Comparative of Forensic DNA Identification Using Cell Lysis Method and Magnetic Beads Method / 法医学杂志

OBJECTIVES@#To compare the effects of cell lysis method and magnetic beads method in forensic DNA identification and to explore these two methods in forensic DNA identification.@*METHODS@#The genome DNA of THP-1 cells in different quantities was extracted by the cell lysis method and magnetic beads method, and the DNA content was quantified by real-time quantitative PCR. The cell lysis method and magnetic beads method were used to type the STR of human blood with different dilution ratios.@*RESULTS@#When the numbers of THP-1 cell were 100, 400 and 800, the DNA content extracted by cell lysis method were (1.219±0.334), (5.081±0.335), (9.332±0.318) ng, respectively; and the DNA content extracted by magnetic beads method were (1.020±0.281), (3.634±0.482), (7.896±0.759) ng, respectively. When the numbers of THP-1 cells were 400 and 800, the DNA content extracted by the cell lysis method was higher than that by the magnetic beads method. The sensitivity of cell lysis method and magnetic beads method was similar in STR typing of human blood at different dilution ratios. Complete STR typing could be obtained at 100, 300 and 500-fold dilutions of blood samples, but could not be detected at 700-fold dilution. STR typing of undiluted human blood could not be detected by cell lysis method.@*CONCLUSIONS@#The cell lysis method is easy to operate and can retain template DNA to the maximum extend. It is expected to be suitable for trace blood evidence tests.

Effect of IgG dimer concentration on the binding ability of IgG Fc fragment in IVIG to THP-1 cell surface receptors / 中国输血杂志

【Objective】 To investigate the effect of immunoglobulin G (IgG) dimer concentration of intravenous immunoglobulin (IVIG) on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Methods】 Firstly, protein purification and high performance liquid chromatography (HPLC) were used to prepare different concentrations of IgG dimers. After that, IgG dimer was added to IVIG to prepare IVIG containing different concentrations of IgG dimer. Finally, based on the method established in our laboratory, we analyzed the effect of IgG dimer concentration in IVIG on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Results】 When the concentration of IgG dimer in IVIG was 1.11%-10.30%, its binding ability to Fc receptors on the surface of THP-1 cell was 97.67%-135.33%, and this binding ability was positively correlated with the concentration of IgG dimer. When the IgG dimer concentration exceeded 13.22%, the binding ability had no correlation with the IgG dimer concentration. 【Conclusion】 A certain concentration of IgG dimer can promote the binding ability of the IgG Fc fragment in IVIG to receptors on the surface of THP-1 cells, which needs further verification from animal experiments and clinical data.

Proteomic characterization of four subtypes of M2 macrophages derived from human THP-1 cells / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Macrophages are widely distributed immune cells that contribute to tissue homeostasis. Human THP-1 cells have been widely used in various macrophage-associated studies, especially those involving pro-inflammatory M1 and anti-inflammatory M2 phenotypes. However, the molecular characterization of four M2 subtypes (M2a, M2b, M2c, and M2d) derived from THP-1 has not been fully investigated. In this study, we systematically analyzed the protein expression profiles of human THP-1-derived macrophages (M0, M1, M2a, M2b, M2c, and M2d) using quantitative proteomics approaches. The commonly and specially regulated proteins of the four M2 subtypes and their potential biological functions were further investigated. The results showed that M2a and M2b, and M2c and M2d have very similar protein expression profiles. These data could serve as an important resource for studies of macrophages using THP-1 cells, and provide a reference to distinguish different M2 subtypes in macrophage-associated diseases for subsequent clinical research.

Effect of miltirone on proliferation and apoptosis of leukemia THP-1 cells / 中国中药杂志

To investigate the toxicity and related mechanism of miltirone to human acute myeloid leukemia THP-1 cells. To be specific, the active components and targets of miltirone were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the target proteins were converted into standard gene names with UniProt. Acute leukemia-rela-ted target genes were screened from GeneCards and DisGeNET. Venn diagram was constructed with Venny 2.1 to yield the common targets of the disease and the drug. The protein-protein interaction(PPI) network was constructed by STRING and Cytoscape 3.8.2. THP-1 cells in the logarithmic growth phase were treated with dimethyl sulfoxide(DMSO), and 2.5, 5, 10, 15, and 20 μmol·L~(-1) miltirone for 24 h, respectively. The proliferation rate of cells was analyzed by carboxyfluorescein diacetate succinimidyl ester(CFSE), apoptosis rate by flow cytometry with Annexin V-PE/7 AAD staining, and cell morphology by acridine orange staining. Real-time quantitative PCR(qPCR) was employed to detect the mRNA levels of nuclear receptor coactivator 2(NCOA2), poly(ADP-ribose) polymerase-1(PARP1), B-cell lymphoma-2(Bcl-2)-associated X protein(Bax), Bcl-2, and cysteine aspartyl protease-3(caspase-3). The effect of miltirone on apoptosis was detected in presence of caspase inhibitor Z-VAD-FMK. A total of 26 targets of miltirone, 1 046 genes related to acute leukemia, and 6 common targets of the two were screened out. Flow cytometry result showed miltirone at 10 μmol·L~(-1) can inhibit proliferation and promote apoptosis of THP-1 cells. The typical manifestations of apoptosis, such as cell shrinkage, nuclear rupture, and chromatin agglomerate were displayed by acridine orange staining. The decreased mRNA levels of NCOA2 and PARP1 and increased Bax/Bcl-2 ratio and the activity of pro-apoptotic protein caspase-3 were observed. Z-VAD-FMK can attenuate the apoptosis-inducing effect of miltirone. This study indicates that miltirone can inhibit the proliferation and promote the apoptosis of THP-1 cells, by down-regulating NCOA2 and PARP1, raising Bax/Bcl-2 ratio, and activating caspase-3.

Effect of IgG fragments in IVIG(pH4)on phagocytosis of sensitized erythrocytes by macrophage / 中国输血杂志

【Objective】 To research the effect of the Fc, Fab and F(ab′)2 fragments of immunoglobulin G, the main components of Human Immunoglobulin(pH4) for Intravenous Injection(IVIG), on the phagocytic function of macrophages derived from THP-1 cells. 【Methods】 First of all, IVIG was digested with papain and pepsin to obtain Fc, Fab and F(ab′)2, and these components were then identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Afterwards, propylene glycol monomethyl ether acetate (PMA) was used to induce THP-1 cells to differentiate into M0 macrophages. Finally, the sensitized erythrocytes were labeled with carboxy fluorescein succinimidyl ester (CFSE), and the effect of the above components on the phagocytic ability of M0 macrophages to engulf sensitized erythrocytes was detected by flow cytometry. 【Results】 The identification results of SDS-PAGE showed that the prepared IgG fragments met the requirements of subsequent experiments. Flow cytometry performs showed that the phagocytosis model of M0 macrophages had been successfully established. When the concentration of Fc increased from 0.1μg/ mL to 10μg/ mL, the phagocytosis rate of erythrocytes sensitized by M0 macrophages decreased from (24.21±0.58) % to (12.27±0.19) %. When the concentration of IVIG protein increased from 0.1 μg/ml to 10 μg/ml, the phagocytosis rate decreased from (20.57±0.39) % to (0.20±0.03) %. Meanwhile, at the same protein concentration (10 μg/ml), the inhibitory effect of Fc on phagocytosis was only half that of IVIG. In addition, Fab, F(ab′)2, and human serum albumin could not inhibit phagocytosis of M0 macrophages. 【Conclusion】 IVIG can effectively inhibit the phagocytosis of THP-1 derived M0 macrophages, which is mainly dependent on the Fc, but not related to the Fab of IgG and F (ab′)2.

Gain of function in Mycobacterium bovis BCG Moreau due to loss of a transcriptional repressor

The Bacille Calmette-Guérin (BCG) vaccine comprises a family of genetically different strains derived by the loss of genomic regions (RDs) and other mutations. In BCG Moreau, loss of RD16 inactivates rv3405c * , encoding a transcriptional repressor that negatively regulates the expression of Rv3406, an alkyl sulfatase. To evaluate the impact of this loss on the BCG and host cell viability and the cytokine profile, THP-1 cells were infected with BCG Moreau (harbouring the empty vector) and a complemented strain carrying a functional copy of rv3405c. Viability of the host cells and bacteria as well as the pattern of cytokine secretion were evaluated. Our results show that the viability of BCG Moreau is higher than that of the complemented strain in an axenic medium, suggesting a possible functional gain associated with the constitutive expression of Rv3406. Viability of the host cells did not vary significantly between recombinant strains, but differences in the profiles of the cytokine secretion (IL-1β and IL-6) were observed. Our results suggest an example of a functional gain due to gene loss contributing to the elucidation of the impact of RD16 on the physiology of BCG Moreau.

Effect of Aspergillus fumigatus on the expression of tumor necrosis factor-α and activation of intracellular signaling molecule p38 mitogen-activated protein kinase by a human acute monocytic leukemia cell line THP-1 / 中华皮肤科杂志

Objective To evaluate the effect of Aspergillus fumigatus on the expression of tumor necrosis factor-α (TNF-oα) and activation of intracellular signaling molecule p38 mitogen-activated protein kinase (p38MAPK) in a human acute monocytic leukemia cell line THP-1.Methods Cultured THP-1 cells (2 x 105/ml) were divided into 4 groups to be treated with Aspergillus fumigatus suspensions at concentrations of 106 and 107 colony-forming units (CFU)/ml (106-and 107-CFU/ml Aspergillusfumigatus groups),100 mg/L β-glucan (a positive stimulus,β-glucan group),culture medium (blank control group) respectively for 1,3 and 6 hours.Real-time fluorescence-based quantitative PCR (qPCR) was conducted to determine the mRNA expression of TNF-α in the THP-1 cells in the above groups.Some other THP-1 cells were treated with 107 CFU/ml Aspergillusfumigatus suspensions (107-CFU/ml Aspergillusfumigatus group),β-glucan (β-glucan group) and culture medium (blank control group) separately for 24 hours,and enzymelinked immunosorbent assay (ELISA) was performed to detect the level of TNF-α in the culture supernatant of THP-1 cells.Western blot analysis was conducted to detect the levels of p38MAPK and phosphorylated p38MAPK in THP-1 cells after 15-,30-and 60-minute treatment with 107 CFU/ml Aspergillusfumigatus suspensions.After 2-hour incubation with the p38MAPK inhibitor SB203580 (20 μmol/L),some THP-1 cells were additionally treated with 107 CFU/ml Aspergillus fumigatus suspensions,β-glucan and culture medium separately for 6 hours,and those without SB203580 treatment served as the control group.Then,qPCR was performed to measure the mRNA expression of TNF-α in the THP-1 cells in the above groups.Results The mRNA expression of TNF-α significantly differed among the 106-and 107-CFU/ml Aspergillus fumigatus groups,β-glucan group and blank control group (F =110.983,P ＜ 0.001),and significantly increased over time (F =701.680,P ＜ 0.001).After 24-hour treatment with 107 CFU/ml Aspergillus fumigatus suspensions,the TNF-α level(6 236.30 ± 437.12 ng/L)significantly increased compared with the blank control group (132.10 ± 0.61 ng/L,P ＜ 0.01).Thirty minutes after the treatment with 107 CFU/ml Aspergillusfumigatus suspensions,the phosphorylated p38MAPK level significantly increased,but started to decrease at 60 minutes.The mRNA expression of TNF-α was significantly lower in the SB203580-treated Aspergillusfumigatus groups (3.83 ± 0.62) than in the SB203580-untreated Aspergillus fumigatus groups (187.23 ± 21.62).Conclusion After the treatment with Aspergillus fumigatus,human THP-1 cells can activate the signal molecule p38MAPK and secrete TNF-α,suggesting that monocytes may participate in the innate immune response to Aspergillusfumigatus infection.

Palmitate triggers inflammatory response by upregulating fatty acid translocase in THP-1 macrophages / 中国病理生理杂志

AIM:To investigate the role of fatty acid translocase(FAT/CD36)on palmitate-induced inflam-mation in human monocyte-derived macrophage THP-1.METHODS:THP-1 cells were treated with palmitate(0,0.1 and 0.2 mmol/L)for 24 h.Transwell chamber assay was used to examine the migration ability of THP-1 cells.The mRNA ex-pression of CD36,tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and monocyte chemotactic protein 1(MCP-1) was measured by real-time PCR.The protein levels of TNF-αand IL-6 in the supernatant of cultured cells were measured by ELISA.The protein level of CD36 was examined by Western blot.Small interfering RNA(siRNA)targeting CD36 (siCD36)was used to inhibit the expression of CD 36 in the THP-1 cells,and the changes of the cell migration and inflam-matory response were monitored as mentioned above.RESULTS:Palmitate increased the expression of CD36 in the THP-1 cells(P<0.05).Palmitate also up-regulated inflammatory cytokine and chemokine levels,and the differences were sta-tistically significant(P<0.05).Compared with control group,palmitate promoted migration of THP-1 cells.siCD36 was transfected into the THP-1 cells and the silencing efficiency was approximately 54%.The protein levels of TNF-αand IL-6 were also decreased in siCD36 group compared with scrambled RNA(scrRNA)group,and the differences were statisti-cally significant(P<0.05).The migrated cells in siCD36 group were significantly less than those in scrRNA group(P<0.05).CONCLUSION:Palmitate promotes migration ability and triggers inflammatory response in the THP-1 macropha-ges by upregulating CD36 expression.

Promoting effect of Ginsenoside Ro on differentiation of THP-1-derived DC / 中国免疫学杂志

Objective:To investigate the promoting effect of Ginsenoside Ro on the differentiation of THP-1-derived dendritic cells (DCs) induced by GM-CSF and IL-4.Methods: Sensitive leukemia-derived DC cell line was screened first.Then,the selected sensitive cell line THP-1 was stimulated to differentiate into DCs by cytokines (GM-CSF and IL-4) and small(5 μmol/L),middle(10 μmol/L),and large (20 μmol/L) dose of Ginsenoside Ro respectively.The expressions of CD1a,MHCⅡ and CD86 of leukemia-derived DCs were detected by flow cytometry.In addition,the transcription levels of CD1a,CD86 and MHCⅡ of leukemia-derived DCs were detected by RT-PCR.ELISA was used to measure the protein levels of TNF-α and IL-6 in the culture supernatant.Results: THP-1 was the sensitive leukemia cell line which could be induced to differentiate into DCs by cytokines.Compared with cytokine stimulation alone,the expression of CD1a,MHCⅡ and CD86 in leukemia-derived cells was significantly increased after the stimulation of Ginsenoside Ro combined with cytokine(P<0.05).The CD1a,CD86 and MHCⅡ mRNA expression was significantly increased after the treatment of Ginsenoside Ro combined with cytokine(P<0.05).Moreover,the protein levels of TNF-α and IL-6 in culture supernatant were significantly increased (P<0.05) after the stimulation of Ginsenoside Ro in combination with cytokines.Conclusion: Ginsenoside Ro can significantly promote the differentiation of leukemia-derived DCs.

Mechanistic study on the role of microRNA-146a in THP-1 cells-associated inflammatory response induced by Cryptococcus neoformans and Cryptococcus gattii / 中国感染与化疗杂志

Objective To analyze the differential expression of microRNA-146a (miR-146a) in monocyte-macrophage cell line (THP-1 cells) after induction by Cryptococcus neoformans (C.neoformans,reference strain WM148) or Cryptococcus gattii (C.gattii,reference strain R265),and investigate the mechanism of miR-146a in regulating the inflammatory response of cryptococcal meningitis.Methods The cultured THP-1 cells were divided into two groups to be induced by C.neoformans or C.gattii,respectively.THP-1 cells were induced with inactivated WN148 (or R265) strains at multiplicity of infection (MOI) of 5 in all experiments.The supematant and the cell pellet were collected separately after incubation.The expression of miR-146a was measured by real-time quantitative PCR (qRT-PCR) technique.The levels of TNF-u and IL-6 release were assayed by ELISA.Results The expression of miR-146a increased significantly in the C.neoformans induction group compared to 0 h.It reached peak at 3 h (P＜0.01),and then declined gradually.The level of TNF-α increased in supematant and reached peak at 12 h.The expression of IL-6 did not change significantly at each time point.The expression of miR-146a and TNF-α increased gradually and reached peak at 12 h in the C.gattii induction group (P ＜0.01),but the change did not reach statistical significance at 3 h,6 h time points.The expression of IL-6 gradually increased,and reached peak at 12 h time point.Conclusions Following stimulation with C.neoformans or C.gattii,the expression ofmiR-146a in THP-1 cells showed different patterns over time.The expression levels of TNF-α and IL-6 showed different patterns.These findings suggest that there may be different regulatory mechanisms in the THP-1 cells-associated inflammatory response after stimulation by inactivated C.neoformans and C.gattii strains.

Effects of interleukin-34 expressed by human bone marrow derived mesenchymal stem cells on THP-1 cells / 生物工程学报

To construct recombinant eukaryotic expression plasmid vector of human IL-34 gene, and to study the effects of IL-34 expressed by human bone marrow-derived mesenchymal stem cells (hBM-MSCs) on THP-1 cells. Full-length IL-34 encoding sequence was amplified by PCR. And this fragment was cloned into the plasmid pIRES2-EGFP. Western blotting and ELISA were used to analyze the expression of IL-34 in hBM-MSCs. THP-1 cells were cultured with hBM-MSCs medium containing IL-34 protein. Real-time PCR detected the effects of IL-34 on the expression of IL-10 and TNFα in THP-1 cells. Restrictive enzyme analysis and sequencing demonstrated that IL-34 eukaryotic expression vector was successfully constructed. IL-34 protein expressed by hBM-MSCs could promote IL-10 and TNFα expression in THP-1 cells. Those results show that IL-34 expressed by hBM-MSCs has regulating effect on THP-1 cells.

The effect of moderate static magnetic fields on secretion of pro-inflammatory factors in THP-1 cells / 天津医药

Objective To investigate the effect of moderate static magnetic fields (SMF) on secretion of inflammato?ry factors tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) in human monocytic leukemic cell line THP-1. Methods THP-1 cells at logarithmic phase were divided into control group and magnetic treatment group. CCK-8 method was used to detect cell proliferation after THP-1 cells were exposed to 60 mT, 200 mT and 400 mT static magnetic fields at 18, 24 and 48 h. Then THP-1 cells were divided into control group, magnetic treatment group, LPS activation group and LPS+SMF treatment group. When magnetic treatment group and LPS+SMF treatment group were ex?posed to SMF at 18, 24 and 48 h, the levels of the cytokines TNF-α, IL-6 and IL-8 were determined by ELISA. Results (1) 60 mT, 200 mT and 400 mT SMF had no significant effects on cell proliferation in THP-1 cells (P>0.05). (2)THP-1 cells secreted more TNF-αand IL-6 in 24 h than 18 h in every group, while IL-8 didn′t change. Compared with 24 h, the secre?tion of TNF-αdecreased and IL-6 didn′t change, while IL-8 increased in 48 h. At three sampled time THP-1 cells of LPS activation group secreted more TNF-α, IL-6, IL-8 than those of control group and magnetic treatment group. After magnetic treatment THP-1 cells of LPS+SMF treatment group secreted less TNF-α, IL-6, IL-8 than those of LPS activation group (P<0.05). Conclusion Static magnetic field may have some inhibitory effects on release of TNF-α, IL-6, IL-8 from THP-1 cells, which can provide basic data for the treatment of rheumatoid arthritis.

Construction and expression of eukaryotic expression vector of human IL-37 b gene / 中国实验动物学报

Objective To construct the eukaryotic expression vector pEGFP-N1/IL-37b and analyze the expression of IL-37 gene in THP-1 cells. Methods Total RNA was extracted from human peripheral blood mononuclear cells ( PB-MCs) and the coding region of IL-37b gene was amplified by RT-qPCR. Then, the gene was cloned into pEGFP-N1 eu-karyotic expression vector. After transfected the recombinant plasmid into THP-1 cells, the expression of IL-37 was detec-ted by RT-qPCR and Western blot. Results Double restriction enzyme digestion and gene sequencing showed that IL-37b gene was correctly inserted into the eukaryotic expression vector pEGFP-N1. RT-qPCR and Western blot showed that the IL-37 expression level was increased significantly (P<0. 01) after transfection in THP-1 cells. Conclusions We successful-ly constructed a novel anti-inflammatory cytokine IL-37 eukaryotic expression vector pEGFP-N1/IL-37b, which lays a foun-dation for further study on IL-37 functions and its association with related diseases.

Human amnion mesenchymal cells inhibit lipopolysaccharide-induced TNF-α and IL-1ß production in THP-1 cells

BACKGROUND: Human amnion mesenchymal cells (hAMCs), isolated from the amniotic membrane of human placenta, are a unique population of mesenchymal stem cells. Recent studies demonstrated that hAMCs could inhibit the activities and functions of several immune cells. However, their effect on inflammatory macrophages is largely unknown. This study investigated the effect of hAMCs on expression of inflammatory cytokines and mitogen-activated protein kinases (MAPKs)/NF-kB pathway in human THP-1 macrophages induced by lipopolysaccharide (LPS). RESULTS: The levels of TNF-α and IL-1ß secreted by LPS- stimulated THP-1 cells were increased significantly compared with those in the control group. After co-culture with different numbers of hAMCs, the levels of TNF-α and IL-1ß in LPS-stimulated THP-1 cells were significantly reduced compared with the LPS group. The mRNA expression of TNF-α and IL-1ß were also markedly inhibited. Moreover, treating LPS-stimulated THP-1 cells with hAMCs supernatants could also suppress TNF-α and IL-1ß production in THP-1 cells. Important signaling pathways involved in the production of TNF-α and IL-1ß were affected by hAMCs co-culture: hAMCs remarkably suppressed NF-kB activation and down-regulated the phosphorylation of ERK and JNK in LPS- stimulated THP-1 cells. CONCLUSIONS: Human amnion mesenchymal cells inhibited the production of TNF-α and IL-1ß secreted by LPS-stimulated THP-1 cells, partly through the suppression of NF-kB activation and ERK and JNK phosphorylation.

Influence of Serum Containing Qingre Chubi Decoction on THP-1 Cell Viability and Interleukin-1βRelease Stimulated by Monosodium Urate Crystals / 广州中医药大学学报

Objective To investigate the influence of serum containing Qingre Chubi Decoction ( QCD) on the THP-1 cell viability and the release of interleukin 1 beta ( IL-1β) stimulated by monosodium urate crystals in vitro. Methods The cultured human monocyte THP-1 strain were divided into blank serum group, model control group, and high-, middle- and low-concentration ( volume fraction being 20%, 10%, 5%) QCD-containing serum groups. Except for the blank serum group , the other groups were all given 500 mg/L of monosodium urate crystals. On culturing hour 0, 12, 24 and 48, THP-1 cell viability was tested by methy1 thiazolyl tetrazolium celorimetry ( MTS) method. On culturing hour 48, the content of IL-1β in the supernatant of THP-1 cells was detected by enzyme-linked immunosorbent assay ( ELISA) . Results The THP-1 cell viability in various groups was increased along with the prolongation of culturing time. The THP-1 cell viability in the model control group was increased as compared with that in the blank serum group at different time points (P<0.05 or P<0.01) . And the content of IL-1β in the model control group was increased significantly as compared with that in the blank serum group on culturing hour 48 (P<0.01) . The THP-1 cell viability in various QCD-containing serum groups on culturing hour 12 and 24, and in high- and middle-concentration QCD-containing serum group on culturing hour 48 was decreased significantly as compared with that of the model control group at the same time point ( P<0.05 or P<0.01) . The content of IL-1β in various serum containing QCD groups was markedly decreased as compared with that in the model control group on culturing hour 48 ( P<0.01) . Conclusion Serum containing QCD can inhibit the viability of THP-1 cells stimulated by monosodium urate crystals, and the possible mechanism is related with the inhibition of IL-1 release.

Influence of leptin on secretion of chemokine in THP1 cells and its mechanism / 吉林大学学报(医学版)

Objective To investigate the effect of leptin on the secretion of chemokine in THP1 cells and explore its related mechanism, and to provide basis for research on the role of leptin in immune response.Methods The expressions of Ob-Rb and Ob-Rt in THP1 cells were detected by RT-PCR and flow cytometry (FCM).The THP1 cells at logarithm growth phase were selected and randomly divided into blank control group and different concentrations(10,50,100,200μg· L-1 )of leptin groups.Transwell chamber assay was performed to detect the number of invated THP1 cells.The THP1 cells were divided into blank control group and 100μg·L-1 leptin group.Western blotting method was carried out to detect the expressions of p-AKT,p-ERK 1/2,and p-STAT3 in THP1 cells.The THP1 cells were divided into blank control group and 100μg·L-1 leptin group,100μg·L-1 leptin+ DMSO group,100μg·L-1 leptin+50μmol·L-1 AG490 group,100μg·L-1 leptin+10μmol·L-1 LY294002 group and 100μg·L-1 leptin+ 10 mol·L-1 PD980590 group.RT-PCR and Western blotting methods were performed to detect the expression of IL-8.Results Ob-Rb and Ob-Rt were highly expressed in THP1 cells. Compared with blank control group,the number of invated THP1 cells was significantly increased in 50,100,and 200μg·L-1 leptin groups (P0.05).Conclusion leptin can up-regulate the expression of chemokine in THP1 cells,which might be associated with PI3K-AKT and MAPK/ERK 1/2 signaling pathways.

Influence of RNA interference on MMP-9 gene expression in THP-1 cell line / 中国免疫学杂志

To study the inhibitory effect of RNA interference (RNAi) on MMP-9 gene expression in THP-1 cell line.To investigate the application of RNAi on the therapy of leukemia.Methods:Small interfering RNA ( siRNA) for MMP-9 gene was designed and transfected into THP-1 cells.MMP-9 mRNA expression was assessed by RT-PCR, and MMP-9 protein expression was tested by Western blot.MTT and trypan blue staining were used to observe the effect on the proliferation of THP-1 cells after RNAi.The changes in cell morphology were observed under the microscope.Results:The expressions of MMP-9 mRNA and protein were inhibited in THP-1 MMP-9 siRNA-transfected cells ,significantly lower than those of control cells.The results of MTT and trypan blue staining in-dicated that the proliferation ability of THP-1 cells obviously decreased after siRNA-transfected 48h and 72h.The growth of cells was in-hibited and the cells survival rate was significantly lower than that of control group ( P<0.05 ).The cells of control groups grew semi-quote wall under inverted microscope.The outline of cells was clear and the shape was uniform.The cells grew vigorously.While the growth of cells in siRNA group was inhibited.The morphology of siRNA group cells changed obviously by the Wright staining.Most cells expressed changes of apoptosis.Conclusion: siRNA for MMP-9 gene can not only reduce the expressions of MMP-9 mRNA and protein,but also inhibit the proliferation and induce apoptosis of THP-1 cells.

Treponema pallidum membrane protein Tpp47 promotes the adhesion ability of vascular endothelial cells in vitro: an experimental study / 中华皮肤科杂志

Objective To evaluate the effect of Treponema pallidum membrane protein Tpp47 on vascular endothelial cells.Methods Human umbilical vein endothelial cells (HUVECs) were classified into multiple groups to be cultured with various concentrations (50,100,200,400 and 800 μg/L) of the recombinant protein Tpp47 or lipopolysaccharide (LPS) for different durations (3,6,12,24 and 48 hours).Then,enzyme-linked immunosorbent assay (ELISA) was performed to determine the levels of intercellular cell adhesion molecule-1 (ICAM-1) and E-selectin in the culture supernatant of,fluorescence-based real-time quantitative PCR to quantify the mRNA expressions of ICAM-1 and E-selectin in,HUVECs.The 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay was used to evaluate the proliferation activity of HUVECs treated with Tpp47 (400 μg/L) and LPS (200 μg/L) respectively for 24 hours.To estimate the effect on adhesion ability,some HUVECs were pretreated with Tpp47 (400 μg/L) and LPS (200 μg/L) respectively for 24 hours followed by coculture with THP-1 human monocytic leukaemia cells for 6 hours,then,the adhesion of HUVECs to THP-1 cells was visualized by fluorescence microscopy.The cells receiving no treatment served as the blank control.Results A significant increase was observed in the supernatant level (expressed as the absorbance value at 450 nm) of ICAM-1 for HUVECs treated with Tpp47 of 400 μg/L for 24 hours (1.28 ± 0.03 vs.0.90 ± 0.01,t =18.28,P ＜ 0.05) and that of E-selectin for HUVECs treated with Tpp47 of 400 μg/L for 12 hours (0.51 ± 0.01 vs.0.13 ± 0.03,t =18.19,P＜ 0.05) compared with untreated HUVECs.The adhesion rate to THP-1 cells was significantly higher in HUVECs pretreated with Tpp47 for 24 hours than in untreated HUVECs (56.1％ ± 1.9％ vs.16.3％ ± 2.1％,x2 =12.65,P ＜ 0.05).The cell proliferation rate was 19.5％ ± 1.7％ in HUVECs treated with Tpp47,significantly higher than that in untreated HUVECs (10.0％ ± 3.1％,x2 =3.92,P＜ 0.05),but lower than that in those treated with LPS (41.2％ ± 3.7％,x2 =10.42,P ＜ 0.05).Conclusions The recombinant membrane protein Tpp47 could enhance HUVECs to proliferate and adhere to monocytic THP-1 cells,suggesting a certain role of Tpp47 in the pathogenesis of syphilis.

Treponema pallidum membrane protein Tpp17 activates vascular endothelial cells in vitro / 中华微生物学和免疫学杂志

Objective To study the effects of Treponema pallidum membrane protein Tpp17 on ac-tivation of human umbilical vein endothelial cells (HUVECs) in vitro and to understand its role in the immu-nopathogenesis of syphilis .Methods HUVECs were co-cultured with recombinant protein Tpp 17.Then the expressions of TNF-α, MCP-1, ICAM-1 and E-selectin at mRNA and protein levels in supernatants were re-spectively detected by enzyme-linked immunosobent assay ( ELISA ) and fluorescent real-time quantitative PCR.The adhesive ability of THP-1 cells was observed by fluorescence microscopy after co-cultured pretrea-ted HUVECs with recombinant protein Tpp 17 with Calcein-AM labeled THP-1.Pretreated HUVECs were cultured in the lower chamber of Transwell with recombinant protein Tpp 17 and monocytic THP-1 cells were cultured in the upper chamber of Transwell .After that, the migration of monocytic THP-1 cells was evalua-ted by using fluorescence microscopy .Results Compared with the blank control group , the expression of TNF-α, ICAM-1, E-selectin, especially the MCP-1, were enhanced by recombinant protein Tpp 17 of Trepo-nema pallidum.Moreover, Tpp17 improved the adhesive ability and migration of monocytic THP-1 cells, es-pecially the latter.Conclusion Treponema pallidum membrane protein Tpp17 might play a certain role in the immunopathogenesis of syphilis by enhancing the expression of TNF-α, MCP-1, ICAM-1 and E-selectin, and by promoting the adherence ability and migration of monocytic THP-1 cells.

The effect of β-hydroxyisovalerylshikonin dimethylether derivative on THP-1 leukemia cell line / 白血病·淋巴瘤

Objective To evaluate the growth inhibition and apoptosis of human monocytic leukemia THP-1 cell line by using 5,8-dimethyl-2-β-hydroxyisovalerylshikonin (SK36) and explore its preliminary mechanism. Methods CCK colorimetric assay and cell counting was used to examine the growth inhibition of shikonin on THP-1 cells. The apoptosis of THP-1 cells was detected by Annexin V/PI double labeling. The activation of Caspase-3 apoptosis pathway was determined by FCM. The apoptosis and the necrosis of THP-1 cells were detected by the laser scanning confocal microscopy. Results When the THP-1 cells were treated with SK36 at 1.02 μg/ml for 24 h and 48 h, the growth inhibition was dose-dependent. The cell apoptotic rate of THP-1 cells treated with 1.02 μg/ml evaluated by FCM with Annexin V/PI double labeling staining were (40.61 ±2.13) % and (67.40±9.15) % at 24 h and 48 h after treatment, respectively, which were significantly higher than that of the control group [(16.97±0.61) %] ([ = 18.444, t = 9.528, P <0.01). SK36 could induce THP-1 cells apoptosis involving the activation of Caspase-3 (F= 323.61, P<0.01). Conclusion SK36 can induce human THP-1 cells to undergo apoptosis, and its primary mechanism was to activate the Caspase-3.