Objective To study the effects of adenosine 2A receptor activation on activation, proliferation, and toxicity of T lymphocytes stimulated by phytohemagglutinin (PHA) in vitro. Methods A model of activated T cells was established by stimulating the cells with PHA. Those T cells were treated with different concentrations of adenosine 2A receptors agonist (0.01 μmol/L, 0.1 μmol/L, 1 μmol/L, and 10 μmol/L CGS21680). The expressions of CD69, CD25 and proliferation of T cells were measured by fluorescent antibody stain and flow cytometry. ELISA method was used to detect IL-2 and INF-γ levels. Results All concentrations of CGS21680 significantly inhibited the expressions of CD25 and CD69 on PHA-stimulated T cells surface and proliferation of T cells （Plt;0.05， Plt;0.01）. IL-2 and INF-γ secreted by T cells were significantly suppressed, too （Plt;0.01）. Conclusion Activation of adenosine 2A receptor can effectively inhibit the activation, proliferation, and toxicity of T cells in vitro.
ObjectiveTo investigate the effect of attenuated expression of neuraminidase 3 (NEU3) via RNA interference on the proliferation and apoptosis in human osteosarcoma MG-63 cells.MethodsMG-63 cells were immunostained to observe the expression of NEU3. The cells were then divided into 5 groups: MG-63 cells in normal control group (group A) were not treated; MG-63 cells in 30, 50, and 100 nmol/L NEU3 RNA interference groups (groups B, C, and D) were transfected with 30, 50, and 100 nmol/L of NEU3 small interfering RNA (siRNA); negative control group (group E), MG-63 cells were transfected with different species negative siRNA (actin siRNA of mice, 50 nmol/L). The expression level of NEU3 mRNA was measured with real-time fluorescence quantitative PCR (qPCR). The proliferation of the cells was measured by cell counting kit 8 (CCK-8). The cell apoptosis rate was detected by flowcytometry (FCM). The expressions of cell apoptosis related proteins (Ras and Bcl-2) were detected by Western blot assay.ResultsNEU3 expressed in the cytoplasm of MG-63 cells under fluorescence microscope. The qPCR results showed that NEU3 mRNA levels were significantly lower in groups B, C, D than that in groups A and E (P<0.05) after 24 hours of transfection; meanwhile, with the increase of siRNA concentration, NEU3 mRNA levels were significantly decreased (P<0.05). The CCK-8 results showed that with the increase of siRNA concentration, the survival rate of MG-63 cells was significantly suppressed (P<0.05) and the apoptosis rate of MG-63 cells was significantly accelerated (P<0.05) after 48 hours of transfection. FCM results showed that after 24 hours of transfection, the number of live MG-63 cells decreased as that of the dead cells increased in groups B, C, D, and showing significant differences between 3 groups (P<0.05). While the apoptosis rate in groups B, C, and D showed significant difference when compared with that of group A (P<0.05); and when compared with group E, the apoptosis rate in groups C and D were significantly reduced (P<0.05), but there was no significant difference between groups B and E (P>0.05). The results of Western bolt assay showed that the protein levels of Ras and Bcl-2 in groups B and C were not significantly different from groups A and E (P>0.05), while the protein levels of Ras and Bcl-2 were significantly decreased in group D (P<0.05).ConclusionAttenuated expression of NEU3 could inhibit the survival of MG-63 cells and accelerate its apoptosis. The results suggest that NEU3 could be a possible target for treating osteosarcoma.
Objective To investigate the effects of cediranib on hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway and proliferation, migration and invasion of liver cancer cells. Methods The hypoxia microenvironment was simulated in vitro, and different doses of cediranib were used to intervene the human hepatoma cell HepG2, MTT assay was used to detect the proliferation of human hepatoma cell HepG2, Transwell chamber assay was used to detect the invasion and migration of human hepatoma cell HepG2, tumor formation in nude mice was used to detect the growth of human hepatoma cell HepG2 in vivo, the angiogenesis of tumor tissue and expression level of HIF-1α/VEGF pathway protein were detected by immunohistochemistry. Results Compared with the control group, the proliferation rate, invasion and migration abilities, and the expression of HIF-1α/VEGF pathway proteins of human hepatoma cell HepG2 were significantly decreased in the different concentration of cediranib treatment group (P<0.05), the tumor volume and microvessel formation of tumor tissues in nude mice were significantly reduced (P<0.05). Conclusion Cediranib may inhibit the proliferation, migration and invasion of liver cancer cells by inhibiting HIF-1α/VEGF signaling pathway.
Objective To measure the concentration of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) prepared from different long bones and to evaluate the osteoinductivity of different DBM on MC3T3-E1 cells. Methods Different bones from the same cadaver donor were used as the initial materials for making DBM, which were divided into ulna group (uDBM), humerus group (hDBM), tibia group (tDBM), and femur group (fDBM) according to the origins, and boiled DBM (cDBM) was taken as the control group. The proteins of DBM were extracted by guanidine hydrochloride, and the concentrations of BMP-2 were determined by ELISA assay. Then the DBM were co-cultured with MC3T3-E1 cells, the proliferation of MC3T3-E1 cells was observed by cell counting kit 8 (CCK-8) assay. The osteogenic differentiation ability of MC3T3-E1 cells was qualitatively observed by alizarin red, alkaline phosphatase (ALP), and Van Gieson staining, and the osteogenic differentiation ability of MC3T3-E1 cells was quantitatively analyzed by ALP content. Linear regression was used to analyze the effect of BMP-2 concentration in DBM on ALP synthesis. ResultsThere were significant differences in the concentration of BMP-2 among the DBM groups (P<0.05). The concentrations of BMP-2 in the lower limb long bone were higher than those in the upper limb long bone, and the concentration of BMP-2 in the fDBM group was about 35.5 times that in the uDBM group. CCK-8 assay showed that the cells in each group continued to proliferate within 5 days of co-culture, and the absorbance (A) values at different time points were in the order of cDBM group<uDBM group<hDBM group<tDBM group<fDBM group. After co-culture for 14 days, the expressions of ALP, calcified nodules, and collagen fibers in each group were consistent with the distribution of BMP-2 concentration in DBM. The order of ALP content from low to high was cDBM group<uDBM group<hDBM group<tDBM group<fDBM group, and the differences between the groups were significant (P<0.05). Linear regression analysis showed that \begin{document}$\hat y $\end{document}=0.361+0.017x, the effect of BMP-2 concentration in DBM on cellular ALP content was significant (t=3.552, P=0.005); for every 1 ng/g increase in BMP-2 concentration, ALP content would increase by 0.017 [95%CI (0.006, 0.027)] U/100 mL. Conclusion The concentration of natural BMP-2 in different long bones varies greatly, and the lower limb long bone is higher than the upper limb long bone. The harvested location of bone material was an important factor affecting the osteoinductivity of DBM.
Objective To investigate the effect of ginkgolide B (GB) on cysteinyl aspartate specific proteinase-3 (Caspase-3)/chromosome 10 deletion phosphatase-tension protein homologue (PTEN)/protein kinase B (Akt) pathway and cell proliferation and apoptosis in hypoxia/reoxygenation cardiomyocytes. Methods H9C2 cells were cultured in vitro. A control group was cultured in serum-free DMEM high glucose medium at 37°C and 5% CO2 for 28 hours. The remaining groups were prepared with hypoxia/reoxygenation models. A GB low-dose group and a GB high-dose group were treated with GB pretreatment with final concentration of 50 μmol/L and 200 μmol/L respectively at 1 h before hypoxia/reoxygenation. A carvedilol group was treated with carvedilol of a final concentration of 10 μmol/L at 1 h before hypoxia/reoxygenation. The proliferation and apoptosis of H9C2 cells were detected, and the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), reactive oxygen species (ROS), PTEN, Akt, phosphorylated Akt (p-Akt) and Caspase-3 in H9C2 cells were also detected. Results Compared with the control group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in other groups decreased, and the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 increased (P<0.05). Compared with the hypoxia/reoxygenation group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in all GB dose groups and the carvedilol group increased; the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 decreased, and the effect of GB was in a dose dependent manner; however, the effect of GB was not as strong as carvedilol (P<0.05). Conclusion GB can inhibit H9C2 cell apoptosis and promote H9C2 cell proliferation by activating Caspase-3/PTEN/Akt pathway.
To study the potential molecular mechanism of tumor angiogenesis in its microenvironment, we investigated the effects of HepG2 conditioned medium on the proliferation of vascular endothelial cell and vascular angiogenesis in our laboratory. Human umbilical vein endothelial EA.hy926 cells were co-cultured with HepG2 conditioned medium in vitro. The proliferation and the tubulogenesis of EA.hy926 cells were detected by teramethylazo salt azole (MTT) and tube formation assay, respectively. The results showed that the survival rate of the EA.hy926 cells was significantly increased under the co-culture condition. HepG2 conditioned medium also enhanced the angiogenesis ability of EA.hy926 cells. In addition, the expressions of intracellular VEGF and extracellular VEGFR (Flk-1) were regulated upward in a time-dependent manner. In conclusion, the proliferation of vascular endothelial cells and Vascula angiogenesis were improved under the condition of indirect co-culture.
ObjectiveTo summarize the progress of study on the relationship between endoplasmic reticulum stress and cell proliferation and provide evidence with reliable evidence-based data to the experiment on the field of tissue damage repair, organ proliferation, and regeneration.MethodThe relevant literatures about the progress of multiple signaling pathways related to the endoplasmic reticulum stress in the cell proliferation and injury repair in recent years were reviewed.ResultsThe endoplasmic reticulum stress participated in the process of proliferation and regeneration in the intestinal epithelial cells, skeletal muscle cells, islet cells, and hepatocytes through different pathways, which involved the three pathways of unfolded protein reaction that interacted with interleukin-6, tumor necrosis factor-α, vascular endothelial growth factor, Wnt, etc.ConclusionsAlthough endoplasmic reticulum stress has been widely debated in the field of determining cell fate, after we reviewed recent studies on endoplasmic reticulum stress in maintaining cell survival and promoting cell proliferation, the complexity, diversity, and importance of the endoplasmic reticulum stress in promoting cell proliferation have been presented in front of us. It not only promotes cell proliferation through the classical signaling pathway with Wnt protein, but also acts to repair tissue and promote proliferation by interacting with Musashi protein independently of the Notch pathway. The complex reaction pathway interacts with different stimulating factors in different cells, providing research directions and exploration possibilities for cell proliferation, injury repair, and organ regeneration, reveales the critical role of endoplasmic reticulum stress in cell proliferation.
ObjectiveTo explored the effect of stromal cell-derived factor 1α (SDF-1α) on promoting the migration ability of rat adipose derived stem cells (rADSCs) by constructed the rADSCs overexpression SDF-1α via adenovirus transfection.MethodsrADSCs were isolated from adipose tissue of 6-week-old SPF Sprague Dawley rats. Morphological observation, multi-directional differentiations (osteogenic, adipogenic, and chondrogenic inductions), and flow cytometry identification were performed. Transwell cell migration experiment was used to observe and screen the optimal concentration of exogenous SDF-1α to optimize the migration ability of rADSCs; the optimal multiplicity of infection (MOI) of rADSCs was screened by observing the cell status and fluorescence expression after transfection. Then the third generation of rADSCs were divided into 4 groups: group A was pure rADSCs; group B was rADSCs co-cultured with SDF-1α at the best concentration; group C was rADSCs infected with recombinant adenovirus-mediated green fluorescent protein (Adv-GFP) with the best MOI; group D was rADSCs infected with Adv-GFP-SDF-1α overexpression adenovirus with the best MOI. Cell counting kit 8 (CCK-8) and Transwell cell migration experiment were preformed to detect and compare the effect of exogenous SDF-1α and SDF-1α overexpression on the proliferation and migration ability of rADSCs.ResultsThe cell morphology, multi-directional differentiations, and flow cytometry identification showed that the cultured cells were rADSCs. After screening, the optimal stimulating concentration of exogenous SDF-1α was 12.5 nmol/L; the optimal MOI of Adv-GFP adenovirus was 200; the optimal MOI of Adv-GFP-SDF-1α overexpression adenovirus was 400. CCK-8 method and Transwell cell migration experiment showed that compared with groups A and C, groups B and D could significantly improve the proliferation and migration of rADSCs (P<0.05); the effect of group D on enhancing the migration of rADSCs was weaker than that of group B, but the effect of promoting the proliferation of rADSCs was stronger than that of group D (P<0.05).ConclusionSDF-1α overexpression modification on rADSCs can significantly promote the proliferation and migration ability, which may be a potential method to optimize the application of ADSCs in tissue regeneration and wound repair.
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.
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.