成纤维细胞静脉注射途径实施血友病B基因治疗的初步探索

将转有报告基因ｌａｃＺ的成纤维细胞３Ｔ３／ＢＡＧ以５×１０＾５量尾静脉注射到Ｂａｌｂ／ｃ小鼠体内，７ｄ后在主要脏器检测到３ＴＥ／ＢＡＧ的表达且至少可持续３０ｄ。将转有人凝血因子Ⅸ小基因的正向表达载体Ｇ１ＮａＣｉ’Ⅸ和反向表达载体Ｇ１ＮａＣｉ’ⅨＲ的成纤维细胞ＰＡ３１７／Ｇ１ＮａＣｉ’Ⅸ和ＰＡ３１７／Ｇ１ＮａＣｉ’ⅨＲ分别以１×１０＾５量尾静脉注射到Ｂａｌｂ／ｃ小鼠体内，ＥＬＩＳＡ测定小鼠血浆中ｈＦ     

电击法转移含人凝血因子IX基因重组质粒的影响因素

反转录病毒载体在基因治疗中可能会产生野生型病毒颗粒而引起安全性问题，本文研究含ＦＩＸｃＤＮＡ重组质粒基因治疗血友病Ｂ的可能，构建了不具反转录病毒载体结构的两重组质粒ｐＳＣＩＸＴＮ和ｐＣＩＸＴＮ，前者合ＳＶ４０早期启动子和ｈＣＭＶ启动子共同控制的ＦＩＸｃＤＮＡ，后者仅含ｈＣＭＶ启动于控制的ＦＩＸｃＤＮＡ，它们都含有ＴＫ启动于驱动的ｎｅｏ基因，通过电击法将基因转移到ＰＡ３１７和ＨＴ１０８０细胞，在ＨＴ１０８０细胞中的ＦＩＸ表达量分别为２２０、２１２ｎｇ／（１０６细胞·２４ｈ）通过与ｐＣＭＶＩＸ共转染靶细胞，可以增加人ＩＸ因子表达１．５～３．５倍，显示了用质粒载体转染靶细胞在血友病Ｂ基因治疗中是一条潜在可行的途径．本项研究用自制的电击仪转移ＰＡ３１７和ＨＴ１０８０等细胞，最高的转移效率达１０－３，并探讨了细胞种类，ＤＮＡ量，ＤＮＡ结构，电压，脉冲时间与转移效率及表达量的关系．     

HARMONIC AC SUSCEPTIBILITY FOR TEXTURED YBa_2Cu_3O_7

ＨＡＲＭＯＮＩＣＡＣＳＵＳＣＥＰＴＩＢＩＬＩＴＹＦＯＲＴＥＸＴＵＲＥＤＹＢａ２Ｃｕ３Ｏ７ＧｅＹｏｎｇ１）ＤｉｎｇＳｈｉＹｉｎｇ２）（１）ＤｅｐａｒｔｍｅｎｔｏｆＰｈｙｓｉｃｓ,ＮａｔｉｏｎａｌＬａｂｏｆＳｏｌｉｄＳｔａｔｅＭｉｃｒｏｓｔｒｕｃｔｕｒ...     

电击法转移含人凝血因子Ⅸ基因重组质粒的影响因素

反转录病毒载体在基因治疗中可能会产生野生型病毒而引起安全性问题，本文研究含ＦＩＸｃＤＮＡ重组质粒基因治疗血友病Ｂ的可能，构建了不具反转录病毒载体结构的两重组质粒ｐＳＣＩＸＴＮ和ｐＣＩＸＴＮ，前者含ＳＶ４０早期启动子和ｈＣＭＶ启动子共同控制的ＦＩＸ ｃＤＮＡ，后者仅含ｈＣＭＶ启动子控制的ＦＩＸｃＤＮＡ，它们都含有ＴＫ启动子驱动的ｎｅｏ基因，通过电击法将基因转移到ＰＡ３１７和ＨＴ１０８０细胞，在ＨＴ１     

关于矩阵方程∑(from i=1 to n) A_iX_iB_i=F的可解性

本文考虑含有ｎ个未知矩阵Ｘ１,Ｘ２,Ｘ３,…,Ｘｎ的非齐次矩阵方程：Ａ１Ｘ１Ｂ１＋Ａ２Ｘ２Ｂ２＋…＋ＡｎＸｎＢｎ＝Ｆ,（１）其中Ａｉ∈Ｃｍ×ｌｉ,Ｂｉ∈Ｃｐｉ×ｑ,Ｘｉ∈Ｃｌｉ×ｐｉ,Ｆｉ∈Ｃｍ×ｑ．我们先给出一个求解的方法;然后利用矩阵的拉直运算和...     

SiC2分子电子光谱的ab initio研究

在ＭＲＳＤＣＩ／ＤＺ＋Ｐ水平上，计算了ＳｉＣ２分子７个电子态Ｘ＾１Ａ１，＾３Ｂ２，＾１Ｂ２，＾３Ｂ１，＾１Ｂ１，＾３Ａ２和１＾Ａ２的平衡构型和激发能。所得Ｘ＾１Ａ２和＾１Ｂ２态的平衡构型以及Ｘ＾１Ａ２→＾１Ｂ２的激发能和实验值符合很好。     

NEPE推进剂中可提取有机组分的分离测定

研究了ＮＥＰＥ推进剂中可提取有机组分（ＨＭＸ，Ｃ２和ＮＧ＋ＢＴＩＮ）的分离和测定方法．试样用ＴＨＦ浸泡２４ｈ后，在５０℃搅拌５ｈ．提取液采用正相高效液相色谱（ＮＰＨＰＬＣ）分离、测定ＨＭＸ，Ｃ２和混合酯．ＨＭＸ，Ｃ２和ＮＧ＋ＢＴＴＮ测定的相对标准偏差分别为０．３１％，１．２０％和２．１９％．     

MAGNETIC RELAXATION AT EARLY TIMES AND FLUX DIFFUSION BARRIER V（J，B，T） FOR Ti－1223 DOPED WITH Pb AND Ba BY COMPLEX AC SUSCEPTIBILITY MEASUREMENTS

ＭＡＧＮＥＴＩＣＲＥＬＡＸＡＴＩＯＮＡＴＥＡＲＬＹＴＩＭＥＳＡＮＤＦＬＵＸＤＩＦＦＵＳＩＯＮＢＡＲＲＩＥＲＶ（Ｊ，Ｂ，Ｔ）ＦＯＲＴｉ－１２２３ＤＯＰＥＤＷＩＴＨＰｂＡＮＤＢａＢＹＣＯＭＰＬＥＸＡＣＳＵＳＣＥＰＴＩＢＩＬＩＴＹＭＥＡＳＵＲＥＭＥＮＴＳＤ...     

ON THE EXPONENT SET OF PRIMITIVE LOCALLY SEMICOMPLETE DIGRAPHS

ＯＮＴＨＥＥＸＰＯＮＥＮＴＳＥＴＯＦＰＲＩＭＩＴＩＶＥＬＯＣＡＬＬＹＳＥＭＩＣＯＭＰＬＥＴＥＤＩＧＲＡＰＨＳ￥ＺｈａｎｇＫｅｍｉｎｇ１）；ＢｕＹｕｅｈｕａ２）（１）ＤｅｐａｒｔｍｅｎｔｏｆＭａｔｈｃｍａｔｉｅｓ，ＮａｎｊｉｎｇＵｎｉｖｅｒｓｉｔｙ，Ｎ...     

TECTONIC DEVELOPMENT OF THE METAMORPHIC CORE COMPLEX OF THE WUGONGSHAN IN THE NORTHERN JIANGXI PROVINCE

ＴＥＣＴＯＮＩＣＤＥＶＥＬＯＰＭＥＮＴＯＦＴＨＥＭＥＴＡＭＯＲＰＨＩＣＣＯＲＥＣＯＭＰＬＥＸＯＦＴＨＥＷＵＧＯＮＧＳＨＡＮＩＮＴＨＥＮＯＲＴＨＥＲＮＪＩＡＮＧＸＩＰＲＯＶＩＮＣＥＳｕｎＹａｎ１）ＳｈｕＬｉａｎｇｓｈｕ１）Ｍ．Ｆａｕｒｅ２）Ｊ．Ｃｈａ...     

Construction and expression of inverted configuration of retroviral vector containing intron 1 of hFIX

Intron was found to play an important role in improving gene expression. To improve the human factor IX(hFIX) expression level in hemophilia B gene therapy study, the retroviral vector containing intron 1 of hFIX gene was constructed in forwarded configuration, but the intron 1 was found spliced in virus particles by RT_PCR detection. So the inverted configuration vector G1NaPAi′IX was suggested and constructed on the basis of SNMBAIXm and transfected into PA317. Then C2C12 cells were transfected using the above virus supernatant and the G418_resistant clones were selected. PCR and RT_PCR detection found that intron 1 structure existed in C2C12 clones and retroviral particles. And the expression level of inverted vector was 3 times higher than that of forwarded vector. These results showed that the inverted configuration vector was in deed able to avoid splicing of intron 1 during the process of retroviral packaging and improved the expression level of hFIX protein.     

RNA干扰沉默PID1基因在C2C12细胞中表达的研究

 构建和筛选对PID1（phosphotyrosine interaction domain containing 1, PID1）基因有RNA干扰作用的PID1-shRNA表达载体。据小鼠PID1 cDNA序列,优化设计了4条shRNA及1条阴性干扰序列,插入pGPU6/GFP/Neo载体中,得到pGPU6/GFP/Neo-PID1-1、pGPU6/GFP/Neo-PID1-2、pGPU6/GFP/Neo-PID1-3、pGPU6/GFP/Neo PID1 4和pGPU6/GFP/Neo-PID1-NC。干扰载体转染C2C12细胞,以RT-PCR和Western blot技术检测shRNA对C2C12细胞中PID1 mRNA和蛋白表达的下调作用。结果表明：靶向PID1基因的4个shRNA重组质粒载体经测序分析,其shRNA编码序列与预期设计的完全一致,经酶切鉴定和测序分析证实,靶向PID1基因的shRNA重组质粒载体构建成功。进一步将构建的4个表达载体分别转染C2C12细胞,24h后细胞中PID1基因mRNA表达水平依次下调 (23.58±1.87)%、(75.44±0.77)%、(70.52±0.41)% 和 (56.60±3.13)%。48 h后细胞中PID1蛋白表达水平依次降低 (30.15±5.05)%、(71.86±4.85)%、(67.93±2.28)% 和 (56.81±2.01)%。所筛选出的pGPU6/GFP/Neo-PID1-2、pGPU6/GFP/Neo-PID1-3和pGPU6/GFP/Neo-PID1-4三个表达载体均能高效地抑制转染细胞PID1 mRNA和蛋白的表达,为进一步研究PID1基因的功能奠定了基础。     

汉滩病毒受体与细胞因子TNFRⅠ真核表达载体的构建及稳定转染细胞系的建立

摘要 目的：构建带有荧光标签的汉滩病毒受体整合素β3与细胞因子TNFRⅠ的稳定转染细胞系。方法：构建目的基因的真核表达载体pIRES2-EGFP-β3和pIRES2-DsRed-TNFRⅠ(EC),并转染CHO细胞,直接荧光观察报告基因和荧光定量RT-PCR检测目的基因mRNA表达。结果：转染重组质粒pIRES2-EGFP-β3的细胞可检测到荧光报告基因和目的基因β3 mRNA的表达,转染pIRES2-DsRed-TNFRⅠ(EC)的细胞可检测到荧光报告基因和目的基因TNFRⅠmRNA的表达。结论：成功构建真核表达整合素β3与TNFRⅠ的稳定细胞系。     

High expression of EPO gene using un-dhfr negative cell

Erythropoietin (EPO) genomic gene was cloned and its expression vector pOP13/EPO was constructed. CHO_K12 cell was transfected by this vector using lipofectin method. A stable expression cell strain C10 cell with the EPO production at 160IU/d in 10\+6 cells were obtained at 400 μg/mL G418. Based on the C10 cell, another vector pHY/dhfr (dihydrofolate reductase) that carries a dhfr gene and a selecting marker of hygromycin B resistant gene was transferred to this cell. Several cell clones were obtained at 200 μg/mL hygromycin B. These cell clones that can express both EPO gene and exogenous dhfr gene were selected under the progressively increased concentration to 1 μmol methotrexate(MTX). Some high EPO expression cell clones were obtained, the highest expression was 2 400 IU/d in 10\+6 cells, 15 times higher than that without MTX pressure. Then, a method of EPO high expression by using un_dhfr negative cell was primarily established. EPO bioactivity was found by using TF_1 cell.     

Identification of a bi-directional promoter from Tomato yellow leaf curl China virus

A bi-directional promoter of Tomato yellow leaf curl China virus (TYLCCNV) was obtained with the total DNA from TYLCCNV isolate Y10 infected tobacco leaves as a template. Plant expression vectors were constructed by fusing the amplified DNA fragment with the gus gene and nopaline terminator in different orientations. The vectors containing promoter fragments were transferred into leaf cells and plant stems of Nicotiana benthamiana by Agrobacterium-mediated method. Transient expression results showed that both the complementary and virion-sense promoters could drive the gus gene to express, and the GUS activity of the complementary-sense promoter was stronger than that of the virion-sense. Co-expression of the vector containing βC1 gene of TYLCCNV DNAβ with the vector containing a bi-directional promoter revealed that the βC1 protein has no impact on expression of either the virion- or the complementary-sense promoter.     

Preparation of rAAV/hFⅨ by HSV/AAV hybrid helper virusand evaluation of its safety

Hemophilia B is a hemorrhagic disease resulting from Factor Ⅸ gene (hFⅨ) mutation as an X-linked recessive inherited trait. The incidence of this disease is 1 in 30000. Clinical treatments depend mainly upon blood transfusions or administration of prothrombin complex so that patients are at the risk of infections with the HIV and hepatitis viruses. Gene therapy offers an attractive alternative in the treatment of hemophilia B by eliminating those risks. In 1991, our lab conducted clinica…     

草鱼生长激素基因在COS7细胞中转染表达的研究

应用RT-PCR技术克隆草鱼生长激素(gcGH)的cDNA,将此cDNA定向插入真核表达载体VR1020中,构建成重组真核表达质粒VgcGH.利用脂质体法使质粒VgcGH转染哺乳动物细胞COS7,对转染后的COS7细胞进行RT-PCR、ELISA和免疫荧光检测,分别在转录和翻译水平证实gcGH基因在COS7细胞中得到了持续和正确的转染表达.     

MiR-218慢病毒表达载体的构建及鉴定

摘要:构建人mir-218-2, pre-miRNA慢病毒表达载体,为研究miR-218在人体的功能及作用机制打下基础。以人hsa-mir-218-2前体序列,设计部分互补的正反向引物,进行引物退火,形成引物二聚体,PCR扩增引物二聚体,酶切后插入到线性化pGCSIL-GFP慢病毒表达载体中,对重组质粒进行双酶切鉴定,并进行慢病毒的包装与滴度检测。用构建好的慢病毒表达载体感染人胃癌细胞MKN-28,qPCR检测细胞内miR-218表达。结果显示,重组质粒经双酶切分析及转化菌液测序,插入序列正确,慢病毒表达载体感染人胃癌细胞后qPCR检测显示能显著增高miR-218的表达。说明本实验成功构建了hsa-mir-218-2慢病毒表达载体,感染人胃癌细胞后能有效提高miR-218的表达。为进一步研究miR-218在人体的功能及作用机制建立了实验基础。     

Efficient expression of human factor Ⅸ cDNA in liver mediated by hydrodynamics-based plasmid administration

Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor IX (hFIX) cDNA in 2.2 mL Ringer‘s solution into mice within 7 s. The peak level of expression of hFIX was 2921 ng/mL in mouse plasma. The hFIX cDNA expression increased with increasing the amount of plasmid DNA injected. The peak level of gene expression declined after repeated injection of plasmid (1459 ng/mL). The hFIX cDNA was detected in various organs, but the highest level of gene expression appeared in liver. Transaminase levels and liver histologicalresults showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage, which was rapidly repaired within 3--10 d. These results suggested that high-level expression of hFIX cDNA can be achieved by hydrodynamics-based plasmid transfer and this method is nowfurther used for gene therapy and gene function study in our lab.     

甲醇酵母表达系统pMIRH型载体研究

在为甲醇酵母（Ｈａｎｓｅｎｕｌａｐｏｌｙｍｏｒｐｈａ）表达系统成功构建高拷贝整合型表达载体ｐＭＩＲＨ－１的基础上,构建了更能满足外源基因表达需要的、大小仅为７３００ｂｐ的高拷贝整合型表达载体ｐＭＩＲＨ－２．有关转化、筛选及传代稳定性试验结果表明,ｐＭＩＲＨ－２亦具有与ｐＭＩＲＨ－１相似的转化和筛选特性及高拷贝整合能力,且二者均可在非选择条件下稳定传代６０代以上．