微囊化牛嗜铬细胞移植对疼痛大鼠行为学及脊髓P物质释放的影响

Effects of transplantation of microencapsulated bovine chromaffin cells on the praxiology and release of P substance in spinal cord in rats with ache

目的:了解微囊化牛嗜铬细胞蛛网膜下腔移植对疼痛大鼠脊髓后角P物质释放的影响,探讨嗜铬细胞移植镇痛的机制。方法:实验于2003-08/2004-09在郑州大学医学院解剖实验室完成。取SD大鼠24只,随机分为3组,空囊组、微囊化细胞组和裸细胞组各8只。分别在空囊组、微囊化细胞组和裸细胞组大鼠的脊髓蛛网膜下腔植入不包裹牛嗜铬细胞的含海藻酸钠-聚赖氨酸-海藻酸钠空囊的Dulbecco改良的Eagle培养液、海藻酸钠-聚赖氨酸-海藻酸钠微囊化牛嗜铬细胞和单纯牛嗜铬细胞。移植8周后,在3组大鼠右侧后爪掌侧皮下注射50μL5mL/L的甲醛溶液,观察其注射后1h内每5min大鼠的疼痛反应时间,即缩腿及舔爪时间之和。在行甲醛处理后1h,将各组大鼠麻醉后处死,制备脊髓切片,免疫组化法染色,通过测量各组大鼠脊髓后角浅层P物质样免疫反应阳性纤维吸光度分析大鼠脊髓后角浅层P物质含量的变化。结果:空囊组和微囊化细胞组分别有1只大鼠移植术后死亡,进入结果分析共22只。①各组大鼠缩腿舔爪时间比较:微囊化细胞组、裸细胞组大鼠显著低于空囊组(F=31.271～122.402,P<0.05),微囊化细胞组大鼠显著低于裸细胞组(F=31.271～122.402,P<0.05)。②各组大鼠两侧脊髓后角浅层P物质样免疫反应阳性纤维吸光度比较:微囊化细胞组及裸细胞组大鼠显著高于空囊组[右侧:0.065±0.003,0.040±0.006,0.034±0.004;左侧:0.066±0.002,0.042±0.004,0.035±0.005(F右侧=156.672,F左侧=155.841,P<0.05～0.01)],而微囊化细胞组显著高于裸细胞组(P<0.01)。结论:牛嗜铬细胞移植对甲醛致痛大鼠具有镇痛作用,可应用于慢性疼痛的治疗;同时还可以抑制甲醛致痛诱发的P物质的释放,增加脊髓后角P物质的含量,提示P物质可能参与了嗜铬细胞移植镇痛过程。其中海藻酸钠-聚赖氨酸-海藻酸钠微囊化的牛嗜铬细胞移植可以明显提高镇痛效果,提示微囊具有良好的免疫隔离作用。

AIM： To know about the effects of transplant of microencapsulated bovine chromaffin cells （BCCs） in subarachnoid space on the release of P substance in spinal cord posterior horn,and to explore the analgesic mechanism of chromaffin cells transplant. METHODS： The experiment was done at the dissection laboratory of Medical College, Zhengzhou University from August 2003 to September 2004. Twenty-four SD rats were selected, and divided randomly into 3 groups： empty capsule group, microcapsule cell group and null cell group with 8 rats in each group. Eagle medium improved by Dulbecco including alginic acid natrium-polylysine-empty capsule of alginic acid natrium without packing the BCCs, alginic acid natrium-polylysine-microcapsule of alginic acid natrium BCCs and pure BCCs were planted in the spinal subarachnoid space of rats in the empty capsule group, microcapsule cell group and null cell group, respectively. Eight weeks after transplantation, the fight hind paws of rats were injected with 50 μL 5 mL/L formaldehyde solution by subcutaneous injection in the three groups. The ache reaction time was observed every 5 minutes within 1 hour after injection, i.e., the summation of the time of drawing back legs and the time of licking claw. After the disposal of formaldehyde for 1 hour, the rats in every group were killed after anesthesia to make the spinal cord section and perform the immunohistochemical method staining. Through measuring the positive fiber absorbance of P substance immunologic reaction in the superficial lamella of spinal cord posterior horn in rats of every group, the changes of P substance content in the superficial lamella of spinal cord posterior horn in rats were analyzed. RE SULTS： One rat died in the empty capsule group and microcapsule cell group, respectively after the transplantation, and totally 22 rats were involved in the result analysis. ①Comparison of time of drawing legs and licking claws of rats in every group： That of the rats in the microcapsule cell group and the null cell group was lower significantly than that in the empty capsule group （F=31.271-122.402, P 〈 0.05）, and that in the microcapsule cell group was lower significantly than that in the null cell group （F=31.271-122.402, P 〈 0.05）.②Comparison of positive fiber absorbance of P substance immunologic reaction in the superficial lamella of spinal cord posterior horn in double sides of rats of every group： That in microcapsule cell group and null cell group was significantly higher than that in the empty capsule group [right side：0.065±0.003, 0.040±0.006, 0.034±0.004; left side： 0.066±0.002,0.042±0.004, 0.035±0.005 （Frogjt sode=156.672, Fleft side=155.841, P 〈 0.05-0.01）], while it was higher in the mierocapsule cell group than that in the null cell group（P 〈 0.01 ）. CONCLUSION： The transplantation of BCCs can produce analgesic effect on ache due to formaldehyde in rats, and can cure the chronic pain; Meanwhile, it also can inhibit the release of P substance induced by ache due to formaldehyde, increase the content of P substance in spinal cord posterior horn, which indicates that the P substance may take part in the process of transplantation analgesia of ehromaffin cells. The alginic acid natrium- polylysine-mieroeapsule of alginie acid natrium BCCs can significantly enhance the effects of analgesia, which indicates that the microcapsule has good effects on immunity and insulation.