摘要
目的：通过慢病毒载体介导的 RNA 干扰（RNAi）技术抑制雌鼠下丘脑 KiSSI 基因表达，分析KiSS1、促性腺激素释放激素（GnRH）mRNA 表达变化，血清性激素水平变化，探讨靶向沉默 KiSSI 基因对性发育的影响及可能的相关机制。方法21日龄 Sprague-Dawley 雌鼠90只，按照随机数字表分为3组：干扰病毒组、病毒对照组、9 g/ L 盐水对照组。干扰病毒组侧脑室注射携带 KiSSI-微 RNA（microRNA）的慢病毒，病毒对照组侧脑室注射无干扰作用的对照病毒，9 g/ L 盐水对照组侧脑室注射9 g/ L 盐水。在青春期前（30日龄）、青春发育早期（35日龄）、青春发育晚期（45日龄）时分别处死各组动物10只，留取下丘脑组织，实时定量（RT） PCR 检测下丘脑 KiSSI、GnRH mRNA 的表达，并同时留取血清采用化学发光法检测卵泡刺激素（FSH）、黄体生成素（LH）、雌二醇（E2）水平。结果干扰病毒组的 KiSSI mRNA 与9 g/ L 盐水对照组及病毒对照组相比明显下调（30日龄0.106±0.018；35日龄0.218±0.025；45日龄0.215±0.033，P 均＝0.000）；干扰病毒组的 GnRH mRNA 表达在30日龄及35日龄显著下降（0.230±0.040、0.407±0.030，P 均＝0.000），45日龄时 GnRH mRNA与9 g/ L 盐水对照组和病毒对照组相比，差异无统计学意义（0.455±0.054，P ﹥0.05）。干扰病毒组 LH 水平[（0.101±0.004）IU/ L]在35日龄低于9 g/ L 盐水对照组及病毒对照组（P ＝0.467）；干扰病毒组 FSH 水平[（0.235±0.014）IU/ L]在35日龄低于9 g/ L 盐水对照组及病毒对照组（P ＝0.015）；干扰病毒组 E2水平在35日龄[（171.750±11.050）IU/ L]及45日龄[（192.310±13.100）IU/ L]时低于9 g/ L 盐水对照组及病毒对照组（P ＝0.000，0.010）。结论侧脑室注射携带 KiSSI-microRNA 的慢病毒载体，可下调 KiSSI mRNA 水平，进而影响 GnRH mRNA 及下游性激素的表达水平、推迟雌鼠的性发育进程。
Objective To explore the possible mechanism for KiSSI gene in control gonadotropin - releasing hormone（GnRH）secretion participating in sexual development onset and normal reproduction regulation by investigating the changes in expression of KiSS1,GnRH in hypothalamus and luteinizing hormone（LH）,follicle stimulating hormone （FSH）,and estradiol（E2 ）in serum by using RNA interference mediated by lentivirus - based vectors,after interfering expression of KiSSI. Methods Ninety female Sprague - Dawley rats of 21 days were randomly divided into 3 groups：interference virus group receiving intracerebroventricular injection of KiSSI - microRNA interference lentivirus;lentivi-rus - control group given intracerebroventricular injection of lentivirus without interference effect;9 g/ L saline control group given intracerebroventricular injection of 9 g/ L saline. Ten rats in each group and the animals were sacrificed at 30 - day - old,35 - day - old,45 - day - old,respectively. Then the expressions of KiSSI and GnRH mRNA were detec-ted in the rat hypothalamus with real - time PCR,the LH,FSH,and E2 in serum was examined with chemiluminescence method. Results The levels of KiSSI mRNA in interference virus group were significantly reduced after being infected with recombinant lentivirus compared with those of 9 g/ L saline control group（ at 30 d：0. 106 ± 0. 018;at 35 d：0. 218 ± 0. 025;at 45 d：0. 215 ± 0. 033,all P = 0. 000）. The level of GnRH mRNA in interference virus group was sig-nificantly reduced after being infected with recombinant lentivirus compared with that of 9 g/ L saline control group（at 30 d：0. 230 ± 0. 040;at 35 d：0. 407 ± 0. 030,all P = 0. 000）. The level of LH in interference virus group was lower than the other 2 groups at 35 d[（0. 101 ± 0. 004）IU/ L,P = 0. 467]. The level of FSH in the interference virus group was lower than that of the other 2 groups at 35 d[（0. 235 ± 0. 014）IU/ L,P = 0. 015]. The level of E2 in the interfe-rence virus group was lower than that of the other 2 groups at 35 d and 45 d[at 35 d：（171. 750 ± 11. 050）nmol/ L, P = 0. 000;at 45 d：（192. 310 ± 13. 100）nmol/ L,P = 0. 010]. Conclusions Lentivirus with KiSSI - microRNA can affect the expression of GnRH mRNA and the levels of sex hormone. Lateral cerebral ventricle microinjection of KiSSI -microRNA lentivirus can delay sexual development of Sprague - Dawley female rats.
出处
《中华实用儿科临床杂志》
CAS
CSCD
北大核心
2015年第20期1545-1548,共4页
Chinese Journal of Applied Clinical Pediatrics
