仔猪胰腺细胞体外培养体系的建立

为了研究体外培养仔猪胰腺细胞的适宜条件,试验选取1日龄的仔猪胰腺组织置于冰上清洗、修剪,对消化酶、离心方式、培养皿铺被方式、培养液及添加不同促生长因子等多种因素进行筛选和优化.结果表明:0.25%胰酶消化所获得的细胞强于0.1%胶原酶消化获得的细胞,细胞清亮,易于生长;采用500r/min离心5 min、多次离心所获得...     

仔猪心肌细胞分离及培养方法研究

为探讨仔猪心肌细胞的分离及培养方法,采用胶原酶Ⅳ型和胰酶同时消化心肌组织,用10%胎牛血清终止消化,并用100目细胞筛过滤后,1200 r/min离心沉淀细胞,再加入培养液重悬细胞。采用差速贴壁法纯化心肌细胞后置CO2培养箱孵育。结果显示,未贴壁时心肌细胞呈圆形,培养12~24 h心肌细胞开始贴壁生长,细胞伸出伪足呈梭形、多角形,3~4 d后形成细胞簇,5~7 d后细胞汇合成片。因此,同时使用胶原酶Ⅳ型和胰酶消化仔猪心肌可简便、快速地获得高活力的心肌细胞。     

牛乳腺上皮细胞体外分离和培养

从健康奶牛乳房无菌采取乳腺组织,通过添加两种不同的培养液来分离、培养、纯化牛乳腺上皮细胞,研究乳腺上皮细胞的体外培养效果。结果表明,使用组织块接种可以得到大量细胞用于体外培养。在以DMEM/F12为基础的普通培养液中进行乳腺上皮细胞体外培养,原代细胞生长较慢,细胞形态不典型。而添加表皮生长因子、胰岛素、氢化可的松所组成的完全培养液中,奶牛乳腺上皮细胞生长良好。并通过细胞形态学观察,细胞染色体核型分析,荧光免疫细胞染色方法鉴定了培养的细胞表达上皮细胞特异的角蛋白K14,K15。结果表明,分离培养的细胞是牛乳腺上皮细胞。     

番鸭胚成纤维细胞的原代分离与培养研究

为建立番鸭胚成纤维细胞(MDEF)的体外培养、传代和冻存技术,选用11胚龄番鸭胚,2.5g/L胰蛋白酶消化,原代MDEF于含100 mL/L血清的DMED培养液中培养;研究热、冷两种消化方法对MDEF消化密度及活率的影响,探讨不同血清浓度对MDEF生长、冻存活性的影响,以及离心对MDEF传代和复苏时细胞活率的影响。结果显示,胰酶消化法获得了MDEF,细胞贴壁紧密,生长良好;冷消化得到的原代MDEF密度和活率均高于热消化;血清浓度在20mL/L~100mL/L范围内,浓度越高MDEF生长速度越快;血清浓度显著影响MDEF复苏的活率;MDEF冻存液中DMSO∶血清∶培养液比例为1∶2∶7冻存效果良好;在MDEF传代和复苏过程中去除离心操作,不仅可简化操作,细胞活率也有一定提高。本研究建立了MDEF的原代分离和培养方法,为适用于番鸭相关研究的体外细胞培养体系奠定基础。     

奶牛卵丘细胞无血清体外培养液的优化

采集奶牛卵巢表面卵泡中的卵丘-卵母细胞复合物,通过胰蛋白酶消化获得卵丘细胞,分别利用对照组DMEM/F12培养液和4个试验组McCoy's5a培养液对卵丘细胞进行原代及传代培养.试验1组用McCoy's5a基础培养液,试验2组在McCoy's5a基础培养液中添加29 ng·mL-1的睾酮,试验3组同时添加29 ng·mL-1的睾酮和15μmol·L-1的绿茶多酚,试验4组在第3组基础上添加转铁蛋白(5 μg·mL-1)、硒(5 ng·mL-1)和胰岛素(10μg·mL-1),构建成优化培养液(Optimized McCoy's5a).结果表明,试验4组优化培养液所培养的卵丘细胞的形态和增殖规律符合正常细胞的一般规律,染色体核型正常,其细胞内SOD、GSH含量及冷冻解冻后的存活率均显著高于对照组,优化培养液可以代替含血清的DMEM/F12培养液用于奶牛卵丘细胞的体外培养.     

猪胎盘绒毛滋养层细胞体外原代培养

本研究旨在建立一种简便有效的猪胎盘绒毛滋养层细胞体外分离纯化方法。采用胰酶DNase I酶联合消化法消化母猪自然分娩后胎盘绒毛组织,以35%、45%2个Percoll密度梯度法进行猪胎盘绒毛滋养层细胞的分离纯化,并通过观察细胞形态、测定生长曲线、免疫荧光染色、透射电镜等鉴定猪胎盘绒毛滋养层细胞。结果表明,分离培养的猪胎盘绒毛滋养层细胞呈上皮样平铺成片生长,形态为多角形或类圆形,培养48h后部分细胞开始融合成多核合胞体滋养层细胞。免疫荧光染色结果显示,细胞角蛋白7染色阳性占91%以上,表明分离获得的猪胎盘绒毛滋养层细胞纯度较高。透射电镜观察显示,所获细胞具有典型滋养层细胞结构特征。综上表明,采用胰酶DNase I酶联合消化法和35%、45%2个Percoll密度梯度法进行分离纯化,可简便有效地获得较高纯度的猪胎盘绒毛滋养层细胞。     

不同方法分离奶牛乳腺上皮细胞体外培养的研究

从荷斯坦奶牛乳房无菌采取乳腺组织,通过不同的方法分离、培养、纯化牛乳腺上皮细胞,研究乳腺上皮细胞的体外培养效果.结果表明:组织块接种、0.25%的胰酶和100 u/mL透明质酸酶的混合液37℃消化组织块3 h.可以得到大量细胞用于原代培养.在以DMEM/F12为基础培养液,在培养液中添加10%的胎牛血清、100μg/mL的双抗、表皮生长因子、胰岛素、转铁蛋白、硒酸钠等,组成的完全培养液中奶牛乳腺上皮细胞生长良好.上皮细胞显微结构显示:奶牛乳腺上皮细胞在体外培养过程中舍有不同的细胞类型,大多数上皮细胞呈多角形,细胞之间连接成片,细胞界限明显,部分细胞界限不明显.     

仔猪胰腺干／祖细胞的生物学特性

分离1日龄长白仔猪的胰腺细胞后,通过显微镜和电镜观察细胞生长形态和超微结构,运用细胞计数法制作细胞生长曲线测定其群体倍增时间,采用免疫组化法测定细胞生长不同时期的表面标志蛋白的表达,用肝细胞因子和尼克酰胺联合诱导其向功能性G细胞分化,并测定了分化细胞对葡萄糖的反应能力。结果显示获得的仔猪胰腺祖／干细胞具有多种形态,以神经样细胞克隆生长占优势,电镜超微结构证实其具有核质比高、细胞器不分化的干细胞态特征。试验测得第1代和第3代群体的倍增时间分别为96．0、307．7h,仔猪胰腺祖／干细胞表达胚胎干细胞标志Oct4、SSEA-1、SSEA-4,也表达胰腺干细胞的标志PDX-1、CK7等。仔猪胰腺干／祖细胞的向β细胞诱导分化细胞对22mmol／L葡萄糖的刺激产生强烈的胰岛素分泌反应,胰岛素分泌量可达321．75mIU／L。结果证实仔猪胰腺千／祖细胞具有干细胞和胰腺祖的表面标志和超微结构,同时具有分化成分泌胰岛素β细胞的能力。     

小鼠精原干细胞的分离纯化及初步鉴定

用两步酶消化法对昆明白小鼠睾丸精原细胞及精原干细胞进行了体外分离、纯化和初步鉴定。经两步酶消化所获细胞悬液内活细胞、死细胞及细胞团的百分比分别为97.03%、2.97%和2.95%;平均每个睾丸可获得4.53×105个细胞。Percoll不连续密度梯度离心初步纯化后得到4个细胞带,精原细胞主要分布在40%~50%Percoll梯度间,得到的精原细胞密度为3.20×106个/mL。因此,采用两步酶消化,结合Percoll不连续密度梯度离心法分离的昆明白小鼠精原细胞能满足体外培养的需要,还可以作为精原干细胞移植的研究材料。     

绿色荧光蛋白转基因小鼠胚胎成纤维细胞的分离和培养

研究了不同胎龄、消化液、培养液对转绿色荧光蛋白(GFP)基因小鼠胚胎成纤维细胞(MEF)分离和培养的影响,并对其生长形态进行观察。结果表明,在实验室条件下,12.5～14.5d胎龄的GFP-MEF分离效果最好,0.25%胰蛋白酶+0.02%EDTA所用时间短,消化后培养细胞贴壁率高;GFP-MEF形态以小梭形为主,呈漩涡状、鱼群状生长;最佳培养液为DMEM添加10%胎牛血清。研究获得了实验室分离制备GFP-MEF的方法,为制作饲养层和进一步构建转基因动物奠定基础。     

饲喂氧化鱼油对新生仔猪肠道黏膜免疫应答的影响及大豆异黄酮的干预作用

本试验旨在通过研究饲喂氧化鱼油对新生仔猪生长性能、肠道黏膜氧化相关指标、免疫因子、氧化和免疫共同相关因子的影响以及添加大豆异黄酮(SI)对其的干预作用,探讨氧化应激对新生仔猪生长、肠道免疫应答的影响机制.试验选用4日龄新生仔猪96头,分成4组,每组6个重复,每个重复4头仔猪,各组饲粮中分别添加5%新鲜鱼油(Ⅰ组)、5%...     

Impaired pancreatic endocrine and exocrine responses in growth-retarded piglets

The alteration of pancreatic endocrine and exocrine secretory responses induced by secretagogues and neural input was investigated in post-weaning growth-retarded (GR) piglets. Blood and pancreatic juice were collected from these animals (6-8-weeks old). Plasma insulin and pancreatic digestive enzymes induced by nutrients, drugs and vagal stimulation were measured biochemically. The pancreas was inspected by immunohistochemical analysis. In GR piglets, the plasma glucose and insulin concentrations at the resting state were very low, and the secretory response was also markedly reduced, with maximum inhibition of 90% by glucose administration and 83% by arginine administration. The insulin secretion was not increased by 2-deoxy-D-glucose administration in GR piglets. The pancreatic juice secretions induced by vagal stimulation and secretagogues in GR piglets were not different from those induced in the control piglets. However, amylase activity in the pancreatic juice and in the pancreas was significantly decreased in GR piglets, although trypsin and chymotrypsin activities were not different. In the immunohistochemical analysis, the numbers of islets and the staining degree for insulin antibody also declined in the pancreases of GR piglets. These results indicated the reduction of insulin and amylase secretions from the pancreas in GR piglets, suggesting that a dysfunction of pancreatic endocrine and exocrine secretion during growth after weaning may be an important factor in the induction of growth retardation in piglets.     

添加胰岛素对胎儿宫内发育迟缓新生仔猪胰腺生长发育的影响

(目的和方法)为研究添加胰岛素对IUGR新生仔猪胰腺重量、酶活性的影响,试验选择初生IUGR仔猪15头,分为新生对照组(N)、牛乳组(M)和试验组即牛乳添加胰岛素(60 IU/L)组(I),人工饲喂3 d后宰杀取样。(结果)结果显示,与N组相比,饲喂3 d后,M组和I组仔猪胰腺重量、胰腺重/体重、蛋白质含量、DNA含量、RNA含量、胰淀粉酶活性、胰脂肪酶总活性、胰高血糖素总含量均不同程度提高,而胰总蛋白水解酶活性、胰蛋白酶活性、胰糜蛋白酶总活性、胰腺胰岛素总含量均不同程度降低,变化较明显。与M组相比,I组胰腺蛋白质含量、蛋白质/DNA比值分别提高了61.43%、60.54%(P0.01);I组胰总蛋白水解酶活性、胰淀粉酶活性、胰脂肪酶总活性、胰腺胰岛素总含量、胰高血糖素含量分别提高了10.59%、10.40%、9.37%、14.30%、12.30%(P0.05);胰腺重、胰腺重/体重、胰腺DNA含量、RNA含量、RNA/DNA比值也不同程度提高(P0.05);而I组与M组间胰蛋白酶活性、胰糜蛋白酶活性差异不显著。(结论)表明IUGR仔猪生后胰腺生长发育较迅速,添加胰岛素能在一定程度上刺激IUGR仔猪胰腺细胞增殖和增大,能显著促进IUGR仔猪胰腺蛋白质合成和沉积,有刺激胰腺内外分泌部发育、提高胰腺酶活性提高、刺激组织激素分泌的趋势,对IUGR仔猪出生后胰腺的生长具有一定的补偿作用。     

Emigration of polymorphonuclear leucocytes into the intestinal lumen of the neonatal piglet in response to challenge with K88-positive Escherichia coli

The emigration of neutrophils from the blood of neonatal piglets into the intestinal lumen in response to a K88-positive strain of Escherichia coli was investigated. The pig herd used was of known genetic susceptibility to K88-positive E coli and had recently experienced an outbreak of neonatal diarrhoea. Neutrophil emigration depended on certain factors. Neutrophils emigrated into ligated loops in susceptible piglets sucking immune colostrum from susceptible dams but not into loops in colostrum deprived resistant piglets or piglets sucking non-immune colostrum from resistant dams. In susceptible, colostrum deprived piglets neutrophils in intestinal contents were only associated with severe lesions. Large numbers of neutrophils which appeared at several foci on the villi were observed in three of six resistant piglets that sucked immune colostrum from susceptible dams. It was concluded that neutrophil emigration into the intestinal lumen of piglets could occur in response to K88-positive E coli and resulted not from the presence or absence of the intestinal K88-receptor but from the ingestion of immune colostrum.     

Effects of dietary changes on the antibacterial activity of pancreatic juice in weaned piglets

At weaning, low feed intake level is a major factor responsible for gut disorders. However, less is known on adaptation of the antibacterial activity of pancreatic juice in response to variations in feed intake level. Moreover bovine colostrum has been demonstrated to decrease coliform counts in duodenal digesta and to improve health status of weaned piglets. Aims of the present study were to evaluate effects of feed intake level and of a bovine colostrum-supplemented diet on the pancreatic secretion of antibacterial activity in piglets at weaning. At 33 days of age, twenty-eight piglets fitted with two permanent catheters (for pancreatic juice collection and reintroduction) were weaned and allocated to one of the following three dietary treatments for 5 days: low feed allocation with a starter diet (L), gradually increased feed allocation with a starter diet (Ctrl), or gradually increased feed allocation with a starter diet supplemented with bovine colostrum (Col). In Ctrl piglets, the post-weaning secretion of antibacterial activity did not significantly differed from pre-weaning values. The pancreatic secretion of antibacterial activity in L piglets was higher than that in Ctrl piglets at day 5 (+ 300%; P < 0.05). Moreover in Col piglets the secretion of the antibacterial activity tended to increase by 55% (P = 0.08) compared to Ctrl piglets. Variations in the pancreatic secretion of antibacterial activity could constitute an adaptive phenomenon to maintain bacterial homeostasis in the upper small intestine during the early post-weaning period.     

Porcine parvovirus- and porcine circovirus 2-associated reproductive failure and neonatal mortality in crossbred Indian pigs

The objective of this study was to detect the presence of porcine parvovirus (PPV) and porcine circovirus 2 (PCV2) in a farm showing reproductive failure and increased mortality in neonatal piglets by histopathological examination, polymerase chain reaction, and demonstration of viral antigen and nucleic acid. Out of 594 piglets farrowed by 70 first-parity gilts, nine (1.51%) mummified fetuses, 13 (2.19%) stillborn, and 572 (96.3%) live-born piglets were recorded. The average litter size at birth was 8.48 piglets per litter. One hundred ninety-four (33.91%) piglets died within 7 days of age. PPV was detected in five litters (7.14%) and two of them revealed coinfection with PCV2. The pathological lesions in the coinfected litters were more severe, indicating a synergistic action between the two viruses. Results of this study suggest for the first time occurrence of PPV and coinfection with PCV2 in crossbred Indian pigs affected with reproductive problem and neonatal mortality.     

Effect of metabolic disorders accompanying gastroenteritis on the pancreatic exocrine function in piglets. B. Disturbances of the pancreatic exocrine function in piglets with gastroenteritis

The study was performed on 90 piglets of both sexes, aged from 21 to 42 days. The control group consisted of 30 healthy piglets, the experimental one included 60 piglets with symptoms of gastroenteritis. Clinical and biochemical tests were performed on all the animals at the age of 21 and 35 days, and 7 days after weaning. Biochemical examinations allowed to determine the total serum activity of amylase and lipase, and the amylase salivary and pancreatic fractions. The total activity of amylase and lipase was also determined in pancreatic homogenates. A biochemical feces analysis (pH and fat content) and a D-xylose test were made in 20 piglets from each of the groups. The results obtained revealed failure of the pancreatic exocrine function in the piglets with gastroenteritis. A decrease in the total amylase activity, resulting from hypoactivity of its pancreatic fraction, was observed in the experimental animals. It was also found that indirect function tests cannot be applied to evaluate the pancreatic exocrine function in piglets with diarrhea.     

Morphological changes in the lungs of meconium-stained piglets.

Meconium staining of the skin is a common event associated with fetal hypoxia, stillbirths, weak-born piglets, and neonatal mortality. Aspiration of meconium leads to meconium aspiration syndrome (MAS). This study was undertaken to assess the relationship between the degree of meconium staining of the skin at birth, meconium aspiration, and pulmonary changes in porcine neonates. A total of 353 farrowing sows and 3,693 born piglets were monitored during parturition and for 15 days after delivery. Umbilical cords were classified as normal or ruptured. Meconium staining in the skin was graded as nonstained, mildly, moderately, and severely stained. Mortality from birth to 15 days of age was 8.4%. The lungs from 60 meconium-stained piglets and 60 lungs from nonstained piglets were collected and microscopically examined for meconium aspiration and inflammation. Rupture of the umbilical cord was significantly higher (P < 0.01) in meconium-stained piglets. Microscopically, 32% and 40% of the lungs had evidence of meconium for the stained and nonstained groups, respectively. The microscopic grade of meconium aspiration and inflammatory cells was not different between nonstained and meconium-stained piglets. Aspiration of meconium induced a granulomatous response in the lungs. It was concluded that the grade of meconium staining is a good indicator of fetal hypoxia, but not a good predictor for meconium aspiration and MAS in piglets.     

宫内生长迟缓仔猪T细胞的发育研究

研究旨在探讨从出生到断奶不同日龄宫内生长迟缓(IUGR)猪T细胞的发育变化情况。选用体重相近和配种期相同的达兰初产妊娠母猪30头,分娩时从每窝仔猪中挑选1头正常体重猪和1头IUGR猪,分别在1、7、14、21、28日龄时屠宰取胸腺,分离T细胞,通过流式细胞仪测定CD3、CD4、CD8T细胞的数量,每个日龄6个重复。结果表明:出生后第1天IUGR仔猪与正常仔猪的CD4+CD8+细胞(双阳性T细胞)占总T细胞的百分比差异显著(P<0.05);出生后第7天IUGR仔猪与正常仔猪的CD4细胞占总T细胞的百分比差异极显著(P<0.01);后期各细胞亚群之间没有差异。结果提示,子宫内各种因素的综合影响能造成出生后早期IUGR仔猪T细胞亚群发育不完善,这就从T细胞亚群的发育方面解释了IUGR仔猪免疫力低的原因。