鼠源VCAM-1真核表达载体构建及在同源细胞中过表达研究

构建鼠源VCAM-1基因的真核表达载体,并使其在C2C12细胞中过表达,为相关病毒受体研究奠定基础.采用RT-PCR方法扩增鼠源VCAM-1基因,构建重组载体pcDNA3.1-VCAM-1.通过脂质体法转染至C2C12细胞内;采用实时荧光定量PCR和Western-blot分别检测目的基因和蛋白的过表达情况.应用间接免疫荧光实验分析VCAM-1在细胞中的定位.构建的重组质粒pcDNA3.1-VCAM-1转染C2C12细胞后,实验组VCAM-1基因和蛋白表达均显著高于对照组细胞(P0.01),且主要表达定位于胞浆中.结果表明,实验成功地构建了鼠源VCAM-1真核表达载体,并在同源细胞中实现过表达.     

基于CRISPR/Cas9技术构建TRPV1基因敲除的CACO-2稳定细胞系

基于CRISPR/Cas9基因编辑技术敲除人结直肠腺癌细胞Caco-2中的辣椒素受体基因TRPV1,构建TRPV1基因敲除的Caco-2稳定细胞系.使用CRISPR/Cas9在线靶点设计程序在TRPV1基因4个转录本的公共CDS区的第一个外显子DNA区域中设计一对gRNA,将gRNA构建到真核重组表达质粒的载体上,电转染待敲除细胞,经嘌呤毒素抗性筛选和基因测序获得敲除TRPV1基因的稳定细胞系.用Western Blot检测TRPV1基因蛋白表达量验证Caco-2中TRPV1基因的敲除效果,CCK-8检测TRPV1基因敲除细胞的生长曲线,与野生型细胞对比检测TRPV1基因敲除对细胞生长的影响.筛选出TRPV1基因敲除的CACO-2稳定细胞系,而且TRPV1基因敲除对Caco-2细胞生长无显著影响.基于CRISPR/Cas9基因编辑技术成功构建了TRPV1基因敲除的CACO-2稳定细胞系,为后续研究辣椒素对肠道脂类吸收影响的机理研究提供了有利的工具.     

rcan1-1l基因的克隆和表达

从小鼠脑组织中通过总mRNA的提取、RT-PCR方法,利用pEASY-T1载体克隆出了鼠源rcan1-1l基因,并构建出了重组有rcan1-1l基因的PET21a原核表达质粒,进行了表达条件的初步摸索,这为今后研究rcan1-1l基因以及蛋白的结构与功能打下了前期实验基础.     

pEGFP-C1-MCH真核表达载体的构建及其在HEK293 细胞系中的表达

目的:构建pEGFP-C1-MCH真核表达载体,并将其转染入HEK293细胞中,筛选阳性细胞克隆,为研究MCH基因在能量代谢中的功能及机制提供细胞模型.方法:提取脑组织总RNA,反转录为cDNA,参照Genbank中提供的序列设计引物扩增MCH基因全长.再将该基因全长cDNA克隆至质粒pEGFP-C1,经菌落PCR筛选及双酶切和DNA测序鉴定,成功构建了含有目的基因MCH的重组质粒pEGFP-C1-MCH.并利用脂质体2000介导其转染HEK293细胞,用荧光显微镜和RT-PCR检测EGFP和MCH在细胞中的表达.结果:克隆的pEGFP-C1-MCH质粒序列中的MCH与Gen Bank相符;细胞转染72 h后,转染成功的细胞在荧光显微镜下表达较强的绿色荧光,MCH基因稳定表达.结论:pEGFP-C1-MCH真核表达载体的构建及其在HEK293细胞中的稳定表达,为研究MCH基因在能量代谢中的功能及作用机制提供了实验模型.     

高迁移率蛋白HmgB3和组蛋白Mlh1维持了四膜虫小核稳定性

高迁移率族蛋白(High Mobility Group protein,HMG)和组蛋白H1结合染色质DNA,在维持染色质的高级结构、基因组基因表达调控以及DNA修复等方面发挥重要作用。嗜热四膜虫细胞含有一个体细胞系的大核和一个生殖系的小核,小核特异定位的高迁移率蛋白HmgB3或组蛋白Mlh1的缺失并未引起生长期细胞的异常表型。本研究通过同源重组的方法构建了HMGB3和MLH1的双敲除细胞突变株ΔHMGB3ΔMLH1。突变细胞在营养生长期能够正常增殖,但对甲基磺酸甲酯较为敏感。缺对PCR检测发现突变株中小核染色体有缺失现象,并且不能完成有性生殖。有性生殖过程中,减数分裂后的小核异常降解。结果表明高迁移率蛋白HmgB3和小核组蛋白Mlh1共同维持了四膜虫小核的稳定性,并可能具有功能上的冗余性。     

CHAC1基因沉默SU-DHL-8细胞株的构建及其对青蒿素细胞毒活性的影响

应用慢病毒短发夹RNA(short hairpin RNA,shRNA)转染的方式构建了阳离子转运蛋白样蛋白1(cation transport regulator-like protein 1,CHAC1)基因沉默SU-DHL-8细胞株模型,用于研究CHAC1基因对青蒿素细胞毒活性的影响.研究了青蒿素对弥散性大B细胞淋巴瘤SU-DHL-8细胞增殖的抑制作用及作用后CHAC1基因及蛋白的表达情况.应用小干扰RNA(small interfering RNA,siRNA)沉默SU-DHL-8细胞株内的CHAC1基因.采用慢病毒转染的方法构建CHAC1基因沉默的SU-DHL-8细胞株.采用实时荧光定量核酸扩增检测系统(real-time quantitative PCR detecting system,qPCR)、3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT)活性测定法和蛋白免疫印迹(Westen blot)的方法检测沉默效果以及CHAC1基因对青蒿素细胞毒活性的影响.成功构建了稳定沉默CHAC1基因的稳转细胞株,并初步探讨了CHAC1基因对青蒿素抑制弥散性大B细胞淋巴瘤SU-DHL-8细胞增殖的影响.     

人工合成启动子特异启动IGF-1真核载体的构建与绵羊成纤维细胞转染

构建了肌肉特异表达IGF-1载体,并转染绵羊成纤维细胞获得了稳定整合外源基因可用于细胞核移植的转基因细胞.通过人工合成骨骼肌特异启动子SP片段,与羊IGF-1相连,最终连接到骨架载体pCDsRed2中构建成骨骼肌特异表达IGF-的真核表达载体pSPICDS.使用脂质体法转染绵羊成纤维细胞,通过红色荧光与G418双重选择,获得了转基因细胞.经PCR检测证实得到的细胞克隆外源基因稳定整合,可以作为供体细胞用于体细胞核移植.     

标记减数分裂遗传重组的免疫荧光染色方法

通过免疫荧光染色方法制备小鼠精母细胞联会复合体, 研究其形态组成与遗传重组特征,展示雄性小鼠遗传重组图谱并分析其重组位点(MLH1位点)的分布特征.4只小鼠共145个精母细胞在平均每个细胞的MLH1位点数为23.3±2.4; 在19个常染色体联会复合体中, 未发现有3个MLH1位点的, 具有1个MLH1位点数的较多, 平均为14.2; 无XY联会复合体的细胞占所有细胞的4.1%, XY联会复合体上有MLH1位点的细胞占30.2%; 联会复合体上有裂缝的细胞占0.7%.通过联会复合体免疫荧光染色可以清晰地分辨出联会复合体(红色)、着丝粒(蓝色)和MLH1位点(绿色), 是遗传重组分析的是一种强有力工具.     

pEGFP-Egr-1表达质粒构建及其表达抑制HepG2细胞增殖

将RT-PCR扩增的Egr-1基因连接到pMD-18载体上并测序鉴定,经测序正确的Egr-1基因亚克隆到绿色荧光蛋白真核表达载体pEGFP-C1上构建成重组质粒pEGFP-Egr-1.重组质粒pEGFP-Egr-1转染HepG2细胞后,运用荧光显微镜及荧光定量PCR技术分析其在HepG2细胞中的表达情况,并进一步运用WST-1法分析其表达对HepG2细胞增殖的影响.结果显示,重组质粒pEGFP-Egr-1构建正确,转染后在荧光显微镜下观察可见HepG2细胞发出绿色荧光信号,荧光定量PCR分析发现转染后HepG2细胞Egr-1基因mRNA水平明显升高.WST-1结果表明,与对照组相比,转染pEGFP-Egr-1质粒的HepG2细胞,其增殖速率明显下降.上述结果表明,pEGFP-Egr-1重组质粒构建成功,外来过表达Egr-1基因能够抑制HepG2细胞增殖.     

人肿瘤细胞株中细小病毒H—1的DNA扩增与非结构蛋白NS—1基因的表达

研究三株人癌细胞和两株对照细胞对细小病毒Ｈ－１杀伤作用敏感性的分子机制．表明了在感染复数ｍｏｉ（ｍｕｌｔｉｐｉｃｉｔｙｏｆｉｎｆｅｃｔｉｏｎ）为５ｐｆｕ（ｐｌａｑｕｅｆｏｒｍｉｎｇｕｎｉｔ）／细胞的情况下，作为Ｈ—１病毒复制受纳细胞的人肝癌细胞株ＯＧＹ－７７０３和人胃癌细胞株ＳＧＣ—７９０１，能够支持病毒ＤＮＡ扩增和非结构蛋白ＮＳ—１基因的表达，这和作为阳性对照的由ＳＶ４０转化的新生儿肾细胞株ＮＢ—Ｋ一样，但对Ｈ—１病毒感染有抗性的人肾癌细胞株ＯＵＲ—１０和它的对照人胎肾细胞株ＨｕＫ—１，并不支持病毒ＤＮＡ扩增和ＮＳ—１蛋白的表达．本文结果指出，细小病毒Ｈ—１的杀伤作用与细胞中的病毒ＤＮＡ扩增及ＮＳ—１基因表达的程度相关．     

人翻译延伸因子eEF1A1促进肺癌细胞H1299对紫杉醇和阿霉素药物耐受的研究

在已知人翻译延伸因子eEF1A的多功能性和与细胞存活相关的基础上,本文进一步研究eEF1A的变体蛋白eEF1A1对癌细胞耐药性的影响.eEF1A1基因的蛋白质编码区经过克隆,并瞬时转染了人肺癌细胞H1299,在确定eEF1A1蛋白的胞内表达后,采用MTT法测定高表达eEF1A1的H1299细胞对抗癌药物紫杉醇和阿霉素的耐受.相对于对照细胞,eEF1A1的高表达可显著提高H1299细胞对阿霉素和紫杉醇的耐受性.通过耐药性测试揭示出eEF1A可能参与肿瘤发生的潜在功能.     

Effects ofPKCα antisense RNA on human breast cancer cell proliferation and expression ofcyclinD1 andCDK4

The role of PKCα in human breast cancer cell proliferation and expression ofcyclinD1 andCDK4 has been investigated using inhibition ofPKCα expression by its antisense RNA. WhenPKCα expression was inhibited the rate of cell proliferation decreased apparently and the levels ofcyclinD1 andCDK4 mRNA were lower than the control. The results showed thatPKCα, a key member of signal transduction system, played an important role in human breast cancer cell proliferation and had a close relationship with expression ofcyclinD1 andCDK4 which control start of cell cycle.     

G1 cell-cycle control and cancer

Before replicating DNA during their reproductive cycle, our cells enter a phase called G1 during which they interpret a flood of signals that influence cell division and cell fate. Mistakes in this process lead to cancer. An increasingly complex and coherent view of G1 signalling networks, which coordinate cell growth, proliferation, stress management and survival, is helping to define the roots of malignancies and shows promise for the development of better cancer therapies.     

Comprehensive diagnostic value of P53, p21WAF1 and proliferating cell nuclear antigen for lung cancer

The expression of P53, p21WAF1 and proliferating cell nuclear antigen (PCNA) was detected in 114 samples of lung cancer patients (with 89 cases benign lung tissue as control) and the diagnostic value of these markers was evaluated. The results show the following: ➀ The positive expression rates of P53, p21WAF1 and PCNA in samples of lung cancer were 47.37%, 75.44% and 80.70%, respectively, which were significantly higher than that in the samples of benign lung diseases ( p < 0.001). The odds ratios were 39.15, 5.75, and 6.76, respectively. This indicates that the expression of P53, p21WAF1 and PCNA was helpful for the diagnosis of lung cancer. ➁ For the diagnosis of lung cancer, the positive likelihood ratio of P53 was 21.08, which were significantly higher than that of p21WAF1 (2.16), PCNA (2.11) and of all the combined tests. This shows that P53 expression was the most valuable for diagnosis of lung cancer. ➂ For the diagnosis of lung cancer, the negative likelihood ratio of P53/p21WAF1/PCNA parallel test was 0.057 1, which was lower than that of other single and combined tests. This indicates that P53/p21WAF1/PCNA parallel test has high diagnostic value for exclusion of lung cancer.     

Proliferation, cell cycle and apoptosis in cancer

Beneath the complexity and idiopathy of every cancer lies a limited number of 'mission critical' events that have propelled the tumour cell and its progeny into uncontrolled expansion and invasion. One of these is deregulated cell proliferation, which, together with the obligate compensatory suppression of apoptosis needed to support it, provides a minimal 'platform' necessary to support further neoplastic progression. Adroit targeting of these critical events should have potent and specific therapeutic consequences.     

RNA干扰Nucleostemin基因对人结肠癌细胞株HT-29细胞增殖及细胞周期的影响

将Nucleostemin(NS)siRNA利用脂质体2000转染人结肠癌细胞株HT 29,分别利用CCK-8试剂盒和流式细胞术检测NS siRNA对HT 29细胞增殖和细胞周期的影响,进一步利用Real-time PCR和Western blotting技术检测NS基因和细胞周期相关基因p21 mRNA和蛋白的表达.结果表明,NS siRNA的转入能有效下调NS mRNA和蛋白的表达(P0.05),并能明显抑制人结肠癌细胞株HT 29的细胞增殖(P0.05),并诱导细胞周期静止在G_0/G_1期,转染NS siRNA后,p21 mRNA和蛋白的表达均明显升高,提示细胞增殖抑制和细胞周期静止与p21基因表达的升高密切相关.