牙龈卟啉单胞菌脂多糖对活性中性粒细胞胞外诱捕网形成的影响

目的:探讨牙龈卟啉单胞菌(P.gingivalis)脂多糖(LPS)对活性中性粒细胞胞外诱捕网(NETs)形成的影响及可能的调控机制。方法:采用25 ng/mL粒细胞-巨噬细胞集落刺激因子(GM-CSF)和0.3μg/mL P.gingivalis LPS刺激人中性粒细胞35 min,免疫荧光显微镜下观察活性NETs的形成,酶标法对胞外DNA定量,PCR法检测构成NETs的DNA来源。检测产生活性NETs的中性粒细胞感染P.gingivalis后胞内外的细菌数量,以明确NETs的杀菌效果;流式细胞术检测细胞产生的活性氧(ROS)水平;Western blot检测丝氨酸/苏氨酸蛋白激酶(Raf)、丝裂原活化的细胞外信号调节激酶(MEK)和细胞外信号调节蛋白激酶(ERK)的磷酸化水平。结果:GM-CSF+P.gingivalis LPS可促进线粒体DNA来源的活性NETs形成和胞外DNA数量的增加(P<0.05),增强中性粒细胞对P.gingivalis的杀菌活性(P<0.05)。与阴性对照组相比,GM-CSF+P.gingivalis LPS刺激的中性粒细胞ROS和Raf、MEK、ERK磷酸化水平均明显增高(P<0.05)。还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶抑制剂DPI处理后,GM-CSF+P.gingivalis LPS组胞外DNA数量降低,ROS水平降低(P<0.01)。结论:GM-CSF+P.gingivalis LPS可能通过Raf、MEK、ERK磷酸化调控ROS生成,促进人中性粒细胞形成活性NETs,进而促进P.gingivalis的清除,调控牙周免疫炎症反应。

中性粒细胞碱性磷酸酶活性在血液病鉴别中的临床应用

目的探讨中性粒细胞碱性磷酸酶(NAP)活性在血液病患者鉴别诊断中的临床应用。方法对260例血液系统疾病患者外周血涂片用化学染色法染色并作积分判断。并以50例健康体检者作正常对照。结果急性髓细胞白血病(AML),慢性粒细胞白血病(CML)慢性期,阵发性睡眠性血红蛋白尿(PNH),骨髓增生异常综合征(MDS-RA)患者的NAP阳性率积分均明显低于正常对照组(P<0.01)。而急性淋巴细胞白血病、骨髓增殖性疾病(如慢性中性粒细胞白血病、骨髓纤维化、真性红细胞增多症、原发性血小板增多症),再生障碍性贫血(AA)、慢性淋巴细胞白血病(CLL)、类白血病反应患者的NAP阳性率积分均明显高于正常对照组(P<0.01)。特发性血小板减少性紫癜(ITP),骨髓增生异常综合征(MDS-RAS),缺铁性贫血与正常对照组比较差异无统计学意义(P>0.05)。结论根据NAP活性特征可以用于血液病的鉴别和诊断。

体外受精-胚胎移植妇女体内颗粒细胞线粒体活性和左旋肉碱水平的关系

目的探讨行IVF-ET妇女体内颗粒细胞线粒体活性与血浆、卵泡液左旋肉碱水平三者的关系,及其与年龄、优胚率、受精率及妊娠结局等的关系。方法应用流式细胞仪法测定颗粒细胞线粒体活性,柱前衍生-高效液相色谱法测定游离左旋肉碱水平。结果妊娠组颗粒细胞线粒体活性高于未妊娠组(P<0.05)。颗粒细胞线粒体活性与年龄呈负相关,与受精率、正常受精率、优胚率呈正相关(P<0.05)。妊娠组卵泡液左旋肉碱水平低于未妊娠组(P<0.05),血浆左旋肉碱水平与年龄呈正相关(P<0.05),血浆左旋肉碱水平与颗粒细胞线粒体活性呈负相关(P<0.05)。结论颗粒细胞线粒体活性的降低可能与年长妇女IVF-ET受精率低、胚胎质量差、妊娠结局差有关。妇女体内颗粒细胞线粒体活性下降时,血浆左旋肉碱水平可能代偿性增加。

失血性休克时雌激素能保护红细胞的变形能力和中性粒细胞活性

最近，美国学者研究了创伤失血性休克（T／HS）时雌激素对血液中细胞的保护作用。激素正常和卵巢切除（OVX）后的雌性大鼠在经历T／HS[剖腹放血至平均动脉压约为30～35mmHg（1mmHg=0．133kPa）并持续90min]或假创伤休克（T／SS）后再灌注3h，

中性粒细胞碱性磷酸酶活性在骨髓增殖性肿瘤中的诊断价值

目的:分析中性粒细胞碱性磷酸酶(NAP)活性在骨髓增殖性肿瘤(MPN)中的诊断价值。方法:选取我院2020年11月11日-2021年4月11日收治的MPN患者23例作为试验组,继发性红细胞增多和继发性血小板增多患者24例作为对照组1,同期健康体检者26例作为对照组2,均应用偶氮偶联法进行NAP测定,分析NAP阳性率和NAP积分在MPN中的诊断价值。结果:试验组骨髓纤维化(MF)、原发性血小板增多症(ET)、真性红细胞增多症(PV)患者NAP阳性率和积分均高于对照组1和对照组2,差异均有统计学意义(P<0.05)。试验组CML患者NAP阳性率和积分均低于对照组1和对照组2,差异均有统计学意义(P<0.05)。试验组中CML、ET、MF患者NAP阳性率和积分均低于PV患者,差异均有统计学意义(P<0.05)。试验组中慢性粒细胞性白血病(CML)患者NAP阳性率和积分均低于ET、MF患者,差异均有统计学意义(P<0.05)。MF患者NAP阳性率和积分均高于ET患者,差异均有统计学意义(P<0.05)。结论:NAP活性在MPN中具有一定的诊断价值,可作为重要的参考指标,用于该类疾病的鉴别诊断。

重组人粒细胞集落刺激因子生物学活性工作标准品的标定

目的用世界卫生组织 (WHO)粒细胞集落刺激因子 (G CSF)生物学活性国际标准品对所制备的重组人粒细胞集落刺激因子 (rhG CSF)生物学活性工作标准品以及惠尔血 针剂 75进行标定。方法用NFS 6 0细胞采用MTT法进行rhG CSF生物学活性的测定 ,(4 ,4)法计算测定结果。结果制备rhG CSF生物学活性工作标准品 3批 ,两批为冻干剂 ,与G CSF生物学活性国际标准品配方一致 ,一批为水剂 ,与惠尔血 针剂 75配方一致 ;用G CSF生物学活性国际标准品对其标定 ,结果依次为 3.0 6 2× 10 6、4.2 76× 10 6IU/支及 1.6 35× 10 7IU/ml,样品为1.880× 10 7IU/ml。标定结果的FL %分别为 5 .5 2 9%、4.2 91%、4.2 44 %及 5 .175 %。结论所标定的rhG CSF生物学活性工作标准品可用于rhG CSF生物学活性测定。

重组人粒细胞集落刺激因子生物学活性标准品的免疫学活性测定

目的测定重组人粒细胞集落刺激因子 (rhG CSF)生物学活性标准品的免疫学活性。方法用深圳晶美公司及美国R&D公司的G CSFELISA试剂盒分别对WHOrhG CSF生物学活性国际标准品、惠尔血针剂 75 (GRAN 75 )及用WHOrhG CSF生物学活性国际标准品标定过生物学活性的工作标准品T1、T2、T3进行免疫学活性测定。结果不同公司的rhG CSFELISA试剂盒对所测定样品的测定结果不同 ,且用不同稀释液稀释样品测定结果亦不同。结论用免疫学方法定量测定rhG CSF的含量是有条件的 ,且免疫反应能识别聚体但不能识别蛋白分子的降解。

红细胞、内毒素对淋病患者粒细胞吞噬功能影响的实验研究

检测红细胞和内毒素对 30例淋球菌感染 (GI)患者变形核粒细胞 (PMN)吞噬功能的影响。实验结果显示 :GI患者PMN吞噬活性 ,红细胞促粒细胞吞噬活性 (ERPN)均明显低于正常人 (P <0 0 5 )。提示淋病发病机制可能与红细胞免疫功能降低有关。

不稳定型心绞痛患者冠状动脉局部炎性细胞因子的变化及临床意义

目的 研究不稳定型心绞痛 (USA)时炎性细胞因子在冠状动脉局部的变化及其临床意义。方法 测定 USA、稳定性心绞痛 (SA)及正常对照组患者右心房、外周静脉中的血浆血小板 α-颗粒膜蛋白 14 0 (GMP-14 0 )含量及中性粒细胞 (PMN)活性。结果 U SA组冠状动脉局部表现为血小板高度活化、PMN活性明显增强 ,与外周血相比 ,GMP- 14 0含量、PMN活性均存在显著差异 (P <0 .0 5 ,P <0 .0 1) ;与 SA组及正常对照组比较 ,U SA组冠状动脉局部及外周血炎性细胞因子活性均明显增强。结论 USA患者的冠状动脉局部存在急性炎症过程 ,炎症反应激活可诱导血管收缩、血栓形成 。

TPOL triggers apoptosis with mitochondrial injury through activating a ROS-dependent p53/p21/p27/Rb/Bax/Cyto C/caspase-mediated signaling

AIM:To explore the influence of ethyl(2,4,6-trimethylbenzoyl)phenylphosphinate(TPOL)on cell apoptosis and its potential mechanism.METHODS:HEK293T cells sensitive to TPOL were treated with different concentrations of TPOL with or without exposure to light radiation,before treatment with various inhibitors,N-acetyl-Lcysteine(NAC),pifithrin-αand Z-DVED-FMK.Cell viability was measured by CCK-8 assay.Annexin V/propidium iodide staining was used to count the number of apoptotic cells.DCFH-DA staining was used to detect reactive oxygen species(ROS)levels,and JC-1 staining was used to assess mitochondrial membrane potential by flow cytometry.The expression of apoptosis-related proteins and cell cycle-regulated molecules was measured by Western blot.RESULTS:TPOL enhanced the apoptosis of HEK293T cells in a dose-dependent manner(P<0.05),with a decrease in Bcl-2 and increases in Bax and cytochrome C(Cyto C),followed by up-regulation of activated caspase-9 and caspase-3,and the cleavage of PARP(P<0.05).The TPOL-enhanced cleavage of caspase-3 and PARP was rescued by Z-DVED-FMK(P<0.01).TPOL also led to a rapid increase in ROS,a reduction in mitochondrial membrane potential,and the release of Cyto C(P<0.01),all of which could be reversed by the ROS scavenger NAC.Moreover,the TPOL-caused alterations in p21,p27,Rb,and CDK2 were also recovered by the p53 inhibitor pifithrin-α(P<0.05).The TPOL-induced changes in Bax,Bcl-2,cleaved caspase-9,activated caspase-3,and cleaved PARP were subsequently rescued by pretreatment with pifithrin-α(P<0.05).CONCLUSION:TPOL can induce cellular apoptosis with ROS-mediated mitochondrial membrane damage through the activation of a ROS-dependent p53/p21/p27/Rb/Bax/Cyto C/caspase-mediated signal axis.

昆虫过敏原对血细胞的影响

为了进一步研究哮喘发病机制,测定蜚蠊,臭虫和尘螨对嗜酸性粒细胞趋化活性的影响,EDA分别为2.072±0.0105,1.531±0.0015,1.630±0.0041。蜚蠊抗原对嗜酸性粒细胞趋化活性影响大于臭虫,尘螨的抗原。(P<0.01)

Liriodendrin protects SH-SY5Y cells from dopamine-induced cytotoxicity

Aim To investigate the effect of liriodendrin, an extract from Fraxinus sielboldiana blume belonging to the Oleaceae family, on dopamine-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Methods Cell viability was processed when treated with 50 μmol·L^-1 of dopamine for 24 h by MTT assay. Early apoptosis, late apoptosis/necrosis were analyzed by flow cytometry using Annexin V-FITC and propidium iodide （PI） double-staining, respectively. Generation of reactive oxygen species （ROS） was assessed by DCFH-DA, an oxidation-sensitive fluorescent probe. To evaluate mitochondrion membrane potential （Δψm） using flow cytometry with the fluorescent dye Rhodamine 123. The transcriptional level of P53 was studied using RT- PCR. Results The dopamine-induced loss of cell viability was significantly attenuated by liriodendrin treatment at the concentration of 10^-8, 10^-7, 10^-6, 10^-5 and 10^-4 mol·L^-1. The protective effects of liriodendrin （10^-7, 10^-6 and 10^-5 mol·L^-1） on dopamine-induced cytotoxicity may be ascribed to its anti-oxidative properties by reducing ROS level and anti-apoptotic effect via protection of Δψm. In addition, the effect of liriodendrin may involve the P53 pathway in apoptosis. Conclusion Liriodendrin may provide a useful therapeutic strategy for the treatment of neurodegenerative diseases such as Parkinson＇s disease （PD）

Expression of Telomerase Activity in Gastric Cancer Cells

Objective To study the relationship between telomerase activity and biological behavior in human gastric cancer cells and appraise the clinical significance of detecting telomerase activity.Methods The telomerase activity in 47 gastric cancer tissue samples , their matched normal tissues, 7 gastric ulcer and 2 gastric cancer cell lines was detected using a PCR-based non-radioisotopic telomeric repeat amplification protocol (TRAP) assay. Results None of the 47 samples from normal gastric tissues expressed telomerase activity. The 41 of 47 cases of gastric cancer presented telomerase activity with an 87.2 % positive rate (P < 0.001). 2/2 gastric cancer cell lines and 0/7 gastric ulcer line were also positive for telomerase activity. The activity of telomerase was associated with the pathological differentiation of gastric cancer. Conclusion Telomerase activity may be related to the biological behavior of gastric cancer and can help in assessing the malignant potential of gastric cancer. Telomerase activity will be a good diagnostic marker for the detection of gastric cancer.

Study of the expressions of p53 and bcl-2 genes, the telomerase activity and apoptosis in GIST patients

AIM： To explore the relationship between clinicobiological behavior and the expression levels of telomerase activity, apoptosis, p53 gene and bcl-2 gene in gastrointestinal stromal tumors （GISTs）. METHODS： The intensity of telomerase activity, apoptosis, p53 and bcl-2 expression in GISTs were detected by telomeric repeat amplification protocol, in situ end-labeling technique, and immunohistochemistry, respectively. RESULTS： The positive rates of telomerase activity of malignant GIST, potential malignant GIST and benign GIST were 85% （17/20）, 22.8% （2/9） and 0 （0/9）, respectively. The apoptosis indices of malignant GIST, potential malignant GIST, and benign GIST were 11.7±5.4, 30.2±5.6 and 45.2 ±7.2, respectively. The intensity of telomerase activity and apoptosis were related to the biological characteristics of GISTs （85% vs 22.8%, 0, 0; P 〈 0.01 or 11.7±5.4 vs 30.2±5.6, 45.2±7.2, 72.1±9.3; P 〈 0.05）. The intensity of telomerase activity was negatively correlated with cellular apoptosis （22.9±8.4 vs 9.5±5.7, P 〈 0.01）. The intensity of telomerase activity was positively correlated with/753, bcl-2 expression （40.0% vs 78.9%, 40.0% vs 84.2%; P 〈 0.05）. CONCLUSION： The detection of telomerase activity, apoptosis and its control genes in GIST will be helpful for the discrimination of the malignant and benign GIST and evaluation of the prognosis.

Effects of Berbamine on PAF Production in Human Neutrophils and on Platelet Aggregation

The effects of berbamine, an alkaloid of dibenzylisoquinoline, on PAF produc tion in human neutrophils and on platelet aggregation induced by PAF were studied and compared with those of the calcium antagonist verapamil. Preincubation with berbamine (50 mmol / L, 100 mmol / L) or verapamil (10 mmol / L, 100 mmol / L) was shown to significantly inhibit A 23187 stimulated PAF synthesis. Berbamine and verapamil were found to inhibit platelet aggregation induced by PAF 70 pmol / L in a dose dependent manner. These results suggest that the inhibitory effects of berbamine and verapamil on A 23187 stimulated PAF synthesis in human neutrophils and PAF induced platelet aggregation are possibly brought about by inhibiting cellular calcium influx.

Using a non-radioisotopic, quantitative TRAP-based method detecting telomerase activities in human hepatoma cells

A non-radioisotopic, quantitative TRAP-based telomerase activity assay was established mainly by using SYBR Green-I staining instead of radioisotope. Comparing with conventional radioisotope based method, it was better in reproducibility and accuracy. Using this method, we found telomerase activities were absent in normal human liver cells, while detected in ail of four human hepatoma cell lines (BEL-7404, SMMC-7721, QGY-7903 and HCCM) without significant differences.

Inhibition of Leukemic Cell Telomerase Activity by Antisense Phosphorothioate Oligodeoxynucleotides

Objective To evaluate the effect of human telomerase reverse transcriptase (hTERT) gene antisense oligodeoxynucleotide (ASODN) ontelomerase activity in K562 cells.Methods Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA) in K562 cellstreated with ASODN and hTERT mRNA expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR).Results The hTERT mRNA level was decreased, and teloraerase activity was significantly inhibited when the K562 cells were treated withASODN for 48 h.Conclusion It is suggested that hTERT ASODN might specifically inhibit telomerase activity of K562 cells at translation level, and it isfurther proved that hTERT gene has significant correlation with telomerase activity.

Clinical significance of telomerase activity in peritoneal lavage fluid from patients with gastric cancer and its relationship with cellular proliferation

AIM： To evaluate the efficacy of telomerase activity assay and peritoneal lavage cytology （PLC） examination in peritoneal lavage fluid for the prediction of peritoneal metastasis in gastric cancer patients, and to explore the relationship between telomerase activity and proliferating cell nuclear antigen expression.METHODS： Telomeric repeated amplification protocol （TRAP）-enzyme-linked immunosorbent assay （ELISA） was performed to measure the telomerase activity in 60 patients with gastric cancer and 50 with peptic ulcer. PLC analysis of the 60 patients with gastric cancer was used for comparison. The proliferating cell nuclear antigen （PCNA） in gastric carcinoma was immunohistochemically examined.RESULTS： The telomerase activity and PLC positive rate in peritoneal lavage fluid from patients with gastric cancer was 41.7% （25/60）, and 25.0% （15/60）, respectively. The positive rate of telomerase activity was significantly higher than that Qf PLC in the group of pT, （15/16 vs 9/16, P 〈 0.05）, P1-3 （13/13 vs 9/13, P 〈 0.05） and diffuse type （22/42 vs 13/42, P 〈 0.05）. The patients with positive telomerase activity, peritoneal metastasis, and serosal invasion had significantly higher levels of average PCNA proliferation index （PI）, （55.00 ± 6.59 vs 27.43 ± 7.72, 57.26 ±10.18 vs 29.15 ±8.31, and 49.82 ± 6.74 vs 24.65 ±7.33, respectively, P 〈 0.05）.CONCLUSION： The TRAP assay for telomerase activity is a useful adjunct for cytologic method in the diagnosis of peritoneal micrometastasis and well related to higher proliferating activity of gastric cancer. The results of this study also suggest a promising future therapeutic strategy for treating peritoneal dissemination based on telomerase inhibition.