沉默MDR1基因增强急性早幼粒白血病耐药细胞HT9药物敏感性

该研究利用短发卡RNA（small hairpinin RNA,shRNA）表达载体沉默HT9急性早幼粒白血病耐药细胞MDRl基因表达,以提高细胞对三尖杉酯碱、阿霉素的敏感性。通过设计合成编码shRNA的DNA模板序列,定向克隆到pSilencer2．1－u6neo质粒,成功构建1个P－gP蛋白基因特异的shRNA表达载体,稳定电转染HT9细胞,实时荧光定量PCR分析MDRlmRYAg表达,Westem blot检测细胞P—gp蛋白表达,流式细胞术检测P—gP蛋白外排泵功能,MTT法检测细胞对药物敏感性。结果显示,成功构建了shRNA表达载体pSilencer2-1-U6neo—MDRl,转染HT9细胞后,PCR检测重组质粒整合到HT9／sh－2－1－1细胞基因组DNA,获得稳定遗传;HT9／sh－2－1—1细胞MDRlmRNA表达降低了78．84％（P〈0．01）,P—gP蛋白表达降低了48．27％（P〈0．05）,细胞内Rh0123相对荧光强度由（10．8±0．58）％升高至（73．56±1．37m;转染细胞对三尖杉酯碱、阿霉素敏感性明显增强,IC50分别由（2．06±0．15）gmol／L降至（0．57±0．01）gmol／L、（4．04±017）gmol／L降至（1．56±0．05）μtmol／L。提示shRNA干扰表达载体pSilencer2．1－U6neo—MDRl能够稳定、持久地抑制MDRI基因表达,并能有效增强HT9细胞对三尖杉酯碱、阿霉素的敏感性。     

Effect of noradrenaline on triacylglycerol synthesis in rat brown adipocytes.

1. The effects of hypothyroidism on the sensitivity of glycolysis and glycogen synthesis to insulin were investigated in the isolated, incubated soleus muscle of the rat. 2. Hypothyroidism, which was induced by administration of propylthiouracil to the rats, decreased fasting plasma levels of free fatty acids and increased plasma levels of glucose but did not significantly change plasma levels of insulin. 3. The sensitivity of the rates of glycogen synthesis to insulin was increased at physiological, but decreased at supraphysiological, concentrations of insulin. 4. The rates of glycolysis in the hypothyroid muscles were decreased at all insulin concentrations studied and the EC50 for insulin was increased more than 8-fold; the latter indicates decreased sensitivity of this process to insulin. However, at physiological concentrations of insulin, the rates of glucose phosphorylation in the soleus muscles of hypothyroid rats were not different from controls. This suggests that hypothyroidism affects glucose metabolism in muscle not by affecting glucose transport but by decreasing the rate of glucose 6-phosphate conversion to lactate and increasing the rate of conversion of glucose 6-phosphate to glycogen. 5. The rates of glucose oxidation were decreased in the hypothyroid muscles at all insulin concentrations.     

Tubulin Beta3 Serves as a Target of HDAC3 and Mediates Resistance to Microtubule-Targeting Drugs

We investigated the role of HDAC3 in anti-cancer drug-resistance. The expression of HDAC3 was decreased in cancer cell lines resistant to anti-cancer drugs such as celastrol and taxol. HDAC3 conferred sensitivity to these anti-cancer drugs. HDAC3 activity was necessary for conferring sensitivity to these anti-cancer drugs. The down-regulation of HDAC3 increased the expression of MDR1 and conferred resistance to anti-cancer drugs. The expression of tubulin β3 was increased in drug-resistant cancer cell lines. ChIP assays showed the binding of HDAC3 to the promoter sequences of tubulin β3 and HDAC6. HDAC6 showed an interaction with tubulin β3. HDAC3 had a negative regulatory role in the expression of tubulin β3 and HDAC6. The down-regulation of HDAC6 decreased the expression of MDR1 and tubulin β3, but did not affect HDAC3 expression. The down-regulation of HDAC6 conferred sensitivity to taxol. The down-regulation of tubulin β3 did not affect the expression of HDAC6 or MDR1. The down-regulation of tubulin β3 conferred sensitivity to anti-cancer drugs. Our results showed that tubulin β3 serves as a downstream target of HDAC3 and mediates resistance to microtubule-targeting drugs. Thus, the HDAC3-HDAC6-Tubulin β axis can be employed for the development of anti-cancer drugs.     

青菜幼苗体内几种保护酶的活性对Pb、Cd、Cr胁迫的反应研究

1 引言自从McCord在1969年第一次从牛红血细胞中发现超氧化物歧化酶(SOD)并且证明其功能是清除超氧化物以后,研究者们在植物细胞中也发现了这种酶,并且证明具有同样的功能.后来又发现植物细胞通过多种途径产生活性氧自由基,同时细胞也存在清除这些自由基的多种途径[5],两者形成平衡体系.但是,许多逆境因子如寒冷、干旱、干燥、水淹、重金属Al等都能影响植物体内活性氧代谢系统的平衡,产生大量的氧自由基,它们能够启动膜脂过氧化或膜脂脱脂作     

Sensitivity of adipocyte lipolysis to stimulatory and inhibitory agonists in hypothyroidism and starvation.

The responsiveness of lipolysis to the stimulatory agonists noradrenaline, corticotropin and glucagon and to the inhibitory agonists N6-phenylisopropyladenosine, prostaglandin E1 and nicotinic acid was investigated with rat white adipocytes incubated with a high concentration of adenosine deaminase (1 unit/ml). The cells were obtained from fed or 48 h-starved euthyroid animals or from fed or starved animals rendered hypothyroid by 4 weeks of treatment with low-iodine diet and propylthiouracil. Hypothyroidism increased sensitivity to and efficacy of all three inhibitory agonists in their opposition of noradrenaline-stimulated lipolysis. Starvation decreased sensitivity to all three inhibitory agonists when opposing basal lipolysis. Hypothyroidism decreased sensitivity to noradrenaline, glucagon and corticotropin by 37-, 4- and 4-fold respectively and decreased the maximum response to these agonists by approx. 50%, 50% and 75% respectively. Starvation reversed decreases in maximum response to these agonists in hypothyroidism. Starvation in the euthyroid state increased sensitivity to glucagon and noradrenaline, but did not alter sensitivity to corticotropin. Cells from hypothyroid rats were relatively insensitive to Bordetella pertussis toxin, which substantially increased basal lipolysis in the euthyroid state.     

Effect of starvation and diabetes on the sensitivity of carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate.

The sensitivity of carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate was decreased markedly in liver mitochondria isolated from either 48 h-starved or streptozotocin-diabetic rats. These treatments of the rat also decreased the sensitivity of fatty acid oxidation by isolated hepatocytes to inhibition by this compound. Furthermore, incubation of hepatocytes prepared from fed rats with N6O2'-dibutyryl cyclic AMP also decreased the sensitivity, whereas incubation of hepatocytes prepared from starved rats with lactate plus pyruvate had the opposite effect on 4-hydroxyphenylglyoxylate inhibition of fatty acid oxidation. The sensitivity of carnitine palmitoyltransferase I of mitochondria to 4-hydroxyphenylglyoxylate increased in a time-dependent manner, as previously reported for malonyl-CoA. Likewise, oleoyl-CoA activated carnitine palmitoyltransferase I in a time-dependent manner and prevented the sensitization by 4-hydroxyphenylglyoxylate. Increased exogenous carnitine caused a moderate increase in fatty acid oxidation by hepatocytes under some conditions and a decreased 4-hydroxyphenylglyoxylate inhibition of fatty acid oxidation at low oleate concentration, without decreasing the difference in 4-hydroxyphenylglyoxylate inhibition between fed- and starved-rat hepatocytes. Time-dependent changes in the conformation of carnitine palmitoyltransferase I or the membrane environment may be involved in differences among nutritional states in 4-hydroxyphenylglyoxylate-sensitivity of carnitine palmitoyltransferase I.     

RNAi Depletion of Deoxycytidine and Deoxyguanosine Kinase in Human Leukemic CEM Cells

Resistance toward nucleoside analogues is often due to decreased activities of the activating enzymes deoxycytidine kinase (dCK) and/or deoxyguanosine kinase (dGK). With small interfering RNA (siRNA), dCK and dGK were downregulated by approximately 70% in CEM cells and tested against six nucleoside analogues using the methyl thiazol tetrazolium assay. SiRNA-transfected cells reduced in dCK activity were 3- to 6-fold less sensitive to CdA, AraC, and CAFdA. The sensitivity to AraG and FaraA was unchanged, while the sensitivity toward gemcitabine was significantly increased. dGK depletion in cells resulted in lower sensitivity to FaraA, dFdC, CAFdA, and AraG, but slightly higher sensitivity to CdA and AraC.     

RNAi depletion of deoxycytidine and deoxyguanosine kinase in human leukemic CEM cells

Resistance toward nucleoside analogues is often due to decreased activities of the activating enzymes deoxycytidine kinase (dCK) and/or deoxyguanosine kinase (dGK). With small interfering RNA (siRNA), dCK and dGK were downregulated by approximately 70% in CEM cells and tested against six nucleoside analogues using the methyl thiazol tetrazolium assay. SiRNA-transfected cells reduced in dCK activity were 3- to 6-fold less sensitive to CdA, AraC, and CAFdA. The sensitivity to AraG and FaraA was unchanged, while the sensitivity toward gemcitabine was significantly increased. dGK depletion in cells resulted in lower sensitivity to FaraA, dFdC, CAFdA, and AraG, but slightly higher sensitivity to CdA and AraC.     

Down-regulation of lipocalin 2 contributes to chemoresistance in glioblastoma cells

Malignant gliomas are the most common primary brain tumor and have a poor clinical prognosis. 1, 3-Bis (2-chloroethyl)-1-nitrosourea (BCNU) is an alkylating agent that is commonly used in glioma therapy. However, BCNU chemotherapy often fails due to drug resistance. To gain better understanding of molecular mechanisms underlying the drug resistance of glioma, a BCNU-resistant variant (C6R) of C6 rat glioma cells was selected and characterized. The established C6R cells were resistant to BCNU-induced cell death and cell cycle arrest as confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide reduction assay and flow cytometric analysis of DNA content. C6R cells showed an increased expression of common drug resistance-related genes such as O6-methylguanine-DNA methyltransferase and multiple drug resistance 1. In contrast, C6R cells showed a decreased expression of glial fibrillary acidic protein, therefore, displaying shorter cellular processes compared with parental C6 cells. More importantly, in conjunction with the morphological changes, the expression of lipocalin-2 (lcn2), a 25-kDa secreted proapoptotic protein, was markedly reduced in the BCNU-resistant C6R cells. However, there was no significant change in the expression of lcn2 receptors. Addition of recombinant LCN2 protein or introduction of lcn2 cDNA significantly increased the sensitivity of C6 cells and human glioma cells to BCNU or other anticancer drugs, while knockdown of lcn2 expression by antisense cDNA transfection decreased the sensitivity. When lcn2 was re-expressed in C6R cells, the BCNU sensitivity was restored. Lcn2 enhanced BCNU-induced Akt dephosphorylation providing a molecular basis of apoptosis sensitization. These results suggest that LCN2 protein may be involved in glioma drug resistance and may provide a new approach to sensitizing glioblastoma to chemotherapy.     

Docosahexaenoic acid affects insulin deficiency- and insulin resistance-induced alterations in cardiac mitochondria

The effect of docosahexaenoic acid (DHA) intake on cardiac mitochondrial function was evaluated in permeabilized fibers in insulin deficiency and insulin resistance in rats. The insulin-deficient state was obtained by streptozotocin injection 2 mo before investigations. Insulin resistance was obtained by feeding a 62% fructose diet for 3 mo. DHA was incorporated in the diet to modify the fatty acid composition of cardiac membranes, including mitochondria. Insulin deficiency decreased mitochondrial creatine kinase (mi-CK) activity and mitochondrial sensitivity to ADP. DHA intake prevented these alterations. Moreover, the insulin-deficient state significantly decreased n-3 polyunsaturated fatty acids (PUFA) and slightly increased n-6 PUFA in both cardiac and mitochondrial membranes, inducing a significant increase in the n-6-to-n-3 ratio. DHA intake maintained high myocardial and mitochondrial DHA content. Insulin deficiency also decreased glutamate- and palmitoylcarnitine-supported mitochondrial respiration, but DHA intake did not prevent these effects. In contrast, insulin resistance did not affect mi-CK activity or sensitivity to ADP. However, insulin resistance influenced the myocardial fatty acid composition with decreased n-6 and n-3 PUFA contents and increased monounsaturated fatty acid content. Only slight alterations were observed in mitochondrial fatty acid composition, and they were corrected by DHA intake. Moreover, insulin resistance decreased the glutamate-supported respiration, and DHA intake did not influence this effect. In conclusion, the impairment of cardiac mitochondrial function was more pronounced in the insulin-deficient state than in insulin resistance. The modification of fatty acid composition of cardiac and mitochondrial membranes by DHA partially prevented the mitochondrial alterations induced in the two models.     

Reactivity of brain benzodiazepine receptors and individual sensitivity of rats to pentylenetetrazole

The individual sensitivity of the male Wistar rats to acute pentylenetetrazole injection was studied, the density and the affinity of benzodiazepine receptors in the cerebellar cortex for 3H-diazepam was measured. It was demonstrated that the reactivity of benzodiazepine receptors underlies the individual sensitivity to pentylenetetrazole. The animals with higher sensitivity were characterized by more intensive reaction than the control and resistant animals, i.e., by an decrease in the receptors density (the initial receptors density being equal in the sensitive, resistant, and control animals). Daily injections of a subconvulsive dose of pentylenetetrazole during 24 days increase the animal sensitivity to this substance, and this was accompanied by an increase in the reactivity of benzodiasepine receptors. Later on, the produced high sensitivity became somewhat lower but persisted for 6 months. The receptors density in this period reduced almost by half. In sensitive rats, a single low dose of pentylenetetrazole injected 6 months after treatment increased the density of benzodiazepine receptors. The age-matched controls, the same acute dose of pentylenetetrazole decreased both the receptor density and affinity of their binding. It is suggested that the increase in reactivity of benzodiazepine receptors is actualized via the intracellular metabotropic feedback mechanism.     

Effects of hyperthyroidism on the sensitivity of glycolysis and glycogen synthesis to insulin in the soleus muscle of the rat.

1. The effects of hyperthyroidism on the sensitivity and responsiveness of glycolysis and glycogen synthesis to insulin were investigated in the isolated incubated soleus muscle of the rat. 2. Hyperthyroidism, which was induced by administration of tri-iodothyronine (T3) to rats for 2, 5 or 10 days, increased fasting plasma concentrations of glucose, insulin and free fatty acids. 3. Administration of T3 for 2 or 5 days increased the rates of glycolysis at all insulin concentrations studied: this was due to increased rates of both glucose phosphorylation and glycogen breakdown, but there was no effect of T3 on the sensitivity of glycolysis to insulin. However, administration of T3 for 10 days increased the sensitivity of the rate of glycolysis to insulin. 4. The concentration of adenosine in the gastrocnemius muscles of the rats was not different from controls after 5 days, but it was markedly decreased after 10 days of T3 administration. If these changes are indicative of changes in the soleus muscle, the increased sensitivity of glycolysis to insulin found after 10 days' T3 administration could be due to the decrease in the concentration of adenosine. 5. Administration of T3 decreased the sensitivity of glycogen synthesis to insulin and the glycogen content of the soleus muscles. This may explain the decreased rates of non-oxidative glucose disposal found in spontaneous and experimental hyperthyroidism in man. 6. The rates of glucose oxidation did not change after 2 days, but they were increased after 5 and 10 days of T3 administration.     

混合单克隆抗体在固相ELISA双夹心法中的应用研究

在进行固相ELISA双夹心法时,要选择两种配对的单克隆抗体(McAb)殊非易事。本文用不同McAb的混合物与另一种McAb进行配对夹心,获得了较好的效果。实验表明,在心肌肌球蛋白轻链(CM—LC)的固相ELISA双夹心体系中,以抗CM-LCMcAb(1G6)铺底,(2B4及2F6)混合物为后续复盖抗体,最低检出量可低达10ng/mL,其检出率较单独2B4或单独2F6作为后续复盖抗体者高5—10倍。而若反之,以(2B4及2F6)混合物铺底,1G6作为后续复盖抗体,则其最低检出量竟高至200ng/mL,还不如以其中之一铺底为佳。在人绒毛膜促性腺激素(HCG)的检测体系中,用多克隆抗体与单克隆抗体配对的研究中,也获得了类似的实验结果。     

Breast sensitivity after vertical mammaplasty

Breast sensation after reduction mammaplasty is a major concern for surgeons and patients. The sensitivity of 80 breasts that were reduced using Lejour's technique (a superior dermoglandular pedicle with resection at the lower quadrants) was assessed in a prospective study. Ten points were selected on each breast for this study, including the nipple, four points on the areola, and five points on the breast skin. The measurements were performed preoperatively and at 3, 6, and 12 months postoperatively. Pressure thresholds were measured with 20 Semmes-Weinstein monofilaments, temperature sensitivity with hot and cold metal probes, vibratory thresholds with the Biotesiometer, and static and moving two-point discrimination tests with a Disk-Criminator. To assess the influence of breast ptosis and hypertrophy on sensitivity, the population was divided into two groups. In group I (19 patients), the sternal notch-to-nipple distance was less than 29 cm, and less than 500 g of tissue per breast was removed. In group II (21 patients), the sternal notch-to-nipple distance was more than 29 cm, and more than 500 g of tissue was resected. The sensitivity on the nipple and areola was significantly decreased at 3 and 6 months postoperatively for all modalities. At 1 year, sensitivity recovered, and no breast or nipple-areola complex was insensitive. Pressure sensitivity was not significantly different from the preoperative measurement in any area of the breast or in either group of patients, except for superior breast skin, for which sensitivity was improved in group II (p = 0.0004). Temperature sensitivity in group I was not different preoperatively and postoperatively, but in group II, a significant decrease was observed in sensitivity for the nipple and areola (p = 0.01 and 0.004, respectively). Vibratory sensitivity was significantly decreased on the nipple, the areola, and the inferior breast skin (p = 0.01, 0.01, and 0.001, respectively) in group II but not in group I.In conclusion, ptotic or moderately hypertrophied breasts that were reduced using Lejour's technique recovered their preoperative level of sensitivity after an initial postoperative decline. However, in large breasts, although pressure sensitivity recovered after 1 year, temperature and vibration sensitivity remained diminished on the nipple-areola complex.     

Effects of eicosapentaenoic acid (EPA) treatment on insulin sensitivity in an animal model of diabetes: improvement of the inflammatory status

In addition to decreased insulin sensitivity, diabetes is a pathological condition associated with increased inflammation. The ω-3 fatty acids have been proposed as anti-inflammatory agents. Thus, the major goal of this study was to analyze the effects of fatty acid supplementation on both insulin sensitivity and inflammatory status in an animal model of type 2 diabetes. Diabetic rats (Goto-Kakizaki model) were treated with eicosapentaenoic acid (EPA) or linoleic acid at 0.5 g/kg body weigh (bw) dose. In vivo incorporation of (14)C-triolein into adipose tissue was improved by the ω-3 administration. In vitro incubations of adipose tissue slices from EPA-treated rats showed an increase in (14)C-palmitate incorporation into the lipid fraction. These observations were linked with a decreased rate of fatty acid oxidation. EPA treatment resulted in a decreased fatty acid oxidation in incubated strips from extensor digitorum longus (EDL) muscles. The changes in lipid utilization were associated with a decrease in insulin plasma concentration, suggesting an improvement in insulin sensitivity. These changes in lipid metabolism were associated with an activation of AMP-activated protein kinase (AMPK) in white adipose tissue. In addition, EPA treatment resulted in a decreased content of peroxisome proliferator-activated receptor-α (PPARα) and PPARδ and in increased GLUT4 expression in skeletal muscle. Moreover, EPA increased 2-deoxy-D-[(14)C]glucose (2-DOG) uptake in C2C12 myotubes, suggesting an improvement in glucose metabolism. Concerning the inflammatory status, EPA treatment resulted in a decreased gene expression for both tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) both in skeletal muscle and adipose tissue. The data suggest that EPA treatment to diabetic rats clearly improves lipid metabolism although the evidences on insulin sensitization are less clear.     

Determination of Herpes simplex virus DNA with the use of solid-phase methods for the detection of PCR products

To determine DNA of herpes simplex virus (HSV), types 1 and 2, the polymerase chain reaction (PCR) method was developed with the subsequent detection of amplification products by means of electrophoresis or the molecular hybridization of nucleic acids (MHNA). Two variants of MHNA have been compared: hybridization in the solution of a biotinylated probe with digoxigenin-labeled PCR with the subsequent sorption of hybridization complexes onto streptavidin-covered plates and solid-phase hybridization of digoxigenin-labeled PCR with a biotinylated probe. Effective hybridization was observed after the denaturation of targets at 95 degrees C in the solution of 50 mM NaOH, but not in neutral solutions. To increase the level of sensitivity of hybridization in solution, the exact selection of the amount of the probe was shown to be necessary, for both its excess and deficiency essentially decreased the method sensitivity. A decrease in the ionic power of hybridization solutions from 6 h SSC to 1 h SSC led to greater specificity of hybridization without a decrease in the method sensitivity. A rise in the temperature of hybridization and subsequent washing to 45 degrees C decreased the sensitivity of the method. The limit of the sensitivity PCR with electrophoretic detection was 30 HSV genome equivalents, and 10 genome equivalents in the presence MHNA in the solution and on the solid phase.     

Nucleotide pools and mutagenic effects of alkylating agents in wild-type and APRT-deficient Friend erythroleukaemia cells

Wild-type Friend mouse erythroleukaemia cells (clone 707) were compared with adenine phosphoribosyltransferase (APRT)-deficient mutant subclones (707DAP8 and 707DAP10) for sensitivity to cell killing and mutagenesis by ethyl methanesulphonate (EMS) and methyl methanesulphonate (MMS). Cells were exposed to 0-300 micrograms/ml EMS and to 0-20 micrograms/ml MMS for a period of 16 h. A slight difference was found between wild-type cells and the two APRT-deficient subclones in terms of sensitivity to cell killing by both mutagens. The APRT-deficient subclones were, however, significantly more sensitive than wild-type cells to mutagenesis to 5-bromo-2-deoxyuridine resistance and 6-thioguanine resistance by EMS and MMS. The APRT-deficient subclones were found to have significantly decreased levels of dATP and dTTP nucleotides and decreased levels of all four ribonucleoside triphosphates (ATP, GTP, CTP and UTP) relative to wild-type cells. Wild-type Friend cells were found to have insignificant levels O6-methylguanine-DNA methyl transferase and it is suggested that the increased mutagen sensitivity of APRT-deficient cells may be due to imbalance of deoxyribonucleoside triphosphate pools during DNA excision-repair processes, or more probably due to deficiency of ATP for ATP-dependent DNA excision-repair enzymes.     

Involvement of a putative response regulator FgRrg-1 in osmotic stress response, fungicide resistance and virulence in Fusarium graminearum

Response regulator (RR) proteins are core elements of the high-osmolarity glycerol (HOG) pathway, which plays an important role in the adaptation of fungi to a variety of environmental stresses. In this study, we constructed deletion mutants of two putative RR genes, FgRRG-1 and FgRRG-2, which are orthologues of Neurospora crassa RRG-1 and RRG-2, respectively. The FgRRG-1 deletion mutant (ΔFgRrg1-6) showed increased sensitivity to osmotic stress mediated by NaCl, KCl, sorbitol or glucose, and to metal cations Li(+) , Ca(2+) and Mg(2+) . The mutant, however, was more resistant than the parent isolate to dicarboximide and phenylpyrrole fungicides. Inoculation tests showed that the mutant exhibited decreased virulence on wheat heads. Quantitative real-time polymerase chain reaction assays indicated that the expression of FgOS-2, the putative downstream gene of FgRRG-1, was decreased significantly in ΔFgRrg1-6. All of the defects were restored by genetic complementation of ΔFgRrg1-6 with the wild-type FgRRG-1 gene. Different from the FgRRG-1 deletion mutant, FgRRG-2 deletion mutants were morphologically indistinguishable from the wild-type progenitor in virulence and in sensitivity to the dicarboximide fungicide iprodione and osmotic stresses. These results indicate that the RR FgRrg-1 of F. graminearum is involved in the osmotic stress response, pathogenicity and sensitivity to dicarboximide and phenylpyrrole fungicides and metal cations.     

Effect of serum on the plasma membrane fluidity of hybridomas: an insight into its shear protective mechanism.

We have previously shown that decreasing the concentration of fetal bovine serum (FBS) increased the fragility of a mouse hybridoma (HB-32) during agitated batch cultivation and that increasing the plasma membrane fluidity (PMF) increased the shear sensitivity during exposure to laminar flow. In this study, the effect of FBS concentration on the PMF of HB-32 was investigated. PMF was evaluated by steady-state fluorescence anisotropy (rs) of 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene. Increasing serum concentration increased the rs of hybridomas, indicating a decrease in their PMF. The effect of cholesterol modulation on the PMF and shear sensitivity was also evaluated. Hybridomas were exposed to turbulent fluid shear after modification of PMF by cholesterol modulation. Direct cholesterol enrichment of the plasma membranes caused a decrease in the PMF and shear sensitivity, while cholesterol depletion caused an increase in PMF and shear sensitivity. Low- and high-density lipoprotein supplementation to cultures in serum-free or complete medium decreased their shear sensitivity. Lipoprotein supplementation to serum-free cultures decreased the PMF. Altogether, these results suggest that the protective mechanism of serum against hydrodynamic damage relies, at least partially, on its ability to decrease the PMF of hybridomas possibly through the transfer of cholesterol from the serum lipoproteins into the plasma membrane.