搜索到1881篇“ KNOCKDOWN“的相关文章
过表达溶质载体家族1成员5和敲低慢病毒载体构建及稳定转染RAW264.7细胞株
2025年
背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。
郭大鑫范苏苏朱振东侯建红张旋
关键词:慢病毒载体过表达RAW264.7细胞
A CRISPR/RfxCas13d-mediated strategy for efficient RNA knockdown in mouse embryonic development
2024年
The growing variety of RNA classes,such as mRNAs,lncRNAs,and circRNAs,plays pivotal roles in both developmental processes and various pathophysiological conditions.Nonetheless,our comprehension of RNA functions in live organisms remains limited due to the absence of durable and effective strategies for directly influencing RNA levels.In this study,we combined the CRISPR-RfxCas13d system with spermlike stem cell-mediated semi-cloning techniques,which enabled the suppressed expression of different RNA species.This approach was employed to interfere with the expression of three types of RNA molecules:Sfmbt2 mRNA,Fendrr lncRNA,and circMan1a2(2,3,4,5,6).The results confirmed the critical roles of these RNAs in embryonic development,as their loss led to observable phenotypes,including embryonic lethality,delayed embryonic development,and embryo resorption.In summary,our methodology offers a potent toolkit for silencing specific RNA targets in living organisms without introducing genetic alterations.
Lin ZhangShi-Meng CaoHao WuMeng YanJinsong LiLing-Ling Chen
Sm-like 5 knockdown inhibits proliferation and promotes apoptosis of colon cancer cells by upregulating p53,CDKN1A and TNFRSF10B
2024年
BACKGROUND The role of Sm-like 5(LSM5)in colon cancer has not been determined.In this study,we investigated the role of LSM5 in progression of colon cancer and the potential underlying mechanism involved.AIM To determine the role of LSM5 in the progression of colon cancer and the potential underlying mechanism involved.METHODS The Gene Expression Profiling Interactive Analysis database and the Human Protein Atlas website were used for LSM5 expression analysis and prognosis analysis.Real-time quantitative polymerase chain reaction and Western blotting were utilized to detect the expression of mRNAs and proteins.A lentivirus targeting LSM5 was constructed and transfected into colon cancer cells to silence LSM5 expression.Proliferation and apoptosis assays were also conducted to evaluate the growth of the colon cancer cells.Human GeneChip assay and bioinformatics analysis were performed to identify the potential underlying mechanism of LSM5 in colon cancer.RESULTS LSM5 was highly expressed in tumor tissue and colon cancer cells.A high expression level of LSM5 was related to poor prognosis in patients with colon cancer.Knockdown of LSM5 suppressed proliferation and promoted apoptosis in colon cancer cells.Silencing of LSM5 also facilitates the expression of p53,cyclin-dependent kinase inhibitor 1A(CDKN1A)and tumor necrosis factor receptor superfamily 10B(TNFRSF10B).The inhibitory effect of LSM5 knockdown on the growth of colon cancer cells was associated with the upregulation of p53,CDKN1A and TNFRSF10B.CONCLUSION LSM5 knockdown inhibited the proliferation and facilitated the apoptosis of colon cancer cells by upregulating p53,CDKN1A and TNFRSF10B.
Cai-Jing MoXiao-Yuan DengRu-Lan MaKun ZhuLei ShiKang Li
关键词:PROLIFERATIONKNOCKDOWN
敲减ARHGAP30基因对宫颈癌Siha细胞增殖及凋亡的影响被引量:1
2024年
目的探究敲减Rho鸟苷三磷酸酶激活蛋白30(Rho GTPase-activating protein 30,ARHGAP30)后,宫颈癌Siha细胞增殖及凋亡的变化。方法设计特异性shARHGAP30引物并连接pLKO.1载体,转化到大肠杆菌感受态细胞中,再与慢病毒辅助质粒转入HEK-293T细胞,收集细胞上清获得的病毒过滤后感染Siha细胞,RT-qPCR和Western blot检测敲减效率,以及转染后Bax及Bcl-2的表达变化;CCK-8法检测敲减后细胞的增殖水平。结果成功构建敲减ARHGAP30基因的慢病毒质粒,并建立Siha稳转细胞,ARHGAP30在Siha细胞中的转录和翻译减少(P<0.01),Bax/Bcl-2明显降低(P<0.01),凋亡减少,细胞增殖水平升高(P<0.01)。结论ARHGAP30参与Siha细胞的增殖与凋亡,调控ARHGAP30基因或将干扰宫颈癌的发生和发展。
彭雅婷刘端孟洁李文超李慧琦郭华牛美兰秦巧红
关键词:SIHA细胞宫颈癌细胞增殖细胞凋亡
YBX1基因肠道特异性敲低小鼠模型的建立
2024年
Y-盒结合蛋白1(Y-box binding protein 1,YBX1)是冷休克蛋白超家族的成员,由YBX1基因编码。YBX1在机体多个组织中高度表达,参与多种生理功能的调节。然而目前YBX1在肠道中的作用尚不清楚。本研究先用CCK-8法在细胞水平上检测过表达/抑制YBX1基因对猪(Sus scrofa)肠上皮细胞IPEC-J2、大鼠(Rattus norvegicus)肠上皮细胞IEC-6增殖的影响。然后利用Cre-loxP基因敲除系统,构建YBX1基因肠道特异性敲低小鼠(Mus musculus)模型。通过YBX1flox/flox小鼠与PVillin-Cre^(+/-)小鼠杂交繁育获得子代小鼠。利用PCR方法鉴定小鼠基因型,采用实时定量PCR技术(real-time quantitative PCR,RT-qPCR)和Western blot法分别从mRNA水平和蛋白水平检测YBX1基因肠道特异性敲低小鼠和对照小鼠YBX1的表达情况,验证基因敲低的效率。结果表明,过表达/抑制YBX1基因不影响细胞增殖,模型小鼠YBX1的mRNA水平下降为对照鼠的62%,蛋白水平下降为对照鼠的63%,证明YBX1基因肠道特异性敲低小鼠构建成功,为深入研究YBX1在肠道中的功能和作用机制提供稳定可靠的动物模型。
齐心语侯梦龙杨兴珍周磊李一星
关键词:C57BL/6J小鼠
Knockdown of neuronal DAF-15/Raptor promotes healthy aging in C.elegans
2024年
The highly conserved target of rapamycin(TOR)pathway plays an important role in aging across species.Previous studies have established that inhibition of the TOR complex 1(TORC1)significantly extends lifespan in Caenorhabditis elegans.However,it has not been clear whether TORC1 perturbation affects aging in a spatiotemporal manner.Here,we apply the auxin-inducible degradation tool to knock down endogenous DAF-15,the C.elegans ortholog of regulatory associated protein of TOR(Raptor),to characterize its roles in aging.Global or tissue-specific inhibition of DAF-15 during development results in various growth defects,whereas neuron-specific knockdown of DAF-15 during adulthood significantly extends lifespan and healthspan.The neuronal DAF-15 deficiency-induced longevity requires the intestinal activities of DAF-16/FOXO and PHA-4/FOXA transcription factors,as well as the AAK-2/AMP-activated protein kinaseαcatalytic subunit.Transcriptome profiling reveals that the neuronal DAF-15 knockdown promotes the expression of genes involved in protection.These findings define the tissue-specific roles of TORC1 in healthy aging and highlight the importance of neuronal modulation of aging.
Xiao ZangQi WangHanxin ZhangYiyan ZhangZi WangZixing WuDi Chen
云南省埃及伊蚊击倒抗性基因流行特征研究被引量:1
2024年
目的 在云南省埃及伊蚊重点分布区,检测埃及伊蚊引起击倒抗性(kdr)的电压门控钠离子通道基因(VGSC)突变并分析,阐明VGSC基因流行特征。方法 收集云南省盈江县、瑞丽市、镇康县、耿马傣族佤族自治县(耿马县)、勐海县、景洪市和勐腊县共7个重点县(市)野生埃及伊蚊成蚊或幼蚊样本,直接测序法扩增VGSC部分基因片段,分析各位点突变率和联合突变构成情况。χ^(2)检验比较不同位点突变率和不同性别成蚊VGSC基因kdr突变的差异等。结果 7个县(市)共计561只(雌蚊276只、雄蚊242只和幼蚊43只)埃及伊蚊样本成功提取蚊DNA基因组并被用于kdr突变的检测,共发现VGSC中4个位点突变。每个县(市)均存在S989P、V1016G和F1534C突变,S989P突变率为71.43%~100%,均值为92.51%[95%置信区间(CI)90.33%~94.70%];V1016G突变率均为100%;F1534C突变率为55.81%~100%,均值为85.38%(95%CI:82.45%~88.32%);首次发现瑞丽市、耿马县和镇康县3个县(市)存在Y1527F位点突变(0~35.45%),均值为8.73%(95%CI:6.39%~11.08%)。同时存在4种联合突变类型:S989P+V1016G、V1016G+F1534C、S989P+V1016G+F1534C和S989P+V1016G+Y1527F+F1534C,未发现单个位点突变情况。除盈江县外的6个县(市)雌蚊与雄蚊分别进行989、1016、1527和1534位点突变率以及联合突变构成比的比较,差异均无统计学意义(均P>0.05)。结论 云南省埃及伊蚊VGSC基因中kdr突变以多位点联合突变为主,且出现Y1527F新突变,应定期开展埃及伊蚊kdr突变监测,及时掌握其对杀虫剂靶标抗性变化规律。
陈丽周克梅吴超赵晓涛董朝良尹小雄王炳辉姜进勇
关键词:埃及伊蚊电压门控钠离子通道联合突变
Targeted knockdown of PGAM5 in synovial macrophages efficiently alleviates osteoarthritis被引量:2
2024年
Osteoarthritis(OA)is a common degenerative disease worldwide and new therapeutics that target inflammation and the crosstalk between immunocytes and chondrocytes are being developed to prevent and treat OA.These attempts involve repolarizing pro-inflammatory M1 macrophages into the anti-inflammatory M2 phenotype in synovium.In this study,we found that phosphoglycerate mutase 5(PGAM5)significantly increased in macrophages in OA synovium compared to controls based on histology of human samples and single-cell RNA sequencing results of mice models.To address the role of PGAM5 in macrophages in OA,we found conditional knockout of PGAM5 in macrophages greatly alleviated OA symptoms and promoted anabolic metabolism of chondrocytes in vitro and in vivo.Mechanistically,we found that PGAM5 enhanced M1 polarization via AKT-mTOR/p38/ERK pathways,whereas inhibited M2 polarization via STAT6-PPARγpathway in murine bone marrow-derived macrophages.Furthermore,we found that PGAM5 directly dephosphorylated Dishevelled Segment Polarity Protein 2(DVL2)which resulted in the inhibition ofβ-catenin and repolarization of M2 macrophages into M1 macrophages.Conditional knockout of both PGAM5 andβ-catenin in macrophages significantly exacerbated osteoarthritis compared to PGAM5-deficient mice.Motivated by these findings,we successfully designed mannose modified fluoropolymers combined with siPGAM5 to inhibit PGAM5 specifically in synovial macrophages via intra-articular injection,which possessed desired targeting abilities of synovial macrophages and greatly attenuated murine osteoarthritis.Collectively,these findings defined a key role for PGAM5 in orchestrating macrophage polarization and provides insights into novel macrophage-targeted strategy for treating OA.
Yuhang LiuRuihan HaoJia LvJie YuanXuelei WangChurong XuDing MaZhouyi DuanBingjun ZhangLiming DaiYiyun ChengWei LuXiaoling Zhang
关键词:OSTEOARTHRITISMETABOLISM
Knockdown of HE4 suppresses tumor growth and invasiveness in lung adenocarcinoma through regulation of EGFR signaling
2024年
It has been shown that the high expression of human epididymis protein 4(HE4)in most lung cancers is related to the poor prognosis of patients,but the mechanism of pathological transformation of HE4 in lung cancer is still unclear.The current study is expected to clarify the function and mechanism of HE4 in the occurrence and metastasis of lung adenocarcinoma(LUAD).Immunoblotting evaluated HE4 expression in lung cancer cell lines and biopsies,and through analysis of The Cancer Genome Atlas(TCGA)dataset.Frequent HE4 overexpression was demonstrated in LUAD,but not in lung squamous cell carcinoma(LUSC),indicating that HE4 can serve as a biomarker to distinguish between LUAD and LUSC.HE4 knockdown significantly inhibited cell growth,colony formation,wound healing,and invasion,and blocked the G1-phase of the cell cycle in LUAD cell lines through inactivation of the EGFR signaling downstream including PI3K/AKT/mTOR and RAF/MAPK pathways.The first-line EGFR inhibitor gefitinib and HE4 shRNA had no synergistic inhibitory effect on the growth of lung adenocarcinoma cells,while the third-line EGFR inhibitor osimertinib showed additive anti-proliferative effects.Moreover,we provided evidence that HE4 regulated EGFR expression by transcription regulation and protein interaction in LUAD.Our findings suggest that HE4 positively modulates the EGFR signaling pathway to promote growth and invasiveness in LUAD and highlight that targeting HE4 could be a novel strategy for LUAD treatment.
YUE ZHANGWENYU YANGXIAOWANG HANYUE QIAOHAITAO WANGTING CHENTIANYING LIWEN-BIN OU
关键词:BIOMARKER
山东省潍坊市2022年白纹伊蚊击倒抗性基因型分布研究
2024年
目的检测山东省潍坊市白纹伊蚊击倒抗性基因突变情况,为科学防控白纹伊蚊提供依据。方法2022年7月用勺捕法在潍坊市5个县(市、区)农村居民区采集伊蚊幼虫,在实验室集中饲养,羽化后经形态学鉴定为白纹伊蚊,单只提取白纹伊蚊DNA,通过PCR扩增电压门控钠离子通道基因部分片段并测序,对白纹伊蚊击倒抗性突变情况进行描述性统计分析。结果共检测96只白纹伊蚊,获得192条DNA序列,测序片段长度约为400 bp。测序结果表明白纹伊蚊3个击倒抗性基因位点均发生突变。3个突变位点均是以野生型纯合子为主,野生/突变型杂合子次之,突变型纯合子所占比例最低。1016位点存在2种等位基因,分别是野生型1016V(95.31%)和突变型1016G(4.69%);2种基因型分别为野生型纯合子1016V/V(90.62%),野生/突变型杂合子1016V/G(9.38%)。1532位点存在2种等位基因,即野生型1532I(79.17%)和突变型1532T(20.83%);3种基因型分别为野生型纯合子1532I/I(61.46%),野生/突变型杂合子I/T(35.42%)和突变型纯合子T/T(3.12%)。1534位点存在3种等位基因,即野生型1534F(85.94%)、突变型1534S(1.56%)和1534C(12.50%);4种基因型分别为野生型纯合子1534F/F(77.08%)、野生/突变型杂合子1534F/C(17.71%)、突变型杂合子1534S/C(3.13%)以及突变型纯合子1534C/C(2.08%)。共得到10种基因型组合,其中3个位点的野生纯合型组合基因型突变率最高,为36.46%。结论潍坊市白纹伊蚊击倒抗性基因突变率高且突变情况复杂,应密切关注白纹伊蚊的抗药性水平,从而科学指导卫生杀虫剂的使用。
霍锡元张晔刘国军苑森梅韩雪峰赵春春孟凤霞
关键词:白纹伊蚊基因突变位点基因型

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