寿光黑鸡成纤维细胞的体外培养与冷冻保存(英文)

[Objective] The aim of this study was to establish the in vitro culture system of chicken fibroblasts.[Method] Tissue explant method and enzymatic digestion method were used to separate and culture chicken skin fibroblasts respectively.The rate of cell growth,cryopreservation and recovery were compared.[Result] The primary chicken fibroblasts prepared by enzymatic digestion grew faster and converged together to form monolayer on 5 d post preparation;the passage cells prepared by these 2 methods grew at similar speed and formed monolayer within 2-3 d;homogeneous fibroblasts could be obtained by trypsin digestion and repeated attachment for 3-4 passages;there were 75%-80% of cells survived after cryopreservation and recovery;the growth curves of embryonic fibroblasts and skin fibroblasts were all normal and the two kind of cells still retained the normal number of chromosomes even at the twelfth passage.[Conclusion] The feeder layer cells needed for establishing ES cell lines could be obtained by culturing chicken fibroblasts through both tissue explant method and enzymatic digestion method.This study provided a basis for the successful establishment of ES cell lines.     

Culture and Identification of Myoblasts Isolated from Duck Embryos

Using embryonic myoblasts to research the formation and de-velopmental mechanisms of skeletal muscle is becoming a research hotspot. This study aimed to establish a method of isolation, culture and identification of my-oblasts in duck embryos. [Method] Pectoral and leg muscle samples were isolated from the embryos of Gaoyou duck at 13 d of hatching, then disassociated with col-lagenase and trypsin and purified via differential adhesion. The isolated cells were cultured in vitro and detected for the expression of Pax7 protein using immunofluo-rescence technique. [Result] Myoblasts were obtained successful y both from pectoral and leg muscles in duck embryos and these cells proliferated strongly and differen-tiated wel . Immunofluorescence staining showed that more than 95% cells could express Pax7 protein. [Conclusion] In summary, we report the successful establish-ment of a complete system for the isolation, purification, identification and culture of myoblasts from duck embryos.     

Effects of Yimu Shenghuasan Preparation on the Cytochrome P450 in Endometrial Cells and Immune Function of Dairy Cows

In order to investigate the mode of action ofYimu Shenghuasan preparation in endometrial cells of dairy cows, the primary cultured endometrial cells in cows were isolated and the inflammatory models were made by lipopolysaccharide （LPS） induction. The inflammatory cells were treated with gradient concentration of herbal medicine preparation, Yimu Shenghuasan for 48 and 72 h. The expression of cytochrome P450 （CYP450） was detected by Western blot. The amounts of IgG and lgA in sera were also detected in the endometritis of dairy cows. The expression level of CYP450 in the endometrial cells of dairy cow was increased gradually, and the amounts of IgG, IgA were increased significantly as compared with those in the control group. The expression level of CYP450 in the inflammatory cells was increased significantly in the treatment of 2 000 μg mL^-1 of Yimu Shenghuasan after 48 h of treatment.     

Study on in vitro Cultural Behaviors of Spermatogonium Stem Cells of New Born Calves

Three methods were adopted in culture spermatogoniums of newly born calf in vitro,such as enzymatic digestion and percoll density gradient centrifugation（MethodⅠ）,tubular fragments culture（MethodⅡ）and tissue culture（MethodⅢ）,and cultural behaviors of cells were observed.The results showed that typical spermatogonium colonies appeard at 144 h of culture by enzymatic digestion-percoll density gradient centrifugation method and tubular fragments culture method,2.5%FBS kept the characteristics of spermatogonium stem cell better than others,produced more mass clones,and FBS of more than 2.5%concentration benefited spermatogonium differentiation and the number of colonies was significantly affected by FBS concentration.After 1 week of culture in method Ⅲ,the diameter of lumens and quantity of sertoli’s cells in tubal wall increased obviously,lumen of seminiferous tubules appeared.Sertoli’s cells kept constant and the number of spermatogoniums decreased obviously after 2 weeks of culture.     

小球藻的分离及其DNA提取方法的研究(英文)

[Objective] The aim of this study is to isolate Chlorella vulgaris(chlorella)and extract its genomic DNA.[Method] Both the dilution method and drip method were employed to isolate chlorella from lake water samples;the conditions for culturing chlorella were optimized and its genomic DNA was extracted by improved CTAB method and SDS method.[Result] The proper conditions for chlorella culture were as following:temperature 20-25 ℃,illumination 4.39-5.86 W/m2 and rotational speed 100-150r/min;improved CTAB method was suitable for extracting genomic DNA from chlorella.[Conclusion] The study is helpful to study the chlorella at molecular level and promote the exploitation and utilization of chlorella resources.     

一种新的非酶法分离荠幼胚的方法(英文)

[Objective] The aim of this study was to manually isolate the microstructures such as ovule and embryo of Capsella bursa-pastoris (L.) Medic. by the non-enzymatic method. [Method] With the gum block and dissecting microscope, its floral characteristics, ovule and embryo were manually isolated and observed by the non-enzymatic method. [Result] C. bursa-pastoris had four calyces, four petals, tetradynamous stamen and silicle, while its embryogenesis contained globular embryo, heart-shaped embryo and torpedo-shaped embryo. [Conclusion] The microstructures such as ovule and embryo of plants become easier to be isolated manually with the viscosity of gum block, which has small damage on embryo or disadvantages of keeping original shapes, and also can used in multi fields studies. This test can also be accomplished in labs with poor experiment facilities.     

小球藻的分离及其DNA提取方法的研究(英文)

[Objective] The aim of this study is to isolate Chlorella vulgaris(chlorella)and extract its genomic DNA.[Method] Both the dilution method and drip method were employed to isolate chlorella from lake water samples;the conditions for culturing chlorella were optimized and its genomic DNA was extracted by improved CTAB method and SDS method.[Result] The proper conditions for chlorella culture were as following:temperature 20-25 ℃,illumination 4.39-5.86 W/m2 and rotational speed 100-150r/min;improved CTAB method was suitable for extracting genomic DNA from chlorella.[Conclusion] The study is helpful to study the chlorella at molecular level and promote the exploitation and utilization of chlorella resources.     

Isolation of Chlorella vulgaris and Its DNA Extraction Methods

[Objective] The aim of this study is to isolate Chlorella vulgaris(chlorella)and extract its genomic DNA.[Method] Both the dilution method and drip method were employed to isolate chlorella from lake water samples;the conditions for culturing chlorella were optimized and its genomic DNA was extracted by improved CTAB method and SDS method.[Result] The proper conditions for chlorella culture were as following:temperature 20-25 ℃,illumination 4.39-5.86 W/m2 and rotational speed 100-150r/min;improved CTAB method was suitable for extracting genomic DNA from chlorella.[Conclusion] The study is helpful to study the chlorella at molecular level and promote the exploitation and utilization of chlorella resources.     

E.tenella吉林株单卵囊的分离及PCR鉴定(英文)

[Objective] In order to get a purified strain to carry out the relative molecular biology research about E.tenella. [Method] The single-oocyst isolation method was improved and the isolated single-oocyst which was put into capsule was fed to chickens. At the same time, the collected oocysts were identified by PCR method. [Result] The oocysts were isolated from feces of 15 chickens among that of 20 chickens and the infection rate was 75%. The PCR results demonstrated that the single-oocyst strain was E.tenella. [Conclusion] The inoculation of single oocyst capsule was simple, besides, this method did not only save time but also declined inoculation difficulty, increased infection rate and provided good materials for biological research of coccidian.     

Isolation, Purification and Cryopreservation of Cells from Neonatal Bovine Testis

The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous epithlium cells were isolated and purified, the cells were cryopreserved after identification, and the effects of different purification and cyopreservation methods on bovine testicular cells were studied. The results showed that there were spermatogonial stem cells and sertoli cells in the neonatal bovine seminiferous tubules, differential adherent selection methods could effectively separate these two cell types. Spermatogonial stem cells were positive after AKP, C-kit, and OCT-4 identification; sertoli cells were positive after oil red O and vimentin identification. Frozen stock solution supplemented with 10% DMSO had the best effect in spermatogonial stem cell cryopreservation, while frozen stock solution supplemented with 10% of ethylene glycol and 0.1 mmol·L-1 trehalose had the best effect in sertoli cells cryopreservation.     

水牛子宫内膜腺上皮细胞和基质细胞的分离·培养和生物学特性研究

1材料与方法1．1材料1．1．1取材。孕早期成年水牛子宫由南宁某屠宰场提供。1．1．2试剂。一次性细胞培养皿地幻自美同BD公司;新生牛血清购A杭州四季青公司;雌二醇购自宁波市激素制品有限公司;离糖型DMEM细胞培养液粉剂、DPBS粉剂和胶原酶I购自美因GIBCO公司;细胞角蛋白单抗和：二抗购白广州深达生物制品公司。另外,青霉素、链霉素、胰蛋门酶、表皮生氐因子（EGF）、     

水牛子宫内膜腺上皮细胞和基质细胞的分离·培养和生物学特性研究

[目的]建立一种高效的水牛子宫内膜腺上皮细胞和基质细胞分离培养方法,为胚胎着床和子宫内膜相关疾病的分子生物学机制研究奠定基础。[方法]采用酶消化、刮取、系列过滤和差速贴壁相结合的方法分离水牛子宫内膜腺上皮细胞和基质细胞,用免疫细胞化学法和台盼兰染色法检测分离细胞的纯度和活率。[结果]成功分离培养出子宫内膜腺上皮细胞和基质细胞;免疫荧光染色和细胞计数表明上皮细胞和基质细胞的纯度均达90％以上;台盼兰染色结果显示,上皮细胞的活率为91％,基质细胞的活率为78％。[结论]采用酶消化、刮取、过滤和差速贴壁相结合的方法可分离出高纯度的永牛子宫内膜腺上皮细胞和基质细胞。     

复方益母生化散对奶牛子宫内膜细胞CYP450表达和免疫功能的影响

【目的】探讨复方益母生化散对奶牛子宫内膜炎的作用机理。【方法】体外实验:分离奶牛子宫内膜细胞,细菌脂多糖(LPS)诱导子宫内膜细胞炎症模型,复方益母生化散处理子宫内膜炎症细胞48h、72h,Western blotting法检测CYP450的表达;体内实验:复方益母生化散栓剂治疗患有子宫内膜炎的奶牛,检测血清中IgG、IgA含量的变化。【结果】复方益母生化散处理后,子宫内膜炎症细胞CYP450的表达随复方益母生化散剂量的增加而呈升高的趋势,血清中IgG、IgA的含量与对照组相比明显升高。【结论】2000μg·mL-1的复方益母生化散作用奶牛子宫内膜炎症细胞48h,CYP450的表达最明显。     

LPS诱导奶牛子宫内膜上皮细胞氧化损伤模型的建立

[目的]采用脂多糖(LPS)作为刺激源,建立奶牛子宫内膜上皮细胞(BEEC)氧化损伤模型.[方法]体外培养BEEC并进行细胞鉴定,用不同浓度的LPS刺激细胞,在不同时间点用CCK-8法测定细胞存活率,以确定BEEC氧化损伤模型条件.倒置显微镜下观察细胞形态学变化,流式细胞术检测细胞凋亡率,DCFH-DA检测细胞内活性氧(ROS)含量,比色法检测细胞中的丙二醛(MDA)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT).[结果]与空白对照组相比,当LPS浓度为80μg/mL,作用时间为12 h时,造成细胞损伤,其细胞凋亡率、ROS产生量和MDA含量明显高于对照组,SOD和CAT活性显著低于对照组.[结论]建立奶牛子宫内膜上皮细胞氧化损伤模型的条件为LPS作用浓度80 μg/mL,作用时间12 h.     

小鼠子宫内膜诱导蜕膜化的研究

[目的]建立体内和体外诱导小鼠子宫内膜蜕膜化的方法。[方法]利用假孕小鼠子宫角注油和激素处理体外分离的4周龄小鼠子宫内膜基质细胞进行诱导蜕膜化,通过半定量PCR和实时定量PCR检测蜕膜化标志分子蜕膜/滋养层泌乳雌激素相关蛋白。[结果]假孕第4天子宫角注油和雌激素与孕酮联合处理小鼠子宫内膜基质细胞都可显著提高蜕膜化标志分子的mRNA水平,可诱导产生蜕膜化。[结论]建立了体内和体外诱导蜕膜化的方法,为验证小鼠蜕膜化芯片奠定了基础。     

脂肪酸结合蛋白4在小鼠子宫蜕膜中的表达

[目的]检测脂肪酸结合蛋白4(FABP4)在小鼠子宫蜕膜细胞中的表达。[方法]获取正常的小鼠子宫蜕膜组织和人工诱导蜕膜化组织,同时利用酶消化分离小鼠子宫内膜基质细胞,进行体外诱导蜕膜化,利用实时定量PCR检测脂肪酸结合蛋白4在子宫蜕膜组织细胞中的表达。[结果]FABP 4在小鼠正常子宫蜕膜组织、人工诱导蜕膜化组织及子宫内膜蜕膜化基质细胞中的表达水平均显著高于对照组。[结论]FABP4可能参与了小鼠子宫内膜蜕膜化过程,为研究蜕膜化机理奠定了基础。     

牦牛子宫内膜腺上皮细胞分离培养方法比较

[目的]建立牦牛子宫内膜腺上皮细胞体外分离培养方法。[方法]分别采用组织块培养法和消化培养法分离、纯化牦牛子宫内膜腺上皮细胞。[结果]组织块培养约8d细胞可汇合成片,经胰蛋白酶分次消化,可得到纯化的子宫内膜上皮细胞;使用2g／L的胶原酶Ⅱ消化2．5h,更换新鲜消化液继续消化2．5h,消化悬液经74斗“滤网过滤,400r／rain离心5min,收集沉淀,重悬后自然沉降,可得到纯净的腺细胞团。免疫细胞化学显示所得细胞角蛋白染色阳性,阳性率达到95％以上。[结论]2种方法均可得到纯净的牦牛子宫内膜上皮细胞。     

兴义维蚋蚋卵(胚胎)原代细胞培养及鉴定

【目的】筛选出最适宜兴义维蚋蚋卵(胚胎)原代细胞的培养条件和最佳培养基组合,并对其原代细胞进行鉴定,为蚋类细胞系的建立打下基础。【方法】采用组织块和单细胞法对兴义维蚋蚋卵(胚胎)进行原代细胞培养,分析培养基、温度、pH、CO2和培养器皿对细胞培养的影响;并测定蚋卵(胚胎)及原代细胞的rDNA-ITS区序列,通过构建基于ITS区序列的蚋类系统发育进化树鉴定其种属来源。【结果】兴义维蚋蚋卵(胚胎)原代细胞最适宜的培养方法为组织块法,培养基为改良型Shields and Sang M3,pH 6.0~6.5,培养温度28℃,恒温生化培养箱和5% CO2培养箱对原代细胞培养无明显差异,培养器皿为经多聚赖氨酸(PLL)处理的一次性塑料培养皿/板。由基于ITS区序列构建的蚋类系统发育进化树可知,蚋卵(胚胎)及其原代培养细胞均来源于兴义维蚋。【结论】成功建立的兴义维蚋蚋卵(胚胎)原代细胞培养方法可在科研生产中进一步推广应用。     

猪FAM213B基因mRNA和启动子的克隆及序列分析

【目的】获得猪FAM213B基因完整mRNA和启动子序列,研究猪FAM213B基因表达,为探讨母猪妊娠的建立和胚胎发育调控机制奠定基础。【方法】通过5'RACE和3'RACE技术,获得基因完整mRNA序列,分析不同物种该基因氨基酸序列相似性;通过PCR克隆启动子区,并通过双荧光素酶报告基因载体系统转染猪子宫内膜细胞,研究其转录活性。【结果】猪FAM213B基因mRNA全长808 bp,其中5'UTR、CDS区和3'UTR长度分别为67、609(含终止密码子)和132 bp(不含poly A序列),在17~106位氨基酸之间存在硫氧还蛋白折叠结构域;与猪FAM213B基因其他2个潜在转录本相比,三者都包含硫氧还蛋白折叠结构域,但蛋白三级结构存在较大差异;猪FAM213B氨基酸序列与山羊、牛和绵羊高度相似,相似性分别为94.03%、93.03%和91.54%。克隆获得2 261 bp(-2 231/+30)的基因启动子序列,将其连接至双荧光素酶报告基因载体,转染猪子宫内膜细胞,发现获得的启动子片段能够启动下游报告基因的转录,在启动子区存在潜在的典型NFκB等转录因子结合位点。【结论】本研究获得猪FAM213B基因转录本长度为808 bp,其蛋白存在硫氧还蛋白折叠功能结构域,其启动子序列(-2 231/+30)在猪子宫内膜细胞中具有较强的转录活性。     

阿魏酸抗LPS诱导的奶牛子宫内膜上皮细胞炎性作用

[目的]为了探明阿魏酸的抗炎机制.[方法]采用组织块培养法获得奶牛子宫内膜上皮细胞.用噻唑蓝(MTT法)检测细胞活力.采用荧光定量PCR方法,分别对对照组、模型组、药物作用高、中、低剂量组在LPS作用3h、6h炎症基因IL-1β、IL-6、IL-8和TNF-α的mRNA变化进行检测.[结果]阿魏酸在一定浓度范围内对细胞增殖无显著影响.药物预处理细胞能够显著降低LPS诱导的IL-1β、IL-6、IL-8和TNF-α的mRNA表达.[结论]阿魏酸能够显著降低LPS刺激细胞后炎症细胞因子的表达,说明阿魏酸是通过抑制炎症因子的表达发挥其抗炎作用.