[Construction of three-dimensional dermoid tissue based on cell sheets technology in vitro].

OBJECTIVE: To explore a new strategy for constructing three-dimensional dermoid tissue in vitro by using cell sheets technology. METHODS: Rabbit bone marrow mesenchymal stem cells (rBMSCs) were isolated from bone marrow of New Zealand white rabbits and cultured by whole bone marrow adherent method. Human dermal fibroblasts (HDFs) were cultured and passaged in vitro. The 2nd generation rBMSCs and the 3rd generation HDFs were cultured in a culture dish for 2 weeks with cell sheets conditioned medium respectively to obtain a monolayer cell sheets. Human umbilical vein endothelial cells (HUVECs) were inoculated on rBMSCs sheet to construct pre-vascularized cell sheet. During the culture period, the morphological changes of the cell sheet were observed under an inverted phase contrast microscope. At 1, 3, 7, and 14 days, HE staining and CD31 immunofluorescence staining were performed to observe the cell distribution and microvascular network formation. The rBMSCs sheet was used as control. The pre-vascularized cell sheet (experimental group) and rBMSCs sheet (control group) cultured for 7 days were placed in the middle of two HDFs sheets, respectively, to prepare three-dimensional dermoid tissues. After 24 hours of culture, CD31 immunofluorescence staining and collagen type Ⅰ and collagen type Ⅲ immunohistochemical stainings were performed to evaluate cell distribution and collagen expression. RESULTS: HDFs and rBMSCs sheets were successfully prepared after 2 weeks of cell culture. After inoculation of HUVECs on rBMSCs sheet for 3 days, HUVECs could be seen to rearrange on rBMSCs sheet and forming vacuoles. The reticular structure was visible at 7 days and more obvious at 14 days. The formation of vacuoles between the cell sheets was observed by HE staining, and the vacuoles became more and more obvious, the thickness of the membranes increased significantly with time. CD31 immunofluorescence staining showed the microvascular lumen formation. However, only the thickness of rBMSCs sheet increasing was observed, with no changes in cell morphology or cavitation structure. The three-dimensional dermoid tissue observation showed that the endothelial cells in the experimental group were positive expressions, and the rBMSCs, HDFs, and HUVECs cells were arranged neatly. The endothelial cells were negative expressions and randomly arranged in the control group. The collagen type Ⅰ and collagen type Ⅲ were positive expression in the experimental group and the control group. But compared with control group, experimental group presented a "honeycomb" network connection, where the matrix was distributed regularly, and cells were arranged tightly. The difference in the expression of collagen type Ⅰ and collagen type Ⅲ between the experimental group and the control group was not significant ( P>0.05). CONCLUSION: Three-dimensional dermoid tissue is successfully constructed by using cell sheet technology. The cell matrix distribution of the pre-vascularized cell sheet constructed by HUVECs and rBMSCs sheet is relatively regular, which has the potential to form tissue engineered dermis.

Whole-lesion histogram analysis metrics of the apparent diffusion coefficient: a correlation study with histological grade of hepatocellular carcinoma.

PURPOSE: The study evaluated the relationship between the histological grade of hepatocellular carcinoma (HCC) and the histogram-derived parameters of apparent diffusion coefficient (ADC) obtained from the whole-lesion assessment of diffusion-weighted magnetic resonance (MR) imaging in the liver. METHODS: A total of 51 patients were included. The parameters were correlated with the Edmondson-Steiner grades by using the Spearman correlation coefficient (ρ). The differences of ADC parameters between different tumor histological grades were compared using the Mann-Whitney U test. The extent to which each parameter aided in differentiating tumors with poor performance (III, IV) and fair performance (I, II) was assessed by using the area under the receiver operating characteristic curve (Az). RESULTS: The 25th percentile ADC exhibits the most negative correlation with histological grade (ρ = - 0.397), followed by the 30th percentile ADC (ρ = - 0.395), the minimum ADC value (ρ = - 0.390) and the 20th percentile ADC (ρ = - 0.385), whereas the minimum ADC value yielded the highest Az (0.763) in the discrimination of tumor foci with poor differentiation from fairly differentiated HCCs. The minimum ADC of 4.15 × 10-3 mm2/s or lower was considered to indicate poorly differentiated performance, and the corresponding sensitivity and specificity were 66.7 and 90.9%, respectively. CONCLUSION: The 25th percentile ADC showed a stronger correlation with the histological grade of HCC than other ADC parameters, and the minimum ADC value might be an optimal metric for determining poor and fair differentiations of HCC in DWI.

Effect of high uric acid on the disposition of metformin: in vivo and in vitro studies.

Metformin is always used as the baseline antidiabetic therapy for patients with type 2 diabetes mellitus (T2DM) and hyperuricemia. Metformin is excreted into urine through active secretion mediated by rOCTs and rMATE1.The aim of this study was to identify the effects of high uric acid on the disposition and its mechanism. For the in vivo study, a hyperuricemic animal model was induced by intraperitoneal injection of potassium oxonate (250 mg/kg) in rats. Metformin (100 mg/kg) was administered orally to investigate the pharmacokinetics in control and hyperuricemic rats, respectively. For the in vitro study, HEK293 and HepaRG cells were used to investigate the effect of uric acid (15 mg/dl) on the expression of OCT1, OCT2 and MATE1 and the disposition of metformin, respectively. The in vivo study showed that the AUC0 â†’ 600 of metformin was significantly decreased by 33.3%, whereas the cumulative urinary excretion of metformin was increased by 25.4% in hyperuricemic rats compared with that in control rats. The renal rOCT1, rOCT2 and rMATE1 and hepatic rMATE1 levels were increased in hyperuricemic rats compared with those in control rats, respectively. The in vitro study showed that uric acid could upregulate the expression of OCT2 and MATE1 in HEK293 cells and MATE1 in HepaRG cells and increase the intracellular metformin concentration in these two cell lines. These results demonstrated that a high uric acid level promoted urinary metformin excretion and decreased the plasma metformin concentration; the in vivo and in vitro studies provided a possible explanation being that high uric acid could upregulate the expression of renal metformin transporters OCTs and MATE1.