FTY720诱导1型1-磷酸鞘氨醇受体内化与其保守基序的关系研究

目的:研究FTY720诱导1型1-磷酸鞘氨醇受体(S1P1)内化与后者保守基序之间的关系.方法:以HA-S1P1(WT)-Myc-EGFP-N1融合表达载体为模板,应用重叠PCR的方法,将S1P1保守基序ERY突变为ENY,构建HA-S1P1(R142N)-Myc-EGFP-N1融合表达载体.测序鉴定后,经Polyfect转染入HEK293细胞.G418筛选出稳定细胞株.100 nmol/L FTY720处理3、6、12小时后,荧光显微镜下观察S1P1在HEK293细胞上的表达情况.结果:HA-S1P1(R142N)-Myc-EGFP-N1载体被成功构建,S1P1(WT)和S1P1(R142N)蛋白表达在HEK293稳定细胞株的表面,FTY720能诱导S1P1(WT)内化,但不能诱导S1P1(R142N)内化.结论:FTY720诱导S1P1内化与ERY保守基序有关.     

蛋白激酶NUAK1的定位及其与NF-κB信号通路关系的探讨

目的:确定蛋白激酶NUAK1的定位,检测其是否参与NF-κB信号通路。方法:构建质粒NUAK1-pEGFP顺转于293细胞,然后用DAPY染核,荧光显微镜下观察NUAK1的定位。构建质粒NUAK1-pcDNA3.1和显性失活突变体NU-AK1(D178N)-pcDNA3.1,以空载pcDNA3.1做对照,分别过表达于带稳转有NF-κB荧光酶素报告基因质粒的293细胞株中,检测在有或没有TNF-α刺激下的NF-κB荧光酶素报告基因的活性,为进一步确认,对NUAK1-pcDNA3.1设了个浓度梯度进行检测。结果:293细胞中,NUAK1定位于细胞核。在TNF-α的刺激下,以空载和显性失活突变体为对照,NU-AK1显著抑制NF-κB的活性,且与其表达量成正相关。结论:由于NUAK1定位于细胞核中,且显著抑制TNFα刺激下NF-κB的活性,所以NUAK1可能参与NF-κB的转录调控。     

SFTSV重组假病毒的构建及Gc糖基化对病毒感染力的影响

目的为探究发热伴血小板减少综合征病毒(SFTSV)中Gc及其N-糖基化位点与病毒感染性的关系, 构建了含有SFTSV Gc糖基化位点突变体的重组假病毒。方法利用定点突变和同源重组技术构建了SFTSV Gc及3个N-糖基化位点突变的真核表达载体pcDNA3.1(+)-Gc、pcDNA3.1(+)-Gc(N291Q)、pcDNA3.1(+)-Gc(N352Q)和pcDNA3.1(+)-Gc(N374Q)。验证其在293T细胞中成功表达后, 感染VSVΔG-Fluc*G假病毒, 构建4株重组假病毒并检测其对细胞感染力的影响。结果双酶切鉴定和序列测定证实成功构建真核表达载体pcDNA3.1(+)-Gc、pcDNA3.1(+)-Gc(N291Q)、pcDNA3.1(+)-Gc(N352Q)和pcDNA3.1(+)-Gc(N374Q)。间接免疫荧光和Western blot结果表明4个重组质粒均成功表达。SFTSV Gc重组假病毒对Vero细胞有感染特异性。糖基化位点突变后假病毒感染能力明显降低, 且352位糖基化位点突变株感染水平最低(P<0.001、P=0.001)。结论 SFTSV G...     

小鼠FasL基因真核表达载体的构建及在HEK293细胞中的表达

目的构建含有小鼠Fas Ligand(FasL)基因的重组真核表达载体,并检测FasL蛋白在其稳定转染的HEK293细胞中的表达情况,为构建表达小鼠Fas L的树突状细胞(DC)模型,深入研究DC联合T细胞防治移植物抗宿主病(GVHD)的新方法奠定基础。方法从小鼠脾脏中提取RNA并逆转录为cDNA,以该cDNA为模板扩增FasL基因,插入pcDNA3.1(+)载体中构建pcDNA3.1(+)-FasL重组质粒,利用脂质体将其转染HEK293细胞,用G418筛选稳定表达细胞系,Western blot确定重组蛋白的表达。结果通过酶切及测序证实FasL序列正确插入表达载体pcDNA3.1(+),Western blot检测证实转染重组质粒的细胞能正确表达FasL蛋白,G418筛选后能够得到稳定表达FasL的抗性细胞株即FasL-HEK293。结论重组质粒pcDNA3.1(+)-FasL和稳定表达FasL的HEK 293细胞成功构建,为后续FasL-DC细胞的研究打下了坚实基础。     

大鼠GLUT1真核表达载体的构建及其在293细胞中的表达

目的：构建大鼠葡萄糖转运体1（GLUT1）的真核表达载体。 方法： 以RT-PCR方法从大鼠脑组织中获取GLUT1全长cDNA片段，将其克隆至真核表达质粒pcDNA3.1(+)中，构建重组真核表达质粒pcDNA3.1(+)-Glut1，随后用lipofectamineTM 2000介导转染HEK293细胞，以RT-PCR法检测重组质粒在mRNA水平的表达，以免疫组化的方法检测重组质粒在蛋白水平的表达。 结果： 以构建的重组真核表达质粒pcDNA3.1(+)-Glut1转染293细胞后，在基因及蛋白表达水平均检测到了葡萄糖转运体1的表达。 结论： 成功构建了携带大鼠葡萄糖转运体1的真核表达载体pcDNA3.1(+)-Glut1，且证实其可在293细胞中成功表达目的基因，为进一步研究外源性GLUT1表达对缺血缺氧脑细胞的保护作用奠定了基础。     

CTLA4.FasL双功能融合基因真核表达载体的构建与表达

目的:构建携带CTLA4.FasL双功能融合基因的真核表达质粒,并在HEK293细胞中表达。方法:PCR扩增得到CTLA4和FasL胞外区编码序列,后用重叠PCR的方法将该两段序列融合,且在两者间插入接头序列(编码一柔性短肽GGSGG),所得的基因片段命名为CTLA4.FasL。XhoⅠ/KpnⅠ双酶切后,定向克隆至质粒pcDNA3.1(-),构建成真核表达载体pcDNA3.1(-)-CTLA4.FasL。转染入HEK293细胞,RT-PCR和Western blot验证融合基因的表达。结果:重叠PCR扩增获得CTLA4.FasL融合基因。真核表达载体pcD-NA3.1(-)-CTLA4.FasL成功构建,并在HEK293细胞表达。结论:成功构建了携带双功能融合基因的真核表达载体,为利用CTLA4.FasL基因修饰肝干细胞用于同种细胞移植治疗奠定了基础。     

S1P受体激动剂FTY720干扰孕鼠体内DC分布从而诱导妊娠免疫耐受

目的探讨S1P受体激动剂FTY720在降低自然流产模型孕鼠胚胎丢失率进程中的作用及其可能的机制。方法以自然流产模型孕鼠为研究对象,观察腹腔注射FTY720对自然流产模型孕鼠胚胎丢失率的影响,RT-PCR检测DC表面S1PR的表达,流式细胞术及免疫组化检测孕鼠外周血及局部组织中DC数量、成熟度及其表面相应趋化因子CCL19及其受体CCR7的表达情况,趋化实验验证FTY720对DC细胞趋化能力的影响。结果 1)FTY720对正常妊娠模型孕鼠的胚胎丢失率无明显影响(P<0.05),过继转移FTY720能明显降低自然流产模型孕鼠的胚胎丢失率(P<0.05);2)DC表面存在S1P受体的广泛表达,S1P受体激动剂FTY720减少了DC表面趋化因子及其受体的表达(P<0.05),致使趋化至母胎界面DC的数量显著减少(P<0.05);3)FTY720对DC细胞的分化及凋亡率均无明显影响(P>0.05)。结论 FTY720可能通过下调DC表面趋化因子受体CCR7的表达而使趋化至母胎界面的DC数量减少,最终诱导母胎免疫耐受。     

miRNA-144抑制小鼠RAW264.7巨噬细胞ABCA1表达

目的构建miR-144真核表达载体,检测miR-144是否调控小鼠RAW264.7巨噬细胞三磷酸腺苷结合盒转运体A1(ABCA1)的表达。方法结合文献,通过预测软件分析,选取评分较高、可作用于ABCA1 mRNA 3'非编码区(ABCA1 mRNA3'UTR)的miR-144。通过分子克隆技术,利用PCR扩增miR-144和ABCA1 mRNA 3'UTR的DNA序列,酶切胶回收后,分别连接至真核表达载体pcDNA3.1(+)载体和pcDNA3.1(+)-luciferase载体上。测序后,运用双荧光素酶报告基因系统检测萤火虫荧光素酶的活性,观察miR-144是否对ABCA1 mRNA 3'UTR具有直接靶向作用。运用LipofectamineTM2000将pcDNA3.1(+)空载体、pcDNA3.1(+)-miR-144转染至小鼠RAW264.7巨噬细胞。通过实时定量PCR(qRT-PCR)检测转染后miR-144的表达含量。通过qRT-PCR和Western blot法检测过表达miR-144后ABCA1在mRNA和蛋白水平的变化。结果 miR-144 PCR产物连接至pcDNA3.1(+)载体上,ABCA1 mRNA 3'UTR连接至pcDNA3.1(+)-luciferase载体上,测序正确。运用双荧光素酶报告基因系统检测发现,和对照组[pcDNA3.1(+)空载体、pcDNA3.1(+)-luciferase-ABCA1 3'UTR和PRL-SV40联合转染]三者相比,实验组[pcDNA3.1(+)-miR144、pcDNA3.1(+)-luciferase-ABCA1 3'UTR和PRL-SV40联合转染]三者可以显著降低萤火虫荧光素酶的活性(P0.05),证明miR-144对ABCA1 mRNA3'UTR具有直接靶向作用。将pcDNA3.1(+)空载体、pcDNA3.1(+)-miR-144转染至小鼠RAW264.7细胞中,qRT-PCR结果显示,转染pcDNA3.1(+)空载体组miR-144表达无明显变化(P0.05);转染pcDNA3.1(+)-miR-144组中miR-144表达量显著增高(P0.01).qRT-PCR结果显示,转染pcDNA3.1(+)空载体和pcDNA3.1(+)-miR-144对ABCA1 mRNA表达水平无明显影响(P0.05),Western blot结果显示转染pcDNA3.1(+)-miR-144可以显著降低ABCA1蛋白水平的表达(P0.01)。结论 miR-144可以在转录后水平降低ABCA1蛋白的表达。     

Trim6真核表达载体的构建及表达

目的:构建人Trim6的真核表达载体pcDNA3.1(+ )-Trim6,并观察其在HEK293T细胞中的表达.方法:提取HeLa细胞总RNA,用RT-PCR方法扩增出Trim6编码区全长基因片段,克隆到pcDNA3.1(+),通过酶切、菌落PCR及测序进行鉴定.将该载体转染HEK293T细胞,用蛋白印迹检测Trim6蛋白的表达.结果:通过酶切、PCR及测序鉴定,成功构建人Trim6表达载体,该载体可以在HEK293T细胞细胞系中表达Trim6.结论:构建Trim6真核表达载体pcDNA3.1(+)-Trim6,为研究Trim6在固有免疫应答中的作用奠定了基础.     

Sirt2基因的克隆及其在HEK293中的表达

目的:克隆大鼠Sirt2 基因, 构建其真核表达载体并在HEK293细胞中表达.方法:利用RT-PCR从大鼠脑组织总RNA中扩增出包含Sirt2编码区的cDNA片段, 产物纯化后T-A克隆连接至pMD20-T载体.以此为模板, 将该基因编码区克隆入真核表达载体pcDNA3.1myc-his(-)中, 转染HEK293细胞检测其表达.结果:测序证实所克隆的Sirt2编码区cDNA正确地插入pcDNA3.1myc-his(-)中, 经免疫荧光检测证实其在HEK293细胞中得到表达.结论:成功克隆了大鼠Sirt2 cDNA, 构建了其真核表达载体, 并在HEK293细胞中得到有效表达, 为进一步研究大鼠Sirt2的功能奠定了基础.     

Role of sphingosine 1-phosphate receptor type 1 in lymphocyte egress from secondary lymphoid tissues and thymus

Circulation of mature lymphocytes between blood and secondary lymphoid tissues plays a central role in the immune system. Homing of lymphocytes from blood into secondary lymphoid tissues beyond high endothelial venules is highly dependent on the interaction between the chemokines CCL19, CCL21, CXCL12, and CXCL13, and their receptors CCR7, CXCR4 and CXCR5. However, the molecular mechanism（s） of lymphocyte egress from secondary lymphoid tissues to lymph remained unclear. We have found a new class of immunomodulator, FTY720 by chemical modification of vegetative wasp-derived natural product, ISP-I （myriocin）. FTY720 has been shown to be highly effective in experimental allograft and autoimmune disease models. A striking feature of FTY720 is the induction of a marked decrease in peripheral blood lymphocytes at doses that show immunomodulating activity in these models. The reduction of circulating lymphocytes by FTY720 is caused by sequestration of lymphocytes into secondary lymphoid tissues and thymus. FTY720 is rapidly converted to （S）-enantiomer of FTY720-phosphate [（S）-FTY720-P] by sphingosine kinase 2 in vivo. （S）-FTY720-P acting as a potent agonist of S1P receptor type 1 （S1P1）, induces long-term down-regulation of S1P1 on lymphocytes, and thereby inhibits the migration of lymphocytes toward S1P. Thus, it is presumed that FTY720-induced lymphocyte sequestration is due to the inhibition of S1P/S1P1-dependent lymphocyte egress from secondary lymphoid tissues and thymus by its active metabolite （S）-FTY720-P. Throughout the analysis of the mechanism of action of FTY720, it is clarified that S1P/S1P1 interaction plays an important role for lymphocyte egress from secondary lymphoid tissues and thymus.     

Local delivery of FTY720 in PCL membrane improves SCI functional recovery by reducing reactive astrogliosis

FTY720 has recently been approved as an oral drug for treating relapsing forms of multiple sclerosis, and exerts its therapeutic effect by acting as an immunological inhibitor targeting the sphingosine-1-phosphate (S1P) receptor subtype (S1P1) of T cells. Recently studies demonstrated positive efficacy of this drug on spinal cord injury (SCI) in animal models after systemic administration, albeit with significant adverse side effects. We hereby hypothesize that localized delivery of FTY720 can promote SCI recovery by reducing pathological astrogliosis. The mechanistic functions of FTY720 were investigated in vitro and in vivo utilizing immunofluorescence, histology, MRI and behavioral analysis. The in vitro study showed that FTY720 can reduce astrocyte migration and proliferation activated by S1P. FTY720 can prolong internalization of S1P1 and exert antagonistic effects on S1P1. In vivo study of SCI animal models demonstrated that local delivery of FTY720 with polycaprolactone (PCL) membrane significantly decreased S1P1 expression and glial scarring compared with the control group. Furthermore, FTY720-treated groups exhibited less cavitation volume and neuron loss, which significantly improved recovery of motor function. These findings demonstrated that localized delivery of FTY720 can promote SCI recovery by targeting the S1P1 receptor of astrocytes, provide a new therapeutic strategy for SCI treatment.     

FTY720, sphingosine 1-phosphate receptor modulator, ameliorates experimental autoimmune encephalomyelitis by inhibition of T cell infiltration

FTY720, a sphingosine 1-phosphate receptor modulator, induces a marked decrease in the number of peripheral blood lymphocytes and exerts immunomodulating activity in various experimental allograft and autoimmune disease models. In this study, we evaluated the effect of FTY720 and its active metabolite, （S）-enantiomer of FTY720-phosphate [（S）-FTY720-P] on experimental autoimmune encephalomyelitis （EAE） in rats and mice. Prophylactic administration of FTY720 at 0.1 to 1 mg/kg almost completely prevented the development of EAE, and therapeutic treatment with FTY720 significantly inhibited the progression of EAE and EAE-associated histological change in the spinal cords of LEW rats induced by immunization with myelin basic protein. Consistent with rat EAE, the development of proteolipid protein-induced EAE in SJL/J mice was almost completely prevented and infiltration of CD4^＋ T cells into spinal cord was decreased by prophylactic treatment with FTY720 and （S）-FTY720-P. When FTY720 or （S）-FTY720-P was given after establishment of EAE in SJL/J mice, the relapse of EAE was markedly inhibited as compared with interferon-β, and the area of demyelination and the infiltration of CD4^＋ T cells were decreased in spinal cords of EAE mice. Similar therapeutic effect by FTY720 was obtained in myelin oligodendrocyte glycoprotein-induced EAE in C57BL/6 mice. These results indicate that FTY720 exhibits not only a prophylactic but also a therapeutic effect on EAE in rats and mice, and that the effect of FTY720 on EAE appears to be due to a reduction of the infiltration of myelin antigen-specific CD4^＋ T cells into the inflammation site. Cellular ＆ Molecular Immunology. 2005;2（6）：439-448.     

Palmitoylation of the sphingosine 1-phosphate receptor S1P1 is involved in its signaling functions and internalization

The lipid mediator sphingosine 1-phosphate (S1P) regulates several cellular processes through binding to its receptors (S1P₁–S1P₅), which are heterotrimeric G protein-coupled receptors. Here, we report that all S1P receptors are palmitoylated. In S1P₁, three Cys residues in the cytoplasmic tail are palmitoylated. We examined the roles of palmitoylation of S1P₁ using model cells in which wild-type S1P₁ or a non-palmitoylated mutant S1P₁ was overproduced. Compared with wild-type S1P₁, the non-palmitoylated S1P₁ exhibited binding affinity similar to the natural ligand S1P but lower to the synthetic ligand FTY720 phosphate (FTY720-P), the active form of the immunomodulator FTY720. However, downstream signaling of non-palmitoylated S1P₁ was similarly affected by S1P and FTY720-P stimulation. Moreover, upon stimulation with S1P, internalization of the mutant non-palmitoylated S1P₁ was retarded, compared with that of the wild-type protein. This effect was much more pronounced with FTY720-P stimulation. Similar differences were observed for the phosphorylation of S1P₁ and its mutant. These findings may provide insights into the molecular mechanisms of the pharmacological effects of FTY720. Finally, palmitoylation of wild-type S1P₁ increased upon treatment with S1P, suggesting that S1P₁ undergoes a palmitoylation/depalmitoylation cycle after stimulation by its ligands.     

FTY720 attenuates hypoxia–reoxygenation-induced apoptosis in cardiomyocytes

FTY720, sphingosine 1 phosphate (S1P) receptor agonist, is a potent immunosuppressive agent. Numerous studies have documented a relationship between S1P and cardioprotection. We therefore hypothesized that a S1P analogue FTY720 would attenuate hypoxia/reoxygenation (H/R) induced cadiomyocyte apoptosis. H9C2 cardiomyocytes were employed to establish an in vitro model of H/R. Cells were treated or not with different doses of FTY720. Cell viability was measured by flow cytometry and TUNEL staining. Western blot was used to analyze downstream signaling pathway. We observed that FTY720 inhibits the expression of cleaved caspase-3 and activates both AKT and ERK1/2 pathways. AKT pathway can be blocked by MEK kinase inhibitor PD98059. ERK1/2 pathway can be blocked by the phosphoinositide-3 kinase inhibitor wortmannin. AKT and ERK1/2 activation can also be inhibited by S1P1/3 receptor antagonist VPC23019, Gi antagonist PTX. The protein levels of TNF-α and IL1ß were upregulated during hypoxia/reoxygenation and were attenuated by FTY720. We conclude that FTY720, via its cargo of S1P, can protect cardiomyocytes against hypoxia/reoxygenation injury. This effect is achieved by inhibiting caspase-3 expression, inflammatory cytokine levels and activating AKT and ERK1/2 signaling pathways. The prosurvival signal activation is dependent on S1P1, 3 subtype receptors and Gi protein.     

The role of the lysophospholipid sphingosine 1-phosphate in immune cell biology

Sphingosine 1-phosphate (S1P) has been shown to be a bioactive lipid mediator intimately involved in mediating a variety of immunological processes. In particular, S1P regulates lymphocyte cell trafficking between the lymphatic system and the blood. The lysophospholipid signals mainly through five related G protein-coupled receptor subtypes, termed S1P₁ to S1P₅. S1P₁ seems to play an essential role in cell trafficking, as this receptor subtype promotes the egress of T and B cells from secondary lymphatic organs. This S1P₁-mediated migratory response is a consequence of different S1P levels in the serum and lymphatic organs. In addition to its direct effects on lymphocyte motility, S1P strengthens cell barrier integrity in sinus-lining endothelial cells, thereby reducing lymphocyte egress out of lymph nodes. Furthermore, S1P modulates cytokine profiles in T and dendritic cells, resulting in an elevated differentiation of T helper-2 cells during the T cell activation process. It is of interest that the mode of molecular action of the novel immunomodulator FTY720 interferes with the signaling of S1P. After phosphorylation, FTY720 shares structural similarity with S1P, but in contrast to the natural ligand, phosphorylated FTY720 induces a prolonged internalization of S1P₁, resulting in an impaired S1P-mediated migration of lymphocytes.     

野生型和突变型PS1-EGFP真核共表达载体的构建与鉴定

为了构建人野生型和突变型PS1(早老素-1, presenilin-1)-EGPF共表达蛋白的真核表达重组质粒pcDNA3.1 /PS1-EG-FP,本研究应用下述方法:对野生型pcDNA3.1 /PS1(WT)质粒,采用一步法定点突变技术构建pcDNA3.1 /PS1(C407G)突变质粒;合成两对引物,利用PCR技术从pEGFP-C1质粒合成EGFP cDNA片断,在EGFP转录起始密码子ATG之前导入一BamHⅠ酶切位点,利用与pcDNA3.1 /PS1质粒共存的另一EcoRⅠ酶切位点,将合成的EGFP片断通过BamHⅠ和EcoRⅠ两个位点分别克隆入野生型及突变型pcDNA3.1 /PS1质粒的多克隆位点中,从而构建了野生型和突变型PS1与增强绿色荧光蛋白共表达载体。经限制性内切酶双酶切鉴定及DNA测序证实此蛋白共表达载体构建成功。利用此重组质粒瞬时转染SH-SY5Y细胞,72 h后荧光显微镜(激发波长488 nm)观察细胞内有绿色荧光蛋白表达。本研究成功构建了人野生型和突变型PS1-EGFP的真核表达载体,为下一步建立细胞模型,研究突变型PS1的生物学功能以及PS1在AD发病机制中的作用奠定了基础。     

Immunomodulator FTY720 induces myofibroblast differentiation via the lysophospholipid receptor S1P3 and Smad3 signaling

The novel immunomodulator FTY720 is an effective immunosuppressive agent in experimental models of transplantation and autoimmunity and is currently undergoing phase III clinical trials for multiple sclerosis. Phosphorylated FTY720 is a structural analogue of sphingosine 1-phosphate (S1P) and therefore acts as a high-affinity agonist at four of the five G protein-coupled S1P receptors. It has been well established that there exists a crosstalk between S1P and transforming growth factor (TGF)-beta signaling. Because TGF-beta is the most prominent inductor of fibrosis and myofibroblasts are primarily responsible for excessive matrix protein formation, we examined whether FTY720, in analogy to TGF-beta, induces differentiation of fibroblasts into myofibroblasts. Indeed, FTY720 provoked myofibroblast differentiation comparable with that of TGF-beta. For biological efficacy, FTY720 required endogenous phosphorylation because inhibition of sphingosine kinase completely prevented FTY720 from inducing the differentiation process. Moreover, we identified the lysophospholipid receptor S1P3 as the crucial receptor subtype for FTY720-induced myofibroblast differentiation because the effect was abolished in fibroblasts isolated from S1P3 knockout mice. Finally, we determined that downstream of S1P3 signaling Smad3 activation is essential for myofibroblast differentiation in response to FTY720. Thus, FTY720 may have adverse fibrotic effects related to its activity on S1P3 signaling.