经睾丸输出小管注射慢病毒对Wistar大鼠生殖系统的影响

doi:10.6040/j.issn.1671-7554.0.2018.1486

摘要/Abstract

摘要： 目的探讨慢病毒高毒性物质稳定转基因技术对雄性Wistar大鼠生殖系统的影响。方法构建PAX2稳定干扰的含有绿色荧光蛋白的慢病毒,将该病毒溶液用台盼蓝标记,通过34 G针头注入16只Wistar大鼠睾丸输出小管。2周后用超声监测睾丸实质,1个月后将大鼠处死,取出睾丸及其附属腺体,免疫荧光染色及HE染色观察组织结构形态。结果大鼠睾丸输出小管注射,共有8只注射成功,均出现不同程度的睾丸曲精小管凋亡坏死,部分小管腔内可见报告基因的高表达,附睾上皮未出现明显的转染情况,而精囊腺上皮细胞出现不同程度的绿色荧光蛋白高表达。结论睾丸和附睾不适合雄性动物的慢病毒转染,并且该处转染容易影响精子的成熟和发育,而精囊腺注射慢病毒建立转基因动物模型具有可行性。

关键词: 睾丸输出小管, 慢病毒, 转基因, 精囊腺, 附睾

Abstract: Objective To investigate the effect of stably transgenic technology of highly toxic lentivirus on the reproductive system of male Wistar rats. Methods The stable interference lentivirus of PAX2 containing green fluorescent protein were constructed. The solution was labeled with Trypan blue. The testicular efferent tubules injections of 16 Wistar rats were carried out with a 34 G needle. The testis parenchyma was monitored by ultrasound after two weeks. The rats were executed after one month. The testis and the accessory glands were removed and fixed, and the tissue structure and morphology were studied by immunofluorescence staining and HE staining. Results Eight rats were successfully injected. Apoptosis and necrosis in testicular convoluted seminiferous tubules were observed in different degrees. High expression of the reporter gene was observed in some tubules, and no obvious transfection was found in the epididymal epithelium. However, there was a high expression of the green fluorescent protein in seminal vesicle glandular epithelial cells. Conclusion Testis and epididymis are not suitable for lentivirus transfection in male animals for making transgenic animals, and the local transfection can easily affect sperm maturation and development. However, it is practical to establish transgenic animals by injecting lentivirus into the seminal vesicle.

Key words: Testicular efferent tubules, Lentivirus, Transgenic, Seminal vesicle, Epididymis

中图分类号:

Q492.4

狄钰,陈东山,阎磊. 经睾丸输出小管注射慢病毒对Wistar大鼠生殖系统的影响[J]. 山东大学学报 (医学版), 2019, 57(5): 67-73.

DI Yu, CHEN Dongshan, YAN Lei. Effects of lentivirus injection via testicular efferent tubules on the reproductive system in Wistar rats[J]. Journal of Shandong University (Health Sciences), 2019, 57(5): 67-73.

参考文献

[1] 罗庆苗, 苗向阳, 张瑞杰, 等. 转基因动物新技术研究进展[J]. 遗传, 2011, 33(5): 449-458. LUO Qingmiao, MIAO Xiangyang, ZHANG Ruijie, et al. An update on the development of transgenic animal technology[J]. Hereditas, 2011, 33(5): 449-458.
[2] 郭宇航, 张守纯. 精子载体转基因技术的研究[J].畜禽业, 2008(8): 8-9.
[3] Russell LD, Ettlin RA, Hikim APS, et al. Histological and histopathological evaluation of the testis[J]. Int J Androl, 1993, 16(1): 83. doi:10.1111/j.1365-2605.1993.tb01156.x.
[4] Sun S, Wang YC, Guo K, et al. Finasteride inhibits microvascular density and VEGF expression in the seminal vesicle of rats[J]. Zhonghua Nan Ke Xue, 2018, 24(5): 387-392.
[5] Estemalik J, Demko C, Bissada NF, et al. Simultaneous detection of oral pathogens in subgingival plaque and prostatic fluid of men with periodontal and prostatic diseases[J]. J Periodontol, 2017, 88(9): 823-829.
[6] 曹碗君, 赵洁, 乔慧莲, 等. PTIP相关蛋白1在小鼠睾丸发育过程中的表达及定位[J]. 中国组织化学与细胞化学杂志, 2018, 27(1): 8-12. CAO Wanjun, ZHAO Jie, QIAO Huilian, et al. Expression and localization of PTIP associated protein 1 during mouse testis development[J]. Chinese Journal of Histochemistry and Cytochemistry, 2018, 27(1): 8-12.
[7] Jafarian A, Lakpour N, Sadeghi MR, et al. Transplantation of spermatogonial stem cells suspension into rete testis of azoospermia mouse model[J]. Urol J, 2018, 15(1): 40-47.
[8] Nistal M, González-Peramato P, Serrano Á. Acquired pathology of the rete testis[M] //Nistal M, González-Peramato P, Serrano Á. eds. Clues in the diagnosis of non-tumoral testicular pathology. Cham: Springer International Publishing, 2017: 279-285. doi:10.1007/978-3-319-49364-0_31.
[9] 廖波, 邓显忠, 代小思, 等. 成年SD大鼠睾丸网的分布[J]. 川北医学院学报, 2010, 25(3): 199-208. LIAO Bo, DENG Xianzhong, DAI Xiaosi, et al. Distribution of the rete testis in the adult SD rat[J]. Journal of North Sichuan Medical College, 2010, 25(3): 199-208.
[10] Martins-Santos E, Pimenta CG, Campos PRN, et al. Atrazine affects the morphophysiology, tissue homeostasis and aromatase expression in the efferent ductules of adult rats with mild alterations in the ventral prostate[J]. Chemosphere, 2018, 193: 958-967. doi:10.1016/j.chemosphere.2017.11.124.
[11] Michaelis M, Sobczak A, Weitzel JM. In vivo microinjection and electroporation of mouse testis[J]. J Vis Exp, 2014(90). doi:10.3791/51802.
[12] 王鹤, 曹文广. 小鼠睾丸输出管注射法建立转基因小鼠的试验研究[J]. 天津农业科学, 2013, 19(4): 34-37. WANG He, CAO Wenguang. Construction of transgenic mice by testicular efferent duct injection[J]. Tianjin Agricultural Sciences, 2013, 19(4): 34-37.
[13] 申复进, 张茨, 王将, 等. 输出小管-睾丸网注射提高小鼠精原干细胞移植效率[J]. 武汉大学学报(医学版), 2009, 30(4): 518-521. SHEN Fujin, ZHANG Ci, WANG Jiang, et al. Efferent duct-rete injection improves the efficiency for transplanting mouse spermatogonial stem cells[J]. Medical Journal Wuhan University, 2009, 30(4): 518-521.
[14] 周之畅, 李嘉诚, 胡宇峰, 等. 睾丸“门”的定位及临床解剖意义[J]. 现代实用医学, 2013, 25(10): 1097-1098. ZHOU Zhichang, LI Jiacheng, HU Yufeng, et al. The location of testis porta and its clinical significance in anatomy[J]. Modern Practical Medicine, 2013, 25(10): 1097-1098.
[15] 覃金洲. 雄性生殖细胞介导的转基因技术的研究[D]. 杨凌:西北农林科技大学,2016.
[16] Ning L, Meng J, Goossens E, et al. In search of an efficient injection technique for future clinical application of spermatogonial stem cell transplantation: infusion of contrast dyes in isolated cadaveric human testes[J]. Fertil Steril, 2012, 98(6): 1443-1448.e1. doi:10.1016/j.fertnstert.2012.08.023.
[17] Pease S. Infecting mouse embryos by coculturing with lentivirus[J]. Cold Spring Harb Protoc, 2018, 2018(3): pdb.prot093922. doi:10.1101/pdb.prot093922.
[18] 孟凡荣, 陈琛, 万海粟, 等. 慢病毒载体及其研究进展[J]. 中国肺癌杂志, 2014, 17(12): 870-876. MENG Fanrong, CHEN Chen, WAN Haisu, et al. Advances of lentiviral vectors[J]. Chinese Journal of Lung Cancer, 2014, 17(12): 870-876.
[19] Hashimoto M, Rockenstein E, Mante M, et al. An antiaggregation gene therapy strategy for Lewy body disease utilizing beta-synuclein lentivirus in a transgenic model[J]. Gene Ther, 2004, 11(23): 1713-1723.
[20] Kane NM, Nowrouzi A, Mukherjee S, et al. Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors[J]. Molecular Therapy, 2010, 18(12): 2139-2145.
[21] Bouchard M, Souabni A, Mandler M, et al. Nephric lineage specification by Pax2 and Pax8[J]. Genes Dev, 2002, 16(22): 2958-2970.
[22] 徐海生, 王长山, 李丽. PAX2基因在肾间质纤维化中的研究进展[J]. 齐齐哈尔医学院学报, 2017, 38(18): 2185-2188.
[23] Sinn PL, Arias AC, Brogden KA, et al. Lentivirus vector can be readministered to nasal epithelia without blocking immune responses[J]. J Virol, 2008, 82(21): 10684-10692.
[24] VandeWoude S, Hageman CL, Hoover EA. Domestic cats infected with lion or puma lentivirus develop anti-feline immunodeficiency virus immune responses[J]. J Acquir Immune Defic Syndr, 2003, 34(1): 20-31.

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