Establishment of Drug-Resistant Cell Lines of Acute Myeloid Leukemia and Correlation of Sirt1 and PGC-1α Expression Levels with Drug Resistance / 中国实验血液学杂志

OBJECTIVE@#To construct cytarabine-resistant acute myeloid leukemia (AML) cell lines, and explore the correlation between Sirt1, PGC-1α expression levels and drug resistance.@*METHODS@#Human acute promyelocytic leukemia Kasumi-1 cells were induced by the method of gradually increasing the concentration of Ara-C drug. The IC50 value of Kasumi-1 cells before and after drug addition was detected by CCK-8 method, so as to construct Ara-C resistant cell lines. The expression levels of Sirt1 and PGC-1α mRNA in Kasumi-1 drug-resistant cell lines and their parental cell lines were detected by real-time fluorescence quantitative PCR, and the expression levels of Sirt1 and PGC-1α protein in kasumi-1 drug-resistant cell lines and their parental cell lines were detected by Western blot.@*RESULTS@#The constructed Kasumi-1 cell line had common morphological characteristics of drug-resistant cell lines under microscope, and the drug resistance index was greater than 5, indicating that Kasumi-1 drug-resistant cells had good drug resistance after the construction. The RT-qPCR and Western blot assays showed that the expression levels of Sirt1 and PGC-1α mRNA and protein in the drug-resistant cell lines were higher than those of the parental cell lines (P<0.001).@*CONCLUSION@#AML cell lines resistant to Ara-C can be successfully induced by the method of gradually increasing the concentration, and the co-high expression of Sirt1 and PGC-1α may mediate the drug resistance of AML cells.

miR-202 promotes the differentiation of 3T3-L1 preadipocyte via inhibiting PGC1β expression / 基础医学与临床

Objective To explore the effect and molecular mechanism of miR-202 on the differentiation of 3T3-L1 preadipocyte .Methods Through lentivirus infected with 3T3-L1 preadipocytes , we set up the AMO-miR-202 group and the random control group , then, these cells were induced to differentiate , nine days later, differentiation was assessed by Oil Red O staining and we examined the mRNA expression of PPARγ2 and aP2 by RT-PCR method. We examined the mRNA expression of PPARγ2,aP2 and PGC1βby Western blot method .Results After packa-ging lentivirus with AMO-miR-202 and random sequence control miRNA through cell line 293T, 80%-90%cells with fluorescence were found under fluorescence microscope; After these two lentivirus respectively infected with 3T3-L1 preadipocytes, About 70%-80%cells with fluorescence were found under fluorescence microscope .Oil Red O staining test showed that these cells with Oil Red O stained bright red fat droplets of AMO-miR-202 group and PPARγ2 and aP2 mRNA expression in the AMO-miR-202 group significantly lower than control groups (P<0.05). Western blot assay showed that the protein expression of PGC 1βin the AMO-miRNA-202 group was significantly increased(P<0.05), but the expression of aP 2 and PPARγ2 was significantly decreased (P<0.01).However, the random control group and the adipocyte group had no significant effect on the above indexes .Conclusions miR-202 can promote the differentiation of 3T3-L1 preadipocyte by inhibiting the protein expression of PGC 1βand im-proving the protein expression of PPARγ2 and aP2.

Process of Hypertrophic Scar Formation: Expression of Eukaryotic Initiation Factor 6 / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hypertrophic scar is one of the most common complications and often causes the disfigurement or deformity in burn or trauma patients. Therapeutic methods on hypertrophic scar treatment have limitations due to the poor understanding of mechanisms of hypertrophic scar formation. To throw light on the molecular mechanism of hypertrophic scar formation will definitely improve the outcome of the treatment. This study aimed to illustrate the negative role of eukaryotic initiation factor 6 (eIF6) in the process of human hypertrophic scar formation, and provide a possible indicator of hypertrophic scar treatment and a potential target molecule for hypertrophic scar.</p><p><b>METHODS</b>In the present study, we investigated the protein expression of eIF6 in the human hypertrophic scar of different periods by immunohistochemistry and Western blot analysis.</p><p><b>RESULTS</b>In the hypertrophic scar tissue, eIF6 expression was significantly decreased and absent in the basal layer of epidermis in the early period, and increased slowly and began to appear in the basal layer of epidermis by the scar formation time.</p><p><b>CONCLUSIONS</b>This study confirmed that eIF6 expression was significantly related to the development of hypertrophic scar, and the eIF6 may be a target molecule for hypertrophic scar control or could be an indicator of the outcomes for other treatment modalities.</p>

The role of arecoline on hepatic insulin resistance in type 2 diabetes rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To explore the effects of arecoline on hepatic insulin resistance in type 2 diabetes rats and to elucidate its possible mechanism.</p><p><b>METHODS</b>Forty five Wistar rats were fed with high fructose diet for 12 weeks to induce type 2 diabetic rat model. rats were randomly divided into 5 groups (n = 8): control group, model group and model group were treated with different dose (0, 0.5, 1, 5 mg/kg) of arecoline. After 4 weeks, the fasting blood glucose, blood lipid and insulin level measured , mRNA expression of liver constitutive androstane receptor (CAR), pregnane X receptor (PXR), glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were detected by reverse transcription polymerase chain reaction (RT-PCR), the protein expression of p-AKT and glucose transporter4 (GLUT4) were detected by Western blot.</p><p><b>RESULTS</b>1.5 mg/kg arecoline could significantly decrease the level of fasting blood glucose, blood lipid, blood insulin level and liver G6Pase, PEPCK, IL-6, TNF-alpha mRNA level in type 2 diabetes rats. 1.5 mg/kg arecoline also could significantly increase CAR, PXR mRNA level and p-AKT and GLUT4 protein expression.</p><p><b>CONCLUSION</b>Arecoline improved hepatic insulin resistance in type 2 diabetes rats by increasing the mRNA levels of CAR and PXR leading to the creased glucose metabolism and inflammation related genes expression.</p>

Biologic effect of nitric oxide on human epidermal stem cells in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the effect of nitric oxide (NO) on adhesion, proliferation, and migration of human epidermal stem cells (ESC) in vitro.</p><p><b>METHODS</b>ESC were isolated and cultured by the modified method of rapid attachment to type IV collagen. (1) Morphology of cells was observed under inverted phase-contrast microscope. Expression levels of integrin β(1) and cytokeratin 19 (CK19) of cells were determined by Western blotting and immunofluorescence staining. (2) After being treated with scratching, ESC adhered to the wall was respectively treated with nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) in the concentration of 1, 10, 100, 500 µmol/L. ESC without treatment of SNAP was used as control. The migration rate of ESC was detected at post scratching hour (PSH) 12 and 24. The chemotaxis of ESC (treated with SNAP in above-mentioned concentration) was tested by Transwell assay, and the transferred cell number was counted. (3) ESC was respectively treated with SNAP in the concentration of 10, 100, 500 µmol/L for 1 h. ESC without treatment of SNAP was used as control. The adhesion of ESC was detected with adhesion test, and the inhibition rate of adhesion was calculated. The proliferation of ESC (denoted as absorbance value) was determined by microplate reader at post-treatment hour (PTH) 0, 12, 24, 48. Data were processed with one-way analysis of variance and Dunnett t test.</p><p><b>RESULTS</b>(1) Small clone formed on post culture days (PCD) 5 to 9. On PCD 10 to 14, cell proliferation sped up. CK19 and integrin β(1) were detected to be expressed in the isolated cells. The cells were identified as ESC. (2) Compared with that of ESC without treatment of SNAP [(35.7 ± 0.3)%, (45.7 ± 5.0)%], migration of ESC treated with SNAP in the concentration from 1 to 100 µmol/L was promoted at PSH 12 and 24. Migration rates of ESC treated with 100 µmol/L SNAP were the highest [respectively (48.8 ± 2.7)%, (82.1 ± 15.8)%, with t value respectively 8.34, 5.10, P values both below 0.01]. The number of ESC transferred to membrane after being treated with 100 µmol/L SNAP was significantly larger than that of ESC without treatment of SNAP (t = 9.24, P = 0.00). (3) Absorbance values of ESC treated with 100, 500 µmol/L SNAP were obviously higher than that of ESC without treatment of SNAP (with t value respectively 4.30, 4.67, P values both equal to 0.00). Proliferation of ESC treated with 100, 500 µmol/L SNAP was obviously stronger than that of cells without treatment of SNAP at PTH 24, 48 (with t values from 2.84 to 8.17, P values all below 0.05).</p><p><b>CONCLUSIONS</b>Exogenous NO in suitable concentration can promote the migration of human ESC. Exogenous NO can inhibit the adhesion and promote the proliferation of human ESC in vitro.</p>

Effects of P 311 on the migration of epidermal stem cells in mice with superficial partial-thickness burn and injured cell model in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study effects of P311 on the migration of epidermal stem cells (ESCs) in mice with superficial partial-thickness burn and injured cell model in vitro and to explore the mechanism.</p><p><b>METHODS</b>(1) Eighteen male C(57) BL/6 mice were used. Fifteen of them were inflicted with superficial partial-thickness burn on the back. In three injured mice wound tissue and skin of wound edge were obtained at post burn hour (PBH) 6, 12, 24, 48, 72 respectively. The rest three mice were used as normal control, and samples were harvested with the same method as above. The expressions of P311 in harvested samples were assessed with biotin-streptavidin-peroxidase (SP) staining. (2) Six newly born C(57) BL/6 mice were intraperitoneally injected with 50 µg/g BrdU (two times a day) for three days for ESCs-labelling. Seven weeks later, the mice were inflicted with superficial partial-thickness burn on the back. Serial slices of burn wound tissue were prepared at PBH 72 and immunohistochemically stained with SP for observation of the co-localization of BrdU-positive ESCs and P311-positive cells. (3) The empty vector pAdEasy-enhanced green fluorescence protein (EGFP) and the adenovirus P311-expressing vector named pAdEasy-EGFP-P311 were constructed and packed. Human ESCs were isolated by the method of rapid adhesion to collagen IV. After being divided into P311 high-expressing group (n = 3) and EGFP control group (n = 3), the ESCs in two groups were respectively infected by pAdEasy-EGFP-P311 and pAdEasy-EGFP. Scratching assay was performed on ESCs in both groups after they were treated by mitomycin C for 2 hours. The remaining area within the fixed range was measured at post scratching hour (PSH) 0, 24, 48, and 72, and the wound-area healing rate was calculated. Data were processed with independent samples t test.</p><p><b>RESULTS</b>(1) Expression amount of P311 was different in different parts of wound at different time points after burn. Expression amount of P311 in the newly formed epidermis and hair follicle of wound increased along with prolongation of time. Expression amount of P311 in the epidermis and hair follicle of wound edge peaked at PBH 12 and then decreased to normal levels at PBH 72. (2) Co-localization of BrdU-positive ESCs and P311-positive cells was observed in the new epidermal layer of wound tissue of mice, where ESCs were labeled by BrdU. (3) At PSH 48 and 72, wound-area healing rate was obviously higher in P311 high-expressing group [(69 ± 31)%, (89 ± 26)%] than in EGFP control group [(35 ± 12)%, (46 ± 31)%, with t values respectively -2.336, -2.611, P values all below 0.05].</p><p><b>CONCLUSIONS</b>P311 may promote the migration of ESCs both in rats with superficial partial-thickness burns and in injured cell model in vitro, and it may play an important role in wound healing.</p>

miRNA expression change of differentiation of mice marrow mesenchymal stem cells into adipocytes / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To explore miRNA expression change of differentiation of mice marrow mesenchymal stem cells (MSCs) into adipocytes, which lay the foundation for further studies on molecular mechanism of miRNA regulating the differentiation of MSCs into adipocytes.</p><p><b>METHODS</b>C57BL/6 mice MSCs were isolated, cultured through the whole bone marrow method, amplified by the differential adherent method. Cell growth was observed by morphology and the expression of superficial antigen CD29, CD44, CD34 were detected through immunohistochemistry. MSCs was induced to differentiation into adipocytes with adipocyte differentiation medium, and adipogenic differentiation of MSCs was analyzed by oil Red O staining. MicroRNA microarray was used to investigate the differentially expressed miRNAs in MSCs and adipocytes.</p><p><b>RESULTS</b>(1) The fifth passage of MSCs had high purity under an inverted m icroscope. Immunohistochemistry staining showed that CD29, CD44 were positive and CD34 was negative in more than 90% MSCs. There were a large number of lipid droplets in cytoplasm after MSCs were induced with adipocyte differentiation medium, Oil O staining was positive. (2) The microarray experiment showed that 75 differentially expressed miRNAs were obtained in adipocytes compared with MSCs, 20 up-regulated and 55 down-regulated miRNAs were observed among them.</p><p><b>CONCLUSION</b>There was a expression change of miRNA of differentiation of MSCs into adipocytes, some miRNAs might play important roles in MSCs adipogenic differentiation.</p>