脂质体法和电穿孔法体外转染SD大鼠视网膜Mller细胞的比较

目的:探讨脂质体法及电穿孔法介导增强型绿色荧光蛋白(EGFP)基因转染体外培养的视网膜Mller细胞的可行性和区别。方法:体外培养出生后7～10d的SD大鼠视网膜Mller细胞,免疫荧光染色法鉴定95%以上为视网膜Mller细胞。分别用阳离子脂质体Lipofectamine2000介导的脂质体转染法和电穿孔法将质粒PEGFP-N1转染视网膜Mller细胞,荧光显微镜下检测转染后1,2,3,4d的转染效率,并持续观察至转染后14d,比较两者基因表达持续时间。结果:荧光显微镜下检测转染后1d,两组均可见少量细胞表达EGFP绿色荧光蛋白。转染后2d,两者转染效率均达到最大,并且电穿孔法介导质粒PEGFP-N1转染Mller细胞效率约为31.0%±2.8%,较脂质体法转染效率(10.5%±2.4%)更高。两者比较有统计学意义(P<0.01)。随后,两者转染效率均逐渐降低。电穿孔转染后的PEGFP-N1可在视网膜Mller细胞内持续表达近14d,而脂质体转染后仅能表达约7d。结论:脂质体法和电穿孔法均适用于视网膜Mller细胞的基因转染,但电穿孔法效率更高,表达时间更长。

脂质转染剂增强基因疫苗诱导的免疫应答效力

目的 研究脂质转染剂 (L ipofect AMINE)提高丙型肝炎病毒 (HCV)核心 (C)基因疫苗的抗病毒免疫应答效力 .方法 人工构建包含 HCV C基因片段的真核表达载体 pc D-NAHCV- C,在证实其可以在真核细胞中表达之后 ,将其用脂质转染剂包裹 ,形成 L ipofect AMINE- pc DNAHCV- C脂质混合物 ,将该混合物或单纯 pc DNAHCV- C直接注射 BAL B/ c小鼠股四头肌 ,以空载体 pc DNA3做对照 ,EL ISA法检测血清中抗体产生水平 .结果 12只实验组小鼠 ,初次免疫 2 wk后 ,血清中均出现了 HCV C抗体 ,而 L ipofect AMINE- pc D-NAHCV- C混合物免疫组 ,抗体表达值明显高于单纯 pc D-NAHCV- C免疫组 (P<0 .0 5 ) .结论 脂质转染剂可以促进基因疫苗的摄取并增强其诱导的抗病毒免疫应答的效力 .

阳离子脂质体介导的A20基因转染人外周血单核细胞的可行性

目的探讨阳离子脂质体介导的A20基因转染人外周血单核细胞的可行性及最佳转染条件。方法采用脂质体Lipofectamine TM 2000包裹pCAGGS-GFP/A20质粒转染人外周血单核细胞,通过荧光显微镜检测GFP报告基因,RT-PCR检测A20基因表达。结果应用Lipofectamine TM 2000介导的A20基因转染人外周血单核细胞,在2×106/L细胞接种浓度,脂质体与质粒的量之比为4μl∶3μg时,转染效果最佳,RT-PCR证实成功获得A20目的基因的表达。结论脂质体转染法可介导A20基因在人外周血单核细胞获得表达,该研究为A20基因转染的相关实验研究提供参考依据。

猪肾细胞脂质磁转染系统的构建与表征

磁性纳米基因载体是一种非病毒基因载体,经过功能性基团修饰后能够连接阳离子转染剂构建细胞转染系统。本文将磁转染技术结合常用的脂质体转染,形成了一种新型动物体细胞转染方法,即称脂质磁转染(Liposomal magnetofection,LMF)。这将为体细胞克隆培育转基因动物提供稳定遗传的细胞系。为构建脂质磁性纳米基因载体复合物系统,本研究利用一种磁性纳米基因载体通过分子自组装与脂质阳离子转染剂结合,用于携带外源基因转染动物体细胞。通过原子力显微镜(AFM)观测、ζ电位-粒度等分析表征手段,研究磁性纳米基因载体的形貌、粒径分布、负载及浓缩DNA的方式。结果表明,通过猪肾(PK)细胞的LMF实验,与脂质体(Lipofectamine2000)介导的转染比较,具有较高的转染率,更重要的是克服了脂质体转染瞬时表达的缺陷。MTT细胞毒性试验结果也显示该方法具有较低的细胞毒性。因此LMF是一种切实可行的高效低毒性的细胞转染方法。

Transfection of the Human Sodium/Iodide Symporter(NIS) Gene with Liposomes and the Expression of the NIS Protein in Human Lung A549 Cancer Cells

OBJECTIVE To examine the possibility of human sodium iodide symporter （hNIS） protein expression in lung cancer cells. METHODS Human lung A549 cancer cells were thawed and cultured in vitro. The cells were divided into an experimental group transfected with a recombinant pcDNA3-hNIS plasmid and a control group transfected only with a pcDNA3 plasmid. The recombinant plasmid vector encoding the hNIS gene （pcDNA3-hNIS） was amplified, purified and identified. The hNIS gene was followed by DNA sequencing. A Western blot and an immunohistochemical assay were applied to detect the hNIS protein expression in the transfected human lung A549 cancer cells. RESULTS Restriction enzyme digestion and DNA sequencing results showed the size and direction of the inserted gene in the recombinant pcD- NA3-hNIS plasmid was correct. The Western blot method and immunohistochemical analysis showed a positive NIS protein expression in the experimental group. The NIS protein was detected mainly in the cell membranes showing a positive rate up to 70.6% with no expression of the NIS protein in the control group. There was a significant difference between two groups （P=0.000）. CONCLUSION The hNIS gene was transfected effectively into human lung A549 cancer cells mediated by Lipofectamine 2000, and was expressed with its protein in vitro.

端粒酶突变体对肝癌细胞端粒酶活性的作用

目的 观察端粒酶突变体对肝癌细胞端粒酶活性和生长抑制的作用。方法 利用质脂体转染法在培养的肝癌细胞中导入端粒酶突变体 ,并与维甲酸和人参皂甙对照 ,利用TRAP 银染方法对不同时期肝癌细胞进行了端粒酶活性的测定 ,并观察各期细胞生长情况。结果 在加入端粒酶突变体后肝癌细胞端粒酶活性明显降低 ,细胞出现明显凋亡现象 ,其作用效果与维甲酸基本相同。结论 端粒酶突变体对肝癌细胞的抑制作用可能是通过抑制端粒酶活性途径实现的。

pCR3.1-bvLDH-C′_4的构建及在体内外的表达

精子特异性乳酸脱氢酶 (LDH C4)在精子的运动和存活等生理活动中起着重要的作用。研究表明 ,LDH C4接种鼠、兔等能够降低动物的生育率。通过RT PCR方法从布氏田鼠睾丸总RNA中克隆出编码抗原决定簇的LDH C4基因片断 ;采用T A克隆的方法将其插入到pCR3 1载体中构建重组载体pCR3 1 bvLDH C′4,测序结果表明克隆的基因片断与已知小鼠相应片断有 83 %的序列同源。通过质脂体法转染HeLa细胞 ,RT PCR证实其可在mRNA水平有效表达 ;通过肌肉接种BALB c小鼠 ,RT

Liposome transfected to plasmid-encoding endostatin gene combined with radiotherapy inhibits liver cancer growth in nude mice

AIM： To evaluate whether intratumoral injection of liposome-endostatin complexes could enhance the antitumor efficacy of radiation theapy in human liver cardnoma （BEL7402） model.METHODS： Recombinant plasmid pcDNA3.End was transfected into human liver carcinoma cell line （BEL7402） with lipofectamine to produce conditioned medium. Then BEL7402 cells and human umbilical vein endothelial cells （HUVECs） were treated with the conditioned medium. Cell cycle and apoptosis were analyzed by flow cytometer and endothelial cell proliferation rates were determined by MTT assay. The antitumor efficacy of endostatin gene combined with ionizing radiation in mouse xenograft liver tumor was observed.RESULTS： Endostatin significantly suppressed the S phase fraction and increased the apoptotic index in HUVECs. In contrast, endostatin treatment had no effect on BEL7402 cell apoptosis （2.1±0.3% vs 8.9±1.3%, t= 8.83, P= 0.009〈0.01） or cell cycle distribution （17.2±2.3% vs 9.8±1.2%, t = 4.94,P = 0.016〈0.05）. The MTT assay showed that endostatin significantly inhibited the proliferation of HUVECs by 46.4%. The combination of local endostatin gene therapy with radiation therapy significantly inhibited the growth of human liver carcinoma BEL7402 xenografts, the inhibition rate of tumor size was 69.8% on d 28 compared to the untreated group. The tumor volume in the pcDNA3.End combined with radiation therapy group （249±83 mm^3） was significantly different from that in the untreated group （823±148 mm^3, t= 5.86, P= 0.009〈0.01） or in the pcDNA3 group （717±94 mm^3, t= 6.46, P= 0.003〈0.01）. Endostatin or the radiation alone also inhibited the growth of liver tumor in vivo, but their inhibition effects were weaker than those of endostatin combined with radiation, the inhibition rates on d 28 were 44.7% and 40.1%, respectively.CONCLUSION： Endostatin not only significantly suppresses tumor growth but also enhances the antitumor efficacy of radiation therapy in human carcinoma xenograft.

Growth-inhibitory effects of MOB2 on human hepatic carcinoma cell line SMMC-7721

AIM:To investigate the growth-inhibiting and apoptosis-inducing effects of the gene MOB2 on human hepatic carcinoma cell line SMMC-7721.METHODS:The full-length cDNA of the MOB2 gene was amplified from human umbilical vein endothelial cells.The correct full-length MOB2 cDNA was subcloned into the eukaryotic expression vector pEGFP-C1.After lipofection of the MOB2 gene into cancer cells,the levels of MOB2 protein in the cancer cells were detected by immunoblotting.To transfect the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells,the cells were cultured in Dulbecco's Modified Eagle'sMedium with 10% fetal calf serum and glutamine,and then mixed with liposomes,Lipofectamine 2000 and the plasmid vector pEGFP-CI-MOB2.RESULTS:We observed the growth and proliferation of SMMC-7721 cells containing pEGFP-CI-MOB2 and analyzed their apoptosis and growth cycle phases by flow cytometry.We successfully transfected the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells and screened for a single clone cell containing MOB2.After transfection,MOB2 enhanced growth suppression,induced apoptosis,increased the ratio of G0/G1,significantly inhibited the advance of cell cycle phase,and arrested cells in G0/G1 phase.CONCLUSION:MOB2 overexpression induces apoptosis and inhibits the growth of human hepatic cancer cells,which may be useful in gene therapy for hepatic carcinoma.

Biological Analysis of HSV-1 Immediate-early Proteins ICP0, ICP22, and ICP27 in Neuroblastoma Cells

The three immediate-early proteins of HSV-1, ICP0, ICP22, and ICP27, have specific and pivotal functions in transcriptional activation and inhibition, multiple regulatory and control processes of viral genes. In this paper, the expression and localization of these three proteins were studied in neuroblastoma cells using biochemical assays, and their possible and potential interactive functions are discussed. The data show that the three proteins are localized in different structures, specifically in the PML-NB-associated structure, which is a specific nuclear structure composed of many protein molecules and bound tightly to the nuclear matrix in neuroblastoma cells. The results suggest that the activating and suppressive functions of ICPs are mostly dependent on their transcriptional and regulatory roles, including the PML-NB-associated structure.

Down-regulation of Bmi-1 by RNA interference in Jurkat cells

Objective： The aim of our study was to investigate the effects of down-regulation Bmi-1 by RNA interference （RNAi） in T Lymphocytic leukemia Jurkat cells. Methods： Two complementary oligonucleotide strands were synthesized based on the siRNA sequence targeting Bmi-1 gene. After annealing, siRNA strands were recombined into the pRNAT- U6.2 vector, and then DNA sequencing was carded out following transformation and amplification. The recombinant was transfected into Jurkat cells with liposomes. Positive colonies were obtained through G418 selection. The mRNA and protein expressions of Bmi-1 were detected by RT-PCR and Western-blot, respectively. Effects of Bmi-1 silence on cell proliferation, cell cycle and cell aging of Jurkat cells were detected by M＇l-r assay, flow cytometry, colony formation assay and SA-β-Gal staining, respectively. Results： The siRNA recombinant targeting Bmi.1 gene was successfully constructed. All three siRNA recombinants could significantly inhibit the expression of Bmi-l. The siRNA targeting 825nt-843nt （GACCAGACCACTACT GAAT） has the strongest inhibitory effect on Bmi-1 expression, with almost complete inhibition on Bmi-1 mRNA and protein expressions. Compared with the non-transfection group and the empty vector group, growth velocity and colony formation ability were significantly decreased, while the proportion of calls in G1 phase and the percentage of senile cells were signifi- cantly increased in highly transfected group （P 〈 0.05）. Conclusion： Down-regulation Bmi-1 by RNA interference （RNAi） could significantly inhibit the growth of Jurkat cells in vitro.

Up-regulation of Raf kinase inhibitor protein enhances chemosensitivity of cervical cancer cell

Objective： The purpose of the study is to investigate the effects of up-regulation of Raf kinase inhibitor protein （RKIP） on the chemosensitivity of cervical cancer Hela cells. Methods： Eukaryotic expression plasmid pcDNA3.1（±）-ssRKIP containing human overall length RKIPcDNA was transfected into cervical cancer Hela cell by lipofectin assay, establishing a stable cell line containing a target gene by G418. Expression of RKIP in Hela cells was measured by Western blot analysis. After treatment with cisplatin of different concentrations and intervals of time, the effect of RKIP on the proliferation of Hela cells was evaluated by MTT method. The flow cytometry was used to investigate whether the RKIP could inhibit apoptosis in Hela cells induced by cisplatin. Results： The expression of RKIP in Hela cells transfected with pcDNA3.1-ssRKIP was increased obviously. After different concentrations of cisplatin treatment cells for 24, 48 and 72 h, the growth inhibition rate in Hela cells transfected with pcDNA3.1-ssRKIP was significantly higher than in control cells （P 〈 0.05）. With 5 pg/mL cisplatin treatment for 24 h, pcDNA3.1-ssRKIP-transfected Hela cells had an obviously higher percentage of apoptosis （23.2 ± 0.24）% than non-transfected cells （12.4 ± 0.31）% and empty vector-transfected cells （13.4 ± 0.47）%. Without treatment of cisplatin, the percentage of apoptosis for Hela cells transfected with pcDNA3.1-ssRKIP was （5.7 ± 0.12）%, which was still higher than those of the non-transfected cells （2.9 ± 0.21）% and empty vector-transfected cells （3 ± 0.08）%. Conclusion： Higher expres- sion of RKIP gene can improve chemosensitivitv of cervical cancer Hela cells to cisplatin.

Optimization on cationic liposome-mediated cell transfection of plasmid DNA

Objective:The development of gene carriers for efficient gene delivery into cells has attracted growing attention in recent years.The aim of this study was to achieve a better outcome of AAV-293 cells transfection by plasmid DNA.Methods:We studied the optimal condition for higher efficiency of cationic lipid-mediated cell transfection.Four experimental groups were set.Plasmid DNA and liposome were mixed in each groups at different ratios(μg:μL),1:2.5,1:3.5,1:4.0 and 1:5.0,respectively.LacZ gene functioned as reporter gene,measuring the transfection efficiency of the four groups using the method of X-gal staining.Results:When the ratio was 1:3.5,the cell transfection rate was the highest.While the ratio of 1:2.5 recommended by product manual achieve the lowest transfection rate.Their difference had statistical significance.Conclusion:In order to obtain a higher transfection efficiency,optimization on conditions of the ratio of plasmid DNA to liposome is necessary in cell transfection.

COX-2 silencing inhibits cell proliferation in A549 cell

Objective: The aim of this study was to explore the effects on malignant proliferation of A549 cell by silencing cy-clooxygenase (COX)-2. Methods: In the present study, we constructed three siRNA vectors producing small interference RNA. The siRNA vectors and the vacant vectors were transfected into A549 cell with lipofectamine respectively and the transfected cell strains were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of lung cancer cells were studied by cell growth curve, clonogenic assay and xenograft assays. Results: The siRNA expression vectors produced marked effects in A549 cell but the inhibited effects were different. The effect of psi-10 was best and the mRNA and protein levels of COX-2 reduced 61.2% and 56.2% respectively in A549-si10 cell in contrast to the control. The growth of A549 cell slowed and the colony formation rate reduced after silencing COX-2. In xenograft assays, the growth speeds of tumor became slow and the numbers of tumor reduced after silencing COX-2. Conclusion: The si10 target of COX-2 has the best silencing effect in A549 cell and the best inhibition effect on malignant proliferation of A549 cell in vivo and in vitro.