消癌解毒方联合顺铂通过调控组蛋白去甲基化酶抑制4T1荷瘤小鼠肿瘤生长

目的:观察消癌解毒方联合顺铂抑制4T1荷瘤小鼠肿瘤生长的作用及机制。方法:BALB/c小鼠皮下接种4T1乳腺癌细胞形成荷瘤模型,分别单独给予顺铂(2 mg·kg-1)、消癌解毒方(1×104mg·kg-1)及两者联合用药进行治疗,给药期间称量并记录小鼠体质量和瘤体积,15 d后脱颈处死,剥离并称量瘤块质量,计算抑瘤率,苏木素-伊红(HE)染色观察心、肝、脾、肾和瘤块的病理情况,原位末端转移酶标记技术(TUNEL)观察肿瘤细胞凋亡情况并统计凋亡率,蛋白免疫印迹法(Western blot)检测肿瘤组织中B淋巴细胞瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax),组蛋白去甲基化酶1(LSD1),组蛋白H3(Histone H3),组蛋白H3第4位赖氨酸二甲基化(Histone H3K4me2),组蛋白H3第9位赖氨酸二甲基化(Histone H3K9me2)的表达水平。结果:与模型组相比,顺铂给药组小鼠体质量均有下降,其中消癌解毒方+顺铂组较单用顺铂组体质量增加(P0.01);消癌解毒方+顺铂较单用顺铂更能缩小移植瘤体积,提高抑瘤率(P0.01);更明显提高了肿瘤细胞凋亡率(P0.05,P0.01);更明显下调肿瘤组织Bcl-2表达水平,上调Bax表达水平(P0.05,P0.01);各给药组均能下调LSD1蛋白的表达,增加组蛋白H3K4,H3K9的二甲基化,消癌解毒方+顺铂组作用最为明显(P0.05,P0.01)。结论:消癌解毒方+顺铂用药对于4T1荷瘤小鼠抑瘤作用明显,其机制可能与下调组蛋白去甲基化酶水平有关。     

组蛋白甲基化修饰与哺乳动物生殖

组蛋白甲基化是表观遗传学研究的热点和难点,研究表明,组蛋白甲基化修饰在哺乳动物配子的发生、胚胎着床和发育以及胎盘的发育过程中有着特殊作用,这些修饰可影响基因的表达,从而动态调节哺乳动物生殖过程中许多生物学进程。但目前研究的组蛋白甲基化修饰相关基因位点多而杂,且多停留在广泛的探索和描述这一层面,这些甲基化修饰发生动态变化的机制,以及相互作用等一系列过程还有待深入研究。     

Melatonin impedes Tet1‐dependent mGluR5 promoter demethylation to relieve pain

Melatonin (N‐acetyl‐5‐methoxytryptamine)/MT2 receptor‐dependent epigenetic modification represents a novel pathway in the treatment of neuropathic pain. Because spinal ten‐eleven translocation methylcytosine dioxygenase 1 (Tet1)‐dependent epigenetic demethylation has recently been linked to pain hypersensitivity, we hypothesized that melatonin/MT2‐dependent analgesia involves spinal Tet1‐dependent demethylation. Here, we showed that spinal Tet1 gene transfer by intrathecal delivery of Tet1‐encoding vectors to naïve rats produced profound and long‐lasting nociceptive hypersensitivity. In addition, enhanced Tet1 expression, Tet1‐metabotropic glutamate receptor subtype 5 (mGluR5) promoter coupling, demethylation at the mGluR5 promoter, and mGluR5 expression in dorsal horn neurons were observed. Rats subjected to spinal nerve ligation and intraplantar complete Freund's adjuvant injection displayed tactile allodynia and behavioral hyperalgesia associated with similar changes in the dorsal horn. Notably, intrathecal melatonin injection reversed the protein expression, protein‐promoter coupling, promoter demethylation, and pain hypersensitivity induced by Tet1 gene transfer, spinal nerve ligation, and intraplantar complete Freund's adjuvant injection. All the effects caused by melatonin were blocked by pretreatment with a MT2 receptor‐selective antagonist. In conclusion, melatonin relieves pain by impeding Tet1‐dependent demethylation of mGluR5 in dorsal horn neurons through the MT2 receptor. Our findings link melatonin/MT2 signaling to Tet1‐dependent epigenetic demethylation of nociceptive genes for the first time and suggest melatonin as a promising therapy for the treatment of pain.     

Ex vivo expanded regulatory T cells CD4+CD25+FoxP3+CD127Low develop strong immunosuppressive activity in patients with remitting-relapsing multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease characterized by defect in regulatory function of CD4⁺CD25⁺ T cells. We demonstrated difference in proportion of regulatory T cells CD4⁺CD25⁺FoxP3⁺CD127^low (Tregs) within the same patients’ relapse and remission. Proportion of peripheral Tregs (pTregs) dropped almost two times in the relapse compare to remission. Levels of pTregs in patients’ remission were lower than in healthy donors. Suppressive ability of pTregs was decreased in MS patients compared to healthy donors. Injections of expanded ex vivo autologous Tregs (eTregs) could be helpful in bringing up the level of Tregs in patients’ blood. We developed a simple method for ex vivo expansion of autologous Tregs within a short period of time. The final pool of cells consisted of 90-95% eTregs. When we started the culture with 10-20?×?10⁶ CD4⁺ T cells, we yield 300-400?×?10⁶ eTregs in a week. Expression of FoxP3 and Helios was calculated by two methods. Expanded ex vivo patients’ and donors’ Tregs were characterized by increased from three to five times expression of FoxP3, as well as almost doubled Helios expression. Peripheral Tregs in MS patients have decreased demethylation of FoxP3 gene promoter in comparison with donors. On the contrary, eTregs showed stable up-regulated demethylation without difference between MS patients and donors. MS patients’ and donors’ eTregs have much more suppressive ability than pTregs. Our data showed that eTregs can be applied as immunotherapy for MS patients and other autoimmune diseases if further investigated.     

组蛋白甲基化与lncRNAs的表达调控

LncRNAs是长度超过200 nt非编码蛋白质的RNA分子,并参与细胞内多种调控过程.LncRNA在发育和基因表达中发挥着复杂精确的调控功能,补充解释了基因组复杂性的生物意义,同时也使人们重新认识了生命过程中基因表达调控网络的复杂性.LncRNA参与了X染色体沉默、基因组印记、染色质修饰、转录激活、转录干扰以及核内运输等多种重要的调控过程.组蛋白修饰是基因表达调控的重要形式,尤其是组蛋白甲基化的调控作用.研究发现组蛋白甲基化对基因异常表达的调控参与多种肿瘤的发生.组蛋白去甲基化酶的发现又提出了一种新的理念,即组蛋白甲基化对lncRNA表达调控作用,这将是基因表达调控研究的新领域.     

Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte

In double fertilization, the vegetative cell of the male gametophyte (pollen) germinates and forms a pollen tube that brings to the female gametophyte two sperm cells that fertilize the egg and central cell to form the embryo and endosperm, respectively. The 5-methylcytosine DNA glycosylase DEMETER (DME), expressed in the central cell, is required for maternal allele demethylation and gene imprinting in the endosperm. By contrast, little is known about the function of DME in the male gametophyte. Here we show that reduced transmission of the paternal mutant dme allele in certain ecotypes reflects, at least in part, defective pollen germination. DME RNA is detected in pollen, but not in isolated sperm cells, suggesting that DME is expressed in the vegetative cell. Bisulfite sequencing experiments show that imprinted genes (MEA and FWA) and a repetitive element (Mu1a) are hypomethylated in the vegetative cell genome compared with the sperm genome, which is a process that requires DME. Moreover, we show that MEA and FWA RNA are detectable in pollen, but not in isolated sperm cells, suggesting that their expression occurs primarily in the vegetative cell. These results suggest that DME is active and demethylates similar genes and transposons in the genomes of the vegetative and central cells in the male and female gametophytes, respectively. Although the genome of the vegetative cell does not participate in double fertilization, its DME-mediated demethylation is important for male fertility and may contribute to the reconfiguration of the methylation landscape that occurs in the vegetative cell genome.