摘要
目的探讨溴隐亭对肝癌多药耐药逆转的作用机制。方法体外实验分亲本细胞HepG2组(A组),耐药细胞HepG2／A13M组(B组)和B组加溴隐亭称为C组。分别检测各组细胞内荧光强度变化,细胞P-糖蛋白、蛋白激酶C α蛋白表达情况,及5种肿瘤药物半数抑制浓度及耐药指数变化。分别将肝癌细胞:HepG2和耐药细胞HepG2／ADM原位种植入裸鼠肝脏内,称A组鼠和B组鼠,另设B组种植鼠给予溴隐亭灌冒治疗称C组鼠。B超观察种植瘤的生长情况,肿瘤大小为1．0cm左右时经腹腔化疗,2周后处死裸鼠计算肿瘤的体积和质量抑制率,观察种植瘤组织多药耐药基因1不敷出 mRNA表达情况,及治疗后肝癌细胞的凋亡指数。单独检测24只种植耐药细胞HepG2／ADM的裸鼠在服用溴隐亭前后分别注射的99mTc-MIBI在肝脏肿瘤部位的潴留情况。结果体外实验结果显示在溴隐亭浓度为10 μmol／L时罗丹明123 在细胞内的潴留率均明显增加,且呈时间依赖性,对阿霉素的耐药逆转以溴隐亭浓度为10μmol／L时最显著,逆转率为 82．6％。蛋白激酶C-α蛋白表达,C组与B组比较差异有统计学意义(q=5．37,P<0．01);但两组P-糖蛋白表达差异无统计学意义(q=1.86,P>0．05)。体内试验结果显示种植瘤的体积和质量抑制率比较,C组鼠明显高于B组鼠(q1= 5．89,q2=4．92,P<0．01),接近于A组鼠(q1=2．47,q2=3．02,P>0．05)。C组鼠与B组鼠多药耐药基因1 mRNA 表达差异无统计学意义(q=3．71,P>0．05)。化疗后肿瘤细胞凋亡率比较,C组鼠明显高于B组鼠(q=3．72, P<0．01)。口服溴隐亭后肝肿瘤组织对99mTc-MIBI的潴留指数明显提高(t=3．58,P<0．01)。结论溴隐亭通过抑制 P-糖蛋白的功能可有效的逆转肝癌多药耐药性。
Objective To investigate the mechanism of reversing multidrug resistance of hepatocarcinoma by bromocriptine （BCT） in vitro and in vivo. Methods Three groups of cultured HepG2 cells were used： HepG2 （group A）, the multidrug resistance HepG2/ADM celts （group B）, and the HepG2/ADM celts treated with BCT （group C）. Rhodamine 123 test was used to detect the function of P-gp protein. MTT assay was performed to examine the IC50. Multidrug resistance index to common five anticaneer drugs of different concentrations of BCT and immunocytochemistry was performed to detect the expression of PKC- α protein. P-gp protein levels of the cells of each group were determined by Western blot. In addition, HepG2 and HepG2/ADM were injected into the livers of the nude mice （NM） （named NM HepG2, NM ADM, NM BCT groups respectively）. The BCT group mice were treated with bromocriptine through gastric feedings. The sizes of the tumor growths in the livers were measured using B ultrasound. The MDRlmRNA levels in these tumor tissues were determined by reverse transcription polymerase chain reaction （RT-PCR） and the apoptosis rates of them were measured with TUNEL assay. ^99mTc-MIBI SPECT was performed to detect the tumor ^99mFc-MIBI accumulation index before and after the BCT treatment. Results The rate of reversing resistance to ADM by BCT was 45.68% shown by using MTT assay, and the intracellular Rho123 accumulation increased more than two times compared with the control group shown by flow cytometric assay at the concentration of 10 μmol/L BCT and the effect was time-dependent. Between group B and group C there was a significant difference in the expression of PKC- α protein by immunocytochemistry detection （q = 5.37, P 〈 0.01 ）, but there was no significant difference in the expression of P-gp protein between the two groups （q = 1.86, P 〉 0.05）.There was no notable difference of growth rates of the transplanted liver tumors among the three NM groups （F = 6.39, P 〉 0.05）, and the inhibition rate of tumor volume and weight in NM BCT was higher than that in NM ADM （q1 = 5.89, q2 = 4.92, P 〈 0.01）, but similar to that in NM HepG2 （q1 = 2.47, q2 = 3.02, P 〉 0.05）. No difference was detected in MDR 1 mRNA between NM ADM and NM BCT using RT-PCR （q = 3.71, P 〉 0.05）. The average number of apoptotic cells per high-power field （25.7 ± 1.8） in NM BCT tumor tissues was higher than that in group ADM （2.7 ± 0.2） （q = 3.72, P 〈 0.01 ）, but similar to that of NM HepG2 （23.9 ± 1.6） （q = 1.43, P 〉 0.05）. The uptake of ^99mTc-MIBI in all the NM after BCT therapy was significantly higher than that before the BCT treatment （t = 3.58, P 〈 0.01）. Conclusions BCT can reverse multidrug resistance of hepatocarcinomas by inhibiting the function of P-gp protein and can enhance the susceptibility of HepG2/ADM cells to cytotoxic drugs.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2006年第5期353-357,共5页
Chinese Journal of Hepatology
基金
卫生部临床学科重点项目(20012434)
关键词
癌
肝细胞
细胞凋亡
小鼠
裸
多药耐药
Carcinoma, hepatocellular
Apoptosis
Mice, nude
Multidmg resistance
