鞘内注射含HPPE基因HSV-1型扩增子载体对大鼠细胞免疫功能的影响

Effect of intrathecal HSV-1 amplicon vector-mediated HPPE gene on cellular immune function in rats

目的观察鞘内注射含人前脑啡肽原（HPPE）基因单纯疱疹病毒1（HSV-1）型扩增子载体对大鼠细胞免疫功能的影响。方法24只成年雄性SD大鼠，采用改良Yaksh法进行鞘内置管后，随机分为3组（n=8）：生理盐水组（NS组）、pHSVIRES-LacZ空白载体组（SHZ组）和pHSVIRES-HPPE-LacZ载体组（SHPZ组）。置管后5d鞘内注射NS、SHZ或SHPZ各8μl，注射后1周行福尔马林实验，评估镇痛效果后处死大鼠，称量体重及脾脏重量，计算脾脏指数，并分离、培养脾脏淋巴细胞，甲基-3^H胸腺嘧啶核苷掺入法检测T淋巴细胞增殖转化水平，乳酸脱氢酶释放法检测自然杀伤（NK）细胞活力，流式细胞仪检测脾脏T淋巴细胞及其亚群和NK细胞表型百分率。结果与NS组和SHZ组比较，SHPZ组在福尔马林给药后5min和15～60min时痛级评分降低（P〈0．05或0．01）。NS组脾脏指数（5．8±0．3）mg／g，SHZ组脾脏指数（5．8±0．9）mg/g，SHPZ组脾脏指数（5．9±0．7）mg／g，3组比较差异无统计学意义（P〉0．05）。与NS组比较，SHPZ组和SHZ组T淋巴细胞增殖转化水平、NK细胞活力及CD3^＋、CD3^＋CD4^＋、CD3^＋CD8^＋、CD161^＋的百分率升高（P〈0．05）；与SHZ组比较，SHPZ组T淋巴细胞增殖转化水平、NK细胞活力和CD3^＋CD4^＋、CD3^＋CD8^＋、CD161^＋的百分率升高（P〈0．05）。结论大鼠鞘内注射含HPPE基因HSV-1型扩增子载体在产生良好的抗伤害作用时，不抑制细胞免疫功能。

Objective To investigate the effect of intrathecal （IT） HSV-1 amplicon vector-mediated HPPE gene on cellular immune function in rats. Methods Twenty-four SD rats weighing 260-320 g were randomly divided into 3 groups （ n = 8 each） ： group Ⅰ received IT normal saline （NS） ; group Ⅱ received IT pHSVIRES- LacZ （SHZ） and group Ⅲ received IT pHSVIRES-HPPE-LacZ （SHPZ）. The animals were anesthetized with intraperitoneal chloral hydrate 300-350 mg/kg. Microspinal catheter was inserted into the subarachnoid space at the lumbar region using modified Yaksh technique. Five days later, NS, SHZ and recombinant HSV-1 amplicon vector SHPZ were administered IT in group Ⅰ , Ⅱ and Ⅲ respectively. Seven days after IT administration, 5 % formalin 50 μl was injected into the plantar surface of left hindpaw. The number of flinches, lickings and total time of licking were recorded for 60 min. Pain intensity scoring system （PIS） （0 = no pain, 3 = severe pain） was used to assess the antinociceptive effect of SHPZ. The animals were killed after evaluation of pain intensity. Body weight and spleen weight were measured. Spleen index （spleen weight/body weight） was calculated. T-lymphocyte function was evaluated based on concanavalin A （COMA） -induced splenocyte proliferation. A modified lactic acid dehydrogenase （LDH） release assay was used to assess NK cell activity. The phenotype expression of cell surface markers ofT-lymphocyte subsets （CD3^＋ , CD3^＋ CD4^＋ , CD3^＋ CD8^＋ ） and NK cell （CD161^＋ ） in the spleen were analyzed by flow cytometry. Results Intrathecal SHPZ had antinociceptive effect on formalin-induced pain compared with SHZ and NS （ P 〈 0.05 or 0.01 ）. There was no significant difference in spleen index among the 3 groups（P 〉 0.05）. T-lymphocyte proliferation, NK cell activity and percentage of CD3^＋ CD4^＋ , CD3^＋ CD8^＋ , CD161^＋ were significantly higher in SHPZ group than in SHZ and NS groups （P 〈 0.05 or 0.01 ）. Conclusion Intrathecal HSV-1 amplicon vector-mediated HPPE gene does not suppress cellular immune function when it has antinociceptive effect.