完全体外化牛细胞核移植的研究

本研究探讨了用体外成熟卵母细胞和体外受精胚胎进行核移植的可能性。本研究结果在我国首次获得牛核移植的成功。从屠宰场回收的卵巢中抽取卵球－卵母细胞复合体，采用以前报道的方法（石德顺等，广西农业大学学报’１９９４：１３：１－５）进行体外成熟（ＩＶＭ）和体外受精（ＩＶＦ），获得ＩＶＭ卵母细胞和ＩＶＦ胚胎，体外成熟２３－２４ｈ后，在含０．１％透明质酸酶的无钙镁ＰＢＳ（ＰＢＳ）内，用细管反复吹打去掉卵丘细胞。选择具有明显第一极体的卵母细胞通过显微操作移去第一极体及其附近约一半的细胞质进行去核，去核的卵细胞在成熟液内继续培养到ＩＶＭ３０ｈ．体外发育到８－３２细胞期的ＩＶＦ胚胎用作核供体．供体胚胎用含０．２５％Ｐｒｏｎａｓｅ（溶于ＰＢＳ￣－）溶解透明带，并用细管吹打将其分散成单个卵裂球，在ＩＶＭ３０ｈ将单个卵裂球显微注入去核卵母细胞的卵周隙内，并在ＩＶＭ３１ｈ用８０Ｖ／ｍｍ４０μｓ两次电脉冲（间隔１秒）来诱导卵裂球与去核卵母细胞的融合。融合卵在颗粒细胞单层培养滴内共培养，体外培养２４ｈ后观察卵裂结果，经体外培养５－８天后发育到桑椹和囊胚的核移植胚胎移植到同期发情的受体．２个月后直检妊娠情况．本实验中共操作１９４枚卵母细胞，     

Purification of platelet-derived endothelial cell growth inhibitor and its characterization as transforming growth factor-β type 1

In 1986, Brown and Clemmons (Proc. natl Acad. Sci. USA83 (1986) 3321) showed that platelets contain a substance, platelet-derived growth inhibitor (PDGI), that inhibits in vitro endothelial cell replication. Although platelets are rich in transforming grwoth factor (TGF-), PDGI was considered not to be related to TGF-, on the basis of its reported properties (extraction from platelets at neutral pH, binding to heparin-Sepharose). However, we purified PDGI to near homogeneity and showed that on the basis of HPLC retention behavior, in vitro growth inhibitory activities with several cell types, receptor binding, and immunoneutralization of growth inhibitory activity with specific anti-TGF- type 1 antibodies, PDGI is most probably identical with TGF- type 1.     

In vitro amiodarone protein binding and its interaction with warfarin

Summary The binding of amiodarone to human plasma protein and to bovine serum, albumin was studied by three different methods, ultracentrifugation, equilibrium dialysis and fluorescence spectroscopy. The fraction of amiodarone bound to plasma protein amounted to 96.3%. The changes in the binding properties of 1-anilino-naphthalene-8-sulfonate for bovine serum albumin using warfarin and amiodarone as independent inhibitors were analyzed in terms of binding site specificity. The findings indicated that amiodarone and warfarin have two different binding sites on bovine serum albumin, so a noncompetitive inhibition mechanism was indicated. On the basis of our data we cannot exclude other mechanisms of interaction besides direct displacement of one drug by another; nevertheless, metabolite interference between amiodarone and coagulation cofactors may better explain the enhancement of warfarin's pharmacological action in association with amiodarone.This work was partially funded by the CNR (National Research Council, Rome, Italy), Program on Clinical Pharmacology and Rare Diseases. The authors would like to thanks Drs E. Marzi and E. riva for their help.     

雌二醇对体外培养内皮细胞抗氧化活力的影响

雌激素对心血管系统的保护作用,是育龄妇女少患此类疾病的重要原因。本文以体外培养的小牛主动脉内皮细胞为实验材料,考察了雌二醇对血管内皮细胞抗氧化系统的影响。结果表明,０．５μｇ／ｍｌ雌二醇作用内皮细胞后,细胞内ＳＯＤ活性显著增加,与此相对应,细胞内过氧化产物ＭＤＡ的含量显著降低。提示该浓度的雌二醇能够提高细胞内的抗氧化能力,因而可能防止活性氧对内皮细胞的损伤,而这种损伤往往是动脉粥样硬化的开端。０．     

牛泡沫病毒BFV3026 gag基因的克隆及其在大肠杆菌中的表达

从牛泡沫病毒BFV3026感染的胎牛肺细胞中提取Hirt DNA,PCR扩增BFV3026　gag基因。序列分析确证后，将其克隆于原核表达载体pET32A，重组质粒转化大肠杆菌BL21（DE3），经IPTG诱导，使Gag蛋白得以高效表达。SDS－PAGE及Western blot证实：表达蛋白占菌体总蛋白的40%，本工作的完成为Gag蛋白功能的研究奠定基础。     

牛血清白蛋白对牛红细胞SOD的化学修饰

应用戊二醛交联法研究了牛血甭白蛋白对牛红细胞ＳＯＤ的化学修饰，修饰酶活力回收率为５１．２％，氨基修饰率为３３．１％，ＳＯＤ经牛血清白蛋白修饰后提高了耐热、耐酸碱稳定性，并保持良好贮藏稳定性。     

MMA/HEA共聚物膜的制备及性能

采用溶液聚合法先制备甲基丙烯酸甲酯(MMA)和丙烯酸羟乙酯(HEA)的共聚物,然后通过溶剂蒸发沉淀制得了聚合物膜。用DSC和SEM对聚合物膜进行了表征,并测定了单体投料比与膜的力学性能之间的关系,用动态接触角研究了聚合物膜表面的亲水性,并对牛血清蛋白(BSA)进行了吸附研究。结果表明:当HEA的投料量wHEA=0.18~0.25时聚合物膜可达到医用膜的力学性能要求;HEA的引入提高了膜表面的亲水性,从而有利于提高膜的生理相容性,同时降低了膜对蛋白质的吸附,有利于提高膜的抗凝血性。     

一种高灵敏度测定血红蛋白的化学发光分析方法

研究了在碱性介质和阳离子表面活性剂CTMAB存在下,牛血红蛋白催化鲁米诺-铁氰化钾的化学发光行为和化学发光反应的条件,提出一种流动注射-化学发光法测定牛血红蛋白的新方法.该方法测定牛血红蛋白的线性范围为5.18×10-10~1.29×10-6mol/L,r2=0.998 5;检出限(RSN=3)为1.2×10-10mol/L;回收率为(100±10)%;dRSD≤5%(n=6).     

IGF-Ⅰ及其受体在牛体外受精早期胚胎中的表达

采用成分明确的体外培养系统产生牛体外受精胚胎,结合荧光免疫定位的方法,利用激光共聚焦成像系统研究了在牛卵母细胞及不同发育阶段早期胚胎中IGF-Ⅰ及其受体的表达.结果表明,IGF-Ⅰ及其受体从未成熟的卵母细胞、成熟的卵母细胞到早期胚胎发育的各个阶段均有表达,在COC的颗粒细胞中也有表达.IGF-I在成熟卵母细胞中主要定位于细胞膜附近,在未成熟卵母细胞、2细胞期胚胎中分布于细胞膜及膜下区域,在8细胞期胚胎和桑椹胚中分布于整个细胞质中,在囊胚阶段主要位于滋胚层,内细胞团细胞有少量的表达.IGF-IR在未成熟卵母细胞和成熟卵母细胞以及2细胞、8细胞期胚胎中集中分布于卵裂球远离胚胎中心的细胞膜上,在桑椹胚期时遍布于整个胚胎细胞的细胞质中,在囊胚期时主要分布在滋胚层,内细胞团细胞有少量表达.     

血管内皮生长因子对牛早期胚胎发育的影响

为了探明血管内皮生长因子VEGF对牛体外受精胚胎早期发育的影响,本试验采用了成分明确的体外发育培养系统.通过在体外发育培养液中分别添加0、1、5、10 ng/mL的VEGF,对868枚胚胎的发育能力进行检测.研究结果显示:对照组与添加1 ng/mL、5 ng/mL、10 ng/mL VEGF的各实验组卵裂率分别为61.99%、75.69%、76.81%、68.92%;8-细胞期发育率分别为51.82%、61.21%、65.41%、63.40%;囊胚期发育率分别为27.74%、24.85%、23.90%、22.88%;囊胚孵化率分别为5.11%、4.85%、5.66%、3.27%.添加1、5、10 ng/mLVEGF与对照组比较,卵裂率、8-细胞期发育率有所增加,囊胚期发育率和囊胚孵化率表现下降趋势,但实验组与对照组间上述几项发育指标差异均不显著(P>0.05).