Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

BACKGROUND: Fetal bovine serum (FBS) has been used to support the growth and proliferation of mammalian cells for decades. Owing to several risk factors associated with FBS, several trials have been conducted to evaluate substitutes to FBS with the same efficiency and the lower risk issues.METHODS: In this study, human platelet lysate (HPL) derived from activated human platelets was evaluated as an alternative to FBS due to the associated risk factors. To evaluate the efficiency of the preparation process, platelet count was performed before and after activation. The concentrations of several growth factors and proteins were measured to investigate HPL efficiency. HPL stability was studied at regular intervals, and optimal heparin concentration required to prevent gel formation in various media was determined. The biological activity of HPL and FBS was compared by evaluating the growth performance of Vero and Hep-2 cell lines.RESULTS: Result of platelet count assay revealed the efficiency of HPL preparation process. Growth factor concentrations in HPL were significantly higher than those in FBS, while the protein content of HPL was lower than that of FBS. Stability study data showed that the prepared HPL was stable for up to 15 months at −20℃. Ideal heparin concentration to be used in different media was dependent on calcium concentration. Results of cell viability assay showed that HPL was superior to FBS in supporting the growth and proliferation of Vero and Hep-2 cells.CONCLUSION: The HPL prepared by the mechanical activation of platelets may serve as an efficient alternative to FBS in cell culture process.

miR-135a knockdown inhibits the malignant biological behaviors and promotes oxaliplatin-sensitivity of human laryngeal carcinoma Hep-2 cells by down-regulation of SOX2 / 中国肿瘤生物治疗杂志

@# Objective: To investigate the effect of miR-135a on the malignant biological behaviors of human laryngeal carcinoma epithelial Hep-2 cells and its sensitivity to oxaliplatin. Methods: Samples of laryngeal carcinoma tissues and para-cancerous tissues were collected from 10 patients who underwent laryngectomy in Nanyang Hospital Affiliated to Zhengzhou University-Nanyang City Center Hospital from January 2018 to June 2018. The expression of miR-135a in laryngeal carcinoma tissues and Hep-2 cells was detected by qPCR.After being transfected with miR-135 inhibitor, cell proliferation viability of Hep-2 cells was measured by CCK-8 assay, cell colony formation ability was detected by colony formation assay, and cell proliferation invasion and migration abilities were detected by Transwell analysis, and the expression of SOX2 protein in Hep-2 cells was detected by WB. Hep-2 cells transfected with miR-135 inhibitor were further treated with various concentrations (0.5, 1.0, 1.5 and 2.0 μmol/L) of oxaliplatin, and the cell proliferation viability was detected by CCK-8 while cell apoptosis was detected by Annexin-V-FITC/PI double staining flow cytometry. miR-135a inhibitor plasmid, control pcDNA empty vector (SOX2-Con) plasmid, and pcDNA-SOX2 (SOX2-OE) plasmid were transfected into Hep-2 cells to construct the miR-135a inhibitor+SOX2-Con group and miR-135a inhibitor+SOX2-OE group, and the cell viability, cell colony formation ability, cell invasion and migration ability in two groups were detected. Results: Compared with para-cancerous tissues, miR135a expression in laryngeal cancer tissues was significantly increased (P<0.01). Compared with normal NHP cells, miR-135a expression in Hep-2 cells was significantly increased (P<0.01). miR-135a inhibitor significantly reduced the expression level of miR-135a in Hep-2 cells (P<0.01). miR-135a knockdown significantly reduced the cell proliferation viability, cell colony number, migration, invasion and SOX2 expression in Hep-2 cells (all P <0.01), but significantly enhanced the sensitivity of Hep-2 cells to oxaliplatin (P<0.01). Compared with miR-135a inhibitor+SOX2-Con group, the cell proliferation viability, cell colony number, migration and invasion of Hep-2 cells in miR-135a inhibitor+SOX2-OE group were significantly increased (P<0.01); Meanwhile, the cells of the 2 groups were treated with different concentrations of oxaliplatin, and the results of CCK-8 assay showed that, compared with the miR-135a inhibitor+ SOX2-Con group, the cell proliferation viability of Hep-2 cells in miR-135a inhibitor+SOX2-OE group was significantly increased (P< 0.01). Conclusion: miR-135a knockdown inhibits the malignant biological behaviors and promotes oxaliplatin-sensitivity of Hep-2 cells possibly by inhibiting the expression of the transcription factor SOX2.

HEp-2 cell line as an experimental model to evaluate genotoxic effects of pentavalent inorganic arsenic / HEp-2 como modelo experimental para evaluar los efectos genotóxicos del arsénico inorgánico pentavalente

Early detection of toxic events induced by xenobiotics is necessary for a proper assessment of human risk after the exposure to those agents. The aim of this work was to evaluate the cell line HEp-2 as an experimental model to determine the genotoxic effects of sodium arsenate. To this end, we determined the metabolic activity cells by the MTT test on seven concentrations of arsenate that range from 27 to 135,000 μM, obtaining the median lethal concentration (LC50), the lowest observed effect concentration (LOEC), and the not observed effect concentration (NOEC) of sodium arsenate at 24 h of exposition. According to the cytotoxic response obtained, we evaluated the genotoxic effect of the 27 and 270 μM concentrations by using the micronucleus assay and chromosomal aberrations test. We found a statistically significant increase (p<0.05) in the frequency of micronuclei between control cultures and those exposed to the highest concentration of sodium arsenate. Furthermore, the frequencies of nucleoplasmic bridges and tripolar mitosis were significantly higher in cell cultures exposed to the above concentrations compared to the control cultures (p<0.05). The participation of the glutathione system as response to the arsenate exposition was also analyzed, and a statistically significant increase in the glutathione content was found in those cells exposed to 27 μM of arsenate. The Glutathione S-transferase activity did not increase in the exposed cells compared to control cells, suggesting that the arsenate reduction involved other metabolic pathways in the HEp-2 cells. These results confirm that, under the conditions carried out in this study, sodium arsenate is genotoxic for HEp-2 cells. Therefore, we suggest that this cell line would be a good model for the assessment of the cytotoxic and genotoxic effects of xenobiotics on human cells.

La detección temprana de eventos tóxicos inducidos por xenobióticos es necesaria para una adecuada evaluación del riesgo humano ante la exposición a dichos agentes. El objetivo de este trabajo fue evaluar a la línea celular HEp-2 como modelo experimental para determinar los efectos genotóxicos del arseniato de sodio. Para ello, se determinó la actividad metabólica de las células mediante el ensayo de MTT, en siete concentraciones de arseniato de sodio en el rango 27-135.000 μM, determinando la concentración letal media (LC50), la menor concentración de efecto observado (LOEC) y la mayor concentración de efecto no observado (NOEC) de arseniato de sodio para una exposición de 24 h. Teniendo en cuenta los datos de citotoxicidad, se evaluó el efecto genotóxico a las concentraciones 27 y 270 μM por medio del ensayo de micronúcleos y aberraciones cromosómicas, encontrando un aumento estadísticamente significativo en la frecuencia de micronúcleos entre el control y la mayor concentración arseniato de sodio ensayada. Además, la presencia de puentes nucleoplasmáticos y mitosis tripolar fue significativamente mayor en ambas concentraciones estudiadas con respecto al control. Se analizó la participación del sistema de glutatión como respuesta a la exposición al arseniato, encontrándose un aumento estadísticamente significativo en el contenido de glutatión en la concentración de arseniato de 27 μM. La actividad de la glutatión S-transferasa no aumentó, lo que sugiere que la reducción del arseniato implicó otra vía metabólica en las células HEp-2. Estos resultados confirman que el arseniato de sodio induce genotoxicidad en células HEp-2 en las condiciones realizadas en este estudio y por lo tanto este tipo de línea celular es un buen modelo para ensayos de citotoxicidad y genotoxicidad en los cuales se quiere evaluar el riesgo humano.

Influence of siRNA interfering USP7 expression on cytobiological characteristics of laryngeal cancer cells / 重庆医学

Objective This study is to investigate the influences of USP7 on the cytobiological characteristics of laryngeal cancer cells by small interfering RNA (siRNA) interfering the USP7 expression in the laryngeal cancer cells .Methods The self‐de‐signed highly efficient siRNA was used to conduct the specific interference on USP7 expression in laryngeal cancer HEP2 cells . Then the influence on the capacity of cell proliferation and migration ,as well as apoptosise after USP7 interference were observed by using the CCK‐8 method ,Transwell chamber migration test and flow cytometry .Results The self‐designed siRNA could effi‐ciently inhibit the expression of USP7 mRNA in laryngeal cancer cells ,furthermore markedly suppressed the proliferation and mi‐gration of laryngeal cancer cells ,enhanced the cell apoptosis in laryngeal cancer HEP2 cells in vitro .Conclusion The siRNA inter‐fering USP7 can inhibit the proliferation and migration capacity of laryngeal cancer cells ,and promoted their apoptosis .

mTOR signaling pathway for regulating inhibition of 1,25 dihydroxyvitamin D3 on Hep-2 cells proliferation in laryngeal carcinoma / 国际检验医学杂志

Objective To explore the inhibition role of 1 ,25 dihydroxyvitamin D3 on laryngeal cancer Hep‐2 cell proliferation and its influence on mTOR signal pathway .Methods Hep‐2 cells were treated with different concentrations of 1 ,25 dihydroxyvitamin D3 (10-8 ,10-7 ,10-6mol/L) for 24 ,48 ,72 h respectively .The proliferation situation of Hep‐2 cells was detected by the MTT meth‐od and the inhibition rate was calculated .The effect of 1 ,25 dihydroxyvitamin D3 on Hep‐2 cell cycle distribution was analyzed by flow cytometry .The influence of 1 ,25 dihydroxyvitamin D3 on mTOR signaling pathway was detected by Western blot .Results Different concentrations of 1 ,25 dihydroxyvitamin D3 could inhibit the proliferation of Hep‐2 cells ,changed the cell cycle distribu‐tion and increased the proportion of Hep‐2 cells in G0/G1 phase .The expressions of TSC1 and TSC2 protein after 1 ,25 dihydroxyvi‐tamin D3 intervention were increased compared with the control group (P<0 .01) ,while the Rheb protein expression was signifi‐cantly decreased(P<0 .01):mTOR protein and phosphorylation level were significantly decreased compared with the control group (P<0 .01) ,the decrease of mTOR protein phosphorylation was especially obvious (P<0 .01);4EBP‐1 protein expression was in‐creased compared with the control group (P<0 .01) .Conclusion 1 ,25‐dihydroxyvitamin D3 alters the Hep‐2 cell cycle distribution , affects the protein expression of mTOR signaling pathway ,thus inhibits the cell proliferation .

Identification and characterization of a potent anticancer fraction from the leaf extracts of Moringa oleifera L.https://docs.google.com/forms/d/1RkQLeYfiMvE7SKcdOXv9Acm5eXRjmvleic5FYjjX1TE/viewform?fbzx=6173639259593088622

Anticancer potential of Moringa oleifera L. extracts have been well established. However, there are no reports on the isolated molecules/fractions from these extracts which are responsible for the anticancer/cytotoxic activity. Thus, in the present study, we explored the same. The n-hexane, chloroform, ethyl acetate, methanol extracts of the M. oleifera leaves and 15 fractions (F1 to F15) of ethyl acetate extract were evaluated for their in vitro and in vivo anticancer activity using Hep-2 cell lines and Dalton’s lymphoma ascites model in mice, respectively. Among the tested samples, the F1 fraction showed potential cytotoxic effect in Hep-2 cell lines with a CTC50 value of 12.5 ± 0.5 µg/ml. In vivo studies with the doses 5 and 10 mg/kg, p.o. demonstrated significant reduction in body weight and increased the mean survival time compared to the control group. These results were also comparable to the standard, 5-Fluorouracil, treated animals. We have also successfully isolated and characterized the anticancer fraction, F1 from the leaves of M. oleifera L.

Identification of Enteroaggregative Escherichia coli in Mongolia / Монголын Анагаах Ухаан

Introduction: Enteroaggregative Escherichia coli (EAEC) is an important agent of acute and persistent diarrhea worldwide. Few cases have been reported in healthy children. EAEC strains are characterized by aggregative adherence (AA) to HEp-2 cells, wherein bacteria are seen in “stacked brick” aggregates attaching to HEp-2 cells and usually to the glass surface between cells. Goal: To identify Enteroaggregative Escherihia coli using multiplex polymerase chain reaction (PCR) and HEp-2 adherence assay in Ulaanbaatar, Mongolia Materials and Methods: A total of 329 E. coli strains were isolated from stool with diarrhea in National Center for Communicable Diseases from July 2012 through September 2014. All specimens were processed by routine microbiological and biochemical tests in the bacteriological laboratories to identify Salmonella spp., Shigella spp. All specimens in our study were negative for these bacterial and parasitic pathogens. The biofilm formation was evaluated by the growth rate of E.coli on plastic surface. PCR assays were used to detect genes of five types of diarrheagenic E.coli (DEC). All of the DEC strains showed mannose-resistant adherence to HEp-2 cells, and aggregative adherence was predominant in these isolates. Bacterial susceptibility to antimicrobial agents determined by the Kirby Bauer disk diffusion method on Muller Hinton agar. Results: EAEC (31.9%) was the most prevalent by PCR and HEp-2 assay comparing with others. EAEC by multiplex PCR in samples (11, 3.3%), followed by enteropathogenic E.coli (EPEC) seen in 2.1%. Enterohemorrhagic E.coli (EHEC) and enteroinvasive E.coli (EIEC) were found in 7 (2.1%) and 1 (0.3%) of the samples. Enterotoxigenic E.coli (ETEC) and diffusely adhering E.coli were detected in 2 (0.6%), respectively. The evaluation of bacterial biofilm formation using 96 well plates showed 309 negative (93%), 15 weak biofilm (4.6%) and 8 moderate biofilm (2.4%) formation for E.coli and no strong biofilm forming strain was detected. Above 50% of antibiotic resistance was observed for ampicillin, trimethoprim/sulfamethoxazole, cefuroxime and cephalotin. Also, 95.4% of isolates were resistant to at least three different classes of antimicrobial agents and considered as multidrug resistance. Conclusion: EAEC is most prevalent pathogen among DEC in our samples. It is necessary to implement EAEC identifying method on Hep-2 assay in our laboratory practice.

Ferulic Acid Reduces Cell Viability through Its Apoptotic Efficacy: An In vitro Approach.

Aim: Ferulic acid, a well known dietary phenolic antioxidant, possesses diverse pharmacological and biochemical effects, including anti-inflammatory, hepatoprotective, antidiabetic and anticancer properties. The present study explores the cytotoxic potential of ferulic acid using Hep-2 cell line by analyzing its effect on cell viability, reactive oxygen species generation, apoptotic induction, nuclear damage, DNA fragmentation and expression of apoptosis related proteins. Materials and Methods: The effect of ferulic acid (2.5, 5, 10, 20 and 40 μg/ml) on Hep-2 cells viability for 24 hr was determined by MTT assay. To substantiate the cytotoxic effect of ferulic acid, the intracellular ROS level was determined using DCFH-DA assay; apoptosis by dual staining; nuclear damage by DAPI staining; DNA fragmentation by using agarose gel electrophoresis; apoptosis related proteins by western blotting. Results: Ferulic acid significantly inhibited the Hep-2 cell growth in a dose dependent manner and ferulic acid treated Hep-2 cells exhibited features of apoptosis and increase in nuclear damage and DNA fragmentation. We also observed excess reactive oxygen species generation in ferulic acid treated Hep-2 cells. Apoptosis related proteins (p53, Bcl-2, Bax, Caspase 3 & Caspase 9) were significantly modulated in favour of programmed cell death in ferulic acid treated cells. Conclusion: We thus conclude that the cytotoxic potential of ferulic acid might be due to its role in apoptosis induction, excessive ROS generation and DNA fragmentation in Hep-2 cells.

Curso de reumatologia: aula 1: significado clínico do FAN anticorpos antinucleares / Course of rheumatology: class 1: clinical significance of antinuclear antibodies ANA

A utilização de células HEp-2 na detecção dos anticorpos antinucleares (FAN-HEp-2) constituiu-se em grande avanço metodológico. Como consequência, o teste tornou-se muito sensível, porém com diminuição da especificidade, fato comprovado pelo crescente número de exames positivos em indivíduos sabidamente hígidos. O objetivo deste artigo é fazer uma abordagem sobre os pontos importantes, para correta valorização dos achados do teste de FAN, que possa auxiliar o clínico na exata identificação de pacientes com doenças autoimunes, particularmente o lúpus eritematoso sistêmico. Os autores discutem a importância do título e do padrão na interpretação de um FAN positivo. Discutem também os possíveis significados do teste positivo em pacientes sem evidências objetivas de doenças imunológicas.

Antinuclear antibody (ANA) tests are frequently used to screen patients for chronic inflammatory diseases such as lupus erythematosus (SLE). However, the methodological advances have brought up a considerable improvement in the test's sensitivity and consequently a decrease in its specificity. This has resulted in an increasing number of positive tests in apparently healthy subjects. The present article brigs an approach on the most important points to be considered in the analyses and evaluation of an ANA test, like titer and immunofluorescence pattern of a positive test. Those points are importants parameters in the evaluation of the significance of a positive ANA-HEp-2 test that might help the identification of patients with autoimmune disease. Also, it is important to consider the possible and alternative significance of a positive ANA test in patients without objective evidence of autoimmune diseases.

Molecular Genetic Characterization of Shiga Toxin-producing E. coli Isolated from Diarrhea Patients and Cattle in Gwangju Area, Korea

Shiga toxin-producing Escherichia coli (STEC) can cause a broad spectrum of human illness ranging from symptom-free to hemolytic uremic syndrom (HUS). Associations between known or putative virulence factors of STEC and diseases in human were investigated. PCR analyses showed that 33 (78.6%) isolates carried an ehxA enterohemolysin gene and 6 (14.3%) isolates possessed an saa autoaggutinating adhesin gene, and 31 (73.8%) isolates carried an eae intimin gene (7 isolates with type beta, 16 with type gamma, and 3 with type epsilon). Twenty-nine (69%) isolates from patients carried eae+, ehxA+, saa- (genotype A) and 68 (86%) isolates from asymptomatic outbreaks and 4 (36%) isolates from bovine possessed eae-, ehxA+, saa+ (genotype C). Neither the bundle-forming pilus gene nor the enteropathogenic E. coli adherence factor plasmid was found. In HEp-2 cell adherence assay, isolates carrying eae gene exhibited a localized adherence phenotype, the other isolates carrying saa showed LC (loose clusters of bacteria) and IS (isolated bacteria). In conclusion, most STEC isolated from cattle feces in Gwangju, Korea showed characteristics different from those isolated from patients. But these results may be useful information for pathogenesis judgement of STEC.

Effect of DRB on the Biological Characteristics of Human Laryngeal Carcinoma Hep-2 Cell Line / 华中科技大学学报（医学）（英德文版）

In order to study the effect of 5, 6-Dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) on the biological characteristics of human laryngeal carcinoma Hep-2 cell line in vitro, Hep-2 cells cultured in vitro were treated with different concentrations of DRB. Changes in cell proliferation, apoptotic rate and invasiveness were detected by MTT assay, flow cytometry (FCM) and matrigel in vitro invasion assay, respectively. It was found that DRB inhibited the proliferation of Hep-2 cells in a dose- and time-dependent manner. After being treated with 0, 10, 20, 40, 80 μmmol/L DRB for 24 h, the apoptotic rate in Hep-2 cells was (0.68±0.19) %, (1.95±0.12)%, (8.51 ±0.26)%, (11.26±0.17) % and (14.99±0.32)%, respectively. The matrigel in vitro invasion assay revealed that DRB began to inhibit the invasion of Hep-2 cells at the concentration of 5 μmmol/L, and with the increase of DRB concentration, the inhibitory effect was enhanced. It was suggested that DRB could influence the essential biological characteristics of Hep-2 cells, inhibit Hep-2 cells proliferation, reduce invasive ability and induce apoptosis of Hep-2 cells.

Synergistic induction of apoptosis of HEP-2 cell line by the combination of TRAIL and 5-FU,DDP / 重庆医科大学学报

Objective:To investigate whether TRAIL can synergize 5-Fu and DDP to kill HEP-2 cell line.Methods:MTT was used to detecet the cytotoxicity of HEP-2 cell line intervented in 5-FU,DDP,TRAIL,5-FU+TRAIL and DDP+TRAIL.Fluorescence microscope and FCM were used to detecet the apoptosis rate of cell.Results:HEP-2 cell line was insensitive in TRAIL,but the apoptosis rate obviously was upgraded after intervented in 5-FU+TRAIL and DDP+TRAIL.Conclusion:TRAIL in combination with subtoxic dose of 5-FU and DDP can effectively kill HEP-2 cell line.The combination of TRAIL,5-FU and DDP induced apoptosis of HEP-2 cell line.

Proliferation inhibition of chlorpromazine combined with taxol on Hep-2 cells / 吉林大学学报(医学版)

Objective To investigate the proliferation inhibition of chlorpromazine combined with taxol on Hep-2 cells (human laryngeal carcinoma cell line) and the effects on cell cycle progression. Methods Hep-2 cells at logarithmic growth phase were divided into taxol groups(3.0,6.0 and 12.0 mg?L-1),chlorpromazine(12.0 mg?L-1) comined with taxol (4 mg?L-1)group and control group (100 mL culture fluid).MTT and flow cytometry were used to detect the proliferation inhibition rates of Hep-2 cells in various groups.Flow cytometry was also used to analyze the cell cycle progression of Hep-2 cells and apoptotic rate after administration.Results The proliferation inhibition rates in 3.0,6.0 and 12.0 mg?L-1 taxol groups were 14.0%,23.9% and 36.7%,respectively,there were significant differences between three groups(P

Inhibitory Effects of Celecoxib and Sc-58125 on Proliferation of Human Carcinoma of Larynx Hep-2 In Vitro / 华中科技大学学报（医学）（英德文版）

The inhibitory effects of two kinds of selective cyclooxygenase-2 inhibitors on the proliferation of human carcinoma of larynx Hep-2 in vitro and their corresponding mechanisms were investigated. Hep-2 cells were cultured with two kinds of selective cyclooxygenase-2 inhibitors (Sc58125 and Celecoxib) at various concentrations for 24 h. Morphological changes were observed under the phase microscopy and the growth suppression was detected by using MTT colorimetric assay. Apoptotic DNA fragments were observed by agarose gel electrophoresis, and the cell cycle andapoptotic rate were detected by flow cytometry (FCM) respectively. Hep-2 cells became roundedand detached from the culture dish after being treated with Celecoxib for 24 h, however, they remained morphologically unchanged with Sc-58125. Sc-58125 could increase G2 phase cells, whereas, Celecoxib rose G1 phase cells. Both of the two effects were dose-dependent. Moreover, the Hep-2 cells cultured with 50μmol/L and 100 μmol/L Celecoxib showed obvious apoptosis, with the nuclear DNA of cells exhibiting characteristic DNA ladder. So Sc-58125 could inhibit the proliferation of Hep-2 cells by altering the G2 phase cells. However, Celecoxib had the same effect by changing the G1 phase cells and inducing apoptosis at higher concentration.