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BACKGROUND: Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell In the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. RESULTS: Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. CONCLUSION: These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.
Subject(s)
Animals , Cell Survival , Apoptosis , Cell Proliferation , Caspase 7/deficiency , Cricetulus , Cricetinae , CHO Cells , Caspase 7/geneticsABSTRACT
Purpose To observe the expression of longchain non-coding RNA-LINC00485 (LINC00485) in lung cancer cell lines and tissues, and to investigate its effect on the proliferation and migration of lung cancer cells and its mechanism.Methods Quantitative real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect differential expression of LINC00485 in four lung cancer cell lines (H1975, A549, HCC827, H1299), normal alveolar epithelial cells HPAEPIC, and in 12 cases lung cancer tissues and adjacent tissues. Bioinformatics methods were used to predict the microRNA (miRNA) that LINC00485 may bind and target gene that miRNA may bind. Small interfering RNAs (siRNAs) that target silencing LINC00485 were transfected into HCC827 cells by liposomes.The expression levels of LINK00485, miR-361-5p, and p21 activated protein kinase 2 (PAK2) mRNA were detected by qRTPCR. The expression level of PAK2 protein was detected by Western blot. The cell proliferation ability was measured by MTS assay. Cell scratch assay was used to detect cell migration. Results Compared with normal alveolar epithelium, LINC00485 was highly expressed in lung cancer cell lines (P < 0.05), and the expression level was highest in HCC827 cells. The expression of LINC00485 in lung cancer tissues was higher than that in adjacent tissues (P < 0.01). After down-regulation of LINC00485 expression in HCC827 cells, the expression of miR-361-5p was up-regulated (P < 0.01), the expression of PAK2 mRNA and protein was down-regulated (P < 0.01), the proliferative capacity of HCC827 cells was decreased (P < 0.05), and the ability of cell migration was decreased (P < 0.01).Conclusion The expression of LINC00485 is increased in lung cancer cell lines and tissues. Down-regulation of LINC00485 can inhibit the proliferation and migration of lung cancer HCC827 cells by regulating the expression of miR-361-5p and PAK2 genes.
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Objective To investigate the effects of silencing Smo gene on proliferation and apoptosis of rat prima-ry chondrocyte in vitro.Methods The primary chondrocyte was obtained by mechanical-enzyme digestion and identified by Immunohistochemical cells ( ColⅡ) .The animals were divided into control group , control siRNA group and Smo siRNA 1 ~3 group.The siRNA was transfected into chondrocytes by lentivirus vector .After 72 h, the cell viability was detected by MTT, Smo expression was detected by RT-PCR and Western blot, and the apoptosis of chondrocyte was assessed by flow cytometry .Results All types of siRNA were transfected into primary chondrocyte by vectors, the Smo siRNA 1 ~3 may inhibit the expression of Smo mRNA and protein in chondrocytes, and Smo siRNA2 had the highest silencing rate ( the expressions of Smo mRNA and protein were 0.19 ±0.03 and 0.39 ±0.07 ) .The cell viability in Smo siRNA2 group was lowest ( 77.38% ±7.19%) , while the apoptosis rate of Smo siRNA2 was highest ( 21.43%±2.97%) .Conclusions Silencing Smo gene in primary chondrocytes may inhibit proliferation and promote apoptosis , Smo may have a protecting role from apop-tosis of the chondrocyte.
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INTRODUÇÃO: Existe um grande interesse no estabelecimento de recursos e terapias a serem utilizados na tentativa de proporcionar um processo de reparo muscular de melhor qualidade e menor duração. O ultrassom terapêutico (US) e o laser de baixa potência (LBP) são recursos muito usados na prática clínica, porém são escassas, e por vezes contraditórias, as evidências científicas que determinam com segurança os parâmetros dosimétricos e metodológicos adequados. OBJETIVOS: O objetivo do estudo foi analisar o efeito do US e do LBP sobre a proliferação celular durante a diferenciação de mioblastos C2C12. MATERIAIS E MÉTODOS: Os mioblastos foram cultivados em meio de cultura de Eagle modificado por Dulbecco, contendo 10% de soro fetal bovino (SFB), sendo induzida a diferenciação pela adição de 2% de soro de cavalo durante 96 horas. Posteriormente, as células foram irradiadas com US pulsado a 20%, 3 MHz de frequência (intensidades de 0,2 e 0,5 W/cm², durante cinco minutos) ou submetidas ao tratamento com LBP (potência de saída de 10 mW, densidade de energia de 3 e 5 J/cm², por 20 segundos). A proliferação celular foi avaliada após 24h e 72h utilizando o método de MTT. Foram realizados três experimentos independentes, em cada condição citada e células não irradiadas serviram como controle. RESULTADOS: Os resultados obtidos foram submetidos à análise estatística utilizando a Análise de Variância (ANOVA), teste Dunnet, para verificar diferenças entre o grupo controle (não tratado) e os grupos tratados com US e LBP, adotando significância de p < 0,05. Os resultados evidenciaram que não houve diferença significativa na proliferação celular entre as células musculares submetidas a tratamento com ambos os recursos terapêuticos e as células controle, nos períodos de 24h e 72h após tratamento. Além disso, foi possível verificar que não houve aumento significativo no número de células após o período de 72h quando comparado a 24h, confirmando o processo de diferenciação celular, conforme esperado. CONCLUSÕES: Conclui-se que o US e o LBP, nos parâmetros avaliados, não alteraram a proliferação de mioblastos em processo de diferenciação.
INTRODUCTION: There is great interest in establishing resources and therapies to be used in an attempt to provide a process of muscle repair better and shorter. Two features commonly used to facilitate the healing process are the therapeutic ultrasound and laser power, however, are still scarce and sometimes contradictory scientific evidence to determine with certainty the parameters and methodology necessary to acquire these goals. OBJECTIVES: Thus, the objective of present study was to evaluate the effect of therapeutic ultrasound (US) and low level laser GaAlAs 660 nm on the proliferation of C2C12 myoblasts. MATERIALS AND METHODS: The myoblasts were cultivated in culture medium of Eagle modified by Dulbecco, containing 10% fetal bovine serum (FBS) and induced to differentiate by addition of 2% horse serum for 96h. Subsequently, cells were irradiated with US (20%, 3 MHz, and intensities of 0.2 and 0.5 W/cm² for five minutes) or with LLL (output power of 10 mW, density of 3 and 5 J/cm² for 20 seconds). The non-irradiated cells serve as controls. Three independent experiments were performed for each condition cited. Cell proliferation was evaluated after 24h and 72h using the MTT method. RESULTS: The results were statistically analyzed using analysis of variance (ANOVA/Dunnet) to verify differences between control cells (untreated) and US/LLL treated groups. The results showed no significant differences in viability and cell proliferation between LLL or US treated cells and control cells after 24h and 72h. Furthermore, it was observed no increase in the number of cells within the evaluation period after treatment, confirming the process of cell differentiation, as expected. RESULTS: In conclusion, US and the LLL, employed under the parameters described does not alter C2C12 proliferation.
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Lasers , Muscle Cells , Myoblasts , Cell Proliferation , UltrasonicsABSTRACT
Objective: To study the effects of BMPs signals on the proliferation of tongue cancer Tca8113 cells. Methods: Th e cDNA of truncated BMP-II receptor was transfected into Tca8113 cells by usin g FuGENE6 transfection kit, the transfected cells were named Tca8113ZR. The pro liferation and DNA synthesis of Tca8113 and Tca8113ZR cells were investigated b y MTT assay,FCM and BrdU analysis. Results: In MTT assay the A value of Tca8113 and Tca8113ZR cells was 0.47?0.01 and 0.35?0.01 (P0.05).Conclus ion: BMPs might be involved in the development of squamous cell carc inoma of tongue.