流式细胞术的临床应用

流式细胞术(flow cytometry,FCM)是20世纪70年代发展起来的对单细胞定量分析的一种新技术.它借鉴了荧光标记技术、激光技术、单抗技术和计算机技术,具有极高的检测速度与统计精确性,而且从单一细胞可以测得多个参数,为生物医学与临床检验提供了全新视角和强有力手段.目前,随着单克隆抗体技术的发展,流式细胞仪检测技术已经广泛使用在基础研究和临床实践的各个方面,在肿瘤学、血液学,免疫学、微生物学、细胞生物学-遗传学及临床检验学等诸多领域内发挥着重要作用[1].本文就其在临床上的应用综述如下.

Objective:To determine the activity and phenotype of decidual natural killer (NK) cells in patients with unexplained habitual abortions (UHA).Methods:A total of 32 patients with UHA were studied, and 20 cases of normal pregnant women were selected as control group. The levels of CD56+CD3- NK cells and their CD56+CD16-, CD56+CD16+ subsets in decidua were detected using two-color flow cytometric analysis.The NK cells activity was measured by a chromium-51(51Cr) release cytotoxicity assay,with K562 human myeloid leukaemia cells as targets.Results:Compared with control group, the proportion of CD56+CD3- NK cells in decidual mononuclear cells(DMC) of UHA patients had no difference, but the CD56+CD16- NK cell subset decreased and the CD56+CD16+ subset increased significantly (P＜0.05). The decidual NK cells activities of UHA patients were higher than those of normal controls (P＜0.05).Conclusions:NK cell is predominant lymphocyte in normal decidua and plays an important role in maintaining successful pregnancy. Abnormally raised activity and disbalanced CD56+CD16+, CD56+CD16- subsets of decidual NK cell are associated with UHA and may play a role in reproductive failure.

TAT-tCNTF对CD诱导HUVEC细胞损伤的作用研究

目的探讨TAT-tCNTF对细胞松弛素D（CD）诱导人脐静脉内皮细胞（HUVEC）损伤作用的影响。方法 CCK-8法检测TAT-tCNTF对CD致HUVEC生长抑制的作用；荧光显微镜检测CD诱导HUVEC的F-actin的变化，以及在TAT-tCNTF作用下对细胞微丝的影响；荧光显微镜和流式细胞术检测CD和TAT-tCNTF作用后细胞内Ca2+浓度；Western blot检测CD和TAT-tCNTF作用后F-actin的蛋白水平的变化。结果在1μg·ml-1的CD作用HUVEC后，细胞表现出明显的生长抑制，而在0.05~10μg·ml-1浓度范围内，TAT-tCNTF促进HUVEC的生长，并可改善CD对HUVEC的损伤，TAT-tCNTF组与CD+TAT-tCNTF组细胞活动度具有明显差异；荧光显微镜下检测到经CD作用后细胞F-actin分布减少，而经TAT-tCNTF作用后细胞F-actin分布密集；采用Fluo 4-AM荧光探针标记细胞内Ca2+浓度，荧光显微镜和流式细胞术检测到TAT-tCNTF能够降低经CD损伤HUVEC后的细胞内Ca2+浓度升高；FCM检测显示，CD组的MFI相对于对照组明显增加，而CD+TAT-tCNTF组MFI较CD组明显降低。Western blot显示各组之间的F-actin表达量没有明显改变。结论TAT-tCNTF对CD致内皮细胞的损伤有改善和逆转作用，其改善机制可能与增加细胞活性和维持细胞骨架有关。

This experiment, using cultured bovine pulmonary artery endothelial cells (BPAEC), was undertaken to investigate roles of endogenous ONOO- in lipopolysaccharide (LPS)-caused injury to endothelial cells. The fluorescent intensity of nitrotyrosine (NT), a marker of ONOO- generation, in BPAEC represented content of endogenous ONOO- generation. The fluorescent intensity of NT and number of NT positive cells were detected with flow cytometry, and the percentage of NT positive cells was calculated. Results were as follows. (1) LPS (1 mg/L, 5 mg/L and 10 mg/L) caused marked increase in fluorescent intensity of NT in a dose dependent manner. The number and percentage of NT positive cells were markedly increased (P＜0.05). Aminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase, inhibited the increase in fluorescent intensity of NT in BPAEC induced by LPS. However, the number and percentage of NT positive cells had tendency to reduce. (2) LPS caused the enhancement of MDA content and activity of LDH in cultured supernatant (P＜0.01). AG reversed the enhancement of MDA content induced by LPS (P＜0.01). In contrast, AG had marginal effect on activity of LDH. (3) LPS induced the increase in apoptotic rate in BPAEC in a dose dependent manner. Some BPAEC stained with fluorescent probe ethidium bromide showed morphological features of apoptosis with chromatin condensation and nuclear fragmentation. AG reduced the apoptotic rate and number of apoptotic cells, both of which were still higher than those of vehicle group (P＜0.05). (4) LPS inhibited mitochondrial respiration. Effect of LPS on mitochondrial membrane potential (ΔΨ) depended on the doses of LPS. 1 mg/L LPS led to a little increase in ΔΨ, while 5 mg/L and 10 mg/L LPS significantly reduced ΔΨ. In conclusion, LPS caused injury to cultured BPAEC and increased production of ONOO-. Cytotoxicity of LPS may be mediated by endogenous ONOO-.

The present study was undertaken to investigate the effect of human PMNs on the production of TNF-α by the human peripheral blood mononuclear cells (PBMCs) and to elucidate its tentative mechanism. Human PMNs and PBMCs were isolated from the venous blood of healthy donors by dextran sedimentation and density gradient centrifugation. In the presence of lipopolysaccharide (LPS), PMNs and PBMCs were cocultured at the ratio of 2:1 for 20 h and the concentration of TNF-α in the supernatant was measured by enzyme-linked immunosorbent assay. The binding rate of monocytes with the fluorescein isothiocyanate-labeled LPS (FITC-LPS) and the mean surface fluorescence intensity of monocytes were analyzed by flow cytometry. Results showed that PMNs were capable of inhibiting the TNF-α release from PBMCs (P＜0.05). PMNs suppressed the TNF-α release from PBMCs by 45% on average when PMNs and PBMCs cocultured at the ratio of 2:1. Paraformaldehyde-fixed PMNs still demonstrated the same inhibition (P＜0.05)，which proved that the inhibition was dependent on cell-to-cell contact and suggested that effector molecules responsible for this effect existed on the cell surface of PMNs. In the presence of PMNs, the binding rate of monocytes with the FITC-LPS and the mean surface fluorescence intensity of monocytes were not affected compared with PBMCs alone (P＞0.05). As incubation time was prolonged, the binding of FITC-LPS to monocytes increased (P＜0.05). Thus PMNs did not block the binding of LPS with monocytes. It was concluded that PMNs suppressed the TNF-α release from PBMCs via cell-to-cell interaction. In a cell-contact dependent manner, PMNs might interfere with the signal transduction pathway through which LPS activated PBMCs, thus attenuating the response of PBMCs to LPS and downregulating the TNF-α release.

Microvascular endothelial cell (MVEC) is one of the target cells of TNFα (TNF effect). The dysfunction of MVEC induced by TNF plays an important role in some cardio-cerebral vascular diseases. ① Cell proliferation kinetic: Using flow cytometry, we found cell count ［(4.30±0.34)×107／L)］ in TNF group (4×105 U/L) was obviously less than that in control ［(5.23±0.50)×107／L, P＜0.01］. The cells of G1 phase were more than those of the control, while the cells of G2, S and M phase became less (P＜0.05). ② Coagulant and anticoagulant: 72 h after MVEC cultued in the media, the content of 6-keto-PGF1α (RIA) and activity of PAI decreased significantly in TNF (4×105 U/L) group (P＜0.01, vs control). The difference between TXB2 content and t-PA activity in groups was not significant (P＞0.05). ③ Adhesive molecule: The effect of low concentration TNF (＜4×105 U/L) on adhesion between cultured MVEC and leukocytes was not signficant, but when the concentration of TNF reached 8×105 U/L or more, 12 h after culture the adhesion rate between MVEC and neutrophil increased 30.8%±4.5%. If adding monoclonal antibody of ICAM-1/CD11 into media, the adhesion rate of leukocytes decreased significantly (from 31.2% to 63.4%). ④ NO: The level of nitrite in culture media (Griess reaction) was higher than that of control (P＜0.05) after pretreatment of TNF (2×106 U/L) for 6 h. Adding L-NMMA, Dexamethasone or Cycloheximide in media could block the increase of nitrite induced by TNF, while L-Arg could enhance it. The expression of iNOS mRNA of PMVEC increased significantly after treated with TNF (2×106 U/L) for 24 h (quantitative RT/PCR). Pretreatment with Dexamethasone or Cycloheximide could block the increase (P＜0.05). Meanwhile, the expression of eNOS mRNA decreased significantly compared with control, the decrease can be blocked by Cycloheximide but not by Dexamethasone. So that TNF can induce the expression of iNOS mRNA in PMVEC, but inhibited the expression of eNOS mRNA. NO production in PMVEC can be time-dependently induced by TNF. ⑤ Apoptosis: Adding high concentration TNF(＞1.2×106 U/L) in culture media for 12 h can induce apoptosis of PMVEC with electron microscopy, flow cytometry (PI/AnnexinV stain), TUNEL and DNA ladder eletrophoresis. Meanwhile, expression of apoptosis-associated gene Bcl-2 mRNA decreased and that of Fas mRNA increased (Northern blot). The expression of FADD, caspase 3 and caspase 8 enhanced too. So that signal of apoptosis induced by TNF may be transmitted following the cascade of Fas→FADD→caspase8→caspase3. In conclusion, it should be paid attention to metabolism inability and dysfunction induced by TNF which can not found easyily using light microscope. High concentration TNF can induce apoptosis of endothelial cells regulated by apoptosis-associated genes. The changes mentioned above are common pathway in pathogenesis of some diseases related to TNF.

AIM:Early calcification of atherosclerotic plaques are colocalized with macrophage and high mobility group box 1 (HMGB1), a cytokine associated with biomineralizing process under physiological and pathological conditions .Our study aims to evaluate whether HMGB1 induces ectopic mineralization via promoting the secretion of matrix vesicles ( MVs) from macrophages .METHODS:HMGB1 was added to the medium of macrophages , the secretion of MVs in the supernatant was tested by flow cytometry analysis .The mineral deposition in calcifying medium was detected by Alizarin Red staining and von Kossa staining .Transmission electron microscopy showed the formation of hydroxyapatite crystals in MVs .Then we subcutaneous injection into mice with MVs to induce regional minera-lization.RESULTS:HMGB1 significantly promoted secretion of MVs from macrophages as raveled by flow cytometry analysis .TNAP activity, considered as a marker of MVs maturation , was higher in HMGB1-induced MVs compared to the control-MVs.HMGB1-MVs also led to mineral deposition in an in vitro MVs-collagen mineralization model .Subcutaneous injection into mice with MVs derived from HMGB1-treated cells showed a greater potential to initiate regional mineralization .Mechanistic experiments revealed that HMGB 1 activated neutral sphingomyelinase 2 ( nSMase2 ) that involved the receptor for advanced glycation end products ( RAGE ) and p38 MAPK (upstream of nSMase2).Inhibition of nSMase2 with GW4869 or p38 MAPK with SB-239063 prevented MVs secretion and min-eral deposition .CONCLUSIONS: HMGB1 induces MVs secretion from macrophages at least in part , via the RAGE/p38 MAPK/nSMase2 signaling pathway .Our findings thus reveal a novel mechanism by which HMGB 1 may participated in the early calcification of atherosclerotic plaques .

Validation and quality control of hematolymphoid neoplasm immunophenotyping by flow cytometry

Flow cytometric detection of immunoglobulin light chain in hematolymphoid immunophenotyping

During B-cell development and maturation,the antigen receptor,which is encoded by the immunoglobulin heavy-(IgH) and light-chain genes,rearrange to associate one of a number of variable,diverse,and joining gene segments.A single mature B-cell expresses an IgH chain and either a kappa or lambda light chain,which is known as allelic or isotypic exclusion.In normal or reactive conditions,lymphoid cells comprise the mixtures of lymphocytes with either kappa or lambda expression.

用流式细胞术监测原位心脏移植病人术后的T细胞及亚群的变化

GM-CSF,IL-3和GM-CSF/IL-3融合蛋白对辐射致髓样白血病细胞株Tf-1凋亡的影响

AIM: To observe the effects of three cytokines on the apoptosis of Tf- 1 cells induced by γ irradiation and investigate the relationship between apoptosis and caspase-3 activity. METHODS: Different cytokines GM-CSF, IL-3 and GM-CS/IL-3 fusion protein were added into the irradiated Tf-1 cells. MTT assay, morphology, flow cytometry,and DNA fragmentation assay were used to observe the effects of cytokines on apoptosis. The caspase-3 activity was determined with a fluorocytometer. RESULTS: Irradiated Tf-1 cells showed typical morphological characteristic of apoptosis demonstrated by transmission electron microscopy and were accumulated in G0/G1 phase. In the groups treated with growth factors after irradiation, three cytokines significantly increased the viability rate, distinctly decreased the apoptosis rate and the proportion of DNA fragmentation. When Tf- 1 cells were irradiated by γ irradiation, caspase-3 activity was increased at different time points. In comparison with the control group in which no growth factor was added after the cells were irradiated, the caspase-3 activity of irradiated Tf-1 cells was significantly inhibited by addition of the above cytokines. Thirty-six hours after irradiation, in the control group,GM-CSF, IL-3, GM-CSF and IL-3 in combination, and two GM-CSF/IL-3 fusion protein groups, the apoptosis rate was 73 %, 11%, 15 %, 13 %, 12 %, and 13 %. The percent of fragmented DNA was 36 %, 19 %, 18 %, 14 %,13 %, and 14 %. The fluorescence intensity was 16923, 5529, 6581, 5322, 5426, and 5485. CONCLUSION:GM-CSF, IL-3, and GM-CSF/IL-3 fusion protein could protect Tf-1 cells from apoptosis induced by γ irradiation.After Tf-1 cells were irradiated, the caspase-3 activity was significantly increased but was dramatically decreasedby the above cytokines. The remarkable inhibition of caspase-3 activity may be one of the mechanisms of thesehematopoietic growth factors exerting their anti-apoptotic effects.

Objective: To investigate the effect and clinical significance of Xuebijing injection on periph-eral T-lymphocyte subpopulations in patients with severe trauma.Methods: Thirty-three patients with severe trauma were randomly divided into a control group (n=16) and a treatment group (n= 17).The patients of two groups were all treated conventionally, and the only difference was that Xuebijing injection was given to patients of the treatment group.The CD_4~+ and CD_8~+ subpopulations of T-lympho-cyte in the peripheral blood were detected respectively on admission, 3rd and 5th days after trauma by double anti-body labeling and flow cytometry.Results: The CD_4~+ T-lymphocytes and CD_4~+/CD_8~+ ra-tio of peripheral blood in patients with severe trauma de-creased markedly on the 3rd and 5th days after trauma.Furthermore, compared with control group, the peripheral CD_4~+ T-lymphocytes and CD_4~+/CD_8~+ ratio of treatment group renewed obviously on the 5th day after trauma, and showed statistical differences (P<0.05).Conclusion: In the treatment of patients with severe trauma, the early use of Xuebijing injection is effective in correcting disorder or suppression of T-lymphocyte sub-populations regulating network, and promoting a more bal-anced profile of immunologic function.