微量NAG酶释放法鉴定牛血清促进细胞生长的活性

目的：检查牛血清促进细胞生长的活性。方法：微量NAG酶释放法。结果：在同一实验条件下，试验血清（990507，990402与对照血清（990405）对促进K562细胞生长的活性没有明显判别，但血清的促进细胞生长活性随着储存时间的增加而降低，如试验血清（97062）。结论：利用微量NAG酶释放法可以筛查新生牛血清促进细胞生长的活性。     

一种新的细胞毒测定法：NAG酶荧光比色法

用NAG酶释放法检测细胞毒，因常规底物PNP－NAG灵敏度低而无法使用。故使用发荧光底物4Mu－NAG，提高了方法灵敏度，同时对诸如靶细胞数、底物浓度、NP－40的影响，适宜的效靶比等一系列因素进行了检测。确定了最佳反应条件，建立了新方法，并用该方法测出了LAK细胞的活性曲线，与文献对比，结果一致。从而证明NAG酶荧光比色法是测定细胞毒的良好方法。     

肿瘤患者外周血NK细胞活性与sIL-2R水平的研究

目的探讨肿瘤患者外周血NK细胞活性与血清sIL-2R的临床意义。方法应用LDH释放法及双抗体夹心酶联免疫吸附试验(ELISA)对40例正常人、114例恶性肿瘤患者进行了外周血NK细胞活性和血清sIL-2R水平检测。结果与正常对照组相比,肿瘤患者外周血NK细胞活性显著降低(P<0.01),血清sIL-2R水平明显增高(P<0.01);上述改变在伴转移患者中更明显,与不伴转移者相比,差异显著(P<0.001)。肿瘤患者外周血NK细胞活性与血清sIL-2R水平呈负相关(r=-0.473,P<0.001)。治疗有效的转移癌患者NK活性明显增强(P<0.05),sIL-2R水平显著降低(P<0.01);无效者变化不明显(P>0.05)。结论血清中异常增高的sIL-2R可能是导致肿瘤患者NK细胞活性低下的原因之一。NK细胞活性和sIL-2R可作为研究肿瘤发生、发展及转移的重要免疫学指标,监测肿瘤患者外周血中两者的变化有助于评估疗效和预后。     

脐血淋巴细胞表型、NK细胞活性和血清sIL-2R水平研究

目的:为脐血临床应用提供免疫学依据,方法:采用碱性磷酸酶抗碱性磷酸酶桥联酶标法(APAAP法)、乳酸脱氢酶(LDH)释放法和双抗体夹心酶联免疫吸附试验(ELISA)对足月正常分娩新生儿脐血进行了淋巴细胞表型、NK细胞活性和血清sIL-2R水平研究.并与健康成人外周血进行了比较.结果:与成人外周血相比,脐血CD3~ 和CD8~ T细胞百分比显著降低,CD4~ /CD8~ 比值明显升高,因而CD4~ T细胞百分比相对增高.CD19~ B细胞百分比亦显著增高,CD16~ NK细胞百分比两者无显著性差别,NK细胞活性显著降低,脐血中仅有CD19~ B细胞百分比女性明显高于男性,其它指标均无性别差异,脐血清sIL-2R水平明显高于成人外周血清.     

NAG酶荧光比色测定细胞毒及其应用研究

细胞毒测定作为免疫学的一项基本技术在肿瘤的生物治疗方面已成为检测NK细胞、LAK细胞、CTL细胞、细胞因子活性、药敏实验等一切杀伤活性的必不可少的手段.我室建立的NAG酶荧光比色法测定细胞毒,较经典的~(51)Cr释放     

精氨酸酶对人APL原代细胞生长和分化的影响

了解精氨酸酶对人急性早幼粒白血病原代细胞生长和分化的影响。方法 :在RPMI 16 40培养液中补加 10 %胎牛血清和精氨酸酶 5 0 0U/L ,与对照组培养液同时置于 37℃孵箱中孵育 2 4h ,以活细胞密度 5× 10 8/L接种来自 18例急性早幼粒白血病病人的原代白血病细胞 ,在 37℃ ,5 %CO2 和饱和湿度下培养。结果 :培养 4d计数活细胞 ,发现其密度为起始密度的 (6 4 2 8± 3 12 ) % ,而对照组为 (10 8 5 6± 12 2 7) % (P <0 0 0 1) ;收集细胞行Wright Giemsa、DNA、POX、NAE及NaF抑制试验、墨汁吞噬试验和NBT还原试验等细胞化学染色 ,油镜下观察发现 ,白血病细胞向成熟分叶核粒细胞分化。结论 :精氨酸酶对APL原代细胞有抑制生长和引导分化作用     

大鼠卵巢颗粒细胞的原代培养与鉴定

背景与目的： 探讨大鼠卵巢高纯度颗粒细胞培养及鉴定的方法,建立一种简便、稳定的细胞模型。 材料与方法： 选用SD雌性大鼠(21～25 d),皮下注射孕马血清促性腺激素(PMSG),48 h后用颈椎脱臼法处死,剖取双侧卵巢,采用机械分离方法释放卵巢颗粒细胞,0.25％胰蛋白酶消化结合低速离心分离细胞,用含15％胎牛血清的DMEM/F12培养基,置于37 ℃,5％ CO2培养箱培养。以HE染色和卵泡刺激素受体(FSHR)免疫组化染色对所培养的卵巢颗粒细胞进行鉴定,用MTT法绘制细胞生长曲线,并检测细胞培养液雌二醇(E2)和孕酮(P)的分泌量。 结果： 分离培养的卵巢颗粒细胞存活率>90％,细胞纯度达到95％以上;体外培养的颗粒细胞对数生长期为48～96 h;颗粒细胞具有正常的分泌雌激素功能,在培养24 h细胞培养液中E2和P的分泌量分别为(10.36±15.89) pg/ml和(77.91± 17.24) pg/ml。 结论： 采用机械分离法结合胰蛋白酶消化及低速离心法分离培养的卵巢颗粒细胞纯度高、活性好,用FSHR表达染色鉴定颗粒细胞是一种简便快速的方法。     

精氨酸酶对人APL原工细胞生长和分化的影响

目的 :了解精氨酸酶对人急性早幼粒白血病原代细胞生长和分化的影响。方法 :在RPMI 16 40培养液中补加 10 %胎牛血清和精氨酸酶 5 0 0U/L ,与对照组培养液同时置于 37℃孵箱中孵育 2 4h ,以活细胞密度 5× 10 8/L接种来自 18例急性早幼粒白血病病人的原代白血病细胞 ,在 37℃ ,5 %CO2 和饱和湿度下培养。结果 :培养 4d计数活细胞 ,发现其密度为起始密度的 (6 4 2 8± 3 12 ) % ,而对照组为 (10 8 5 6± 12 2 7) % (P <0 0 0 1) ;收集细胞行Wright Giemsa、DNA、POX、NAE及NaF抑制试验、墨汁吞噬试验和NBT还原试验等细胞化学染色 ,油镜下观察发现 ,白血病细胞向成熟分叶核粒细胞分化。结论 :精氨酸酶对APL原代细胞有抑制生长和引导分化作用     

嵌合抗原受体T细胞治疗相关细胞因子释放综合征小鼠模型的建立

目的建立嵌合抗原受体T(CAR-T)细胞治疗相关细胞因子释放综合征(CRS)的小鼠模型。方法通过分子克隆及慢病毒转染技术, 构建靶向人CD19分子的CAR-T细胞, 采用流式细胞术检测CAR-T细胞转染效率, 酶联免疫吸附试验(ELISA)法及流式细胞术检测CAR-T细胞特异性杀伤靶细胞的能力。通过尾静脉注射CAR-T细胞至荷瘤重症联合免疫缺陷裸鼠体内, 小鼠分为磷酸缓冲液组、低负荷组(注射1×105个人淋巴瘤细胞Raji-Luc2细胞)和高负荷组(注射5×105个Raji-Luc2细胞)。采用动物活体成像法检测肿瘤治疗效果, ELISA法检测小鼠血清中细胞因子人白细胞介素2(IL-2)、人γ-干扰素(IFN-γ)、鼠IL-6、鼠粒细胞-巨噬细胞集落刺激因子(GM-CSF)的水平。结果经尾静脉注射人T细胞和T细胞+OKT-3抗体后, T细胞组和T细胞+OKT-3组小鼠的健康得分分别为(1.15±0.08)分和(2.90±0.15)分, 差异有统计学意义(P<0.001)。T细胞+OKT-3组小鼠血清中人IL-2、人IFN-γ、人IL-15、鼠IL-6、鼠GM-CSF水平分别为(1...     

转染CD40配体的卵巢癌细胞株OVHM抗卵巢癌肝转移机制的研究

目的 探讨转染CD40配体(CIMOL)的卵巢癌细胞株OVHM(CD40L-OVHM)体内抗卵巢癌肝转移的可能机制.方法 6～8周龄的C57BL/6N×C3H/He杂交一代(B6C3F1)雌性小鼠5只,经小鼠脾脏内接种OVHM,应用HE染色法验证小鼠肝脾转移模型是否建立成功.将OVHM细胞、空载体DNA-pMKITneo-OVHM细胞和CD40L-OVHM细胞接种于B6C3F1小鼠的脾脏,应用流式细胞术,分析荷瘤小鼠脾细胞CD11c分子的表达情况;应用四甲基偶氮唑蓝(MTT)法,检测荷瘤小鼠脾脏细胞毒性T淋巴细胞(CTL)的杀伤活性;应用酶联免疫吸附试验(ELISA),检测荷瘤小鼠外周血血清中干扰素(IFN)-γ、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-12、IL-4和IL-10的含量,并且观察小鼠脾脏和肝脏的成瘤情况及小鼠生存时间.结果 经病理学证实,卵巢癌肝脾转移动物模型建立成功.CD40L-OVHM组中小鼠脾细胞CD11c阳性细胞率明显高于OVHM组和转染空载体DNA-pMKITneo-OVHM组.CD40L-OVHM组小鼠脾脏内CTL的特异性杀伤活性明显增强,小鼠外周血血清中IFN-γ、TNF-α和IL-12的含量明显升高,而IL-4和IL-10的含量则明显降低,与OVHM组和转染空载体DNA-pMKITneo-OVHM组相比,差异均有统计学意义(P<0.05).CD40L-OVHM组小鼠的肝脏和脾脏重量均明显低于OVHM组和转染空载体DNA-pMKITneo-OVHM组,并且小鼠的生存期也明显延长(P<0.05).结论 转染CD40L cDNA的卵巢癌细胞可促进脾脏树突状细胞(DC)的成熟和分化,增强脾脏CTL的特异性杀伤活性,诱导Th1型细胞因子的分泌,抑制Th2型细胞因子的分泌,这可能是CD40L基因体内抗卵巢癌肝转移的机制之一.     

Long-term cultivation of a human breast cancer cell line, MCF-7, in a chemically defined medium. Effect of estradiol

The human breast cancer cell line, MCF-7, has been adapted to long-term growth in chemically defined medium without loss of estrogen and progesterone receptors or tumorigenicity in athymic mice. An estrogen reversible inhibition of cell proliferation is exerted by newborn calf serum (NCS) 10%, athymic mouse serum (AMS) 2% or tamoxifen 0.1 to 1.0 microM. The mamma-related hormones, hydrocortisone, progesterone, prolactin, and insulin could not mimic this growth inhibitory effect, and estradiol alone or combined with these hormones did not stimulate cell proliferation in chemically defined medium.     

Indirect mechanism of oestradiol stimulation of cell proliferation of human breast cancer cell lines

The human breast cancer cell line MCF-7 requires oestrogen to produce and promote growth of tumours in athymic mice. In vitro, however, MCF-7 cells proliferate rapidly without supply of oestrogen (Briand & Lykkesfeldt, 1984). Oestrogen stimulation of proliferation of MCF-7 cells can be achieved when the cells are grown at high concentration of newborn calf serum (NCS, 10%) or oestrogen deprived foetal calf serum (10%). The stimulation involves an abolishment of inhibitory activity present in the serum. The oestradiol stimulated cultures grow rapidly for a much longer time period and attain a much higher cell density than the unstimulated cultures. Oestrogen is specific for the promotion of cell proliferation and only oestrogen receptor positive cell lines with a functional oestrogen receptor mechanism can be stimulated. We assume that oestradiol acts directly on the cells and via the oestrogen receptor mechanism induces the synthesis of a substance which abolishes the inhibitory activity in serum. We call this mechanism of action an indirect stimulation of cell proliferation. A similar mechanism may exist in vivo since we find that serum from athymic mice contains a growth inhibitory activity towards MCF-7 cells and the inhibitory effect can be abolished by oestradiol.     

晚期糖基化终产物在子宫内膜癌患者血清中的表达及临床意义

目的:通过研究晚期糖基化终产物(AGEs)在子宫内膜癌(EC)患者血清中的浓度来研究其在子宫内膜癌患者血清中的表达,并探讨其浓度变化对于子宫内膜癌临床辅助诊断的意义.方法:应用ELISA法对106例子宫内膜癌人群、45例糖尿病人群,41例健康人群的血清进行检测,观察血清中晚期糖基化终产物浓度的变化.结果:子宫内膜癌人群血清中晚期糖基化终产物的浓度均较糖尿病人群及健康人群升高(P<0.05).糖尿病人群血清中晚期糖基化终产物浓度较健康人群升高(P<0.05).在子宫内膜癌人群中,合并糖尿病人群血清中晚期糖基化终产物浓度高于单纯子宫内膜癌人群(P<0.05),且与血糖控制是否良好无关(P>0.05).晚期糖基化终产物浓度与分化程度相关(在低分化子宫内膜癌中浓度明显升高,与高分化及中分化相比P<0.05),而与临床分期、肌层浸润、淋巴结转移无关(P>0.05).结论:晚期糖基化终产物在子宫内膜癌人群及糖尿病人群血清中表达升高,在子宫内膜癌合并糖尿病人群中升高更为明显,在低分化子宫内膜癌中表达较高分化及中分化升高.晚期糖基化终产物有望成为早期筛查子宫内膜癌的生物学标志.     

Cell cycle analysis of estrogen stimulated growth of the human breast cancer cell line,MCF-7

The estrogen receptor positive human breast cancer cell line, MCF-7, can be growth inhibited by high concentrations of newborn calf serum (NCS). MCF-7 cells grown with high concentration of NCS can be growth stimulated by 10⁻⁸ M estradiol, and the growth stimulation seems to involve the abolishment of the effect of inhibitory activity in serum. Flow cytometry has been used to determine cell cycle parameters such as distribution of cells in the different phases of the cell cycle and growth fraction. The cell cycle analysis revealed that addition of 10% NCS to cultures grown with 0.5% fetal calf serum (FCS) increased the doubling time by elongating the G1 transit time. Estradiol stimulation occurred through a shortening of the G1 transit time. An effect on growth fraction was observed neither during growth inhibition with high NCS concentration nor during growth stimulation with estradiol. Cells which are growth stimulated by estradiol have an activated estrogen receptor mechanism as indicated by the presence of filled nuclear estrogen receptors and high level of progesterone receptors. We suppose that a possible mechanism for this estrogen stimulation could be induction of synthesis of growth factors which annul the effect of the inhibitory activity present in NCS.     

Regulation of DNA synthesis and growth of cells derived from primary human meningiomas

We have studied the effects of insulin, epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor, and steroid hormones (estradiol, progesterone, and cortisol) on human meningioma cell proliferation and DNA synthesis in a serum-free culture system. The growth factors, particularly EGF and FGF, increased DNA synthesis in a dose-dependent manner as measured by [3H]thymidine incorporation, and they stimulated submaximal cell proliferation. No individual factor or combination of factors was able to successfully reproduce the effects of 10% fetal calf serum (FCS) on cell growth, although a combination of platelet-derived growth factor (5 units/ml) and EGF (10 ng/ml) synergistically stimulated DNA synthesis to near maximal levels. In addition, serum dependency was observed in studies involving the mitogenic effects of insulin, EGF, or FGF. Both EGF and FGF (10 ng/ml) maximally stimulated cell growth in the presence of 5% FCS. The effects of steroid hormones on cell proliferation, individually or in combination with growth factors or charcoal-treated FCS, were also evaluated. Estradiol (100 nM) significantly increased cell number over control values only in the presence of charcoal-treated FCS; no effects of progesterone or cortisol on cell proliferation were observed. In conclusion, both EGF and FGF stimulated cell proliferation and DNA synthesis in human meningioma cultures in a serum-free system, whereas steroid hormones were without effect. It appears that additional serum components are required for both estradiol-stimulated growth and for maximal proliferation of human meningioma cells under serum-free conditions.     

Control of proliferation of MCF-7 breast cancer cells in a commercial preparation of charcoal-stripped adult bovine serum

Summary A commercial preparation of charcoal-stripped adult bovine serum was used to culture MCF-7 cells in estrogen-free media. Use of this stripped adult bovine serum represents an alternative to calf serum which is in more limited supply, and saves charcoal-stripping of serum in the laboratory, which can be a rate-limiting step in the preparation of materials for estrogen-free tissue culture. MCF-7 cell proliferation was controlled by estrogens, epidermal growth factor (EGF) and lithium chloride in adult bovine serum as well as in standard media prepared with charcoal-stripped calf serum, and approximately the same fold-increase in response to the tested agents was observed in the two sera. Although the growth rates were lower in media prepared with adult bovine serum, MCF-7 cells in both media exhibited the same sensitivities in dose-responses to these three mitogens. Levels of estrogen and progesterone receptors, and the magnitude of estrogen-dependent stimulation of the progesterone receptors, were similar in cells maintained in both sera. Therefore, a commercially stripped adult bovine serum can be used to replace calf serum in the study of estrogenic responses and the control of proliferation in MCF-7 breast cancer cells.     

Netilmicin effect on urinary retinol binding protein (RBP) and N-acetyl-beta-D-glucosaminidase (NAG) in preterm newborns with and without anoxia

The study aim was to evaluate urinary excretion of Retinol Binding Protein (RBP), compared with urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG), in preterm infants with anoxia and netilmicin treatment. Urinary RBP and NAG were evaluated in 83 preterm newborns divided in 4 groups: 37 healthy preterm newborns (controls); 14 with neonatal anoxia; 16 treated with ampicillin + netilmicin; 16 with neonatal anoxia and treated with ampicillin + netilmicin. RBP was determined by an automated nephelometric technique and NAG by a colorimetric method on 5-h urine samples in the first week of life. Results showed that urinary excretion of RBP (average from first week values) was 1.06+/-0.67 g/mol creatinine (mean +/- SD) in controls, 1.99+/-1.41 in antibiotic-treated newborns, 3.99+/-4.57 in anoxic newborns and 3.75+/-3.48 in anoxic newborns under antibiotic treatment. When gestational age was not considered, a marked effect of anoxia (P<0.001) and a borderline effect of netilmicin (P<0.059) on RBP excretion were detected by ANOVA. However when gestational age was also considered by analysis of covariance, it appeared as the strongest predictor of RBP excretion (P<0.001), while the effect of netilmicin was no longer significant (P=0.181). The effect of anoxia persisted, although less remarkable (P=0.010). Conversely anoxia did not affect urinary NAG excretion, which was rather correlated with gestational age and netilmicin administration. The authors conclude that RBP and NAG urinary excretion may be used to discriminate between neonatal anoxia and netilmicin treatment, respectively as etiologic factors of renal tubular damage in the newborn.     

Renal tubular dysfunction and urinary zinc excretion in breast cancer patients treated with anthracycline-based combination chemotherapy

The survival of breast cancer patients has significantly improved through the treatment with anthracyclines. Although anthracyclines are known to produce renal disease in experimental animals, little is known about the toxicity of anthracyclines at clinically relevant doses in humans. In a previous study on cancer patients we have observed an increase in the urinary activity of N-acetyl-beta-D-glucosaminidase (NAG), an indicator of renal tubular cell dysfunction that was accompanied by increased urinary zinc loss. Because an increase in NAG activity was reported after the treatment with anthracyclines, we hypothesized that an increase in urinary NAG activity in breast cancer patients treated with anthracycline-based regimens will be accompanied by hyperzincuria and hypozincemia. Urinary and serum zinc, urinary NAG and serum creatinine were examined during chemotherapy in 26 breast cancer patients treated with anthracycline-based chemotherapy. A trend for increased NAG activity, as compared to baseline, was observed throughout the first 4 cycles of treatment. NAG activity was significantly elevated compared to pretreatment levels one week after the first, third and fourth dose of chemotherapy. Serum creatinine concentrations decreased significantly after the second cycle of therapy. On the other hand, urinary and serum zinc levels did not change significantly during the treatment. In conclusion, our data confirm the presence of mild renal tubular cell dysfunction in breast cancer patients treated with doxorubicin-based chemotherapy. Increased urinary NAG is accompanied by a decrease in serum creatinine which is consistent with hyperfiltration. These changes are not associated with abnormalities of renal zinc handling or a decrease in serum zinc concentrations.     

Effects of Phorbol Esters and Cytokines (Interleukin-2,-4, and -6) on the Proliferation and Surface Phenotype of Epstein-Barr Virus Immortalised Human B Lymphocytes

Epstein-Barr virus (EBV)-induced in vitro infection of peripheral blood mononuclear cells (PBMCs) leads to a polyclonal proliferation and immortalisation of B lymphocytes. In the present study we determined the effects of three different cytokines, interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-6 (IL-6), and the tumour promoting phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on EBV-immortalised B lymphocytes. These factors have known activities on normal B cells. IL-4 and IL-6 increased significantly EBV-B cell proliferation after 3 and 5 days of culture, where IL-2 had no effect. The effect of IL-4 and IL-6 on EBV-B cells was abolished after pre-incubation with anti-IL-4 and anti-IL-6 neutralising antisera, respectively. TPA induced a dose dependent inhibition of proliferation both in serum free and 10% fetal calf serum (FCS) supplemented culture medium. Combinations of TPA and interleukins did not restore lymphoblastoid cell proliferation to background levels. All possible combinations of the three cytokines showed no synergistic or antagonistic effect on proliferation. TPA induced significant phenotypic changes of EBV immortalised B lymphocytes, by increasing IL-2 receptor (IL-2R) expression and decreasing CD20 and CD23 antigen expression. Other B cell differentiation antigens; HLA-DR, CD19, and transferrin receptor (CD71), did not demonstrate significant changes. A dose dependent inhibition of CD21 and increase in CD22 expression was observed in 2 out of 3 lymphoblastoid cell lines tested.     

Netilmicin Effect on Urinary Retinol Binding Protein (RBP) and N-Acetyl-Beta-D-Glucosaminidase (NAG) in Preterm Newborns with and without Anoxia

Abstract

The study aim was to evaluate urinary excretion of Retinol Binding Protein (RBP), compared with urinary excretion of N-acetyl-β-D-glu-cosaminidase (NAG), in preterm infants with anoxia and netilmicin treatment. Urinary RBP and NAG were evaluated in 83 preterm newborns divided in 4 groups: 37 healthy preterm newborns (controls); 14 with neonatal anoxia; 16 treated with ampicillin + netilmicin; 16 with neonatal anoxia and treated with ampicillin + netilmicin. RBP was determined by an automated nephelometric technique and NAG by a colorimetric method on 5-h urine samples in the first week of life. Results showed that urinary excretion of RBP (average from first week values) was 1.06±0.67 g/mol creati-nine (mean ± SD) in controls, 1.99±1.41 in antibiotic-treated newborns, 3.99±4.57 in anoxic newborns and 3.75±3.48 in anoxic newborns under antibiotic treatment. When gestational age was not considered, a marked effect of anoxia (P<0.001) and a borderline effect of netilmicin (P<0.059) on RBP excretion were detected by ANOVA. However when gestational age was also considered by analysis of covariance, it appeared as the strongest predictor of RBP excretion (P<0.001), while the effect of netilmicin was no longer significant (P=0.181). The effect of anoxia persisted, although less remarkable (P=0.010). Conversely anoxia did not affect urinary NAG excretion, which was rather correlated with gestational age and netilmicin administration. The authors conclude that RBP and NAG urinary excretion may be used to discriminate between neonatal anoxia and netilmicin treatment, respectively as etiologic factors of renal tubular damage in the newborn.