| Charpter I The Possible Mechanisms Responsible for the Effects of the Extracellular Nucleotides on the Proliferation of Human Umbilical Vein Endothelial CellsPart I The Effects of Extracellular Nucleotides on the Proliferation of Human Umbilical Vein Endothelial CellsObjective:To observe the effects of extracellualr nucleotides on the proliferation、apoptosis and autophagy of human umbilical vein endothelial cells.Methods:HUVEC-Cs were treated with extracellular nucleotides at concentrations of 0 to 100μM sequentially for 2 days, and then cell numbers were measured by CCK-8 assay. HUVEC-Cs were treated with ATP/ADP at different concentrations, cell apoptosis was detected by the PI/Annexin V staining and the phase of cell cycle was tested by PI staining. After transfected with a plasmid containing the DNA sequence coding for the human LC3 and GFP fusion protein, HUVEC-Cs were treated with l00μM ATP or ADP, the GFP puncta cells were photographed and counted under fluorescence microscope, Western-blot was further performed to detect the autophagy of HUVEC-Cs.Results:Compared with the control group, ATP at low concentrations of 1,5 10μM did not affect cell growth, only ADP at concentration of 1μM promote HUVEC-Cs proliferation. But at high concentrations of 50 and 100μM, both ATP and ADP significantly inhibit cell proliferation in a time-dependent manner, while UTP and UDP did not affect cell growth; The results of flow cytometric analyses showed that neither ATP nor ADP at high concentrations of 50 and 100μM induced cell apoptosis in HUVEC-Cs, but significantly decreased the proportion of cells in G2/M phage, and significantly increased the proportion of cells in S phase; GFP-LC3 Assay showed that ATP and ADP induced the formation of GFP puncta in endothelial cells and the puncta positive cells were significantly increased in both ATP and ADP treated cells; Western blot results showed that all ATP or ADP non-treated or treated endothelial cells expressed high levels of LC3-I and LC3 II proteins.Conclusions:ATP and ADP at high concentrations of 50 and 100μM inhibit HUVEC-Cs proliferation by inducing cell cycle arrest in S phase and inducing autophagic cell death, while cell apoptosis was not involved in nucleotides induced inhibition of cell proliferation. Part II The Mechanism of ATP Induced Inhibition of Human Umbilical Vein Endothelial Cells ProliferationObjective:To elucidate the mechanism of ATP induced inhibition of the proliferation in human umbilical vein endothelial cells.Methods:We detected the gene expression of P2Y receptors in endothelial cells at rest by RT-PCR; Moreover, RT-PCR and Western-blot was performed to detect the effects of ATP at different concentrations on the expression of P2Y2 and P2Y11 receptors at both mRNA and protein levels; In order to prove if the P2Y receptors are involved in the inhibition of cell proliferation induced by ATP, HUVEC-Cs were pre-treated with 100μM suramin, a non-specific P2 receptor inhibitor for 1h, and then stimulated with 100μM ATP for 48 h. Cell proliferation was measured by CCK-8 assay. Furthermore, HUVEC-Cs was transfected with shRNA plasmid containing the sequence targeting human P2Y2 or P2Y11, RT-PCR was performed to detect the transfect efficiency and after transient transfection, HUVEC-Cs were treated with 100μM ATP sequentially for 2 days, then cell numbers were measured by CCK-8 assay; In order to confirm if the inhibition of cell proliferation induced by ATP is primed by ERK activation, the phosphorylation of ERK was detected by Western blot. We also detected the effect of ERK 1/2 inhibitor U0126 and PD98095 on the proliferation of HUVEC-Cs treated or left untreated with ATP. After interfered with or without P2Y11 shRNA, HUVEC-Cs were stimulated with ATP and/or cisplatin for 24h, cell death was measured by Annexin V/PI staining. RT-PCR and Western blot was performed to detect the effect of ATP on the gene expression of Bcl-2 family members.Results:1. The results of RT-PCR showed that immortalized HUVEC-Cs expressed high levels mRNA for P2Y2 and P2Y11 receptor and low levels mRNA for P2Yl，4.13,14 receptors; Primary human aortic endothelial cells (HAEC) expressed high level mRNA for P2Y11 receptor and low level mRNA of P2Y2,4,13,14 receptors. Moreover, the treatment of HUVEC-Cs with ATP at concentrations of 5,10,50, and 100μM up-regulated the gene expression of P2Y2and P2Y11. ATP also up-regulated the protein expression of P2Y2 and P2Y11 in pHAEC in a dose dependent manner;2. CCK-8 assay revealed that suramin, a non-specific P2 receptor inhibitor, did not regulate cell proliferation alone, but it block the inhibition of cell proliferation induced by ATP at high concentrations;3.24h after shRNA plasmid transfected, RT-PCR results showed that the gene expression of P2Y2 and P2Y11 was down-regulated significantly by RNA interference; CCK-8 assay suggested that the inhibition of cell proliferation was induced by ATP (l00μM) in wild type group, sequence non-specific transfected group and P2Y2 shRNA transfected group, but was blocked in P2Y11 shRNA transfected group;4. The Western-blot results showed that 100μM ATP induced the activation of ERK and the phospholyration of ERK lasted from 1 to 8 minutes after ATP treatment, which could be blocked by ERK inhibitor U0126. Moreover, both U0126 and PD98095 could inhibit HUVEC-Cs proliferation alone or synergized the inhibition of ATP on cell proliferation.5. ATP increased cisplatin-induced cell death by down-regulating the gene expression of Bcl-2 and its family members such as Bcl-w、A1 and Mcl-1, all of which were promoting growth factors, while P2Y11 shRNA transfection could partially reverse this process.Conclusions:The inhibition of proliferation of HUVEC-Cs induced by ATP at high concentrations of 50 and 100μM was mediated by the P2Y11 receptor, which was not primed by the activation of ERK signall-ing pathway. ATP could augment cisplatin-induced cell death by down-regulating the gene expression of Bcl-2 and its family members via P2Y11 receptor. Charpter II The Possible Mechanisms Responsible for the Effects of the Extracellular Nucleotides on the Physiological Characteristics of Human Umbilical Cord Blood-Derived Endothelial Progenitor CellsPart I The Effects of Extracellular Nucleotides on the Proliferation of Human Umbilical Cord Blood-Derived Endothelial Progenitor CellsObjective:To observe the effects of extra-cellualr nucleotides on the proliferation of human umbilical cord blood-derived endothelial proge-nitor cells (EPCs).Methods:Mononuclear cells were isolated from fresh cord blood by density gradient centrifugation and cultured in EGM-2 medium supplemented with VEGF and bFGF. The biological features of the isolated cells were observed at different time point and identified by morphology, immunofluorescence staining and RT-PCR. Furthermore, RT-PCR was performed to detect the mRNA expression of P2 receptor subtypes in EPCs at rest. After treated with extracellular nucleotides at different concentrations sequentially for 3 days, cell numbers were measured by CCK-8 assay.Results:1. The attached cell population at day 4-10 is usually mixed with both round and spindle cells that exhibit many characteristics of EPC, but little proliferative capacity. Late EPC colonies typically sprout around days 11～18, and are comprised of larger cells with mature endothelial cell culture characteristics (cobblestone monolayer, contact inhibition, and rapid proliferation). Adherent EPCs were characterized by DiLDL uptake and concomitant lectin binding, and by expressing of AC 133, CD34 and CD31, which are markers of stem cells.2. RT-PCR results shown that rest EPCs expressed high levels of P2X4,6,7 and P2Y2,4,11 receptors, and low levels of P2Y13,14 receptors. Moreover,5 and 50μM ATP up-regulated the mRNA expression of P2Y2 receptor, while UTP up-regulated the mRNA expression of P2Y2 only at concentration of 5μM, suggesting that EPCs expressed functional P2Y receptors.3.50 and 100μM ATP significantly inhibited EPCs proliferation, while ADP, UTP and UDP at indicated concentrations have no effect on EPCs proliferation.Conclusions:High purity of EPCs could be isolated from human umbilical cord blood. ATP at high concentrations of 50 and 100μM significantly inhibit EPCs proliferation, however, which subtype of P2 receptor mediated this process need to be further elucidated. Part II The Possible Mechanism Responsible for the Effects of ATP/UTP on the Expression of Cytokines in EPCs Induced by LPSObjective:To observe the effects of ATP and UTP on the expression of cytokines in EPCs induced by LPS and to elucidate the possible mechanism.Methods:Using RT-PCR, We first detected the gene expression of TLRs in EPCs at rest, and the effect of 1mg/mL LPS on the mRNA expression of cytokines in EPCs; Then, we detected the effects of ATP and UTP at different concentrations on the mRNA expression of TLR4, the TLR4 co-receptor CD 14 and the adaptor protein MyD88; Besides, the effects of ATP/UTP at concentration of 5μM on the expression of cytokines induced by LPS was also detected by RT-PCR. Furthermore, RT-PCR and Western-blot was performed to detect the effects of ATP/UTP at low concentrations on the expression of TLR4, CD 14 and MyD88 induced by LPS. In order to prove whether NF-KB-mediated signaling was involved in this process, Western-blot was performed to detect the phosphorylation of NF-κB.Results:1. The RT-PCR results indicate that EPCs expressed high levels of TLR2,3,4,10, and low levels of TLR 1,5-9. When we treated EPCs with 1 mg/ml LPS for 24 h, we found that LPS up-regulated the gene expression of IL-1β, MCP-1, ICAM-1, VCAM-1, E-selectin, IFN-a and TNF-a;2. After treated with different concentrations of ATP and UTP for 24 h, the RT-PCR results show that both ATP and UTP could significantly down-regulate the mRNA expression of TLR4, CD 14 and MyD88 in EPCs;3. We stimulated EPCs with 1 mg/ml LPS in the presence or absence of 5μM ATP/UTP for 24 h and found that both ATP and UTP inhibited LPS-induced production of MCP-1, VCAM-1, IFN-a and TNF-a, but had little effect on LPS induced IL-1p and ICAM-1 expression;4. ATP and UTP at low concentrations significantly down-regulated the expression of TLR4, CD14 and MyD88 at mRNA level induced by 1 mg/ml LPS as well as the LPS-induced expression of CD14 and MyD88 at protein level;5. The Western-blot results showed that ATP and UTP at concentrations of 1 and 5μM significantly inhibited the phosphorylation of NF-κB p65.Conclusions:ATP and UTP at low concentrations could inhibit the LPS-induced expression of inflammatory factors and cytokines in EPCs, which was probably mediated by P2Y2 receptor by negatively regulating the TLR4 signaling. |
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