Objective.To establish a flow cytometric internal standard method for counting platelet-derived microparti-cles(PMPs)and to study its clinical significance. Methods. PMPs suspension(platelet poor plasma,PPP) was extra...Objective.To establish a flow cytometric internal standard method for counting platelet-derived microparti-cles(PMPs)and to study its clinical significance. Methods. PMPs suspension(platelet poor plasma,PPP) was extracted by gradual centrifugation. According to the size of PMPs,3 μm and 0.8μm latex beads were used as internal standards for the quantitation. PMPs were counted by adjusting flow cytometric discrimination and voltage of forward scatter and side scatter. Results. In 30 healthy donors,the average concentration of resting PMPs was(1.2×105±5.7×104 )/ml and that of activated PMPs was(1.6×106±9.1×105)/ml. Compared with healthy donors,PMPs mean value was significantly higher(P< 0.001)in 18 patients with coronary artery disease,12 with acute cerebral infraction and 23 with chronic renal failure[the average PMPs concentration,( 6.1×105±2.5×105 )/ml, ( 6.8×105±3.4×105)/ml and(5.9×105±3.1×105)/ml respectively]. However,no significant difference in PMPs concentration was observed in 25 patients with acute leukemia and severe thrombocytopenia during the aplastic phase after chemotherapy [1.3×105±6.1×104)/ml,(P >0.05)] .Conclusions. PMPs is a useful indicator in monitoring platelet activation,and plays an important role in thrombotic disease. By flow cytometric internal standard method,PMPs can be counted rapidly and accurately,which may be very helpful in interlaboratory comparative studies.展开更多
Analyzing the ploidy levels of plants is important for identifying species, selecting parental lines, identifying the relationships between species, and determining evolutionary patterns. The genus Chrysanthemum is wi...Analyzing the ploidy levels of plants is important for identifying species, selecting parental lines, identifying the relationships between species, and determining evolutionary patterns. The genus Chrysanthemum is widely distributed throughout the world and exhibits different ploidy levels. We used flow cytometry to analyze the ploidy levels of nine species of Chrysanthemum L. collected from different regions and geographical locations in China. Three diploids from Henan and Wuhan provinces corresponded to Chrysanthe- mum lavandulifolium and two species of C. nankingense, while three tetraploids from various regions corresponded to C. indicum and two species of C. chanetii. Two hexaploids corresponding to C. vestitum were collected at Funiu moun- tain （Henan province）, and C. zawadskii was collected at Huangshan mountain （Anhui province）. We found that OTTO extraction buffer was suitable for extracting nuclei from most species, apart from C. zawadskii. Flow cytometry proved to bea simple, rapid, and highly accurate method for identifying ploidy levels in Chrysanthemum species.展开更多
Objective:To analyze the antifungal effects of Chinese herb monomers,i.e.berberine,baicalin,eugenol and curcumin,on Candida albicans.Methods:After Candida albicans strain Y01-09 was incubated for 48 h in YEPD broth wh...Objective:To analyze the antifungal effects of Chinese herb monomers,i.e.berberine,baicalin,eugenol and curcumin,on Candida albicans.Methods:After Candida albicans strain Y01-09 was incubated for 48 h in YEPD broth which contained different concentrations of Chinese herb components,the cell cycle,fluorescent intensity and the size of cell volume were detected by flow cytometry.Results:The 4 Chinese herb monomers could affect the cell cycle of Candida albicans in different ranges.The ratio of cells in S-G2-M period decreased as the agents concentration increased,indicating that the cell division was inhibited.The fluorescent intensity of Candida albicans cells became weaker after being incubated,which reflected the loss of DNA fragments.The higher the concentration was,the weaker the fluorescent intensity became.The cell size,cell diopter and particle size changed much as the agents concentration increased.Conclusion:Chinese herb monomers play the antifungal role in inhibiting cell division.FCM could be used to determine the susceptibility of antifungal agents.展开更多
To quantitatively analyze apoptotic and secondary necrotic cells unde r apoptosis conditions. Methods. The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1 .0 μmol/L dexamethasone (DEX) for 2, 4 an...To quantitatively analyze apoptotic and secondary necrotic cells unde r apoptosis conditions. Methods. The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1 .0 μmol/L dexamethasone (DEX) for 2, 4 and 8 h respectively, then stained with Annexin V FITC (fluorescein isothiocyanate conjugated) which was used to detec t the exposed phosphatidylserine (PS) on the epimembrane resulting from a loss o f phospholipid asymmetry in the early stage of apoptosis, and also stained with propidium iodide (PI) which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably represent late stage of apoptosi s, then apoptotic cells were quantified by flow cytometry (FCM). Furthermore, An nexin+/PI and Annexin+/PI+ cells were sorted by fluoresence activated cell sorter (FACS), and identified by electron microscopy (EM) and DNA gel electroph oresis. Results. The percentage of apoptotic cells was found to increase with the incuba tion time (r=0.97). This method was sensitive with low detection limit (0.02%), and was reproducible with low coefficient variance (CV)(4.2%). Meanwhile, the Annexin+/PI and Annexin+/PI+ cells were identified as apoptotic and necroti c cells under EM, and DNA extracted from the Annexin+/PI cells was characteri zed by “ladder pattern”. Conclusions. Annexin V assay is a specific, sensitive, accurate, reproductive and quantitative method for analyzing apoptotic cells.展开更多
Doubled haploid(DH)plants have been widely used for breeding and biological research in crops.Pop ulus spp.have been used as model woody plant species for biological research.However,the induction of DH poplar plants ...Doubled haploid(DH)plants have been widely used for breeding and biological research in crops.Pop ulus spp.have been used as model woody plant species for biological research.However,the induction of DH poplar plants is onerous,and limited biological or breeding work has been carried out on DH individuals or populations.In this study,we provide an effective protocol for poplar haploid induction based on an anther culture method.A total of 96 whole DH plant lines were obtained using an F1hybrid of Populus simonii×P.nigra as a donor tree.The phenotypes of the DH population showed exceptionally high variance when compared to those of half-sib progeny of the donor tree.Each DH line displayed distinct features compared to those of the other DH lines or the donor tree.Additionally,some excellent homozygous lines have the potential to be model plants in genetic and breeding studies.展开更多
BACKGROUND： The effect of pituitary adenylate cyclase activating polypeptide （PACAP） during traumatic brain injury （TBI） and whether it can modulate secondary injury has not been reported previously. The present ...BACKGROUND： The effect of pituitary adenylate cyclase activating polypeptide （PACAP） during traumatic brain injury （TBI） and whether it can modulate secondary injury has not been reported previously. The present study evaluated the potential protective effects of ventricular infusion of PACAP in a rat model of TBI.METHODS： Male Sprague Dawley rats were randomly divided into 3 treatment groups （n=6, each）： sham-operated, vehicle （normal saline）＋TBI, and PACAP＋TBI. Normal saline or PACAP （1 μg/5 μL） was administered intracerebroventricularly 20 minutes before TBI. Right parietal cortical contusion was produced via a weight-dropping method. Brains were extracted 24 hours after trauma. Histological changes in brains were examined by HE staining. The numbers of CD4＋ and CD8＋ T cells in blood and the spleen were detected via flow cytometry.RESULTS： In injured brain regions, edema, hemorrhage, inflammatory cell infiltration, and swollen and degenerated neurons were observed under a light microscope, and the neurons were disorderly arrayed in the hippocampi. Compared to the sham group, average CD4＋ CD8＋ lymphocyte counts in blood and the spleen were significantly decreased in rats that received TBl＋vehicle, and CD4- CD8＋ were increased. In rats administered PACAP prior to TBI, damage was attenuated as evidenced by significantly increased CD4＋, and decreased CD8＋, T lymphocytes in blood and the spleen.CONCLUSION： Pretreatment with PACAP may protect against TBI by influencing periphery T cellular immune function.展开更多
Objective：To compare the phenotype characteristics of rat annulus fibrosus （AF） cells cultured on flexible silicone membranes and those in plastic plates. Methods：The morphology of AF cells cultured in different s...Objective：To compare the phenotype characteristics of rat annulus fibrosus （AF） cells cultured on flexible silicone membranes and those in plastic plates. Methods：The morphology of AF cells cultured in different suhstrates was examined. Proteoglycan was stained by toluidine blue. Contents of collagen type Ⅰ, collagen type Ⅰ and aggrecan mRNAs were determined by reverse transcription-polymerase chain reaction （RT-PCR）. The expression of integrin β1 was monitored by flow cytometry. By using propidium iodide （PI）, the cell cycle in AF cells was analyzed. Cell adhesion to silicone membrane was also measured. Results：The AF cells cultured on different suhstrates were morphologically undistinguishable. Toluidine blue staining showed that there was also no difference between AF cells cultured on these 2 substrates. They still had the same expression levels of collagen type Ⅰ , collagen type Ⅰ, aggrecan mRNAs, and integrin β1. No significant difference was observed in the distribution of the cell cycle. AF cells grew well on silicone membrane. Conclusion：AF cells cultured on flexible silicone membrane maintain the stability of phenotype and may he appropriate for further studying the metabolic responses to mechanical stimuli at the cellular level.展开更多
Background:One-third of veterans returning from the 1990–1991 Gulf War reported a myriad of symptoms including cognitive dysfunction,skin rashes,musculoskeletal discomfort,and fatigue.This symptom cluster is now refe...Background:One-third of veterans returning from the 1990–1991 Gulf War reported a myriad of symptoms including cognitive dysfunction,skin rashes,musculoskeletal discomfort,and fatigue.This symptom cluster is now referred to as Gulf War Illness(GWI).As the underlying mechanisms of GWI have yet to be fully elucidated,diagnosis and treatment are based on symptomatic presentation.One confounding factor tied to the illness is the high presence of post-traumatic stress disorder(PTSD).Previous research efforts have demonstrated that both GWI and PTSD are associated with immunological dysfunction.As such,this research endeavor aimed to provide insight into the complex relationship between GWI symptoms,cytokine presence,and immune cell populations to pinpoint the impact of PTSD on these measures in GWI.Methods:Symptom measures were gathered through the Multidimensional fatigue inventory(MFI)and 36-item short form health survey(SF-36)scales and biological measures were obtained through cytokine&cytometry analysis.Subgrouping was conducted using Davidson Trauma Scale scores and the Structured Clinical Interview for Diagnostic and statistical manual of mental disorders(DSM)-5,into GWI with high probability of PTSD symptoms(GWIH)and GWI with low probability of PTSD symptoms(GWIL).Data was analyzed using analysis of variance(ANOVA)statistical analysis along with correlation graph analysis.We mapped correlations between immune cells and cytokine signaling measures,hormones and GWI symptom measures to identify patterns in regulation between the GWIH,GWIL,and healthy control groups.Results:GWI with comorbid PTSD symptoms resulted in poorer health outcomes compared with both healthy control(HC)and the GWIL subgroup.Significant differences were found in basophil levels of GWI compared with HC at peak exercise regardless of PTSD symptom comorbidity(ANOVA F=4.7,P=0.01)indicating its potential usage as a biomarker for general GWI from control.While the unique identification of GWI with PTSD symptoms was less clear,the GWIL subgroup was found to be delineated from both GWIH and HC on measures of IL-15 across an exercise challenge(ANOVA F>3.75,P<0.03).Additional differences in natural killer(NK)cell numbers and function highlight IL-15 as a potential biomarker of GWI in the absence of PTSD symptoms.Conclusions:We conclude that disentangling GWI and PTSD by defining trauma-based subgroups may aid in the identification of unique GWI biosignatures that can help to improve diagnosis and target treatment of GWI more effectively.展开更多
文摘Objective.To establish a flow cytometric internal standard method for counting platelet-derived microparti-cles(PMPs)and to study its clinical significance. Methods. PMPs suspension(platelet poor plasma,PPP) was extracted by gradual centrifugation. According to the size of PMPs,3 μm and 0.8μm latex beads were used as internal standards for the quantitation. PMPs were counted by adjusting flow cytometric discrimination and voltage of forward scatter and side scatter. Results. In 30 healthy donors,the average concentration of resting PMPs was(1.2×105±5.7×104 )/ml and that of activated PMPs was(1.6×106±9.1×105)/ml. Compared with healthy donors,PMPs mean value was significantly higher(P< 0.001)in 18 patients with coronary artery disease,12 with acute cerebral infraction and 23 with chronic renal failure[the average PMPs concentration,( 6.1×105±2.5×105 )/ml, ( 6.8×105±3.4×105)/ml and(5.9×105±3.1×105)/ml respectively]. However,no significant difference in PMPs concentration was observed in 25 patients with acute leukemia and severe thrombocytopenia during the aplastic phase after chemotherapy [1.3×105±6.1×104)/ml,(P >0.05)] .Conclusions. PMPs is a useful indicator in monitoring platelet activation,and plays an important role in thrombotic disease. By flow cytometric internal standard method,PMPs can be counted rapidly and accurately,which may be very helpful in interlaboratory comparative studies.
基金supported by the National Natural Science Foundation of China(No.31470699)the Fundamental Research Funds for the Central Universities(No.130420003)
文摘Analyzing the ploidy levels of plants is important for identifying species, selecting parental lines, identifying the relationships between species, and determining evolutionary patterns. The genus Chrysanthemum is widely distributed throughout the world and exhibits different ploidy levels. We used flow cytometry to analyze the ploidy levels of nine species of Chrysanthemum L. collected from different regions and geographical locations in China. Three diploids from Henan and Wuhan provinces corresponded to Chrysanthe- mum lavandulifolium and two species of C. nankingense, while three tetraploids from various regions corresponded to C. indicum and two species of C. chanetii. Two hexaploids corresponding to C. vestitum were collected at Funiu moun- tain （Henan province）, and C. zawadskii was collected at Huangshan mountain （Anhui province）. We found that OTTO extraction buffer was suitable for extracting nuclei from most species, apart from C. zawadskii. Flow cytometry proved to bea simple, rapid, and highly accurate method for identifying ploidy levels in Chrysanthemum species.
文摘Objective:To analyze the antifungal effects of Chinese herb monomers,i.e.berberine,baicalin,eugenol and curcumin,on Candida albicans.Methods:After Candida albicans strain Y01-09 was incubated for 48 h in YEPD broth which contained different concentrations of Chinese herb components,the cell cycle,fluorescent intensity and the size of cell volume were detected by flow cytometry.Results:The 4 Chinese herb monomers could affect the cell cycle of Candida albicans in different ranges.The ratio of cells in S-G2-M period decreased as the agents concentration increased,indicating that the cell division was inhibited.The fluorescent intensity of Candida albicans cells became weaker after being incubated,which reflected the loss of DNA fragments.The higher the concentration was,the weaker the fluorescent intensity became.The cell size,cell diopter and particle size changed much as the agents concentration increased.Conclusion:Chinese herb monomers play the antifungal role in inhibiting cell division.FCM could be used to determine the susceptibility of antifungal agents.
文摘To quantitatively analyze apoptotic and secondary necrotic cells unde r apoptosis conditions. Methods. The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1 .0 μmol/L dexamethasone (DEX) for 2, 4 and 8 h respectively, then stained with Annexin V FITC (fluorescein isothiocyanate conjugated) which was used to detec t the exposed phosphatidylserine (PS) on the epimembrane resulting from a loss o f phospholipid asymmetry in the early stage of apoptosis, and also stained with propidium iodide (PI) which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably represent late stage of apoptosi s, then apoptotic cells were quantified by flow cytometry (FCM). Furthermore, An nexin+/PI and Annexin+/PI+ cells were sorted by fluoresence activated cell sorter (FACS), and identified by electron microscopy (EM) and DNA gel electroph oresis. Results. The percentage of apoptotic cells was found to increase with the incuba tion time (r=0.97). This method was sensitive with low detection limit (0.02%), and was reproducible with low coefficient variance (CV)(4.2%). Meanwhile, the Annexin+/PI and Annexin+/PI+ cells were identified as apoptotic and necroti c cells under EM, and DNA extracted from the Annexin+/PI cells was characteri zed by “ladder pattern”. Conclusions. Annexin V assay is a specific, sensitive, accurate, reproductive and quantitative method for analyzing apoptotic cells.
基金supported by the National Key R&D Program of China(2021YFD2200203)Heilongjiang Province Key R&D Program of China(GA21B010)+1 种基金Heilongjiang Touyan Innovation Team Program(Tree Genetics and Breeding Innovation Team)Heilongjiang Postdoctoral Financial Assistance(LBH-Z21097)。
文摘Doubled haploid(DH)plants have been widely used for breeding and biological research in crops.Pop ulus spp.have been used as model woody plant species for biological research.However,the induction of DH poplar plants is onerous,and limited biological or breeding work has been carried out on DH individuals or populations.In this study,we provide an effective protocol for poplar haploid induction based on an anther culture method.A total of 96 whole DH plant lines were obtained using an F1hybrid of Populus simonii×P.nigra as a donor tree.The phenotypes of the DH population showed exceptionally high variance when compared to those of half-sib progeny of the donor tree.Each DH line displayed distinct features compared to those of the other DH lines or the donor tree.Additionally,some excellent homozygous lines have the potential to be model plants in genetic and breeding studies.
文摘BACKGROUND： The effect of pituitary adenylate cyclase activating polypeptide （PACAP） during traumatic brain injury （TBI） and whether it can modulate secondary injury has not been reported previously. The present study evaluated the potential protective effects of ventricular infusion of PACAP in a rat model of TBI.METHODS： Male Sprague Dawley rats were randomly divided into 3 treatment groups （n=6, each）： sham-operated, vehicle （normal saline）＋TBI, and PACAP＋TBI. Normal saline or PACAP （1 μg/5 μL） was administered intracerebroventricularly 20 minutes before TBI. Right parietal cortical contusion was produced via a weight-dropping method. Brains were extracted 24 hours after trauma. Histological changes in brains were examined by HE staining. The numbers of CD4＋ and CD8＋ T cells in blood and the spleen were detected via flow cytometry.RESULTS： In injured brain regions, edema, hemorrhage, inflammatory cell infiltration, and swollen and degenerated neurons were observed under a light microscope, and the neurons were disorderly arrayed in the hippocampi. Compared to the sham group, average CD4＋ CD8＋ lymphocyte counts in blood and the spleen were significantly decreased in rats that received TBl＋vehicle, and CD4- CD8＋ were increased. In rats administered PACAP prior to TBI, damage was attenuated as evidenced by significantly increased CD4＋, and decreased CD8＋, T lymphocytes in blood and the spleen.CONCLUSION： Pretreatment with PACAP may protect against TBI by influencing periphery T cellular immune function.
文摘Objective：To compare the phenotype characteristics of rat annulus fibrosus （AF） cells cultured on flexible silicone membranes and those in plastic plates. Methods：The morphology of AF cells cultured in different suhstrates was examined. Proteoglycan was stained by toluidine blue. Contents of collagen type Ⅰ, collagen type Ⅰ and aggrecan mRNAs were determined by reverse transcription-polymerase chain reaction （RT-PCR）. The expression of integrin β1 was monitored by flow cytometry. By using propidium iodide （PI）, the cell cycle in AF cells was analyzed. Cell adhesion to silicone membrane was also measured. Results：The AF cells cultured on different suhstrates were morphologically undistinguishable. Toluidine blue staining showed that there was also no difference between AF cells cultured on these 2 substrates. They still had the same expression levels of collagen type Ⅰ , collagen type Ⅰ, aggrecan mRNAs, and integrin β1. No significant difference was observed in the distribution of the cell cycle. AF cells grew well on silicone membrane. Conclusion：AF cells cultured on flexible silicone membrane maintain the stability of phenotype and may he appropriate for further studying the metabolic responses to mechanical stimuli at the cellular level.
基金suppor ted by the US Depar tment of Defense Congressionally Directed Medical Research Program (CDMRP)awards (http://cdmrp.army.mil/) W81XWH-16-1-0632 (Craddock PI),W81XWH-16-1-0552 (Craddock PI),W81XWH-18-1-0549 (Sullivan PI),W81XWH-13-2-0072 (Sullivan PI),and W81XWH-09-2-0071 (Klimas PI)a Veterans Affairs Merit Award (4987.69) to Dr.Nancy Klimas。
文摘Background:One-third of veterans returning from the 1990–1991 Gulf War reported a myriad of symptoms including cognitive dysfunction,skin rashes,musculoskeletal discomfort,and fatigue.This symptom cluster is now referred to as Gulf War Illness(GWI).As the underlying mechanisms of GWI have yet to be fully elucidated,diagnosis and treatment are based on symptomatic presentation.One confounding factor tied to the illness is the high presence of post-traumatic stress disorder(PTSD).Previous research efforts have demonstrated that both GWI and PTSD are associated with immunological dysfunction.As such,this research endeavor aimed to provide insight into the complex relationship between GWI symptoms,cytokine presence,and immune cell populations to pinpoint the impact of PTSD on these measures in GWI.Methods:Symptom measures were gathered through the Multidimensional fatigue inventory(MFI)and 36-item short form health survey(SF-36)scales and biological measures were obtained through cytokine&cytometry analysis.Subgrouping was conducted using Davidson Trauma Scale scores and the Structured Clinical Interview for Diagnostic and statistical manual of mental disorders(DSM)-5,into GWI with high probability of PTSD symptoms(GWIH)and GWI with low probability of PTSD symptoms(GWIL).Data was analyzed using analysis of variance(ANOVA)statistical analysis along with correlation graph analysis.We mapped correlations between immune cells and cytokine signaling measures,hormones and GWI symptom measures to identify patterns in regulation between the GWIH,GWIL,and healthy control groups.Results:GWI with comorbid PTSD symptoms resulted in poorer health outcomes compared with both healthy control(HC)and the GWIL subgroup.Significant differences were found in basophil levels of GWI compared with HC at peak exercise regardless of PTSD symptom comorbidity(ANOVA F=4.7,P=0.01)indicating its potential usage as a biomarker for general GWI from control.While the unique identification of GWI with PTSD symptoms was less clear,the GWIL subgroup was found to be delineated from both GWIH and HC on measures of IL-15 across an exercise challenge(ANOVA F>3.75,P<0.03).Additional differences in natural killer(NK)cell numbers and function highlight IL-15 as a potential biomarker of GWI in the absence of PTSD symptoms.Conclusions:We conclude that disentangling GWI and PTSD by defining trauma-based subgroups may aid in the identification of unique GWI biosignatures that can help to improve diagnosis and target treatment of GWI more effectively.
