Objective To establish a stable colorectal cancer model in liver specific insulin-like growth factor (IGF)-1 deficient (LID) mice and examine the potential relationship between IGF-1 level and risk of mice constitutional colorectal cancer. Methods ①Establishment of a colorectal cancer model: The LID mice, in which IGF-1 level in circulation was 25% of BALB/c mice. Induction of colorectal cancer was achieved by using the 1,1 Dimethylhydrazine (DMH) with hypodermic injection at transverse part. ②Eighty fresh samples of cancer tissues and adjacent tissues were obtained from LID mice (experimental group) and BALB/c mice (control group). The expression of IGF-1 was studied by immunohistochemical assay (SP method). Results ①Weight loss occurred in both experimental group and control group after injection. Compared with the body weight before injection on 18 weeks and 24 weeks in each group, there were significant differences after injection at the same phase in each group (P＜0.05). ②The results of IGF-1 expression in cancer tissues and adjacent tissues: IGF-1 got a diffuse distribution in cancer cell cytoplasm. The positive expressions of IGF-1 in the cancer tissues and their adjacent cancer tissues were 6/7, 2/7 and 13/16, 7/16 respectively in experimental group and control group. There were significant differences between the cancer tissues and adjacent tissues inside both groups (P＜0.05). There were no significant differences inside both of cancer tissues and adjacent tissues respectively between experimental group and control group (Pgt;0.05). Conclusion In the established colorectal cancer model by DMH, IGF-1 plays an important role in the development and progression of colorectal cancer.
Objective To observe the expressions of CXC chemokine receptor 4 (CXCR4) in muscle satell ite cells in situ of normal and cardiotoxin-intoxicated muscle tissues so as to further investigate the molecular mechanism involving inmuscle regeneration such as progressing muscular dystrophy (PMD) for seeking the way to cure muscle retrogression. Methods The muscle injured model of 12 C57 male mice was made by injecting cardiotoxin (5 μg per mouse) in left quadriceps femoris, their right quadriceps femoris was used as control without any injection. The histological, immunohistochemical analysis and RT-PCR were done to investigate the expression of CXCR4 in the quadriceps femoris in situ after 1 day, 4 days, 1 week, 2 weeks, 4 weeks and 6 weeks. Results HE staining results demonstrated that the muscle tissues experienced the process from muscle injury, repair to regeneration. The result of immunohistochemistry showed that the expressions of CXCR4 in injured muscle tissue were 1 955.6 ± 150.3, 2 223.2 ± 264.3, 2 317.6 ± 178.7, 3 066.5 ± 269.6, 1 770.9 ± 98.7 and 1 505.7 ± 107.1 at 1 day, 4 days, 1 week, 2 weeks, 4 weeks and 6 weeks after injection of cardiotoxin, there was significant difference when compared with normal muscle (640.3 ± 124.0, P lt; 0.001). The RT-PCR showed that the expressions of CXCR4 mRNA in injured muscle tissue were0.822 ± 0.013, 0.882 ± 0.025, 1.025 ± 0.028, 1.065 ± 0.041, 0.837 ± 0.011 and 0.777 ± 0.015 at 1 day, 4 days, 1 week, 2 weeks, 4 weeks and 6 weeks after injection of cardiotoxin, there was significant difference when compared with normal muscle (0.349 ± 0.006, P lt; 0.001). Conclusion CXCR4 may be the critical protein in the process of muscle impairment and reparation.
ObjectiveTo study the effect and mechanism of lipopolysaccharide (LPS) on osteoclasts formation and its bone resorption function.MethodsBone marrow-derived macrophages (BMMs) were extracted from the marrow of femur and tibia of 4-week-old male C57BL/6 mice. Flow cytometry was used to detect BMMs. The effect of different concentrations of LPS (0, 100, 200, 500, 1 000, 2 000 ng/mL) on BMMs activity was examined by cell counting kit 8 (CCK-8) activity test. In order to investigate the effect of LPS on osteoclastogenesis, BMMs were divided into macrophage colony-stimulating factor (M-CSF) group, M-CSF+receptor activator of nuclear factor κB ligand (RANKL) group, M-CSF+RANKL+50 ng/mL LPS group, M-CSF+RANKL+100 ng/mL LPS group. After the completion of culture, tartrate resistant acid phosphatase (TRAP) staining was used to observe the formation of osteoclasts. In order to investigate the effect of LPS on the expression of Connexin43, BMMs were divided into the control group (M-CSF+RANKL) and the LPS group (M-CSF+RANKL+100 ng/mL LPS); and the control group (M-CSF+RANKL), 50 ng/mL LPS group (M-CSF+RANKL+50 ng/mL LPS), and 100 ng/mL LPS group (M-CSF+RANKL+100 ng/mL LPS). The expressions of Connexin43 mRNA and protein were detected by Western blot and real-time fluorescent quantitative PCR, respectively. In order to investigate the effect of LPS on osteoclast bone resorption, BMMs were divided into M-CSF group, M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group. Bone absorption test was used to detect the ratio of bone resorption area.ResultsThe flow cytometry test confirmed that the cultured cells were BMMs, and CCK-8 activity test proved that the 100 ng/mL LPS could promote the proliferation of BMMs, showing significant differences when compared with the 0, 200, 500, 1 000, and 2 000 ng/mL LPS (P<0.05). TRAP staining showed no osteoclast formation in M-CSF group. Compared with M-CSF+RANKL group, the osteoclasts in M-CSF+RANKL+50 ng/mL LPS group and M-CSF+RANKL+100 ng/mL LPS group were larger with more nuclei, while the osteoclasts in M-CSF+RANKL+100 ng/mL LPS group were more obvious, and the differences in the ratio of osteoclast area between groups were statistically significant (P<0.05). Western blot result showed that the relative expression of Connexin43 protein in LPS group was significantly higher than that in control group (P<0.05). Real-time fluorescent quantitative PCR showed that the relative expression of Connexin43 mRNA in control group, 50 ng/mL LPS group, and 100 ng/mL LPS group increased gradually, and the differences between groups were statistically significant (P<0.05). Bone resorption test showed that osteoclast bone resorption did not form in M-CSF group, but the ratio of bone resorption area increased gradually in M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group, and the differences between groups were statistically significant (P<0.05).ConclusionLPS at concentration of 100 ng/mL can promote the expression of Connexin43, resulting in increased osteoclastogenesis and enhanced osteoclastic bone resorption.
Objective To investigate the serum levels of endostatin and vascular endothelial growth factor ( VEGF) at different therapy stages of mouse Lewis lung carcinoma, and elucidate the relation to the progress and prognosis of tumor. Methods Forty-four Lewis lung carcinoma-bearing C57BL/6 mice were randomly divided into 4 groups, ie. a non-therapy group, a chemotherapy group, a gene therapy group, and a combination therapy group ( chemotherapy plus gene therapy) . Eleven healthy mice were included as normal control group. Serum was collected on the 0th, 5th, 19th day after therapy for measurement of endostatin and VEGF by ELISA. The correlations were analysed between endostatin and VEGF levels in each group. Results ( 1) The serum endostatin levels had no significant difference in all groups on the 0th day,but increased significantly on the 5th day in the gene and combination therapy groups than those in other three groups ( all P lt;0. 01) . Then the endostatin level decreased on the 19th day in the gene and combination therapy groups, but still higher than those in the chemotherapy group and the normal group. ( 2 ) On the contrary, serum VEGF levels of the gene and combination therapy groups decreased significantly on the 5th day and increased little on the 19th day, which were both significant lower than those in chemotherapy group on the 5th and 19th day( all P lt; 0. 05) . There were significant diferences between the three therapy groups and the non-therapy group( all P lt;0. 05) . ( 3) Negative correlations between VEGF and endostatin levels were revealed in the gene and combination therapy groups ( r = - 0. 77 and - 0. 761 respectively) .Correlation was not found in the non-therapy and chemotherapy groups. Conclusion The serum levels of endostatin and VEGF might be used as monitoring indices of antiangiogenesis therapy.
ObjectiveTo investigate the mechanism of G protein coupled receptor kinase interacting protein 1 (GIT1) affecting angiogenesis by comparing the differentiation of bone marrow mesenchymal stem cells (BMSCs) differentiated into endothelial cells between GIT1 wild type mice and GIT1 gene knockout mice.MethodsMale and female GIT1 heterozygous mice were paired breeding, and the genotypic identification of newborn mice were detected by PCR. The 2nd generation BMSCs isolated from GIT1 wild type mice or GIT1 gene knockout mice were divided into 4 groups, including wild type control group (group A), wild type experimental group (group A1), GIT1 knockout control group (group B), and GIT1 knockout experimental group (group B1). The cells of groups A1 and B1 were cultured with the endothelial induction medium and the cells of groups A and B with normal cluture medium. The expressions of vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, and phospho-VEGFR-2 (pVEGFR-2), and pVEGFR-3 proteins were detected by Western blot. The endothelial cell markers [von Willebrand factor (vWF), platelet-endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial cadherin (VE-Cadherin)] were detected by flow cytometry. The 2nd generation BMSCs of GIT1 wild type mice were divided into 4 groups according to the different culture media: group Ⅰ, primary cell culture medium; group Ⅱ, cell culture medium containing SAR131675 (VEGFR-3 blocker); group Ⅲ, endothelial induction medium; group Ⅳ, endothelial induction medium containing SAR131675. The endothelial cell markers (vWF, PECAM-1, and VE-Cadherin) in 4 groups were also detected by flow cytometry.ResultsWestern blot results showed that there was no obviously difference in protein expressions of VEGFR-2 and pVEGFR-2 between groups; and the expressions of VEGFR-3 and pVEGFR-3 proteins in group A1 were obviously higher than those in groups A, B, and B1. The flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group A1 than in groups A, B, and B1 (P<0.05), and in group B1 than in groups A and B (P<0.05); but no significant difference was found between groups A and B (P>0.05). In the VEGFR-3 blocked experiment, the flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group Ⅲ than in groupsⅠ, Ⅱ, and Ⅳ, and in group Ⅳ than in groups Ⅰ and Ⅱ (P<0.05); but no significant difference was found between groups Ⅰ and Ⅱ (P>0.05).ConclusionGIT1 mediates BMSCs of mice differentiation into endothelial cells via VEGFR-3, thereby affecting the angiogenesis.
Objective To determine the effects of lensspecific overexpression of OSM on the eye development. Methods A truncated mouse OSM c DNA (661 bp) was linked to the αA-crystallin promoter. Transgenic mice were characterized by routine histological and immunohistochemical techiniques. TUNEL assays were used to de tect cell death. The mRNA expression of caspase-3 was detected by in situhybridization, Rabbit anti-cleavage caspase-3 antibody was used to detectactive capase-3. Results At embryonic day (E) 14.5 and 17.5, expression of the OSM transgenic protein was detected specifically in lens fiber cells. The onset of retinal degeneration in the mid portion of the transgenic retinae was observed started from E17.5. By the time of birth 50% or more of the retinal cells were missing. The OSM transgenic retinal cells underwent apoptosis indicated by TUNEL assays. Most strikingly, activation of caspase-3 protein were observed throughout the transgenic retinas. Conclusions Lens-specific overexpression of OSM activate caspase-3, leading to abnormal eye development,apoptosis and widespread retinal degeneration. (Chin J Ocul Fundus Dis，2003，19：201-268)
ObjectiveTo investigate the effects of human placental mesenchymal stem cells (hPMSCs) transplantation on pulmonary vascular endothelial permeability and lung injury repair in mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI).MethodsThe hPMSCs were isolated from the human placental tissue by enzyme digestion and passaged. The cell phenotype of the 3rd generation hPMSCs was detected by flow cytometry. Twenty-four 6-week-old healthy male C57BL/6 mice were randomly divided into 3 groups (n=8). The mice were instilled with LPS in the airway to prepare an ALI model in the ALI model group and the hPMSCs treatment group, and with saline in the control group. At 12 hours after LPS infusion, the mice were injected with 3rd generation hPMSCs via the tail vein in hPMSCs treatment group and with saline in the ALI model group and the control group. At 24 hours after injection, the lung tissues of all mice were taken. The pathological changes were observed by HE staining. The wet/dry mass ratio (W/D) of lung tissue was measured. The Evans blue leak test was used to detect the pulmonary vascular endothelial permea bility in mice. The expression of lung tissue permeability-related protein (VE-cadherin) was detected by Western blot.ResultsFlow cytometry examination showed that the isolated cells had typical MSCs phenotypic characteristics. Mice in each group survived. The alveolar structure of the ALI model group significantly collapsed, a large number of inflammatory cells infiltrated, and local alveolar hemorrhage occurred; while the alveolar structure collapse of the hPMSCs treatment group significantly improved, inflammatory cells infiltration significantly reduced, and a few red blood cells were in the interstitial lung. W/D and exudation volume of Evans blue stain were significantly higher in the ALI model group than in the control group and the hPMSCs treatment group (P<0.05), in the hPMSCs treatment group than in the control group (P<0.05). The relative protein expression of VE-cadherin was significantly lower in the ALI model group than in the control group and the hPMSCs treatment group (P<0.05), and in the hPMSCs treatment group than in the control group (P<0.05).ConclusionIntravenous injection of hPMSCs can effectively reduce the increased pulmonary vascular endothelial permeability mediated by LPS, relieve the degree of lung tissue damage, and play a therapeutic role in ALI mice.
ObjectiveTo investigate the effects of resveratrol on airway remodeling in mice with chronic asthma. MethodsTwenty-four female BALB/c mice were randomly divided into three groups (8 mice in each group), namely a control group, an asthma group and a resveratrol (RV) group. All mice were sensitized with ovalbumin (OVA). The sensitized mice were then challenged with OVA while the control group were challenged with phosphate-buffered saline. The mice in the RV group were intraperitoneally injected with RV 30 min before OVA challenge, while the mice in the control and the asthma group were intraperitoneally injected with equal volume of dimethylsulfoxide. Periodic acid-Schiff (PAS) staining was performed to evaluate goblet cell hyperplasia, and Masson-trichrome staining was used to evaluate the deposition of collagen matrix. In addition, immunohistochemical analysis of the α-smooth muscle actin (α-SMA) was applied to examine airway smooth muscle cell hyperplasia and hypertrophy. The positive staining with PAS, Masson, α-SMA areas (μm 2/μm) of per bronchial basement membrane perimeter was used to indicate the degree of airway remodeling. ResultsIn the asthma group and the RV group, the degree of the goblet cell hyperplasia was significantly higher than that in the control group (5.44±1.13, 4.18±0.85vs. 0.00±0.00,P<0.01), and the level of goblet cell hyperplasia in the RV group was lower than that in the asthma group (P<0.05). The Masson staining showed that the deposition of collagen in the asthma group and the RV group was significantly higher than that in the control group (9.80±2.78, 5.71±0.68vs. 1.67±0.65,P<0.01), and the collagen deposition in the RV group was further lower than that in the asthma group (P<0.01). The α-SMA immunohistochemical analysis demonstrated that the expression of α-SMA in the asthma group and the RV group was significantly higher than that in the control group (10.39±1.65, 7.57±1.98vs. 2.41±1.06,P<0.01), and the level of α-SMA in the RV group was also lower than that in the asthma group (P<0.05). ConclusionThese findings suggest that resveratrol has an inhibitory effect on the process of airway remodeling in mice with chronic asthma.
ObjectiveTo explore the feasibility of establishment of a artificial joint aseptic loosening mouse model by cobalt-chromium particles stimulation.MethodsTwenty-four 8-week-old male severe combined immunodeficient (SCID) mice were divided into experimental group (n=12) and control group (n=12). The titanium nail was inserted into the tibial medullary cavity of mouse in the two groups to simulate artificial joint prosthesis replacement. And the cobalt-chromium particles were injected into the tibial medullary cavity of mouse in experimental group. The survival of the mouse was observed after operation; the position of the titanium nail and the bone mineral density of proximal femur were observed by X-ray film, CT, and Micro-CT bone scanning; and the degree of dissolution of the bone tissue around the tibia was detected by biomechanical test and histological staining.ResultsTwo mice in experimental group died, and the rest of the mice survived until the experiment was completed. Postoperative imaging examination showed that there was no obvious displacement of titanium nails in control group, and there were new callus around the titanium nails. In experimental group, there was obvious osteolysis around the titanium nails. The bone mineral density of the proximal tibia was 91.25%±0.67%, and the maximum shear force at the tibial nail-bone interface was (5.93±0.85) N in experimental group, which were significantly lower than those in control group [102.07%±1.87% and (16.76±3.09) N] (t=5.462, P=0.041; t=3.760, P=0.046). Histological observation showed that a large number of inflammatory cells could be seen around the titanium nails in experimental group, while there was no inflammatory cells, and obvious bone tissue formation was observed in control group.ConclusionThe artificial joint aseptic loosening mouse model can be successfully established by cobalt-chromium particles stimulation.
Objective Lots of metal ions accumulation and over-expression of receptor activator of NF-κB l igand (RANKL) around the prosthesis could be found in revision of total hip arthroplasty. To investigate the relationship between metal ions and aseptic loosening by observing the effects of Co2+ and Cr3+ ions on the expression of RANKL and osteoprotegerin(OPG) from osteoblast. Methods Osteoblasts were cultured in vitro at the density of 1 × 105 cells/mL, and were divided into 2 groups according to different culture solutions. In control group, osteoblasts were cultured with normal medium without CoCl2 and CrCl3. In experimental group, osteoblasts were cultured with the medium including CoCl2 (10 mg/ L) and CrCl3 (150 mg/L) solutions. The RT-PCR and ELISA methods were appl ied to detect the mRNA expression of RANKL and OPG and protein level at 24 and 48 hours after co-cultured, respectively. Results RT-PCR revealed that the mRNA expression of RANKL and OPG could be found in two groups at 24 and 48 hours after co-cultured, the expression was higher in the experimental group than in control group, especially the expression of RANKL, showing significant difference (P lt; 0.05). At 24 and 48 hours after co- cultured, the ratios of RANKL mRNA to OPG mRNA in the experimental group were 0.860 and 1.232, respectively, which were significantly higher than those in the control group (0.695 and 0.688,P lt; 0.05). ELISA revealed that the protein level of RANKL and OPG in experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion Co2+ and Cr3+ can stimulate the mRNA expressions of RANKL, OPG and secretion of those protein from osteoblasts, especially increase of the RANKL, which promotes the formation and activation of osteoblasts and the generation of aseptic loosening.