Objective To solve the shortage of hepatocytes for l iver tissue engineering, to explore the possibil ity of prol iferation of rat bone marrow mesenchymal stem cells (BMSCs) and the feasibil ity of differentiation of BMSCs into hepatocyteswith a culture system containing cholestatic rat serum and hepatocyte growth factor (HGF) in vitro. Methods Myeloid cellsof femur and tibia were collected from the female healthy Wistar rats at the age of 6 weeks, the BMSCs were isolated, purified and identified. Normal and cholestatic rat serum were prepared from 40 healthy Wistar rats at the age of 12-14 weeks. The 3rd passage of BMSCs were harvested and added different cultures according to the following grouping: group A, DMEM plus 10%FBS; group B, hepatocyte growth medium (HGM) plus 5%FBS; group C, HGM plus 5% normal rat serum; group D, HGM plus 5% cholestatic rat serum; group E, HGM plus 5% cholestatic rat serum plus 25 μg/L HGF. The changes of cell morphology were observed, MTT assay was used to measure cell growth; the expression of alpha-fetoprotein (AFP) and cytokeratin 18 (CK18) were detected by immunocytochemistry; the glycogen deposit was examined by periodic acid-schiff (PAS) staining; and the urea content in culture supernatant was determined by glutamate dehydrogenase. Results Polygonal cells and binuclear cells were observed in groups D and E, while the shapes of cells in groups A, B, and C did not obviously change. The cell growth curve demonstrated that the speed of cells proliferation in group C was the fastest, the one in group B was the slowest; showing significant differences when compared with groups A, D, and E (P lt; 0.05). On the 7th day in groups D and E, the positive expressions of AFP and CK18 emerged, on the 14th day the positive expression of glycogen emerged. At the same period, the expression ratio was higherin group E than in group D (P lt; 0.05). The urea concentration increased gradually with induction time in groups D and E, the concentration was higher in group E than in group D (P lt; 0.05). No expressions of AFP, CK18, glycogen, and change of the urea concentration were observed in groups A, B, and C. Conclusion Normal rat serum can obviously promote the growth of BMSCs; cholestatic rat serum which promote the growth of BMSCs can induce to differentiate into hepatocyte; and a combination of cholestatic serum and HGF can increase the differentiation ratio.
In order to explore further the regulatory factors to the potentiality in inducing osteogenesis by fibroblasts, the fibroblasts were isolated, and purified from human skin, and were grown in incubation in the media of EGF, IL-6, TNF-alpha and BMP2 at different concentrations for two weeks, then, the markers for osteogenic features were investigated by biochemistry, histochemistry and electron microscopic observations. It was found that the combined use of TNF-alpha and BMP2 could stimulate fibroblasts to secrete alkaline phosphatase, osteocalcin and collagen, and the morphological changes of the fibroblasts were also very striking. In the extracellular matrix, the collagen fibrils, with or without periodicity, were arranged regularly or randomly oriented, and numerous minute calcium granules were interspersed among them. The fibroblasts were interwoven one on top of another in the form of multilayer structure and on the surface, there were secreting granules and piling up of calcium crystals which coalessed steadily and increased in size in forming bony nodules. It was considered that TNF-alpha and BMP2 were capable of inducing the fibroblasts to form bone.
Objective To explore the in vitrodifferentiation of the rat mesenchymal stem cells (MSCs ) into the skeletal muscle cells induced by the myoblast differentiation factor (MyoD) and 5-azacytidine. Methods The MSCs were taken from the rat bone marrow and the suspension of MSCs was made and cultured in the homeothermia incubator which contained 5% CO2at 37℃. The cells were observed under the inverted phase contrast microscope daily. The cells spreading all the bottom of the culture bottle were defined as onepassage. The differentiation of the 3rd passage of MSCs was induced by the combination of 5-azacytidine, MyoD, transforming growth factor β1, and the insulin like growth factor 1. Nine days after the induction, the induced MSCs were collected, which were analyzed with the MTT chromatometry, theflow cytometry, and the immunohistochemistry. Results The primarily cultured MSCs grew as a colony on the walls of the culture bottle; after the culture for 5-7 days, the cells were shaped like the fibroblasts, the big flat polygonal cells, the medium sized polygonal cells, and the small triangle cells; after the culture for 12 days, the cells were found to be fused, spreadingall over the bottle bottom, but MSCs were unchanged too much in shape. After the induction by 5-azacytidine, some of the cells died, and the cells grew slowly. However, after the culture for 7 days, the cells grew remarkably, the cell volume increased gradually in a form of ellipse, fusiform or irregularity. After theculture for 14 days, the proliferated fusiform cells began to increase in a great amount. After the culture for 18-22 days, the myotubes increased in number and volume, with the nucleus increased in number, and the newly formed myotubes and the fusiform myoblst grew parallelly and separately. The immunohistochemistry for MSCs revealed that CD44 was positive in reaction, with the cytoplasm ina form of brown granules. And the nucleus had an obvious border，and CD34 was negative. The induced MSCs were found to be positive for desmin and specific myoglobulin of the skeletal muscle. The flow cytometry showed that most of the MSCs and the induced MSCs were in the stages of G0/G1，accounting for 79.4％ and 62.9％，respectively; however, the cells in the stages of G2/S accounted for 20.6％ and 36.1％. The growth curve was drawn based on MTT，which showed that MSCs weregreater in the growth speed than the induced MSCs. The two kinds of cells did not reach the platform stage，having a tendency to continuously proliferate.ConclusionIn vitro,the rat MSCs can be differentiated into the skeletal muscle cells with an induction by MyoD and 5-azacytidine, with a positive reaction for the desmin and the myoglobulin of the skeletal muscle. After the induction, the proliferation stage of MSCs can be increased, with a higher degree of the differentiation into the skeletal muscle.
ObjectiveTo evaluate the superiority of nasopharyngeal airway on obesity patients during general anesthesia induction period. MethodForty-two trachea cannula and general anesthesia obesity patients treated from June to November in 2013 were chosen and divided equally into two groups:nasopharyngeal airway group (group A) and control group (group B). Mean arterial pressure (MAP), heart rate (HR), pulse oxygen saturation (SpO2), arterial blood partial pressure of carbon dioxide (PaCO2) were recorded when the patients entered the operation room, three minutes after man-made positive pressure ventilating and five minutes after intubation. Peak voltage (Ppeak) of man-made positive pressure ventilation for three minutes was also observed, and intubation frequency and time, mouth mucosa bleeding, and sore throat examples were compared between the two groups. ResultsCompared with group B, MAP, HR, PaCO2 and Ppeak three minutes after man-made positive pressure ventilating were lower (P<0.05), but SpO2 was higher in group A (P<0.05). Intubation frequency and time, mouth mucosa bleeding, and sore throat examples of group A were less than those in group B (P<0.05). ConclusionsNasopharyngeal airway is better for obesity patients during general anesthesia induction period, which also improves anesthesia safety level.
ObjectiveTo probe into the clinical effects of intra-amniotic injection of ethacridine with Cook cervical ripening balloon in terminating mid-pregnancy. MethodsA total of 150 mid-pregnant women who required induction of labor from January 2011 to December 2012 were randomly divided into two groups: observation group (intra-amniotic injection of ethacridine with Cook cervical ripening balloon) and control group (intra-amniotic injection of ethacridine). ResultsThe time of labor induction was obviously shorter in the observation group than the control group [the time from using ethacridine to contraction: （29.68±4.17） vs (33.60±5.38) hours, P<0.05; total process: (7.63±2.30) vs (9.86±3.20) hours, P<0.05], and the residual rate of placental membranes [28.6% (10/35) vs 56.4%(22/39), P<0.05] was significantly lower. But there was no significant difference in postpartum hemorrhage [(81.60±17.64) vs (83.82±15.08 ) mL, P>0.05] and rate of success [100.0% (35/35) vs 94.9% (37/39), P>0.05]. ConclusionTerminating mid-pregnancy by intra-amniotic injection of ethacridine with Cook cervical ripening balloon has the advantages of shorter time and less pain, which deserves clinical application widely.
Objective To investigate the curve correlation between ventilation pressure and tidal volume in assisted mechanical ventilation with facemask during anesthesia induction. Methods Between January and August 2015, 120 patients, American Society of Anesthesiology Ⅰ-Ⅱ, undergoing selective gynecological surgery were randomly divided into four groups: groups P5, P10, P15 and P20, with 30 patients in each group. Mask ventilation pressure for the four groups were respectively 5, 10, 15 and 20 cm H2O (1 cm H2O=0.098 kPa). Patients were ventilated by preset ventilation pressure and frequency based on different groups after loss of consciousness. Mean ventilation volume (mean value of three tidal volumes) and end-tidal carbon dioxide pressure (PetCO2) were recorded for analysis. Results There was no significant difference among the four groups in patient’s general condition (P>0.05). The tidal volume of assisted mechanical ventilation increased with ventilation pressure degrees, and the differences among the four groups were significant (P<0.05). After curve regression analysis, tidal volume and ventilation pressure showed a positive linear correlation when ventilation pressure was set at 5-20 cm H2O, and the correlation equation was: tidal volume = 33.612×ventilation pressure-53.155. PetCO2 in P5 group was lower than those in the other three groups (P<0.05), while there were no significant differences among groups P10, P15 and P20 (P>0.05). Conclusion When ventilation pressure is set at 5-20 cm H2O in assisted mechanical ventilation with facemask during anesthesia induction, tidal volume and ventilation pressure show a positive linear correlation.
Objective To analyze the changes of gene expression profiles during the process that human bonemarrow mesenchymal stem cells (hBMSCs) are induced to differentiate into cardiomyogenic cells with 5-azacytidine (5-aza). Methods hBMSCs were isolated from marrow of obsolete ribs and induced with 5-aza. Then immunocytochemicalstaining was used to detect the expressions of α-actin, cardiac troponin T (cTnT), and connexin 43, and the percentage ofcTnT positive cells was tested with flow cytometry. In the process of differentiation, variation of gene expression was screenedwith Genechi ps Operating System of human gene expression profiles. And the differentially expressed genes were functionallyanalyzed and hierarchical clustered. Results When BMSCs were induced in vitro with 5-aza, part of the cells turnedinto myogenic cells morphologically. Before induction, immunocytochemical staining for α-actin and cTnT showed sl ightpositive and for connexin 43 showed negative. While after 3 weeks of induction, immunocytochemical staining for α-actin,cTnT, and connexin 43 showed all positive. With flow cytometry, the percentage of cTnT positive cells was 7.43% ± 0.02%before induction, but it was 49.64% ± 0.05% after induction. During differentiation, 1 814 differentially expressed geneswere reported by gene chi ps. Of them, 647 genes were divided into 5 groups with hierarchical clustering. They had variousbiological functions, involving signal transduction, cell metabol ism, prol iferation, differentiation, development, andtopogenesis. Conclusion hBMSCs can differentiate into cardiomyogenic cells with the induction of 5-aza in vitro. Multi plegenes related with signal transduction, transcri ption, and growth factors are involved during this process.
Objective To explore the regulator factor of osteogenes is induced by the fibroblast in vitro so as to provide enough seeding cells for the bon e tissue engineering. Methods The fibroblasts were isolated and purified from granu lation of New Zealand rabbits, and they were incubated in the media offibronectin (FN) 10, 20, 40, 60 and 80 μg/ml, respectively, in the experimenta l grou ps 1- 5，but there was no FN in the control group. The markers for osteogenic features were investigated by fibroblast morphogenesis,calcium nodules formationratios,labeling of tetracycline fluorescence, labeling of 3H-TdR, determination of o steocaline, and labeling of 3H-proline within 2 weeks. Results The morphologic al changes of the fibroblasts were manifested as transference from a long spindle to a round or multiple form, shifted nucleus increased in number, confluenced and formed multilayered structure. There was a piling-up of calcium crystals that were gradually merged into foggy substances. The foggy substances increased and formed nodules. The calcium nodules formation ratios were as follows: 15.35％± 3.45％in the control group, and 53.73％± 9.49％, 75.21％± 9.80％, 98.34％± 15.2 0％, 61.83％± 10.04％, and 45.11％± 8.70％ in the experimental groups 1.5 ,respectively. There was a significant difference between the control group and the 5 experimental groups at 14 days (Plt;0.05), and a significant differenc e be tween the experimental group 3 and the other experimental groups at 14 days (Plt;0.05). The histochemical study on the nodules with the specific labeling of tet racycline fluorescence indicated that the nodules were composed of new bones. Conclusion Fibronectin can stimulate the fibroblast to prolifer ate, secrete osteocaline, and synthesize collagen fibrils. Fibronectin, in an optimal dose of 40 -60 μg/ml, is capable of inducing the fibroblast to form the bone.
Objectives To evaluate the clinical effectiveness and safety of combined induction therapy of interferon (IFN) with chemotherapy for survival of the patients with advanced non-small cell lung cancer (NSCLC) by meta-analysis. Methods All clinical trials of addition of IFN plus chemotherapy versus chemotherapy alone for induction therapy to advanced NSCLC patients in MEDLINE (1966-2006), EMBASE (1984-2006.1) and The Cochrane Library (Issue 1,2006) were identified. The references of related studies and Education Books of ASCO and ESMO meeting were handsearched. The quality of included trials was evaluated. Data were extracted by two reviewers independently with a designed extraction form. RevMan 4.2.7 software was used for data analysis. Results Five randomized controlled trials involving 360 patients were included. The pooled result of 3 studies showed that IFN plus chemotherapy induction treatment did not improve 1-year survival rate with RR 0.76, 95%CI 0.46 to 1.26. The pooled result of 5 studies showed that IFN plus chemotherapy induction treatment did not improve response rate with RR 1.40, (0.83 2.34). The pooled result showed that IFN plus chemotherapy induction treatment might significantly increase leukopenia and thrombocytopenia with RR 2.61,95%CI1.70 to 3.99) and RR 4.78,95%CI 1.87 to 12.19 respectively . Conclusion Insufficient data exists to state whether IFN plus chemotherapy induction treatment can improve 1-year survival rate and response rate. IFN plus chemotherapy may increase occurrence of leucopenia and thrombocytopenia. Further studies are warranted.
Objective To investigate the feasibil ity of inducing canine BMSCs to differentiate into epithel ial cells in vitro with epithel ial cell conditioned medium (ECCM). Methods Five mL BMSCs were obtained from il iac spine of a healthy adult male canine with weighing 10 kg, and then isolated and cultured. The oral mucosa was harvested and cut into 4 mm × 4 mm after the submucosa tissue was el iminated; ECCM was prepared. BMSCs of the 2nd passage were cultured and divided into two groups, cultured in ECCM as experimental group and in L-DMEM as control group. The cell morphological characteristics were observed and the cell growth curves of two groups were drawn by the continual cell counting. The cells were identified by immunohistochemical staining through detecting cytokeratin 19 (CK-19) and anti-cytokeratin AE1/AE3 on the21st day of induction. The ultra-structure characteristics were observed under transmission electron microscope. Results The cells of two groups showed long-fusiform in shape and distributed uniformly under inverted phase contrast microscope. The cell growth curves of two groups presented S type. The cell growth curve of the experimental group was right shifted, showing cell prol iferation inhibition in ECCM. The result of immunohistochemical staining for CK-19 and anti-cytokeratin AE1/AE3 was positive in the experimental group, confirming the epithel ial phenotype of the cells; while the result was negative in the control group. The cells were characterized by tight junction under transmission electron microscope. Conclusion The canine ECCM can induce allogenic BMSCs to differentiate into epithel ial cells in vitro.