细胞周期调控基因——p21^CIP1／WAF1

Ｐ２１＾ＣＩＰ１／ＷＡＦ１基因编码的产物是一种细胞周期蛋白依赖性激酶的抑制蛋白。它通过调控细胞周期的进程,参与细胞的生长,衰老及死亡。在细胞ＤＮＡ损伤反应及肿瘤的发生、发展中也占且席之地。本文对Ｐ２１基因的发现,基因及蛋白南结构、生物化学特性、生物学特性以及研究前景概述。     

外源p21^WAF1转染对人成纤维细胞调亡的影响

构建了有义和反义ｐ２１＾ＷＡＦ１逆转录病毒表达载体,分别经脂质体包裹后转染人胚肺二倍体成纤维细胞（２ＢＳ）。Ｓｏｕｔｈｅｒｎ印迹杂交证实转染细胞中源ｐ２１＾ＷＡＦ１ｃＤＮＡ已整合入基因组中,与空载体转染细胞相比,有义转染细胞的ｐ２１＾ＷＡＦ１ｍＲＮＡ表达上升;细胞增殖速度明显减慢;对丁酸钠诱导调亡的敏感性降低,表现在细胞存在活率升高,核ＤＮＡ梯状断裂片段出现的时间滞后,断裂片段浓度下降,流式细胞计     

Epstein—Barr病毒BCRF1基因重组质粒的构建及其在真核细胞中的表达

应用基因重组技术,把编码ＥＢ病毒早期蛋白的ＢＣＲＦ１基因重组于真核表达载体ｐＳＧ５中,并使该基因在乳地鼠肾（ＢＨＫ）传代细胞中获得良好表达,表达率为０．５％。血清学实验证实,鼻咽癌、类风湿性关节炎病人和正常人血清中均不同程度地含有ＩｇＧ／ＢＣＲＦ１抗体,抗体阳性率分别为９２％、８６％和７７％,几何平均滴度（ＧＭＴ）分别是１：１６．３５、１：１４．７２和１：１０．１５。两组病人和正常人血清中ＩｇＡ／ＢＣＲＦ１抗体阳性率和滴度之间有较大差别,它们的阳性率分别是７４％、７１％和１２％,ＧＭＴ分别是１：１２．３２,１：１０．５６和１：２．３５。还证实,鼻咽癌和类风湿性关节炎病人血清中ＩｇＡ／ＢＣＲＦ１和ＩｇＡ／ＥＡ（早期抗原）抗体阳性率和滴度间有很好的相关性,此为首次报导。重组表达质粒ｐＳＧ５－ＢＣＲＦ１的构建和表达为进一步研究ＢＣＲＦ１基因在病毒感染和肿瘤免疫中的作用创造了条件。本文就质粒的构建和３组血清中ＩｇＡ／ＢＣＲＦ１、ＩｇＡ／ＢＣＲＦ１、ＩｇＡ／ＥＡ和ＩｇＧ／ＢＣＲＦ１抗体间的关系和有关问题进行了讨论。     

外源p21~(WAF1)转染对人成纤维细胞凋亡的影响

构建了有义和反义ｐ２１ＷＡＦ１ 逆转录病毒表达载体, 分别经脂质体包裹后转染人胚肺二倍体成纤维细胞（２ＢＳ）。Ｓｏｕｔｈｅｒｎ 印迹杂交证实转染细胞中外源ｐ２１ ＷＡＦ１ｃＤＮＡ 已整合入基因组中。与空载体转染细胞相比, 有义转染细胞的ｐ２１ＷＡＦ１ ｍ ＲＮＡ 表达上升; 细胞增殖速度明显减慢; 对丁酸钠诱导凋亡的敏感性降低, 表现在细胞存活率升高, 核ＤＮＡ 梯状断裂片段出现的时间滞后, 断裂片段浓度下降, 流式细胞计检测的凋亡峰面积缩小。而反义转染细胞的ｐ２１ＷＡＦ１ ｍ ＲＮＡ表达下降; 细胞增殖速度较快; 对丁酸钠诱导凋亡的敏感性上升, 有关表现与有义转染细胞相反。说明２ＢＳ细胞内ｐ２１ＷＡＦ１ 的表达量与其被丁酸钠诱导凋亡的能力呈负相关。     

丙型肝炎病毒非结构区NS4b基因片段的克隆和表达

采用聚合酶链反应（ＰＣＲ）技术和ＤＮＡ体外重组方法,克隆出５７９ｂｐ的丙型肝炎病毒（ＨＣＶ）ＮＳ４ｂ基因片段,插入到原核高效表达载体ｐＥＴ－２８ａ中,构建重组质粒ｐＥＴ／ＮＳ４ｂ,转化大肠杆菌ＢＬ２１（ＤＥ３）菌株,经ＩＰＴＧ诱导培养后,获得了目的蛋白的高效表达。ＳＤＳ－ＰＡＧＥ分析显示在３０ｋＤ处有一条表达的目的蛋白区带。通过固定化金属配体亲和层析（ＩＭＡＣ）纯化目的的蛋白,ＥＬＥＳＡ检测结果表     

人I型胶原基因第一内含子调节转录的研究

人Ｉ型胶原α１（Ｉ）链（ＣＯＬＩＡ１）基因内含子序列在不同细胞内有不同的转录调节活性,报道了含人ＣＯＬＩＡ１基因内含子Ｉ不同区段（＋５４４～＋８５５和＋８２０～＋１０９３）的重组质粒ｐＳＣＥＰ－ＣＡＴ和ｐＳＣＩＰ－ＣＡＴ的构建并转染人胚肌腱成纤维细胞和Ｔｃａ８１１３舌癌细胞,地高辛标记抗ＣＡＴ－ＥＬＩＳＡ检测结果显示：ｐＳＣＥＰ－ＣＡＴ在两种细胞均获表达;ｐＳＣＩＰ－ＣＡＴ在人成纤维细胞未表达,但     

粒细胞—巨噬细胞集落刺激因子／白细胞介素—3融合蛋白的表达研究

利用ＰＣＲ扩增得到粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）、白细胞介素－３（ＩＬ－３）完整基因片段,将其分别克隆ｐＧＥＭ－Ｔ构建成ＧＭ－ＣＳＦ／ＩＬ－３融合蛋白基因,ＤＮＡ序列与设计预期一致。将得到的融合蛋白基因克隆对７２ＲＮＡ聚合酶表达载体ｐＴ７ｚｚ,得到表达质粒ｐＦｕ,经转化至表达宿主Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）,在ＩＰＴＧ诱导下获得融合蛋白目的产物的直接表达。经ＳＤＳ－ＰＡＧＥ电泳鉴     

丙型肝炎病毒核酸疫苗的免疫效果观察

将丙型肝炎病毒Ｃ＋Ｅ１区基因克隆到不同的真核细胞表达载体ｐＣＤＮＡ３．１（＋）和ｐＳＶＬ中,通过肌肉注射和皮下注射免疫ＢＡＬＢ／Ｃ及Ｆ１小鼠后,产生了抗ＨＣＶ／Ｃ区抗体。其中核酸疫苗ｐｃＤＮＡ３．１－ＨＣＶ／Ｃ＋Ｅ１的免疫高峰的出现早于ｐＳＶＬ－ＨＣＶ／Ｃ＋Ｅ１,Ｆ１小鼠的抗体应答强于ＢＡＬＢ／小鼠。肌肉注射优于皮下注射。     

玉米细胞质分子伴侣Hsp70的ATPase活性

玉米胚乳细胞中纯化的细胞质Ｈｓｐ７０蛋白有低水平的ＡＴＰａｓｅ活性,它在５０℃、ＰＨ５．８、２０ｍｍｏｌ／Ｌ的ＫＣｌ条件下活性最高,Ｃａ＾２＋和Ｍｇ＾２＋抑制其活性。大肠杆菌ＤｎａＪ蛋白能将玉米细胞质Ｈｓｐ７０的ＡＴＰａｓｅ活性提高６倍,而ＧｒｐＥ蛋白对其影响很小。８种不同的人工合成多肽均能刺激该蛋白折ＡＴＰａｓｅ活性,增加幅度从２．５倍到１０倍痫水性不同的氨基酸对Ｈｓｐ７０的ＡＴＰａｓｅ活性影响     

大肠杆菌cai DE的基因克隆与表达

从Ｅ．ｃｏｌｉ ＭＣ４１００菌体的染色体ＤＮＡ中利用鸟枪法克隆含编码肉碱消旋酶及相关因子的ｃａｉＤＥ基因片段,并经序列分析验证,由重组质粒ｐｌＸ３９３亚克隆得到ｐＤＳＷ２重组表达质粒,后者转入Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）菌株中是丙基硫代半乳糖苷（ＩＰＴＧ）诱导,在聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）上分子量为３０ｋＤ和２４ｋＤ附近可见明显的表达蛋白带。     

STAT3 regulates the growth and immunoglobulin production of BCL(1) B cell lymphoma through control of cell cycle progression

STAT3 is constitutively phosphorylated on tyrosine(705) in self-renewing, CD5(+) murine B-1 lymphocytes. Nuclear extracts from untreated primary B-1 or CD5(+) BCL(1) B lymphoma cells were found to contain immunoreactive STAT3 protein that binds to a sis-inducible element present in the promoter of the p21(waf1/cip1) tumor suppressor gene and is constitutively phosphorylated on serine(727). To determine the functional significance of constitutive STAT3 activation in B lymphoma cells, a specific STAT3 antisense oligonucleotide was developed and used to examine basal BCL(1) cell growth and IgM production. Abrogating STAT3 expression in BCL(1) cells inhibited their proliferative capacity and induced a corresponding decrease in secretion of IgM. Cell cycle analysis showed a block in progression through G1 in BCL(1) cells treated with the STAT3 antisense oligonucleotide. These results indicate that STAT3 controls cell growth and immunoglobulin secretion by enhancing progression through the G1 phase of the cell cycle in BCL(1) B cell lymphoma.     

Effect of epiberberine from Coptis chinensis Franch on inhibition of tumor growth in MKN-45 xenograft mice

Background and purposeGastric cancer is one of the major malignancies worldwide. Epiberberine (EPI) is a major alkaloid from Coptis chinensis Franch and the antitumor property of EPI remains poorly understood.MethodThe inhibition on gastric cancer cells was observed by MTT assays and colony formation experiments. The apoptosis, cell cycle, and reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) in gastric cancer cells were analyzed by Flow cytometry. The anti-tumor effect of EPI was evaluated with the MKN-45-beraring nude mice, and the potential mechanisms were explored by RNA-seq, qPCR, siRNA silencing and western blotting.ResultsEPI inhibited the proliferation of human gastric cancer cell lines MKN-45 (harboring wild-type p53) and HGC-27 (harboring mutant p53) in a dose dependent manner. EPI induced the apoptosis and cell cycle arrest in these two cell lines, of which MKN-45 cells are more sensitive to EPI than HGC-27 cells. Further experiments indicated that EPI induced the accumulation of ROS and decreased of ΔΨm in MKN-45 cells. The significant differentially expressed genes obtained by RNA-seq were distinctly enriched in the p53 signaling pathway. The apoptosis induced by EPI in MKN-45 cells would be effectively inhibited with the treatment of p53 siRNA and p53 inhibitor PFT-α. Western blotting demonstrated that EPI diminished the expression of Bcl-2 and XIAP, and increased those of p53, Bax, p21, p27, Cytochrome C and Cleaved-caspase 3. Animal experiments confirmed that EPI significantly alleviated tumor growth in MKN-45 xenograft mice via p53/Bax pathway.ConclusionsThese data indicated that EPI could be a novel anti-tumor candidate against MKN-45-related gastric cancer via targeting p53-dependent mitochondria-associated pathway.     

Amlodipine inhibits cell proliferation via PKD1-related pathway

Human coronary artery smooth muscle cell (hCASMC) proliferation is involved in the progression of coronary artery disease. Amlodipine, a widely used antihypertensive drug, exerts antiproliferative effects by increasing the expression of p21^(Waf1/Cip1). Polycystic kidney disease 1 (PKD1) is also involved in cell cycle inhibition via p21^(Waf1/Cip1) up-regulation. We clarified the involvement of PKD1-related signaling on hCASMCs. Cultured hCASMCs, which constitutively express PKD1, were stimulated with 5% serum. Amlodipine increased p21^(Waf1/Cip1) expression in a dose- and time-dependent manner, resulting in reduced hCASMC proliferation. The inhibitory effect of amlodipine was mimicked by overexpression of PKD1 and was reversed by a dominant-negative version of PKD1 (R4227X). Immunoblot analysis showed that phosphorylated JAK2 was increased by amlodipine treatment or PKD1 overexpression. A luciferase assay revealed that the overexpression of PKD1 induced STAT1 enhancer activity. These data suggest that PKD1 contributes to the antiproliferative effect of amlodipine on hCASMCs via JAK/STAT signaling and p21^(Waf1/Cip1) up-regulation.