Inhibitory effect of Ophiopogon japonicus on rat cardiac fibroblasts

AIM：To investigate the inhibitory effect of Ophiopogon japonicus on rat cardiac fibroblast (CFs) and the underlying mechanism. METHODS：Cultured CFs from Sprague-Dawley (SD) rats were randomized into 4 groups: control group (normal rat cardiac fibroblasts), Ophiopogon japonicus of 10 μg/L group, Ophiopogon japonicus of 20 μg/L group and Ophiopogon japonicus of 30 μg/L group. Cell vitality, ［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3 in CFs were determined. RESULTS：Compared with control, the cell vitality, ［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3 were significantly decreased in Ophiopogon japonicus of 10 μg/L group. Compared with Ophiopogon japonicus of 10 μg/L group, the cell vitality, ［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3 were significantly decreased in Ophiopogon japonicus of 20 μg/L group. Compared with Ophiopogon japonicus of 20 μg/L group, the cell vitality,［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3 were significantly decreased in Ophiopogon japonicus of 30 μg/L group. CONCLUSION：Ophiopogon japonicus may inhibit CFs. These actions are related to the changes of ［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3.     

带有绿色荧光蛋白外源DNA转染绵羊成纤维细胞的研究

为了优化建立转染绵羊成纤维细胞系的外源基因转染体系,本研究利用带有绿色荧光蛋白（GFP）的外源基因,采用阳离子脂质体法,探讨了DNA用量、脂质体用量和转染时间等因素对转染绵羊成纤维细胞的影响。结果表明,在500 μL转染培养基中成纤维细胞达到85%汇合时,添加由50 μL DMEM中含有0.2 μg DNA和50 μL DMEM中含有2.0 μL LipofectamineTM 2000的两种溶液混合构成的转染液并转染12 h,可获得最高的转染效率。当转染12 h后,用基础培养液培养至第24小时,细胞核周区出现强绿色荧光,而当转染后48～64 h观察时,细胞核周区的荧光强度明显弱,而远离核周区的胞质中的荧光强度增加。     

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.     

创伤愈合中P物质的免疫组化观察

目的：动态分析创伤愈合中中性粒细胞、成纤维细胞(fibroblast，FB)和P物质(Substance P，SP)阳性细胞的时空分布以及创伤局部SP物质的分布与变化，阐述上述变化与创伤愈合的关系。方法：取家兔皮肤创伤组织进行H．E．、免疫组化染色，分别观察局部组织与细胞的变化。结果：中性粒细胞于创后第1天最高，然后回落；FB在第3天即开始大量增殖，在上皮化接近完成，真皮重建活跃的第7天到达峰值；SP阳性物质在刨伤局部呈现强阳性反应，SP阳性细胞数在创后第1天，第3天，第5天时持续显著上升，维持在较高水平直到第15天后才回落。结论：中性粒细胞在创伤愈合早期起作用，不直接参与创伤修复，FB为刨伤修复的主要细胞，SP参与了创伤愈合的全过程。     

Effects of short-chain acyl-CoA dehydrogenase on collagen expression and proliferation of rat cardiac fibroblasts

AIM: To investigate the effect of short-chain acyl-CoA dehydrogenase (SCAD) on collagen expression and proliferation of rat cardiac fibroblasts and to explore the relationship between SCAD and cardiac fibrosis. METHODS: The model of proliferation and collagen expression of rat cardiac fibroblasts induced by angiotensin II was established. After treatment with siRNA-1186, the expression of SCAD at mRNA and protein levels, fatty acids beta oxidation rate, ATP, the enzyme activity of SCAD and free fatty acids in the rat cardiac fibroblasts were determined. RESULTS: The mRNA and protein expression of SCAD was decreased in the rat cardiac fibroblasts induced by angiotensin II compared with the control cells, and the expression of collagen I and collagen III was significantly upregulated. Compared with negative control group, SCAD expression and activity, fatty acid beta-oxidation rate and ATP significantly decreased in siRNA-1186 group, but the content of free fatty acids were obviously increased in the rat cardiac fibroblasts, and the expression of collagen I and collagen III was significantly up-regulated. CONCLUSION: The expression and synthesis disorder of collagen may be triggered by down-regulation of SCAD. SCAD may be a promising therapeutic target for myocardial fibrosis.     

Effects of interleukin-10 on the calcineurin activity in cultured cardiac fibroblasts induced by arginine vasopressin

AIM: The purpose of the present study was to investigate the effect of interleukin-10 (IL-10) on the proliferation and calcineurin (CaN) activity in cultured cardiac fibroblasts (CFs) induced by arginine vasopressin (AVP).METHODS: The CFs of left ventricle in neonatal Sprague-Dawley rats were isolated and cultured by trypsin digestion and selective plating technique. Then the proliferation rates of cells were determined by using the MTT ［3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide］ assay (A490 value). Cell cycle distribution was determined with flowcytometry technique. The CaN activity was measured by ultra-violet spectrophotography.RESULTS: (1) MTT colorimetry showed that 10-7 mol/L AVP significantly increased A490 value of CFs in comparison with control group (P<0.01). IL-10 attenuated the A490 value of AVP group in a concentration dependent manner. The A490 value of the 10-8, 10-7, 10-6, 10-5 g/L IL-10+10-7 mol/L AVP groups was 0.201±0.007, 0.187±0.006, 0.173±0.010 and 0.157±0.029 respectively, all data significantly lower than those in the presence of AVP alone (P<0.05 or P<0.01). (2) The percentage of the cells in S stage and proliferation index were markedly increased in 10-7mol/L AVP group compared with the control (P<0.01, respectively). In the 10-6 g/L IL-10+10-7 mol/L AVP group, the percentage cells in S stage and proliferation index were significantly lower than those in AVP group (P<0.01, respectively). IL-10 itself had no effect on fibroblast proliferation, but reduced AVP-induced fibroblast proliferation. (3) There was a significantly increase in CaN activity in AVP group compared with control (P<0.01). In the 10-8, 10-7, 10-6 and 10-5 g/L IL-10＋10-7 mol/L AVP groups, the CaN activity was 3.22±0.04, 3.06±0.06, 2.53±0.04 and 2.22±0.04, respectively. IL-10 dose-dependently down-regulated the CaN activity induced by AVP (P<0.01, respectively). However, the CaN activity was still higher in IL-10+AVP group than that in control group (P<0.05 or P<0.01).CONCLUSION: Our data indicate that IL-10 regulates the CaN activity of CFs in the cell proliferation induced by AVP, suggesting that IL-10 plays a role in the regression of cardiac remodeling.     

猪SMYD3基因的克隆、序列分析及其对猪成纤维细胞增殖的影响

试验旨在克隆猪SMYD3(SET and MYND domain-containing protein 3)基因并对其进行序列分析,研究其对猪成纤维细胞增殖的影响。首先克隆猪SMYD3基因,根据其他物种SMYD3基因siRNA和shRNA序列,经同源性比对分析,获得两条猪SMYD3基因shRNA序列,分别构建pSicoR-GFP-SMYD3 shRNA1/shRNA2表达载体,转染HEK293T细胞,利用实时荧光定量PCR分析干扰效率,筛选出抑制效率较好的shRNA,并构建pLVX-IRES-ZsGreen1-SMYD3及pSicoR-GFP-SMYD3 shRNA真核表达载体,同时分析SMYD3基因对猪成纤维细胞的增殖作用,检测细胞Nanog、DNMT1及DNMT3a基因表达情况。结果显示,试验克隆得到1 404 bp的猪SMYD3基因编码区序列,生物信息学分析发现,德保猪SMYD3基因与野猪、山羊和野耗牛相应氨基酸序列的同源性分别为99.5%、93.8%和92.9%。shRNA1/shRNA2均能显著抑制SMYD3基因表达(P<0.05),抑制效果分别是34%和54%,选择pSicoR-GFP-SMYD3 shRNA2进行后续研究。通过脂质体转染法将构建的pLVX-IRES-ZsGreen1-SMYD3及pSicoR-GFP-SMYD3 shRNA真核表达载体导入HEK293T细胞,均可观察到清晰的绿色荧光。慢病毒感染细胞及实时荧光定量PCR结果显示,与空白对照组及阴性对照组相比,过表达SMYD3基因促进猪成纤维细胞增殖,Nanog和DNMT1基因表达显著升高(P<0.05);抑制SMYD3基因表达,细胞增殖受到抑制,Nanog、DNMT1、DNMT3a基因表达显著降低((P<0.05),说明SMYD3基因的表达与猪成纤维细胞的增殖显著相关。     

4℃保存的小鼠胎体皮肤成纤维细胞继代培养及冻存

为了探讨利用死亡动物皮肤组织获取成纤维细胞的方法,研究将小鼠胎体或皮肤分别在4℃的mPBS液中保存24~120 h,然后通过原代培养、冷冻复苏和继代培养,观察了皮肤成纤维细胞的生长增殖能力。结果表明:与新鲜皮肤(对照组)原代培养成纤维细胞达到85%以上汇合需要8 d相比,4℃保存24~120 h后的皮肤(试验组)需要的时间延长(9~21 d),但仍能贴壁生长并达到85%以上汇合,且皮肤保存法优于胎体保存法;各试验组经3~4 d继二代培养即可达到85%汇合(对照组为3 d);冷冻复苏的各试验组原代培养细胞,经继二代培养即可在3~4 d内达到85%以上汇合。说明动物死亡后,若及时采集其皮肤并在4℃的mPBS液中保存一段时间,经原代培养和继代培养,或经原代培养、冷冻复苏和继代培养,可获得具有正常继代能力的成纤维细胞。     

鸭胚成纤维细胞核糖体S12蛋白编码基因的克隆与序列分析

本研究通过鸭胚成纤维细胞（Duck Embryo Fibroblasts，DEF）cDNA文库的构建及酵母双杂交系统筛选，获得了能与鹅细小病毒（Goose parvovirus，GPV）结构蛋白VP1互作的核糖体蛋白S12（RPS12）。RPS12是核糖体小亚基40S的组成部分，属于核糖体蛋白S17Ae家族，主要存在于真核生物中。本研究根据已报道的禽类及部分哺乳动物的RPS12基因相关信息设计引物，运用RT-PCR技术，从鸭胚成纤维细胞总RNA中成功扩增了RPS12基因的表达序列，对其进行克隆、测序及分析。结果表明，鸭胚成纤维细胞核糖体蛋白S12基因的开放阅读框（ORF）长399 bp，编码132个氨基酸，相对分子质量14.474 kDa，pI为7.121。进一步分析发现，鸭胚成纤维细胞RPS12基因与已报道的禽类及部分哺乳动物的基因序列及其编码的氨基酸序列都有很高的同源性。     

Leptospiral lipopolysaccharide stimulates the expression of toll‐like receptor 2 and cytokines in pig fibroblasts

Pigs throughout the world are afflicted with leptospirosis, causing serious economic losses and potential hazards to human health. Although it has been known that leptospiral lipopolysaccharide (L‐LPS) is involved in an immunological reaction between an antigen and a host cell, little is known about how the immune system of pigs can respond to L‐LPS. Here, we stimulated pig fibroblasts by L‐LPS and then quantitatively measured gene and protein expression levels of two toll‐like receptors (TLRs), TLR2 and TLR4, by real‐time PCR and Western blotting. As a result, expression of TLR2 was found to be significantly up‐regulated within 24 h after L‐LPS stimulation whereas induction of TLR4 expression was relatively weak. We also revealed that of myeloid differentiation primary response gene 88 (MyD88), interleukin 6 (IL‐6) and IL‐8 gene expressions were markedly up‐regulated by L‐LPS stimulation. These results may suggest that the pig cell can activate TLR2 rather than TLR4 by L‐LPS stimulation, thereby inducing expression of cytokines.