牛胎儿成纤维细胞分离与体外培养

本研究以 4 5d牛胎儿为材料 ,使用 0 .2 5 %胰酶 + 0 .0 4 %EDTA消化液 ,分离得到牛胎儿成纤维细胞 ,体外培养传代至第 2 8代。本实验描绘出牛胎儿成纤维细胞生长曲线 ,并发现α MEM是一种适合牛胎儿成纤维细胞生长的培养基。分离得到的牛胎儿成纤维细胞传至第 1 8代时核型仍然正常 ,冷冻、解冻后细胞可继续传代     

Effect Comparison of Adenovirus Vector Transfer into Different Buffalo Cells

Buffalo mammary epithelial cell,cumulus cell and fibroblasts were transfected by adenovirus vectors and compared their transfection efficiency.293 cells were transfected with pBHGloxdelE13cre and pDC316-eGFP by liposome,the virus was collected and titer was detected.Buffalo mammary epithelial cell,cumulus cell and fibroblasts were exposed to different multiplicity of infection (MOI) of adenovirus vectors.After 72 h,the cells were observed with inverted fluorescence microscope,and transfection efficiency was calculated.When the MOI was 25,50,100,200 and 400,the transfection efficiency of fibroblasts were 0.7%,7.0%,9.0%,12.5% and 34.0%,the transfection efficiency of cumulus cells were 42.5%,55.3%,57.4%,76.0% and 80.0%,the transfection efficiency of mammary epithelial cells were 88.7%,100%,100%,100% and 100%.The results showed that the transfection efficiency of mammary epithelial cell was the best,followed by cumulus cell,and fibroblast was poor.     

p300/p53/Smad3 pathway involved in aging-related atrial fibrosis in human atrial fibroblasts

AIM To investigate the role of p300 in aging-related atrial fibrosis in human atrial fibroblasts （HAFs） and its potential mechanism. METHODS HAFs were obtained from human left atrial tissue, and the senescence model was established by cell passage. Senescence-associated β-galactosidase （SA-β-Gal） staining was used to detect the cell senescence, and Western blot was used to determine the protein levels of p300, p53, Smad3 and other senescence and fibrosis associated proteins in HAFs. RESULTS Compared to passage 3 HAFs, the proportion of senescent cells, and the protein levels of p300, p53, p-Smad3 and other senescence and fibrosis associated proteins were increased in HAFs at passage 7 and 11 （P<0.05）. After treated with curcumin （a p300 inhibitor） or transfection with p300 small-hairpin （sh） RNA plasmid, the protein levels of p300, and the senescence and fibrosis associated proteins were decreased in HAFs at passage 7（P<0.05）. Up-regulation of p300 by transfection with p300 over-expression plasmid increased the protein levels of p53, Smad3 and MMP-2 in HAFs at passage 3 （P<0.05）. CONCLUSION p300/p53/Smad3 signaling pathway plays an important role in aging-related atrial fibrosis in HAFs.     

A study on the mechanism of doxorubicin promoting IL-1β and collagen type I secretion in cardiac fibroblasts

AIM To observe the effect of adriamycin/doxorubicin （DOX） on the production of inflammatory cytokines and collagen in cardiac fibroblasts and its mechanism. METHODS Neonatal SD rat cardiac fibroblasts were isolated， cultured， and identified by immunofluorescence staining with monoclonal antibodies against vimentin observed under a confocal laser-scanning microscope. The Cell Counting Kit-8 assay was used to detect the toxicity of DOX on cardiac fibroblasts， and flow cytometry with annexin V-FITC/PI double staining was used to detect apoptosis. ELISA was used to detect the release of inflammatory factors in the supernatant of cultured cells. Immunofluorescence labeling assay was used to detected α-smooth muscle actin （α-SMA） expression and mitochondrial reactive oxygen species （mROS） in the cells. Western blot was used to detect the expression of nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） inflammasome-related proteins in cardiac fibroblasts. RESULTS （1） Compared with the control group， DOX inhibited the proliferation of cardiac fibroblasts （P<0.05）， but had no significant effect on apoptosis （P>0.05）. （2） Treatment with DOX promotes the release of proinflammatory factors interleukin-1β （IL-1β） and IL-6 in cardiac fibroblasts （P<0.05）. （3） The expression of α-SMA， collagen type I and transforming growth factor-β in DOX treatment group increased significantly compared with control group （P<0.05）. （4） Compared with the control group， the levels of mROS， cellular NLRP3 and cleaved caspase-1 in cardiac fibroblasts increased significantly after DOX treatment. CONCLUSION Doxorubicin promotes cardiac fibroblasts to secrete IL-1β and collagen type I by promoting mROS production and activating NLRP3 inflammasome.     

Comparison of two methods for culturing neonatal rat cardiac fibroblasts

AIM:To establish and compare the methods for culturing neonatal rat cardiac fibroblasts culture. METHODS:Neonatal rat hearts were isolated by collagenase+trypsin or trypsin digestion. The cardiomyocytes and cardiac fibroblasts were isolated by different attachment techniques. The cellular morphology, purity and reactions to the reagent were tested. RESULTS:For morphology and purity, no difference between the 2 methods was observed, though more myocytes were harvested by the method of collagenase+trypsin digestion. For the cellular responses to reagent, the cardiac fibroblasts harvested with trypsin digestion had more potent proliferative ability than those with collagenase+trypsin digestion. CONCLUSION:There is no difference of cellular morphology and purity in the cardiac fibroblasts isolated by collagenase+trypsin digestion and trypsin digestion, but the fibroblasts with trypsin digestion have more potent proliferative ability.     

Schisandrin B protects against solar irradiation-induced oxidative injury in BJ human fibroblasts

The effects of schisandrin B (Sch B) and its analogs on solar irradiation-induced oxidative injury were examined in BJ human fibroblasts. Sch B and schisandrin C (Sch C) increased cellular reduced glutathione (GSH) level and protected against solar irradiation-induced oxidative injury. The photoprotection was paralleled by decreases in the elastases-type protease activity and matrix-metalloproteinases-1 expression in solar-irradiated fibroblasts. The cytochrome P-450-mediated metabolism of Sch B or Sch C caused ROS production. The results suggest that by virtue of its pro-oxidant action and the subsequent glutathione antioxidant response, Sch B or Sch C may offer the prospect of preventing skin photo-aging.     

猪胎儿成纤维细胞制作方法的改进及应用

实验以猪胎儿为研究材料 ,对胎儿成纤维细胞的分离与培养方法进行改进 ,采用室温消化的方法制作猪胎儿成纤维细胞。结果表明 ,室温消化方法的细胞获得量 ,细胞活力和贴壁细胞的形态均优于常规的热消化方法 ,而且操作简单 ,速度快     

Effects of serum from patients with atrial fibrillation on chemotaxis of cardiac fibroblasts

AIM: To investigate the effects of the serum from the patients with atrial fibrillation (AF) on the chemotaxis of rat cardiac fibroblasts.METHODS: Cardiac fibroblasts were isolated from the ventricles of neonatal Sprague-Dawley rats and primarily cultured with digestion and differential adhesion. The cells in 3 to 4 passages were used for Transwell chamber assay to determine the chemotatic effects of the sera.RESULTS: Compared with control group, the cells that migrated through the polycarbonate membrane were obviously increased in AF group. The strongest chemotaxis was induced by the serum from the patients with persistent atrial fibrillation(pers-AF).The number of migrated cells in non-AF atrial arrhythmia(AA) group was higher than that in paroxysmal atrial fibrillation(paro-AF) group, and that in control group was the lowest. The results of multiple Logistic regression analysis showed that the migrated cells were related to AF and left atrial diameter.CONCLUSION: The chemotactic effect of AF serum is obviously higher than that of control serum. The differences of AF sera among groups show that myocardial fibrosis is a chronic, insidious and delayed process. The migration and infiltration of cardiac fibroblasts indirectly reflect the presence, severity and extent of the myocardial damage. The changes of migrated cells precede the changes of left atrial diameter, indicating that the cause of fibrosis is more important, and the positive correlation between AF and left atrial diameter may not be the direct causality.     

新生广西巴马小型猪肾成纤维细胞体外培养体系的建立

利用微量液体组织块贴壁法原代培养分离新生广西巴马小型猪肾成纤维细胞,进行常规传代培养、冷冻和复苏;通过接触抑制和解除抑制研究该类细胞的增殖潜力,免疫荧光进行鉴定;并通过脂质体介导对分离到的新生广西巴马小型猪肾成纤维细胞分别进行EGFP-N1和DsRed-N1基因转染。结果成功分离到新生广西巴马小型猪肾成纤维细胞,体外可以大量传代和冷冻,目前已经传至50代以上,生长良好;冷冻复苏和接触抑制解除后仍可以正常培养和传代;细胞免疫荧光检测,波形蛋白表达阳性,角形蛋白表达阴性;转染48h后荧光显微镜下可以观察到绿色荧光和红色荧光,表明瞬时转染新生广西巴马小型猪肾成纤维细胞可以获得成功,其中转染DsRed-N1基因的新生广西巴马小型猪肾成纤维细胞已经传至60代以上。结果表明,本试验成功建立了新生广西巴马小型猪肾成纤维细胞的体外培养体系,在体外能稳定培养和传代,并可以成功获得转基因的新生广西巴马小型猪肾成纤维细胞。