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OBJECTIVE: To investigate liposome-mediated cytosine deaminase gene transfecting rabbit BMSCs and its gene expression.

目的:探讨脂质体介导胞嘧啶脱氨酶基因转染兔骨髓间充质干细胞及其基因的表达。

OBJECTIVE: To investigate transfection and expression of liposome-mediated cytosine deaminase gene in rabbit BMSCs.

目的:观察脂质体介导胞嘧啶脱氨酶基因转染兔骨髓间充质干细胞及其表达。

By the electropositive Liposome 2000, in the 6-plate for cell culture, pEGFP-P80 above is transfected into the PK-15 cells from the kidney of pigs and expressed.

利用阳性脂质体L2000 介导,在六孔细胞培养板中,将已经构建好的pEGFP-P80重组表达质粒转染到PK-15 细胞中进行表达。

Methods Eukaryotic expression plasmid pCMVCD was constructed , and identified by re2 striction endoenzyme digestion. CD gene was transfected into NSCs from new2born Wistar rats using Lipofec2tamine2000. Positive clones (named NSCs/ CD cells) were screened by G418 presence. 52Fluorocytosine (52FC) ad2ministration of different concentrations were incubated with NSCs/ CD cells. NSCs/ CD cells viability ratios were mea2 sured by MTT assay.

通过构建真核表达质粒pCMVCD ,限制性内切酶消化鉴定后,采用Lipofectamine 2000 脂质体介导法转染新生大鼠室管膜下区神经干细胞(Neural stemcells , NSCs),G418 筛选阳性克隆,加入不同浓度的52氟胞嘧啶(52Flourocytosine , 52FC),MTT比色法测定NSCs的生存率。

An MFGM-derived phospholipid fraction was used to produce liposomes via a high-pressure homogenizer.

一个MFGM源性磷脂小部分被用来制造脂质体经高压均质。

Methods A two stage pressure homogenizer was used to prepare Venenum Dufonis lipid microsphere for injection. The effects of oil phase,emulsifier,pH and sterilization method on the stability of lipid microsphere were investigated separately.

采用两级高压均质法制备蟾酥脂质微球注射液,分别考察了油相组成、乳化剂用量、pH、灭菌方式对制剂灭菌稳定性的影响。

Methods:Marrow stromal cells were obtained from the dual ilium of three adult dogs.

从狗的髂骨穿刺骨髓分离骨髓基质细胞,应用脂质体转染VEGF165基因,以RT-PCR检测转染的基因表达。

Rat glioma cell C6 was infected by the deficiency recombinant retrovirus (named C6TK). The sensitivity of C6TK cells to GCV or ACV increased 450 or 10 times than that of C6 cells respectively. Rat C6 glioma model was successfully built, and SD rats inoculated intracerebrally C6TK cells had normal gliomas. The average survival periods of the rats were about 15 days. However, after treated with ACV, the growth of C6TK tumors obviously regressed, and the survival periods extended to 57.8±8.1 days, especially in rats injected with mixed PA317TK and C6 cells or in situ PA317TK cells, which the tumors nearly disappeared after ACV administration and the survival periods extended to over 120 or 71.4±36.1 days respectively, P<0.001.

结果:构建了带有HSV-tk基因的反转录病毒载体GINaTK,应用脂质体转移方法将GINaTK导入反转录病毒包装细胞PA317,成为产病毒细胞PA317TK,用带有HSV-tk基因的复制缺陷型反转录病毒感染C6细胞,命名为C6TK细胞,对GCV和ACV的敏感性分别高于亲本450倍和10倍;成功地建立了大鼠脑胶质瘤模型,并证实转染细胞C6TK的成瘤效应未改变,存活期约为15天;而经ACV治疗后,含有C6TK细胞的肿瘤生长明显被抑制,大鼠生存期延长为57.8±8.07天,尤其是采用PA317TK细胞混和治疗组和原位治疗组,肿瘤基本消失,大鼠生存期显著延长,混和治疗组存活120天以上,原位治疗组存活至71.4±36.1天。t检验,P值均小于0.001。

Results: GINaTK retroviral vector containing HSV-tk was constructed and transduced into PA317 retrovirus packaging cell by lipofectin (named PA317TK). Rat glioma cell C6 was infected by the deficiency recombinant retrovirus (named C6TK). The sensitivity of C6TK cells to GCV or ACV increased 450 or 10 times than that of C6 cells respectively. Rat C6 glioma model was successfully built, and SD rats inoculated intracerebrally C6TK cells had normal gliomas. The average survival periods of the rats were about 15 days. However, after treated with ACV, the growth of C6TK tumors obviously regressed, and the survival periods extended to 57.8±8.1 days, especially in rats injected with mixed PA317TK and C6 cells or in situ PA317TK cells, which the tumors nearly disappeared after ACV administration and the survival periods extended to over 120 or 71.4±36.1 days respectively, P <0.001.

结果:构建了带有HSV-tk基因的反转录病毒载体GINaTK,应用脂质体转移方法将GINaTK导入反转录病毒包装细胞PA317,成为产病毒细胞PA317TK,用带有HSV-tk基因的复制缺陷型反转录病毒感染C6细胞,命名为C6TK细胞,对GCV和ACV的敏感性分别高于亲本450倍和10倍;成功地建立了大鼠脑胶质瘤模型,并证实转染细胞C6TK的成瘤效应未改变,存活期约为15天;而经ACV治疗后,含有C6TK细胞的肿瘤生长明显被抑制,大鼠生存期延长为57.8±8.07天,尤其是采用PA317TK细胞混和治疗组和原位治疗组,肿瘤基本消失,大鼠生存期显著延长,混和治疗组存活120天以上,原位治疗组存活至71.4±36.1天。t检验,P值均小于0.001。

It is reported that locust lipids are well worth researching and utilizing based on their quantity and quality.

据有关资料显示,蝗虫脂质无论在量上还是在质上都有其可资研究和利用的特点。

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