蛋氨酸脑啡肽联合低剂量纳曲酮治疗胰腺癌的实验研究

Experimental study on the therapy of pancreatic cancer by combining methionine encephalin with low dose naltrexone

目的:通过单独应用MENK、NTX与联合应用MENK与NTX的体内外实验,探讨MENK、NTX及二者联合时对胰腺癌的抑制作用。方法:体外实验中,MTS方法检测各实验组中胰腺癌增殖细胞的抑制情况;体内实验中,通过建立胰腺癌荷瘤小鼠模型,检测各实验处理组中胰腺癌的抑制情况。WB法检测荷瘤小鼠胰腺癌组织阿片受体蛋白表达量;流式细胞术检测荷瘤小鼠淋巴细胞亚群(CTL、Treg、NK、γδT)的增殖情况。结果:在体外实验中,MENK在浓度为10^(-6)mol/L时对胰腺癌细胞的抑制作用最强;NTX在浓度为10^(-5)mol/L时对胰腺癌细胞的抑制作用最强;二者相比较,MENK的作用更加显著,具有统计学意义(P<0.05);在体内实验中,MENK与NTX联合应用在48小时后与对照组相比较,具有统计学意义(P<0.05),在96小时,与MENK组和NTX组相比较抑癌率升高,具有统计学意义(P<0.05);在体内实验中,在1次/24 h的给药方式下,MENK在剂量为20 mg/kg时抑癌效果最显著;NTX在剂量为5 mg/kg时抑癌效果最显著;MENK组与NTX组相比较作用更加显著,具有统计学意义(P<0.0 5);MENK与NTX联合应用时,MENK在剂量为20 mg/kg 1次/24 h与NTX在剂量为5 mg/kg 1次/96 h给药方式时,抑癌效果最显著,与对照组相比具有统计学意义(P<0.05);Western-blot技术检测荷瘤小鼠肿瘤组织阿片受体的蛋白的表达含量,NTX组与MENK+NTX组肿瘤组织阿片受体蛋白表达量增加,NTX组高于MENK+NTX组,具有统计学意义(P<0.05);流式细胞技术检测荷瘤小鼠淋巴细胞亚群增殖情况,MENK组、NTX组与MENK+NTX组都可以促进淋巴细胞亚群(CTL、NK、γδT)增殖,抑制Treg增殖,与对照组相比具有统计学意义(P<0.05)。结论:MENK在体内外能够抑制胰腺癌细胞增殖,NTX在体内外能够抑制胰腺癌细胞增殖,两者相比MENK作用更加显著;MENK与NTX联合应用,在NTX给药72小时后具有协同作用;NTX在体内能够上调肿瘤组织阿片受体的蛋白表达;MENK和NTX均能在体内能够促进CTL、NK、γδT细胞的增殖,抑制Treg细胞的增殖,二者联合具有协同作用。

Objective: Compare MENK,LDN and the combine of MENK and NTX in vivo and in vitro,evaluate the effect of MENK,LDN and combining MENK with NTX on the inhibition of pancreatic cancer. Methods: In vitro,MTS method for detecting the inhibition of pancreatic cell proliferation in different experimental groups. In vivo,by the model of the pancreatic tumor-bearing mice,evaluated the effect of different experimental groups on inhibition of pancreatic cancer. Assay the expression of opioid receptor protein in pancreatic tissue of the model of the pancreatic tumor-bearing mice by the mothed of WB. Analyze the proliferation of cytometry lymphocyte subsets( CTL,Treg,NK,γδT) of tumor-bearing mice by FCM. Results: In vitro,it’s strongest of MENK to inhibit pancreatic cancer cells at a concentration of 1 0-6 mol/L. It ’s strongest of NTX to inhibit pancreatic cancer cells at a concentration of 10-5 mol/L. Comparatively,MENK has a more significant role. In vivo,MENK combining with NTX group compared with control group,tumor suppressor rate increased after 48 hour sand after 96 hours,got the highest tumor suppressor rate comparing with MENK and NTX group. In vivo,in once every 24 hours of delivery mode,the best inhibiting tumor dose of MENK was 20 milligrams per kilogram of body weight. In once every 96 hours of delivery mode,the best inhibiting tumor dose of NTX was 5 milligrams per kilogram of body weight. MENK in once every 24 hours of delivery mode,at a dose of 20 milligrams per kilogram of body weight,combining with NTX in once every 96 hours of delivery mode,at a dose of 5 milligrams per kilogram of body weight had the highest tumor suppressor rate. Detect the opioid receptor protein expression of tumor tissue by WB. The opioid receptor expression of NTX and MENK combining with NTX group in tumor tissue increased. Compared with MENK combing with NTX group,NTX group had a higher opioid receptor expression. Detect lymphocyte subsets proliferation of tumor-bearing mice by FCM. MENK,NTX and MENK combining with NTX group can all promote lymphocyte subsets( CTL,NK,γδT) proliferation,compared with the control group. MENK,NTX and MENK combining with NTX group can all inhibit lymphocyte subsets( CTL,NK,γδT)proliferation,compared with the control group. Conclusion: In vitro,both MENK and NTX can inhibit pancreatic cancer cell proliferation,comparatively,MENK has a more significant role. MENK combined with NTX to inhibit pancreatic cancer cell proliferation,NTX administered in a synergistic effect after 72 hours. NTX can increase the protein expression of opioid receptors in tumor tissue of tumor-bearing mice. In vivo,both MENK and NTX can promote the proliferation of CTL,NK and γδT cell. Both MENK and NTX can inhibit the proliferation of Treg cells. MENK combining with NTX have synergistic effect on the proliferation of CTL,NK and γδT cell.