流感疫苗病毒株在MDCK细胞中的培养条件

 目的   研究4价流感疫苗所用病毒株在MDCK细胞中的扩增条件。方法 在6孔细胞培养板中以不同感染复数（multiplicity of infection,MOI）（0.100 0、0.010 0、0.001 0、0.000 1）和对甲苯磺酰-L-苯丙氨酸氯甲基酮（TPCK）-胰蛋白酶（胰酶）浓度（0、2、4、8 μg/ml）进行病毒接种和扩增,感染后72 h收获细胞上清液,检测病毒血凝素效价,确定病毒最佳扩增条件。随后,在搅拌瓶中进行MDCK细胞微载体悬浮培养,研究不同起始细胞接种密度（1.5×10⁵、2.0×10⁵、3.0×10⁵ 个/ml）和微载体浓度（3、5、10 g/L）对MDCK细胞生长的影响,确定细胞最佳扩增条件。根据确定的最佳扩增条件,在搅拌瓶培养体系中扩增4种病毒。结果 6孔板中4种流感病毒的最佳接种条件分别是,A/Michigan/45/2015(H1N1) pdm09：MOI 0.010 0、TPCK-胰酶浓度2 μg/ml;A/Hongkong/4801/2014(H3N2)：MOI 0.010 0、TPCK-胰酶浓度4 μg/ml;B/Brisbane/60/2008：MOI 0.001 0、TPCK-胰酶浓度4 μg/ml;B/Phuket/3073/2013：MOI 0.010 0、TPCK-胰酶浓度4 μg/ml。在搅拌瓶中以3.0×10⁵ 个/ml初始细胞密度接种,3 g/L微载体浓度培养,MDCK细胞能够实现较好的扩增,最高密度可达2.1×10⁶ 个/ml;搅拌瓶悬浮培养,以最佳接种条件接种后,4种病毒的血凝素效价为：6.75~8.42log₂ 血凝素单位/50 μl。结论   通过摸索病毒接种最佳MOI、TPCK-胰酶浓度及优化MDCK细胞微载体悬浮培养条件,能够在MDCK细胞微载体悬浮培养体系中有效扩增4价流感疫苗用病毒株,为后期工艺放大研究奠定基础。     

长时间甲醛固定的大鼠视网膜组织胰蛋白酶消化方法探讨

目的探讨长期甲醛固定的大鼠视网膜胰蛋白酶消化的最佳消化时间和最佳胰蛋白酶浓度。方法健康♂SD大鼠,颈椎脱臼处死,摘取眼球,放入4%甲醛固定液,固定时间为2 d、2周、4周、3月、5月、7月。分离固定不同时间的视网膜,放入3%、6%、9%胰蛋白酶消化,37℃恒温箱孵育。分别记录固定不同时间的视网膜在3%、6%、9%胰蛋白酶中消化完全所需时间。铺片操作,行PAS染色。显微镜下记录视网膜消化情况。结果新鲜眼球常规固定48 h,3%胰蛋白酶37℃消化3.5 h,视网膜消化完全。对于甲醛固定2周及1月的大鼠眼球,采用常规胰蛋白酶浓度(3%),仅延长胰蛋白酶的消化时间至9 h及14 h,可以达到常规固定组织的消化效果。对于固定3月的大鼠眼球,采用常规浓度的胰蛋白酶仅延长消化时间至22 h,同样可以得到完整的视网膜血管铺片,且提高胰蛋白酶浓度对于缩短视网膜消化时间的影响不大。而对于固定5⁷月的大鼠眼球,6%和9%浓度的胰蛋白酶可以在一定程度上缩短视网膜消化的时间至22 h和28 h,有利于得到相对完整的视网膜血管。结论固定时间短于3月的眼球组织,采用3%胰蛋白酶消化,延长消化时间可达到最佳消化效果。对于固定57月的大鼠眼球,6%和9%浓度的胰蛋白酶可以在一定程度上缩短视网膜消化的时间至22 h和28 h,有利于得到相对完整的视网膜血管。结论固定时间短于3月的眼球组织,采用3%胰蛋白酶消化,延长消化时间可达到最佳消化效果。对于固定5⁷月的视网膜组织,不仅需要延长消化时间,还需要通过提高胰蛋白酶浓度至6%,才可以获得最佳消化效果的视网膜血管。     

不同培养基和胰蛋白酶对Vero细胞培养及消化效果的比较

目的 比较不同公司生产的无血清培养基对病毒性疫苗生产用Vero细胞培养效果,及基因工程胰蛋白酶的细胞消化效果,以判断是否适用。方法 实验组用VirusPro Vero-A培养基进行Vero细胞的培养,用Trpzyme消化细胞。对照组用VP-SFM培养基进行细胞培养,用TrypLE Select消化细胞。两组Vero细胞以相同密度接种在T175培养瓶中,以相同的培养条件和传代方法在T175瓶和细胞工厂中各培养3代。培养期间观察细胞的上清液、形态以及汇合度,消化后检测细胞活率并计算细胞收获量。采用配对t检验比较两组消化液的pH值、总消化时长、37 ℃消化孵育时长、细胞活率以及细胞收获量。结果 两组细胞生长状态均良好。配对t检验显示,实验组消化液的pH值（6.99）、总消化时长（17.28 min）和37 ℃消化孵育时长（6.93 min）均值均大于对照组消化液的（分别为6.75、12.34 min、3.30 min）,细胞活率（94.79%）及细胞收获量（T175瓶：3.91×10⁷个;细胞工厂：1.90×10⁹个）均值均高于对照组的（分别为90.20%、3.33×10⁷个、1.26×10⁹个）,且差异有统计学意义（t值分别为9.17、3.46、2.98、2.31和4.38,P值均<0.05）。结论 实验组无血清培养基培养效果较好,基因工程胰蛋白酶细胞消化液温和、细胞损伤小,均适用于Vero细胞。     

转移性肾细胞癌药物治疗方法的研究

肾细胞癌在我国的发病率逐年升高,且对于放化疗不敏感,免疫治疗效果有限,尤其是转移性肾细胞癌的预后较差。该文就转移性肾细胞癌的药物治疗方案的作用机制、现状及展望进行综述。     

动物细胞培养技术在流感疫苗研发中的应用进展

流感是由流感病毒引发的急性呼吸道传染病,每年可引起季节性流行。疫苗接种是预防和控制流感流行和大流行期间病毒感染的主要方法,目前流感疫苗生产主要仍依赖鸡胚培养技术,但近年来,使用动物细胞基质代替鸡胚培养已成为流感疫苗技术创新的重要趋势。随着贴壁培养及无血清全悬浮培养等培养技术在生物制药领域的发展,已有多种动物细胞系用于流感疫苗生产。本文简要综述近年来动物细胞培养技术在流感疫苗研发中的应用进展。     

人用疫苗生产用细胞基质使用现状及研究进展

疫苗是预防控制传染性疾病的最有效手段, 传染性疾病中约80%是由病毒引起的。传统的病毒类疫苗需要经过病毒培养、抗原收获、抗原浓缩、纯化过滤等过程制备而成。由于病毒的生长和繁殖必须在细胞内进行, 因此细胞基质的优劣直接影响疫苗的质量。此文对各类细胞基质使用情况及优缺点进行综述, 旨在为选择合适的人用疫苗生产用细胞基质提供参考。     

多发性肾细胞癌临床诊治

目的 探讨多发性肾细胞癌发生的分子生物学机制以及多发性肾细胞癌的诊断与治疗情况.方法 回顾性分析我院2000年至今诊治的9例多发性肾细胞癌病例.结果 9例患者均治愈出院.随访4个月～7年,8例患者肾脏功能正常,而且无肿瘤复发,1例因肿瘤复发而死亡.结论 多发性肾细胞癌的发病机制目前还不清楚,但是多发性肾细胞癌在临床上并不少见,而且有逐年增多的趋势,所以必须引起足够的重视.对于多发性肾细胞癌患者来说,根据患者的不同情况选择恰当的手术方式以保留足够的肾单位、肾功能是必要的.     

Transport of poly amidoamine dendrimers across Madin-Darby canine kidney cells

The objective of this study was to determine the permeability of a series of poly amidoamine (PAMAM) dendrimers of generations 0-4 (G0-G4) across MDCK (Madin-Darby Canine Kidney) cell line. PAMAM dendrimers with incremental increase in size and molecular weight were labeled by fluorescein isothiocyanate (FITC) and the least polydisperse fractions were collected by size exclusion chromatography. MDCK cells were grown on Transwell filters for four days. The conjugates were detected by HPLC equipped with fluorescence detector. The permeability of the dendrimers across MDCK cells was determined in the apical to basolateral direction. The rank-order permeability of the PAMAM dendrimers was G4 > G1 approximately G0 > G3 > G2. The permeability of mannitol in the presence of G4 increased by nine-fold. Results suggest that the transepithelial transport of PAMAM dendrimers is effected by both the polymer size, and the modulation of the cell membrane by the cationic dendrimers.     

Parathyroid hormone-stimulated cadmium accumulation in Madin-Darby canine kidney cells.

Although most renal cadmium transport occurs in proximal tubules indirect evidence suggests that distal tubules may also transport this heavy metal. Since the distal nephron is the site at which parathyroid hormone (PTH) regulates calcium absorption, we evaluated the effects of PTH on Cd2+ accumulation in Madin-Darby canine kidney (MDCK) cells. MDCK cells express a distal-like phenotype including PTH-sensitive adenylyl cyclase and stimulation of calcium transport. MDCK cells were grown to confluence in phenol red-free Dulbecco's modified Eagle's medium. PTH increased 109CdCl2 accumulation in a concentration-dependent manner over the range of 10(-11)-10(-9) M bPTH[1-34]. At 10(-9) M, PTH increased Cd2+ accumulation maximally by 205%. The PTH antagonist, bPTH[3-34], failed to augment 109Cd2+ accumulation. The dihydropyridine agonist, Bay k 8644, in the presence of PTH, increased 109Cd2+ uptake by 200% over vehicle-treated controls and by approximately 100% over PTH or Bay k 8644 alone. The apparent Km for Bay k 8644 activation was 1.3 microM. Bay k 8644-augmented 109Cd2+ uptake was competitively inhibited by the calcium channel antagonist nifedipine. No voltage dependence of Bay k 8644-amplified 109Cd2+ uptake could be detected. Based on these observations we conclude: (1) MDCK cells accumulate Cd2+; (2) PTH increases Cd2+ uptake into MDCK cells; and (3) Cd2+ entry in kidney epithelial cells is mediated, at least in part, by dihydropyridine-sensitive calcium channels.     

Zinc alters actin filaments in Madin-Darby canine kidney cells.

Exposure of semiconfluent cultures of Madin-Darby canine kidney cells to 10 microM zinc leads to a change in the organization of the actin filament system. Most of the stress fibers at the basal end of the cell are lost and the actin associated with the lateral membrane and junctional regions appears to retract into the cytoplasm. In addition, at the base of the cell in regions of cell-substratum contact, dense, actin-rich plaques appear. These alterations in actin filaments are associated with a change in cell shape. Microtubules were unaffected by exposure to 10 microM zinc. At zinc concentrations greater than or equal to 50 microM the microtubules depolymerized. Exposure to cadmium alters the actin filaments as well but the effect is different from the change seen with zinc. When the cells are exposed simultaneously to zinc and cadmium the cells appear the same as they would if exposed to zinc alone. Exposure of MDCK cells to either metal, individually or in combination, results in a significant and similar increase in F-actin content as determined spectrofluorometrically. The changes in organization and amount of F-actin are associated with a reduction in the ability of the cells to remain attached to the substrate, a toxic effect of these metals with regard to epithelial function. The results indicate that zinc, an essential metal, and cadmium, a highly toxic metal, interact with the actin cytoskeleton in intact cells.     

Fluoxetine-induced Ca2+ signals in Madin-Darby canine kidney cells

The effect of fluoxetine on Ca2+ signaling in Madin-Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca2+ probe. Fluoxetine increased [Ca2+]i concentration-dependently between 5 microM and 200 microM with an EC50 value of 40 microM. The response was reduced by external Ca2+ removal by 30%40%. In Ca2+-free medium pretreatment with 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, abolished 100 microM fluoxetine-induced Ca2+ release. Addition of 3 mM Ca2+ to Ca2+-free medium increased [Ca2+]i when cells were pretreated with 100 microM fluoxetine. Suppression of 1,4,5-trisphosphate (IP3) formation by 2 microM U73122 (a phospholipase C inhibitor) did not affect 100 microM fluoxetine-induced Ca2+ release. Fluoxetine (5-100 microM) also increased [Ca2+]i in neutrophils, prostate cancer cells and bladder cancer cells from human and rat glioma cells.     

Purinoceptor-stimulated phosphoinositide hydrolysis in Madin-Darby canine kidney (MDCK) cells

Extracellular nucleotides, acting through P₂-purinoceptors, have been implicated in the regulation of ion transport in epithelia, including Madin-Darby canine kidney (MDCK) cells. In this study, experiments were conducted to characterize the P₂-purinoceptor subtype on MDCK cells responsible for stimulating inositol phosphate (IP) accumulation using a range of nucleotide analogues. In Ca²⁺- and Mg²⁺-free Krebs-Henseleit solution (KHS), ATP, UTP, and ATPγS caused an increase in IP accumulation as a function of concentration with comparable kinetics. The order of potency for the nucleotide analogues was UTP = ATPγS > ATP = 2-chloro ATP (Cl-ATP) >> α,β-methylene ATP (α,β-MeATP) = 2-methylthio ATP (2MeSATP). Selective agonists for P₁-, P_2X- and P_2Y-purinoceptors, such as N⁶-cyclopentyl adenosine, AMP, α,β-MeATP, and 2MeSATP, had little effect. Stimulation of MDCK cells with maximally effective concentrations of ATP and UTP showed no additive effect and furthermore, ATP, UTP, and ATPγS induced cross-desensitization of the IP response, suggesting that ATP and UTP act upon a common nucleotide receptor, i.e. a P_2U-purinoceptor. In Ca²⁺- and Mg²⁺-containing KHS, the concentration-response curves of ATP, UTP, and ATPγS were shifted to the right of those obtained in Ca²⁺- and Mg²⁺-free buffer, and asymptotic maxima were not reached, indicating that ATP^4- and not MgATP^2- or CaATP^2- was the active agonist. Pretreatment of MDCK cells with pertussis toxin (PTX) inhibited ATP- and UTP-induced IP accumulation in a concentration-dependent fashion but did not completely abolish the IP accumulation, indicating that a PTX-sensitive G protein was partially involved in the IP response. In conclusion, ATP- and UTP-stimulated IP accumulation in MDCK cells appears to be mediated through the activation of P_2U-purinoceptors coupled to a G protein that is partially sensitive to PTX. A form of nucleotide uncomplexed with divalent ions such as ATP^4- seems to be the preferential agonist form for the purinoceptors on MDCK cells. Received: 25 June 1996 / Accepted: 4 April 1997     

Effect of cadmium on F-actin and microtubules of Madin-Darby canine kidney cells

The effect of cadmium on F-actin and microtubules of Madin-Darby Canine Kidney cells was studied by cytochemical methods. A 6-hr exposure to cadmium (10 microns) in a buffered salt solution resulted in the breakdown of actin filaments, particularly those associated with both the stress fibers and the lateral membranes in areas of intercellular contact. Microtubules were not dramatically altered during this exposure period and cell viability, determined by trypan blue exclusion, was similar to controls. The effect of cadmium on actin was reversible if the cells were returned to culture medium. The results indicate that one possible mechanism of cadmium toxicity is via an effect on the organization of actin filaments.     

5-Hydroxytryptamine 5-HT1D receptors mediating inhibition of cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells

5-Hydroxytryptamine 5-HT_1B/5-HT_1D receptors are members of the same receptor subfamily, but display a different pharmacology (Hartig et al. (1992) Trends Pharmacol Set 13:152–159). Whereas several cell lines have been reported to contain 5-HT_1B receptors, none has been described, however, that endogenously expresses well-characterized 5-HT_1D receptors. The present study deals with the identification of 5-HT_1D receptors inhibiting cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells. 5-HT (1 nM– 10 M) induced a concentration-dependent inhibition of the cyclic AMP accumulation stimulated by prostaglandin E₁ (1 M) in MDCK cells. The maximal effect of 5-HT averaged 50% inhibition and was abolished after a pre-treatment of the cells with pertussis toxin. Other agonists mimicked the effects of 5-HT, with the following rank order of potency (pEC₅₀ ± SEM, n 3): 5-carboxamidotryptamine (8.36 ± 0.48) > PAPP (p-aminophenylethyl-m-trifluoromethylphenyl piperazine, 7.89 ± 0.23) > 5-HT (7.35 ± 0.05) > sumatriptan (6.65 ± 0.27). PAPP behaved as a partial agonist. 8-OH-DPAT (8-hydroxy-2(di-n-propylamino)tetralin) was less potent, its maximal effect being not reached at 0.1 mM. Methiothepin, GR127935, (–)propranolol, rauwolscine and ketanserin were all devoid of intrinsic activity (up to 10 M or 0.1 mM). Methiothepin (10 nM, 0.1 M and 1 M) antagonized 5-HT effect (pA₂ 8.57 ± 0.44, Schild slope 1.17 ± 0.21, n = 3). GR127935 (1 nM, 10 nM and 0.1 M) shifted the curve of 5-HT to the right, but the antagonism was not fully surmountable (apparent pK_B value, 9.80 ± 0.16, n = 9). From the shifts obtained with rauwolscine (1 M) and (–)propranolol (10 M), respective pK_B values were estimated 6.68 ± 0.30 and 5.4 (n = 3 each). PAPP, when tested as an antagonist at 1 M, also shifted the curve of 5-HT to the right, with a pK_B of 8.27 ± 0.16 (n = 3). Finally, ketanserin (10 M) also antagonized the effects of 5-HT, the pK_B being 6.54 ± 0.16 (n = 9). The rank orders of agonist and antagonist potencies strongly suggest 5-HT receptors mediating inhibition of cyclic AMP accumulation in MDCK cells to be 5-HT_1D receptors. This is the first report of a cell line expressing endogenous, well-characterized, 5-HT_1D receptors. With regard to the 5-HT_1D receptor subtype involved, the relatively high potency of ketanserin would suggest it to be a 5-HT_1D subtype or a mixture of 5-HT_1D/5-HT_1D\ subtypes. However, caution must be exercised here, owing to the poor knowledge of canine 5-HT_1D receptor subtypes.     

Predicting ocular irritancy and recovery from injury using Madin-Darby canine kidney cells

Two promising cell culture assays, using Madin-Darby canine kidney cells, for predicting eye irritancy, the fluorescein leakage assay and the neutral red release assay, have been adapted to try and assess the ability of damaged cells to recover from chemical-induced injury. The fluorescein leakage and neutral red release protocols are similar but measure injurious effects on different parts of the cells, namely the tight junctions and the cell membrane, respectively. Both endpoints have previously given equivalent rankings of chemicals in order of their eye irritancy potential. Sixteen compounds of varying irritancy potential and chemical nature were tested using the two assays. In both assays, little or no cell recovery was measured 72 hr after a mildly injurious exposure, although using the fluorescein leakage assay, five test agents displayed substantial recovery and two displayed significant deterioration of the cell layer after removal of the test material. Comparing these in vitro results with in vivo data suggests that the fluorescein leakage assay, in its current format, does not predict the likely recovery rate of ocular tissue after chemical damage.     

Effect of triethyltin on Ca2+ movement in Madin-Darby canine kidney cells

The effects of the environmental toxicant, triethyltin, on Ca2+ mobilization in Madin-Darby canine kidney (MDCK) cells have been examined. Triethyltin induced an increase in cytosolic free Ca2+ levels ([Ca2+]i) at concentrations larger than 2 microM in a concentration-dependent manner. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was partly reduced by removing extracellular Ca2+. In Ca(2+)-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor, reduced 50 microM triethyltin-induced [Ca2+]i increase by 80%. Conversely, pretreatment with triethyltin abolished thapsigargin-induced Ca2+ release. Pretreatment with U73122 (2 microM) to inhibit phospholipase C-coupled inositol 1,4,5-trisphosphate formations failed to alter 50 microM triethyltin-induced Ca2+ release. Incubation with triethyltin at a concentration (1 microM) that did not increase basal [Ca2+]i for 3 min did not alter ATP (10 microM)- and bradykinin (1 microM)-induced [Ca2+]i increases. Collectively, this study shows that triethyltin altered Ca2+ movement in renal tubular cells by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner, and by inducing Ca2+ influx.