【Abstract】 Objective To reconstruct three different kinds of tympanic membrane in vitro by tissue engineeringtechnique, and to examine their histological structures and mechanical properties. Methods The skin and dura of pig(weight 30 kg) were processed with high satuated sal ine and enzymes to make extracellular matrix. Meanwhile, fibroblasts(1×106 /mL,0.2 mL) were seeded on the surface of these two scaffolds and collagen. The composite tissues were cultured in vitro for 1 week and examined in histological structure and mechanical properties. Results Fibroblasts cultured were spindle–shaped and could grow and attach to these scaffolds with a arrangement of sarciniform and parallel. The reconstructed tissue of ECM and collagen appeared to integrate well and had better bio-compatibil ity. The mean thickness of the collagen, the skin and the dura (all covered with fibroblasts) were 9.4, 10.0 and 10.4 μm respectively. The tension of the collagen was (1.417±0.030) N/mm2, of the acellular dermal matrix was (24.500±2.040) N/mm2(being close to the tension of normal tympanic membrane, 26.700 N/ mm2),of the acellular dura was (53.300±2.600) N/mm2. Conclusion The results suggest that the tension and the thinkness of acellular dermal matrix is similar to the normal tympanic membrane of guinea pig, it is an ideal material for tympanoplasty.
【Abstract】 Objective To investigate the effect of IGF-1 on the growth of primary human embryonicmyoblasts. Methods The method of incorporation of 3H-TdR was used to evaluate the abil ity of prol iferation of myoblasts.The count per minute (CPM) values of myoblasts at different concentrations(1, 2, 4, 8, 16 and 32 ng/mL) of IGF-1 were measured,and dose-effect curves were drawn to choose the optional concentration of IGF-1 to promote the prol iferation. Then theexperimental group of myoblasts received the addition of the optional concentration of IGF-1 in the growth medium, the controlgroup just received the growth medium. The flow cytometry was used to detect the cell cycle . The method of incorporation of3H-TdR was used to measure the peak-CPM. The myotube fusion rate was measured in myoblasts with different concentrations(0, 5,10, 15, 20, 25 and 30 ng/ mL) of IGF-1 in fusion medium, the dose-effect curves were also drawn, so as to decided the optional concentrationof IGF-1 in stimulating differentiation. Fusion medium with optional concentration of IGF-1 was used in experimentalgroup, and the control group just with fusion medium. The fusion rate of myotube and the synthesis of creatine kinase(CK) weredetected in both groups. Results The optional concentration of 5 ng/mL IGF-1 was chosen for stimulating prol iferation . It was shown that the time of cell cycle of control was 96 hours, but that of the experimental group was reduced to 60 hours. The results of flow cytometry showed that the time of G1 phase, S phase and G2M phase was 70.03, 25.01 and 0.96 hoursrespectively in control group, and were 22.66, 16.47 and 20.87 hours respectively in experimental group. The time-CPM value curves showed that the peak-CPM emerged at 96 hours in control group and 48 hours in experimental group, which was in agreementwith the results of the flow cytometry. The optional concentration stimulating prol iferation was 20 ng/mL IGF-1. Compared with control, the quantity of CK was increased by 2 000 mU/mL and the fusion rate was elevated by 30% in experimental group. Conclusion The concentrations of 20 ng/mL IGF-1 can elevat obviously the fusion rate and the quantity of CK. IGF-1 can enhance the prol iferation and differentiation of myoblasts via inducing the number of myoblasts at G1 phase and increasing the number of myoblasts at S and G2M phases.
【Abstract】 Objective To observe the effect of cytokines and combinations in the inducement of human umbil icalcord blood-derived CD34+ cells into hepatocyte-l ike cells. Methods The mononuclear cells (MNCs) were derived by density gradient centrifugation and the CD34+ cells were sperated from MNCs. The human umbil ical cord blood-derived CD34+ cells were cultivated through 49 different combinations of cytokines including leukemia inhibitor factor (LIF), oncostatin M, bFGF, aFGF, hepatocyte growth factor, EGF and stem cell factor for 28 days, and the concentrations of the cytokines were 10, 10, 10, 10, 20, 20 and 50 ng/mL, respectively. The mRNAs of cytokeratin 19 (CK-19), CK-18, glutamine synthetase (GS), human albumin (ALB) and α-fetoprotein (AFP) were detected every seven days. The ALB secretion abil ity, detoxification abil ity and hepatin synthesis abil ity of the induced cells were detected by immunofluorescence assay, indocyanine green (ICG) and periodic acid-schiff assay, respectively. The fresh umbil ical cord blood-derived CD34+ cells were detected at the same time as a control. Results The mRNAs of CK-19, CK-18 and GS could be transcribed in all the induced cells, but the transcription of the mRNAs of ALB and AFP which was the special mark of mature hepatocyte and l iver stem cell, respectively, was not found. All the mRNAs could not be found in freshly isolated umbil ical cord blood-derived CD34+ cells. All the cells induced in vitro could not release ALB, and not help the detoxification of ICG which was the fundamental function of mature l iver cells. These results were the same in the control group. The hepatin synthesis abil ity of all the induced cells increased by comparison to the fresh ones. Conclusion Though some mRNAs of proteins which are transcribed in hepatocytes can be found in the induced cells, umbil ical cord blood-derived CD34+ cells could not be transdifferentiated into hepatocyte-l ike cells through cytokines in vitro.
Objective To investigate the influence of the exogenouscollagen on the function of cells in construction of artificial biotendon.Methods Three materials including human hair, carbon fiber(CF) and polyglycolic acid (PGA) were combined with exogenous collagen and co-cultured with standard transferred human embryonic tenocytes at a concentration of 3×106/mm3 in vitro. The cell number and morphology were observed under inverted microscope and scanning electron microscope after 2 hours, 3 days and 5 days.Results In the artificial biotendon combined with collagen, the cells concentrated around the materials and the cells adhering to the materials turned into round after 2 hours. After 3 days, the adhering cells increased. After 5 days, the shape of the cells changed from round to spindle.ConclusionExogenous collagen will facilitate the cells to adhere onto materials and proliferate.
ObjectiveTo observe the protective effect of etomidate (ET) on cultured retinal ganglion cells (RGC) with mechanical injury in vitro. MethodsNew Sprague-Dawley rat RGC was cultured in vitro and identified by double immunofluorescent labeling of Thy1.1 and microtubule associated protein 2. The cultured primary cells were randomly divided into control group, RGC scratch group, ET low dose group (1 μmol/L), ET medium dose group (5 μmol/L) and ET high dose group (10 μmol/L). The RGC mechanical injury model was established by using iris knife to culture cells in RGC scratch group and ET group with different concentration. Seven days after modeling, the RGC survival rate of each group was detected by cell count Kit 8 proliferation assay. The apoptosis rate of RGC was detected by Annexin Ⅴ/propyl iodide double staining. Single factor analysis of variance was used to compare the groups. The pairwise comparison between groups was tested by the least significant difference method. ResultsThe survival rates of RGC in RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (72.60±2.97)%, (73.73±1.14)%, (79.19±1.79)% and (83.88±0.94)%, respectively. The RGC apoptosis rates of control group, RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (5.08±0.17)%, (18.67±1.24)%, (17.96±0.74)%, (15.11± 0.56)% and (11.67±1.32)%, respectively. Comparison of RGC survival rate between groups: compared with RGC scratch group, the cell survival rate of ET low-dose group, ET medium-dose group and ET high-dose group was increased, and the difference between RGC scratch group and ET low-dose group was not statistically significant (P=0.728); the differences between RGC scratch group, ET medium dose group and ET high dose group were statistically significant (P＜0.001); the difference between ET medium dose group and ET high dose group was statistically significant (P=0.002). Comparison of apoptosis rate of RGC among groups: the apoptosis rate of RGC scratch group was significantly higher than that of control group, the difference was statistically significant (P＜0.001). Compared with RGC scratch group, the apoptosis rate of ET low-dose group, ET medium-dose group and ET high-dose group was decreased, and there was no statistical significance between RGC scratch group and ET low-dose group (P=0.869). The differences of apoptosis rate between RGC scratch group, ET medium dose group and ET high dose group were statistically significant (P＜0.05). The difference of apoptosis rate between ET medium dose group and ET high dose group was statistically significant (P=0.007). ConclusionET has neuroprotective effect on RGC cultured in vitro with mechanical injury, and the protective effect increases with the increase of ET dose in a certain range.