Objective: To observe the effect of emodin on the biological behavior of human fibroblasts (FB) in culture of kidney in patients with lupus nephritis (LN). Methods: FB were isolated from kidney culture of LN patients, and the effect of emodin on 3　H-TdR incorporated rate of FB was observed. The apoptosis and c-myc gene expression were detected in the same way by flow cytometry. Results: Emodin could markedly inhibit the proliferation of human kidney FB, and inducing cell apoptosis through up-regulating c-myc gene expression in human renal FB. Conclusion: Emodin can inhibit proliferation and promote apoptosis of FB, which may be important in ameliorating interstitial fibrosis, and thus improve prognosis of LN.

流式细胞术的研究进展及临床应用前景

自从商品化的流式细胞仪在20世纪70年代进入市场以来,流式细胞术(flow cytometry,FCM)检测的项目不断地由科研转向临床应用领域,并在临床免疫学、血液肿瘤学及微生物学等疾病诊断和治疗等方面起到不可或缺的作用.现对该技术的进展概述如下.

再谈尿流式有形成分分析在尿路感染诊断中的应用

编辑同志:拜读贵刊2009年第6期刊载的<尿流式有形成分及干化学分析在尿路感染诊断中的应用评价>[1]、[2]及2010年第4期顾兵等[3]的读者来信<对尿流式有形成分用于尿路感染诊断的思考>,收获很大.笔者就尿流式有形成分在尿路感染(UTT)诊断中的应用及顾兵等来信中提出的诸多问题,浅谈一下自己的看法和观点,仅供广大读者参考.

对尿流式有形成分用于尿路感染诊断的思考

编辑同志:对贵刊2009年第6期齐杰的<尿流式有形成分及干化学分析在尿路感染诊断中的应用评价>~([1])一文所得出的"联合UF1000i流式尿有形成分分析及尿干化学分析可在早期尿路感染筛查诊断中发挥重要作用;同时对尿细菌培养为阴性的UTI患者的明确诊断具有重要价值"的结论我们有不同的看法,阐述如下.

AIM To explore the therapeutic potential of antisense oligodeoxynucleotides on hepatcellular cacinoma(HCC).METHODS Four antisense phosphorothioated oligodeoxynucleotides (asON), complementary to differentsites of HBV, were synthesized and assayed for their anti-HBV activity in HepG22. 2.15 cells with ELISA.The most effective asON was chosen for the following study: FACSCAN, TRAP and immuno-staining wereused respectively for checking apoptosis, telomerase activity and expression of oncogene p21ras and p62C-myc inHepG2.2.15 cells after treated by asON.RESULTS The oligomer directed against the initiator of pre-S2 was the most effective one with aninhibitory rate of 66% on HBsAg and 91% on HBeAg (P<0.02). Two inhibitory peaks (bimodal)appeared. Telomerase activity as well as the expression of p21fas and p62C-myc decreased drastically 3 days afterasON-HBpreS2 treatment. Meanwhile, apoptosis appeared in the experiments.CONCLUSION The inhibitory effects of as-preS2 on the HBV gene expression and the reversion of somemalignant behaviour in HepG2.2.15 cells were the significant, effective therapy against HBV infection andhepatocellular carcinoma.

AIM To explore the anti-tumor effect of indomethacin (IN) on human colon adenocarcinoma cells anddetermine the influence of indomethacin on cancer cell proliferation and apoptosis and elucidate the anti-tumor mechanism of Indomethacin.METHODS Human colon adenocarcinoma HCT116 cell line were cultured separately in vitro. Indomethacin(final concentration 100 μm - 800 gm) was administered alone or altogether with 5-Fu (50 μm). Agarose gelelectrophoresis, MTT, and Flow cytometry were used to study cell proliferation and apoptosis in humancolon carcinoma cell RT-PCR, western blot were used to detect the expression level of Bcl-2, bax gene andcdk4 protein expression in HCT116 cell lines after treated with IN for 24 hours.RESULTS Indomethacin can inhibit significantly the proliferation of HCT116 cell, change the morphology,and cause the cells to accumulate in the G0/Gl phase of the cell cycle, and induce apoptosis. The apoptosis oftumor cells was confirmed by DNA ladder formation on gel electrophoresis and sub-Gl peak on flowcytometry. These responses were time-and concentration-dependent. A synergic effect of inhibiting cancercell proliferation was observed when combined with Indomethacin and 5-Fu. RT-PCR results showed that INdown-regulated Bcl-2 mRNA expression, and did not change Bax mRNA expression. Western blot resultsconfirmed that IN inhibited Bcl-2 protein expression. No influence was found in the translation of Baxprotein. In inhibited cdk4 protein expression.CONCLUSION Our study results indicate that IN induce apoptosis of HCT116 cell by down-regulating Bcl-2expression and inhibiting cdk4 protein expression partially. This explains the mechanisms of antitumoractivity of the Indomethacin.

It has been reported in many studies that electroacupuncture (EA) can positively regulate erythrocytic immunity and T-lymphocytic subgroups[1-8].Nevertheless, its mechanism remains to be explored. In the present study, a multi-group, multi-stepped and multiindexed observation was conducted on the effects of EA on erythyrocytic immunity and T-lymphocytic subgroups. A simultaneous assay of the changes in immunoreactivesubstance-P (ir-SP) content in the pituitary gland and peripheral blood was also carried out. The objective of the study was to investigate the regulatory effects of the immune system and their possible mechanism in the treatment of relevant diseases with EA.

用流式细胞仪分析两例同胞脐血移植后的早期免疫重建

脐血造血干细胞移植(CBT)作为临床治疗的一个手段,已成功地治疗了一些遗传缺陷性疾病、白血病、恶性实体肿瘤等疾病.2001年5月～2002年5月,我们利用流式细胞仪,对2例急性淋巴细胞白血病(ALL)患儿同胞脐血移植后3个月内免疫重建的过程进行了研究,现报道如下.

Objective: To study the effect of active compound 6F and A from Pteris semipinnata L.(PsL) on the activities of DNA topoisomerase (TOPO) I and II, activities of cytosolic and membrane TPK, and expression of oncogene c-myc in lung adenocarcinoma cells. Methods: The effect of compound 6F and A on activities of cytosolic and membrane TPK was measured by scintillation counting; the effect of compound A on expression of oncogene c-myc was determined by flow cytometry indirect fluorimetry. Results: compound 6F and A could inhibit the activities of TOPO I, and they strongly inhibited the TOPO II in 0.01 mg/L and 10.0 mg/L respectively. Compound A slightly inhibited the activities of membrane TPK, but not the cytosolic one. Compound A could inhibit the expression of oncogene c-myc. Conclusion: Topoisomerases are target of compound 6F and A. Compound A could slightly inhibit the activities of TPK, and showed an inhibitory effect on the expression of oncogene c-myc.

After hypoxia, ischemia, or inlfammatory injuries to the central nervous system, the damaged cells release a large amount of adenosine triphosphate, which may cause secondary neuronal death. Autophagy is a form of cell death that also has neuroprotective effects. Cell Counting Kit assay, monodansylcadaverine staining, lfow cytometry, western blotting, and real-time PCR were used to determine the effects of exogenous adenosine triphosphate treatment at different concentrations (2, 4, 6, 8, 10 mmol/L) over time (1, 2, 3, and 6 hours) on the apoptosis and autophagy of SH-SY5Y cells. High concentrations of extracellular adenosine triphosphate induced autophagy and apoptosis of SH-SY5Y cells. The enhanced autophagy ifrst appeared, and peaked at 1 hour after treatment with adenosine triphosphate. Cell apoptosis peaked at 3 hours, and persisted through 6 hours. With prolonged exposure to the adenosine triphosphate treatment, the fraction of apoptotic cells increased. These data suggest that the SH-SY5Y neural cells initiated autophagy against apoptosis within an hour of adenosine triphosphate treatment to protect themselves against injury.

A preliminary study from our research group showed that picroside II inhibited neuronal apop-tosis in ischemic penumbra, reduced ischemic volume, and improved neurobehavioral function in rats with cerebral ischemia. The aim of the present study was to validate the neuroprotective effects of picroside II and optimize its therapeutic time window and dose in a rat model of ce-rebral ischemia. We found that picroside II inhibited cell apoptosis and reduced the expression of neuron-speciifc enolase, a marker of neuronal damage, in rats after cerebral ischemic injury. The optimal treatment time after ischemic injury and dose were determined, respectively, as fol-lows:(1) 2.0 hours and 10 mg/kg according to the results of toluidine blue staining;(2) 1.5 hours and 10 mg/kg according to early apoptotic ratio by lfow cytometry;(3) 2.0 hours and 10 mg/kg according to immunohistochemical and western blot analysis;and (4) 1.5 hours and 10 mg/kg according to reverse transcription polymerase chain reaction. The present ifndings suggest that an intraperitoneal injection of 10 mg/kg picroside II 1.5-2.0 hours after cerebral ischemic injury in rats is the optimal dose and time for therapeutic beneift.

外周血淋巴细胞γ-H2 AX的RL／G值分析：一种新的用于辐射事故伤员分类诊断和剂量估算的方法

在应对大规模的核与辐射事故过程中，对伤员进行快速分类诊断和快速剂量估算，是核事故医学救援的重要问题之一。目前，以染色体畸变为代表的生物剂量估算方法并不能满足大规模核与辐射事故中批量伤员的分类诊断和快速剂量估算的需要。近年来的研究报道表明，外周血淋巴细胞中γ-H2AX分析是一种非常有潜力的生物剂量估算方法。目前，对于外周血淋巴细胞中γ-H2AX的分析主要有两种方法：①荧光显微镜分析细胞中γ-H2AX聚集点。这种方法准确度高，灵敏度高，但是分析速度慢；②采用流式细胞术结合细胞免疫荧光方法分析细胞中γ-H2AX含量，这种方法分析速度快，但是实验重复性差，存在个体间和个体内差异大的问题，降低了其实际应用价值。王治东等的“Ratio of γ-H2AX level in lymphocytes to that in granulocytes detected using flow cytometry as a potential biodosimeter for radiation exposure”一文建立了一种基于流式细胞快速分析外周血淋巴细胞中γ-H2AX含量的新方法。该方法保留了流式细胞分析的快速、高通量等优点，同时解决了目前流式细胞分析外周血淋巴细胞γ-H2AX方法存在个体间和个体内差异大的问题，该研究对生物剂量计领域具有重要的意义。

Objective. To evaluate the role of glucose transporter-l (GLUT1) in the glucose uptake of glomerular mesangial cells. Methods. Cultured C57/SJL mouse mesangial cells were used in the study. The expression of GLUT1 mRNA was detected by RT-PCR. The expression of GLUT1 protein was detected by immunofluorescence and flow cytometry. The uptake of glucose and its kinetics were determined by 2-deoxy-[3H] -D-glucose uptake. Results. Both GLUT1 mRNA and protein were found in mouse glomerular mesangial cells. 2-deoxy-D-glucose uptake and kinetics assay showed that this glucose transporter had high affinity for glucose and the glucose uptake specificity was further confirmed by phloretin. Conclusion. Functional GLUT1 did present in mouse mesangial cells cultured in vitro and it might be the predominant transporter mediated the uptake of glucose into mesangial cells.

大鼠颈椎间盘退行性变后软骨细胞凋亡及形态学改变

背景:椎间盘软骨终板是椎间盘营养渗透的主要途径,又是维持脊柱生物力学的重要结构之一.对其重要性的认识及相关研究日益深入,但对椎间盘退行性变的确切原因仍不清楚.目的:动态观察大鼠颈部动静力失去平衡后颈椎间盘软骨细胞凋亡的形态学改变以及凋亡率.设计:采用完全随机对照设计.地点和对象:实验在上海中医药大学脊柱病研究所完成,对象为8月龄清洁级SD大鼠60只,雌雄各30只.干预:将雌雄大鼠分别按随机数字表法分为3,5,7月对照组与模型组,每组10只,雌雄各5只.取大鼠颈背部正中纵向切口,切开皮肤后,充分游离各层肌肉,横向切断深群颈夹肌和头、颈、寰长肌,完全切除颈髂肋肌与头半棘肌,然后再依次切断C2～C7棘上和棘间韧带,建立的动静力失衡性颈椎间盘退行性变模型.主要观察指标:3,5,7月后椎间盘软骨细胞凋亡程度.结果:退行性变椎间盘内有典型凋亡的软骨细胞.与对照组比较,各个模型组软骨细胞凋亡指数明显升高(P＜0.01);模型组间比较,TUNEL法5,7月软骨细胞凋亡指数分别为36.59±5.93和36 36±5.13较3月(27.73±4.12)明显升高(P＜0.01),流式细胞仪PI法5,7月细胞凋亡指数分别为37.56±3.82和28.02±3.48较3月(21.45±2.23)明显升高(P＜0.01).结论:退行性变椎间盘软骨终板内软骨细胞凋亡数目增多,这可能是椎间盘退行性变的重要机制之一.

不同功率密度氦氖激光照射对培养瘢痕成纤维细胞生长的影响

Objective To explore the relationship between power density of He－Ne laser with the growth of fibroblasts in hypertrophic scar(HS).Methods The cultured fibroblasts in HS were irradiated with He－Ne laser(Wavelenth 632.8 nm),various power densities such as 50mW/cm2,100mW/cm2 and 150mW/cm2 were respectively adopted once a day for 10 minutes.After 1,3 and 5 times of He－Ne laser for dose rate irradiation separatedly,the cell count and cell circle analysis were counted and examined by trypan blue staining and flow cytometry .Results The amount of cell after 1 times of 50mW/cm2 laser irradiation was markedly more than the control(P<0.01).The amount of cells after 5 times of 100mW/cm2,3 and 5 times of 150 mW/cm2 laser irradiation was less than the controls(P< 0.05).The result of cell circle analysis was corresponded with that of the cell count.Conclusion Both stimualtion and inhibition of He－Ne laser on the growth of scar fibroblasts can be obtained with various power densities.Power density of He－Ne laser associates with the effects on the growth of scar fibroblasts.

多参数流式细胞术在多发性骨髓瘤诊疗中的应用

多发性骨髓瘤(multiple myeloma,MM) 是血液系统常见的恶性肿瘤之一.近年来,多参数流式细胞术(Multiparameter flow cytometry,MP-FCM)在多发性骨髓瘤诊断、治疗以及疗效观察等方面的价值日益突出,本研究应用多参数流式细胞术,对MM患者骨髓标本中的浆细胞与瘤细胞表面相关抗原进行检测,以期探讨多参数流式细胞术在本病的诊断、治疗中的应用价值.

目的：观察低剂量辐射（LDR）诱导胸腺细胞凋亡及细胞周期进程适应性反应的基本规律。方法：用X射线照射昆明系雄性小鼠，其诱导剂量（D1）及其后攻击剂量（D2）分别是75 mGy 和1.5 Gy。D1和D2间隔时间分别是3、6、12、24和60 h。D2照射后18 h胸腺细胞培养4、20 和44 h用流式细胞仪检测胸腺细胞凋亡小体（TAB）和细胞周期进程的变化。结果：当D1和D2间隔3、6和12 h，在D2照射后胸腺细胞培养4和20 h，D1 + D2组 TAB 百分数明显低于D2组（P<0.05），G0/G1和G2+M期细胞百分数也不同程度地低于D2组, 而S期细胞百分数却明显高于D2组（P<0.05或P<0.01）。结论：D1和D2分别是75 mGy（剂量率，12.5 mGy/min）和1.5 Gy（剂量率，0.287 Gy/min），D1和D2间隔3～12 h, 在小鼠全身照射后其胸腺细胞培养4和20 h可诱导细胞凋亡和细胞周期进程的适应性反应。

人类免疫缺陷病毒感染者不同疾病阶段中自然杀伤细胞和γδT细胞的改变

目的 了解NK细胞和.γδT细胞在HIV/AIDS患者不同疾病阶段中的改变特点,探讨其在AIDS发病机制中的作用.方法 以311例未接受过抗HIV治疗的HIV/AIDS患者为研究对象,经流式细胞仪检测患者外周血CD4+T淋巴细胞、NK细胞和γδT细胞比例及计数,根据CD4+T淋巴细胞<0.20×109L、(0.20～0.35)×109L及>0.35×109/L.将病例分为低、中、高CD4+T淋巴细胞组,进行组间比较.Mann-WhitneyU检验和Kruskal-Wallis检验进行两组和多组独立样本秩和检验,Spearman和Pearson检验进行相关性分析.结果 HIV/AIDS患者外周血NK细胞比例及计数的中位数分别为8.4%和103×106L,γδT细胞比例及计数中位数分别为3.4%和41×106L,均明显低于健康对照组(Z=-5.029,Z=-7.723,Z=-2.437,Z=-6.063;均P<0.01).低、中、高CD4+T淋巴细胞组CD4+T淋巴细胞计数中位数分别为0.062×109L、0.276×109L、0.482×109L,NK细胞计数分别为89×106L、97×106L、146×106L,低、中CD4+T淋巴细胞组间的NK细胞计数差异无统计学意义,但均显著低于高CD4+T淋巴细胞组(Z=-3.392,P=0.001;Z=-4.849,P<0.01).低、中、高CD4+T淋巴细胞组γδT细胞计数中位数分别为29×106L、43×106L、59×106L,两两之间比较均差异有统计学意义(P<0.05).结论 HIV感染后外周血NK细胞和γδT细胞数量降低程度存在差异.

Th17细胞、IL-17A mRNA及IL-23R mRNA与寻常性银屑病的关系

目的 探讨寻常性银屑病(PV)皮损中IL-17A mRNA及IL-23R mRNA水平、外周血CD4+IL-17+T细胞数量及它们与疾病严重程度的关系.方法 PV组25例,对照组14例.对PV皮损面积和严重指数( PASI)评分,用RT-PCR检测皮肤中IL-17A mRNA及IL-23R mRNA的水平,并用流式细胞仪测定20例PV患者及10例对照组外周血CD4+IL-17+T细胞的数量.结果 PV组IL-17A mRNA的水平明显高于对照组(0.996±0.231比0.437±0.096,t=10.57,P＜ 0.05),IL-23R mRNA水平也明显高于对照组(1.006±0.339比0.491±0.196,t=6.02,P＜0.05).PV皮损中IL-17A mRNA与IL-23R mRNA的水平与PASI均呈线性正相关(分别为rs=0.67,P＜0.05；rs=0.70,P＜0.05).外周血CD4+IL-17+T细胞数量在两组差异无统计学意义.PV组中,外周血中CD4+IL-17+T细胞数量与皮损中IL-17A mRNA、IL-23RmRNA水平无相关性.结论 PV皮损中IL-17A mRNA及IL-23R mRNA的水平升高,与疾病严重程度相关,外周血Th17细胞数量无改变.

流式细胞术的临床应用

流式细胞术(Flow Cytometry,FCM)是一种可以对细胞或亚细胞结构进行快速测量的新型分析技术和分选技术.它以高能量激光照射高速流动状态下被荧光色素染色的单细胞或微粒,测量其产生的散射光和发射荧光的强度从而对细胞(或微粒)的物理、生理、生化、免疫、遗传、分子生物学性状及功能状态等进行定性或定量检测的一种现代细胞分析技术.其主要临床应用现综述如下: