【摘要】 目的 构建一种新型的更接近疾病规律的胰腺癌吉西他滨耐药细胞及动物模型。方法 选择胰腺癌细胞株PANC-1通过体外细胞培养,裸鼠皮下成瘤后瘤块移植构建胰腺癌动物模型,腹腔吉西他滨化疗,选取瘤体最大的动物模型,获取瘤块,进行分离提取瘤体细胞进行培养,用于下一循环的动物模型构建,如此循环2次,获得耐药细胞株及动物模型。将原代细胞和耐药细胞分别进行体外及体内吉西他滨的耐药试验,分析验证耐药模型。通过微卫星序列测定鉴定耐药模型与亲代细胞的同源性。结果 建立胰腺癌吉西他滨耐药的细胞及动物模型,第一循环原位移植成功率为73.3%,吉西他滨腹腔灌注后死亡率为18.2%,瘤体最大的5个均值为(1.76±0.87)g,远处转移率11.1%;第二循环原位移植成功率80%,吉西他滨腹腔灌注后死亡率为8.3%,瘤体最大的5个均值为(2.16±1.12)g,远处转移率36.3%。两循环比较,瘤体大小及远处转移率差异有统计学意义(P<0.05)。将第2循环后培养的胰腺癌吉西他滨耐药命名为PANC-1/R2。在5μg/dl、10μg/dl和20μg/dl浓度的吉西他滨作用下PANC-1和PANC-1/R2的抑制率分别是21.8%、37.1%、46.1%和12.6%、25.3%、36.8%。PANC-1和PANC-1/R2裸鼠皮下移植后3周成瘤,吉西他滨240mg/kg腹腔注射,1次/周,共4次,35d后瘤体大小分别是(1.87±0.69)g和(2.09±0.71)g,差异有统计学意义(P<0.05)。人类基因组微卫星D14S68、D18S69、D20S199检测结果显示,亲代胰腺癌细胞、吉西他滨耐药胰腺癌细胞三个位点微卫星扩增大小完全相同的片段,而荷瘤裸鼠胰腺组织没有扩增出相应的带型。结论 应用体外细胞培养,体内化疗药物诱导的方法是可行的,该体系可用于各种耐药细胞的筛选,与原代细胞相比,筛选后的细胞明显耐药性增强。且构建的细胞株更符合机体多因素复杂环境下的耐药产生过程,是理想的耐药模型。且该方法构建的耐药细胞与亲代细胞具有相同的遗传位点,具有遗传同源性。但构建过程长,需要熟练和细致的操作,提高成功率,增加循环次数或可筛选更具耐药特性的细胞模型。更多还原

【Abstract】 Objective To establish new cell and animal gemcitabine-resistant models for pancreatic carcinoma, which mimic the pathologic processes of gemcitabine-resistance of pancreatic carcinoma in clinic. Methods The pancreatic cancer cell line PANC-1 cells were cultured in vitro,which were injected to subskin of nude mice. After formation of tumor mass,the mass was transplanted to tail of pancreas of nude mice to establish the animal models of pancreatic cancer. The mice were dealt with gemcitabine intraperitoneal chemotherapy, the mice with the largest tumor were selected and then digested and separated to gain tumor cells. The cells were used to establish the animal models of pancreatic cancer. The progresses above were recycled by two times to obtain resistant cell lines and animal models. In vivo and in vitro tests were applied to verify resistance of models to gemcitabine.The drug-resistant models and parental cells were identified by homologous microsatellites sequencing. Results The cells and animal gemcitabineresistant models of pancreatic carcinoma were established successfully. In the first cycle, the successfiil rate of pancreatic carcinoma was 73.3%(11/15),mortality after gemcitabine intraperitoneal chemotherapy was 18.2%( 2/11). The mean weight of five largest tumor was( 1.76 ± 0.87) g,and the distal metastatic rate was 11.1%( 1/9). In the second cycle, the successful rate of pancreatic carcinoma was 80%( 12/15), mortality after gemcitabine intraperitoneal chemotherapy was 8.3%( 1/12). The mean weight of five largest tumor was( 2.16 ± 1.12) g,and the distal metastatic rate was 36.3%(4/11). The weight of five largest tumor and distal metastatic rate between two groups were significantly different( P<0.05). The cell line which was screened by two cycles was named PANC-1/R2. The inhibition rates of PANC-1 and PANC-1/R2 were 21.8%, 37.1%, 46.1%and 12.6%, 25.3%,36.8% individually under dealing with gemcitabine on 5 μg/dl, 10μg/dl and 20μg/dl concentration respectively. Subcutaneous tumor of PANC-1 and PANC-I/R2 were formed for three weeks, gemcitabine 240 mg/kg intraperitoneal injection per week and for 4 times, after 35 days, the tumor size were( 1.87±0.69) g and(2.09±0.71) g,the difference was statistically significant(P<0.05).The results of human genome microsatellite D14 S68,D18 S69, D20 S199 showed that parental pancreatic cancer cells, gemcitabine-resistant pancreatic cancer cells in three microsatellite loci amplified fragments of the same size, and pancreatic tissue of nude mice did not amplify the corresponding banding patterns. Conclusions The recycle-screening technique of establishing gemcitabine-resistant models for pancreatic carcinoma is feasible. The cells screened by the technique obtain significantly enhances resistance, compared with the primary cells. The models mimic the process of the body’s resistance with influence of multi-factor complex environment.And the cell models are constructed with the same parent cell genetic loci and genetic homology. But the process is complicated and takes a long time.Skilled and careful operation benefits the success rate. Increasing the times of recycles may enhance the resistant characteristics.更多还原