ca²⁺ content of rabbit PASMC (pulmonary artery smooth muscle cells) under hypoxia. METHODS: Techniques of cell culture, MTT test, [³H][³H][³H]-TdR incorporation, fluo-3 and confocal laser scanning microscopy were used. RESULTS: The A value of MTT and [³H][³H]-TdR incorporation of PASMC increased significantly by 62% (P＜0.05) and 138% (P＜0.01) after 24 h hypoxia. Salidroside (32×10^-5 mol/L) inhibited the action of hypoxia on the proliferation of PASMC, the A value of MTT and [³H][³H]-TdR incorporation declined significantly by 29% (P＜0.05) and 37% (P＜0.01) compared with hypoxia group. A calcium channel blocker, verapamil could also inhibit the accelerative effect of hypoxia on the proliferation of PASMC. The intracelluler Ca²⁺ content of PASMC raised markedly under hypoxia, but the effect of hypoxia on the intracelluler Ca²⁺ content could be inhibited by Salidroside. CONCLUSION: Salidroside inhibited the proliferation, DNA synthesis of PASMC induced by hypoxia. The inhibitory action of Salidroside on the increase in intracellular Ca²⁺ concentration under hypoxia might be one of the mechenisms.     

Effect of trichosanthes injection on expression of proliferating cell nuclear antigen of vascular smooth muscle cell in rabbit

AIM: To observe the effect of thichosanthes injection on the expression of proliferating cell nuclear antigen (PCNA) of vascular smooth muscle cell (SMC). METHODS: The expression of PCNA of cultured rabbit aortic SMC was examined with LSAB immunohistochemical technique, and [³H]-thymidine( [³H]-TdR) incorporation data of SMC and the contents of superoxide dismutase (SOD), lipid peroxide (LPO), prostacyclin (PGI₂) and cyclic AMP (cAMP) in medium were simultaneously determined. RESULTS: Thichosanthes injection has an effects of increasing SOD activity, decreasing LPO, elevating PGI₂ and cAMP, reducing [³H]-TdR incorporation and expression of PCNA (all P＜0.05,P＜0.01). CONCLUSION: Thichosanthes could inhibit SMC proliferation.     

Effect of interferon-inducible protein p204 on proliferation of vascular smooth muscle cells in rats

AIM: To study the effect of interferon-inducible protein p204 on the proliferation of vascular smooth muscle cells (VSMCs) in rats. METHODS: Cultured VSMCs were treated with interferon alpha (IFN-α) and p204 gene (Ifi204) small interfering RNA (siRNA) in vitro instantaneously. The cell vitality was detected by MTT me-thod,and the cell cycle was analyzed by flow cytometry. The expression of mRNA and proteins was determined by real-time qRT-PCR and Western blotting,respectively.RESULTS: IFN-α induced the increase in the expression of p204 at mRNA and protein levels, reduced the cell vitality, inhibited the cell cycle of G₁/S transition, and down-regulated the expression of Ras protein in VSMCs. Meanwhile, the phosphorylation levels of Raf and ERK were decreased. Transfection of Ifi204 siRNA restrained the expression of p204, increased the cell vitality and promoted the cell cycle of G₁/S transition in VSMCs. The up-regulation of Ras protein expression and the increased phosphorylation levels of Raf and ERK were also observed.CONCLUSION: The expression of p204 restrains the proliferation of VSMCs in rats by inhibiting the activation of Ras/Raf/MEK/ERK signal pathway.     

Effects of IL-32γ on proliferation and cell cycle of rat vascular smooth muscle cells

AIM: To observe the effects of interleukin-32γ (IL-32γ)on the proliferation and cell cycle of rat vascular smooth muscle cells (VSMCs). METHODS: The VSMCs were isolated from the thoracic aorta of SD rats by the method of tissue-piece inoculation. The cells were cultured and treated with different concentrations of IL-32γ. The proliferation of the cells was examined by MTT assay. The cell cycles were analyzed by flow cytometry. The protein levels of NF-κB p65 and cyclin D1 were detected by Western blotting. The expression of proliferating cell nuclear antigen (PCNA)was examined by immunocytochemical staining. RESULTS: Administration of IL-32γ at the concentrations of 10~50 μg/L for 24~48 h significantly promoted the proliferation of VSMCs in a dose- and time-dependent manner. After stimulation with IL-32γ at the concentration of 50 μg/L for 24 h, the cell cycle transition from G₁ phase to S/G₂ phase was accelerated and the expression levels of NF-κB p65, cyclin D1 and PCNA increased as compared with those in control group. CONCLUSION: IL-32γ promotes the proliferation of rat VSMCs and accelerates the cell cycle transition via upregulating the expression of NF-κB p65 and cyclin D1.     

Inhibitory effect of hydrogen sulfide (H2S) on cultured rat aortic smooth muscle cells

AIM:To explore the effects of hydrogen sulfide (H₂S) on proliferation of vascular smooth muscle cells (VSMC) stimulated by endothelin (ET-1, 10^-7mol/L) and mitrogen-activated protein kinase (MAPK) activity in VSMCs.METHODS:Cultured VSMCs were divided into six groups: (1) control group, (2) serum group, (3) endothelin group, (4) NaHS groups, (5) serum+NaHS group, and (6) endothelin+NaHS group. VSMC proliferation was measured by[³H]-TdR incorporation and MAPK activity in VSMC was determined by radioactivity assay.RESULTS:ET-1 increased VSMC[³H]-TdR incorporation by 2.39 times (P＜0.01) and MAPK activity by 1.62 times(P＜0.01), as compared with control. H₂S (5×10^-5-5×10^-4mol/L) decreased VSMC[³H]-TdR incorporation and MAPK activity by 16.8%-37.4% and 7.4%-33.6%, respectively (P<0.05 or P＜0.01).CONCLUSION:This study demonstrates that H₂S inhibits ET-1-induced proliferation of VSMC, which might be mediated by the inhibition of MAPK.     

Role of leukotriene D4 in promoting proliferation of cultured human airway smooth muscle cells

AIM: To investigate whether leukotriene D₄(LTD₄) would stimulates proliferation of cultured human airway smooth muscle (ASMC). METHOD: Human ASMC were isolated and subcultured, varying concentration of LTD₄ were added to the media. Cell counts were obtained, -thymidine([³H]-TdR) incorporation and inositol 1, 4, 5-trisphosphate (IP₃) accumulation were measured. RESULTS: LTD₄(0.1nmol·L^-1~10 nmol·L^-1) increased cell number and also increased incorporation of[³H]-TdR and accumulation of IP₃ in a concentration dependent manner(P＜0.01). The latter response was blocked by phospholipase C inhibition with neomycin (1 μmoL·L^-1(P＜0.01). However, neomycin had no effect on the promitogenic action of LTD₄. CONCLUSION: LTD₄ stimulates proliferation of cultured human ASMC and may play a role in airway remodeling of asthma.     

Effect of HMBA on proliferation and expression of KLF6 and related proteins in human tongue carcinoma Tca8113 cells

AIM: To study the effect of hexamethylene bisacetamide(HMBA) on the proliferation and expression of KLF6 and related proteins in human tongue carcinoma Tca8113 cells. METHODS: After cultured with HMBA, the growth of the Tca8113 cells was assayed by MTT method, and the morphology of the cells was observed under microscope. The cell cycle was determined by flow cytometry. The mRNA expression of KLF6 was detected by RT-PCR. The protein levels of KLF6, p53, cyclin D1 and c-Jun were measured by the method of immunohistochemistry. RESULTS: The number of adherent cells obviously decreased along with the concentration of HMBA, and the growth inhibition of Tca8113 cells was in a concentration/time-effect relationship after treated with HMBA. Some reversal features of the Tca8113 cells developed to normal cells in morphology after induced by HMBA. The proportion of the cells in G₁ phase was (52.00?0.02)% before treating with HMBA. The proportion of the cells in S phase was (34.00?0.08)%, and (14.00?0.10)% of G₂ phase cells. After treated with HMBA, the cell number in G₁ phase significantly increased with the exposure time going on, while the cell number in S phase significantly reduced, so did the cell number in G₂ phase. The cell cycle was significantly arrested in G₁ phase (P<0.05). The apoptosis peak also appeared. The mRNA expression of KLF6 significantly increased after induced by HMBA (P<0.05), so did the protein levels of KLF6 and p53 (P<0.05), while the expression of cyclin D1 and c-Jun was significantly decreased (P<0.05). CONCLUSION: HMBA inhibits the proliferation of Tca8113 cells by arresting the cell cycle in G₀/G₁ phase and resuming Tca8113 cells to normal and apoptosis at last.     

Effects of β3 integrin on adhesion and migration of vascular smooth muscle cells induced by growth factor

AIM: To investigate the effect of platelet-derived growth factor (PDGF) on β₃ integrin gene expression and the role of β₃ integrin on adhesion, migration and proliferation of vascular smooth muscle cells (VSMC) induced by PDGF. METHODS: β₃ integxin gene expression was detected by RT-PCr. After β₃ integrin extracellular do-main was blocks, VSMC adhesio, migration and proliferation were measured by adhesion assay awound-culture model an [³H]-TSR incorporation respectively.RESULTS: After the interaction between β₃ integrin and extracellular matrix was blocked, VSMC proliferation was inhibited in some degree and the rate of [³H]-TdR incorporation into VSMC decreased 39%. The cell adhesion and migration were significantly inhibited when 10 mg/L anti-β₃ integrin antibody was added (P＜0.05). When VSMC were treated by PDGF for 6 hours, the expression of β₃ integrin gene was 87% higher than that of control. CONCLUSION: PDGF significantly induces expression of β₃ integrin gene in VSMC, and the interaction between β₃ integrin and ECM protein may play an important role in VSMC adhesion and migration.     

Effect of calcitonin gene-related peptide on proliferation of vascular smooth muscle cells

AIM: To elucidate the effect of calcitonin gene-related peptide (CCRP) in the therapy of atherosclerosis.METHODS:Effect of CGRP on cell cycle kinetics of cultured vascular smooth muscle cells(HA-VSMC) was investigated by flow cytometry. The expression of cyclins D₁ and E required for initiation of S phase were also studied by immunochemistry method. RESULT: CGRP was shown to arrest VSMC in the G₀/G₁ phase of cell cycle and reduced expression of cyclins D₁ and E. CONCLUSION:CGRP inhibits proliferation of HA-VSMC by arresting cells in G₁ phase via limiting accumulation of cyclin D₁ and E. It might play a role in the therapy of atherosclerosis.     

Effect of Bmi-1 gene overexpression on proliferation of human gastric mucosal epithelial cell line GES-1

AIM: To investigate the effect of B lymphoma Moloney murine leukemia virus insertion region 1 ( Bmi-1 ) gene overexpression on the proliferation of a human normal gastric epithelial cell line GES-1. METHODS: The plasmid containing Bmi-1 gene or empty plasmid was transfected into GES-1 cells by retroviral mediation. The expression of Bmi-1 at mRNA and protein levels was detected by quantitative real-time PCR (qRT-PCR) analysis and Western blotting, respectively. The effect of Bmi-1 gene overexpression on the cell cycle of GES-1 cells was evaluated by flow cytometry. The proliferation of the stably transfected cells was measured by Cell Counting Kit-8. RESULTS: The results of qRT-PCR analysis and Western blotting demonstrated that stably transfected cell line was successfully established. The results of flow cytometry analysis showed that overexpression of Bmi-1 reduced the G₀/G₁ phase, arrested the cells in G₂/M phase and S phase. The growth curve showed that overexpression of Bmi-1 resulted in increased growth speed. CONCLUSION: Increase in Bmi-1 gene expression regulates the cell cycle and promotes the proliferation of GES-1 cells.     

Effects of adrenomedullin on proliferation of vascular smooth muscle cells induced by urotensin Ⅱ

AIM:To observe the effect of adrenomedullin(ADM)on proliferation of vascular smooth muscle cells(VSMC) induced by urotensin Ⅱ(UⅡ). METHODS:DNA synthesis of cultured rat aortic VSMC was measured by [³H]-TdR incorporation. The activities of mitogen activated protein kinase(MAPK) were determined by isotope tagged with [γ-³²P]-ATP. RESULTS:UⅡ(10^-8mol/L) significantly increased [³H]-TdR incorporation of VSMC and MAPK activities by 38%(P＜0.05) and 260%(P＜0.01) respectively compared with control group. Compared with UⅡ group, 10^-10,10^-9,10^-8mol/L ADM decreased [³H]-TdR incorporation of VSMC by 7%(P＞0.05), 32%(P＜0.05)and 41%(P＜0.01),respectively, and diminished MAPK activities by 24%(P＞0.05), 32%(P＜0.05)and 36%(P＜0.05),respectively. CONCLUSION:ADM inhibits proliferation of VSMC induced by urotensin Ⅱ through inhibiting MAPK activation.