Evaluation of rat osteoarthritis chondrocyte models induced by interleukin-1beta / 中国组织工程研究

BACKGROUND:Establishing a chondrocyte model of osteoarthritis is of great significance for further explaining the pathological process of osteoarthritis and evaluating and screening the therapeutic drugs of osteoarthritis. OBJECTIVE:To evaluate the effect of interleukin-1β to induce osteoarthritis models in rat chondrocyte models,thereby providing a reference for further exploration of drug treatment of osteoarthritis. METHODS:Chondrocytes were isolated from the hip cartilage of 3-week-old Sprague-Dawley rats by mechanical shearing and enzymatic digestion,and then identified.Chondrocytes were randomly divided into three groups:control group,5 ng/mL interleukin-1β-induced group,10 ng/mL interleukin-1β-induced group,with induction times of 24 and 48 hours.Chondrocyte proliferation activity was detected by MTT.Real-Time PCR was used to detect the expression of type Ⅱ collagen,Aggrecan,sex-determining region Y-box protein 9(Sox9),matrix metalloproteinase 13,and a disintegrin and metaloproteinase with thrombospondin-like motifs-5(Adamts5)mRNA.Western blot was used to detect the expression of type Ⅱ collagen,Sox9,matrix metalloproteinase 13 and Adamts5. RESULTS AND CONCLUSION:Primary rat chondrocytes were successfully isolated and cultured.Induction of chondrocytes by interleukin-1β at 10 ng/mL for 24 hours could significantly reduce cell proliferation and viability(P＜0.05),while the 5 ng/mL interleukin-1β-induced group required 48 hours of induction to cause a significant decrease in cell proliferation and viability.Real-Time PCR results showed that compared with the control group,5 or 10 ng/mL interleukin-1β induction for 24 and 48 hours significantly reduced the expression levels of type Ⅱ collagen,Aggrecan,Sox9 mRNAs(P＜0.05)and increased the expression levels of matrix metalloproteinase 13 and Adamts5 mRNAs(P＜0.05).Compared with the 10 ng/mL interleukin-1β-induced group,5 ng/mL interleukin-1β induction significantly increased the mRNA expression of matrix metalloproteinase 13 and Adamts5 in chondrocytes after 48 hours of induction(P＜0.05).Western blot results showed that compared with the control group,10 ng/mL interleukin-1β induction for 24 hours and 5 ng/mL interleukin-1β induction for 48 hours significantly decreased the protein expression of type Ⅱ collagen and Sox9 in chondrocytes(P＜0.05),and significantly increased the protein expression of matrix metalloproteinase 13 and Adamts5(P＜0.05).To conclude,compared with 5 ng/mL interleukin-1β,10 ng/mL interleukin-1β may have more obvious effects on chondrocytes for 24 hours,while 5 and 10 ng/mL interleukin-1β have similar effects after 48 hours of intervention.

Expression of centromere protein-H in adrenocortical carcinoma and its impact on viability and migration of adrenocortical carcinoma cells / 中国病理生理杂志

AIM:To investigate the expression of centromere protein-H(CENP-H)in adrenocortical carcino-ma(ACC)and its relationship with disease progression and prognosis,and to explore the impact of CENP-H gene knock-down on the viability and migration of ACC cells.METHODS:The mRNA expression level of CENP-H in 76 ACC pa-tients and 128 healthy controls,and its correlations with tumor stages and prognosis were analyzed by GEPIA2 database.The mRNA expression of CENP-H in different stages of ACC and its correlation with disease prognosis were further ana-lyzed by ULCAN database.The protein expression of CENP-H was examined by immunohistochemical staining of paraffin-embedded ACC and normal adrenal gland specimens.Knockdown of CENP-H by siRNA(siCENP-H)was performed in human ACC cell line H295R.The viabilty of H295R cells transfected with siCENP-H or siNC was measured by CCK-8 as-say,the cell migration was detected by wound-healing assay,and the protein levels of CENP-H,p-ERK1/2,t-ERK1/2,p-P38,t-P38,p-JNK1/2 and t-JNK1/2 were detected by Western blot.RESULTS:The mRNA level of CENP-H was signifi-cantly higher in ACC than that in normal controls,and was correlated with tumor stages and prognosis.The protein level of CENP-H was significantly higher in ACC specimens than that in normal adrenal gland.Knockdown of CENP-H in H295R cells resulted in decreased cell viability and migration.The protein levels of p-P38 and p-JNK1/2 were decreased in si-CENP-H group.CONCLUSION:CENP-H is highly expressed in ACC,and is correlated with tumor stages and poor prognosis.Knockdown of CENP-H can inhibit the viability and migration of ACC cells,and its mechanism may related to inactivation of P38 and JNK signaling pathways.

Effect of hydrofluoric acid concentration and aging on the bond strength ceramics to a resin cement

Abstract This study evaluated the influence of hydrofluoric acid (HF) concentration and thermal cycling on the microshear bond strength (µSBS) of a resin luting agent to IPS e.max® CAD and Rosetta® SM. Ceramic specimens (12.0 x 14.0 x 1.5mm) were randomized into 8 groups (n=10) according to HF concentration, commercial brand, and aging. Immediately after polishing, and etching, all specimens were silanized and a layer of adhesive was applied. A PVS mold of 3 mm thickness and 10mm diameter with (four) 1.0mm holes was fabricated, placed on each specimen, and then filled with a resin luting agent. Half of the specimens were subjected to the µSBS test using an Instron at a speed of 1.0 mm/min, following a 24-hour storage in deionized water at 37ºC. The remaining specimens were subjected to thermal cycling (5ºC-55ºC, 30 seconds per bath) and µSBS. The data were evaluated utilizing a three-way ANOVA and Tukey's post-hoc test (α=0.05). Significant differences were found for HF concentration and aging (p<0.0001). No significant difference in µSBS was found for commercial brands (p=0.085). The interaction between brand and HF concentration (p=0.358), brand and aging (p=0.135), and HF concentration and aging (p=0.138) were not statistically significant. The triple interaction among these factors was not statistically significant (p=0.610). In conclusion, the bond strength is affected by the HF concentration. No statistical difference was observed between the two ceramics. Thermal cycling significantly reduced µSBS.

Resumo Este estudo avaliou a influência da concentração do ácido fluorídrico (AF) e da ciclagem térmica na resistência de união ao microcisalhamento (RUµC) de um cimento resinoso para IPS e.max® CAD e Rosetta® SM. Espécimes cerâmicos (12,0 x 14,0 x 1,5mm) foram divididos em 8 grupos (n=10) de acordo com concentração do HF, marca comercial e envelhecimento. Imediatamente após o polimento e condicionamento ácido, todos os espécimes foram silanizados e uma camada de adesivo foi aplicada. Um molde PVS de 3 mm de espessura e 10 mm de diâmetro com (quatro) orifícios de 1,0 mm foi confeccionado, colocado em cada espécime e preenchido com o cimento resinoso. Metade dos espécimes foi submetida ao teste RUµC na Instron a velocidade de 1,0 mm/min, após 24 horas de armazenamento em água deionizada a 37ºC. Os espécimes restantes foram submetidos a ciclagem térmica (5ºC-55ºC, 30 segundos por banho) e a RUµC. Os dados foram avaliados por ANOVA de três fatores e ao teste post-hoc de Tukey (α=0,05). Diferenças significativas foram encontradas para concentração de HF e envelhecimento (p<0,0001). Nenhuma diferença significativa na RUµC foi encontrada para cada marca comercial (p=0,085). A interação entre marca comercial e a concentração do HF (p=0,358), marca comercial e envelhecimento (p=0,135) e concentração do HF e envelhecimento (p=0,138) não foram estatisticamente significativas. A tripla interação entre esses fatores não foi estatisticamente significativa (p=0,610). Concluindo, a resistência de união é afetada pela concentração de HF. Não foi observada diferença estatística entre as duas cerâmicas. A ciclagem térmica reduziu significativamente a resistência de união ao microcisalhamento.

Cytotoxic effects on human dental pulp stem Cells after exposure to adhesive bonding agents

Abstract Studies regarding cytotoxic effects attributed to the use of adhesive bonding agents on pulp tissue are not conclusive. To point out whether these materials are safe for clinical use, in vivo exposure of dental pulp to adhesive bonding agents was simulated using an experimental setup in which Human Dental Pulp Stem Cells (hDPSC) are exposed to the action of two kinds of adhesives: self-etching adhesives and two-step bonding agents through a dentine barrier. Cytotoxic effects on these cells were evaluated by MTT assay protocol and fluorescence microscopy, and their results were contrasted to those obtained through Raman spectra taken on single hDPSCs. Overall, no significant cytotoxic effects were observed by combining all the techniques, and cell viability close to 90% was achieved for a dentine barrier of at least 1 mm thick. Moreover, Raman spectroscopy was able to detect structural DNA damage in some dental pulp cells when exposed to two-step bonding agents, suggesting that this technique could be considered a complementary tool with the potential to evaluate cell toxicity beyond cell viability.

Resumo Os estudos sobre os efeitos citotóxicos atribuídos ao uso de agentes de união adesivo no tecido pulpar não são conclusivos. Para determinar se esses materiais são seguros para uso clínico, a exposição in vivo da polpa dentária a agentes de união adesiva foi simulada por meio de uma configuração experimental na qual as células-tronco da polpa dentária humana (hDPSC) são expostas à ação de dois tipos de adesivos: adesivos autocondicionantes e agentes de união de duas etapas por meio de uma barreira de dentina. Os efeitos citotóxicos nessas células foram avaliados pelo protocolo de ensaio MTT e microscopia de fluorescência, e seus resultados foram contrastados com os obtidos por meio de espectros Raman obtidos em hDPSCs individuais. De modo geral, não foram observados efeitos citotóxicos significativos com a combinação de todas as técnicas, e a viabilidade celular próxima a 90% foi obtida para uma barreira de dentina de pelo menos 1 mm de espessura. Além disso, a espectroscopia Raman foi capaz de detectar danos estruturais ao DNA em algumas células da polpa dentária quando expostas a agentes de colagem de duas etapas, sugerindo que essa técnica poderia ser considerada uma ferramenta complementar com potencial para avaliar a toxicidade celular além da viabilidade celular.

Cytotoxic effects of bioceramic materials on stem cells from human exfoliated deciduous teeth (SHED) / Efeitos citotóxicos de materiais biocerâmicos em células-tronco de dentes decíduos esfoliados humanos (SHED)

Objective: This study aimed to evaluate stem cell from human deciduous teeth (SHED) viability after exposure to different bioceramic materials. Material and Methods: Discs were constructed to obtain the material extracts according to the following groups: G1 - Bio-C Repair, G2 - MTA Repair HP, G3 - TheraCal LC, and G4 ­ Biodentine. Positive and negative control group were respectively maintained with αMEM + 10% FBS and αMEM + 1% FBS. SHED obtained through primary culture were in contact with material extracts for 24, 48, and 72h. MTT assay evaluated cell viability. Groups were plated in triplicate and the cell viability assay were repeated three times. Data were analyzed by two-way ANOVA followed by Tukey test (p<0.05). Results: The treatment and period comparisons showed statistically significant differences (p<0.000). G2 (MTA Repair HP) had greater cell viability values than the other experimental groups and negative control. MTA Repair HP and the control groups exhibited a similar behavior with cell viability values decreasing from 24h to 48h and increasing from 48h to 72h. Bio-C Repair, Biodentine, and Theracal LC did not show statistically significant differences among periods. Conclusions: SHED increased viability values after contact with MTA Repair HP in comparison with other bioceramic materials.(AU)

Objetivo: O objetivo desse estudo foi avaliar a viabilidade de células-tronco de dentes decíduos humanos (SHED) após o contato com diferentes materiais biocerâmicos. Material e Métodos: Foram confeccionados discos para obtenção dos extratos dos materiais de acordo com os seguintes grupos: G1 - Bio-C Repair, G2 - MTA Repair HP, G3 - TheraCal LC e G4 - Biodentine. Grupo de controle positivo e negativo foram mantidos respectivamente com αMEM + 10% FBS e αMEM + 1% FBS. SHED obtidas por cultura primária entraram em contato com os extratos de materiais por 24, 48 e 72h. O ensaio MTT avaliou a viabilidade celular. Os grupos foram semeados em triplicata e o ensaio de viabilidade celular foi repetido três vezes. Os dados foram analisados por ANOVA a dois critérios seguido pelo teste de Tukey (p<0,05). Resultados: As comparações de tratamentos e períodos mostraram diferenças estatisticamente significativas (p<0,000). O G2 (MTA Repair HP) apresentou maiores valores de viabilidade celular que os demais grupos experimentais e controle negativo. O MTA Repair HP e os grupos controle exibiram um comportamento semelhante com os valores de viabilidade celular diminuindo de 24h para 48h e aumentando de 48h para 72h. Bio-C Repair, Biodentine e Theracal LC não apresentaram diferenças estatisticamente significativas entre os períodos. Conclusões: SHED aumentou os valores de viabilidade após o contato com o MTA Repair HP em comparação com outros materiais biocerâmicos (AU)

Antimicrobial effect of phytosphingosine in acrylic resin

Abstract This study evaluated color stability (CS), anti-adherence effect (AAE), and cell viability of microorganisms on acrylic resin (AR) surface, treated associated or not with sodium percarbonate (SP). AR specimens were prepared, and color analysis was performed before and after the treatments and the CS was calculated. For analysis of AAE, the samples were sterilized by radiation in a microwave oven. Then samples were randomly distributed: phosphate-buffered saline (PBS - control), 0.5% sodium hypochlorite (SH), phytosphingosine (PHS), and phytosphingosine + SP (PHS+Na2CO3). The specimens remained in contact with solutions for 30 minutes and were later contaminated by Candida albicans. Aliquots were seeded in Petri dishes with Sabouraud Dextrose agar and incubated at 37°C for 24 hours. After the incubation, the number of colonies was counted. The cell viability of adhered microorganisms on the AR was evaluated and 20 fields were observed under an epifluorescence microscope, and the percentage of adhered viable cells was calculated. Data were compared (One-way ANOVA, Tukey, p<.05). As for CS, PHS+ Na2CO3 (0.4±0.1) resulted in less change than PBS (0.9±0.2), similar to the other groups (SH [1.0±0.3)]; PHS [0.9±0.2)]). There was no difference for all tested solutions regarding the ability to avoid microorganism adherence (p>0.05), but PHS (11.2±4.1) resulted in a smaller area of adhered viable cells, statistically different from SH (18.2±7.6) and PBS (26.4±10.8). It was concluded that PHS resulted in lower adhered viable cells and when associated with Na2CO3, also shows a lower effect on the CS of AR.

Resumo Este estudo avaliou estabilidade de cor (EC), efeito antiaderente (EAA) e viabilidade celular de microrganismos em superfície de resina acrílica (RA), tratada com solução de fitoesfingosina, associada ou não ao percarbonato de sódio (PS). Espécimes RA foram preparados e análise de cor foi realizada antes e após os tratamentos e EC foi calculada. Para análise de EAA, as amostras foram esterilizadas por radiação em forno de micro-ondas. Então foram distribuídas aleatoriamente: solução salina tamponada com fosfato (PBS - controle), hipoclorito de sódio 0,5% (SH), fitoesfingosina (PHS) e fitoesfingosina + SP (PHS+Na2CO3). Os espécimes permaneceram em contato com as soluções por 30 minutos e posteriormente foram contaminados por Candida albicans. Alíquotas foram semeadas em placas de Petri com ágar Sabouraud Dextrose e incubadas a 37°C por 24 horas. Após a incubação, o número de colônias foi contado. A viabilidade celular dos microorganismos aderidos na RA foi avaliada e 20 campos foram observados em microscópio de epifluorescência, e a porcentagem de células viáveis aderidas foi calculada. Os dados foram comparados (One-way ANOVA, Tukey, p<0,05). Quanto a EC, o PHS+ Na2CO3 [0,4 (0,1)] resultou em menor alteração que o PBS [0,9 (0,2)], semelhante aos demais grupos (SH [1,0 (0,3)]; PHS [0,9 (0,2)]). Não houve diferença para todas as soluções testadas quanto à capacidade de evitar a aderência de microorganismos (p>0,05), mas o PHS [11,2 (4,1)] resultou em uma área menor de células viáveis aderidas, estatisticamente diferente do SH [18,2 (7,6)] e PBS [26,4 (10,8)]. Concluiu-se que o PHS resultou em menor número de células viáveis aderidas e, quando associado ao Na2CO3, também apresenta menor efeito sobre o EC da RA.

A PRECLINICAL STUDY ON EVALUATION OF ANTICANCER ACTIVITY OF SNUHI KSHARA IN HUMAN COLON CANCER CELL LINE HCT-15

Snuhi (Euphorbia neriifolia Linn.) is a conventional herb used broadly in several disease conditions as indicated in classical texts of Ayurveda. As per literature review ascertained, no literature was accessible regarding anticancer activity of Snuhi Kshara. Thus, present work was designed to evaluate the anticancer activity of Snuhi Kshara in HCT-15 (Human Colon Cancer cell line). Anticancer activity was evaluated using MTT assay by % cell viability and IC50. Anticancer activity was compared with standard drug capecitabine. A positive correlation between Concentration and % cell viability was noticed. Lowest cell viability was noted at 5000 µg concentration. Results obtained through the study indicates towards anticancer activity of Snuhi Kshara.

Phenolic derivatives from root bark of Schisandra sphenanthera / 中国中药杂志

This paper aimed to study the chemical constituents from the root bark of Schisandra sphenanthera. Silica, Sephadex LH-20 and RP-HPLC were used to separate and purify the 80% ethanol extract of S. sphenanthera. Eleven compounds were identified by ~1H-NMR, ~(13)C-NMR, ESI-MS, etc., which were 2-[2-hydroxy-5-(3-hydroxypropyl)-3-methoxyphenyl]-propane-1,3-diol(1), threo-7-methoxyguaiacylglycerol(2),4-O-(2-hydroxy-1-hydroxymethylethyl)-dihydroconiferylalcohol(3), morusin(4), sanggenol A(5), sanggenon I(6), sanggenon N(7), leachianone G(8),(+)-catechin(9), epicatechin(10), and 7,4'-dimethoxyisoflavone(11). Among them, compound 1 was a new compound, and compounds 2-9 were isolated from S. sphenanthera for the first time. Compounds 2-11 were subjected to cell viability assay, and the results revealed that compounds 4 and 5 had potential cytotoxicity, and compound 4 also had potential antiviral activity.

FKBP38 regulates apoptosis of dopaminergic neurons / 中国药理学通报

Aim To investigate the role of FKBP38 in inhibiting apoptosis in a rotenone-induced Parkinson's disease(PD)cell model. Methods In vivo experiments:MPTP-induced PD in vivo models were constructed,and the expressions of α-synuclein,TH and FKBP38 in brains of PD mice were detected. In vitro experiments:Dopaminergic neuron MN9D cells were stimulated with rotenone to construct an in vitro model of PD; Western blot was used to detect the expression levels of α-synuclein,TH,Tom20 and FKBP38 in PD in vitro model; FKBP38 lentivirus was transferred into MN9D cells to construct stable overexpression and FKBP38 knockdown cell lines; CCK-8 assay was used to detect the cell viability of FKBP38 overexpression and knockdown cells stimulated by rotenone; Western blot was used to detect anti-apoptotic protein Bcl-2 and apoptosis protein in PD cell model expression levels of Bax. Results The expression level of FKBP38 was significantly down-regulated in both in vitro and in vivo models of PD(P<0.01). Knockdown of FKBP38 aggravated the decline of dopaminergic neuron cell viability caused by rotenone(P<0.05),while overexpression of FKBP38 significantly ameliorated the decline of dopaminergic neuron cell viability caused by rotenone(P<0.05). Western blot results showed that overexpression of FKBP38 could significantly up-regulate the expression level of anti-apoptotic protein Bcl-2 and increase the ratio of Bcl-2/Bax in PD dopaminergic neurons(P<0.05). Conclusion In the PD cell model regulation of FKBP38 can improve the apoptosis of dopaminergic neurons.

Effects of orlistat on the viability of human gallbladder cancer cells / 中华消化外科杂志

Objective:To investigate the effects of orlistat on the viability of human gall-bladder cancer (GBC) cells.Methods:The experimental study was conducted. The human GBC NOZ cells with high expression of FSAN was screened out through in vitro cultivating human GBC-SD, SGC-996 and NOZ cells. The cell proliferation assay, clone formation assay and protein detection experiment were used to analysis of the effects of orlistat on the viability of human GBC cells. Cell grouping: NOZ cells cultured with medium were set as the control group, cultured with medium + 10 μmol/L orlistat were set as the low-dose orlistat group, cultured with medium + 100 μmol/L orlistat were set as the high-dose orlistat group, respectively. Observation indicators: (1) expression of FASN protein in human GBC cells; (2) effects of orlistat on the proliferation of human GBC NOZ cells; (3) effects of orlistat on apoptosis of human GBC NOZ cells. Measurement data with normal distribution were represented as Mean± SD, the ANOVA test was used for comparison between groups and the least significant difference method was used for pairwise comparison. Results:(1) Expression of FASN protein in human GBC cells. Results of western blot showed that the relative expression of FASN protein in human GBC NOZ, GBC-SD and SGC-996 cells was 0.57±0.06, 0.12±0.04 and 0.10±0.02, respectively, showing a significant difference among them ( F=115.67, P<0.05). There were significant differences between the NOZ cells and the GBC-SD or the SGC-996 cells ( P<0.05), and there was no significant difference between the GBC-SD cells and the SGC-996 cells ( P>0.05). (2) Effects of orlistat on the proliferation of human GBC NOZ cells. ① Results of cell proliferation assay showed that the absorbance value of NOZ cells was 2.34±0.12, 1.57±0.08 and 1.07±0.13 in the control group, low-dose orlistat group and high-dose orlistat group, respectively, showing a significant difference among them ( F=205.88, P<0.05). ② Results of clone formation assay showed that the number of NOZ cells clones was 257±23, 153±11 and 83±11 in the control group, low-dose orlistat group and high-dose orlistat group, respectively, showing a significant difference among them ( F=92.64, P<0.05). ③Results of western blot showed that the relative expression of Cyclin-D1 protein of NOZ cells was 2.31±0.10, 1.52±0.05 and 1.23±0.11 in the control group, low-dose orlistat group and high-dose orlistat group, respectively, showing a significant difference among them ( F=120.73, P<0.05). The relative expression of CDK-4 protein of NOZ cells was 1.58±0.04, 1.21±0.02 and 1.19±0.04 in the control group, low-dose orlistat group and high-dose orlistat group, respectively, showing a signifi-cant difference among them ( F=110.45, P<0.05). (3) Effects of orlistat on apoptosis of human GBC NOZ cells. Results of western blot showed that the relative expression of Bcl-2 protein of NOZ cells was 1.07±0.03, 0.36±0.03 and 0.15±0.02 in the control group, low-dose orlistat group and high-dose orlistat group, respectively, showing a significant difference among them ( F=1 242.93, P<0.05). The relative expression of Bax protein of NOZ cells was 0.51±0.03, 0.38±0.05 and 1.38±0.04 in the control group, low-dose orlistat group and high-dose orlistat group, respectively, showing a signifi-cant difference among them ( F=583.51, P<0.05). Conclusion:Orlistat can inhibit the growth of human GBC NOZ cells and promote their apoptosis.

Effects of long non-coding RNA RP11-1212A22.4 on the cell viability and invasive ability of ovarian cancer cell lines through targeting miRNA-483-5p / 肿瘤研究与临床

Objective:To investigate the effects of long non-coding RNA (lncRNA) RP11-1212A22.4 on the cell viability and invasive ability of esophageal cancer cell lines by targeting miRNA-483-5p (miR-483-5p).Methods:The expression of RP11-1212A22.4 in esophageal cancer tissues was analyzed by using GEPIA online database. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of RP11-1212A22.4 in human esophageal cancer cell lines EC9706, KYSE30, TE-13, Eca109 and normal esophageal epithelial cell line HET-1A. The lowest expression level of EC9706 cell line in RP11-1212A22.4 was divided into RP11-1212A22.4 group (transfected with pcDNA-RP11-1212A22.4 plasmid) and the control group (transfected with pcDNA-NC plasmid). The cell viability of EC9706 cell was analyzed by using methyl thiazolyl tetrazolium (MTT) method, and the invasion ability of EC9706 cell was detected by using Transwell assay. The targeting relationship between RP11-1212A22.4 and miR-483-5p was verified by using StarBase database prediction and dual luciferase reporter assay. The relative expression level of miR-483-5p of EC9706 cell in two groups was detected by using qRT-PCR. Western blot was used to detect the expressions of cyclin-dependent kinase 6 (CDK6), matrix metalloproteinase 2 (MMP-2), cyclin-dependent kinase 4 (CDK4), and matrix metalloproteinase 9 (MMP-9) proteins in two groups.Results:In GEPIA online database, compared with adjacent tissues, the relative expression level of RP11-1212A22.4 in esophageal cancer tissues was decreased, and the difference was statistically significant ( P < 0.001). The relative expression levels of RP11-1212A22.4 in esophageal cancer cell lines EC9706, KYSE30, TE-13, Eca109 and normal esophageal mucosal epithelial cell line HET-1A were 0.11±0.08, 0.32±0.09, 0.72±0.09, 0.59±0.13 and 0.97±0.12, and the difference was statistically significant ( F = 40.42, P < 0.001). The relative expression levels of RP11-1212A22.4 in EC9706 cells of RP11-1212A22.4 group and the control group were 11.9±2.4 and 1.0±0.3, respectively, and the difference was statistically significant ( t = 8.89, P < 0.001). Compared with the control group, the cell viability of EC9706 cell in RP11-1212A22.4 group was decreased (all P < 0.05). The number of invasive cells in RP11-1212A22.4 group was lower than that in the control group (48±12 vs. 106±22, t = 4.63, P < 0.001). StarBase database prediction and dual luciferase reporter assay both showed that RP11-1212A22.4 targeted miR-483-5p. The relative expression level of miR-483-5p in RP11-1212A22.4 group was lower than that in the control group (0.24±0.11 vs. 1.02±0.23, t = 5.98, P = 0.001). Compared with the control group, the expressions of CDK6, MMP-2, CDK4 and MMP-9 proteins in the RP11-1212A22.4 group were decreased. Conclusions:RP11-1212A22.4 is lowly expressed in esophageal cancer tissues and cell lines, and it inhibits the cell viability and invasive ability of esophageal cancer cells by targeting miR-483-5p.

Progress in research on hydrogel and medium permeability / 中国生物制品学杂志

@#As a new research field,gel has been paid more attention and widely used for studies on tissue engineering,drug delivery and biosensor. Hydrogel is the carrier of cells,while the cell survival and death are keys to the construction of tissues and organs. However,the cell viability and biological behavior are limited by the exchange of hydrogel and nutrients in medium. This review summarizes the types of hydrogel,exchange mode of hydrogel and nutrients in medium and the relevant influencing factors,which will provide a reference for the development and research of tissue bioengineering.

Telomerase inhibitors TMPyP4 and thymoquinone decreased cell proliferation and induced cell death in the non-small cell lung cancer cell line LC-HK2, modifying the pattern of focal adhesion

G‐quadruplexes (G4) are structures formed at the ends of telomeres rich in guanines and stabilized by molecules that bind to specific sites. TMPyP4 and thymoquinone (TQ) are small molecules that bind to G4 and have drawn attention because of their role as telomerase inhibitors. The aim of this study was to evaluate the effects of telomerase inhibitors on cellular proliferation, senescence, and death. Two cell lines, LC‐HK2 (non-small cell lung cancer - NSCLC) and RPE‐1 (hTERT-immortalized), were treated with TMPyP4 (5 μM) and TQ (10 μM). Both inhibitors decreased telomerase activity. TMPyP4 increased the percentage of cells with membrane damage associated with cell death and decreased the frequency of cells in the S‐phase. TMPyP4 reduced cell adhesion ability and modified the pattern of focal adhesion. TQ acted in a concentration-dependent manner, increasing the frequency of senescent cells and inducing cell cycle arrest in G1 phase. Thus, the present results showed that TMPyP4 and TQ, although acting as telomerase inhibitors, had a broader effect on other signaling pathways and processes in cells, differing from each other. However, they act both on malignant and immortalized cells, and further studies are needed before their anti-cancer potential can be considered.

Antimicrobial and toxicity properties of crystalline potassium tetrachlorocuprate (II) dehydrate

In the current scenario, microbial infections are major concern and they multiply so rapidly throughout the world; also, they cause serious bio illness and can even results in death among humans. Complexes framed by crystalline metal ions have been used as medications, because of a wide range of organic activities against harmful microorganisms. In the present study, greenish blue potassium tetrachlorocuprate (II) dihydrate K2CuCl4.2H2O crystalline precipitates were prepared by solvent evaporation method at ambient temperature. This complex was found in nature as a rare mineral Mitscherlichite. For finding the crystalline parameters of the sample, a Single-crystal XRD analysis was used and the data confirms. K2CuCl4.2H2O crystallizes into a tetragonal structure. The formation of bonds and the presence of functional groups in the complex were determined from Surface morphology studies, the very clear morphology of the complex has smoothing surface, defect-free, small microstructures and stacklike shapes of a well-defined crystalline pattern. Also, addition, optical analysis supports that its possibility in antimicrobial applications. The sample shows comparatively good results in positive control of antibacterial and antifungal activities, namely Gentamicin, Chloramphenicol and Clotrimazole. IC50 and optical density (OD) values were used to determine the cytotoxicity and cell viability, respectively. In vitro cytotoxicity of MTT assay shows in graphical abstract.

The Relationship between PPP2R5C and Molt-4 Cell Viability, HSP90-GR Signal in Childhood Acute T Lymphocytic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of PPP2R5C to the activity of Molt-4 cells in childhood acute T lymphocytic leukemia and its mechanism.@*METHODS@#The small interfering RNA (siRNA) technology targeting PPP2R5C gene was used to down-regulate the expression of PPP2R5C in Molt-4 cells. At the same time, a blank control group, a negative control group and a 17-DMAG group were set up. The cells in the negative control group were transfected with siRNA-NC, the cells in 17-DMAG group were treated with the HSP90 inhibitor 17-DMAG at a final concentration of 6.4 μmol/L for 48 h. Real-time fluorescent quantitative PCR (RT-qPCR) and Western blot were used to detect transfection efficiency; CCK-8 method was used to detect the proliferation activity of the cells in each group, EdU was used to detect the proliferation level of the cells in each group, flow cytometry was used to detect the cell cycle distribution ratio of the cells in each group, Annexin V-FITC/PI staining was used to detect the apoptosis of the cell, RT-qPCR and Western blot were used to detect the expression changes of heat shock protein 90 (HSP90) and glucocorticoid receptor (GR) of the cells in each group.@*RESULTS@#After Molt-4 cells were transfected with siRNA-PPP2R5C, the expression of PPP2R5C mRNA and protein in the cells were down-regulated significantly compared with those in the blank control group and the si-NC group (P<0.05); compared with cells in the blank control group and the si-NC group, the proliferation activity of the cells in the siRNA-PPP2R5C group and the 17-DMAG group significantly decreased (P<0.05), and the rate of EdU positive cells was significantly reduced (P<0.05); the proportion of the cells in G1 phase decreased while the proportion of the cells in G2 phase increased (P<0.05), the apoptosis rate of the cells also increased significantly (P<0.05); in addition, the expression of PPP2R5C mRNA and protein of the cells in siRNA-PPP2R5C group was significantly down-regulated compared with those in the blank control group and si-NC group (P<0.05). The expressions of PPP2R5C mRNA and protein in the 17-DMAG group were also significantly down-regulated compared with those in the blank control group and si-NC group (P<0.05).@*CONCLUSION@#Down-regulation of PPP2R5C gene expression can inhibit Molt-4 cell activity in childhood acute T lymphocytic leukemia, block the cells in G2 phase, and promote cell apoptosis, the mechanism may be related to the inhibition of HSP90-GR signaling pathway.

Morphological alterations study in the Golgi apparatus of skin fibroblasts in spinocerebellar ataxia type 3 patients / 中华神经科杂志

Objective:To identify the morphological alterations in the Golgi apparatus of skin fibroblasts in spinocerebellar ataxia type 3 (SCA3) patients.Methods:In this study, 3 SCA3 patients and 3 healthy volunteers were obtained in the First Affiliated Hospital of Zhengzhou University from 2016 to 2020. The cytosine, adenine, and guanine repeats of 3 SCA3 patients were 14/76, 20/80 and 21/82, respectively. Tissue mass culture was used to amplify skin fibroblasts derived from SCA3 patients and healthy volunteers. Cell viability and apoptosis were detected using cell counting kit-8 assay and flow cytometry, respectively. Western blotting and immunofluorescence assay were used to detect the protein expression of ataxin-3, Golgi reassembly stacking protein 2 (GORASP2), and Golgi matrix protein 130 (GM130) in the skin fibroblasts. The morphology of the Golgi apparatus in skin fibroblasts was detected using transmission electron microscopy.Results:Tissue culture of skin fibroblasts of both SCA3 patients and healthy volunteers was successfully established. The patient-derived dermal fibroblasts expressing mutant ataxin-3 protein exhibited reduced cell viability ( t=5.06,P=0.007), increased apoptosis ( t=3.77, P=0.020), fragmentation of the Golgi apparatus, increased expression of GM130 ( t=5.23, P=0.006), and decreased expression of GORASP2 ( t=4.35, P=0.012). Transmission electron microscopy revealed that the Golgi apparatus was disorganized in skin fibroblasts. Conclusion:Fragmentation of the Golgi apparatus occurs in the skin fibroblasts of SCA3 patients, and abnormal morphology and structure of the Golgi apparatus may be involved in the pathogenesis of SCA3.

Comparative research on separation and purification methods of neural stem cells / 中国药理学通报

Aim To explore and optimize the different separation and purification methods of neural stem cells (NSCs) , and compare the cell viability and purity after separation and purification.Methods (X) Separation of NSCs: The brains of ICR mice born within 24 h were isolated ( the cerebellum and olfactory bulb removed) , chopped and digested by mechanical pipetting, trypsin digestion, and PDD enzyme digestion.After sieving and centrifugation, the brain tissues were inoculated at a concentration of 1 X 10 • L , and cultured in serum-free medium to observe and compare the activity, proliferation and number of miscellaneous cells.(2) Purification of NSCs: The brain tissues of mice born within 24 h were isolated, and NSCs were purified by differential centrifugation and sieving, and the purity was identi-fied by immunofluorescence.Results NSCs separated by PDD enzyme digestion had high survival rate and rapid proliferation rate; the Nestin positive rate of NSCs in both Sieving assay and Differential centrifugation assay groups was higher than that of primary NSCs (59.14% ± 0.16% , P <0.05) , and the former (93.54% ± 0.02%) was higher than the latter (86.12% ± 0.04% ) .Conclusions The three separation methods can obtain a large amount of NSCs.Among them, the PDD enzymatic digestion method has little damage to the NSCs, and the neuro-sphere proliferation speed is fast; the NSCs obtained by the sieving assay have higher purity.

Synthesis of eicosapentaenoic acid and hyaluronic acid graft copolymer and its anti-hepatoma activity / 中国药科大学学报

@#In this study, the conjugate of eicosapentaenoic acid (EPA) and hyaluronic acid (HA) was synthesized and the anti-hepatoma activities in vitro were evaluated.The hyaluronic acid-eicosapentaenoic acid (HA-EPA)nanoparticle was synthesized by linking eicosapentaenoic acid with hyaluronic acid with cystamine.The structure of HA-EPA was characterized by nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy (FT-IR).Laser particle sizer and Zeta potential analyzer were used to detect the size and potential of HA-EPA.MTT assay was used to detect the anti-proliferative effect of HA-EPA on HepG2, Huh-7 and LX-2 cells in vitro.The effects of HA-EPA nanoparticles on the proliferation and apoptosis of HepG2 cells in vitro were investigated by EdU staining and TUNEL staining. The apoptosis was further confirmed by flow cytometry.The effect of HA-EPA nanoparticles on the migration and invasion of HepG2 cells was demonstrated by transwell and invasion experiments.The results of 1H NMR showed that HA-EPA was successfully synthesized, and the grafting rate of EPA on HA was (40 ± 5) %. The structure of HA-EPA was further confirmed by FT-IR.The particle size was (162.5 ± 10.2) nm, and the potential was -(4.47 ± 0.31) mV.MTT results showed that, with the prolongation of drug treatment time, HA-EPA showed a better inhibitory effect on the activity of HepG2 and Huh-7 cells than EPA under the same EPA content.After treated for 48 hours, the toxicity of HA-EPA to LX-2 cells was less than that of EPA.The results of 24-hour proliferation, apoptosis, migration and invasion of HepG2 showed that, the graft of hyaluronic acid improved the ability of EPA to inhibit proliferation, promote apoptosis, migration and invasion of HepG2 cells (P < 0.001), indicating that grafting of HA can significantly enhance the inhibitory effect of EPA on liver cancer with some role in reducing toxicity.

In Vitro Cytotoxicity Analysis of Bioceramic Root Canal Sealers on Human Gingival Fibroblast Cells

ABSTRACT@#This study evaluated the cytotoxicity of four bioceramic root canal sealers (bioceramic sealers): GuttaFlow Bioseal (GB), MTA Fillapex, CeraSeal Bioceramic root canal sealer (CS), and iRoot SP root canal sealer (iRSP). The viability of human gingival fibroblast (HGF) cells was used to evaluate the cytotoxicity of these bioceramic sealers. HGF cells were cultured and exposed to bioceramic sealer extracts for 24 hours, 48 hours and 72 hours at 37°C in an incubator humidified with 5% CO2. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide or MTT assay was conducted to determine cell viability at each incubation period and compared among all bioceramic sealers. The Kruskal-Wallis test revealed statistically significant differences between the positive control group and MTA Fillapex, MTA Fillapex and GB, and between GB and iRSP with p < 0.05. However, no statistical differences were found in cell viability for each material across all the incubation periods. GB was the least cytotoxic bioceramic sealer with cell viability exceeding 90% throughout the 72-hour incubation followed by CS, iRSP, and MTA Fillapex with non-cytotoxicity after 72-hour incubation, mild cytotoxicity after 72-hour incubation, and mild cytotoxicity after 72-hour incubation, respectively. However, iRSP showed moderate cytotoxicity, and MTA Fillapex was severely cytotoxic (< 30% cell viability) after 24-hour incubation.

Fucoxanthin suppresses OxLDL-induced inflammation via activation of Nrf2 and inhibition of NF-κB signaling

@# Objective: To explore the impact of fucoxanthin on oxidized low-density lipoprotein (OxLDL)-induced stress and inflammation in human endothelial cells and its underlying mechanisms. Methods: HUVECs were treated with OxLDL and/or fucoxanthin for a range of time points and concentrations. We evaluated the effects of fucoxanthin on OxLDL-induced HUVECs using the MTT assay, reactive oxygen species accumulation assay, ELISA, RT-PCR, immunofluorescence, and Western blotting. Results: Fucoxanthin enhanced the cell viability in a dose dependent manner after OxLDL exposure. Furthermore, fucoxanthin pretreatment significantly decreased OxLDL-induced reactive oxygen species production and prevented the activation of the nuclear factor kappa-B pathway, which led to substantial suppression of pro-inflammatory gene expressions. OxLDL-induced upregulation of interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, interleukin-1β, monocyte chemotactic protein-1, cyclooxygenase-1, and tumor necrosis factor-α was significantly reduced by fucoxanthin. Conclusions: Fucoxanthin can inhibit OxLDL-induced vascular inflammation and oxidative stress in HUVECs by targeting Nrf2 signaling pathways.