CAJ | 학술논문

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。目的：通過慢病毒載體介導的 RNA 榦擾（RNAi）技術抑製雌鼠下丘腦 KiSSI 基因錶達，分析KiSS1、促性腺激素釋放激素（GnRH）mRNA 錶達變化，血清性激素水平變化，探討靶嚮沉默 KiSSI 基因對性髮育的影響及可能的相關機製。方法21日齡 Sprague-Dawley 雌鼠90隻，按照隨機數字錶分為3組：榦擾病毒組、病毒對照組、9 g/ L 鹽水對照組。榦擾病毒組側腦室註射攜帶 KiSSI-微 RNA（microRNA）的慢病毒，病毒對照組側腦室註射無榦擾作用的對照病毒，9 g/ L 鹽水對照組側腦室註射9 g/ L 鹽水。在青春期前（30日齡）、青春髮育早期（35日齡）、青春髮育晚期（45日齡）時分彆處死各組動物10隻，留取下丘腦組織，實時定量（RT） PCR 檢測下丘腦 KiSSI、GnRH mRNA 的錶達，併同時留取血清採用化學髮光法檢測卵泡刺激素（FSH）、黃體生成素（LH）、雌二醇（E2）水平。結果榦擾病毒組的 KiSSI mRNA 與9 g/ L 鹽水對照組及病毒對照組相比明顯下調（30日齡0.106±0.018；35日齡0.218±0.025；45日齡0.215±0.033，P 均＝0.000）；榦擾病毒組的 GnRH mRNA 錶達在30日齡及35日齡顯著下降（0.230±0.040、0.407±0.030，P 均＝0.000），45日齡時 GnRH mRNA與9 g/ L 鹽水對照組和病毒對照組相比，差異無統計學意義（0.455±0.054，P ﹥0.05）。榦擾病毒組 LH 水平[（0.101±0.004）IU/ L]在35日齡低于9 g/ L 鹽水對照組及病毒對照組（P ＝0.467）；榦擾病毒組 FSH 水平[（0.235±0.014）IU/ L]在35日齡低于9 g/ L 鹽水對照組及病毒對照組（P ＝0.015）；榦擾病毒組 E2水平在35日齡[（171.750±11.050）IU/ L]及45日齡[（192.310±13.100）IU/ L]時低于9 g/ L 鹽水對照組及病毒對照組（P ＝0.000，0.010）。結論側腦室註射攜帶 KiSSI-microRNA 的慢病毒載體，可下調 KiSSI mRNA 水平，進而影響 GnRH mRNA 及下遊性激素的錶達水平、推遲雌鼠的性髮育進程。
목적：통과만병독재체개도적 RNA 간우（RNAi）기술억제자서하구뇌 KiSSI 기인표체，분석KiSS1、촉성선격소석방격소（GnRH）mRNA 표체변화，혈청성격소수평변화，탐토파향침묵 KiSSI 기인대성발육적영향급가능적상관궤제。방법21일령 Sprague-Dawley 자서90지，안조수궤수자표분위3조：간우병독조、병독대조조、9 g/ L 염수대조조。간우병독조측뇌실주사휴대 KiSSI-미 RNA（microRNA）적만병독，병독대조조측뇌실주사무간우작용적대조병독，9 g/ L 염수대조조측뇌실주사9 g/ L 염수。재청춘기전（30일령）、청춘발육조기（35일령）、청춘발육만기（45일령）시분별처사각조동물10지，류취하구뇌조직，실시정량（RT） PCR 검측하구뇌 KiSSI、GnRH mRNA 적표체，병동시류취혈청채용화학발광법검측란포자격소（FSH）、황체생성소（LH）、자이순（E2）수평。결과간우병독조적 KiSSI mRNA 여9 g/ L 염수대조조급병독대조조상비명현하조（30일령0.106±0.018；35일령0.218±0.025；45일령0.215±0.033，P 균＝0.000）；간우병독조적 GnRH mRNA 표체재30일령급35일령현저하강（0.230±0.040、0.407±0.030，P 균＝0.000），45일령시 GnRH mRNA여9 g/ L 염수대조조화병독대조조상비，차이무통계학의의（0.455±0.054，P ﹥0.05）。간우병독조 LH 수평[（0.101±0.004）IU/ L]재35일령저우9 g/ L 염수대조조급병독대조조（P ＝0.467）；간우병독조 FSH 수평[（0.235±0.014）IU/ L]재35일령저우9 g/ L 염수대조조급병독대조조（P ＝0.015）；간우병독조 E2수평재35일령[（171.750±11.050）IU/ L]급45일령[（192.310±13.100）IU/ L]시저우9 g/ L 염수대조조급병독대조조（P ＝0.000，0.010）。결론측뇌실주사휴대 KiSSI-microRNA 적만병독재체，가하조 KiSSI mRNA 수평，진이영향 GnRH mRNA 급하유성격소적표체수평、추지자서적성발육진정。
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.