Objective To investigate the effect of machine-enzyme digestion method on the residual quantity of small intestinal submucosa (SIS) cell and the content of growth factors. Methods Fresh jejunum of pig within 4 hours after harvesting was prepared into SIS after machine digestion (removing placenta percreta, mucosa, and muscular layer), degrease,trypsinization, abstergent processing, and freeze drying. Samples were kept after every preparation step serving as groups A, B, C, D, and E, respectively (n=4 per group). And the fresh jejunum served as control group (group F, n=4). The histological alteration in each preparation process was reviewed with HE staining and scanning electron microscope (SEM). Nest-polymerase chain reaction (PCR) was used to determine the content of death associated protein 12 (DAP12), and enzyme-linked immunosorbent assay (ELISA) was appl ied to detect the content of vascular endothel ial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β), tumor necrosis factor α (TNF-α). Results HE staining and SEM observation showed that there were residual cells in groups A and B, and there were no residual cells in groups C, D, and E. Nest-PCR test revealed the occurrence of DAP12 in each group. The contents of DAP12 in groups A, B, C, D, E, and F were (18.01 ± 9.53), (11.87 ± 2.35), (0.59 ± 0.27), (0.29 ± 0.05), (0.19 ± 0.04), and (183.50 ± 120.13) copy × 106/cm2. The content of DAP12 in group F was significant higher than that of other groups (P lt; 0.05), groups A and B was higher than groups C, D, and E (P lt; 0.05), there were significantdifferences among groups C, D, and E (P lt; 0.05), and there was no significant difference between groups A and B (P gt; 0.05). The ELISA test showed the content of VEGF, bFGF, TGF-β, and TNF-α in group A was significantly higher than that of groups B, C, D, and E (P lt; 0.05), and there was no significant difference among groups B, C, D, and E (P gt; 0.05). Conclusion SIS prepared by simple mechanical method has more residual cells, while the machine-enzyme digestion method can effectively remove the cells and significantly reduce the DAP12 content. This approach can not obviously reduce the growth factor content in SIS.
【Abstract】Objective To investigate the effects of human growth hormone (GH) on colonic cancer cells to provide experimental evidence about the GH safety in colonic cancer therapy. Methods The nude mouse model of colonic carcinoma induced with SW480 cell line was established to observe the effects of GH on the transplanted carcinoma. GH and 5-FU were administered to SW480 cells cultured in vitro to observe the cell growth with MTT method. Results The volume, average diameter and weight of the transplanted carcinoma in GH group were significantly higher than those in control group（P＜0.05）. In vitro, the value of A in GH group was significantly higher than control group （P＜0.01), but the value of A in 5-FU+GH group was lower than control group（P＜0.01）. Conclusion GH can promote colonic cancer cell growth; GH combined with cell cycle specific chemotherapeutic drugs is safe in colonic cancer therapy and may be used as a promoter of chemotherapy.
Objective
To review the research progress of growth factor sustained-release microspheres in fat transplantation.
Methods
The recently published 1iterature at home and abroad related the growth factor sustained-release microspheres in fat transplantation was reviewed and analyzed.
Results
The sustained-release microsphere carrier materials include natural polymer materials and synthetic polymer materials.The sustained-release complexes of different microsphere materials with different growth factors can promote the vascularization of transplanted fat in a timely manner, improve the survival rate of grafts, and reduce the incidence of complications such as liquefaction, calcification, and necrosis.
Conclusion
The growth factor sustained-release microspheres have the characteristics of persistence and controllability, which is a research hotspot in the field of fat transplantation and has broad application prospects.
Thirty native goats were equally divided into two groups at random.A transverse fracture wasmade at the middle of the femur and tibia on the same side .The c;pver-shaped pin was used to fix thefemur and the Kirshners wire for the tibia.The experimental animals were given L-Dopu tablers.The animals were undergone the gross examination,roentgenographic eXamination, histolgical study,electron microseopic scanning and the examination of blood chemistry at 2,4, 6,8and 12 weeks afteroperatio...
Objective To introduce growth and differentiation factor 5 (GDF-5) gene into hBMSCs using recombinant adenovirus vector and to investigate the effect of GDF-5 gene expression on hBMSCs osteogenic differentiation. Methods Recombinant adenovirus GDF-5 (Ad-GDF-5) containing green fluorescent protein (GFP) and Ad-GFP were amplifiedand tittered. hBMSCs at passage 3 were infected with two viruses at different titers. At 2 days after intervention, GFP expression was observed using fluorescence microscope, and GDF-5 expression in hBMSCs was detected by RT-PCR. Adherent hBMSCs at passage 3 were randomly divided into 4 groups: experimental group (GDF-5 gene transfection), osteogenic induction group, Ad- GFP infection group, and control group. Cell differentiation was detected by inverted phase contrast microscope observation, fluorescence microscope observation, reverse transcription fluorescence quantitative PCR, immunofluorescence staining, and von Kossa staining at different time points after intervention. Results The titer of Ad-GDF-5 and Ad-GFP was 1.0 × 109 pfu/mL and 1.2 × 109 pfu/mL, respectively. hBMSCs was efficiently infected by Ad-GDF-5 and Ad-GFP, and expressed target gene and GFP gene. At 1-7 days after intervention, morphology and growth pattern of the hBMSCs in the experimental group and the osteogenic induction group were transformed into osteoblast-l ike cells, whereas the cells in the other two groups were still maintained their original morphology and growth pattern. Reverse transcription fluorescence quantitative PCR detection: at 4 days after intervention, GDF-5 expression in the experimental group was obviously higher than that of other groups (P lt; 0.05); ALP, Col I, and OC gene expression in the experimental and the osteogenic induction group were superior to those of theAd-GFP infection and the control group (P lt; 0.05); Col I gene expression in the osteogenic induction group was greater than that of the experimental group (P lt; 0.05). Immunofluorescence staining: at 4 days after intervention, the cells in the osteogenic induction group and the experimental group expressed and secreted Col I, and no expression of Col I was evident in the other two groups. At 10 days after intervention, the cells in the osteogenic induction and the experimental group were positive for von Kossa staining, and the results of the other two groups were negative. Conclusion GDF-5 gene can be transferred into hBMSCs via adenovirus vector and be expressed stably. It can facil itate the osteogenic differentiation of the hBMSCs and lay a foundation for the further study of this kind of gene transferred hBMSCs effect on bone tissue repair.
Objective To investigate the effect of chondroitinase ABC (ChABC) on the expression of growth associated protein 43 (GAP-43) and gl ial fibrillary acidic protein (GFAP) after spinal cord injury (SCI) in rats. Methods A total of 150 adult female SD rats, weighing 250-300 g, were randomly divided into ChABC treatment group (group A), sal ine treatment group (group B), and sham operation group (group C) with 50 rats in each group. In groups A and B, the rats were made the SCI models and were treated by subarachnoid injection of ChABC and sal ine; in group C, the rats were not treated as a control. At 1, 3, 7, 14, and 21 days after operation, the Basso, Beattie, and Bresnahan (BBB) score system was used toevaluate the motion function, and immunofluorescent histochemical staining was used to observe the expressions of GAP-43 and GFAP. Results At different time points, the BBB scores of groups A and B were significantly lower than those of group C (P lt; 0.05); there was no significant difference in BBB score between groups A and B after 1, 3, and 7 days of operation (P gt; 0.05), but the BBB score of group A was significantly higher than that of group B after 14 and 21 days of operation (P lt; 0.01). At different time points, the GAP-43 and GFAP positive neurons of groups A and B were significantly higher than those of group C (P lt; 0.05). After 14 and 21 days of operation, the GAP-43 positive neurons of group A were more than those of group B (P lt; 0.01). After 7, 14, and 21 days of operation, the GFAP positive neurons of group A were significantly less than those of group B (P lt; 0.01). Conclusion ChABC can degrade gl ial scar, improve the microenvironment of the injured region and enhance the expression of GAP-43, which promotes axonal growth and extension.
Objective To review research progress of corneal tissueengineering.Methods The recent articles on corneal tissue engineering focus on source and selection of corneal cells, the effects of growth factors on culture of corneal cells in vitro. The preparation and selection of three-dimensional biomaterial scaffolds and their b and weak points were discussed. Results The corneal tissue engineering cells come from normal human corneal cells. The embryo corneal cell was excellent. Several kinds of growth factors play important roles in culture, growth and proliferation of corneal cell, and incroporated into matrix.Growth factors including basic fibroblast growth factor, keratinocyte growth factor, transforming growth factor β1 and epidermal growth factor was favor to corneal cell. Collagen, chitosan and glycosaninoglycans were chosen as biomaterial scaffolds. Conclusion Human tissue engineering cornea can be reconstructed and transplanted. It has good tissue compatibility and can be used as human corneal equivalents.
Objective
To investigate the impact of photodynamic therapy (PDT) with verteporfin on the expression of pigment epithelial derivative factor (PEDF) mRNA and vascular endothelial growth factor (VEGF) mRNA in adult retinal pigment epithelial (RPE) cells in vitro.
Methods
The changes of cellular viability before and after PDT were assessed by methyl thiazolyl tetrazolum (MTT) colorimetric assay. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was conducted to detect the expression of PEDF and VEGF mRNA in RPE cells before and after PDT.
Results
PDT caused the death of RPE cells. The cellular mortality was positively correlated with the power of photocoagulation and the concentration of verteporfin.
Conclusion
PDT could downregulate the expression of PEDF and VEGF mRNA in adult RPE cells in vitro, which may relate to the cure or relapse of subfoveal choroidal neovascular membrane after PDT.
(Chin J Ocul Fundus Dis, 2006, 22: 256-260)
Objective To observe the effects of neural stem cells(NSCs) transplantation on the glial cell line-derived neurotrophic factor (GDNF) and growth associated protein 43(GAP-43) after the spinal cord injury(SCI), and to investigate the mechanism of repairing the SCI by NSCs transplantation. Methods The neural stem cells from the hippocampus of rats’ embryo were cultured and identified by immunocytochemistry. The SCI model was made by the modified Allen device. Sixty adult Wistar rats were randomly divided into three groups: spinal cord injury was treated with transplantation of NSCs (group A, n=24), with DMEM solution(group B, n=24) and normal control group without being injured(group C, n=12). Seven days after the operation of SCI, the NSCs were transplanted into the injured site. Then GAP-43 and GDNF expressions were tested by RT-PCR and immunohistochemistry. Results Compared with group B, the GDNF mRNA expression of group A increased by 23.3％ on the 1st day, by 26.8% on the 3rd day and by 32.7% on the 7th day; the GAP-43 mRNA expression increased by 19.5% on the 1st day, 21.6% on the 3rd day and 23.1% on the 7th day. There were statistically significant differences(Plt;0.05). Conclusion The transplantation of NSCs can change the microenvironment injured site and promote the regeneration of axon by enhancing the expressions of GDNF mRNA and GAP-43 mRNA. It is one of the mechanisms of repairing the SCI by NSCs transplantation.
ObjectiveTo investigate the effectiveness of epidermal growth factor receptor antagonist (AG-1478) on chronic proliferative cholangitis (CPC), so as to investigate new treatment approach for hepatolithiasis associated with CPC. MethodsForty-six SD rats were divided into 5 groups: CPC model group (n=10), only made models. AG-1478 treatment group (divided into 3 mg/kg， 6 mg/kg， and 12 mg/kg groups, n=10 per group), the common bile ducts in CPC animal model received an intralumenal administration of AG-1478 at the meantime of modeling, followed by intraperitoneal AG-1478 injection of 1.5 mg/(kg·d) for 7 days. Sham operation group (SO group, n=6). Subsequently, histopathological observation, immunohistochemistry, real time PCR， and Western blot were used to evaluate the mRNA expression and influence of AG-1478 on the hyperplasia （EGFR， ki-67， BrdU， collagen Ⅰ protein） and lithogenic potential (Mucin 5AC) of CPC. ResultsCompared with CPC model group, the expressions of EGFR, ki-67， and BrdU were obviously decreased in the AG-1478 treatment group. Also, the inhibition of hyperplasia of biliary epithelium and collagen fibers were confirmed by histopathological observation. Additionally, the expressions of Mucin 5AC mRNA and collagen Ⅰ protein remarkable decreased in the AG-1478 treatment group (Plt;0.05). Conclusions EGFR inhibitor (AG-1478) could shows inhibitory effectivenss on the CPC-mediated hyperplasia and lithogenic potential, and therefore holds promise as the new treatment approach for CPC.
