Functionalized liposomes loaded with siRNAs targeting ion channels in effector memory T cells as a potential therapy for autoimmunity

Effector memory T cells (T_M) play a key role in the pathology of certain autoimmune disorders. The activity of effector T_M cells is under the control of Kv1.3 ion channels, which facilitate the Ca²⁺ influx necessary for T cell activation and function, i.e. cytokine release and proliferation. Consequently, the knock-down of Kv1.3 expression in effector T_M's may be utilized as a therapy for the treatment of autoimmune diseases. In this study we synthesized lipid unilamellar nanoparticles (NPs) that can selectively deliver Kv1.3 siRNAs into T_M cells in vitro. NPs made from a mixture of phosphatidylcholine, pegylated/biotinylated phosphoethanolamine and cholesterol were functionalized with biotinylated-CD45RO (cell surface marker of T_M's) antibodies via fluorophore-conjugated streptavidin (CD45RO-NPs). Incubation of T cells with CD45RO-NPs resulted into the selective attachment and endocytosis of the NPs into T_M's. Furthermore, the siRNA against Kv1.3, encapsulated into the CD45RO-NPs, was released into the cytosol. Consequently, the expression of Kv1.3 channels decreased significantly in T_M's, which led to a remarkable decrease in Ca²⁺ influx. Our results can form the basis of an innovative therapeutic approach in autoimmunity.     

内质网应激在Bim介导缺氧致心肌细胞凋亡中的作用

 目的：探讨内质网应激在Bim介导缺氧致心肌细胞凋亡中的作用。方法：在体外原代培养出生1~3 d大鼠心肌细胞,并用抗α-横纹肌肌动蛋白免疫组化法进行鉴定。设计并化学合成3对靶向bim的siRNA,用脂质体法将siRNA转染心肌细胞,筛选沉默效率最高的siRNA。实验分组：（1）空白对照组;（2）缺氧组;（3）缺氧+脂质体组;(4)缺氧+阴性对照siRNA组;(5)缺氧+Bim-siRNA组。MTT法观察细胞活性;流式细胞术检测细胞凋亡率及细胞内钙离子浓度变化情况;Western blotting检测内质网应激标志分子caspase-12和三磷酸肌醇（IP3）的表达情况。结果：免疫组化鉴定证实大鼠心肌细胞原代培养成功。在荧光显微镜下,转染了阴性对照siRNA组的细胞中观察到绿色荧光,即转染成功;Western blotting 结果显示,Bim-siRNA转染均能有效降低Bim蛋白的表达,其中第2对沉默效率最高,达到86.73%。缺氧损伤导致心肌细胞活性明显下降（P<005）,转染Bim-siRNA后细胞活性较阴性对照组升高。缺氧细胞凋亡率较对照组明显增加（P<0.01）,细胞内钙离子浓度明显增高,而沉默bim的表达能降低细胞凋亡率和细胞内钙离子浓度。缺氧导致内质网应激标志分子caspase-12和IP3表达较空白对照组明显上调（均P<005）,而抑制Bim表达后caspase-12和IP3表达明显降低。结论：沉默bim的表达能有效抑制缺氧导致心肌细胞凋亡的作用,内质网应激标志分子caspase-12和IP3可能参与了Bim介导缺氧致心肌细胞凋亡的过程。这有望为临床心肌缺血缺氧损伤的治疗提供新思路。     

Systems Biology Analyses Show Hyperactivation of Transforming Growth Factor-β and JNK Signaling Pathways in Esophageal Cancer

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负性调节葡萄糖转运对糖尿病小鼠视网膜微血管病变的抑制作用

目的 观察通过抑制视网膜上葡萄糖转运蛋白1 (glucose transporter-1,GLUT1)表达而减少葡萄糖转运进入视网膜对糖尿病小鼠微血管病变的影响.方法 36只8周龄C57BL/6小鼠按随机数字表法分为正常对照组、糖尿病对照组和GLUT1小干扰核糖核酸(siRNA)治疗组,每组12只.腹腔注射链脲佐菌素建立糖尿病模型后,GLUT1siRNA组予以玻璃体腔注射1μL靶向GLUT1的siRNA,正常对照组和糖尿病对照组注射等量非靶向性siRNA.建模成功后第21周,对3组小鼠行免疫印迹法检查视网膜GLUT1的表达并测定比较视网膜的含糖量,通过测定视网膜ICAM-1和TNF-α的表达及检查视网膜血管白细胞黏滞和血-视网膜内屏障渗漏以比较微血管病变程度.结果 GLUT1siRNA组GLUT1在神经视网膜层的表达较正常对照组表达约下降77.00％,仅为糖尿病对照组的8.07％,差异有统计学意义(P＜0.01).尽管糖尿病对照组和GLUT1siRNA组视网膜组织含糖量均高于正常对照组,但GLUT1siRNA组小鼠视网膜含糖量为糖尿病对照组的50.05％,差异有统计学意义(P＜0.01);糖尿病对照组和GLUT1siRNA组小鼠视网膜ICAM1和TNF-α的表达均高于正常对照组(P＜0.01),但这两个炎症因子在GLUT1siRNA组表达仅分别为糖尿病对照组的66.14％(P＜0.05)和54.76％(P＜0.01);同时我们发现糖尿病对照组较GLUT1siRNA组有更多的白细胞黏附在视网膜血管中,视网膜铺片发现糖尿病对照组较GLUT1siRNA组其荧光渗漏区域较多,且渗漏面积也较大.结论 GLUT1 siRNA通过抑制GLUT1表达,限制葡萄糖转运进入视网膜,从而降低视网膜含糖量,减轻了糖尿病视网膜的炎症反应和血-视网膜内屏障渗漏,对糖尿病视网膜病变可产生缓解作用.     

JARID1B deletion induced apoptosis in Jeko-1 and HL-60 cell lines

Aims: To investigate the involvement of JARID1B histone methyltransferase in the epigenetic change of euchromatic promoter in mantle cell lymphoma (MCL) and acute leukemia. Methods: We retrospectively analyzed the protein of JARID1B and tri-methylated histone H3 lysine 4 (H3K4), histone H3 lysine 9 (H3K9), and cyclin D1 and Ki67 in 30 cases of MCL by immunohistochemistry. JARID1B was depleted by small interfering RNA (siRNA), and cell apoptosis and cell proliferation were detected by flow cytometry and MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], histone tri-methylated H3K4 and histone acetylated H3, H4, cyclin D1, Bcl-2, procaspase-3, C-myc were studied by Western blot. Results: We demonstrated that JARID1B was upregulated and histone tri-methylated H3K4 was downregulated in MCL compared to proliferative lymphadenitis, P < 0.05. The expression of histone methylated H3K9 was similar in both. Histone methylation of H3K4 was positively correlated with Ki67 in MCL (Kappa = 0.757, P < 0.05). This study showed that depletion of JARID1B cleavage apoptotic proteins of Bcl-2, procaspase-3, C-myc and resulted in loss cell viability and inducing apoptosis in Jeko-1 and HL-60 cell lines. JARID1B siRNA improved tri-methyl H3K4 and histone acetylated H3 and inhibited cyclin D1, but did not affect histone acetylated H4. Conclusions: This study revealed hyper JARID1B expression and hypo histone H3K4 tri-methylation in MCL. We identify depletion JARID1B as a demethylase which is capable of removing three methyl groups from H3K4 and up-regulating histone acetylation of H3 in both cell lines. Interestingly, depletion of JARID1B inhibits Cyclin D1, which is one of the genes contributes to MCL pathogenesis. JARID1B might be one of therapeutic targets in acute leukemia and MCL.     

Down-regulation of TET2 in CD3+ and CD34+ cells of myelodysplastic syndromes and enhances CD34+ cells proliferation

Aims and background: To investigate the expressions of TET2 mRNA in bone marrow CD3⁺ and CD34⁺ cells of the patients with myelodysplastic syndromes (MDS) and to study the effect of silencing TET2 by small interfering RNA (siRNA) on the biological characteristics of CD34⁺ cells. Methods: CD3⁺ and CD34⁺ cells were sorted by magnetic activated cell-sorting system from bone marrow of MDS patients and controls. The mRNA expressions of TET2 in bone marrow CD3⁺ and CD34⁺ cells of 28 MDS patients and 20 controls were detected by qPCR. The silencing effect of RNA interference (RNAi) on TET2 expression in CD34⁺ bone marrow cells of normal control was identified by qPCR and Western blot analysis. The cell cycle kinetics and cell apoptosis were then detected by flow cytometry. Results: The expression of TET2 mRNA in CD3⁺ and CD34⁺ cells was down-regulated in MDS compared with that in controls [(0.16±0.11) vs. (1.05±0.32) (P<0.001); (0.58±0.26) vs. (1.25±0.94) (P<0.005)]. The siRNA targeting TET2 suppressed the expression of TET2 in normal CD34⁺ cells. Meanwhile, the proliferation activity was significantly enhanced [G0/G1: (87.82±8.25)% vs. (92.65±7.06)% and (93.60±5.54)%, P<0.05; S: (11.50±8.31)% vs. (6.92±7.04)% and (5.95±5.53)%, P<0.05] and the apoptosis rate was declined [(21.28±9.73)% vs. (26.17±9.88)% and (26.20±9.78)%] in the cells which transfected with TET2 siRNA as compared to those in the cells transfected with scrambled siRNA and control cells. Conclusions: The TET2 expression of in CD3⁺ and CD34⁺ cells of MDS patients was decreased. Suppression of TET2 expression renders the CD34⁺ cells harboring more aggressive phenotype. This preliminary finding suggests that CD34⁺ cells lowering expression of TET2 may play an oncogenic role on myeloid tumor and CD3⁺ T cells of MDS patients may be derived from the malignant clone.