SCL基因重组慢病毒对DCP膀胱组织中ICC形态学变化的探究

发布时间：2018-02-11 21:42

本文关键词： 干细胞白血病基因发慢病毒 Cajal样间质细胞糖尿病膀胱病　出处：《石河子大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:1.观察含绿色荧光蛋白(GFP)基因的干细胞白血病基因(SCL)重组慢病转染体外高糖环境下膀胱Cajal样间质细胞的形态学变化。2.观察经尿道灌注含绿色荧光蛋白(GFP)基因的SCL基因重组慢病毒转染豚鼠DCP(diabetic cystopathy,DCP)膀胱对Cajal样间质细胞的形态学影响。方法:1.构建SCL基因重组慢病毒且标定滴度;2.体外分离培养豚鼠膀胱ICC(interstitial cells of Cajal,ICC),分别用5mmol/L,15mmol/l和25mmol/l葡萄糖浓度处理24小时,然后每个浓度下分为空白对照组(加1%PBS),空慢病毒对照组(加空慢病毒),慢病毒转染组(加携带人SCL基因慢病毒),转染2d、3d、5d时,用激光共聚焦显微镜观测各组ICC的状态和数量变化及GFP表达。3.构建DCP豚鼠模型;观察经尿道灌注SCL基因重组慢病毒对豚鼠DCP膀胱组织中ICC数量、分布及超微结构的影响;结果:1.病毒滴度测定是5×108TU/ml;2.显微镜下可见典型的膀胱ICC形态及高葡萄糖浓度(15 mmol/L和25 mmol/L)受损的细胞形态,免疫荧光法可见特异性c-kit受体成功表达;慢病毒转染成功导入SCL基因至ICC中;在15 mmol/L葡萄糖浓度下,SCL基因的导入可陆续恢复形态损伤的ICC。在25 mmol/L葡萄糖浓度下,能够改善却没能恢复形态损伤的ICC,此外,SCL基因未使ICC数量得到恢复或增殖。3.糖尿病豚鼠诱导成功率为47.7%;诱导DCP豚鼠27只;激光共聚焦显微镜下观察发现:实验组中随7d、14d、28d时间的延长,ICC数量逐渐增多(P0.05),分布逐渐密集,绿色荧光逐渐减弱。空白对照组和阳性对照组中随7d、14d、28d时间的延长,ICC数量逐渐减少(P0.05),分布稀疏,因无GFP表达,未见绿色荧光;透射电镜下观察发现:实验组中随7d、14d、28d时间的延长,ICC的细胞器数量增多、形态趋于正常、细胞内空泡减少、胞周突起延长变多,受损的ICC的超微结构在不断的恢复之中。空白对照组和阳性对照组中随7d、14d、28d时间的延长,ICC的胞器数量减少、形态不规则、胞内空泡样变增多、胞周突起稀少,ICC的超微结构未见改善。结论:1.SCL基因重组慢病毒载体转染体外高糖环境下培养的ICC,可将SCL基因导入ICC中,能够恢复或改善受损的ICC形态。2.经尿道灌注SCL基因重组慢病毒转染豚鼠DCP膀胱后,对ICC形态和数量的恢复有一定收效,对DCP达到一定程度的治疗效果。能进一步为使用SCL基因重组慢病毒对DCP的基因治疗提供思路。
[Abstract]:Objective 1. To observe the morphological changes of bladder Cajal like interstitial cells transfected with recombinant stem cell leukemia gene (SCL) containing green fluorescent protein (GFP) gene in vitro. 2. To observe the transurethral perfusion of GFP gene containing green fluorescent protein (GFP). The effects of recombinant lentivirus of SCL gene on the morphology of Cajal-like interstitial cells in guinea pig DCP(diabetic cystopathyte) bladder. Methods 1. Construction of recombinant lentivirus of SCL gene and calibration titer 2. Isolation and culture of ICC(interstitial cells of Cajalalus in vitro, 5 mmol / L / 15 mmol / L and 25 mmol / 1, respectively. Glucose concentration was treated for 24 hours. Then each concentration was divided into two groups: blank control group (add 1 PBSX), empty lentivirus control group (plus empty lentivirus), lentivirus transfection group (plus lentivirus carrying human SCL gene), transfection of human SCL gene lentivirus for 3 days or 5 days after transfection, The changes of ICC and the expression of GFP in each group were observed by confocal laser microscope. The DCP guinea pig model was established, and the effects of transurethral perfusion of SCL gene recombinant lentivirus on the quantity, distribution and ultrastructure of ICC in DCP bladder were observed. Results 1. The virus titer was 5 脳 108 TU / ml 2.The typical ICC morphology of bladder and the damaged cells with high glucose concentration of 15 mmol/L and 25 mmol / L were observed under microscope, and the specific c-kit receptor was successfully expressed by immunofluorescence. SCL gene was successfully transfected into ICC by lentivirus transfection, and the morphological damage could be recovered at 15 mmol/L glucose concentration. The number of ICC was not recovered or proliferated by ICC gene. The successful rate of induction was 47.7% in diabetic guinea pigs and 27 in DCP guinea pigs. The results of laser confocal microscopy showed that the number of ICC increased gradually with the extension of 14 d and 28 d in the experimental group, and the distribution of ICC increased gradually. The number of GFP decreased gradually in the blank control group and the positive control group with the extension of 14d ~ 28d, and the distribution was sparse. There was no GFP expression and no green fluorescence was found in the control group. The results of transmission electron microscope showed that the number of organelles increased, the morphology of ICC tended to be normal, the vacuoles decreased, and the pericellular processes became longer in the experimental group with the extension of 14d ~ 28d. The ultrastructure of the damaged ICC was recovering continuously. The number of organelles in the control group and the positive control group decreased with the prolongation of 14d ~ 28d, and the morphology was irregular, and the vacuolation became more and more in the control group and the positive control group. Conclusion: 1. The recombinant lentivirus vector of SCL gene can be transfected into ICC in high glucose environment, and the SCL gene can be transferred into ICC. After transfection of SCL gene recombinant lentivirus into guinea-pig DCP bladder, the morphology and quantity of ICC recovered to some extent. The therapeutic effect on DCP can be achieved to a certain extent, which can further provide ideas for the gene therapy of DCP using SCL gene recombinant lentivirus.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R694

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