1.Influence of S1PR5 Defect on the Lymphocyte Distribution in Mice.
Zhen-Yang GU ; Xiao-Li ZHAO ; Nan YAN ; Li WANG ; Fei-Yang WANG ; Li-Li WANG ; Chun-Ji GAO
Journal of Experimental Hematology 2016;24(4):1168-1172
BACKGROUNDThe sphingosine 1-phosphate (S1P) receptors (S1PRs) are a group of G protein-coupled receptors expressed on the surface of lymphocytes. The interaction between S1P and S1PRs plays a significant role in the migration and distribution of lymphocytes.
OBJECTIVETo investigate the influence of S1PR5 defect on the lymphocytes distribution in mice.
METHODSThe distribution of different subsets of lymphocyte in the mice with S1PR5 defect was examined by flow cytometry.
RESULTSCompared with wild type mice, the number of NK cells in the peripheral blood (PB) and spleen (SP) from the mice with S1PR5 defect decreased very significantly 〔PB: 6.4±0.45% vs 2.2±0.47(P<0.01,n=3);SP: 3.0±0.91% vs 0.68±0.14%(P<0.05,n=3)〕. However, the NK cell number in the bone marrow (BM) and lymphonodes (LN) of the mice with S1PR5 defect increased very significantly 〔BM: 0.97±0.20 % vs 2.6±0.35% (P<0.01, n=3); LN: 0.35±0.16% vs 1.7±0.15% (P<0.01, n=3)〕. The percentages of CD3(+) lymphocyte in peripheral blood, spleen and lymph node were not statistically significantly different between these 2 types of mice 〔PB: 17.3±7.9% vs 17.0±4.6% (P>0.05, n=3); SP: 33.0±6.0% vs 27.4±1.8% (P>0.05, n=3); LN: 42.3±10.7% vs 51.2±2.7% (P>0.05, n=3)〕.
CONCLUSIONS1PR5 defect can significantly influence the NK cell distribution.
Animals ; Bone Marrow ; Cell Count ; Flow Cytometry ; Lymphocytes ; Lysophospholipids ; Mice ; Receptors, Lysosphingolipid ; Sphingosine ; analogs & derivatives
2.Sphingosine-1-phosphate receptors respond differently to early myocardial ischemia and ischemia-reperfusion in vivo.
Geng-Qian ZHANG ; Zheng LIANG ; Xiao-Jia ZHANG
Acta Physiologica Sinica 2014;66(2):169-174
Sphingosine-1-phosphate (S1P) has been demonstrated to be a mediator and marker of heart diseases. We hypothesized that the expression of S1P receptors is involved in the S1P-mediated cardioprotection in vivo and may serve as a biomarker of ischemic heart disease. In vivo models of myocardial ischemia (MI) and ischemia-reperfusion (IR) were established by ligation of the left anterior descending artery (LAD) of rat heart, the mRNA expressions of S1PR1-3 were detected using real time PCR at different time intervals after ischemia (LAD for 15 min, 30 min, and 1 h) and IR. The results showed that mRNA expression of S1PR3, but not S1PR1 and S1PR2, increased greatly after IR. No statistical difference was found in any of the three S1P receptors after MI within 1 h. Regarding the studies of lipid concentration changes in myocardiopathy, we conclude that S1P receptors are not early response biomarkers for MI. There are different mechanisms when S1P plays a protection role in heart during MI and IR. The cooperation of lipid content and S1P receptor expression appears to form a regulation network during MI and IR.
Animals
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Lysophospholipids
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physiology
;
Myocardial Reperfusion Injury
;
physiopathology
;
Rats
;
Receptors, Lysosphingolipid
;
physiology
;
Sphingosine
;
analogs & derivatives
;
physiology
3.Decreased Expression of Sphingosine-1-Phosphate Receptor 1 in the Blood Leukocyte of Rheumatoid Arthritis Patients.
Hyun Suk CHOI ; Kyong Hoon KIM ; Seohoon JIN ; Jinhyun KIM ; Inseol YOO ; Seung Pil PACK ; Un Hwan HA ; Tae Won PARK ; Soo An CHOI ; Soon Hong YUK ; Seong Wook KANG ; Yong Woo JUNG
Immune Network 2018;18(5):e39-
Sphingosine-1-phosphate (S1P) plays an important role in trafficking leukocytes and developing immune disorders including autoimmunity. In the synovium of rheumatoid arthritis (RA) patients, increased expression of S1P was reported, and the interaction between S1P and S1P receptor 1 (S1P1) has been suggested to regulate the expression of inflammatory genes and the proliferation of synovial cells. In this study, we investigated the level of S1P1 mRNA expression in the blood leukocytes of RA patients. In contrast to the previous reports, the expression level of this gene was not correlated to their clinical scores, disease durations and ages. However, S1P1 was transcribed at a significantly lower level in the circulating leukocytes of RA patients when compared to age-, and sex-matched healthy controls. Since these data may suggest the participation of S1P1, further studies are needed to determine the role of this receptor in the pathogenesis of RA.
Arthritis, Rheumatoid*
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Autoimmunity
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Humans
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Immune System Diseases
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Leukocytes*
;
Receptors, Lysosphingolipid*
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RNA, Messenger
;
Synovial Membrane
4.Inhibitory Effect of S1PR2 Antagonist JTE-013 on Proliferation of Chronic Myeloid Leukemia Cells.
Meng PANG ; Fang LI ; Jing WANG ; Hong-Mei JING
Journal of Experimental Hematology 2020;28(4):1081-1085
OBJECTIVE:
To investigate the effect of sphingosine-1-phosphate receptor 2 (S1PR2) specific antagonist JTE-013 on the proliferation of human chronic myeloid leukemia (CML) cell line K562.
METHODS:
K562 cells were treated with JTE-013 (0, 0.5, 1, 5, 10, 20 μmol/L) for 24 and 48 hours respectively, CCK8 assay was used to detect the cell viability. K562 cells were treated with JTE-013 (0, 5, 10, 20 μmol/L) for 24 hours, propidium iodide (PI) DNA staining was used to analyze the cell cycle, Western blot was used to determine the levels of P21 and Cyclin D1 protein expression.
RESULTS:
JTE-013 inhibited the proliferation of CML cell line K562 in a dose dependent manner (r=-0.971). The proliferation rate of CML cells showed that the activity of CML cells decreased gradually with the increase of JTE-013 concentration (r=-0.971). The detection demonstrated that JTE-013 suppressed tumor cell proliferation through cell cycle arrest in G/G phase. Further detection of the protein expressions of G phase regulators showed that level of P21 increased, and expression of Cyclin D1 decreased.
CONCLUSION
JTE-013, a S1PR2 antagonist, can inhibit the proliferation of human CML K562 cells, which may be achieved by arresting the cells in G/G phase.
Apoptosis
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Cell Proliferation
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Humans
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K562 Cells
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Leukemia, Myelogenous, Chronic, BCR-ABL Positive
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Pyrazoles
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Pyridines
;
Receptors, Lysosphingolipid
;
Sphingosine-1-Phosphate Receptors
5.Bone marrow stem/progenitor cell mobilization in C57BL/6J and BALB/c mice.
Hakmo LEE ; Jeong Hwan CHE ; Ju Eun OH ; Sung Soo CHUNG ; Hye Seung JUNG ; Kyong Soo PARK
Laboratory Animal Research 2014;30(1):14-20
Bone marrow (BM) has been considered as a reservoir of stem/progenitor cells which are able to differentiate into ectodermal, endodermal, and mesodermal origins in vitro as well as in vivo. Following adequate stimulation, such as granulocyte stimulating factor (G-CSF) or AMD3100, BM resident stem/progenitor cells (BMSPCs) can be mobilized to peripheral blood. Several host-related factors are known to participate in this mobilization process. In fact, a significant number of donors are resistant to G-CSF induced mobilization protocols. AMD3100 is currently used in combination with G-CSF. However, information regarding host-related factors which may influence the AMD3100 directed mobilization is extremely limited. In this study, we were to get some more knowledge on the host-related factors that affect the efficiency of AMD3100 induced mobilization by employing in vivo mobilization experiments. As a result, we found that C57BL/6J mice are more sensitive to AMD3100 but less sensitive to G-CSF which promotes the proliferation of BMSPCs. We excluded S1P as one of the host related factor which influences AMD3100 directed mobilization because pre-treatment of S1P receptor antagonist FTY720 did not inhibit BMSPC mobilization. Further in vitro experiments revealed that BALB/c mice, compared to C57BL/6J mice, have less BMSPCs which migrate in response to host related factors such as sphingosine-1-phosphate (S1P) and to CXCL12. We conclude that AMD3100-directed mobilization depends on the number of BMSPCs rather than on the host-related factors. These results suggest that the combination of AMD3100 and G-CSF is co-operative and is optimal for the mobilization of BMSPCs.
Animals
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Bone Marrow*
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Ectoderm
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Endoderm
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Granulocyte Colony-Stimulating Factor
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Granulocytes
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Humans
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Mesoderm
;
Mice*
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Receptors, Lysosphingolipid
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Tissue Donors
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Fingolimod Hydrochloride
6.Effect of rhG-CSF Mobilization on S1P5 Expression in T Lymphocyte Subsets of Allo-HSCT Donors.
Meng LI ; Li-Xun GUAN ; Zhen-Yang GU ; Sha-Sha ZHAO ; Fei-Yan WANG ; Hua-Ping WEI ; Li WANG ; Hong-Hua LI ; Jian BO ; Chun-Ji GAO
Journal of Experimental Hematology 2016;24(4):1132-1136
OBJECTIVETo explore the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) mobilization on S1P5 expression in T lymphocyte subsets of allo-HSCT donors.
METHODSThe peripheral blood was collected from 10 allo-hematopoietic stem cell transplantation (allo-HSCT) donors before and after mobilization with rhG-CSF for 4 days. The flow cytometry was used to detect S1P5 expression in T lymphocyte subsets.
RESULTSThere was no S1P5 expression on the surface of T-lymphocytes both before and after rhG-CSF mobilization. After fixation with permeabilization agent, S1P5 expression could be detected in lymphocytes after rhG-CSF mobilization, which indicates S1P5 may be located in cells. Compared with level before rhG-CSF mobilization, S1P5 expression was significantly increased in T lymphocyte subsets after rhG-CSF mobilization, CD3(+)T cells (57.92±2.32)% vs (7.94±1.47)%(P<0.05), CD4(+)T cells (72.58±1.73)% vs (5.48±0.82)%(P<0.05), CD8(+)T cells(51.79±3.57)% vs (6.46±1.01)%(P<0.05),CD3-/CD56(+)NK cells(40.00±1.47)% vs(4.97±0.74)%(P<0.05). The up-regulated level of S1P5 expression in CD4(+)T cells was most high(P<0.05).
CONCLUSIONS1P5 expression significantly increases in T lymphocyte subsets after rhG-CSF mobilization, and the up-regulated level of S1P5 expression in CD4(+)T cells is the most high.
Flow Cytometry ; Granulocyte Colony-Stimulating Factor ; Hematopoietic Stem Cell Transplantation ; Humans ; Receptors, Lysosphingolipid ; Recombinant Proteins ; T-Lymphocyte Subsets ; Transplantation, Homologous
7.Application of CRISPR/Cas9 Gene Editing Technique to Establish S1PR5 Gene Knockout Mice.
Zhen-Yang GU ; Xiao-Li ZHAO ; Nan YANG ; Li WANG ; Fei-Yan WANG ; Li-Li WANG ; Chun-Ji GAO
Journal of Experimental Hematology 2016;24(4):1155-1162
OBJECTIVETo establish the S1PR5 gene knockout mouse model by using CRISPR/Cas9 gene editing technique so as to provide the tool for studying the regulating role of sphingosine-1-phosphate receptor 5 (S1PR5) in allogeneic hematopoietic stem cell transplantation.
METHODSSingle guide RNA (sgRNA) plasmids against the exon 3 of S1PR5 were designed and constructed. Then the sgRNA and hCas9 were transcribed by T7 RNA polymerase in vitro. Cas9 mRNA and sgRNA were mixed and microinjected into fertilized eggs of C57BL/6 mice. T7E1 digestion and gene sequencing were used to detect the mutations of S1PR5. Quantitative PCR (qPCR) and Western blot were used to detect the expression of S1PR5.
RESULTSFinally 2 kinds of F2 generation of homozygous S1PR5 deficent mice (S1PR5-170/-170 mice and S1PR5-215/-215 mice) were gained, and in these 2 model mice the S1PR5 did not express at mRNA and protein levels.
CONCLUSIONA mouse model with S1PR5 dificiency has been successfully established, which shall lay a foundation for future investigation of S1PR5.
Animals ; CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Microinjections ; Mutation ; Plasmids ; RNA, Guide ; Receptors, Lysosphingolipid ; Zygote
8.Impact of rhG-CSF on Sphingosine 1-phosphate receptor 1 expression in CD4+ T cells of donor peripheral blood.
Hai-Yan ZHU ; Wan-Ming DA ; Chun-Ji GAO ; Meng LI ; Wei-Hua CHEN ; Li YÜ ; Wen-Rong HUANG
Journal of Experimental Hematology 2010;18(2):427-430
CD4+ T cells mainly interact with Sphingosine 1-phosphate (S1P) to regulate immune function through Sphingosine 1-phosphate receptor 1 (S1P1). This study was aimed to investigate the effects of recombinant human granulocyte-colony-stimulating factor (rhG-CSF) mobilization on S1P1 expression in CD4+ T cells of donor's peripheral blood. The CD4+T cells of peripheral blood were isolated by magnetic beads from 17 allo-hematopoietic stem cell transplantation (allo-HSCT) donors before and at fourth day of mobilization with rhG-CSF. The S1P1 expression was detected by real time quantitative PCR in the RNA extracted from CD4+ T cells collected before and after rhG-CSF mobilization. The results showed that the expression of S1P1 was found in CD4+ cells before and after rhG-CSF mobilization, but the expression level of SIP1 in CD4+ cells after rhG-CSF mobilization was significantly lower than that before rhG-CSF mobilization (p<0.01). It is concluded that the mobilization with rhG-CSF obviously down-regulates the expression of S1P1 in CD4+ T cells of donor's peripheral blood.
CD4-Positive T-Lymphocytes
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drug effects
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metabolism
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Granulocyte Colony-Stimulating Factor
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pharmacology
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Hematopoietic Stem Cell Mobilization
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methods
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Hematopoietic Stem Cell Transplantation
;
Humans
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Receptors, Lysosphingolipid
;
metabolism
;
Recombinant Proteins
9.Expressions of S1P1-3 in the corpus cavernosum of castrated male rats.
Xue-qin CHEN ; Ji-yi XIA ; Bo CHENG ; Rui JIANG
National Journal of Andrology 2016;22(5):393-400
OBJECTIVETo investigate the expressions of sphingosine-1-phosphate receptors 1-3 (S1P1- 3) in the corpus cavernosum of castrated male rats and its relationship with the NOS/NO/cGMP and RhoA/Rho kinase signaling pathways.
METHODSWe equally randomized 18 eight-week-old healthy male SD rats into a sham-operation control, a castration, and a testosterone replacement (TR) group and harvested the bilateral testes and epididymides from the rats in the latter two groups, followed by 4 weeks of subcutaneous injection of testosterone propionate at 3 mg per kilogram of the body weight per day for those in the TR group and that of plant oil for those in the control and castration groups. At the age of 12 weeks, we measured the serum testosterone (T) level and maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP) of the animals and determined the expressions of SlP1-3, eNOS, P-eNOS, ROCK1, and ROCK2 in the corpus cavernosum by Western blot and immunohistochemistry.
RESULTSThe serum T level was significantly decreased in the rats of the castration group as compared with those of the control and TR groups ([0.41 ± 0.04] vs [16.01 ± 1.02] and [15.84 ± 1.32] nmol/L, P < 0.01), with no statistically significant difference between the latter two groups. The ICPmax/MAP at 0 V, 3 V, and 5 V electric stimulation was remarkably lower in the rats of the castration group (0.088 ± 0.014, 0.323 ± 0.014, and 0.432 ± 0.012) than in those of the control group (0.155 ± 0.011, 0.711 ± 0. 010, and 0.819 ± 0.024) and TR group (0.153 ± 0.012, 0.696 ± 0.017, and 0.763 ± 0.027) (P < 0.01), with no significant difference between the latter two groups. With GAPDH as internal control, the animals of the castration group showed markedly reduced expressions of S1P1 ([49.99 ± 3.39]%), eNOS ([46.82 ± 3.81]%) , and P-eNOS ([45.42 ± 4.35]%) in comparison with those in the control group ([72.57 ± 3.06], [89.76 ± 3.98], and [82.53 ± 8.92] and TR group ([71.77 ± 4.43], [87.19 ± 4.23], and [79.82 ± 7.38]%) (P < 0.01) , while the expressions of S1P2, S1P3, ROCK1, and ROCK2 were significantly upregulated in the castration group ([82.35 ± 4.13], [61.03 ± 5.14], [74.50 ± 4.02], and [69.83 ± 5.75]%) as compared with those in the control group ([41.67 ± 1.68], [31.66 ± 2.67], [35.69 ± 5.56], and [39.85 ± 7.17]%) and TR group ([42.80 ± 3.87], [32.25 ± 4.22], 38.06 ± 5.21], and [42.36 ± 4.44]%) (P < 0.01).
CONCLUSIONAndrogen deficiency induces significant reduction of ICPmax/ MAP in male rats, which is possibly associated with the decline of S1P1 in the corpus cavernosum, inhibition of the eNOS/NO/cGMP signaling pathway, increased expressions of S1P2 and S1P3, and activation of the RhoA/Rho kinase signaling pathway.
Animals ; Male ; Nitric Oxide Synthase Type III ; metabolism ; Orchiectomy ; Penis ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Lysosphingolipid ; metabolism ; Testosterone ; blood ; pharmacology ; rho-Associated Kinases ; metabolism
10.Influence of NK cell S1PR5 expression on graft versus host disease in allogeneic hematopoietic stem cell transplantation.
Li-Xun GUAN ; Bo CAI ; Li GAO ; Xiao-Hong LI ; Meng LI ; Li-Li WANG ; Chun-Ji GAO
Journal of Experimental Hematology 2012;20(2):412-415
Natural killer (NK) cells can suppress the development of graft vs host disease (GVHD) while retaining antitumor response in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Sphingosine-1-phosphate receptor 5 (S1PR5) can regulate NK cell migration and distribution in vivo by interacting with sphingosine-1-phosphate (S1P). This study was aimed to investigate S1PR5 expression change of NK cells in allo-HSCT and to explore the relationship between S1PR5 change and frequency of acute/chronic graft-versus-host disease (aGVHD/cGVHD). The S1PR5 expression was detected by real time quantitative PCR in the RNA extracted from blood NK cells of 17 couples of donor and recipient one month after allo-HSCT. The results showed that S1PR5 mRNA level variations in NK cells of donors and recipients post-allo-HSCT were not statistically significant (0.235 ± 0.191 vs 0.330 ± 0.261, P > 0.05). S1PR5 expression of NK cells was significantly lower in patients with aGVHD than those in patient without aGVHD (0.973 ± 0.834 vs 6.166 ± 5.32, P < 0.05). Compared with the corresponding donor, S1PR5 expression levels of patient declined by more than 10 that caused the high incidence of aGVHD. No significant correlation was found between S1PR5 expression of NK cells and cGVHD (3.401 ± 2.324 vs 2.762 ± 1.972, P > 0.05). It is concluded that the decreased expression level of NK cells S1PR5 is associated with aGVHD occurrence. Possible mechanism is due to S1PR5 low expression affecting distribution of NK cells in vivo, so affecting the regulation of NK cells for aGVHD.
Graft vs Host Disease
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blood
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epidemiology
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metabolism
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Hematopoietic Stem Cell Transplantation
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adverse effects
;
methods
;
Humans
;
Killer Cells, Natural
;
metabolism
;
Receptors, Lysosphingolipid
;
metabolism
;
Transplantation, Homologous
;
adverse effects