Allogeneic mouse model of peritoneal heart transplant is a microscopic surgery on small animal with complex techniques. For a beginner, a learning curve of this surgical technique has to be experienced. The learning curve contains three stages:(1) to be familiar with the local anatomy of either donor or recipient mouse; (2) to be capable of collecting donor heart and well preparing the major peritoneal vessels of recipient; (3) to be skillful in the anastomosis of major vessels. The bottleneck of the learning curve is the valid skill of vascular anastomosis. The stepwise essentials are to "understand, be familiar, be accurate, and be quick" in the learning curve.
ObjectiveTo compare the effects on the osteogenesis of bone marrow mesenchymal stem cells (BMSCs) between hypoxia and hypoxia mimetic agents dimethyloxalylglycine (DMOG) under normal oxygen condition. MethodsBMSCs were isolated and cultured from healthy 3-4 weeks old Kunming mouse. Cell phenotype of CD29, CD44, CD90, and CD34 was assayed with flow cytometry; after osteogenic, adipogenic, and chondrogenic induction, alizarin red staining, oil red O staining, and toluidine blue staining were performed. The passage 3 BMSCs were cultured under normal oxygen in control group (group A), under 1%O2 in hypoxia group (group B), and under normal oxygen and 0.5 mmol/L DMOG in DMOG intervention group (group C). BMSCs proliferation was estimated by methyl thiazolyl tetrazolium assay at 1, 2, 3, and 4 days. Alkaline phophatase (ALP) expression was determined at 7 and 14 days after osteogenic induction. Western blot was employed for detecting hypoxia inducible factor-1α(HIF-1α) at 24 hours. Real time fluorescence quantitative PCR was employed for detecting the mRNA expression of runt-related transcription factor 2 (RUNX2) and Osterix at 3 and 7 days. Alizarin red staining was applied to assess the deposition of calcium tubercle at 21 days. ResultsThe BMSCs presented CD29(+), CD44(+), CD90(+), and CD34(-); and results of the alizarin red staining, oil red O staining, and toluidine blue staining were positive after osteogenic, adipogenic, and chondrogenic induction. No significant difference in BMSCs proliferation was observed among 3 groups at 1 day (P>0.05); compared with group A, BMSCs proliferation was inhibited in group C at 2, 3, and 4 days, but no significant difference was observed (P>0.05); compared with group A, BMSCs proliferation was significantly promoted in group B (P < 0.05). At each time point, compared with group A, the ALP expression, HIF-1αprotein relative expression, and mRNA relative expressions of RUNX2 and Osterix were significantly up-regulated in groups B and C (P < 0.05); compared with group B, the ALP expression, the RUNX2 and Osterix mRNA relative expression were significantly up-regulated in group C (P < 0.05); compared with group C, the HIF-1αprotein relative expression was significantly up-regulated in group B (P < 0.05). The alizarin red staining showed little red staining materials in group A, some red staining materials in group B, and a large number of red staining materials in group C. ConclusionHypoxia can promote BMSCs proliferation, DMOG can not influence the BMSCs proliferation; both hypoxia and DMOG can improve osteogenic differentiation of BMSCs, and DMOG is better than hypoxia in improving the BMSCs osteogenesis.
Objective To investigate the intervention effect of 3-phosphoinositede dependent protein kinase-1 (PDK1) inhibitor on prostaglandin E2 (PGE2) in smoking-induced chronic obstructive pulmonary disease (COPD) mice. Methods Fifty C57BL/6 male mice were randomly divided into normal control group, smoking group, smoking +low dose PDK1 inhibitor group, smoking + medium dose PDK1 inhibitor group and high dose PDK1 inhibitor group with 10 mice in each group. The mice in the normal control group inhaled phosphate-buffered saline twice a day for 12 weeks, and the mice in the smoking group were fumigated twice a day, 5 days per week for 12 weeks, and the other three groups were given intraperitoneal injection of low-dose PDK1 inhibitor OSU-03012 (0.25 mg/kg), medium-dose PDK1 inhibitor (0.5 mg/kg) and high-dose PDK1 inhibitor (1.0 mg/kg) respectively before smoking. After smoking, lung function was tested, the bronchoalveolar lavage fluid (BALF) of each mouse was taken for cell count, the PGE2 in serum and BALF of mice was determined by enzyme linked immunosorbent assay, and the lung tissue of mice was sectioned with paraffin and stained by hematoxylin-eosin (HE), and pathological changes were observed under microscope. Results Compared with the control group, FEV100/FVC and FEV200/FVC of the mice in each smoking group were significantly decreased (P<0.05); The number of cells in BALF of smoking group was significantly higher than that of normal control group (P<0.05). There was no significant difference in the total number of BALF cells, the proportion of neutrophils and macrophages between the smoking + low-dose PDK1 inhibitor group and the smoking group. However, the total number of BALF cells and the proportion of neutrophils in the smoking + medium dose PDK1 inhibitor group and the high dose PDK1 inhibitor group gradually decreased, while the proportion of macrophages gradually increased, compared with the normal control group, the PGE2 concentrations of serum and BALF in the smoking group and the smoking + PDK1 inhibitor group were significantly higher than those in the control group. Compared with the smoking group, the PGE2 concentrations of serum and BALF in the middle and high dose PDK1 inhibitor groups were significantly lower than those in the smoking group. HE staining of lung tissue showed that there were a large number of inflammatory cell infiltration, alveolar cavity dilatation, alveolar wall rupture and fusion, alveolar formation, significant decrease in the number of alveoli and other pathological changes in the smoking group, which were consistent with the pathological changes of COPD. The inflammatory cell infiltration, mucus obstruction and alveolar dilatation were slightly alleviated in the smoking + low-dose PDK1 inhibitor group, while the inflammatory cell infiltration, alveolar wall thinning and alveolar dilatation were improved in both the medium-dose inhibitor group and the high-dose inhibitor group, and the improvement was more obvious in the high-dose inhibitor group. Conclusion The lung function of the smoked COPD mouse decreases, the airway inflammation is obvious, and the secretion of PGE2 is also increased, while the use of PDK1 inhibitor could reduce the secretion of PGE2, reduce airway inflammation and pathological changes, and improve lung function in a dose-dependent manner.
ObjectiveTo discuss the effects of coix seed extract injection on rate of tumor of C57 mice liver cancer model, tumor size, and serum IL-6. MethodsUsing chemical carcinogens diethyl nitrosamine (DEN) to establish the mice model of liver cancer, liver cancer mouse model to coix seed extract was given observation of C57 mice liver cancer model come tumor formation rate, tumor growth, and the change of serum IL-6. ResultsC57 mice after intraperitoneal injection of coix seed extract injection model of liver cancer tumor rate (55.6%) significantly lower than the DEN group (87.5%), P < 0.01; tumor diameter[(0.3±0.05) cm] was lower than that in group DEN[(0.8±0.06) cm], P < 0.01. The serum level of IL-6 in C57 mice after treated with coix seed extract significantly lower than that in group DEN (P < 0.01). ConclusionCoix seed extract can effectively inhibit the tumor rate and the growth of tumor in hepatocellular carcinoma model of C57 mice, and decrease the level of serum IL-6.
Objective To investigate the possibility of gene therapy of osteolysis around artificial joint prosthesis by constructing the recombinant adenovirus which can silence tumor necrosis factor α (TNF-α). Methods The primer of small interfering RNA (siRNA) coding sequence of silent TNF-α was designed and amplified, and then RAPAD adenovirus packaging system was used to load the sequence to adenovirus, and the recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP which lacked both E1 and E3 regions was constructed. Then 64 female BABL/C mice (weighing, 20-25 g) were randomly divided into 4 groups (n=16): blank control (group A), positive control (group B), simple adenovirus (group C), and treatment group (group D). The prosthetic-model was established in group A, and the prosthetic-loosening-model in groups B, C, and D. At 2 weeks after modeling, PBS solution was injected first, and then the same solution was injected 24 hours later in group A; titanium particle solution was injected, and then PBS solution, Ad5 E1-CMVeGFP (1 × 109 PFU/mL), and Ad5-TNF-α-siRNA-CMVeGFP (1 × 109 PFU/mL) were injected, respectively in groups B, C, and D 24 hours later, every 2 weeks over a 10-week period. The general condition of mice was observed after operation. The tissues were harvested for histological observation, and the expression of TNF-α was detected by Western blot at 12 weeks after operation. Results The positive clones were achieved by enzyme digestion and confirmed by DNA sequencing after loading the target genes into adenovirus vector, and then HEK293 cells were successfully transfected by recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP. All mice survived to the completion of the experiment. Histological observation showed that there were few inflammatory cells and osteoclasts in group A, with a good bone formation; there were a large number of inflammatory cells and osteoclasts in groups B and C, with obvious bone destruction; inflammatory cells and osteoclasts in group D was less than those in groups B and C, with no obvious bone destruction. Significant difference was found in the limiting membrane thickness and the number of osteoclasts (group A lt; group D lt; group B lt; group C, P lt; 0.05). Western blot showed that the TNF-α expression levels were 0.235 ± 0.022, 0.561 ± 0.031, 0.731 ± 0.037, and 0.329 ± 0.025 in groups A, B, C, and D respectively, showing significant difference among 4 groups (P lt; 0.05). Conclusion The recombinant adenovirus for silencing TNF-α is successfully constructed, which can effectively inhibit osteolysis by silencing TNF-α expression in the tissues around prosthesis in mice.
Objective To investigate the effects of removing microglia from spinal cord on nerve repair and functional recovery after spinal cord injury (SCI) in mice. MethodsThirty-nine 6-week-old female C57BL/6 mice were randomly divided into control group (n=12), SCI group (n=12), and PLX3397+SCI group (n=15). The PLX3397+SCI group received continuous feeding of PLX3397, a colony-stimulating factor 1 receptor inhibitor, while the other two groups were fed a standard diet. After 14 days, both the SCI group and the PLX3397+SCI group were tested for ionized calcium binding adapter molecule 1 (Iba1) to confirm that the PLX3397+SCI group had completely depleted the spinal cord microglia. The SCI model was then prepared by clamping the spinal cord in both the SCI group and the PLX3397+SCI group, while the control group underwent laminectomy. Preoperatively and at 1, 3, 7, 14, 21, and 28 days postoperatively, the Basso Mouse Scale (BMS) was used to assess the hind limb function of mice in each group. At 28 days, a footprint test was conducted to observe the gait of the mice. After SCI, spinal cord tissue from the injury site was taken, and Iba1 immunofluorescence staining was performed at 7 days to observe the aggregation and proliferation of microglia in the spinal cord. HE staining was used to observe the formation of glial scars at the injury site at 28 days; glial fibrillary acidic protein (GFAP) immunofluorescence staining was applied to astrocytes to assess the extent of the injured area; neuronal nuclei antigen (NeuN) immunofluorescence staining was used to evaluate neuronal survival. And 5-hydroxytryptamine (5-HT) immunofluorescence staining was performed to assess axonal survival at 60 days. Results All mice survived until the end of the experiment. Immunofluorescence staining revealed that the microglia in the spinal cord of the PLX3397+SCI group decreased by more than 95% compared to the control group after 14 days of continuous feeding with PLX3397 (P<0.05). Compared to the control group, the BMS scores in the PLX3397+SCI group and the SCI group significantly decreased at different time points after SCI (P<0.05). Moreover, the PLX3397+SCI group showed a further decrease in BMS scores compared to the SCI group, and exhibited a dragging gait. The differences between the two groups were significant at 14, 21, and 28 days (P<0.05). HE staining at 28 days revealed that the SCI group had formed a well-defined and dense gliotic scar, while the PLX3397+SCI group also developed a gliotic scar, but with a more blurred and loose boundary. Immunofluorescence staining revealed that the number of microglia near the injury center at 7 days increased in the SCI group than in the control group, but the difference between groups was not significant (P>0.05). In contrast, the PLX3397+SCI group showed a significant reduction in microglia compared to both the control and SCI groups (P<0.05). At 28 days after SCI, the area of spinal cord injury in the PLX3397+SCI group was significantly larger than that in SCI group (P<0.05); the surviving neurons significantly reduced compared with the control group and SCI group (P<0.05). The axonal necrosis and retraction at 60 days after SCI were more obvious. ConclusionThe removal of microglia in the spinal cord aggravate the tissue damage after SCI and affecte the recovery of motor function in mice, suggesting that microglia played a neuroprotective role in SCI.
Objective To determine whether fluoxetine, a commonly used selective serotonin reuptake inhibitors (SSRIs), could exacerbate bleeding in a intracerebral hemorrhage (ICH) mouse model. Methods Forty two 12-14 month old female specific pathogen free C57BL/6 mice were selected. Mice were randomly divided into fluoxetine group (fluoxetine pre-treatment) and control group, with 21 mice in each group. After treated with fluoxetine for 7 days, ICH was induced by injecting collagenase Ⅶ-S into the right striatum of middle-aged female mice. Effects of fluoxetine on exacerbating bleeding were evaluated by a combination of histologic, molecular, cellular, and behavioral assessments. Results On the third day after ICH, the hemorrhage volumes of the control group and fluoxetine group were (4.59±1.80) mm3 and (6.09±1.08) mm3, respectively. In middle-aged female mice subjected to collagenase-induced ICH, fluoxetine pre-treatment significantly exacerbated neurological deficit, cerebral hemorrhage volume, myelin damage, hemoglobin and iron deposition, neuronal degeneration, and brain edema (P<0.05). Although there was no significant difference in tail bleeding time between the two groups, fluoxetine pre-treatment might increase tail bleeding time [(276.73±211.06) vs. (438.00±236.79) s; t=−1.686, P=0.055]. Conclusions The use of fluoxetine and more generally of SSRIs, which inhibits platelet aggregation, may exacerbate bleeding after ICH. Thus, patients with depression after ICH may avoid concomitant use of such drugs when choosing an antidepressant.
Objective To construct specifically expressed vascular endothelial growth factor (VEGF)165 gene in retina. Methods Rho promoter, specifically expressed in retina, was amplified by polymerase chain reaction (PCR) from the genomic DNA of a BLAB/C rat, then it was cut with restriction enzymes and cloned into the plasmid pcDNA3.1+-VEGF165 to form recombinant plasmid pcDNA3.1+-rho-VEGF165. The correct recombinant plasmid pcDNA3.1+-rho-VEGF165 was identified by restriction enzymes and PCR, and was transferred by jetPEI into cultured human navel vein endothelial cells and human retinal pigment epithelial (RPE) cells. The expression of VEGF protein in human navel vein endothelial and RPE cells was detected by immunocytochemical staining and protraction of the growth curve of the cells. Results In human RPE cells, the expression of VEGF protein was more in recombinant plasmidpcDNA3.1+-rho-VEGF165 than that in plasmidpcDNA3.1+-rho-VEGF165 ; in human navel vein endothelial cells, no obvious difference of the expression of VEGF protein between recombinant plasmid pcDNA3.1+-rho-VEGF165 and plasmid pcDNA3.1+-rho-VEGF165 was found. Conclusions The construction of pcDNA3.1+-rho-VEGF165 carrier may provide the basic material for the study of the nosogenesis of VEGF in retinal neovascularization, and establish the foundation to set up the model of transgenic mice with VEGF specific expressing in retina. (Chin J Ocul Fundus Dis, 2005,21:106-108)
ObjectiveTo establish the system of isolation, cultivation, and identification of the neural stem cells (NSCs) from subventricular zone (SVZ) of neonatal mice so as to seek for the appropriate seed cells for potential therapeutic interventions of neurological disorders. MethodsNSCs were isolated enzymatically and mechanically from SVZ of neonatal mice and cultured. The cellular morphology was observed by inverted microscopy. Immunocytochemical stainings of anti-Nestin and anti-SOX-2 were used to identify NSCs of passage 3. To study the differentiation of NSCs, NSCs were plated into 24-wells in the medium supplemented without epidermal growth factor (EGF) and basic fibroblastic growth factor (bFGF) for 3 or 7 days. To compare the differentiation and proliferation potential of NSCs with different cultivation time, the BrdU pulse-labeling method and MTT test were used. To identify neurons and astrocytes, the anti-β-tubulin Ⅲ (Tuj-1) and anti-glial fibrillary acidic protein (GFAP) staining were used. ResultsThe cells of the SVZ can be isolated and cultured in vitro, and these cells began to form neurospheres after cultured for 3 days at primary passage. While cultured for 7 days, these cells formed more neurospheres, and the volume of the neurospheres became bigger than neurospheres cultured for 3 days. In addition, after cultured for 7 days, the phenomena of fusion of neurospheres and adherent differentiation of neurospheres were observed under inverted microscope. These cells were provided with the typical phenotype of NSCs. The immunofluorescence staining results revealed that these cells showed positive immunoreactivity to Nestin and SOX-2. During the 4 hours BrdU pulse, the number of proliferated NSCs cultured for 3 days (75.817±2.961) was significantly higher than that of NSCs cultured for 7 days (56.600±4.881) (t=3.366, P=0.028). The results of MTT assay revealed that the absorbance (A) value of NSCs cultured for 3 days (0.478±0.025) was significantly higher than that of NSCs which were cultured for 7 days (0.366±0.032)(t=2.752, P=0.011). After cultivated without EGF and bFGF, the percentage of Tuj-1 and GFAP positive cells in NSCs was 23.1%±3.7% and 23.7%±3.8% for 3 days and was 40.1%±3.6% and 37.1%±4.5% for 7 days, respectively, all showing significant differences (t=3.285, P=0.030; t=3.930, P=0.017). ConclusionThe NSCs from SVZ of neonatal mice have potentials of self-renewal and multipotential differentiation in vitro. With different cultivation time, the potentials of proliferation and differentiation of NSCs are different.
In this study, the role of newcastle disease virus (NDV) combined thermic solidified tumor vaccine in inhibiting growth of tumor and immune control was investigated, and rate of inhibiting tumor and cellular immunity were measured. The results showed that rate of inhibiting tumor in experimental group Ⅰ and Ⅱ were 24.8% and 41.1% respectively; average weight of tumor was significantly lower in both experimental groups than in control group, and activity of natural killing (NK) cells in experimental groups was higher than that in control group (Ｐ＜0.01). This suggests that NDV combined thermic solidified tumor vaccine can inhibit growth of tumor and improve activity of NK cells, and their effects are better than that of NDV.