新生牛血清促细胞生长效应的研究

为探索我国新生牛血清的质量标准,本文着重在新生牛血清促进人外周血淋巴细胞和羊水细胞生长效应方面与美国GIBCO胎牛血清进行了比较实验研究。取30个批号的混合新生牛血清,每批号分别配制RPMI1640和HAMF—10两种培养基。通过外周血淋巴细胞培养,测定细胞分裂指数和分裂指数比;通过羊水细胞培养,测定细胞附着平和相对附着率。结合其他物理、化学、生物等项检测标准,最后确定新生牛血清等级。     

Sf9昆虫细胞作为流行性乙型脑炎疫苗生产用细胞基质的研究

对Ｓｆ９细胞作为制备乙脑疫苗生产用细胞基质的可能性进行了探讨。用抗胸腺细胞血清处理过的新生小白鼠，检查证明Ｓｆ９昆虫细胞无致肿瘤原性。将在有血清培养液中乙型脑炎病毒（ＪＥＶ）Ｐ３株持续感染的Ｓｆ９细胞，逐步适应到无血清培养液中，此感染细胞持续感染的特性不变。以悬浮培养的方式，无论在有或无血清培养液中培养感染Ｐ３株病毒的Ｓｆ９细胞，其病变细胞数目比例较静止培养均明显增多，病毒滴度形成高峰时间缩短。将感染的Ｓｆ９细胞，经液氮保存后复苏可连续传代，其感染特性未改变。用有血清培养液培养感染细胞第６、１１、２６周和无血清培养液培养的感染细胞上清制备实验性ＪＥＶ疫苗，动物实验证明其效力均能达到疫苗生产规程的要求。Ｓｆ９细胞有望成为大规模悬浮培养制备ＪＥＶ疫苗的细胞基质。     

自体血清与胎牛血清培养兔关节软骨细胞的生物学特性

背景：目前培养软骨细胞多使用胎牛血清。但近年异种血清培养组织向临床应用的安全性受到质疑,因此自体血清培养越来越受到重视。 目的：比较体积分数10%兔自体血清与体积分数10%胎牛血清培养兔关节软骨细胞生物学特性的差异。 方法：兔自体血清培养液制备后,分离培养兔关节软骨细胞,分别在体积分数10%自体血清和体积分数10%胎牛血清中进行单层传代培养至1,3,5代,采用光镜观察细胞形态变化,绘制生长曲线评估细胞增殖速度,观察甲苯胺蓝染色、Ⅰ,Ⅱ型胶原免疫组织化学染色结果,以流式细胞仪分析细胞Ⅰ,Ⅱ型胶原以及CD26,CD44的表达变化。 结果与结论：①在细胞形态上自体血清和胎牛血清培养的软骨细胞差异不大。②自体血清培养的软骨细胞较胎牛血清培养的软骨细胞生长速度更快。③甲苯胺蓝染色结果示,无论是自体血清还是胎牛血清所培养的细胞,染色随代龄的增加逐渐变浅,细胞传至第5代时两组几乎均无异染。对于1代和3代细胞而言,自体血清培养的软骨细胞较胎牛血清所培养的软骨细胞较为深染。④Ⅰ型胶原的表达随代数的增加而增加,而Ⅱ型胶原的表达则随代数的增加而减少。在3代时自体血清培养的软骨细胞Ⅰ型胶原的表达水平低于胎牛血清培养的软骨细胞(P < 0.05);在1代和3代时自体血清培养的软骨细胞Ⅱ型胶原的表达水平高于胎牛血清培养的软骨细胞(P < 0.05)。⑤CD26表达呈现先升高后降低的趋势,而CD44的表达不随传代数的增加而改变。提示同异体体积分数10%的胎牛血清相比,体积分数10%的自体血清培养的兔软骨细胞生长速度快,且在大部分指标上可以较好地保持细胞表型。     

人血清和初生牛血清对冻存人淋巴细胞免疫功能影响的比较研究

本实验比较了人AB血清和初生牛血清对冻存的人外周血淋巴细胞的回收及其功能活性的影响。实验发现,两种血清均可使收获率和存活率明显提高。两者进一步比较发现,冻存于含人血清介质的淋巴细胞其转化能力明显高于初生牛血清组;而初生牛血清组其冻存细胞的花环形成和细胞膜受体的稳定性则较前者为好.     

谷氨酸脱羧酶65重组杆状病毒表达载体的构建及其在昆虫细胞中的表达

目的构建谷氨酸脱羧酶GAD65基因的杆状病毒重组供体质粒,包装重组GAD65的杆状病毒,感染昆虫细胞进行表达。方法先将已克隆的GAD65 cDNA与线性化的pFASTBACHTb进行连接,使pFASTBACHTb-GAD65与DH10BAC感受菌进行转座重组,利用抗性及蓝白斑筛选重组Bacmid/GAD65克隆,提取Bacmid/GAD65转染昆虫细胞Sf 9包装杆状病毒,利用病毒感染Sf9细胞进行蛋白表达,通过免疫荧光、SDS PAGE和Western blot检测表达情况。结果获得了重组GAD65的杆状病毒,细胞能表达出与GAD65单抗结合的蛋白,相对分子质量为65kDa左右,细胞可直接分泌GAD65蛋白至上清。结论GAD65能在昆虫细胞中获得良好表达为研究GAD65的作用及免疫治疗奠定基础,。     

两种具长效性重组人血清白蛋白／干扰素α融合蛋白表达工程菌的构建及融合蛋白的理化特性研究

目的对两种长效干扰素α融合蛋白原料药的理化特性多项指标开展分析和进行比较。方法利用基因工程技术分别构建了可高效表达重组人血清白蛋白／干扰素α2a融合蛋白（rHSA／IFNα2a）或重组人血清白蛋白,干扰素α2b融合蛋白（rHSA／IFNα2b）的毕赤酵母工程菌。融合蛋白直接分泌到组分简单的无机盐培养基中,并经特别建立的高效分离纯化工艺进行纯化,随后对两个融合蛋白进行详尽的理化特性分析。结果获得的融合蛋白纯度在98％以上;N端前15个氨基酸序列与理论序列相同;质谱分子量分别为85640．8D和85781．5D;圆二色光谱分析比对蛋白空间构象未变;等电点DI约在5．1左右;为非糖基化蛋白;紫外光谱呈典型蛋白质光谱;自由巯基含量测定值为1．4;肽图批次问一致;肽质量指纹图谱可匹配分别达83％和82％;对制备的2个融合蛋白原料药中的细菌内毒素、宿主蛋白质、外源性DNA、甲醇和甘油的残留量检测符合标准要求;融合蛋白的免疫学鉴别为阳性;体外细胞生物比活性约为2．5×10^5IU／mg,并且2个融合蛋白生物比活性测定值无不同,由此,获得了符合临床用药标准的原料药。结论研究所获得的结果可以作为指导《注射用重组人血清白蛋白／干扰素α2a融合蛋白》和《注射用重组人血清白蛋白／干扰素α2b融合蛋白》2个新药的原料药生产质量标准的建立和检定方法的确定。     

胎牛血清外泌体对成骨细胞增殖的作用

背景:研究表明外泌体具有促进骨再生的能力,但从胎牛血清中提取的外泌体是否可以促进骨形成仍存在争议。目的:观察胎牛血清外泌体对成骨细胞增殖能力的影响,从而为临床治疗骨破坏提供新思路。方法:通过超速离心法从胎牛血清中提取外泌体,采用透射电子显微镜和Western blot法验证外泌体是否提取成功;然后用10 mg/L胎牛血清外泌体干预成骨前体细胞MC3T3-E1,通过CCK-8实验检测外泌体对成骨细胞增殖能力的影响,Western blot检测外泌体对成骨细胞骨形态发生蛋白2和骨桥蛋白表达的影响。以不含外泌体的胎牛血清培养的MC3T3-E1细胞为对照组。结果与结论:①胎牛血清外泌体具有典型的脂质双层膜结构,大小在30-150 nm之间,外泌体表面标记因子CD81表达呈阳性,而微囊表面标记物CD40表达呈阴性;②外泌体组的增殖能力明显高于对照组,差异有显著性意义(P<0.05);外泌体组成骨标志性因子骨形态发生蛋白2和骨桥蛋白的表达水平明显高于对照组,差异有显著性意义(P<0.05);③结果表明,胎牛血清外泌体对成骨细胞的增殖起促进作用,可为临床治疗骨破坏提供新思路。     

EGF和bFGF对脐血单个核细胞τ蛋白及微管相关蛋白-2 mRNA表达的影响

目的：探讨脐血单个核细胞在细胞因子诱导作用下τ蛋白及微管相关蛋白-2(MAP2) mRNA的表达。方法：用密度梯度离心法分离脐血单个核细胞，接种于含20%胎牛血清H-DMEM培养瓶内。以不加任何生长因子的培养为对照组，其余3组分别加入细胞因子EGF+bFGF、bFGF、EGF,终浓度均为20 μg/L。倒置显微镜下观察细胞形态变化，RT-PCR方法检测脐血单个核细胞培养前后τ蛋白及MAP2 mRNA，免疫细胞化学方法检测τ蛋白及MAP2阳性细胞。结果：培养前，脐血单个核细胞胞体小呈圆形；培养后EGF+bFGF组细胞胞体较大，突起粗而长，EGF组细胞胞体呈梭形，有1-2个长突触，bFGF组细胞胞体较小、有多个细长突起，形似星形细胞，对照组细胞形态类似于bFGF组，但数量较少。RT-PCR检测未培养脐血单个核细胞MAP2 mRNA表达阳性而τ蛋白mRNA呈阴性，培养后MAP2 mRNA表达增强，τ蛋白mRNA亦呈阳性；免疫细胞化学方法可检测MAP2及τ蛋白阳性细胞在EGF+bFGF组、EGF组、bFGF组、对照组分别为33.5%、25.6%、19.6%、14.4%和29.8%、21.4%、15.3%、13.5%。结论：脐血单个核细胞中存在的神经元特异性分子，经培养和生长因子调控后表达增强，联合使用bFGF、EGF具有协同作用。     

具长效性重组人血清白蛋白/粒细胞刺激因子融合蛋白的理化特性研究

目的对符合制药要求的高纯度重组人血清白蛋白/粒细胞刺激因子融合蛋白原料药开展理化特性研究。方法利用基因工程技术构建了可高效表达重组人血清白蛋白/粒细胞刺激因子融合蛋白（rHSA/GCSF）的毕赤酵母工程菌,rHSA/GCSF直接分泌到组分简单的无机盐培养基中,而后经特别建立的高效分离纯化工艺进行分离纯化。通过理化分析手段,首次获得rHSA/GCSF的多项理化特性指标和结果。结果 rHSA/GCSF原料药的蛋白质纯度达到98%以上;肽质量指纹图谱匹配率为84%;N-端氨基酸序列前15个氨基酸与理论序列相符;质谱分子量测定结果为85305D,与理论分子量85215D值相近;自由巯基含量为2.4;融合蛋白经圆二色光谱分析比对表明HSA和GCSF各自的空间构象未变;等电点（pI）约为5.8;融合蛋白为非糖基化蛋白、紫外光谱呈现典型蛋白质光谱;原料药中的细菌内毒素、宿主蛋白质、外源性DNA、甲醇和甘油的残留量符合作为药物的要求。融合蛋白的免疫学鉴别为阳性;体外细胞学生物比活性测定结果值为1.5×107IU/mg,与单体rGCSF的等摩尔比没有显著差异。结论研究获得的方法和结果可作为指导《注射用重组人血清白蛋白/粒细胞刺激因子融合蛋白》原料药的质量标准和制检规程的基础。     

PD-L1重组蛋白的表达及生物学活性分析

目的 构建细胞程序死亡因子1配体1(PD-L1)的重组表达质粒,在原核系统中表达并分析其生物学活性.方法 经密码子优化后合成PD-L1全基因序列,构建硫氧还原蛋白-pET43b/(PD-L1)重组表达质粒并在大肠埃希菌中表达;用ELISA验证表达产物与其受体结合的生物学活性.结果 正确构建了PD-L1重组表达质粒及其大肠埃菌基因工程菌,能稳定、高效地表达目的 蛋白,相对分子质量为47×103,目的 蛋白能与其受体特异性结合.结论 成功获得有生物学活性的PD-L1重组蛋白,为研制单克隆抗体及其与病毒感染慢性化的关系提供基础条件.     

Oxidative stress and DNA damage induced by spinosad exposure in Spodoptera frugiperda Sf9 cells

Spinosad, a neurotoxic insecticide, is widely used for crop protection. In order to elucidate the effects of spinosad on oxidative stress and genotoxicity in Sf9 cells, the levels of lipid peroxidation, the activity of antioxidative enzymes, and DNA damage were measured. The results showed that spinosad caused a time-dependent increase in the formation of malondialdehyde and decrease in the activity of superoxide dismutase and catalase. Further studies confirmed that spinosad induced 8-oxoguanine accumulation in Sf9 cells, which is accompanied by increased expression of DNA repair enzymes (OGG1 and MTH1). The neutral comet assay revealed that spinosad induced significant time-related increases of DNA double-strand breaks in Sf9 cells. Our results indicate that spinosad effectively induced oxidative stress and DNA damage in Sf9 cells.     

重组病毒--St疫苗研究进展

重组St疫苗是一种新型病毒疫苗。本文综述了St菌株的特征、St减毒株构建和重组St疫苗构建及其作用机 制等方面的研究进展。     

Clusters of Cl− channels in CFTR-expressing Sf9 cells switch spontaneously between slow and fast gating modes

The Sf9 insect Spodoptora frugiperda cell line was used for heterologous expression of the cloned human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, or the cloned β-galactosidase gene, using the baculovirus Autographa califonica as the infection vector. Using application of the patch-clamp technique, evidence for functional expression of CFTR was obtained according to the following three criteria. Firstly, whole-cell currents recorded 2 days after infection with CFTR revealed a statistically significant increase of membrane conductance, ≈25 times above that of mock-infected control cells, with the reversal potential of the major current component being governed by the chloride equilibrium potential (E _Cl). Secondly, in contrast to uninfected cells and cells infected with β-galactosidase, the membrane conductance to chloride of CFTR-injected cells was stimulated by cytosolic adenosine 3′,5′-cyclic monophosphate (cAMP), which was raised by exposing the cells to 10 μM forskolin. Thirdly, recordings of currents through single channels in excised outside-out membrane patches of CFTR-infected cells revealed channels which were clearly different from the native insect chloride channel. Excised outside-out patches of CFTR-infected and forskolin-stimulated cells exhibited wave-like gating kinetics of well-resolved current transitions. All-point Gaussian distributions revealed contributions from several (five to nine) identical channels. Such channels, in excised outside-out patches, studied with a pipette [Cl⁻] = 40 mM and a bath [Cl⁻] = 150 mM, rectified the current in agreement with simple electrodiffusion and with a single-channel Goldman-Hodgkin-Katz permeability, P _Cl = 1.34⋅10⁻¹⁴ ± 0.23⋅ 10⁻¹⁴cm³/s (n = 5), corresponding to a physiological single-channel conductance of 2.8 ± 0.5 pS (V _M = E _Cl) and a limiting conductance, γ_150/150, = 7.7 ± 1.3 pS ([Cl⁻]_Bath = [Cl⁻]_Cell = 150 mM). Currents recorded from multichannel excised outside-out patches could shift from the above mode of resolvable unitary conductance transitions to one which was too fast to reveal the dwell-times of closed and open states. During periods characterized by noisy currents, the variance (σ²) of current fluctuations about their stationary mean value depicted a U-shaped function of membrane potential, with a minimum value at a pipette potential where the chloride current was shown to be zero. Thus, it can be concluded that the current fluctuations are caused by fast gating of channels specific for chloride ions. Switching back and forth between the two gating modes of clusters of chloride channels occurred from moment to moment in excised patches when the membrane potential was held at a constant value indicating cooperative gating as a result of interaction between neighbouring chloride channels. Received: 6 November 1995/Received after revision: 1 February 1996/Accepted: 23 February 1996     

Effect of human recombinant erythropoietin on human hemopoietic progenitor cells in vitro

Summary Recombinant human erythropoietin (rhEpo), now available, might become increasingly more important for clinical use, e.g., in the treatment of anemia of chronic renal failure. As a prerequisite of clinical trials, we analyzed the stimulatory and suppressive effects of rhEpo on human hemopoiesis by adding rhEpo to in vitro cultures of nonadherent low-density bone marrow cells obtained from normal persons and from patients undergoing hemodialysis for chronic renal failure. rhEpo was shown to be an effective stimulus for erythroid and multilineage colony formation. The dose-response curve was similar for erythroid progenitors BFU-E from normal controls and patients with chronic renal failure. rhEpo had no effect on megakaryocytic colony formation nor on the megakaryocytic differentiation of multilineage stem cells. Because of a good stimulatory activity on erythroid and multilineage stem cells and lack of toxic effects, rhEpo might be useful in the treatment of certain kinds of anemia.Abbreviations BFU-E burst-forming unit-erythroid - CFU-E colony forming unit-erythroid - CFU-GEMM CFU-granulocyte, erythrocyte, macrophage, megakaryocyte - CFU-GM CFU-granulocyte, macrophage - CFU-Mk CFU-megakaryocyte - IMDM Iscove's modified Dulbecco's medium - Mo-CM Mo-cell line conditioned medium - rhEpo recombinant human erythropoietin     

损伤对上皮细胞及上皮下成纤维细胞内皮素的影响

目的: 观察损伤的气道上皮细胞内皮素-1(ET-1)的合成变化以及气道上皮损伤后对上皮下成纤维细胞ET-1释放的影响,探讨ET-1与基质金属蛋白酶-9(MMP-9)的相互关系在气道重塑中的可能地位。方法:用ELISA法检测机械划伤和或LPS刺激离体培养的气道上皮细胞,与机械损伤上皮复合培养的上皮下成纤维细胞,以及转染MMP-9真核表达质粒的上皮细胞培养上清中ET-1水平;明胶聚丙烯酰胺凝胶电泳酶谱法检测稳定转染ET-1转换酶(ECE)反义核酸上皮细胞培养上清MMP-9酶活性。结果:(1)气道上皮细胞经机械+LPS损伤后,培养上清ET-1水平较对照组明显增加;(2)损伤上皮诱导了上皮下成纤维细胞释放ET-1;(3)转化MMP-9真核表达质粒的气道上皮ET-1的释放较对照转化空载体组显著增高;(4)酶谱分析显示,表达反义内皮素转换酶RNA的气道上皮细胞损伤后MMP-9的活性水平明显低于对照上皮细胞组。结论:ET-1与MMP-9的相互影响在介导哮喘气道炎症与组织重塑的联系中起着重要的作用。     

Construction of the recombinant expression vector for CD80-IgG fusion gene and its expression in Chinese hamster ovary cells

Itiswellestablishedthatoptimalactivationof na veTcellsrequiressignalingthroughtheT cell receptoraswellasthroughothercostimulatorypathways.Na veTcellswillbecomenonrespons ive,anergicorapoptoticifthetumorantigenispr esentedtothemwithoutthecostimulatorysignal,whichmakestumorcellsescapefromtheimmuno surveillance.Oneofthemostimportantcostimula torysignalsofthiskindcanbeprovidedbythein teractionofB7.1(CD80)onantigen presenting cells(APC)withCD28onTcells.Acutemyelogenousleukemia(AML)cellsarenot…     

Immunological properties of thymus cell subpopulations: rat thymic dendritic cells are potent accessory cells and stimulators in a mixed leukocyte culture

Rat thymus cells were fractionated by centrifugation on a discontinuous bovine serum albumin gradient into two subpopulations: one of high density that accounted for>90% of the recovered cells, and a minor low-density subpopulation containing 4 to 10% of the total cells. The high-density subpopulation consisted mainly of uniform small-sized thymocyteS, whereas the low-density subpopulation contained mostly larger-sized cells. High-density thymus cells did not function either as stimulators in a mixed leukocyte reaction or as accessory cells required for T-cell response to mitogens, Con A and sodium periodate, as determined by ³H-thymidine incorporation. Dense thymus cells also responded poorly to allogeneic and to mitogenic stimulation, even when accessory cells were added. In contrast, the low-density thymus cells responded well to allogeneic stimulation and to both mitogens. In addition, low-density thymus cells possessed stimulatory activity in mixed leukocyte cultures, as well as accessory activity for mitogenic responses. Both activities were found to reside in dendritic cells that were purified extensively (70-90% of the preparation) with good yield. When tested as accessory cells for T-cell responses to periodate, thymic dendritic cells were as potent as lymph node dendritic cells on a per cell basis. A small number of thymic dendritic cells was able to cause marked enhancement in T-lymphocyte proliferation in response to stimulation. By immunofluorescence thymic dendritic cells were shown to be Ia-positive, but Thy 1.1-negative.     

Culturing of BHK-21 cells in a medium containing adult bovine serum and pituitary extract

Summary To achieve a comparable protein and cell production by culturing of BHK-21 cells in monolayer, in a medium containing adult bovine serum (ABS) and in a medium containing fetal bovine serum (FBS), 1.5 times (v/v) more ABS than FBS has to be added. At a volume fraction in the medium of less than 2% ABS, no proliferation is observed. For a fixed protein and cell production, the amount of required protein in a medium containing ABS can be reduced by addition of a protein extract from pituitary glands from calves (PEP). The relative cell density after 7 days in a culture medium containing 2 or 5% (v/v) ABS is 3 times higher when 0.2 g/liter pituitary extract is added. This indicates a synergistic interaction between components from ABS and from the protein extract. The addition of a pituitary extract to a medium with ABS also induces a prolonged cell proliferation period. A combined addition to a medium with ABS of 0.02 g/liter PEP and 0.15 g/liter heparin yields almost the same cell density as the addition of 0.2 g/liter PEP alone, indicating the presence of fibroblast growth factor (FGF) in the pituitary extract. However, the enhanced cell production by the addition of a pituitary extract to a medium containing ABS cannot be explained only by the presence in this crude extract of a- or bFGF.Abbreviations ABS adult bovine serum - BE protein extract from whole bovine brains - FBS fetal bovine serum - PEP protein extract from pituitary glands from calves - PEX purchased bovine pituitary extract - PDT population doubling time     

大鼠重组骨桥蛋白在中国仓鼠卵巢细胞中的稳定表达和纯化

目的 构建包含大鼠骨桥蛋白(OPN)的真核表达载体,获得稳定表达重组蛋白的中国仓鼠卵巢(CHO)细胞株并对蛋白进行纯化,为OPN在大鼠模型实验中的进一步功能学研究奠定基础.方法 通过脂质体lipofectamine 2000将重组质粒pLVX-OPN转染到CHO细胞中,嘌呤霉素筛选出稳定克隆细胞.Real-time PCR法检测转染后CHO细胞中OPN的mRNA表达水平,酶联免疫吸附实验法(ELISA)检测上清中OPN分泌水平.取CHO细胞72 h无血清培养上清进行镍柱亲和层析纯化,ELISA法和Western blot法分别检测产量和纯度.结果 pLVX-OPN表达载体经DNA琼脂糖电泳鉴定为阳性,且质粒测序成功.经质粒pLVX-OPN转染的CHO细胞中OPN mRNA表达水平是转染前的上千倍,上清分泌蛋白水平较转染前增加.上清分泌的OPN蛋白通过镍柱亲和层析纯化后纯度有所提高,产量在30％以上.结论 pLVX-OPN真核表达载体的成功构建和在CHO细胞中的稳定表达及纯化为OPN的功能学研究提供了有效工具.