Effect of Raldh2 silence on the differentiation of P19 cells into cardiomyocyte-like cells / 复旦学报（医学版）

Objective To study the effect of retinaldehyde dehydrogenase 2 (Raldh2) on the differentiation of P19 cell to cardiomyocyte-like cells and explore the potential mechanism.Methods A miRNA expression plasmid specific to Raldh2 was packaged and constructed by RNA interference (RNAi) method.The P19 stable cell line carried Raldh2 miRNA expression was selected by adding blasticidin and induced to differentiate towards cardiomyocyte-like cells.The mRNA levels of myocardium development-related markers were determined by qPCR at different stages during the differentiation process.Results The miRNA expression plasmid specific to Raldh2 could effectively suppress Raldh2 expression,and the MiRaldh2 group,a P19 stable cell line was established successfully in which the knockdown efficiency on Raldh2 was 91％ (t =25.52,P＜0.000 1,95％ CI:0.81-1.01).When compared with P19 group,the mRNA levels of cardiac transcription factors were generally decreased in the MiRaldh2 group during the whole differentiation process.In detail,on the 7th day,the relatively low expression rates of these cardiac markers including Gata4,Tef-1,N-myc,α-mhc and Ctnt was0.16±0.01 (t=17.29,P＜0.000 1),0.51 ±0.02 (t=3.564,P=0.023 5),0.23 ±0.01 (t=13.17,P =0.000 2),0.20 ± 0.02 (t=17.76,P＜0.000 1) and 0.59 ± 0.06 (t =3.642,P =0.021 9) in MiRaldh2 group when compared with the P19 group.Conversely,the mRNA levels of Nkx2.5 and Hand2 were dramatically increased in MiRaldh2 group on day 2 to 7 and the expression rates on the 7th day was 2.25 ± 0.35 (t =3.526,P =0.024 3) compared with the P19 group while Hand2 was 3.58 ± 0.20 (t =9.214,P =0.011 6).Conclusions Knockdown of Raldh2 inhibits the P19 cells differentiated into cardiomyocyte-like cells,which suggests that Raldh2 may play a potential role in early development of heart.The low expression of Raldh2 might be an explanation of the cardiac malformations associated with retinoic acid deficiency.

NgR1 Expressed in P19 Embryonal Carcinoma Cells Differentiated by Retinoic Acid Can Activate STAT3

NgR1, a Nogo receptor, is involved in inhibition of neurite outgrowth and axonal regeneration and regulation of synaptic plasticity. P19 embryonal carcinoma cells were induced to differentiate into neuron-like cells using all trans-retinoic acid and the presence and/or function of cellular molecules, such as NgR1, NMDA receptors and STAT3, were examined. Neuronally differentiated P19 cells expressed the mRNA and protein of NgR1, which could stimulate the phosphorylation of STAT3 when activated by Nogo-P4 peptide, an active segment of Nogo-66. During the whole period of differentiation, mRNAs of all of the NMDA receptor subtypes tested (NR1, NR2A-2D) were consistently expressed, which meant that neuronally differentiated P19 cells maintained some characteristics of neurons, especially central nervous system neurons. Our results suggests that neuronally differentiated P19 cells expressing NgR1 may be an efficient and convenient in vitro model for studying the molecular mechanism of cellular events that involve NgR1 and its binding partners, and for screening compounds that activate or inhibit NgR1.

Effects of microRNA -30c knockdown on proliferation and differentiation of P19 cells / 中华实用儿科临床杂志

Objective To explore the effects of microRNA(miRNA)- 30c knockdown on proliferation,diffe-rentiation of P19 cells. Methods miRNA - 30c knockdown plasmid(miRNA - 30c knockdown group)or no - load vector(negative control group)was transfected into P19 cells by lipo2000 and stable cell lines were selected by Blastici-din;Dual luciferase reporter gene system was used to confirm miRNA - 30c knockdown. Cell counting kit - 8(CCK - 8) assay was adopted to detect cell proliferation activity. An inverted microscope was used to observe morphological chan-ges of P19 cell differentiation. Cells were induced to differentiated to myocardiocyte with dimethyl sulfoxide(DMSO). Differentiation marker genes including cTnT,NKX2. 5,GATA4 relative mRNA expression levels were detected with real - time quantitative polymerase chain reaction,respectively. Results Observation of green fluorescent protein ex-pression under a fluorescence microscope indicated similar transfection efficiencies,and miRNA - 30c knockdown re-leased the activity of target gene Gli2. As a result,miRNA - 30c knockdown vector was constructed successfully(P ﹤0. 001). During differentiation of mouse P19 cells into myocardial cells,the beating cell clusters in miRNA - 30c knockdown cells were much lower than those in the control cells,and cTnT,NKX2. 5,GATA4 in miRNA - 30c knock-down cells showed significantly lower expression than those in the control cells( all P ﹤ 0. 05). Conclusions miRNA - 30c inhibits the P19 cell proliferation and differentiation. This study gives us a new insight of heart develop-ment and we need more efforts on exploring the deep function of heart diseases.

Expression pattern of microRNA-379 during the embryo heart development and differentiation of P19 cells / 中华实用儿科临床杂志

Objective To observe the expression changes in microRNA (miR)-379 in the developmental process of the mouse heart and during the differentiation of P19 cells into cardiac myocytes,and to explore the possible relationship between miR-379 and the differentiation of cardiacmyocytes.Methods Heart tissues were collected from fetal mice in pregnant ones at their gestational age (8.5,11.5,14.5 and 18.5 days) respectively.Heart tissue sections of the fatal mice were obtained to observe the heart development process.Then total RNA was isolated from heart tissues by using the TRIzol method.Complementary DNA was synthesized from 1 μg total RNA by using a Reverse Transcriptase Kit.Finally,real-time PCR (RT-PCR) was employed to detect the expression of miR-379.At the same time,P19 cells were cultured with 10 mL/L Dimethyl sulfoxide in suspension for 4 days to form cell aggregation,and these aggregations were transferred into 6-wells plate for culturing by adherence.Beating cells were detected with microscopy on the 10th day after induction.Afterwards,total RNA was extracted from cultured P19 cells at different time points.Reverse transcription was executed to get DNA.At last,RT-PCR was used to explore the expression of miR-379 on 0,4,6,10 days after aggregation.Results The expression level of miR-379 was down-regulated gradually in the developing heart (at gestational age of 8.5,11.5,14.5,16.5 days,respectively),and there were significant differences on the different days (F =21.13,P ＜ 0.05).On the other hand,myocardial markers of troponin T represented an increasing trend during the process of P19 cells induction,which demonstrated that P19 cells were successfully induced into cardiomyocyte-like cells.Meanwhile,miR-379 showed a low expression on day 0 of P19 cells aggregation.On day 4,miR-379 demonstrated a higher level.Afterwards,miR-379 proved to be down-regulated gradually.Conclusions miR-379 plays a role in the process of the heart development,but the specific mechanisms need further research.

Nkx2-5 gene promotes expression of cardiac markers in 5-azacytidine-induced monolayer P19 cells / 第二军医大学学报

Objective: To study the role of Nkx2-5 gene in 5-azacytidine-induced differentiation of monolayer P19 cells into myocardial cells. Methods: The experiment was divided into two groups: an experimental group and a control group. The cells in the experimental group were P19 cells stably expressing Nkx2-5 gene, and cells in the control group were P19 cells. Under the monolayer-culture condition, the cells of two groups were induced by 5-azacytidine (1 μmol/L). The growth of cells were observed by inverted microscope. On the 4th day, 8th day, 12th day and 16th day after induction, RT-PCR was used to detect the expression of GATA-4, α-MHC and ANP gene. Results: In control group, there was no ANP expression after induction; GATA-4 expression was seen on the 8 th day, 12th day, and 16th day after induction; and α-MHC expression was found on the 12th day and 16 th day. In experimental group, the expression of GATA-4 was detected on the 4th day, 8th day, 12th day and 16 th day after induction; Alpha-MHC and ANP expression was noticed on the 8th day, 12th day and 16th day after induction. RT-PCR results showed that the expression of GATA-4 and α-MHC in the experimental group was earlier than that in the control group. And at all time points of observation, the expression of GATA-4 and α-MHC in the experimental group was significantly increased compared with that in the control group (P<0. 05,P<0. 01), except for α-MHC expression on the 12 th day. Conclusion: Nkx2-5 gene can promote 5-azacytidine-induced differentiation of monolayer P19 cells into myocardial cells.

Cardiomyocyte Differentiation from P19 Cells Induced by NKX2-5 / 中国医科大学学报

Objective To identify the rale of NKX2-5 gene in cardiomyocyte differentiation and its mechanism.Methods P19 cells were divided into transfected and non-transfected groups.In the transfected group,P19 cells were with stable expression of NKX2-5 gene.The P19 cells were cultured in suspension for 4 days,and the formed aggregates were transferred to Petri dish for adherent culture.On days 4,8,12,and 16 of the adherent culture,the expressions of ct-saicomeric actin(α-SA)and cardiac troponin T(cTnT)were detected with double-labeling immunofluorescence and Western blot.The ultrastruetural changes were observed on day 16.Results In the transfected group,no expression of α-SA and cTnT was found on day 4,and the expression of these 2 proteins or co-expression existed on days 8,12,and 16.There were early cell junction and myofilament-like structure in the cytoplasm of some cells in the transfected group.In the non-transfected group,these 2 proteins were negative,and no differentiated cell was found.Conclusion Stable expression of NKX2-5 gene can induce cardiomyocyte differentiation from P19 cells,but the P19 cells with stable expression of JVKX2-5 gene is not suitable to be an in vitro model of cardiac development.

Neuronal Differentiatin of P19 Cells / 대한해부학회지

P19, murine embryonal carcinoma (EC) cells can be induced to differentiate into neurons in the presence of retinoic acid (RA). To investigate neuronal differentiation of P19 cells in details, P19 aggregates were obtained in the presence or absence of RA, ascorbic acid (AA) and 2-mercaptoethanol (2-ME) in bacteriological Petri dishes. When the aggregates were transferred into the serum depleted medium, P19 cells exhibited dramatic morphological changes. Cells contained long and thin processes as detected in differentiated neurons. Western blot analysis showed that treatment of RA and AA induced expression of neuron-specific markers such as NCAM, NSE and Tuj1. Expression of GFAP was not detected, suggesting that P19 cells differentiate into neurons under our experimental condition. Immunocytochemical studies also revealed that treatment of RA and AA increased expression of NCAM and Tuj1. On the contrary, 2-ME was ineffective in the neuronal differentiation of P19 cells, which is consistent the results from the western blot analysis. These results suggest that differentiated P19 cells have similar characteristics to those of typically differentiated neurons. This study also suggests that P19 cells may provide useful tools to study neuronal differentiation in vitro.

Induction of Embryonal Teratocarcinoma Cell(P19 cell) to Differentiate into Smooth Muscle Cell

BACKGROUND: Understanding the regulatory mechanisms of the smooth muscle cells differentiation is one of the central issues in researches of atherogenesis where smooth muscle cells undergo dedifferentiation and regain embryonic phenotype. Smooth muscle myosin heavy chain isoforms(SM1,SM2) are important molecular markers to define the stage of smooth muscle cell differentiation. METHODS: In order to establish an in vitro model of smooth muscle cell differentiation using a pluripotent murine embryonal teratocarcinoma cell line(P19 cell), we first isolated cDNA clone of mouse SM1 and then tried various chemicals to induce P19 cells to differentiate into smooth muscle cells, The expression of Sm1 and alpha-smooth muscle actin was examined using RNase protection assay, Western blotting and indirect immunofluorescence. RESULTS: In the presence of luM retinoic acid, a small proportion of P19 cells could be in duced to differentiate into smooth muscle cells expressing SM1 as well as alpha-smooth muscle actin since day 8 after treatment. By blocking the Brain-2 expression, thus inhibiting neuronal differentiation, we could obtain more abundant differentiated smooth muscle cells. Sequential immunofluorescencc demonstrated that it took weveral days for smooth muscle myosin heavy chain to organize completely in differentiation smooth muscle cells. On the other hand, 10nM retinoic acid, 1% dimethyl sulfoxide or 2mM hexamethylene bisacetamide could not indduce P19 cell to differentiate to smooth muscle cells. CONCLUSION: In conclusion, P19 cells can be induced to differentiate into smooth muscle cells and this in vitro system will be useful in understanding the regulation of SM1 gene expression as well as of angiogenesis.

INDUCTION OF MYOCARDIUM CELLS FROM P19 CELLS USING DIMETHYL SULFOXIDE IN VITRO / 解剖学报

Objective Observing the process of cell aggregation and differentiating into myocardium after the P19 cells treated with different methods and dimethyl sulfoxide(DMSO) inducer.Methods P19 cells were cultivated with DMSO in suspension for 7 days in Petri dishes or dishes containing a thin layer of soft agar to form cell aggregation.Then the aggregates were plated on gelatin-coated culture dishes,and cultured without DMSO to 19 days.The beating of cells was observed.?-sarcomeric actin and cardiac Troponin T(cTnT) immunocytochemical stains were used to identify cell differentiation.Results Cell aggregations were cohering-cultured with growth culture medium after DMSO induced for 7days,spontaneous and rhythmically beating cells were present within the aggregation outgrowths at day 15,which were ?-sarcomeric actin-positive and cTnT-positive.By day 19,positive rates were about 26% and 15% respectively.Conclusion By using DMSO exposure combination prior aggregation can induce P19 cells differentiate into cardiac myocytes which can spontaneously and rhythmically beat.