罗格列酮对人子宫肌瘤细胞凋亡和雌激素受体表达的影响

目的:探讨罗格列酮对人子宫肌瘤细胞增殖和凋亡的影响及可能机制.方法:不同浓度的罗格列酮(10-8,10-7和10-6 mol/L)处理原代培养的人子宫肌瘤细胞24,48和72 h.用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)法测定细胞生长曲线,用流式细胞术测定细胞凋亡率,采用放射...     

Concentration-dependent effects of a selective estrogen receptor modulator raloxifene on proliferation and apoptosis in human uterine leiomyoma cells cultured in vitro

BACKGROUND: This study was conducted to elucidate the effects of raloxifeneon proliferation and apoptosis in cultured human uterine leiomyomacells. METHODS: The monolayer cultures were treated with graded concentrations(10^–9, 10^–8 and 10^–7 M) of raloxifeneand 10^–7 M 17-estradiol (E₂). Cell viability, percentageof proliferating cell nuclear antigen (PCNA)-positive cells,percentage of terminal deoxynucleotidyl transferase-mediated2'-deoxyuridine 5'-triphosphate nick-end labelling (TUNEL)-positivecells and the expression of PCNA and Bcl-2 proteins were assessedby 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxylphenyl)-2-(4-sulphophenyl)-2H-tetrazolium assay, immunocytochemistry, TUNEL assay and western blotanalysis, respectively. RESULTS: Compared with untreated cultures, the number of viable culturedcells, percentage of PCNA-positive cells and PCNA protein expressionwere significantly decreased by treatment with 10^–9 Mraloxifene, but increased by treatment with either 10^–8 Mor 10^–7 M raloxifene. In contrast, the percentageof TUNEL-positive cells was significantly increased and Bcl-2protein expression was significantly decreased by treatmentwith 10^–9 M raloxifene, whereas they were not affectedby treatment with either 10^–8 or 10^–7 M raloxifene. CONCLUSIONS: In cultured leiomyoma cells, low concentration (10^–9 M)of raloxifene may inhibit the growth of leiomyoma cells, whereashigh concentrations (10^–8 M, 10^–7 M) ofraloxifene may promote their growth.     

左炔诺孕酮下调survivin基因促进人子宫肌瘤细胞凋亡

目的探讨左炔诺孕酮(LNG)诱导人子宫肌瘤细胞(UtLMC)凋亡过程中survivin的表达变化。方法原代培养人子宫肌瘤细胞传代后,加入不同浓度LNG,以AO/EB双染法区分早、晚期凋亡细胞和坏死细胞;RT-PCR检测抑凋亡基因survivin mRNA的表达,Western blot测定survivin蛋白的表达。结果 10 mg/L LNG作用UtLMC后早期凋亡细胞多于阴性对照组,随着LNG剂量的增加,晚期凋亡细胞也逐渐增多,核浓聚、偏位,被染成桔红色;在10 mg/L以上LNG诱导的UtLMC凋亡中,survivin mRNA表达显著下降,蛋白表达由对照组的33.82±0.02下降至10.37±0.03(P<0.05)。结论一定浓度的左炔诺孕酮所诱导的人子宫肌瘤细胞凋亡,可能与抑制survivin抗凋亡活性相关。     

Prostanoid receptors in human uterine myocytes: the effect of reproductive state and stretch

In the human, prostanoids are known to be important mediators of uterine relaxation and contraction during pregnancy and parturition. We have previously shown that stretch of uterine smooth muscle cells increased prostaglandin H synthase 2 (PGHS-2) mRNA expression, PGHS-2 peptide synthesis and activity, however, the net effect on uterine contractility of this increase in prostaglandin synthesis would be determined by the expression of the different prostanoid receptors. Therefore, the aims of this study were to establish the expression of prostanoid receptor mRNA in uterine myocytes obtained from women in different reproductive states and to test the hypothesis that stretch of uterine myocytes alters prostanoid receptor mRNA expression to promote uterine contractility. Myocytes were isolated from women undergoing hysterectomy (NP) and pregnant women undergoing LSCS either before (NL) or after the onset of labour (L) and were subjected to 11% stretch for 1 h (n = 6 in all cases). Copy numbers of the individual receptors varied widely with reproductive state but followed the pattern: FP > IP = DP = EP-4 > TP = EP-3 = EP-2 > EP-1. FP mRNA expression was significantly lower in the NL group compared to the NP group and EP-3, EP-4 and TP mRNA expression was significantly lower in both NL and L groups compared to NP group levels. The level of mRNA expression of EP-1, EP-2, DP and IP did not differ between NP, NL and L groups. Stretch of cells derived from the NP group resulted in a significant decrease in EP-4 mRNA expression alone and of the NL group a significant decrease in EP-2 mRNA expression alone. Stretch had no effect on cells derived from the L group. These data show that pregnancy is associated with a significant reduction in 3 of 4 pro-contraction associated prostanoid receptor mRNA expression and 1 of 4 pro-relaxant. Stretch elicited no consistent change in prostanoid receptor mRNA expression.     

Dual repressive effect of angiotensin II-type 1 receptor blocker telmisartan on angiotensin II-induced and estradiol-induced uterine leiomyoma cell proliferation

BACKGROUND: Although uterine leiomyomas or fibroids are the most common gynecological benign tumor and greatly affect reproductive health and well-being, the pathophysiology and epidemiology of uterine leiomyomas are poorly understood. Elevated blood pressure has an independent, positive association with risk for clinically detected uterine leiomyoma. Angiotensin II (Ang II) is a key biological peptide in the renin-angiotensin system that regulates blood pressure. METHODS: In this study, we investigated the potential role of Ang II (1-1000 nM) in the proliferation of rat ELT-3 leiomyoma cells in vitro. RT-PCR and western blot analysis with cell proliferation and DNA transfection assays were performed to determine the mechanism of action of Ang II. RESULTS: Ang II induced ELT-3 leiomyoma cell proliferation (P < 0.01) and the expression of Ang II type 1 receptor (AT(1)R) and AT(2)R mRNA and protein was confirmed. Regarding the intracellular signaling pathway, the Ang II-induced cell proliferation was AT(1)R-, epidermal growth factor receptor-, extracellular-regulated kinase- and protein kinase C-dependent but was not dependent on the AT(2)R or phosphatidylinositol-3 kinase or JAK kinase. The AT(1)R blocker telmisartan, effectively repressed Ang II-induced and estradiol-induced cell proliferation (P < 0.01). AT(1)R, but not AT(2)R, plays a role in Ang II-induced ELT-3 cell proliferation. CONCLUSIONS: These experimental findings in vitro highlight the potential role of Ang II in the proliferation of leiomyoma cells.     

Comparative effects of heparin-binding epidermal growth factor-like growth factor on the growth of cultured human uterine leiomyoma cells and myometrial cells

BACKGROUND: The objective of this study was to investigate the comparative effects of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on the growth of cultured human leiomyoma cells and myometrial cells. METHODS: Isolated cells were subcultured in Phenol Red-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions for an additional 24 and 48 h in the presence or absence of graded concentrations of HB-EGF (0.1, 1, 10 and 100 ng/ml). These cells were used for immunocytochemical analysis for Ki67, western blot analysis for proliferating cell nuclear antigen (PCNA) and human EGF receptor (HER1), and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay. RESULTS: Treatment with HB-EGF at concentrations >1 ng/ml significantly increased the Ki67-positive rate of cultured leiomyoma cells and myometrial cells. Treatment with HB-EGF also resulted in a dose-dependent increase in PCNA expression in both cells compared with untreated control cultures. A significant increase in PCNA expression in cultured myometrial cells was noted following treatment with HB-EGF at concentrations >1 ng/ml, whereas an increase in PCNA expression in cultured leiomyoma cells was noted following treatment with HB-EGF at concentrations >10 ng/ml. HER1 expression was significantly higher in untreated myometrial cells than in untreated leiomyoma cells. A significant increase in HER1 expression in myometrial cells was observed when treated with HB-EGF at concentrations >10 ng/ml, whereas a significant increase in HER1 expression in leiomyoma cells was noted only by the treatment with HB-EGF at concentrations >100 ng/ml. Treatment with HB-EGF decreased the TUNEL-positive rate of those cells with no significant differences between the two cell types. CONCLUSIONS: The results obtained suggest that HB-EGF plays a role in stimulating the proliferation of leiomyoma cells and myometrial cells and in inhibiting apoptosis of those cells through augmentation of HER1 expression. Since the proliferative potential of myometrial cells responded better to HB-EGF than that of leiomyoma cells, HB-EGF may play a more vital role in myometrial growth than leiomyoma growth.     

Vascular smooth muscle cell proliferation in arterioles of the human endometrium

Menorrhagia affects approximately 15% of all women, often without identifiable cause. Endometrial spiral arterioles are believed to play a major role in controlling menstruation, and are a major site of menstrual loss. We postulate that alterations in the growth and development of spiral arterioles, particularly the vascular smooth muscle cells (VSMC), may contribute to menorrhagia. We examined VSMC proliferation around endometrial arterioles in control and menorrhagic tissues and the possible roles of transforming growth factor beta (TGF-beta) and endothelin in this process. Proliferating VSMC were located immuno-histochemically, then evaluated using computer-aided image analysis. VSMC proliferation was low and constant during the early stages of the menstrual cycle, increasing at the mid to late secretory stages (P < 0.002). Menorrhagic women had significantly reduced VSMC proliferation in their spiral arterioles at the mid and late secretory stages (P < 0.02). VSMC around straight arterioles proliferated at similar rates across the cycle, apart from a significant decrease in VSMC proliferation in menorrhagic women at the late secretory stage (P < 0.002). Endothelin concentrations decreased significantly in the epithelium of menorrhagic women (P = 0.05), while TGF-beta demonstrated no significant differences in the mid to late secretory tissues studied. The results indicate a significant functional difference between the spiral arterioles of control and menorrhagic women that may play a role in menorrhagia, while leaving the roles of endothelin and TGF-beta undetermined.     

Uterus and endometrium: In-vitro model of uterine leiomyomas: formation of ball-like aggregates

To clarify the biological characteristics of uterine leiomyomas,cells explanted and cultured from uterine leiomyomas and fromnormal myometrlal tissue were observed by time- lapse cinemicrographyand phase-contrast microscopy. The histological characteristicswere evaluated by electron microscopy and immunofluorescencemicroscopy, and these observations revealed significant differences.By time-lapse cinemicrography, the cells cultured from leiomyomasand myometriuin differed in their behaviour. Cells from themyometrium started to grow in parallel with the cell's majoraxis and formed topographically uniform hills and valleys byday 21 of culture. In contrast, the cells from leiomyomas startedto grow irregularly, as if having no contact inhibition, andformed ball-like aggregates of cells by day 21 of culture. Theaggregates resembled the nodules of leiomyoma in vivo. Ultrastructurally,cells from both leiomyomas and myometrium had typical featuresof smooth muscle. Immunofluorescently, a different distribution of -smooth muscle actin-positive filaments and differentstaining of cellular fibronectin and N-cadherin between thecells from Ieiomyomas and myometrium were observed, which maycontribute in part to the different behaviour of the cells.Given that the explant cell culture system resembles the featuresof uterine leiomyomas in vivo, this suggests that it can beused as an in-vitro model.     

阿司匹林对高糖和高胰岛素诱导的血管平滑肌细胞增殖的影响

目的：探讨阿司匹林对高糖和高胰岛素诱导的大鼠血管平滑肌细胞(VSMCs)增殖的影响及相关机制。方法：组织贴块法培养原代大鼠胸主动脉平滑肌细胞, 用高糖和高胰岛素诱导细胞增殖。实验设对照组、高糖和高胰岛素组(HGI)、HGI +阿司匹林（0.5,1.25,2.50 mmol/L）组。采用四甲基偶氮唑盐(MTT)比色法测定VSMCs增殖;一氧化氮(NO)试剂盒检测培养基上清液NO含量;流式细胞仪检测细胞周期。结果：阿司匹林呈剂量依赖性地抑制高糖和高胰岛素诱导的动脉VSMCs增殖,最大抑制作用出现在2.50 mmol/L阿司匹林培养5 d时(P<0.01);阿司匹林（2.50 mmol／L）干预可使高糖和高胰岛素导致下降的NO含量上升 (P<0.01);与高糖和高胰岛素组比较,阿司匹林(2.50 mmol/L)可使细胞静止期/DNA合成前期(G0/G1期)的VSMCs所占比例显著升高(P<0.05),而DNA合成期(S 期)细胞所占比例显著降低(P<0.05)。结论：阿司匹林能呈剂量依赖性地抑制高糖和高胰岛素诱导的动脉VSMCs的增殖,抑制细胞在G0/G1期,促进动脉平滑肌细胞NO的产生。     

The influence of extracellular matrix proteins on T-cell proliferation and apoptosis in women with endometriosis or uterine leiomyoma

PROBLEM: Interactions between the extracellular matrix (ECM) and peripheral blood T cells in women with endometriosis and leiomyoma are hardly unknown. We have investigated the influence of two major ECM components, collagen IV (C-IV) and fibronectin (Fn), on T-cell proliferation and apoptosis in women with endometriosis and uterine leiomyoma. beta1 integrin expression, responsible for interactions with ECM proteins, was also studied. METHOD OF STUDY: Peripheral blood lymphocytes were obtained from 53 women (17 with uterine leiomyomas, 18 with endometriosis, and 18 from healthy donors). T cells were exposed to ECM proteins co-immobilized with monoclonal antibody anti-CD3 for 72 hr. Apoptosis and S phase of the cell cycle of the T cells were studied by DNA analysis using flow cytometry. The proliferation of T cells was evaluated by MTT assay. The percentage of CD3+ cells expressing CD29 (beta1 integrin chain) was evaluated by double-color flow cytometry. Results were analyzed statistically using the Mann-Whitney test. RESULTS AND CONCLUSIONS: (1) A general increase in the percentage of T cells in S phase could be seen in women with endometriosis and uterine leiomyoma in all culture conditions what may suggest general activation of T cells. (2) A significant increase in the percentage of cells in S phase was shown only in the case of T cells exposed to anti-CD3 + C-IV in both women with uterine leiomyoma and endometriosis. (3) However, no apoptotic cells were observed. (4) T cells from patients with uterine leiomyoma exhibited significantly increased level of proliferation after culture with anti-CD3 + C-IV. (5) More T cells expressed beta1 integrin in women with endometriosis or uterine leiomyoma than in healthy donors. Our data may suggest that increased beta1 integrin expression may enhance T-cell-ECM interactions, which may be responsible for the increased proliferation of T cells but not for apoptosis. Therefore, it is possible that interactions of T cells with ECM proteins, especially with C-IV, may contribute to the pathogenesis of endometriosis and uterine leiomyoma.     

Growth dynamics of human leiomyoma cells and inhibitory effects of the peroxisome proliferator-activated receptor-gamma ligand, pioglitazone

Uterine leiomyomas (fibroids) are the most frequent tumour of the female reproductive tract and are the primary cause of hysterectomies in women worldwide. Effective treatment options are few. In a search for alternative treatments, we have established primary cultures of human leiomyoma cells and adjacent myometrial tissues, and documented their growth dynamics in response to estradiol (E2) and pioglitazone (PIO), a peroxisome proliferation-activated receptor-gamma (PPARgamma) ligand, currently in clinical use for type II diabetes mellitus. Human uterine primary cell cultures display morphology and desmin content consistent with their smooth muscle origin. Surprisingly, leiomyoma cells exhibited slower proliferation patterns relative to matched myometrial cells, both in the absence and presence of E2, suggesting that tumour genesis may not be because of increased growth potential but could be related to suppression of growth-inhibiting factors in vivo. PIO significantly inhibited the cell proliferation of both myometrial and leiomyoma cells in a dose-dependent manner. Our results suggest the possibility of using PPARgamma ligands, such as PIO, as therapeutic agents for the conservative management of uterine fibroids.     

Proliferative effects of eosinophil lysates on cultured human airway smooth muscle cells

BACKGROUND: The hypertrophy/hyperplasia of airway smooth muscle (ASM) cells is one of the characteristic features of bronchial asthma. This structural change leads to the thickening of airway walls resulting in the amplification of airway narrowing. However, the pathogenesis of this structural change has not yet been determined. Eosinophils, which play a pathogenic role in asthma, have been demonstrated to have proliferative effects on fibroblasts and vascular smooth muscle cells. OBJECTIVE: We attempted to investigate the potential of eosinophils to induce the proliferation of ASM cells. METHODS: We examined the effect of lysates of eosinophils purified from peripheral blood of healthy donors on cultured human ASM cell proliferation. RESULTS: Eosinophil lysates significantly induced ASM cell proliferation in time- and dose-dependent manners, reaching a maximum on day 6 at 50% of eosinophil lysates (6.0 +/- 0.7 x 104 [mean +/- SD] /well, n = 5 vs. 4.5 +/- 1.1 x 104/well, n = 5; P < 0.05). This proliferative activity was heat-sensitive and recovered in the soluble fraction of the eosinophil lysates. Furthermore, the molecular weight of the mitogenic activity in the soluble fraction was identified as lower than 10 kDa. The inhibitory activity to ASM cell proliferation was also found in the insoluble fraction of the lysates. CONCLUSION: These results indicate that circulating eosinophils store mitogenic activity for ASM cells, suggesting that eosinophils might contribute to the development of the hyperplasia of ASM cells in asthmatics through the release of the stored mitogenic activity upon stimulation at the site of inflammation.     

Acute and chronic effects of genistein, tyrphostin and lavendustin A on steroid synthesis in luteinized human granulosa cells

BACKGROUND: Phytoestrogens, including genistein and other inhibitors of tyrosine kinases (TKs), inhibit specific steroidogenic enzymes. This study was designed to compare the effects of genistein, with two other TK inhibitors, on steroid synthesis in human granulosa luteal (GL) cells and to identify which steroidogenic enzymes they may affect. METHODS: GL cells, obtained from women undergoing IVF procedures, were cultured for various periods of time with and without substrates for progesterone and estradiol synthesis, in the presence or absence of the TK inhibitors. RESULTS: The TK inhibitors significantly suppressed progesterone and estradiol synthesis in a dose-dependent manner over a 48 h culture period. Progesterone production in the presence of 10(-7) mol/l pregnenolone during a 4 h period was inhibited by both acute (4 h) and chronic (24 h) exposure of GL cells to 50 micromol/l genistein (P < 0.05) whilst no significant effects of 50 micromol/l tyrphostin A23 were observed. Genistein (4 and 24 h exposure) inhibited the production of estradiol using 10(-7) mol/l estrone as a substrate, but inhibition of estradiol synthesis using androstenedione or testosterone as substrates was only observed after a 24 h exposure. In contrast, tyrphostin acutely stimulated estradiol synthesis when androstenedione and testosterone were used as substrates (P < 0.05) but not estrone. CONCLUSIONS: Genistein directly inhibits 3 and 17beta-hydroxysteroid dehydrogenase activity, whilst tyrphostin has an acute stimulatory effect on aromatase activity. Over a longer time (24 and/or 48 h period), both TK inhibitors suppress steroid synthesis.     

Leiomyoblastoma of the uterus: an immunohistochemical and electron microscopic study of distinctive tumours with immature smooth muscle cell differentiation mimicking fetal uterine myocytes

AIMS: We present three distinctive uterine tumours which exhibited immature smooth muscle differentiation mimicking smooth muscle cells of the fetal uterus. METHODS AND RESULTS: The patients were 45, 46 and 49 years old, and all of them had simple hysterectomies. Grossly, all tumours were present in the uterine body, and two of the three tumours were well demarcated 60-mm and 85-mm lesions, and the other tumour was a small 25-mm incidental lesion within multiple conventional leiomyomas. The tumours had varied histological features and were composed of round epithelioid, rhabdoid and large vacuolated cells intermingled with spindle-shaped cells to various degrees. Although their round vesicular nuclei showed mild to moderate variation in size, prominent nuclear atypia was not seen. Necrosis and mitotic figures suggesting biological aggressiveness were not present in any of the tumours. Immunohistochemically, tumour cells were intensely positive for desmin and alpha-smooth muscle actin, whereas positivity for heavy molecular weight caldesmon was restricted. In addition, two cases were positive for non-muscle myosin heavy chain (SMemb). Ultrastructurally, most tumour cells contained various amounts of intermediate filaments which were occasionally abundant and aggregated as in rhabdoid cells. Well-developed myofilaments with focal densities were observed in only a few tumour cells. Intermediate filaments and bundles of thin filaments without dense bodies were often intermingled and they occasionally formed distinctive complexes with many irregular dense body-like structures and crystalloid bodies. Other cytoplasmic organelles including rather rich mitochondria, some rough endoplasmic reticulum and free ribosomes were also common. CONCLUSIONS: These findings support their immature smooth muscle cell differentiation which mimics the mesenchymal cells of fetal uterus during 14-26 weeks of gestation. The term 'uterine leiomyoblastoma' is thought to be appropriate for describing these distinctive immature smooth muscle tumours.     

Effect of human endometrial stromal cell-derived conditioned medium on uterine natural killer (uNK) cells' proliferation and cytotoxicity

Citation Chen Y, Zhuang Y, Chen X, Huang L. Effect of human endometrial stromal cell‐derived conditioned medium on uterine natural killer (uNK) cells’ proliferation and cytotoxicity. Am J Reprod Immunol 2011; 65: 589–596 Problem Human endometrial stromal cells are involved in the regulation of immune cell proliferation, apoptosis, differentiation, and function. In the endometrium, uNK cells are in close contact with stromal cells. The aim of the study was to investigate the effects of human endometrial stromal cells on uNK‐cell proliferation and uNK‐cell cytotoxicity. Method of study The conditioned medium was derived from the endometrial stromal cells in the proliferative phase, secretory phase, and early pregnancy. The effects of stromal cell‐derived conditioned medium on uNK‐cell proliferation and cytotoxicity were detected by mitochondrial lactate dehydrogenase‐based MTS staining and flow cytometry. Results The stromal cell‐derived conditioned medium in both secretory phase and early pregnancy significantly promoted uNK‐cell proliferation. Compared with the control group, the uNK‐cell cytotoxicity were significantly reduced by conditioned medium in the proliferative, secretory, and decidua groups, but there were no significant differences among these different physiological stages in the inhibiting ability. Conclusion Human endometrial stromal cells may be involved in the regulation of uNK‐cell functions through influencing proliferation and cytolytic activity.     

三氧化二砷抑制大鼠血管平滑肌细胞增殖并促进细胞凋亡

目的研究三氧化二砷(As2O3)对大鼠血管平滑肌细胞(VSMCs)的抑制增殖、促进凋亡及诱导细胞周期停滞的作用。方法经组织块贴壁法原代培养的大鼠VSMCs,应用MTT法、台盼蓝拒染法3、H-胸腺嘧啶胞苷掺入法测定VSMCs的细胞活力、生长及增殖;应用流式细胞仪、DNA梯带法分析细胞周期分布及凋亡;应用Western blot法检测凋亡相关基因产物P53及细胞周期蛋白激酶抑制蛋白P21waf1/cip1。结果1~16μmol/L As2O3对VSMCs细胞活力无明显影响,但可显著抑制细胞生长及DNA合成(P<0.05),并呈时间和剂量依赖性。8μmol/L As2O3可使细胞周期S期减少(P<0.05)、G0G1期增加(P<0.05),并出现sub-G1期凋亡峰。16μmol/L As2O3对VSMCs作用不同时间后出现典型的凋亡梯带样DNA片段。8μmol/L As2O3可显著上调VSMCs的P53及P21waf1/cip1蛋白产物(P<0.05),且呈时间依赖性。结论As2O3对VSMCs具有明确的抗增殖、促凋亡、阻滞细胞周期进程的作用,并且与P53、P21waf1/cip1表达上调密切相关。     

Airway smooth muscle proliferation and survival is not modulated by mast cells

Background Airway smooth muscle (ASM) hyperplasia and mast cell localization within the ASM bundle are important features of asthma. The cause of this increased ASM mass is uncertain and whether it is a consequence of ASM–mast cell interactions is unknown. Objective We sought to investigate ASM proliferation and survival in asthma and the effects of co‐culture with mast cells. Methods Primary ASM cultures were derived from 11 subjects with asthma and 12 non‐asthmatic controls. ASM cells were cultured for up to 10 days in the presence or absence of serum either alone or in co‐culture with the human mast cell line‐1, unstimulated human lung mast cells (HLMC) or IgE/anti‐IgE‐activated HLMC. Proliferation was assessed by cell counts, CFSE assay and thymidine incorporation. Apoptosis and necrosis were analysed by Annexin V/propidium iodide staining using flow cytometry and by assessment of nuclear morphology using immunofluorescence. Mast cell activation was confirmed by the measurement of histamine release. Results Using a number of techniques, we found that ASM proliferation and survival was not significantly different between cells derived from subjects with or without asthma. Co‐culture with mast cells did not affect the rate of proliferation or survival of ASM cells. Conclusion Our findings do not support a role for increased airway smooth proliferation and survival as the major mechanism driving ASM hyperplasia in asthma. Cite this as: D. Kaur, F. Hollins, R. Saunders, L. Woodman, A. Sutcliffe, G. Cruse, P. Bradding and C. Brightling, Clinical & Experimental Allergy, 2010 (40) 279– 288.     

Mulberry polyphenols ameliorate atherogenic migration and proliferation by degradation of K-Ras and downregulation of its signals in vascular smooth muscle cell

Extra-proliferation and increased migration of vascular smooth cells con-tribute to the formation of atherosclerosis. Ras small G proteins play a critical role in the prolif-eration and migration of a wide range of cells. Mulberry, an economic fruit in Asia, exhibits anti-inflammation, anti-migration, and anti-oxidant properties. The mechanisms of action of mulberry extracts on K-Ras small G protein-induced proliferation and migration of vascular smooth muscle cell have not been extensively investigated. In this study, we explored the effects of mulberry polyphenol extracts (MPE) on the proliferation and migration of K-Ras-overexpressing A7r5 smooth muscle cells. The overexpression of K-Ras enhanced the ex-pression and activity of matrix metalloproteinase (MMP)-2, promoted vascular endothelial growth factor (VEGF) production, and eventually triggered the migration of A7r5 cells. Treatment with MPE attenuated K-Ras-induced phenomenon. In addition, MPE blocked K-Ras-induced actin fibril stress. MPE dose-dependently diminished K-Ras-induced Rho A, Rac1, CDC42, and phosphorylated focal adhesion kinase (FAK) expression. MPE elevated Rho B ex-pression. Phosphorylated AKT and glycogen synthase kinase (GSK) induced by K-Ras were also repressed by MPE treatment. MPE enhanced the interaction of IκB with NFκB. MPE restored the G0/G1 population and p21 and p27 expressions, which were repressed by K-Ras. Finally, MPE triggered the degradation of K-Ras by ubiquitination. MPE inhibited the migration and proliferation of vascular smooth cell through K-Ras-induced pathways and eventually pre-vented atherosclerosis.     

转染TFPI基因对人前列腺平滑肌细胞增殖的影响

目的良性前列腺增生（BPH）是严重危害老年男性健康的常见疾病,本研究旨在研究组织因子途径抑制因子（TFPI）基因对前列腺平滑肌细胞生长的影响,为良性前列腺增生的基因治疗提供参考依据。方法取前列腺增生患者手术切除的前列腺组织,采用酶消化法分离前列腺平滑肌细胞;免疫组化方法进行细胞鉴定;pIRES—TFPI基因转染前列腺平滑肌细胞,以pIRES基因作为基因转染阴性对照;用细胞计数法和四氮唑蓝MTT法观察细胞增殖情况;采用RT-PCR检测细胞内TFPI基因的表达情况。结果SMA免疫组化染色和MASSON染色显示：经过5次传代后,前列腺平滑肌细胞的纯度达到95％以上;TFPI基因转染后,前列腺平滑肌细胞内的TFPImRNA水平明显提高,是未转染组的7倍、阴性对照基因转染组的3．5倍;基因转染4d后,TFPI基因转染组的前列腺平滑肌细胞数明显低于阴性对照基因转染组。结论提示TFPI基因对前列腺平滑肌细胞的增殖具有调控作用,有必要对其作用机理进行进一步研究。