滋阴泻火中药合剂对青春期大鼠垂体生长激素和长骨干骺端胰岛素样生长因子1基因表达的调节

Effect of ziyin xiehuo Chinese herbal mixture on growth hormone in hypophysis and expression of insulin-like growth factor 1 gene in metaphysis of adolescent rats

目的:观察滋阴泻火中药合剂对青春期大鼠垂体生长激素、长骨干骺端胰岛素样生长因子1基因表达的影响,探讨中药对垂体、长骨干骺端促生长机能的调节作用。方法:实验于2002-09/2003-03在上海瑞金医院完成。将青春期SD雌性大白鼠共22只分为对照组和实验组,每组11只。对照组:喂饲生理盐水1个月,5mL/次,1次/d;实验组:喂饲滋阴泻火中药合剂(由生地12g,炙龟板12g,黄柏9g,知母9g等组成;均由上海复旦大学附属儿科医院中药制剂室煎制成浓缩合剂,每毫升约含生药3g。)1个月,5mL/次,1次/d。疗程结束后,动物处死,取出垂体及左后肢骨干骺端,提取总mRNA。采用实时荧光RT-PCR定量检测技术,检测垂体生长激素mRNA,长骨干骺端胰岛素样生长因子1mRNA表达水平。实时荧光RT-PCR定量检测方法:①垂体、长骨干骺端总RNA提取采用一步法。并逆转录合成cDNA,PCR扩增产物经15g/L琼脂糖电泳后,纯化,制备标准曲线,并扩增。②垂体生长激素扩增条件:先94℃2min,然后94℃变性10s,58℃退火30s,72℃延长20s,3个步骤作为1个循环,共扩增40个循环。长骨干骺端胰岛素样生长因子1扩增条件:先94℃3min,然后94℃变性10s,60℃退火20s,72℃延长20s,3个步骤作为1个循环,共扩增40个循环。经过t检验处理,比较两组间垂体生长激素及长骨干骺端胰岛素样生长因子1基因表达水平的变化,观察滋阴泻火中药合剂对上述指标基因表达的影响。结果:实验动物22只,生理盐水和中药灌胃时误灌入气管窒息而死,各1只,进入结果分析20只。①实验组生长激素cDNA的复制数明显低于对照组犤(7.08±1.20)×107,(19.38±6.69)×107;t=5.728,P<0.05犦,且扩增片段为1条长度为533bp的条带,未见其他非特异性条带。②实验组干骺端胰岛素样生长因子1cDNA复制数明显低于对照组犤(2.28±0.65×102),(21.41±5.85×102);t=10.268,P<0.05犦;电泳图显示干骺端胰岛素样生长因子1cDNA扩增片段为一条长度为202bp的条带,未见其他非特异性条带。结论:滋阴泻火中药合剂可在转录水平调节腺垂体生长激素及长骨干骺端软骨细胞胰岛素样生长因子1的基因表达,从而调整生长激素-胰岛素样生长因子1轴的功能活动。

AIM： To observe the effect of ziyin xiehuo Chinese herbal mixture on expression of growth hormone （GH） in hypophysis and insulin-like growth factor 1 （IGFI） in metaphysis, and probe into the regulative effect of Chinese herbal medicine on growing function of GH and IGFI. METHODS： The experiment was completed in Ruijin Hospital of Shanghai from September 2002 to March 2003. Totally adolescent female SD rats were randomly divided into control group and experimental group with 11 in each group. Control group： Rats in control group were fed with saline for 30 days with 5 mL/time and once per day. Experimental group： Mice in experimental group were treated with ziyin xiehuo Chinese herbal mixture （shengdi 12 g, zhiguiban 12 g, huangbai 9 g and zhimu 9 g; provided by Department of Traditional Chinese Medicine of Children Hospital Affiliated to Shanghai Fudan University; 3 graw drag in per milliliter mixture） for 30 days with 5 mL/time and once per day. After the course, the rats were killed. Total mRNA was extracted in hypophysis and metaphysis. The expressions of GH and IGF1 mRNA were measured with real-time RT-PCR. Real-time RT-PCR： ① Total RNA in hypophysis and metaphysis was extracted with one-way method, and synthesized into cDNA reversally. PCR amplificated product was electrophoresized with 15 g/L agarose, purified for standard curve, and amplificated. ② Amplificated condition of GH in hypophysis： Maintain at 94℃ for 2 minutes; degenerate at 94℃for 10 s; anneal at 58 ℃ for 30 s; elongate at 72 ℃ for 20 s; and three procedures regarding as a cycle in total of 40 cycles. Amplificated condition of IGF1 in metaphysis： Maintain at 94 ℃ for 3 minutes; degenerate at 94 ℃ for 10 s; anneal at 60 ℃ for 20 s; elongate at 72 ℃ for 20 s; and three procedures regarding as a cycle in total of 40 cycles, Expressive changes of GH and IGF1 in hypophysis and metaphysis were compared with t test between groups so as to observe effect of ziyin xiehuo Chinese herbal mixture on gene expression mentioned above. RESULTS： Two rats died because of peffusing trachea with saline and Chinese herb respectively, so 20 rats entered the final analysis. ① Copied numbers of GH cDNA in experimental group were lower than those in control group [（7.08±1.20） ×10^7, （19.38±6.69） ×10^7; t=5.728, P 〈 0.05]; amplifieated section was a strap with 533 bp in length, and other unspecific straps were observed. ② Copied numbers of IGF1 cDNA in experimental group were lower than those in control group [（2.28±0.65） ×10^2, （21.41 ±5.85）×10^2; t=10.268, P 〈 0.05]. Eleetrophoregram showed that amplifieated section of IGF1 cDNA was a strap with 205 bp in length, and other unspecific straps were observed.CONCLUSION： Ziyin xiehuo Chinese herbal mixture can regulate the gene expression of GH in hypophysis, IGF1 in metaphysis at the level of the transcription, thereby it can modulate the functional activity of the hypothalamic-pituitary- IGF-1 axis.