1.Roles of STIM2 and TRPC3 in the CaR mediated Ca2+ entry and NO generation in human umbilical vein endothelial cells.
Jing WANG ; Hua ZHONG ; Hui ZHAO ; La-Mei WANG ; Li-Juan PANG ; Zhi-Ping SUN ; Fang HE
Chinese Journal of Applied Physiology 2014;30(4):327-332
OBJECTIVETo study the roles of stromal interaction molecule 2 (STIM2) and transient receptor potential canonical 3 (TRPC3) in extracellular Ca(2+)-sensing receptor (CaR)-induced extracellular Ca2+ influx and the production of nitric oxide (NO) in human umbilical vein endothelial cells (HUVEC).
METHODS(1) The interaction of STIM2 and TRPC3 was determined using the immunofluorescence technique. (2) The expressions of STIM2 and TRPC3 genes were silenced in HUVEC by transfection constructed STIM2 and TRPC3 RNA interference plasmids. The interference efficiency of STIM2, TRPC3 protein and mRNA levels were determined by Western blot and real time RT-PCR, respectively. (3) The second to fifth passage of HUVEC were divided into: STIM2-002 short hairpin RNA (STIM2-002 shRNA ) + spermine + Ca2+ group and TRPC3-004 short hairpin RNA (TRPC3-004 shRNA ) + spermine + Ca2+ group; control group (spermine + Ca2+ group) and vehicle+ spermine + Ca2+ group. The four groups of cells were incubated with CaR agonist spermine, the intracellular Ca2+ concentration ([Ca2+]i) was detected using the fluorescence Ca2+ indicator Fura-2/AM, and the production of NO was determined by DAF-FM (NO fluorescent probe) of each group in HUVEC.
RESULTS(1) Immunofluorescence technique results showed that STIM2 and TRPC3 proteinswere present in the cytoplasm of HUVEC. (2) The results of transfection constructed STIM2 and TRPC3 RNA interference plasmids demonstrated that shRNA targeted to the STIM2 and TRPC3 genes decreased STIM2 and TRPC3 mRNA levels by 88.2% and 74.0%, respectively (P < 0.05), simultaneously, the STIM2 and TRPC3 protein levels were decreased by 79.9% and 71.8%, respectively (P < 0.05). (3) Compared with spermine + Ca2+ group, the [Ca2+]i and the net NO fluorescence intensity of spermine + Ca(2+) + ShSTIM2-002 group, spermine + Ca(2+) + ShTRPC3-004 group and spermine + Ca2+ Vehicle group were not changed (P > 0.05).
CONCLUSIONSTIM2 and TRPC3 do not participate in CaR-mediated Ca2+ influx and NO production individually.
Calcium ; metabolism ; Cell Adhesion Molecules ; physiology ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; physiology ; Humans ; Nitric Oxide ; metabolism ; Stromal Interaction Molecule 2 ; TRPC Cation Channels ; physiology
2.Hematopoietic inhibitors elaborated by bone marrow endothelial cells.
Journal of Experimental Hematology 2002;10(6):485-491
UNLABELLEDIn this study, the roles of hematopoietic inhibitors elaborated by bone marrow endothelial cells in the proliferation and differentiation of hematopoietic progenitors were investigated. Murine bone marrow endothelial cell conditioned medium (BMEC-CM) was collected and the components with > 10 kD and < 10 kD were obtained by centrifugal ultrafiltration. The effect of BMEC-CM and its components on proliferation of hematopoietic progenitors was evaluated by CFU-GM and HPP-CFC assay and antibody neutralization test. The expression of the inhibitors in BMEC and BMEC-CM was detected by RT-PCR and Western blot, and change of proliferation and differentiation-related genes during expansion of hematopoietic progenitors was examined by membrane hybridization technique.
THE RESULTS(1) When BME C-CM and its components directly were added to CFU-GM and HPP-CFC culture system, BMEC-CM had no effect on colony formation, > 10 kD component enhanced and < 10 kD component inhibited the formation of CFU-GM and HPP-CFC. (2) When BMEC-C M and its components were added to liquid culture system of marrow cells, after 24 hours incubation, CFU-GM decreased and HPP-CFC increased significantly in B MEC-CM group, CFU-GM increased and HPP-CFC had no significant change in > 10 kD component group; and both CFU-GM and HPP-CFC reduced in < 10 kD group. (3) MIP-2, MIP-1 alpha, MSP, TGF-beta, TNF-alpha, IFN-gamma and T beta 4 were expressed in murine marrow endothelial cells, and MIP-2, MIP-1 alpha, MSP, TGF-beta, TNF-alpha and T beta 4 were existed in BMEC-CM. (4) Antibody neutralization test results demonstrated that TGF-beta, MSP, MIP-1 alpha, IFN-gamma and T beta 4 existed in BMEC-CM had significant suppressive effects on CFU-GM and HPP-CFC. (5) T beta 4 combined with 5 hematopoietic cytokines (SCF, IL-3, IL-6, GM-CSF and EPO) added to CD34(+) cells expansion culture system, HPP-CFC significantly increased compared with 5 cytokines group. T beta 4 could downregulated the expression of proliferation and differentiation-related genes and signal transduction-related genes. It is concluded that BM EC-CM promotes the proliferation of early hematopoietic progenitor cells, and this effect is related with the inhibitors existed in BMEC-CM and it could be executed via influencing cell proliferation and differentiation-related genes and signal-related genes.
Animals ; Bone Marrow Cells ; metabolism ; Cell Division ; drug effects ; Cytokines ; genetics ; pharmacology ; Endothelium ; cytology ; metabolism ; Hematopoietic Stem Cells ; drug effects ; physiology ; Mice ; RNA, Messenger ; analysis ; Thymosin ; pharmacology
3.MiR-21 Suppresses Anoikis through Targeting PDCD4 and PTEN in Human Esophageal Adenocarcinoma
Meng-Ya ZHAO ; La-Mei WANG ; Jing LIU ; Xing HUANG ; Jing LIU ; Ya-Fei ZHANG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2018;38(2):245-251
Anoikis is a form of apoptosis induced upon cell detachment from extracellular matrix.It has been determined that acquisition of resistance to anoikis is a critical step for tumor cell metastasis.MiR-21,the most prominent oncomiR,plays an important role in tumor progression.In this study,we revealed that up-regulation of miR-21 in human esophageal adenocarcinoma (EA) is associated with lymph node metastasis and poor survival rate.Because of the established anti-apoptosis effect of miR-21,it is tempting to speculate that miR-21 might contribute to tumor metastasis by regulating anoikis,qRT-PCR analysis demonstrated that miR-21 expression in OE33/AR cells (subpopulation of human EA OE33 cells that acquired resistance to anoikis) was significantly increased.Also,transfection of miR-21 mimics provided OE33 cells resisting to anoikis.By luciferase assays,we verified that PDCD4 and PTEN were the functional targets of miR-21.In mouse model,via tail vein injection experiment,we showed that the metastasis formation of OE33 cells in vivo could be mediated by changing the miR-21 expression pattern.Taken together,our findings suggested that miR-21 was involved in the regulation of anoikis in human EA cells.Targeting miR-21 may provide a novel strategy to prevent metastasis.
4.Damage of the expression of NF-kappa B, myeloperoxidase and tissue factor in liver tissue and disorder of blood coagulation following liver ischemia-reperfusion injury in rats.
Lan WANG ; Hong-mei WANG ; Jian-long ZHANG ; Mi-la KA ; Zhan SUN
Chinese Journal of Hepatology 2005;13(8):607-608
Animals
;
Blood Coagulation Tests
;
Liver
;
blood supply
;
metabolism
;
Male
;
NF-kappa B
;
biosynthesis
;
genetics
;
Peroxidase
;
biosynthesis
;
genetics
;
RNA, Messenger
;
biosynthesis
;
genetics
;
Rats
;
Rats, Wistar
;
Reperfusion Injury
;
metabolism
;
Thromboplastin
;
biosynthesis
;
genetics
5.Inducing effects of macrophage stimulating protein on the expansion of early hematopoietic progenitor cells in liquid culture.
Li-xia MA ; Yan-hong HUANG ; La-mei CHENG ; Jun LEI ; Qi-ru WANG
Chinese Medical Journal 2007;120(13):1192-1197
BACKGROUNDMacrophage stimulating protein (MSP) is produced by human bone marrow endothelial cells. In this study, we sought to observe its effects on inducing the expansion of early hematopoietic progenitor cells which were cultured in a liquid culture system in the presence of the combination of stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), erythropoietin (EPO) (Cys) and MSP or of Cys and bone marrow endothelial cell conditioned medium (EC-CM).
METHODSHuman bone marrow CD34(+) cells were separated and cultured in a liquid culture system for 6 days. Granulocyte-macrophage colony forming unit (CFU-GM) and colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) were employed to assay the effects of different treatment on the proliferation of hematopoeitic stem/progenitor cells. The nitroblue tetrazolium (NBT) reductive test and hoechest 33258 staining were employed to reflect the differentiation and apoptosis of the cells respectively.
RESULTSMSP inhibited the proliferation of CFU-GM and CFU-GEMM in semi-solid culture and the inhibitory effect on CFU-GEMM was stronger than on CFU-GM. MSP inhibited the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators. Bone marrow (BM) CFU-GEMM was 2.3-fold or 1.7-fold increase or significantly decreased in either Cys + EC-CM, Cys + MSP or Cys compared with 0 hour control in liquid culture system after 6 days.
CONCLUSIONMSP, a hematopoietic inhibitor, inhibits the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators and makes the early hematopoietic progenitor cells expand in a liquid culture system.
Antigens, CD34 ; analysis ; Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chemokine CCL3 ; Chemokines, CC ; pharmacology ; Hematopoietic Stem Cells ; drug effects ; Hepatocyte Growth Factor ; pharmacology ; Humans ; Proto-Oncogene Proteins ; pharmacology
6.Effects of endothelial cells from cord blood on the amplification of human early hematopoietic cells from cord blood in vitro.
Li-Ming YIN ; La-Mei CHENG ; Qi-Ru WANG ; Meng-Qun TAN
Journal of Central South University(Medical Sciences) 2007;32(2):304-308
OBJECTIVE:
To observe the effects of endothelial cells from umbilical cord blood (UCB) on the amplification of human early hematopoietic cells from UCB in vitro.
METHODS:
Endothelial cells from UCB were cultured by the optimized medium of endothelial cells. There were 2 experiment groups: cytokines group (SCF+IL-3+IL-6+GM-CSF, CKs group) and noncontact group (endothelial cell layer with CKs without contacting the CD34+ cells group). CD34+ cells from UCB were isolated by MiniMACS. After the cells in the CKs group and the noncontact group were cultured for 7 days, the amplifying folds of early hematopoietic cells were assayed.
RESULTS:
Early hematopoietic cells from UCB were expanded in the CKs group or the noncontact group. The amplifying folds of the noncontact group on early hematopoietic cells were significantly more than those of the CKs group.
CONCLUSION
The amplification effect of the noncontact group on early hematopoietic cells is superior to that of the CKs group.
Antigens, CD34
;
analysis
;
Cell Proliferation
;
drug effects
;
Coculture Techniques
;
Culture Media
;
pharmacology
;
Cytokines
;
pharmacology
;
Endothelial Cells
;
cytology
;
metabolism
;
Fetal Blood
;
cytology
;
metabolism
;
Flow Cytometry
;
Hematopoietic Stem Cells
;
cytology
;
metabolism
;
Humans
;
Immunohistochemistry
7.Survey of infection of Toxoplasma gondii in infertile couples in Suzhou countryside.
Yong-Hua ZHOU ; Yong-Juan LU ; Rui-Bing WANG ; La-Mei SONG ; Fang SHI ; Qing-Feng GAO ; Ya-Fang LUO ; Xing-Feng GU ; Pei WANG
National Journal of Andrology 2002;8(5):350-352
OBJECTIVESTo determine the level of anti-Toxoplasma antibody in serum of infertile couples to explore the relationship between toxoplasma infection and infertility.
METHODSEnzyme-linked immunosorbent assay (ELISA) was applied to detect the anti-Toxoplasma antibody, antisperm antibody (AsAb) and anticardiolipin antibody (ACA) in serum of 178 couples with infertility and 190 couples who had normal pregnant history.
RESULTSThe positive result of Toxoplasma infection in the infertile couples was significantly higher than that in fertile couples which was 34.83% vs 12.11% (chi 2 = 26.72, P < 0.01) with the odds ratio 3.88. The positive result of serum AsAb in the Toxoplasma infected group was significantly higher than that in the no Toxoplasma infected group (32.50% vs 15.94%, chi 2 = 10.76, P < 0.01) with the odds ratio 2.54.
CONCLUSIONSToxoplasma infection was related to infertility. The Toxoplasma infection and was posibly related to the antisperm antibodies which can be involved in the pathogenisis of infertility.
Animals ; China ; epidemiology ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Infertility, Male ; epidemiology ; etiology ; parasitology ; Male ; Toxoplasma ; Toxoplasmosis ; complications ; epidemiology ; parasitology
8.Promoting effects of serum-free murine bone marrow endothelial cell conditioned medium on the growth of bone marrow endothelial cells.
Xiao-Ying ZHOU ; Qi-Ru WANG ; Yan-Hong HUANG ; La-Mei CHENG ; Meng-Qun TAN
Acta Physiologica Sinica 2005;57(2):199-204
To study the effects of serum-free murine bone marrow endothelial cell conditioned medium (mBMEC-CM) on the growth of bone marrow endothelial cells, mBMEC-CM was collected and ultrafiltrated by Centriprep-10. The retentate of mBMEC-CM [molecular weight (MW)>10 kDa] and the filtrate of mBMEC-CM (MW<10 kDa) were obtained. The effect of bone marrow conditioned media, their components and exogenous cytokines on the formation of endothelial cell colonies were observed. The effect of bone marrow conditioned media, their components and exogenous cytokines on the proliferation of murine bone marrow endothelial cells were determined by [(3)H]-thymidine incorporation. The method of hybridizing to the Atlas cDNA array was used to determine the expression of cytokine mRNAs in bone marrow endothelial cells. The results obtained are as follows: vWF was expressed in bone marrow endothelial cells. The original mBMEC-CM and MW>10 kDa component of mBMEC-CM promoted the proliferation of bone marrow endothelial cell colonies and increased [(3)H]-thymidine incorporation of bone marrow endothelial cells. The MW<10 kDa component did not affect the production of endothelial cell colonies and did not increase [(3)H]-thymidine incorporation of endothelial cells. Six cytokines (IL-6, IL-11, SCF, GM-CSF, VEGF, bFGF) promoted the proliferation of bone marrow endothelial cell colonies. VEGF, bFGF and SCF increased [(3)H]-thymidine incorporation of bone marrow endothelial cells. According to the results of the Atlas cDNA array, GM-CSF,TGF-beta,BMP-2, bFGF, SCF, endothelin-2, thymosin beta10, MSP-1, connective tissue GF, PDGF-A chain, MIP-2 alpha, PlGF, neutrophil activating protein ENA-78, INF-gamma, IL-1, IL-6, IL-13, IL-11, inhibin-alpha mRNAs were expressed in endothelial cells. These results suggest that murine bone marrow endothelial cell conditioned medium promotes the proliferation of bone marrow endothelial cells.
Animals
;
Bone Marrow Cells
;
cytology
;
Cell Line
;
Cell Proliferation
;
drug effects
;
Cells, Cultured
;
Culture Media, Conditioned
;
pharmacology
;
Culture Media, Serum-Free
;
pharmacology
;
Endothelial Cells
;
cytology
;
Hematopoiesis
;
physiology
;
Mice
9.Biological macrofeatures and criterion thereof for fracture immobilization in Chinese Mongolian traditional osteopathy
Na-mu-la ZHAO ; Mei WANG ; Xue-en LI
Journal of Medical Biomechanics 2011;26(2):E189-E192
Objective To study the biological macrofeatures and criterion thereof for fracture immobilization in Chinese Mongolian traditional osteopathy. Method The principles and methods of modern physiological psychology and biomechanics were used in this study to explore the biological macrofeatures and criterion thereof for fracture immobilization, based on the view of the harmony of human and nature (including a unity of body and function) in Chinese Mongolian traditional osteopathy. Results Chinese Mongolian traditional osteopathy implies the biological macrofeatures and criterion thereof for “dynamic immobilization” in fracture treatment, including the stability of structure and force catched, state of static and dynamic, forming and destroying of bone, physical and psychological stability. Therefore, it is a kind of non invasive and non shelter fixing method. Conclusions The biological macrofeature and criterion thereof for the fracture dynamic immobilization in fracture treatment, in Chinese Mongolian traditional osteopathy is not only the fundamental support for its inheritance up to now, but also could be a new attempt in modern fracture immobilization.
10.Relationship of I/D polymorphism of angiotensin converting enzyme gene with hypertension in Xinjiang Kazakh isolated group.
Xiao-feng WANG ; Shi-zhen WANG ; Ren-yong LIN ; Zu-heng CHENG ; Jian-bin DING ; Mi-la JIA ; Hao WEN ; Gui-zhen WU ; Xiao-mei LU
Chinese Journal of Medical Genetics 2003;20(3):253-255
OBJECTIVETo investigate whether the insertion/deletion(I/D) polymorphism in the angiotensin converting enzyme(ACE) gene is associated with essential hypertension in Xinjiang Kazakh isolated population.
METHODSThe study covered 201 hypertensives and 151 normotensive controls in Xinjiang Barlikun Kazakh population. The I/D polymorphism of ACE gene was determined by polymerase chain reaction.
RESULTSThe frequencies of D and I in the hypertensive group (0.44 and 0.56, respectively) were not significantly different from the controls(0.39 and 0.61, respectively, P=0.16). The frequencies of ACE genotypes of DD, ID, and II were 0.18, 0.52, 0.30 in hypertensives respectively and 0.17, 0.43, 0.40 in control group respectively. There was no significant difference in genotypes between hypertensive group and normotensive group (P=0.14).
CONCLUSIONThe results suggested that the I/D polymorphism of ACE gene might not be associated with hypertension in the Kazakh population of Xinjiang Barlikun area.
Asian Continental Ancestry Group ; genetics ; Blood Pressure ; genetics ; China ; ethnology ; Female ; Gene Frequency ; Humans ; Hypertension ; genetics ; INDEL Mutation ; Male ; Middle Aged ; Peptidyl-Dipeptidase A ; genetics ; Polymorphism, Genetic ; Population Groups