pEGFP-N2-BMP2转染诱导P19细胞分化为心肌样细胞

目的 探讨真核表达质粒pEGFP-N2-BMP2转染P19细胞后能否诱导其分化为心肌样细胞.方法 实验分转染组和对照组,转染组以真核表达质粒pEGFP-N2-BMP2转染P19细胞并使骨形态发生蛋白2(BMP2)基因稳定表达,然后结合细胞聚集培养,对照组为培养的P19细胞.两组细胞聚集培养4d,形成聚集体后贴壁培养,于贴壁培养的4d、8d、12d、16d,免疫细胞化学和Western blotting方法检测心肌标志物心肌肌钙蛋白T (cTnT)和心肌肌动蛋白α;半定量RT-PCR检测心脏早期转录因子GATA-4和NKx2.5基因的表达.结果 转染组心肌肌钙蛋白T和心肌肌动蛋白α两种蛋白形成聚集体贴壁4d时无表达,8d、12d、16d出现表达,其表达随时间延长逐渐增强.取材各时间点之间有显著差异(P＜0.01).在转染诱导后4d时GATA-4、NKx2.5两个基因即出现弱的表达,随着时间延长,8d、12d、16d两个基因的表达逐渐增强.取材各时间点之间有显著差异(P＜0.01).对照组未发现心肌肌钙蛋白T和心肌肌动蛋白α两种蛋白的表达,亦未发现GATA-4、NKx2.5两个基因的表达.结论稳定表达BMP2基因的P19细胞可以分化为心肌样细胞,BMP2可能通过调控转录因子GATA-4、NKx2.5进而影响心肌肌钙蛋白T和心肌肌动蛋白α的表达.     

The crosstalk between TLR2 and NOD2 in Aspergillus fumigatus keratitis

Innate immunity is considered to be critical in the pathogenesis of fungal keratitis. Pattern recognition receptors (PRRs) recognize conserved microbial structures called pathogen-associated molecular patterns (PAMPS), thereby initiating the innate immunity. Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (leucine-rich repeat-containing receptors, NLRs) are two major PRR families. The crosstalk between TLR2 and NOD2 is not completely understood, and their interrelationship in Aspergillus fumigates keratitis is still unclear. To our surprise, we found herein that NOD2 and TLR2 were increased by A. fumigatus conidia in immortalized human corneal epithelial cells (HCECs). In addition, NOD2 expression was up-regulated by its agonist muramyl dipeptide (MDP), along with receptor interacting protein 2 (RIP2), nuclear factor κB (NFκB)-p65, inhibitor of NFκB (IκB)-α, and multiple inflammatory cytokines, including interleukin-6 (IL-6), IL-8 and tumor necrosis factor α (TNF-α). Interestingly, zymosan, a TLR2 agonist, promoted the expression of NOD2 and RIP2 in a TLR2-dependent manner. Furthermore, we demonstrated that the increased expression of NOD2 and RIP2 caused by A. fumigatus conidia occurred in part through a TLR2-dependent pathway. However, zymosan pretreatment decreased NOD2 and RIP2 expression along with the MDP induced secretion of inflammatory cytokines in HCECs. In agreement, NOD2 knockdown by small interfering RNA (siRNA) reduced the release of IL-6, IL-8 and TNF-α induced by A. fumigatus conidia. These findings suggest the existence of complex interactions between TLR2 and NOD2 in HCECs inflammatory response against A. fumigatus infection.     

NDRG2对肝癌细胞HepG2凋亡的诱导作用

目的：探讨NDRG2对肝癌细胞HepG2凋亡的诱导作用。方法：用RT-PCR获取NDRG2基因。测序判断正确后，将其克隆入含绿色荧光蛋白(GFP)基因的真核表达载体pIRES2-EGFP中，并转染NDRG2表达阴性的HepG2细胞。用光镜观察转染细胞的形态变化；荧光显微镜观察NDRG2蛋白的定位；流式细胞仪分析细胞周期的改变；透射电镜进一步观察细胞超微结构的改变。结果：获得了NDRG2基因，成功地构建了pIRES2-EGFP-NDRG2真核表达载体。转染HepG2细胞后，细胞生长受抑，结构破坏并死亡。荧光显微镜观察到NDRG2在胞质中表达，流式细胞仪检测出现G1期阻滞和凋亡峰，透射电镜观察到典型的细胞凋亡特征。结论：NDRG2基因具有诱导HepG2细胞凋亡的作用，其机制有待进一步研究。     

Functional involvement of Annexin-2 in cAMP induced AQP2 trafficking

Annexin-2 is required for the apical transport in epithelial cells. In this study, we investigated the involvement of annexin-2 in cAMP-induced aquaporin-2 (AQP2) translocation to the apical membrane in renal cells. We found that the cAMP-elevating agent forskolin increased annexin-2 abundance in the plasma membrane enriched fraction with a parallel decrease in the soluble fraction. Interestingly, forskolin stimulation resulted in annexin-2 enrichment in lipid rafts, suggesting that hormonal stimulation might be responsible for a new configuration of membrane interacting proteins involved in the fusion of AQP2 vesicles to the apical plasma membrane. To investigate the functional involvement of annexin-2 in AQP2 exocytosis, the fusion process between purified AQP2 membrane vesicles and plasma membranes was reconstructed in vitro and monitored by a fluorescence assay. An N-terminal peptide that comprises 14 residues of annexin-2 and that includes the binding site for the calcium binding protein p11 strongly inhibited the fusion process. Preincubation of cells with this annexin-2 peptide also failed to increase the osmotic water permeability in the presence of forskolin in intact cells. Altogether, these data demonstrate that annexin-2 is required for cAMP-induced AQP2 exocytosis in renal cells.     

BMP-2和FGF-2对小鼠骨髓间充质干细胞向成骨细胞分化的影响

目的:观察骨形成蛋白(BMP-2)和成纤维细胞生长因子2(FGF-2)对小鼠骨髓间充质干细胞(MSCs)向成骨细胞分化的影响.方法:取3～18月雄性C57BL/6J小鼠(共50只)的骨髓细胞, 分离贴壁培养后, 用免疫磁珠法纯化, 并鉴定为MSCs后, 再进行贴壁培养24 h后, 分别在成骨细胞诱导培养液中加入100 μg/L BMP-2和0.5 nmol/L FGF-2持续诱导7、 14、 21 d后, 进行碱性磷酸酶染色、碱性磷酸酶活性检测, Vonkossa染色以及茜素红染色, 并用递转录荧光定量PCR法检测向成骨细胞分化的标志性基因(Runx2/cbfa1、 Alp、 collagen-1、 osteocalcin)的表达情况.结果:BMP-2刺激组的ALP活性以及钙化结节明显高于对照组, Runx2/cbfa1, ALP, collage-1, osteocalcin的mRNA呈高表达; FGF-2刺激组的ALP活性以及钙化结节也高于对照组, Runx2/cbfa1、 Alp、 collagen-1、 osteocalcin的mRNA表达量也高于对照组, 但不如BMP-2明显. 结论:BMP-2和 FGF-2在不同程度上促进体外培养的小鼠MSCs向成骨细胞方向分化.     

PTCH2基因

PTCH 2基因是新发现的与 Patched基因 (PTCH )有相似的结构和功能的基因 ,在胚胎发育和肿瘤形成中起作用。本文对国外关于 PTCH2基因的研究进展 ,特别对 PTCH2的基因定位、基因结构、功能及表达调控机制进行了综述     

Investigating the functional role of CD2BP2 in T cells

The adaptor protein CD2-binding protein 2 (CD2BP2) confers binding to proline-rich sequences (PRS) via its GYF domain. In addition to the cytoplasmic domain of CD2, several other proteins were identified as interaction partners of CD2BP2, but the in vivo significance of these findings is unclear. We now show that CD2BP2's nuclear localization is not changed when CD2 and CD2BP2 are co-expressed in HeLa cells, indicating that other PRS compete effectively for CD2BP2 binding in the nucleus. Since the CD2BP2-binding motifs of CD2 are known to be involved in cytokine signaling, we tested the effect of CD2BP2 knockdown in PBMCs on the expression of T-cell cytokines. No major difference in cytokine expression can be observed for primary cells transfected with CD2BP2-specific small interfering RNA. We conclude that CD2 signaling is at least partially independent of its in vitro binding partner CD2BP2.     

P2Y2受体激动剂2-硫代-UTP及拮抗剂苏拉明对Hela细胞活性影响

目的 研究P2Y2受体激动剂2-硫代-UTP及其拮抗剂苏拉明对Hela细胞活性的影响,从而在细胞水平探究P2Y2受体在人子宫颈癌病理生理过程中的作用.方法 应用CCK-8法检测不同时间点各处理组Hela细胞的活性;ELISA法分别检测细胞上清液中TNF-α和IL-6的水平;qPCR和Western blot法分别检测细胞P2Y2mRNA和蛋白的表达变化.结果 2-硫代-UTP可明显提高Hela细胞的增殖能力.且经2-硫代-UTP处理后的细胞IL-6的水平明显增加,P2Y2mRNA和蛋白表达水平均有所提高;苏拉明可明显抑制Hela细胞增殖,经其处理后的细胞TNF-α水平有所提高,P2Y2mRNA和蛋白表达水平无明显变化.结论2-硫代-UTP可通过激活P2Y2受体的活化细胞,引起细胞过表达P2Y2受体,从而促进Hela细胞增殖.苏拉明可通过阻断P2Y2受体及其下游活动,抑制Hela细胞增殖.以上结果表明,P2Y2受体可能成为治疗子宫颈癌等疾病的新靶点,这将为探索子宫颈癌新型治疗手段等方面提供新思路.     

过氧化氢对白癜风患者黑素细胞的氧化损伤

目的探讨过氧化氢(H2O2)对体外培养黑素细胞的影响及氧化应激与白癜风发病的关系。方法培养的黑素细胞中加入H2O2,用MTT法检测黑素细胞增殖,NaOH法检测黑素合成。结果H2O2可以抑制黑素细胞增殖及黑素合成,且具有浓度和时间依赖性。结论氧化应激可以抑制黑素细胞增殖及黑素合成,提示氧化损伤不足以诱发白癜风皮损。     

Inhibitory role of peroxiredoxin 2 in LRRK2 kinase activity induced cellular pathogenesis

Parkinson's disease (PD) is a major neurodegenerative disease. One of the known genetic contributors to PD pathogenesis is leucine-rich repeat kinase 2 (LRRK2) whose mutations with elevated kinase activity could lead to both familial and sporadic PD. However, how the pathogenic kinase activity of LRRK2 is regulated remains largely unclear. Here we report that peroxiredoxin 2 (Prx2) was identified as a novel interacting protein to LRRK2 with preferential expression in dopaminergic neurons over other Prx proteins. We also confirmed that Prx2 interacted with LRRK2 through its COR domain and its overexpression significantly decreased the kinase activity of mutant LRRK2. Functionally, overexpressed Prx2 rescued the transfected cells from LRRK2 mutant induced apoptotic processes. Importantly, overexpressed Prx2 reversed the altered subcellular distribution of cation-independent mannose 6-phosphate receptor (CI-M6PR) induced by PD-mutant LRRK2. Our results suggest that, by interacting with LRRK2, Prx2 may play an inhibitory role in the LRRK2 mediated cellular toxicity in PD by inhibiting its kinase activity.     

Role of HIV-2 envelope in Lv2-mediated restriction

We have characterized envelope protein pseudotyped HIV-2 particles derived from two HIV-2 isolates termed prCBL23 and CBL23 in order to define the role of the envelope protein for the Lv2-mediated restriction to infection. Previously, it has been described that the primary isolate prCBL23 is restricted to infection of several human cell types, whereas the T cell line adapted isolate CBL23 is not restricted in these cell types. Molecular cloning of the two isolates revealed that the env and the gag gene are responsible for the observed phenotype and that this restriction is mediated by Lv2, which is distinct from Ref1/Lv1 (Schmitz, C., Marchant, D., Neil, S.J., Aubin, K., Reuter, S., Dittmar, M.T., McKnight, A., Kizhatil, K., Albritton, L.M., 2004. Lv2, a novel postentry restriction, is mediated by both capsid and envelope. J. Virol. 78 (4), 2006-2016). We generated pseudotyped viruses consisting of HIV-2 (ROD-ADeltaenv-GFP, ROD-ADeltaenv-RFP, or ROD-ADeltaenv-REN) and the prCBL23 or CBL23 envelope proteins as well as chimeric proteins between these envelopes. We demonstrate that a single amino acid exchange at position 74 in the surface unit of CBL23-Env confers restriction to infection. This single point mutation causes tighter CD4 binding, resulting in a less efficient fusion into the cytosol of the restricted cell line. Prevention of endosome formation and prevention of endosome acidification enhance infectivity of the restricted particles for GHOST/X4 cells indicating a degradative lysosomal pathway as a cause for the reduced cytosolic entry. The described restriction to infection of the primary isolate prCBL23 is therefore largely caused by an entry defect. A remaining restriction to infection (19-fold) is preserved when endosomal acidification is prevented. This restriction to infection is also dependent on the presence of the point mutation at position 74 (G74E).     

2型糖尿病与血小板P2Y12受体

ADP-P2Y12受体途径在血小板激活、血栓形成中发挥重要作用。2型糖尿病血小板P2Y12受体途径异常可能是血小板功能亢进的重要原因之一。在正常人，胰岛素对P2Y12途径有抑制作用，而2型糖尿病由于血小板存在胰岛素抵抗，导致P2Y12途径的高敏感性。许多2型糖尿病对普通剂量的抗血小板药物反应性降低，但是加大P2Y12阻断剂的剂量后可能会克服这种耐药现象。     

Smad3基因敲除小鼠肾Smad2和p-Smad2表达的变化

目的:观察Smad3基因敲除小鼠肾Smad2和p-Smad2表达的变化,探讨Smad3基因敲除小鼠肾是否有Smad2和p-Smad2代偿性增加.方法:4只Smad3基因敲除小鼠,10只野生型小鼠,应用免疫组织化学显色技术,检测Smad2和p-Smad2蛋白的定位和表达情况,并用Motic病理图像分析系统对图像进行半定量分析.结果:野生型小鼠肾Smad2蛋白在肾远端小管和肾集合管细胞胞质中有广泛弱表达,p-Smad2蛋白在肾远端小管和肾集合管细胞胞质、胞核中也有弱表达,而Smad3基因敲除小鼠的Smad2和p-Smad2的表达比野生型小鼠有显著升高.结论:Smad3基因敲除能引起小鼠肾Smad2和p-Smad2表达的代偿性增加.     

TGF-β2 and PGE2 in Rabbit Blastocoelic Fluid Can Modulate GM-CSF Production by Human Lymphocytes

PROBLEM: During normal pregnancy, major changes occur in the production of Th2/Th1 cytokines at the feto-maternal interface. Th2 cytokines such as interleukin-4 (IL-4) or interleukin-10 (IL-10) are predominantly produced locally in the uterine and placental tissues, whereas the production of Th1 cytokines such as tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) are decreased. Because these modulations might be induced by the embryo, the current study was carried out to test the effect of rabbit blastocoelic fluid on the production of Th2/Th1 cytokines by lymphocytes, and to investiate the possible implication of transforming growth factor β₂ (TGF-β₂) prostaglandin E₂ (PGE₂) as modulators of the production of these cytokines. METHOD OF STUDY: Human peripheral blood lymphocytes (PBL) were cultured along with ConcanavalinA (Con A), and rabbit blastocoelic fluid was collected on day 12 of gestation (BF d-12). Concentrations of cytokines in culture media were determined by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: Addition of BF d-12 in the culture medium induced a strong inhibition of IL-2, TNF-α, IL-10, and granulocyte-macrophage colony-stimulating factor (GM-CSF) production. However, an initial pretreatment of the lymphocytes with BF d-12, followed by a Con A stimulation, led to a marked increase in GM-CSF production, whereas IL-2, TNF-α, and IL-10 secretions were inhibited. It was also demonstrated, for the first time, that a pretreatment of the lymphocytes with TGF-β₂ and PGE₂ increased GM-CSF production to the same level reached after the addition of BF d-12. Furthermore, removal of TGF-β₂ and PGE₂ from BF d-12 by affinity chromatography reduced the effect of BF d-12 on GM-CSF production. CONCLUSIONS: Taken together, these findings suggest that the embryo, in modulating harmful and beneficial cytokine production locally, plays an active role in its protection against maternal immune cellular assault. These results also emphasize the importance of growth factors for successfully maintaining pregnancy.     

抑郁症患者IL-2及sIL-2R的检测及临床意义

目的　探讨白介素 -2 ( IL-2 )及可溶性白介素 -2受体 ( s IL-2 R)在抑郁症发病中的作用及临床意义。方法　用酶联免疫吸附法检测 30例抑郁症患者和 30例正常人血清 IL-2及 s IL-2 R水平 ,并比较二者的差异。结果　抑郁症患者 IL-2及 s IL-2 R分别为 95 1 .2± 1 1 0 .5 ngl/L、( 389.6± 2 1 1 .1 ) U/ml,高于对照组的 384 .1± 72 .5 ng/L、2 83.6± 1 4 6 .7U/ml,二者比较差异有显著性 ( P<0 .0 1 )。结论　抑郁症患者 IL-2及 s IL-2 R水平增高。     

Gab2在人骨肉瘤细胞U2-OS中的表达及意义

目的:探讨Grb2协同结合蛋白2(Gab2)在人骨肉瘤细胞系中的表达及其与人骨肉瘤细胞侵袭转移的关系。方法:应用小RNA干扰技术,将合成的小RNA干扰质粒转染给人骨肉瘤U2-OS细胞,采用Western blotting和RT-PCR法检测转染后Gab2的蛋白和mRNA表达水平;体外细胞趋化运动实验和侵袭实验检测细胞的迁移和侵袭能力。结果:Gab2在人骨肉瘤U2-OS细胞系中高表达,且转染Gab2 siRNA后的细胞(Si Gab2/U2-OS)中Gab2蛋白及mRNA的表达水平低于转染scramble siRNA细胞(Scr/U2-OS)和U2-OS;趋化运动实验发现在10μg/L表皮生长因子(EGF)的诱导下Si Gab2/U2-OS细胞的迁移能力较U2-OS和Scr/U2-OS细胞明显下降,差异有统计学意义(P0.01);Si Gab2/U2-OS细胞侵袭并穿透Matrigel膜基质的细胞数量均比Scr/U2-OS细胞和U2-OS细胞少,Si Gab2/U2-OS细胞的体外侵袭能力显著降低(P0.01)。结论:利用siRNA干扰技术降低Gab2的表达对人骨肉瘤U2-OS细胞的迁移和侵袭能力有明显的抑制作用。     

Nkx2．2对髓母细胞瘤细胞恶性表型的调控作用

目的：探究转录因子NKX2．2（NK2 homeobox 2）在小鼠髓母细胞瘤（medulloblastoma, MB）中的表达及其对人源MB细胞系Daoy增殖、迁移能力的影响。方法采用实时定量PCR检测Nkx2．2在MB和野生型小鼠小脑组织中mRNA水平的表达差异,并通过免疫组化进一步检测其差异;设计针对Nkx2．2的小干扰RNA,分别通过实时定量 PCR 和 Western 印迹检测 Nkx2．2的干涉效率;干涉 Daoy 细胞中的Nkx2．2后采用CCK?8、平板克隆形成实验检测Nkx2．2基因沉默对Daoy细胞增殖和迁移能力的影响,流式细胞术检测细胞凋亡,划痕实验评价细胞迁移能力。结果 NKX2．2在小鼠MB中的表达明显低于正常组织;沉默Nkx2．2能显著促进MB细胞的增殖且抑制其凋亡,但不影响细胞迁移。结论 Nkx2．2能抑制MB细胞的增殖并促进其凋亡,其下调可能参与MB的发生。     

Na-K-2Cl共同转运蛋白(NKCC2)基因研究进展

Na-K-2Cl共同转运体在维持肾小管髓袢升支粗段对NaCl 的重吸收,机体容量控制方面起重要作用,NKCC2基因突变可使其编码蛋白质结构、功能改变, 使体内血压调节的稳态发生变化。