经尿道灌注干细胞白血病基因慢病毒转染豚鼠糖尿病膀胱病变的效果研究

摘要

Objective To prepare guinea pig model of diabetic cystopathy( DCP)of bladder and transurethrally perfuse stem cell leukemia(SCL)gene lentivirus in order to evaluate the transfection efficiency and stability. Methods From November 2014 to June 2015,a total of 60 guinea pigs were selected from 75 Holland male guinea pigs by random number table method and given streptozotocin(STZ)(200 mg/ kg)by a single intraperitoneal injection. The models of diabetic were screened according to the blood glucose level > 11. 1 mmol/ L after 6 - week feeding. Then the models of DCP were screened by urodynamic testing after 4 - week feeding. The left 15 guinea pigs were injected with the same dose of buffer STZ and fed in the same way(the control group). Fifteen guinea pigs were selected from the successful DCP models and divided into Ⅰ group( n = 3)and experimental group(n = 12)by random number table method. The Ⅰgroup was not treated,and the experimental group was transurethrally perfused with SCL lentivirus(1. 6 × 107 TU)twice. At the 2th,7th,14th,28th day after the second perfuse,3 guinea pigs were killed by cervical dislocation and marked as Ⅱ,Ⅲ,Ⅳ and Ⅴ group,respectively. The Ⅰ group was killed at the same time with Ⅱ group. The bladder tissue was frozen in liquid nitrogen. The distribution of green fluorescence in the tissue was observed under laser confocal microscopy after glycerol sealing. The expression level of SCL mRNA was detected using qRT - PCR method. Results During the preparing the DCP model,18 guinea pigs were discarded in the diabetic model screening and 19 guinea pigs were discarded in the urodynamic testing,and finally 23 DCP models were obtained(DCP model group). The maximal detrusor pressure in DCP model group of guinea pigs was lower than control group,the maximum bladder capacity was larger than control group(P < 0. 01). There was no green fluorescence in the bladder tissue layer of Ⅰ group. The green fluorescence was not obvious in the bladder tissue layer of Ⅱ group. A super strong green fluorescence was observed throughout the whole bladder tissue layer in Ⅲgroup. There was strong green fluorescence in the whole bladder tissue layer of Ⅳgroup. There was still visible green fluorescence in the bladder tissue layer of Ⅴgroup. The expression of SCL mRNA in Ⅰ group was(1. 00 ± 0. 00). The expression of SCL mRNA in Ⅱgroup was(1. 22 ± 0. 06)times higher than Ⅰ group. The expression of SCL mRNA in Ⅲ group was(50. 45 ± 3. 17)times higher than Ⅰ group. The expression of SCL mRNA in Ⅳgroup was(24. 75 ± 6. 49)times higher than Ⅰ group. The expression of SCL mRNA in Ⅴ group was(3. 27 ± 1. 00)times higher than Ⅰgroup. Conclusion The lentiviral vector of SCL gene could successfully transfect into the DCP bladder of guinea pig and stably express,which might bring a new way for the clinical treatment of DCP.%目的：制备糖尿病膀胱病变（DCP）豚鼠模型，经尿道灌注干细胞白血病（SCL）基因慢病毒，观察其转染效果及稳定性。方法2014年11月—2015年6月，采用随机数字表法在75只普通级荷兰种雄性豚鼠中选取60只单次腹腔注射链脲佐菌素（200 mg/ kg），常规饲养6周后按血糖水平>11.1 mmol/ L 筛选糖尿病模型，再常规饲养4周后通过尿流动力学检测仪筛选 DCP 模型；另15只作为对照组，单次注射配制的链脲佐菌素缓冲液，饲养时间相同。采用随机数字表法从成功造模的 DCP 模型中选取15只后，再采用随机数字表法分为Ⅰ组（3只）和实验组（12只），Ⅰ组不予处理，实验组经尿道分2次灌注 SCL 基因慢病毒（1.6×107 TU），于2次灌注结束后第2、7、14、28天分别采用颈椎脱臼法处死实验组豚鼠3只，记为Ⅱ、Ⅲ、Ⅳ、Ⅴ组，Ⅰ组与Ⅱ组一同处死，取膀胱组织冷冻于液氮中，甘油封固后激光共聚焦显微镜下观察组织中绿色荧光分布情况。采用 qRT - PCR 检测 SCL mRNA 表达水平。结果 DCP模型造模过程中，糖尿病模型筛选时弃去18只，尿流动力学检测仪筛选时弃去19只，最终得到 DCP 模型豚鼠23只，记为 DCP 模型组。DCP 模型组豚鼠最大逼尿肌压低于对照组，最大膀胱容量大于对照组（P <0.01）。Ⅰ组豚鼠膀胱组织未见绿色荧光分布，Ⅱ组豚鼠膀胱组织绿色荧光不明显，Ⅲ组豚鼠膀胱组织可见全层遍布极强绿色荧光，Ⅳ组豚鼠膀胱组织可见全层强绿色荧光，Ⅴ组豚鼠膀胱组织仍可见绿色荧光存在。Ⅰ组豚鼠膀胱组织 SCL mRNA 表达水平为（1.00±0.00），Ⅱ组豚鼠膀胱组织 SCL mRNA 表达水平是Ⅰ组的（1.22±0.06）倍，Ⅲ组豚鼠膀胱组织 SCL mRNA 表达水平是Ⅰ组的（50.45±3.17）倍，Ⅳ组豚鼠膀胱组织 SCL mRNA 表达水平是Ⅰ组的（24.75±6.49）倍，Ⅴ组豚鼠膀胱组织 SCL mRNA 表达水平是Ⅰ组的（3.27±1.00）倍。结论 SCL 基因慢病毒载体转染豚鼠 DCP 膀胱成功，并能稳定表达，有可能为 DCP 的临床治疗带来新的研究方式。