三种方法建立的人肝癌多药耐药细胞系Bel-7402/ADM模型比较
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摘要
目的
比较体外浓度梯度递增诱导、裸鼠肝脏和皮下移植诱导三种方法建立人肝癌多药耐药细胞系Bel-7402/ADM模型的生物学特性。
方法
分别采用体外浓度梯度递增诱导、裸鼠肝脏和皮下移植诱导建立人肝癌阿霉素多药耐药细胞亚系Bel-7402/ADMV,Bel-7402/ADML和Bel-7402/ADMS后,利用相差显微镜观察细胞形态,MTT法检测三种细胞对多种化疗药物的敏感性,细胞计数法绘制生长曲线并用公式法计算倍增时间,流式细胞仪测定细胞ADM的摄入和外排以及P糖蛋白(P-gp)、多药耐药相关蛋白(MRP)、谷胱甘肽硫转酶系统(GSH/ GST )的表达。
结果
1.细胞形态学观察:相差显微镜下见三组耐药细胞生长速度变慢,形态差别不大,均不规则,比亲代体积略增大,细胞核略皱缩,裸鼠体内移植耐药细胞外形更接近亲本。
2.对抗癌药的敏感性:三组耐药细胞对ADM、CDDP都产生了交叉耐药性,较亲本Bel-7402 IC50值差异有显著性(P<0.01),而Bel-7402/ADMV IC50值最高,因此具有更强的耐药特性。对MMC、MTX和5-FU均无交叉耐药。
3.生长曲线与倍增时间:耐药细胞较亲本细胞的倍增时间明显延长,Bel-7402、Bel-7402/ADMS、Bel-7402/ADML和Bel-7402/ADMV倍增时间分别为:39h、45h、46h和65h。
4.ADM的摄入和外排:在相同条件下,Bel-7402/ADMV的ADM外排率最高, Bel-7402/ADML及Bel-7402/ADMS次之,亲本最少。四者对ADM的外排率分别为:81.06%、66.56%、61.56%和34.14%。
5.流式细胞技术测定P-gp、MRP、GSH/GST表达的变化:三组耐药细胞P-gp、MRP表达较亲本细胞显著提高(P<0.01),而GSH/GST的表达无明显变化(P>0.05)。
结论
1.三种方法建立的人肝癌阿霉素多药耐药细胞亚系Bel-7402/ADM模型均有稳定的耐药性。
2.体外浓度梯度递增法建立的耐药细胞模型耐药指数及稳定性均较高,缺点是细胞增殖能力低,同时诱导耐药过程耗时长,诱导过程容易引起污染。
3.采用裸鼠肝脏和皮下移植诱导法建立的人肝癌阿霉素多药耐药细胞系耐药性稍差,优点是诱导耗时短,污染可能小,细胞增殖能力较强,更接近临床耐药现象,尤其是肝脏移植法更能高度模拟人肝癌,它是具有近似人肝癌生物学和抗癌药物动力学特征的较理想模型。
Objective
To compare biological characteristics of three types of human hepatic carcinoma multi- drug resistant cell line models Bel-7402/ADM established by vitro concentration gradient increased progressively induction, nude mice liver implanted and subcutaneous implanted induction.
Methods
Established human hepatic carcinoma adriamycin multi-drug resistant cell line models Bel-7402/ADMV,Bel-7402/ADML and Bel-7402/ADMS by three methods of vitro concentration gradient increased progressively induction, nude mice liver implanted and subcutaneous implanted induction respectively. phase contrast micro- scope was used to observed the shape of cells, MTT assay was used to evaluate several kinds of cells drugs sensitivity, growth curves were drawn by cell counting and doubling time of cell lines were calculated according to formula, flow cytometric assay was used to examine cells adriamycin influx and efflux and the expression of p-glycoprotein(P-gp), multi- drug resistance protein (MRP) and glutathione S-transferase (GSH/GST).
Results
1. Cells morphology observation. Under the phase contrast microscope, growth speed about three kinds of multi-drug resistant cells reduced, the shape were not different obviously, all were irregular, the volume was bigger slightly than the parental genera- tion, the cell nucleus shrinked lightly, nude mice implanted resistant cells shape closer to the parental generation.
2. Cells sensitivity on anticarcinogens. Three groups of cells were resistant to ADM and CDDP, compared with the parental generation, IC50 were significant difference (P< 0.01), and the IC50 of Bel- 7402/ADMV was the highest, so it had greater resistance. All the cells were not resistant to MMC, MTX and 5-Fu.
3. growth curve and doubling time. The doubling time of resistant cells were extended obviously to the parental generation, Bel-7402, Bel-7402/ADMS, Bel-7402/ADML and Bel-7402/ADMV doubling time were: 39h, 45h, 46h and 65h.
4. Cells ADM influx and efflux. in the same conditions, the Bel-7402/ADMV had max- imum ADM efflux rate, Bel-7402/ADML and Bel-7402/ADMS were Secondly, the par- ental generation was minimum. The ADM discharge rate of four cells were: 81.06%, 66.56%, 61.56% and% 34.14%.
5. Flow cytometry determine the expression of P-gp, MRP and GSH/GST. Compared with the parental generation, three kinds of multidrug resistant cells P-gp、MRP were significantly improve (P<0.01), and the expression of GSH/GST without obvious changes (P>0.05).
Conclusions
1. Three multidrug resistant cell line models of human hepatic carcinoma Bel-7402/ ADM established by three different methods separately have stable resistance.
2. The multidrug resistant cell model established by vitro concentration gradient increased progressively induction had higher resistance index and stability, but the disadvantage was that the proliferation ability is lower, induced-process is time-consuming, and easily lead to pollution.
3. The resistance index of the multidrug resistant cell lines established by nude mice liver implanted and subcutaneous implanted induction were lower slightly, its advantage was the time-expended is shorter, the chance of pollution is smaller, the proliferation ability is stronger, so this two cell lines closer to clinical drug resistance, the nude mice liver implanted one highly simulated human hepatocarcinoma especially, it was a better model because of approximating human hepatic carcinoma biology and anticancer pharmacokinetics characteristics.
比较体外浓度梯度递增诱导、裸鼠肝脏和皮下移植诱导三种方法建立人肝癌多药耐药细胞系Bel-7402/ADM模型的生物学特性。
方法
分别采用体外浓度梯度递增诱导、裸鼠肝脏和皮下移植诱导建立人肝癌阿霉素多药耐药细胞亚系Bel-7402/ADMV,Bel-7402/ADML和Bel-7402/ADMS后,利用相差显微镜观察细胞形态,MTT法检测三种细胞对多种化疗药物的敏感性,细胞计数法绘制生长曲线并用公式法计算倍增时间,流式细胞仪测定细胞ADM的摄入和外排以及P糖蛋白(P-gp)、多药耐药相关蛋白(MRP)、谷胱甘肽硫转酶系统(GSH/ GST )的表达。
结果
1.细胞形态学观察:相差显微镜下见三组耐药细胞生长速度变慢,形态差别不大,均不规则,比亲代体积略增大,细胞核略皱缩,裸鼠体内移植耐药细胞外形更接近亲本。
2.对抗癌药的敏感性:三组耐药细胞对ADM、CDDP都产生了交叉耐药性,较亲本Bel-7402 IC50值差异有显著性(P<0.01),而Bel-7402/ADMV IC50值最高,因此具有更强的耐药特性。对MMC、MTX和5-FU均无交叉耐药。
3.生长曲线与倍增时间:耐药细胞较亲本细胞的倍增时间明显延长,Bel-7402、Bel-7402/ADMS、Bel-7402/ADML和Bel-7402/ADMV倍增时间分别为:39h、45h、46h和65h。
4.ADM的摄入和外排:在相同条件下,Bel-7402/ADMV的ADM外排率最高, Bel-7402/ADML及Bel-7402/ADMS次之,亲本最少。四者对ADM的外排率分别为:81.06%、66.56%、61.56%和34.14%。
5.流式细胞技术测定P-gp、MRP、GSH/GST表达的变化:三组耐药细胞P-gp、MRP表达较亲本细胞显著提高(P<0.01),而GSH/GST的表达无明显变化(P>0.05)。
结论
1.三种方法建立的人肝癌阿霉素多药耐药细胞亚系Bel-7402/ADM模型均有稳定的耐药性。
2.体外浓度梯度递增法建立的耐药细胞模型耐药指数及稳定性均较高,缺点是细胞增殖能力低,同时诱导耐药过程耗时长,诱导过程容易引起污染。
3.采用裸鼠肝脏和皮下移植诱导法建立的人肝癌阿霉素多药耐药细胞系耐药性稍差,优点是诱导耗时短,污染可能小,细胞增殖能力较强,更接近临床耐药现象,尤其是肝脏移植法更能高度模拟人肝癌,它是具有近似人肝癌生物学和抗癌药物动力学特征的较理想模型。
Objective
To compare biological characteristics of three types of human hepatic carcinoma multi- drug resistant cell line models Bel-7402/ADM established by vitro concentration gradient increased progressively induction, nude mice liver implanted and subcutaneous implanted induction.
Methods
Established human hepatic carcinoma adriamycin multi-drug resistant cell line models Bel-7402/ADMV,Bel-7402/ADML and Bel-7402/ADMS by three methods of vitro concentration gradient increased progressively induction, nude mice liver implanted and subcutaneous implanted induction respectively. phase contrast micro- scope was used to observed the shape of cells, MTT assay was used to evaluate several kinds of cells drugs sensitivity, growth curves were drawn by cell counting and doubling time of cell lines were calculated according to formula, flow cytometric assay was used to examine cells adriamycin influx and efflux and the expression of p-glycoprotein(P-gp), multi- drug resistance protein (MRP) and glutathione S-transferase (GSH/GST).
Results
1. Cells morphology observation. Under the phase contrast microscope, growth speed about three kinds of multi-drug resistant cells reduced, the shape were not different obviously, all were irregular, the volume was bigger slightly than the parental genera- tion, the cell nucleus shrinked lightly, nude mice implanted resistant cells shape closer to the parental generation.
2. Cells sensitivity on anticarcinogens. Three groups of cells were resistant to ADM and CDDP, compared with the parental generation, IC50 were significant difference (P< 0.01), and the IC50 of Bel- 7402/ADMV was the highest, so it had greater resistance. All the cells were not resistant to MMC, MTX and 5-Fu.
3. growth curve and doubling time. The doubling time of resistant cells were extended obviously to the parental generation, Bel-7402, Bel-7402/ADMS, Bel-7402/ADML and Bel-7402/ADMV doubling time were: 39h, 45h, 46h and 65h.
4. Cells ADM influx and efflux. in the same conditions, the Bel-7402/ADMV had max- imum ADM efflux rate, Bel-7402/ADML and Bel-7402/ADMS were Secondly, the par- ental generation was minimum. The ADM discharge rate of four cells were: 81.06%, 66.56%, 61.56% and% 34.14%.
5. Flow cytometry determine the expression of P-gp, MRP and GSH/GST. Compared with the parental generation, three kinds of multidrug resistant cells P-gp、MRP were significantly improve (P<0.01), and the expression of GSH/GST without obvious changes (P>0.05).
Conclusions
1. Three multidrug resistant cell line models of human hepatic carcinoma Bel-7402/ ADM established by three different methods separately have stable resistance.
2. The multidrug resistant cell model established by vitro concentration gradient increased progressively induction had higher resistance index and stability, but the disadvantage was that the proliferation ability is lower, induced-process is time-consuming, and easily lead to pollution.
3. The resistance index of the multidrug resistant cell lines established by nude mice liver implanted and subcutaneous implanted induction were lower slightly, its advantage was the time-expended is shorter, the chance of pollution is smaller, the proliferation ability is stronger, so this two cell lines closer to clinical drug resistance, the nude mice liver implanted one highly simulated human hepatocarcinoma especially, it was a better model because of approximating human hepatic carcinoma biology and anticancer pharmacokinetics characteristics.
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