Effect ofP15 INK4b/MTS2 on the proliferation of human hepatoma cells SMMC-7721

The full length cDNA coding forP15 ^INK4b, which is a cyclin-dependent kinase inhibitor, was cloned to plasmid PXJ41-neo (Eco R I /Xho I site) and the new constructed plasmid pXJp15 was obtained. pXJp15 was transferred into the human hepatoma SMMC-7721 cetls by lipofectine reagent. After G418 setection, a series of cetl lines stably expressing high levets ofP15 (named SHT) and the clone containing vector PXJ41-neo only (named SVXJ) were obtained by Northern and Western analysis. The results showed that the proliferation of SHT cells is inhibited compared with that of SVXJ cetls. Cell cycle analysis indicated that overexpressing ofP15 inhibited the growth of SHT cetls by decreasing progrssion of cetls from G1 to S and G2 to M phases. The levets ofc-Myc andc-Fos were obviously decreased in SHT cells compared with control cetls by Western blotting. The decreased expression of oncogene may be one of the molecular mechanisms of the effect ofP15 on the proliferation of in SHT cetls.     

cdk2反义RNA对乳腺癌细胞增殖及致瘤性的抑制作用

为了研究cdk2对乳腺癌细胞生长及cyclinA,cyclinB1和ckdl（cdc2)mRNA表达水平的影响,利用直接表达载体p XJ41-neo构建了表达cdk2反义RNA的重组载体,并用此载体转染了人乳腺癌细胞系Bcap37,获得了ckd2受到抑制的细胞模型Bcap37-CDK2AS,然后将Bcap37-CDK2AS细胞的生长能力及cyclinA,cyclinB1和cdk1 mRNA折水平与转入空载体的对照细胞进行了对比分析,结果显示,cdk2表达受到抑制时,细胞生长速率下降,根据测定出的细胞生长曲线,细胞培养至第7天时,细胞生长抑制率为64％,在流式细胞术的分析结果中,G1期细胞中的百分比从39％增加到47％,S期细胞由51％下降到39％,裸鼠接种的实验表明,Bcap37－CDK2AS的致瘤性明显减弱,在对cyclinA,cyclinB1和cdk1mRNA折分析中发现,Bcap37－CDK2AS中这3种基因的mRAN水平均有不同程度的下降,依据这些结果可以推测,ckd2反义RNA可使乳腺癌细胞生长及致瘤性受到抑制,并且cdk2表达的抑制将cyclinA,cyclinB和cdk14的表达水平。     

Effect of P15INK4b expression on the cell cycle and G1 phase related regulatory genes in human melanoma cells

Using the transfeetion teehnique. P15INK4b was introduced into P15INk4b gene deleted human melanoma A375 cells,and a cell model MLED6 overexpressing P15INK4b WAS CONSTRUCTED.Comparing with the control cells MLC2,MLEK6cells in G1phase increased by 11%,but those in Sphase decreased by 15%by FCM.By the method of thymidine(TdR)and N2O arresting,the proportions of synchronized Mphase cells of MLEK6 ana MLC23 were measured and found to be 89.1% and 76.8%respectively ,and the cells in G1phase were 74.3% for MLID6 AND 76. 4% forMLC2.The result of3 H-TdR incorporation indicated that the transition of G1/Sof MLEK6 cell was delayed 2h as compared with that of MLC2 cells,and incorporation rate also decreased.The observation on exprissions of some G1/ S-resates relatory rigusating genes showed that in MLIK6 cells the protein leves of P27KIPI increased with the decreasing expressions of cyclinD1,cyclinE and c-myc,especially cyclinD1 in late G1phade.The expression of cyclinE obviously decreased at G1/S transition ,and c-myc wad inhibited throughout all the process of G1 S phase.All the risults suggest that P15INK4b can delayG1/S transition of MLEK6 cells by inhibiting the cell cycle engine ,and by increasing the expression of Cdk ingibitor P27KIPI in different stages of G1 phase.     

糖氧剥夺对大鼠脂肪干细胞自噬和成脂的影响

目的 研究大鼠脂肪干细胞( adipose derived stem cells,ADSC)及糖氧剥夺下,细胞自噬和对诱导分化成脂的影响。 方法常规制备 ADSC, 设 置 对 照 组 ( G 组 ) 、 少 糖 组 ( SG 组 ) 、 缺 氧 组 ( G + CoCl2 组 ) 、 缺氧少糖组( SG+CoCl2 组)和自噬阳性对照组( EBSS 组) 。 设置不同培养时间,MTT 检测各组 ADSC 存活情况;Western blot 分析细胞自噬及成脂诱导油红“ O”染色与比色。 结果 不同培养时间,ADSC 细胞均可增殖。 培养 24 h,各组细胞之间无差别( P>0. 05) ,但培养 48 h 和 72 h,G 组细胞增殖明显高于其它三组( SG 组、G + CoCl2 组和 SG+CoCl2 组) ,P<0.05;Western blot 分 析, EBSS 组、G 组 和 SG 组 的自噬相关蛋白 Ⅰ 型 ( LC3-Ⅰ ) 明显弱于自噬相关蛋白Ⅱ 型( LC3-Ⅱ ) ,且 G+CoCl2 组和 SG+CoCl2 组的 LC3-Ⅰ 明显高于 EBSS 组;成脂诱导实验中,G 组油红“ O” 比色值最高,与其它组比较均有统计学差异( P<0. 05) 。 结论 ADSC 具有较强的细胞自噬作用,推测在缺糖缺氧条件下,通过抑制细胞自噬,降低 ADSC 存活率以及诱导分化成脂率。     

反义PARP基因真核表达重组体的构建及其HeLa转化细胞系的建立

利用DNA体外重组技术将PARP基因cDNA部分序列反向克隆到真核表达载体pMAMneo上,构建成重组质粒pMAMneo－C1．4和pMAMneo－C0．3．将重组质粒pMAM－neo－C1．4转染HeLa细胞,经G418筛选,建成细胞系HeLa－C1．4－neo,Southern杂交结果表明,外源PARP基因cDNA部分序列已稳定整合到受体细胞基因组中,该细胞系的建立为研究聚ADP核糖基化作用在细胞内的功能打下了基础．     

表达HIV-1复合多表位基因的p815细胞稳定株的建立和评价

建立稳定表达HIV-1p24MEG复合多表位基因的p815细胞克隆.设计引物,以HIV-1标准株全长cDNA序列为模板,PCR扩增获得p24基因片段;合成改造后的多个表位基因MEG并且与p24片段相连接,克隆入pcDNA3.1(+).在阳离子聚合物作用下重组真核质粒转染的p815(H-2d)细胞, 以G418压力筛选,RT-PCR检测mRNA表达,间接免疫荧光检测蛋白表达.通过PCR获得了HIV-1 p24片段,获得了与多表位基因连接后的p24MEG融合基因,成功构建了p24MEG基因的重组真核表达载体pcDNA3.1(+)/p24MEG.转染p815细胞后, RT-PCR检测到融合蛋白mRNA表达,间接免疫荧光显示 p815细胞内有融合蛋白的表达.结论:建立了稳定表达HIV-1p24MEG融合蛋白的p815细胞克隆,为评价多表位抗原p24MEG在BALB/c小鼠体内诱导的细胞免疫应答奠定基础.     

Partial inhibition of CDK4 gene expression leads to the extension of G1 phase of V79-8 cells

V79-8 is an abnormal cell line which does not have detectable G1 and G2 phases in its cell cycle. This cell line is derived from V79 cell line which has Gl phase but lacks G2 phase. By using an anti-sense approach, CDK4 gene expression was partially inhibited to find whether CDK4 might contribute to the lack of Gl phase in V79-8 cells. Anti-CDK4 anti-sense plasmid was constructed and used to transfect V79-8 cells. Clones of transfected cells (V79-8-asCDK4) were examined, in comparison with V79-8 cells, to determine its growth curve, cell doubling-time (GT), the level of CDK4 gene expression and the levels of expression of some other growth related genes. V79-8-asCDK4 cells showed a slower growth rate with a doubling time 2.5-h longer than that of V79-8 cells. A flow cytometry (FCM) analysis demonstrated that the 2.5 h increase of the doubling time of V79-8-asCDK4 cells was mainly due to the appearance of Gl phase because its G2 + M phase was not significantly different from that of V79-8 cells. The decrease of CDK4 gene expression in V79-8-asCDK4 cells was shown by Northern-blot. Changes in the expression levels of the growth-related genes TGF-β, cyclin D1 and Rb were also detected in V79-8-asCDK4 cells. CDK4 functions mainly in G1 and at the transition between G1 and S phases. Expression of an anti-sense CDK4 gene fragment reduces the levels of endogenous CDK4, CDK4/cyclinD kinase activity and the phosphorylation of Rb. These events may postpone the inactivation of the check-point leading to the delay of entry into S phase and the reappearance of G1 phase in V79-8-asCDK4 cells.     

pEgr-Endostatin重组质粒联合电离辐射抗肿瘤效应的实验研究

目的研究pEgr-Endostatin辐射诱导肿瘤的表达特性及其抗肿瘤作用,为提高临床肿瘤放疗疗效提供实验证据.方法 Western blotting法检测pEgr-Endostatin重组质粒联合电离辐射诱导黑色素瘤B16细胞蛋白表达;建立动物模型,用ELISA法检测不同处理组Endostatin的剂量水平,比较不同处理方式的差异.结果 Western blotting法检测结果显示:重组质粒转染的黑色素瘤B16细胞上清中均有Endostatin蛋白表达,而未转染重组质粒的B16细胞和转染空质粒的B16细胞上清中未见Endostatin蛋白表达.ELISA法检测结果表明:体外接受2~10 Gy X射线照射,Endostatin表达水平明显增加,且照射剂量为4 Gy时Endostatin的表达水平最高.2 Gy照射8~72 h后,Endostatin表达水平与假照射组比较明显增加,在48 h时Endostatin表达水平最高.动物实验表明:荷瘤+25 Gy照射组、荷瘤+pEgr-Endostatin照射组、荷瘤+pc DNA3.1+25 Gy照射组和荷瘤+pEgr-Endostatin+25Gy照射组肿瘤生长率显著低于单纯荷瘤组(P0.05#0.01),荷瘤+pEgr-Endostatin+25 Gy照射组亦明显低于荷瘤+25 Gy照射组和荷瘤+pEgr-Endostatin组(P0.05#0.01).结论 pEgr-Endostatin重组质粒联合电离辐射能使Endostatin蛋白表达增加,从而抑制血管内皮细胞生长、发挥抗肿瘤作用.     

X连锁凋亡抑制蛋白对肺癌细胞生长和细胞周期的影响

为了研究靶向X连锁凋亡抑制蛋白的发夹状RNA对肺癌细胞中抗肿瘤作用,构建XIAP基因的shRNA表达载体,并设计阴性对照(psiRNA-Con)质粒,分别转染A549细胞;实时定量PCR和免疫印记法分别检测XIAP的mR-NA和蛋白的表达;四甲基偶氮唑盐试验(MTT)检测A549细胞的增殖;流式细胞术检测细胞周期分布。实验结果表明:转染psiRNA-XIAP组细胞XIAP mRNA和蛋白水平均低于正常对照组,生长慢于正常组(t=16.82和t=12.13,均P<0.01),G2/M期细胞所占比例增高(t=3.78,P<0.05),并出现亚G1峰。而阴性对照质粒psiRNA-Con未能下调XIAP的表达水平,对A549细胞增殖和细胞周期亦无明显影响。结论:针对XIAP的RNA干扰质粒特异性地抑制了其RNA和蛋白水平,使肺癌细胞A549生长减慢,诱导凋亡,并使其发生G2/M期阻滞,有望发展为新的抗肿瘤药物。     

结核分枝杆菌Hsp16.3单克隆抗体的制备及其特性鉴定

制备抗结核分枝杆菌Hsp16.3的单克隆抗体,并对其生物学特性进行鉴定。将含目的基因的表达载体pProEXHTb-Hsp16.3,通过E.coli DH5α诱导表达,获得含有6譎is的Hsp16.3蛋白,采用Ni-NTA纯化试剂盒进行目的蛋白的纯化,并用透析方法进行蛋白复性。将复性的蛋白免疫BALB/c小鼠,利用杂交瘤技术进行细胞融合,间接ELISA筛选阳性杂交瘤细胞株。将获得的Hsp16.3阳性杂交瘤细胞株分别利用间接ELISA法和Western blot方法进行效价、相对亲和力及特异性的测定。获得了三株Hsp16.3的单克隆抗体,分别命名为3H6F9、1D5E1和4H8G6,其效价分别为1:1?07、1:1?06和1:1?06,相对亲和力分别为0.0001 mg/mL、0.001 mg/mL和0.001 mg/mL,并且无交叉反应性。所获得的结核分枝杆菌Hsp16.3的单克隆抗体效价高、特异性强,为进一步研究Hsp16.3在结核分枝杆菌潜伏感染中的作用提供了有效的工具。     

碱裂解提取质粒DNA的改进方法

实验室中常用的质粒提取方法是碱裂解法.一般碱裂解法提取质粒相对耗时长,而且需要冰浴,条件较为严格,本文尝试对碱裂解法进行改进,即在控制菌体量的前提下连续加入溶液Ⅰ,Ⅱ,Ⅲ,进行质粒提取,简化了操作步骤,缩短了实验时间(由150 min左右缩短为110 min以内),质粒收率和质量不变.     

基于会话异构的结构化对等网络拓扑模型

针对分布式哈希表(DHT)拓扑模型在动态网络中性能下降的问题,提出了一种改善的会话异构拓扑(SHT)模型.SHT模型利用了对等网络节点存在的会话异构性,将动态节点聚簇在稳定节点,从而降低了网络动态节点对于DHT拓扑的强干扰性.仿真实验显示,SHT模型能有效减少对等网络系统的拓扑维护开销,提高系统稳定性和数据可用性.     

Effects of enhancing p21 waf1 on proliferation and expression of G1cyclins andCDKs in human breast cancer cells

The cyclin-dependent kinase inhibitor p21( waf1/cip1/sdil) is an important negative regulator in control of cell cycle. Its functions of inhibiting cancer cell growth and its effects on expression of G1 phase cyclins and related CDKs are a worthy topic for study. The plasmid expressing p2l with high level was transformed to human breast cancer cells, and the expression of p2l in cells was enhanced, then the cell growth rate, anchorage-independent growth and tu-morigenecity were tested, at the same time the expression levels of cyclinD1, CDK4, cyclinE and CDK2 were analyzed by Northern blot. The results showed that since the expression of p21 was enhanced in the cell, the rate of cell growth and anchorage-independent growth was inhibited, tumorigenecity was suppressed, the level of expression of cyclinE and CDK2 decreased while that of cyclinDl and CDK4 was not affected. It is suggested that the enhanced expression of p21 markedly inhibits the proliferation and lessens the tumorigenecity of breast cancer cells, and that p2l expression is not related to that of cyclinDl and CDK4, but affects the expression of cyclinE and CDK2 .     

P137G点突变对嗜热细菌木糖异构酶酶活性及热稳定性的影响

利用重叠延伸法对嗜热细菌（Thermus thermphilus）的木糖异构酶基因xylA进行体外P137G定点突变，通过酿酒酵母表达载体XM204将突变基因xylA137引入酿酒酵母H158中，得到重组菌株H158 XI137. 酶活分析表明，H158 XI137在中温条件下的酶活有很大的提高，与对照菌株H158 XI相比，30?℃时的酶活提高1倍，并且拓宽了最适反应pH，但是其热稳定性大大降低. 结果表明，嗜热细菌木糖异构酶137位的Pro对维持其热稳定性起到重要的作用，并且对其酶学性质有很大的影响.     

非小细胞肺癌中转化生长因子βⅠ型受体基因的缺陷表达

转化生长因子β(TGF-β)信号转导通路的紊乱可使细胞逃避TGF-β介导的生长抑制效应,从而导致多种肿瘤的发生.本研究探讨了转化生长因子βⅠ型受体(TGFβRⅠ)在非小细胞肺癌(NSCLC)发生中的作用.采用免疫组化进行TGFβRⅠ基因的表达分析:采用单链构象多态性(SSCP)分析TGFβRⅠ基因结构的变异.结果发现37.2%NSCLC中TGFβRⅠ的表达下降,表明TGFβRⅠ在NSCLC发生中起着重要的作用.同时在TGFβRⅠ基因的第7号内含子中发现了一个单核苷酸多态性(SNP),该SNP与TGFβRⅠ的表达下降无显著关系(P>0.05).     

hSOD基因在大白鼠肺上皮细胞中的表达

本文以哺乳动物细胞表达载体ｐＲｃ／ＣＭＶ为运载体,构建了受控于ＣＭＶ启动子的ｈＳＯＤ基因的表达载体ｐＲｃ／ＣＭＶ＿ＳＯＤ,用脂质体转染法转染大白鼠肺上皮细胞Ｌ２细胞后,经抗生素Ｇ４１８选择性培养,获得与细胞染色体ＤＮＡ稳定整合的ｈＳＯＤｃＤＮＡ导入细胞,经ＳｏｕｔｈｅｒｎＢｌｏｔｓ和ＷｅｓｔｅｒｎＢｌｏｔｓ分析,结果表明,ｈＳＯＤ在细胞中得到稳定表达,同时测定细胞ＳＯＤ酶活性,ｈＳＯＤｃＤＮＡ导入细胞的ＳＯＤ酶活性是空载体导入细胞和父本细胞的约２倍,并且在选择细胞克隆之后的６０天内是持续的．     

结核分枝杆菌HSP65-hIL-2融合基因稳定转染细胞系的建立

建立可稳定表达结核分枝杆菌HSP65-hIL-2融合蛋白的稳定转染P815细胞系. 在阳离子脂质体作用下,将HSP65-hIL-2真核表达质粒转染与BALB/c 遗传背景一致的P815细胞(H-2d).G418筛选阳性克隆,RT-PCR和间接免疫荧光法检测目的蛋白的转录和表达. 阳性克隆细胞经RT-PCR检测到HSP65-hIL-2融合基因特异性的mRNA表达;用鼠抗人的IL-2 mAb进行间接免疫荧光检测,可在转染的P815细胞浆中观察到较强的绿色特异性荧光,而未转染细胞则为阴性.成功获得稳定表达HSP65-hIL-2融合蛋白的稳定转染细胞系,为其疫苗的CTL研究提供了合适的靶细胞.     

HCV感染通过Erk信号的激活上调PTTG1的表达

 为探讨丙型肝炎病毒(HCV)感染导致肝细胞癌发生的分子机制,利用前期研究建立的体外HCV细胞培养体系,将HCV JFH-1 RNA转染CD81-Huh7细胞,确定能够获得感染性HCV后,收集HCV转染后10,50,100和150d的细胞,采用Real time PCR及Western Blot法检测不同时间点细胞内垂体瘤转化基因1(PTTG1)基因和蛋白水平的表达情况,同时检测总MAPK/Erk1/2,磷酸化Erk1/2(p-Erk1/2)蛋白的表达。随后,用干扰素抑制HCV的感染,再检测PTTG1及MAPK/Erk1/2的表达情况,以及用MAPK/Erk抑制剂(PD98059)阻断MAPK/Erk 1/2的磷酸化后,检测PTTG1的表达情况。结果显示,HCV转染的细胞与未转染细胞比较,PTTG1的mRNA和蛋白表达均显著增加,p-Erk1/2蛋白显著升高(P＜0.05);抑制HCV感染显著降低PTTG1的表达和MAPK/Erk1/2的磷酸化(P＜0.05);MAPK/Erk1/2抑制剂显著降低了PTTG1基因和蛋白的表达(P＜0.05)。体外HCV感染可以导致MAPK/Erk信号通路的磷酸化,进一步导致原癌基因PTTG1的表达增加,这可能是慢性HCV感染导致HCV相关性肝细胞癌发生的分子机制之一。     

微血管内皮细胞与肺动脉平滑肌细胞增殖的相互调节

研究常氧下微血管内皮细胞(MVEC)与肺动脉平滑肌细胞(PASMC)增殖的相互调节。先滤膜培养大鼠肺微血管内皮细胞(LMVEC)后,再与PASMC复合培养,采用流式细胞仪检测PASMC周期的变化。复合培养后,正常情况下使PASMC的G1期细胞数减少,S＋G2／M期细胞数增多,即促进细胞生长;使LMVEC田C的G1期细胞数增多,S＋G2／M期细胞数减少,即抑制细胞生长。可见,MVEC与PASMC复合培养后,常氧下促进PASMC细胞生长,抑制LMVEC的增生,存在相互作用。     

Effects of PP4 suppression on the proliferation of MCF7 cells

In mammalian cells, reversible phosphorylation of protein substrates is a major mechanism for many cel-lular processes. Protein kinases and protein phosphata- ses, which catalyze protein phosphorylation and dephosphorylation respectively, together with th…