Low-dose all-trans retinoic acid enhances cytotoxicity of cisplatin and 5-fluorouracil on CD44+ cancer stem cells

Cis-diamminedichloridoplatinum(II)(CDDP)-based combination chemotherapy is frequently used in gastrointestinal cancer. The synergistic mechanism of all-trans retinoic acid (ATRA), cisplatin (CDDP) and 5-fluorouracil (5-FU) in combination remains unclear. Despite their potent antitumor properties, resistance to CDDP and 5-FU develops frequently in tumors. To clarify this mechanism, we determined the sensitivity to each drug and their combination in two gastrointestinal cancer stem cells (CSCs) subpopulation.Here, we report the identification and separation of CD44⁺ cells from human gastric carcinoma (AGS) and human esophageal squamous cell carcinoma (KYSE-30) cancer cell lines by magnetic activated cell sorting (MACS). We allowed the CD44^± cells to grow 6 days at a subtoxic concentration of ATRA and then treated with different concentration of CDDP and 5-FU for 24 h. The cytotoxicity was examined by cell proliferation MTT assay. Additionally, AO/EB staining was used for detection of apoptotic cells. In order to determine whether the growth inhibition was also associated with changes in cell cycle distribution, cell cycle analysis was performed using flow cytometry.Low concentration of ATRA (1 μM, 6days) followed by 5-FU and CDDP was found to be more effective than either drugs alone, thus resulting in synergistic cytotoxicity in Kyse-30 and AGSCD44^± cells. Furthermore, there was an indication that the combination of ATRA with 5FU and CDDP caused an increase in cell cycle arrest in G2/M and G0/G1.We conclude that low concentration of ATRA enhances the cytotoxicity of CDDP and 5FU by facilitating apoptosis and cell cycle arrest in gastrointestinal CSCs and provide a rational basis for the design of novel, well-tolerated CDDP- and 5FU-based chemotherapy in human gastrointestinal carcinoma.     

小鼠pLCK-CD69-IRES-EGFP表达载体构建及CD69转基因小鼠的建立

目的构建携小鼠细胞表面活化蛋白CD69和增强型绿色荧光蛋白(EGFP)在细胞内核糖体切入位点(IRES)的表达载体pLCK-CD69-IRES-EGFP, 并基于此创建CD69转基因小鼠。方法首先通过小鼠肺组织提取RNA, 逆转录成为cDNA, 经过PubMed搜索设计PCR引物, PCR法扩增mCD69片断, 接着将该DNA片段经测序验证后接入pInsulater-LCK-IRES-EGFP质粒, 构建pLCK-CD69-IRES-EGFP转基因载体; 再将其转染到293T细胞中, 通过荧光显微镜技术确定其在293T细胞中的表达状况; 最后将载体显微注射入受精卵并移植入假孕母小鼠, 出生小鼠取尾经PCR鉴定获得阳性首建鼠, 并通过荧光显微镜和流式细胞术确定淋巴细胞上CD69的表达。结果经酶切、DNA测序鉴定证实pLCK-CD69-IRES-EGFP表达载体构建成功; 荧光倒置显微镜检查证实其在转染的293T细胞内的蛋白表达; 小鼠取尾PCR鉴定明确CD69转基因小鼠创建成功; CD69转基因小鼠有外周血淋巴细胞的减少及静息状态下CD69的表达。结论成功构建pLCK-CD69-IRES-EGFP表达载体及制备CD69转基因小鼠, 为CD69在炎症性疾病中作用的整体模型研究奠定了基础。     

Comprehensive Profiling of an Aging Immune System Reveals Clonal GZMK+ CD8+ T Cells as Conserved Hallmark of Inflammaging

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一例X-连锁高IgM综合征合并进行性多灶性脑白质病的基因变异分析

目的对1例临床拟诊为X-连锁高IgM综合征(X-linked hyper-IgM syndrome,XHIM)并发进行性多灶性脑白质病变(progressive multifocal leukoencephalopathy,PML)的患儿CD40L基因及人类嗜神经多瘤病毒(Jamestown Canyon virus,JCV)进行检测,明确致病原因,为临床诊断提供依据。方法采集患儿及父母外周血提取基因组NDA,设计扩增CD40L基因5个外显子及外显子-内含子连接区的特异性引物并进行PCR扩增,产物测序结果与GenBank中CD40L基因序列分析对比确定有无变异。用两对JCV特异性引物对患儿外周血DNA行巢式PCR扩增,目的条带PCR产物测序结果与GenBank中JCV序列对比确定患儿是否存在JCV感染。结果测序结果显示患儿为CD40L c.506 A>C(p.Tyr169Ser)错义变异半合子,该变异位于CD40L肿瘤坏死因子同源区结构域,引起CD40L蛋白疏水作用及结构稳定性丧失,经PolyPhen2及SIFT蛋白功能预测软件预测分别为很可能有害变异(probably damaging,score=1.00)及有害变异(deleterious,score=-8.868),该变异尚未见文献报道。患儿母亲携带CD40L c.506 A>C(p.Tyr169Ser)半合子变异,父亲未检测到该变异。患儿外周血DNA巢式PCR产物经凝胶电泳后发现JCV目的条带,测序结果与GenBank中JCV基因序列比对同源性达到99%,表明患儿外周血DNA中含有JCV,有JCV感染。结论CD40L基因分析及JCV检测结果确诊患儿为XHIGM,其并发的PML与JCV感染有关。     

The KRAS/Lin28B axis maintains stemness of pancreatic cancer cells via the let‐7i/TET3 pathway

Increasing evidence demonstrates that Lin28B plays critical roles in numerous biological processes including cell proliferation and stemness maintenance. However, the molecular mechanisms underlying Lin28B nuclear translocation remain poorly understood. Here, we found for the first time that KRAS promoted Lin28B nuclear translocation through PKCβ, which directly bound to and phosphorylated Lin28B at S243. Firstly, we observed that Lin28B was upregulated in pancreatic cancer, contributing to cellular migration and proliferation. Furthermore, nuclear Lin28B upregulated TET3 messenger RNA and protein levels by blocking the production of mature let‐7i. Subsequently, increased TET3 expression could also promote the expression of Lin28B, thereby forming a Lin28B/let‐7i/TET3 feedback loop. Our results suggest that the KRAS/Lin28B axis drives the let‐7i/TET3 pathway to maintain the stemness of pancreatic cancer cells. These findings illuminate the distinct mechanism of Lin28B nuclear translocation and its important roles in KRAS‐driven pancreatic cancer, and have important implications for development of novel therapeutic strategies for this cancer.

Abbreviations

CCK‐8

cell counting kit‐8

CSC

cancer stem cells

IP

immunoprecipitation

MUT

mutant type

NLS

nuclear localization signal

PC

pancreatic cancer

PCSC

pancreatic cancer stem cells

PKC

protein kinase C

WT

wild‐type

     

黑素细胞及相关细胞群参与白癜风发病的研究进展

【摘要】 白癜风发病机制复杂，黑素细胞缺失是核心。目前认为除黑素细胞自身缺陷外，表皮和真皮细胞群的功能异常及其与黑素细胞的交互作用对白癜风的发病同样重要，充分而准确地了解不同病期白癜风皮肤全层细胞、组织的病理生理状态，对认识和治疗白癜风有重要作用。本文旨在综述黑素细胞及相关细胞群在白癜风发病中作用的研究进展。     

急性脑梗死患者血浆CD147水平与神经功能缺损程度的关系

目的探讨急性脑梗死(acute cerebral infarction,ACI)患者血浆CD147的水平及其与神经功能缺损的关系。方法选择2015年6月至2016年6月期间作者医院住院治疗的ACI患者79例(ACI组),另设对照组37例,选自门诊体检者,入选者均无脑血管病证据和(或)头颅MRI/CT检测正常者。以ELISA法检测两组血浆CD147水平,并比较两组CD147水平;以美国国立卫生研究院脑卒中量表(NIHSS)评分评估ACI组神经功能缺损情况;并分析其与CD147水平的关系。结果ACI组血浆CD147水平明显高于对照组〔(635.80±187.63)pg/mL比(352.70±91.32)pg/mL;t=8.693,P=0.000〕。ACI组患者NIHSS评分<8分组明显低于≥8分组〔(526.48±143.02)pg/mL比(761.02±150.56)pg/mL;t=-7.103,P=0.000〕。相关分析显示CD147水平与NIHSS评分成正相关(r=0.749,P=0.000)。结论ACI组患者血浆CD147水平明显升高,并且与神经功能缺损程度成正相关。