Objective To investigate the effect of ursolic acid on the proliferation and apoptosis of human osteosarcoma cell line U2-OS and analyze its mechanism. Methods Human osteosarcoma cell line U2-OS was divided into 4 groups, which was cultured with ursolic acid of 0, 10, 20, and 40 μmol/L, respectively. At 0, 24, 48, and 72 hours after being cultured, the cell proliferation ability was detected by cell counting kit 8 (CCK-8). At 48 hours, the effects of ursolic acid on cell cycle and apoptosis of U2-OS cells were measured by flow cytometry. Besides, the expressions of cyclin D1 and Caspase-3 were detected by real-time fluorescent quantitative PCR and Western blot. Results CCK-8 tests showed that the absorbance (A) value of each group was not significant at 0 and 24 hours (P>0.05); but the differences between groups were significant at 48 and 72 hours (P<0.05). Flow cytometry results showed that, with the ursolic acid concentration increasing, the G1 phase of U2-OS cells increased, the S phase and G2/M phase decreased, and cell apoptosis rate increased gradually. There were significant differences between groups (P<0.05). Compared with the 0 μmol/L group, the relative expressions of cyclin D1 mRNA and protein in 10, 20, and 40 μmol/L groups significantly decreased (P<0.05); whereas, there was no significant difference in relative expression of Caspase-3 mRNA between groups (P>0.05). However, with the ursolic acid concentration increasing, the relative expressions of pro-Caspase-3 protein decreased and the relative expressions of activated Caspase-3 increased; there were significant differences between groups (P<0.05). Conclusion Ursolic acid can effectively inhibit the proliferation of osteosarcoma cell line U2-OS, induce the down-regulation of cyclin D1 expression leading to G0/G1 phase arrest, increase the activation of Caspase-3 and promote cell apoptosis.
Objective To observe the clinical features of congenital hypertrophy of retinal pigment epithelium （CHRPE）. Methods The clinical data of 13 CHRPE patients including visual acuity, slit-lamp microscope examination, indirect ophthalmoscope examination and fundus fluorescein angiography (FFA) were retrospectively analyzed. The patients, 9 males and 4 females, with the mean age of 27.8 years. Results All patients were unilateral, without systemic diseases and no subjective symptoms in majority. Only 30.77% of initial diagnosis was correct, other diagnosis include choroidal nevi, old chorioretinopathy or no diagnosis. The round or oval black lesion was found in ocular fundus of all patients, 7.69% was located on the optic disk, 46.15% was located on the inferior temporal retina, 30.77% was located on the superior temporal retina, 15.39％ was located on the inferior nasal retina. 92.31％ was pigmented CHRPE and 7.69％ was non-pigmented CHRPE. FFA showed blocked fluorescence and transmitted fluorescence in the lesion, few eyes were found dilated capillary vessel and fluorescent leakage on the late stage of FFA, most eyes had normal retinal vessels. Conclusion The isolated CHRPE is round or oval black lesion in ocular fundus which lack of subjective symptoms, mostly located on the peripheral retina; the FFA characteristics showed blocked fluorescence and transmitted fluorescence, and CHRPE often misdiagnosed as other disease, it should be combine the ocular fundus manifestation with the FFA to diagnose properly.
摘要:目的:探索槐耳清膏对体结肠癌SW480细胞增殖能力影响及其机制。方法:采用噻唑蓝(MTT)比色法检测槐耳清膏对SW480细胞增殖能力的作用,并探求最佳作用浓度;将体外培养细胞随机分为常氧组(NC组)、低氧组(HC组)和低氧槐耳组(HH组),逆转录聚合酶链反应（RTPCR）检测各组血管内皮生长因子(VEGF) mRNA表达水平,Western blot检测蛋白表达水平。结果:槐耳清膏对SW480细胞抑制率随药物浓度增加而上升,1 mg/mL时抑制率最大(66.7%),与氟尿嘧啶组(浓度为10 μg/mL)相比无统计学意义。HH组和HC组VEGF mRNA表达均显著高于NC组,分别为4.71±0.07,4.54±0.02和1.19±0.03(P＜0.05),但HH组与HC组比较差异无统计学意义。HC组VEGF蛋白表达显著高于NC组,分别为0.66±0.03和0.38±0.02(P＜0.05),HH组较HC组VEGF蛋白表达均显著下降,分别为0.37±0.03和0.66±0.03(P＜0.05)。结论:槐耳清膏可抑制SW480细胞增殖,1 mg/mL时抑制率最大。其机制为槐耳清膏下调细胞内VEGF蛋白表达,从而抑制肿瘤生长。Abstract: Objective: To investigate the effect of Huaier cream on proliferation of colon cancer cells SW480 and its mechanism. Methods: The proliferation was analyzed by MTT. SW480 cells were randomly divided into normoxic group (NC group), hypoxia group (HC group) and hypoxia group treated by Huaier (HH group). Levels of mRNA and protein expression of VEGF were detected by RTPCR and Western blot, respectively． Results: Huaier cream induced a dosedependent inhibition of SW480 cells. The maximum percentage of growth inhibition was 66.7% at a concentration of 1.0 mg/mL, but no significant difference was found compared to the positive control (5FU 10 μg/mL). VEGF mRNA levels were significantly higher in HC group and HH group than in NC group (4.71±0.07, 4.54±0.02 vs 1.19±0.03, all Plt;0.05), but not significantly different between HC group and HH group. VEGF protein expression was higher in HC group than NC group (0.66±0.03 vs 0.38±0.02, Plt;0.05). In HH group, VEGF protein was inhibited remarkably compared with HC group (0.37±0.03 vs 0.66±0.03, Plt;0.05). Conclusion: Huaier cream can significantly inhibit SW480 cells and the top inhibition concentration is 1.0 mg/mL. Huaier cream plays a role in inhibiting tumor through downregulating protein expression of VEGF.
目的 提高对成人系统性Epstein-Barr（EB）病毒阳性T细胞淋巴组织增殖性疾病（ASEBV+T-LPD）肠道病变的认识。 方法 报道2012年3月－10月我院收治的2例以肠道病变为首发表现的ASEBV+T-LPD，并结合3例文献报道进行分析讨论。 结果 2例成人患者以腹泻为主要表现，伴有发热、淋巴结长大，初期分别误诊为结核及肠道感染，淋巴结病检提示多克隆EBER+的T淋巴细胞浸润，确诊ASEBV+T-LPD。例1经3 周期GLIDE方案（吉西他滨+门冬酰胺酶+异环磷酰胺+地塞米松+依托泊苷）化学疗法（化疗）后疾病进展，死于肺部感染。例2经干扰素联合抗病毒药物治疗，病情稳定。复习文献目前仅3例以肠道病变为首发表现的ASEBV+T-LPD被报道，早期均被误诊为炎性肠病或感染，3例均出现肠道穿孔或大出血等并发症，其中2例死亡。 结论 成人系统性EB病毒阳性T细胞淋巴组织增殖性疾病的肠道病变较为罕见，容易误诊为结核及炎性肠病。部分患者病情进展迅速，可出现肠道穿孔及消化道大出血等致死性并发症，死亡率高，预后差。确诊本病需密切结合临床和病理学检查。常规化疗及抗病毒治疗仅部分有效，不能维持长期缓解。
ObjectiveTo investigate the influences of lactic acid (LA), the final degradation product of polylactic acid (PLA) on the prol iferation and osteoblastic phenotype of osteoblast-l ike cells so as to provide theoretical basis for bone tissue engineering. MethodsRos17/2.8 osteoblast-l ike cells were harvested and divided into 3 groups. In groups A and B, the cells were cultured with the medium containing 4, 8, 16, 22, and 27 mmol/L L-LA and D, L-LA, respectively. In group C, the cells were cultured with normal medium (pH7.4). The cell prol iferation was determined with MTT method after 1, 3, and 5 days. The relative growth ratio (RGR) was calculated, and the cytotoxicity was evaluated according to national standard of China. In addition, the alkal ine phosphatase (ALP) activity of cells cultured with medium containing 4 mmol/L L-LA (group A), 4 mmol/ L D, L-LA (group B), and normal medium (group C) after 1 and 5 days were detected with ALP kits, and the relative ALP ratio (RAR) was calculated; after 21 days, the calcium nodules were tested with von Kossa staining method, and were quantitatively analyzed. ResultsWhen LA concentration was 4 mmol/L, the mean RGR of both groups A and B were all above 80%, and the cytotoxic grades were grade 0 or 1, which meant non-cytotoxicity. When LA concentration was 8 mmol/L and 16 mmol/ L, groups A and B showed cytotoxicity after 5 days and 3 days, respectively. When LA concentration was above 22 mmol/L, cell prol iferations of groups A and B were inhibited evidently after 1-day culture. At each LA concentration, RGR of group A was significantly higher than that of group B at the same culture time (P<0.05) except those at 4 mmol/L after 1-day and 3-day culture. After 1 day, the RAR of group A was significantly higher than that of group B on 1 day (144.1%±3.2% vs. 115.2%±9.8%, P<0.05) and on 5 days (129.6%±9.8% vs. 78.2%±6.9%, P<0.05). The results of von Kossa staining showed that the black gobbets in group A were obviously more than those of groups B and C. The staining area of group A (91.2%±8.2%) was significantly higher than that of groups B (50.3%±7.9%) and C (54.2%±8.6%) (P<0.05). ConclusionThe concentration and composition of LA have significant effects on the cell proliferation and osteoblastic phenotype of osteoblast-l ike cells.
Objective To observe the effects of Galectin-3 on proliferation of vascular endothelial cells derived from peripheral blood endothelial progenitor cells. Methods The cultured peripheral blood endothelial progenitor cells in vitro were isolated and purified from human peripheral blood, and the cells were differentiated into vascular endothelial cells. Then the cells were cultivated with the galectin-3 of different concentrations, and to observe the proliferation of endothelial cells derived from peripheral blood endothelial progenitor cells. Results The abilities of proliferation of endothelial cells derived from peripheral blood endothelial progenitor cells of 0.1, 1.0, 2.5, 5.0, and 10.0 μg/ml groups were higher than that of 0 μg/ml group, there were not statistic significance of the differences between the 0.1，1.0, 2.5, and 0 μg/ml groups （P＞0.05）. But the abilities of proliferation of 5.0 and 10.0 μg/ml groups were obviously higher than that of 0, 0.1, 1.0, and 2.5 μg/ml groups （P＜0.05）, and the abilities of proliferation of 10.0 μg/ml group was also higher than that of 5.0 μg/ml group （P＜0.05）. Conclusion Galectin-3 can promote the proliferation of endothelial cells derived from peripheral blood endothelial progenitor cell.
Objective To observe the effect of epidermal growth factor (EGF) on integrin alpha;5 expression and its influence on human retinal pigment epithelium (RPE) cells.Methods Human RPE cells were treated in vitro with 0.1,1.0,10.0,20.0 and 100.0 ng/ml of EGF, the mRNA and protein of integrin alpha;5 was measured by reverse transcription polymerase chain reaction（RT-PCR）and flow cytometry. Human RPE cells were cultured under 4 conditions including DMEM/F12,DMEM/F12+10 ng/ml EGF, DMEM/F12+10 ng/ml EGF+rabbit antihuman integrin alpha;5 antibody (1∶100)，DMEM/F12+10 ng/ml EGF+rabbit antihuman vimentin antibody (1∶100), and their proliferation and migration were measured by methylthiazole tetrazolium（MTT）and Boyden chamber.Results The integrin alpha;5 mRNA level of human RPE cells was not changed after 12 hours of EGF stimulation (F=0.618, P=0.687), however it was induced in a dosedependent manner after 24 and 48 hours of EGF stimulation (F=465.303, 212.340; P=0.000,0.000).The protein level of integrinalpha;5 was higher in 10 ng/ml EGF stimulation compared with the control group and 0.1 ng/ml group（P＜0.01）.MTT and Boyden chamber showed that the integrin alpha;5 expression increased the proliferation and migration of human RPE cells. Conclusion EGF can induce integrin alpha;5 expression,thus increase the proliferation and migration of human RPE cells.
ObjectiveTo study the effects of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) on the proliferation and differentiation of human bone marrow mesenchymal stem cells (hBMSCs). MethodshBMSCs at passage 4 were divided into 4 groups according to different culture conditions:cells were treated with complete medium (α-MEM containing 10%FBS, group A), with complete medium containing 10 ng/mL LIF (group B), with complete medium containing 10 ng/mL bFGF (group C), and with complete medium containing 10 ng/mL LIF and 10 ng/mL bFGF (group D). The growth curves of hBMSCs at passage 4 in different groups were assayed by cell counting kit 8; cellular morphologic changes were observed under inverted phase contrast microscope; the surface markers of hBMSCs at passage 8 including CD44, CD90, CD19, and CD34 were detected by flow cytometry. ResultsThe cell growth curves of each group were similar to the S-shape; the cell proliferation rates in 4 groups were in sequence of group D > group C > group B > group A. Obvious senescence and differentiation were observed very early in group A, cells in group B maintained good cellular morphology at the early stage, with slow proliferation and late senescence; a few cells in group C differentiated into nerve-like cells, with quick proliferation; and the cells in group D grew quickly and maintained cellular morphology of hBMSCs. The expressions of CD44 and CD90 in groups A and C at passage 8 cells were lower than those of groups B and D; the expressions of CD19 and CD34 were negative in 4 groups, exhibiting no obvious difference between groups. ConclusionLIF combined with bFGF can not only maintain multiple differentiation potential of hBMSCs, but also promote proliferation of hBMSCs.
ObjectiveTo investigate the effects of thrombospondin-1 active fragment (TSP-1) synthetical peptide VR-10 on proliferation and migration of rhesus choroidal-retinal endothelial (RF/6A) cell and the expressions of apoptosis relative genes in RF/6A cell. MethodsThe survival rate of RF/6A cell were detected by methyl thiazolyl tetrazolium, and migration ability was measured by transwell chamber after exposure to 1.0 μg/ml TSP-1 and synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml) for different times (6, 12, 24, 48 hours). Caspase-3 and factor associated suicide (FAS) protein levels were measured by Western blot. The mRNA level of bcl-2 and FAS ligand (FASL) were measured by reverse transcription-polymerase chain reaction (RT-PCR). ResultsThe survival rate of RF/6A cells was determined by the treatment time and concentration of TSP-1(1.0 μg/ml) and the synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml). The lowest survival ratio of RF/6A was 78% (P < 0.001) when cells were treated by 10 μg/ml synthetic peptide VR-10 after 48 hours. TSP-1 and synthetic peptide VR-10 could inhibit migration of RF/6A cells in transwell chamber (P < 0.001). 10.0 μg/ml synthetic peptide VR-10 had the strongest effect, 1.0 μg/ml TSP-1 was the next. Migration inhibition rate was increase with the increase of the concentration of VR-10 (P < 0.001). There was no significant differences between 0.1 μg/ml and 1.0 μg/ml VR-10 (P=0.114). Western bolt showed that RF/6A cell in control group mainly expressed the 32×103 procaspase-3 forms. To 10.0 μg/ml VR-10 treated group, it showed decreased expression of procaspase-3 (32×103) and concomitant increased expression of its shorter proapoptotic forms (20×103). Compared with control group, expression of FAS peptides were significantly increased in 10.0 μg/ml VR-10 treated group. Compared with control group, expression of FasL mRNA was significantly increased in 10.0 μg/ml VR-10 treated group(t=39.365, P=0.001), but the expression of bcl-2 mRNA was decreased(t=-67.419, P=0.000). ConclusionTSP-1 and synthetic peptide VR-10 had the ability to inhibit proliferation and migration of endothelial cell, and also induce apoptosis by increasing FAS/FASL expression and repressing bcl-2 expression.
This paper aims to study the effects of traditional Chinese medicine Euphorbia esula on multidrug resistant human gastric cancer cells in the cell proliferation, migration, invasion and apoptosis, and to study the apoptosis-inducing pathway. Different dilutions of Euphorbia esula extract were used to process human multidrug resistant gastric cancer SGC7901/ADR cells. Cell proliferation inhibition phenomenon was determined by MTT experiment. Nuclear morphological changes of apoptotic cells and apoptotic indexes were observed and determined by Hochest33528 staining followed with fluorescence microscope observing. Flow cytometry was used to detect cell apoptosis rate. Cell migration and invasion ability were observed and determined by Transwell method. Spectrophotometry was used to detect caspase-3 and caspase-9 enzyme activity. Western blotting was used to detect subcellular distribution of cytochrome c. The results showed that Euphorbia esula extract had obvious inhibition effect on proliferation of gastric cancer multidrug resistant SGC7901/ADR cells, which was time- and concentration-dependent. After processing multidrug resistant gastric cancer SGC7901/ADR cells with Euphorbia esula extract, the apoptotic index and apoptosis rate were significantly increased than those in the control group, which showed a time- and dose-dependent mode; but if a caspase inhibitor was added, apoptosis index was not obviously increased. Transwell method showed that migration and invasion ability of the Euphorbia esula extract-processed SGC7901/ADR cells dropped significantly. Spectrophotometry showed that in Euphorbia esula extract-processed SGC7901/ADR cells, caspase-3 and caspase-9 expression were increased, which had significant differences with the control group. Western blotting test showed that the distribution of cytochrome c decreased in mitochondria, while increased in the cytoplasm (i.e., cytochrome c escaped from mitochondria to the cytoplasm). In conclusion, Euphorbia esula extract could inhibit the proliferation, migration and invasion, and induce apoptosis in human gastric cancer multidrug resistant SGC7901/ADR cells; and cytochrome c, caspase-9 and caspase-3 might be involved in cell apoptosis induced by Euphorbia esula extract, suggesting endogenous or mitochondrial apoptotic pathway.