共表达生长激素释放激素(GHRH)与垂体腺苷酸环化酶(PACAP)对动物生长的影响

生长激素释放激素(GHRH)与垂体腺苷酸环化酶(PACAP)在序列及功能方面均相似，且同为PACAP/胰高血糖素超家族成员．研究了这二者对生长激素释放的刺激作用，以及对动物生长的影响．构建了3个表达载体，pIRES1- GHRH-PACAP(P-G-P)，pIRES1-GHRH(P-G) 及 pIRES1-PACAP(P-P)，并转染到CHO细胞中，进行RT-PCR，Dot-ELISA以及Westen-blot检测．此外，给大鼠注射细胞上清表达产物，检测其生物学活性．注射8 h后，注射表达P-G-P上清的大鼠血清中IGF-Ⅰ浓度显著高于其他组(P < 0.05)．用PLGA微球包裹各种质粒，并注射到家兔后肢胫前肌．观察家兔生长情况，并于注射后0，15，30，45天时分别采集家兔血液，检测血液中IGF-Ⅰ浓度．结果显示，三质粒注射组动物体重变化及血液中IGF-Ⅰ浓度均高于对照组．注射后30天时，P-G-P组增重较对照组提高81% (P < 0.01)，P-G组比对照组提高15%(P > 0.05)，P-P组比对照组高7%(P > 0.05)．另一方面，P-G-P组动物血液中IGF-Ⅰ含量比分别比P-G、P-P及对照组提高16.68% (P > 0.05)，17.14%(P > 0.05)，50.46%(P < 0.05)．以上结果揭示：给动物注射PLGA微球包裹的共表达GHRH与PACAP质粒，可以增强动物体内生长激素(GH)的分泌，并促进动物生长．通过上述研究发现，肌肉注射PACAP表达质粒可以促进家兔的生长，PACAP和GHRH 共表达可以起到协同作用．这可能为动物的促生长研究提供新的方法．

Ectopic Co-expression of Growth Hormone Releasing Hormone and Pituitary Adenylate Activating Peptide in Skeletal Muscle Enhance Animal Growth

Growth hormone (GHRH) and pitutary adenylate cyclase activating peptide (PACAP) are the members of the PACAP/Glucagon superfamily,who are related in both sequence and function.Their stimulation of GH secretion and animal growth is concerned.A series of expression plasmid,pIRES1-GHRH-PACAP (P-G-P),plRESI-GHRH (P-G) and plRESI-PACAP(P-P),were constructed,extracted and purified,then transfected into CHO cell line with Lipofectamine.The expression was examined by RT-PCR,dot-ELISA and Western blotting.The biological activity of expression products was detected in rats.At 8 h after injection of transfection supematant,serum IGF-I concentrations in P-G-P group were significantly higher than that in other groups(P ＜ 0.05).PLGA encapsulating plasmid microspheres were prepared and injected intramuscularly into rabbit legs.Growth behavior and IGF-1 level were measured at day 0,15,30 and 45 after injection.Greater body weights gain and higher serum 1GF- levels were observed in three plasmid microsphere injection groups,compared with control group.At day 30,the body weight gain in P-G-P group was greater than saline group (81%,P＜ 0.01),P-G mierosphere group (15%,P＜ 0.05) and P-P microsphere group (7%,P＞ 0.05),serum IGF-I concentration in P-G-P microsphere group showed a 16.68% increase to P-G microsphere (P ＞ 0.05),a 17.14% increase to P-P microsphere(P ＞ 0.05) and a 50.46% increase to control (P ＜ 0.05).These results suggest that co-expression of GHRH and PACAP in one expression plasmid might exert an additive stimulation of GH secretion and growth when delivered into rabbit skeletal muscle with PLGA mierosphere.The results may provide a new approach to regulate animal growth.