Spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells maintain pluripotency of stem cells by regulating hypoxia-inducible factors

BACKGROUND: Spontaneous spheroid culture is a novel three-dimensional (3D) culture strategy for the rapid and efficient selection of progenitor cells. The objectives of this study are to investigate the pluripotency and differentiation capability of spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells (AB-MSCs); compare the advantages of spontaneous spheroids to those of mechanical spheroids; and explore the mechanisms of stemness enhancement during spheroid formation from two-dimensional (2D) cultured cells. METHODS: AB-MSCs were isolated from the alveolar bones of C57BL/6 J mice. Spontaneous spheroids formed in low-adherence specific culture plates. The stemness, proliferation, and multi-differentiation capacities of spheroids and monolayer cultures were investigated by reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, alkaline phosphatase (ALP) activity, and oil-red O staining. The pluripotency difference between the spontaneous and mechanical spheroids was analyzed using RT-qPCR. Hypoxia-inducible factor (HIFs) inhibition experiments were performed to explore the mechanisms of stemness maintenance in AB-MSC spheroids. RESULTS: AB-MSCs successfully formed spontaneous spheroids after 24 h. AB-MSC spheroids were positive for MSC markers and pluripotency markers (Oct4, KLF4, Sox2, and cMyc). Spheroids showed higher Ki67 expression and lower Caspase3 expression at 24 h. Under the corresponding conditions, the spheroids were successfully differentiated into osteogenic and adipogenic lineages. AB-MSC spheroids can induce neural-like cells after neurogenic differentiation. Higher expression of osteogenic markers, adipogenic markers, and neurogenic markers (NF-M, NeuN, and GFAP) was found in spheroids than in the monolayer. Spontaneous spheroids exhibited higher stemness than mechanical spheroids did. HIF-1α and HIF-2α were remarkably upregulated in spheroids. After HIF-1/2α-specific inhibition, spheroid formation was significantly reduced. Moreover, the expression of the pluripotency genes was suppressed. CONCLUSIONS: Spontaneous spheroids from AB-MSCs enhance stemness and pluripotency. HIF-1/2α plays an important role in the stemness regulation of spheroids. AB-MSC spheroids exhibit excellent multi-differentiation capability, which may be a potent therapy for craniomaxillofacial tissue regeneration.

Assessment of Prognostic Factors of Racial Disparities in Testicular Germ Cell Tumor Survival in the United States (1992-2015) / 生物医学与环境科学（英文）

Objective@#Testicular germ cell tumors (TGCT) are the most common cancer among men aged 15 to 39 years. Previous studies have considered factors related to TGCT survival rate and race/ethnicity, but histological type of the diagnosed cancer has not yet been thoroughly assessed.@*Methods@#The data came from 42,854 eligible patients from 1992 to 2015 in the Surveillance Epidemiology and End Results 18. Frequencies and column percent by seminoma and nonseminoma subtypes were determined for each covariates. We used Cox proportional hazard regression to assess the impact of multiple factors on post-diagnostic mortality of TGCT.@*Results@#Black males were diagnosed at a later stage, more commonly with local or distant metastases. The incidence of TGCT in black non-seminoma tumors increased most significantly. The difference in survival rates between different ethnic and histological subtypes, overall survival (OS) in patients with non-seminoma was significantly worse than in patients with seminoma. The most important quantitative predictor of death was the stage at the time of diagnosis, and older diagnostic age is also important factor affecting mortality.@*Conclusion@#Histological type of testicular germ cell tumor is an important factor in determining the prognosis of testicular cancer in males of different ethnic groups.

Research progress of tissue culture of Aconitum / 中国中药杂志

Aconitum is a kind of important medicinal plant, which has been used in China for more than 2 000 years, with both a good medicinal and ornamental value. However, due to the lack of effective breeding methods and low seed and root propagation coefficients, the comprehensive development and utilization of Aconitum were greatly restricted. Tissue culture is an important basis for seed selection, germplasm conservation and genetic engineering. Therefore, this paper summarized the research on tissue culture of Aconitum, put forward the main problems and corresponding countermeasures, and provided important references for accelerating the seedling breeding of Aconitum and conducting the basic research of molecular biology.

Evaluation of the efficacy and prognostic factors for colorectal liver metastases treated with transcatheter arterial chemoembolization / 中华肿瘤杂志

<p><b>OBJECTIVE</b>The aim of this study was to evaluate the therapeutic efficacy and to determine the prognostic factors of TACE in patients with colorectal liver metastases (CRLM).</p><p><b>METHODS</b>The clinical data of 183 patients with unresectable CRLM treated with TACE from Jan. 2002 to Dec. 2008 were retrospectively reviewed. Log-rank method was used for univariate analysis and Cox proportional hazard model was used for multivariate analysis of the prognostic factors.</p><p><b>RESULTS</b>The median survival time was 22 months, and the 0.5-, 1-, 2-, 3-, 5-year survival rates were 93.9%, 81.1%, 39.8%, 18.2%, and 3.9%, respectively. Multivariate analysis showed that tumor involved more than one lobe of the liver, and elevated CEA and CA19-9 levels were independent risk factors for the overall survival (P < 0.01). Females, more times of TACE, combination with regional therapy and received phase II resection were related with a good survival (P < 0.01) in CRLM patients after TACE treatment.</p><p><b>CONCLUSIONS</b>Transcatheter arterial chemoembolization is an effective therapy for unresectable colorectal liver metastases. Patients with tumor spread more than one lobe of the liver, high CEA and CA19-9 levels are independent poor prognostic factors. Females, patients received more times of TACE, combined with regional therapy and received phase II resection may have a good survival.</p>

Effect of let-7c on the proliferation of human hepatocellular carcinoma cell HCCLM3 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate let-7c's effect on the proliferation of human hepatocellular carcinoma cell HCCLM3 by transient transfection and the mechanism inside.</p><p><b>METHODS</b>Lipofectamine 2000 was used to transfect miRNAs into HCCLM3 cells. The cells were divided into three groups, let-7c group: let-7c was transfected, negative control group: negative control miRNA was transfected, blank control group: nothing was transfected. The proliferation of HCCLM3 cells was evaluated using Cell Counting Kit-8 (CCK-8). The cell cycles of each group were assayed by flow cytometry. Western blot and Real time PCR were used to analyze the protein and mRNA expressions of cyclin D1. Statistical analysis was performed with SPSS 17.0.</p><p><b>RESULTS</b>The absorbances of let-7c group were 0.70 ± 0.05, 0.77 ± 0.09 at 48 h and 72 h after transfection, lower than that of blank control group (0.97 ± 0.10, 1.21 ± 0.12) and negative control group (0.91 ± 0.07, 1.12 ± 0.09), 48 h: F = 14.431, P < 0.05, 72 h: F = 21.146, P < 0.05. The flow cytometry at 72 h after transfection revealed that let-7c increased the percentage of cells in G1 phase. The percentage of blank control group was 43.53% ± 0.86%, the negative control group was 44.82% ± 0.77%, and the let-7c group was 54.52% ± 0.13%, F = 240.739, P < 0.05. let-7c suppressed expressions of cyclin D1 at both protein and mRNA levels. The protein levels of cyclin D1 were 0.48 ± 0.09, 0.47 ± 0.06 and 0.23 ± 0.06 (F = 11.316, P < 0.05) in blank control group, negative control group and let-7c group, respectively. The mRNA levels were 1.03% ± 0.29%, 1.01% ± 0.11% and 0.63% ± 0.14% (F=6.315, P < 0.05) in the above three groups, respectively.</p><p><b>CONCLUSION</b>Let-7c can inhibit proliferation of HCCLM3 cells and increase the proportion of cells in G1 phase. The mechanism may be that let-7c represses the expressions of cyclin D1 at both protein and mRNA levels.</p>

TNFα induced IL-8 production through p38 MAPK- NF-kB pathway in human hepatocellular carcinoma cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To identify the role of p38 MAPK- NF-kB signaling pathway in TNF-α induced IL-8 production in human hepatocellular carcinoma cells.</p><p><b>METHODS</b>The concentrations of IL-8 from MHCC-97H cells were measured by an enzyme-linked immunosorbent assay (ELISA). The phosphorylation of p38 MAPK was analyzed by Western blot and immunofluorescence. NF-kB p65 protein nuclear translocation was determined by non-radioactive NF-kB p50 / p65 transcription factor activity kit and immunofluorescence.</p><p><b>RESULTS</b>The IL-8 production from MHCC-97H cells challenged with TNFa significantly increased in a time-dependent (F = 144.04, P < 0.01) and dose-dependent (F = 364.14, P < 0.01) manners, as compared with those without TNFa challenge. TNFa up-regulated the phosphorylation levels of p38 MAPK and increased the translocation of NF-kB p65 protein into the nucleus, also proved by immunofluorescence staining. p38 MAPK inhibitor (SB203580) could significantly inhibit IL-8 production in a dose-dependent manners (F = 65.47, P < 0.01), and partially inhibited NF-kB p65 nuclear translocation in a dose-dependent manner (F=141.20, P < 0.05).</p><p><b>CONCLUSION</b>TNF-α could increase the production of IL-8 in MHCC-97H cells and p38 MAPK- NF-kB pathways seem to play a central role in the regulation of IL-8 production.</p>

The protective effect of nerve growth factor on PC12 cells to lipopolysaccharide injury / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effect of nerve growth factor (NGF) on lipopolysaccharide (LPS) injury and activation of nuclear factor-kappa B in PC12 cells.</p><p><b>METHODS</b>In order to set injury models, the PC12 cells were incubated in different concentration of LPS. Cells were cultured in the culture and were reduced by LPS, and then cells were treated by NGF of various concentrations. The cell viability was determined by methyl thiazolyl tetrazolium (MTT) assay, cellular morphology was observed under inverted microscope and fluorescence microscope, and the content of NF-kappaB was assessed by RT-PCR.</p><p><b>RESULTS</b>(1) The viability of PC12 cell was decreased with concentration of LPS increasing. (2) The cellular morphology change showed that NGF had an ability to reduce LPS injury. (3) The result of RT-PCR showed that the content of NF-kappaB in LPS injury was more than the normal and treated cell, and the treated one was close to the normal one.</p><p><b>CONCLUSION</b>The reports about NGF in brain cells repair after inflammatory are very small. And our study is about that NGF can protect the PC12 cell from LPS injury, and this mechanism possible bears on the activation of NF-kappaB.</p>

Effects of sinomenine on intracellular free calcium concentration and the activity of protein kinase in cultured rabbit aortic smooth muscle cells / 中国应用生理学杂志

<p><b>AIM</b>To explore the effects of sinomenine(Sin) on intracellular free calcium ([Ca2+]i) and the activity of PKC (protein kinase C) of the cultured aortic vascular smooth muscle cells (VSMC) during ischemia and hypoxia.</p><p><b>METHODS</b>The effect of Sin on changes in [Ca2+]i were determined in cultured rabbit VSMC after exposure to high K+, norepinephrine (NE) and caffeine (Caf). Fluorescent Ca2+ -indicater fura-2/AM was used. The effects of Sin were compared with that of verapamil (Ver). The hypoxia model was made, then the activity of PKC was measured by y scintillation counting instrument.</p><p><b>RESULTS</b>Sin (10 x 10(-6) mol x L(-1), 3 x 10(-5) mol x L(-1) 10(-4) mol x L(-1)) inhibited the elevation of [Ca2+]i induced by high K+ -depolarization in a concentration dependent manner. In addition, Sin inhibited the elevation of [Ca2+]i induced by NE in the presence of extracellular Ca2+. In the absence of extracellular Ca2+, Sin (3 x 10(-5) mol.L(-1)) also had no blocking effect on the NE-induced [Ca2+]i increase. It was found that the activity of PKC treated with Sin in VSMC cytoplasm and cell membrane in normal condition increased, the activity of PKC in cytoplasm in ischemia and hypoxia situation increased, but the activity of PKC in cell membrane decreased. When VSMC was treated with Sin, the activity of PKC in cytoplasm decreased and that of cell membrane increased.</p><p><b>CONCLUSION</b>The results suggest that Sin might decrease the[Ca2+] i of VSMC by blocking both VDC and ROC, could regulate the PKC activities induced by ischemia and hypoxia.</p>

Cellular uptake behavior of antisense oligodeoxynucleotides polymethacrylate submicroparticles / 药学学报

<p><b>AIM</b>To survey the uptake behavior and subcellular distribution of antisense oligodeoxynucleotide polymethacrylate submicroparticles (AS-ODN-SMP) and infer its mechanism in MGC cell lines.</p><p><b>METHODS</b>MGC cells were incubated at certain concentration of AS-ODN-SMP or AS-ODN for 8 h at 4 degrees C or 37 degrees C. Then the fluorescence oligodeoxynucleotide- labeled cells were counted by flow cytometer and the intracellular fluorescence intensity was determined after incubated with chloroquine for 2 h.</p><p><b>RESULTS</b>Cellular uptake of oligodeoxynucleotides was significantly increased following application of AS-ODN-SMP and total intracellular fluorescence intensity was enhanced by 683 folds with the vehicle concentration of 20 microg x mL(-1). AS-ODN-SMP entranced to cells profoundly with temperature-dependent manner. Rare cells took on fluorescence when incubated at 4 degrees C, while 37 degrees C they were significantly increased. But the intracellular fluorescence intensity appeared same level in present or absent of chloroquine.</p><p><b>CONCLUSION</b>With the help of polyacrylate submicroparticles, oligonucleotides efficiently entranced the cells via endocytosis and could successfully escape the degradation in lysosome.</p>

Prospective randomized trial of RFA and chemotherapy for unresectable small hepatocellular carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the clinical safety and effect on local recurrence in unresectable small hepatocellular carcinoma treated by radiofrequency ablation (RFA) with and without chemotherapy through a prospective randomized trial.</p><p><b>METHODS</b>Thirty-eight unresectable small hepatocellular carcinoma patients with diameter </= 3 cm were selected, of which 27 patients have been followed up for 1 year. Through a prospective randomized trial, 12 patients were in the RFA group and 15 patients in the RFA combined with systemic chemotherapy group. RFA was given image-guided. The regimen of systemic chemotherapy: EADM 50 mg on day 1, 3; CDDP 40 mg on day 1, 3 and FUDR 500 mg on day 1, 2, 3. After RFA treatment, liver function, WBC count and complications were observed on day 1, 4, 7; CT scan was performed in 1, 6, 12 months. The safety and local recurrence were analyzed.</p><p><b>RESULTS</b>There was no local recurrence of the tumor in the two groups 1 month after RFA treatment. The 6- and 12-month local recurrence rates were significantly lower in the combined group than that in RFA group alone (P < 0.01). There were no severe complications in the two groups, and nor was there any significant difference in liver function and WBC count.</p><p><b>CONCLUSION</b>RFA combined with systemic chemotherapy is safe, and it can reduce the local recurrence of unresectable small hepatocellular carcinoma </= 3 cm in diameter.</p>