小鼠大脑皮质神经元电穿孔转染条件的优化

目的:以原代培养的小鼠大脑皮质神经元为研究工具,优化电穿孔转染条件。方法:通过设置不同的电压和脉冲时间组合等电转染条件,将表达绿色荧光蛋白(GFP)的shNC质粒导入小鼠大脑皮质神经元,24 h后观察并比较神经元的转染效率,确定最佳电转染条件。结果:200 v电压,25 ms脉冲时间或250 v电压,15 ms脉冲时间的转染条件下,小鼠大脑皮质神经元可获得较好的转染效率,分别为(31.15±1.89)%和(29.10±1.93)%。结论:电穿孔转染是一种高效的基因转染方法,通过条件的优化,可以提高小鼠大脑皮质神经元的电转染效率。     

DNA－转染体细胞系产生单克隆抗体

本文研究淋巴细胞转染体的产生和转染体单克隆抗体的抗原结合特性。以小鼠宫颈癌细胞系Ｕ１４为抗原免疫小鼠。将制备的Ｕ１４和小鼠骨髓瘤细胞系ＳＰ２／０ＤＮＡ分别转染经免疫的小鼠原代脾细胞。将所形成的转染体细胞进行克隆，建成２株淋巴细胞转染体细胞系ＵＤ和ＳＤ，它们能连续生长于含血清和无血清培养液中，但不能在同基因小鼠体内生长。ＵＤ和ＳＤ细胞系所产生的单克隆抗体不仅能与小鼠宫颈癌细胞表面抗原及其可溶性抗原发生特异反应，且能识别人宫颈癌组织和宫颈癌病人血清中的肿瘤抗原决定簇。本实验结果进一步证明了我们提出的肿瘤“进化抗原”理论，并提示，瘤细胞ＤＮＡ转染造成淋巴细胞永生化，可能是产生人源性单克隆抗体具有前景的途径。     

DNA－转染体细胞系产生单克隆抗体

本文研究淋巴细胞转染体的产生和转染体单克隆抗体的抗原结合特性．以小鼠宫颈癌细胞系Ｕ１４为抗原免疫小鼠。以制备的Ｕ１４和小鼠骨髓瘤细胞系Ｓｐ２／０ＤＮＡ分别转染经Ｕ１４免疫的小鼠原代脾细胞．将所形成的转染体细胞进行克隆，建成２株淋巴细胞转染体细胞系ＵＤ和ＳＤ．它们能连续生长于含血清和无血清培养液中，但不能在同基因小鼠体内生长．ＵＤ和ＳＤ细胞系所产生的单克隆抗体不仅能与小鼠宫颈癌细胞表面抗原及其可溶性抗原发生特异反应，且能识别人宫颈癌组织和宫颈癌病人血清中的肿瘤抗原决定簇．本实验结果进一步证明了我们提出的肿瘤＂进化抗原＂理论，并提示瘤细胞ＤＮＡ转染造成的淋巴细胞永生化，可能是产生人源性单克隆抗体具有前景的途径．     

PIPP~(H557A)对小鼠受精卵有丝分裂期的调节作用

目的研究脯氨酸丰富的肌醇多磷酸5-磷酸酶(proline-rich inositol polyphosphate 5-phosphatase,PIPP)组氨酸557位点突变为丙氨酸时PIPPH557A在小鼠1-细胞期受精卵中对有丝分裂促进因子(MPF)的调节作用。方法将HA-PIPPH557A、HA-PIPP和/或HAvector转染到小鼠1-细胞期受精卵中,用激酶活性分析法和WesternBlotting方法检测小鼠受精卵中MPF活性以及pCdc2 Tyr15表达的变化。结果 HA-PIPPH557A对小鼠受精卵MPF活性影响较小,但可以降低pCdc2Tyr15的表达,与HA-PIPP的作用不同。结论 HA-PIPPH557A在小鼠受精卵中通过失活PIPP降低pCdc2 Tyr15的表达调节小鼠受精卵的有丝分裂。     

小鼠心脏移植保存期间经体外灌注行重组腺病毒基因转染的研究

目的:探讨小鼠心脏移植中,通过在体外保存期间对供心进行灌注,进行重组腺病毒基因转染的方法及效率。方法: 利用小鼠异位心脏移植,在供心保存期间行重组腺病毒灌注,在移植后7 d检测移植物内标记基因的表达情况,并通过免疫组织化学染色检测移植物内免疫细胞的浸润,通过流式细胞仪检测受体淋巴细胞的激活状态。结果: 小鼠供心通过体外灌注进行重组腺病毒基因转导,并在4 ℃保存2 h,复跳率达100%,移植后1周,标记基因表达强度到达峰值。移植物组织结构完整,无明显细胞浸润。受体淋巴细胞的激活状态与空白对照组相比,无明显差异。结论: 小鼠心脏移植保存期间体外灌注可应用于腺病毒基因转染等移植物处理过程。     

体内IFN-γ基因转染小鼠腹腔巨噬细胞抗肿瘤作用的研究

采用磷酸钙－ＤＮＡ共沉淀法，将ｐＲＥＰ－８－ＩＦＮ－表达质粒直接注射至小鼠腹腔，可成功地转染腹腔巨噬细胞（ＭΦ），并表达其基因产物，能有效地抑制肿瘤生长；再次基因转染，可获得增强的抗肿瘤效应。还发现转基因ＭΦ所分泌的激活因子能激活未转基因的ＭΦ；增加腹腔ＭΦ的数量；增强ＭΦ的杀瘤活性；并使ＭΦ分泌一定水平的ＮＯ、ＩＬ－１和ＴＮＦ等，进一步增强抗肿瘤作用。转基因ＭΦ及其表达产物可使脾细胞ＮＫ活性和内源性ＬＡＫ活性增强，提高机体抗肿瘤功能，为肿瘤的细胞因子基因治疗提供了一种简便而有效的抗肿瘤新途径。     

IL-18基因转染对小鼠卵巢癌OVHM细胞免疫生物学特性的影响

目的 分析转染IL-18基因后,小鼠卵巢癌OVHM细胞的IL-18 mRNA及蛋白表达、培养上清中IL-18含量、IFN-γ诱生能力等免疫生物学特性的变化,初步探讨IL-18基因转染治疗卵巢癌的应用价值.方法 将逆转录病毒携带的小鼠活性IL-18基因转染至OVHM(OVHM/IL-18),以空载体转染的OVHM(OVHM/LXSN)和野生型OVHM作为对照.分别采用RT-PCR及免疫细胞染色法检测3组细胞的IL-18 mRNA及蛋白表达;ELISA方法测定细胞培养上清中IL-18的含量、及其诱生小鼠脾细胞产生IFN-γ的能力.结果 OVHM/IL-18细胞中可检测到IL-18 mRNA及蛋白的表达,而OVHM/LXSN和野生型OVHM细胞中未见表达.OVHM/IL-18细胞可分泌IL-18,于培养24 h时达高峰(138.25 pg/mL±12.36 pg/mL),之后逐渐减弱;OVHM/LXSN和野生型OVHM细胞上清中未检测出IL-18.0VHM/IL-18细胞培养上清诱生IFN-γ的水平明显高于VHM/KXSN和野生型OVHM细胞(分别为33.84 pg/mL±2.36 pg/mL、18.80 pg/mL±1.06 pg/mL、18.43 pg/mL±2.64 pg/mL,P<0.01),VHM/LXSN与野生型OVHM细胞之间未见显著差异(P0.05).结论 IL-18基因转染可有效提高小鼠卵巢癌细胞IL-18mRNA的表达及其蛋白的合成释放、诱导小鼠脾细胞产生IFN-γ.IL-18基因转染可纠正肿瘤细胞的IL-18低表达状态,诱导IL-18的抗瘤效应,可能对抑制肿瘤生长、促进肿瘤消退有一定的应用价值.     

小鼠Prohibitin真核表达质粒的构建及在293T细胞中的表达

目的构建小鼠Prohibitin基因真核表达质粒,在293T细胞中进行表达鉴定,为研究小鼠Prohibitin基因功能奠定基础。方法以小鼠Prohibitin基因cDNA序列为模板,设计特定引物,应用PCR的方法扩增其编码序列全长,并将其克隆到真核表达载体pEFP-C2中,将获得的重组表达质粒转染293T细胞,应用RTPCR、Western blot方法检测其表达。结果成功构建了pEGFP-Prohibitin真核表达质粒,将其成功转染293T细胞,pEGFP-Prohibitin转染组Prohibitin蛋白和mRNA在293T细胞的表达量比转染空载转染组明显增加。结论成功构建了真核表达质粒pEGFP-Prohibitin,并在真核细胞表达良好,为研究小鼠Prohibitin的基因功能奠定了基础。     

重组质粒pcDNA3-β-NGF的构建及其转染小鼠骨髓间充质干细胞生物学活性的研究*

 目的：构建人神经生长因子β亚基(β-NGF)真核表达载体,转染荧光小鼠骨髓间充质干细胞(MSCs)并研究其生物学活性。方法：全骨髓贴壁培养法分离、培养和纯化荧光小鼠MSCs,构建真核表达载体pcDNA3-β-NGF,并转染MSCs;免疫组织化学染色检测β-NGF的表达,观察β-NGF阳性的MSCs对小鼠海马神经元生长的作用。结果：荧光小鼠MSCs可发出明亮的绿色荧光,且荧光不随培养时间延长和传代次数增加而衰减。重组质粒pcDNA3-β-NGF转染MSCs后β-NGF阳性率为(37.12±2.14)%,高于MSCs组［(2.36±0.62)%］和空白对照组［(1.43±0.76)%］,差异有统计学意义(P<0.05)。pcDNA3-β-NGF转染MSCs上清液培养的新生小鼠海马神经元的突起长度［(31±3)μm］较pcDNA3转染MSCs组［(23±4)μm］明显增加,差异有统计学意义(P<0.05),表明β-NGF基因转染MSCs培养上清液中的产物能促进小鼠海马神经元的突起生长,具有明显的生物学活性。结论：成功构建了pcDNA3-β-NGF真核表达载体,其转染的荧光小鼠MSCs能正确、稳定表达和分泌有生物学活性的β-NGF。     

STOP基因慢病毒载体转染BTBR小鼠少突胶质前体细胞的方法

目的 为在体外考察微管结合蛋白STOP对孤独症谱系障碍BTBR小鼠少突胶质细胞髓鞘形成的影响,建立一种较高纯化率的BTBR小鼠大脑皮质少突胶质前体细胞的原代培养方法,利用慢病毒载体建立一种高转染效率的BTBR小鼠大脑皮质少突胶质前体细胞过表达STOP基因的转染方法。方法 以BTBR乳鼠作为实验对象,每次取6～10只,独立重复3次,0.25%胰蛋白酶消化制备成单细胞悬液,采用免疫磁珠细胞分选法获得原代少突胶质前体细胞。取原代培养的少突胶质前体细胞,利用我们前期构建的STOP基因载体进行转染实验,转染72～96 h后,在荧光显微镜下观察细胞携带荧光的显色情况。结果 采用免疫磁珠细胞分选法提取的BTBR小鼠大脑皮质原代少突胶前体细胞在48 h后基本完全贴壁,细胞生长状态好,培养第5天,免疫荧光法鉴定显示,细胞纯度极高可达95%。建立了一种转染效率较高的BTBR小鼠原代少突胶质前体细胞的慢病毒转染方法,高内涵显微镜拍摄后显示,细胞荧光表达明显,将少突胶质前体细胞的慢病毒转染率提高到60%～70%。结论 成功分选培养获得纯度较高的BTBR小鼠大脑皮质原代少突胶质前体细胞,成功建立了一种转染率较高...     

Protein kinase A regulates cell cycle progression of mouse fertilized eggs by means of MPF.

Cell cycle of one-cell stage mouse fertilized eggs was accompanied by fluctuation in the concentration of adenosine 3'5'-monophosphate (cAMP) and in the activity of free catalytic subunit of cAMP-dependent protein kinase (PKA). The concentration of cAMP and the activity of free catalytic subunit of PKA decreased at the onset of mitosis and increased at the transition between mitosis and G1 phase. Stimulation of PKA by microinjection of cAMP into one-cell stage mouse embryos at G2 phase induced interphase arrest and prevented the activation of M-phase promoting factor (MPF). Upon blockage of the activation of PKA by microinjecting a thermostable PKA inhibitor (PKI) into one-cell stage mouse embryos at G2 phase, the increase in the MPF activity occurred 30 min earlier than in control group. When a high dose of PKI was microinjected, a transition into interphase was prevented, and the activity of MPF remained high. Western blot analysis showed that Cdc2 remained phosphorylated in cAMP microinjected embryos by the time when control embryos were at metaphase and showed dephosphorylated Cdc2; conversely, Cdc2 dephosphorylation was accelerated in PKI-microinjected embryos. At the same time, Cdc2 was phosphorylated at Tyr15 at G2 phase and even at M phase when cAMP was microinjected but was dephosphorylated when PKI was microinjected. PKI microinjection also prevented cyclin B degradation and sustained MPF activity, thus delaying the transition from metaphase to anaphase. Our results show that PKA, by inhibiting MPF, regulates cell cycle progression of fertilized eggs.     

The gnu mutation of Drosophila causes inappropriate DNA synthesis in unfertilized and fertilized eggs

Drosophila melanogaster embryos whose mothers are homozygous for the maternal effect lethal mutation gnu (GNU embryos) under DNA synthesis but no nuclear division; this leads to the formation of a small number of giant nuclei in the syncytial blastoderm. We have shown previously that many components of the mitotic apparatus are present and functional in GNU embryos, and the primary lesion of the gnu mutation has therefore remained obscure. Here, we report that fertilization is not necessary for GNU eggs to develop. Giant nuclei originate from the products of female meiosis, and we see autonomously replicating centrosomes that must be maternally derived. If GNU eggs are inseminated, however, the male pronucleus also undergoes DNA replication. Our observations suggest that the GNU cytoplasm permits DNA synthesis in a relatively unregulated manner. In embryos from females homozygous for gnu and the female sterile haploid mutation mh, we find replication of DNA derived from the male pronucleus. This contrasts with embryos from mothers homozygous for mh alone, in which this does not occur. We propose that the gnu gene product participates in the repression of DNA synthesis found in unfertilized eggs.     

Generation of an antibody specific to Xenopus fertilized eggs by subtractive immunization

Here we report the generation and characterization of a monoclonal antibody, mAb 5H7-G1, which recognizes egg antigens in the animal cortex of fertilized, but not unfertilized, Xenopus eggs. The mAb 5H7-G1 was generated by subtractive immunization of mice: primary immunization with unfertilized egg extract followed by immunosuppression treatment with cyclophosphamide and repeated immunization with fertilized egg extract. In immunoblotting analysis, mAb 5H7-G1 recognizes multiple protein bands of fertilized (but not unfertilized or the ionophore-activated) Xenopus eggs. N-linked polysaccharide is most likely the target of mAb 5H7-G1 because immunoreactivity of mAb 5H7-G1 is effectively diminished when protein samples are treated with N-glycosidase F. Moreover, mAb 5H7-G1 recognizes some, but not all, tyrosine-phosphorylated proteins in eggs treated with H2O2, an artificial activator of the egg tyrosine kinase Src, suggesting that these proteins also contain N-linked sugars. When microinjected into fertilized Xenopus embryos, mAb 5H7-G1 causes a retardation or complete inhibition of first cell cleavage, suggesting that the mAb 5H7-G1-reactive antigens play an important role in this event. These results demonstrate that mAb 5H7-G1 is useful to analyze differential proteome display during fertilization and early development. More generally, subtractive immunization may work as a strategy to uncover cellular events that operate during different cellular conditions of interest.     

Mucosal delivery of human papillomavirus pseudovirus-encapsidated plasmids improves the potency of DNA vaccination

Mucosal immunization may be important for protection against pathogens whose transmission and pathogenesis target the mucosal tissue. The capsid proteins of human papillomavirus (HPV) confer tropism for the basal epithelium and can encapsidate DNA during self-assembly to form pseudovirions (PsVs). Therefore, we produced mucosal vaccine vectors by HPV PsV encapsidation of DNA plasmids expressing an experimental antigen derived from the M and M2 proteins of respiratory syncytial virus (RSV). Intravaginal (IVag) delivery elicited local and systemic M–M2-specific CD8+ T-cell and antibody responses in mice that were comparable to an ∼10,000-fold higher dose of naked DNA. A single HPV PsV IVag immunization primed for M–M2-specific-IgA in nasal and vaginal secretions. Based on light emission and immunofluorescent microscopy, immunization with HPV PsV-encapsidated luciferase- and red fluorescent protein (RFP)-expressing plasmids resulted in transient antigen expression (<5 days), which was restricted to the vaginal epithelium. HPV PsV encapsidation of plasmid DNA is a novel strategy for mucosal immunization that could provide new vaccine options for selected mucosal pathogens.     

Gene delivery via DNA incorporation within a biomimetic apatite coating

Integrating inductivity with conductivity in a material may advance tissue engineering. An organic/inorganic hybrid was developed by incorporating plasmid DNA encoding for the β-gal gene complexed with Lipofectamine 2000^® (DNA–Lipoplex) within apatite via coprecipitation. It was hypothesized that this system will result in enhanced transfection efficiency compared to DNA–Lipoplexes adsorbed to the mineral surface and DNA coprecipitated without Lipofectamine 2000^®. PLGA films were cast onto glass slips and apatite and DNA were coprecipitated in modified simulated body fluid (mSBF). DNA–Lipoplex presence in mineral, DNA–Lipoplex stability (vs. coprecipitation time), and transfection efficiency (determined with C3H10T1/2 cells) as a function of coprecipitation time, DNA–Lipoplex concentration, and DNA incorporation method were studied. DNA–Lipoplex presence and spatial distribution on apatite were confirmed through fluorescence. Transfection efficiency was highest for 6 h of DNA–Lipoplex coprecipitation. Differences in transfection efficiency were found between the DNA concentrations, with the highest efficiency for coprecipitation being 40 μg/ml (p ≤ 0.009 relative to other coprecipitation concentrations). Significant differences in transfection efficiency existed between incorporation methods (p < 0.05) with the highest efficiency for DNA–Lipoplex coprecipitation. This hybrid material system not only integrates inductivity provided by the DNA and conductivity provided by the apatite, but it also has significant implications in non-viral gene delivery due to its ability to increase transfection efficiency.     

Eosinophilia in mouse regulated by multiple factors following repeated stimulations with ascaris eggs into the peritoneal cavity

Repeated injections of viable eggs of Ascaris suum into the peritoneal cavity of BALB/c mice induced marked peritoneal eosinophilia (9.3 X 10(6) cells/mouse, 52.8%) without infection. Extracted antigen (Asc) and eggs that had been killed also were able to induce marked eosinophilia; however, their levels were significantly lower (p less than 0.001). This experimental system was able to demonstrate how various multiple factors cumulatively affected the degree of peritoneal eosinophilia. These factors included: mechanical stimulation, caused by the repeated peritoneal lavages (4.4 X 10(5) cells/mouse), against a background level of peritoneal eosinophils of 4.5 X 10(4) cells/mouse; T-cell-independent stimulation, observed when the viable eggs were injected repeatedly into athymic nude mice (nu/nu; 1.6 X 10(6) cells/mouse), while on the other hand, neither eggs that had been killed nor Asc displayed any T-cell-independent augmentation of eosinophilia; and, T-cell-dependent augmentation of peritoneal eosinophilia, observed in heterozygous athymic mice (nu/+), in thymocyte-reconstituted nu/nu, and in normal BALB/c mice. Egg culture supernatant could not act on bone marrow cells to support eosinophil colony formation. In conclusion, only viable eggs were considered prime inducers, consisting of T-independent and T-dependent parts exerting cumulative effects.     

Nuclear transfer alters the DNA methylation status of specific genes in fertilized and parthenogenetically activated mouse embryonic stem cells

Recent cloning technology has been demonstrated successfully using nuclear transfer (NT) techniques to generate embryonic stem (ES) cells. Mice can be cloned from adult somatic cells or ES cells by NT, and such cloned embryos can be used to establish new NT-ES cell lines. However, ES cells derived from parthenogenetic embryos show epigenetic disorders and low potential for normal differentiation unless used to produce subsequent generations of NT-ES lines. Thus, enucleated oocytes can initialize epigenetic modification, but the extent and efficacy of this remain unclear. In this study, our goal was to clarify why the contribution rate of ES cells derived from parthenogenetic embryos (pES) cells appears to improve after NT. We compared gene expression profiles between pES and NT-pES cell lines using DNA microarray analysis and allele-specific DNA methylation analysis. Although changes in expression level were observed for 4% of 34,967 genes, only 81 (0.2%) showed common changes across multiple cell lines. In particular, the expression level of a paternally expressed gene, U2af1-rs1, was significantly increased in all NT-pES cell lines investigated. The methylation status at the upstream differentially methylated region of U2af1-rs1 was also changed significantly after NT. This was observed in NT-pES cells, but also in conventionally produced NT-ES cells, which has never been reported previously. These results suggest that NT affects the epigenetic status of a few gene regions in common and that a change in the methylation status of U2af1-rs1 could be used as a genetic marker to investigate the effects of NT.