中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2014年
7期
517-522
,共6页
汤小晗%卢美松%李翠萍%邓锁%李萌
湯小晗%盧美鬆%李翠萍%鄧鎖%李萌
탕소함%로미송%리취평%산쇄%리맹
卵巢肿瘤%胞间信号肽类和蛋白质类%细菌蛋白质类%紫杉酚%抗药性,肿瘤%细胞系,肿瘤
卵巢腫瘤%胞間信號肽類和蛋白質類%細菌蛋白質類%紫杉酚%抗藥性,腫瘤%細胞繫,腫瘤
란소종류%포간신호태류화단백질류%세균단백질류%자삼분%항약성,종류%세포계,종류
Ovarian neoplasms%Intercellular signaling peptides and proteins%Bacterial proteins%Paclitaxel%Drug resistance,neoplasm%Cell line,tumor
目的:检测肝素结合表皮生长因子(HB-EGF)在紫杉醇耐药卵巢上皮性癌(卵巢癌)细胞和组织中的表达水平,并探讨HB-EGF与卵巢癌紫杉醇耐药性的关系。方法(1)体外实验:蛋白印迹法检测卵巢癌细胞株A2780(紫杉醇敏感)和A2780/Taxol(紫杉醇耐药)细胞中HB-EGF蛋白的表达。体外细胞实验分为4组,A2780+CRM197组、A2780/Taxol+CRM197组、A2780组、A2780/Taxol组,采用四甲基偶氮唑蓝(MTT)比色法检测4组细胞的增殖能力[以吸光度(A)值表示],流式细胞仪检测4组细胞的细胞周期比例,酶标仪检测4组细胞中半胱氨酸天冬氨酸蛋白酶3(caspase-3)蛋白的活性[以4硝基苯胺(pNa)浓度表示]。(2)体内实验:将A2780和A2780/Taxol细胞分别接种于裸鼠腋下,建立卵巢癌裸鼠移植瘤模型;免疫组化SP法检测裸鼠移植瘤组织中HB-EGF蛋白的表达。体内动物实验分为4组,方法同体外细胞实验分组,每组5只裸鼠,观察4组裸鼠移植瘤的生长情况,并计算抑瘤率。结果(1)体外实验结果:A2780/Taxol细胞中HB-EGF蛋白的表达水平为2.11±0.41,明显高于A2780细胞的0.75±0.20(P<0.01)。A2780+CRM197组、A2780/Taxol+CRM197组细胞增殖的抑制作用随CRM197浓度的增加而增强,呈明显的浓度依赖性(P<0.01),且当CRM197浓度≥1μg/ml时,A2780/Taxol+CRM197组细胞增殖的抑制作用明显高于A2780+CRM197组细胞(P<0.05);A2780+CRM197组细胞G0/G1期比例[(67±4)%]高于A2780组[(54±6)%],A2780/Taxol+CRM197组细胞G0/G1期比例[(72±4)%]高于A2780/Taxol组[(24±8)%],分别比较,差异均有统计学意义(P<0.01);A2780+CRM197组细胞中pNa浓度[(40±6)μmol/L,即caspase-3蛋白活性]高于A2780组[(6±6)μmol/L],A2780/Taxol+CRM197组细胞中pNa浓度[(66±12)μmol/L]高于A2780/Taxol组[(9±6)μmol/L],分别比较,差异均有统计学意义(P<0.01)。(2)体内实验结果:接种A2780和A2780/Taxol细胞的裸鼠移植瘤组织中HB-EGF蛋白的表达水平分别为(5.0±2.2)、(10.8±3.3)分,两者比较,差异有统计学意义(P<0.01)。A2780/Taxol+CRM197组肿瘤体积和肿瘤质量分别为(546±85)mm3和(0.56±0.09)g,明显低于A2780/Taxol组的(1840±145)mm3和(1.76±0.23)g,A2780+CRM197组肿瘤体积和肿瘤质量分别为(818±63)mm3和(0.74±0.15)g,明显低于A2780组的(1355±119)mm3和(1.31±0.27)g,分别比较,差异均有统计学意义(P<0.05)。A2780/Taxol+CRM197组的抑瘤率为68%,高于A2780+CRM197组的43%;A2780/Taxol+CRM197组的抑瘤率为68%,高于A2780+CRM197组的43%,分别比较,差异均有统计学意义(P<0.01)。结论紫杉醇耐药的卵巢癌细胞和组织中HB-EGF蛋白均高表达,与卵巢癌对紫杉醇的耐药性相关。抑制HB-EGF蛋白的表达可有效促进紫杉醇耐药卵巢癌细胞凋亡,抑制其生长。
目的:檢測肝素結閤錶皮生長因子(HB-EGF)在紫杉醇耐藥卵巢上皮性癌(卵巢癌)細胞和組織中的錶達水平,併探討HB-EGF與卵巢癌紫杉醇耐藥性的關繫。方法(1)體外實驗:蛋白印跡法檢測卵巢癌細胞株A2780(紫杉醇敏感)和A2780/Taxol(紫杉醇耐藥)細胞中HB-EGF蛋白的錶達。體外細胞實驗分為4組,A2780+CRM197組、A2780/Taxol+CRM197組、A2780組、A2780/Taxol組,採用四甲基偶氮唑藍(MTT)比色法檢測4組細胞的增殖能力[以吸光度(A)值錶示],流式細胞儀檢測4組細胞的細胞週期比例,酶標儀檢測4組細胞中半胱氨痠天鼕氨痠蛋白酶3(caspase-3)蛋白的活性[以4硝基苯胺(pNa)濃度錶示]。(2)體內實驗:將A2780和A2780/Taxol細胞分彆接種于裸鼠腋下,建立卵巢癌裸鼠移植瘤模型;免疫組化SP法檢測裸鼠移植瘤組織中HB-EGF蛋白的錶達。體內動物實驗分為4組,方法同體外細胞實驗分組,每組5隻裸鼠,觀察4組裸鼠移植瘤的生長情況,併計算抑瘤率。結果(1)體外實驗結果:A2780/Taxol細胞中HB-EGF蛋白的錶達水平為2.11±0.41,明顯高于A2780細胞的0.75±0.20(P<0.01)。A2780+CRM197組、A2780/Taxol+CRM197組細胞增殖的抑製作用隨CRM197濃度的增加而增彊,呈明顯的濃度依賴性(P<0.01),且噹CRM197濃度≥1μg/ml時,A2780/Taxol+CRM197組細胞增殖的抑製作用明顯高于A2780+CRM197組細胞(P<0.05);A2780+CRM197組細胞G0/G1期比例[(67±4)%]高于A2780組[(54±6)%],A2780/Taxol+CRM197組細胞G0/G1期比例[(72±4)%]高于A2780/Taxol組[(24±8)%],分彆比較,差異均有統計學意義(P<0.01);A2780+CRM197組細胞中pNa濃度[(40±6)μmol/L,即caspase-3蛋白活性]高于A2780組[(6±6)μmol/L],A2780/Taxol+CRM197組細胞中pNa濃度[(66±12)μmol/L]高于A2780/Taxol組[(9±6)μmol/L],分彆比較,差異均有統計學意義(P<0.01)。(2)體內實驗結果:接種A2780和A2780/Taxol細胞的裸鼠移植瘤組織中HB-EGF蛋白的錶達水平分彆為(5.0±2.2)、(10.8±3.3)分,兩者比較,差異有統計學意義(P<0.01)。A2780/Taxol+CRM197組腫瘤體積和腫瘤質量分彆為(546±85)mm3和(0.56±0.09)g,明顯低于A2780/Taxol組的(1840±145)mm3和(1.76±0.23)g,A2780+CRM197組腫瘤體積和腫瘤質量分彆為(818±63)mm3和(0.74±0.15)g,明顯低于A2780組的(1355±119)mm3和(1.31±0.27)g,分彆比較,差異均有統計學意義(P<0.05)。A2780/Taxol+CRM197組的抑瘤率為68%,高于A2780+CRM197組的43%;A2780/Taxol+CRM197組的抑瘤率為68%,高于A2780+CRM197組的43%,分彆比較,差異均有統計學意義(P<0.01)。結論紫杉醇耐藥的卵巢癌細胞和組織中HB-EGF蛋白均高錶達,與卵巢癌對紫杉醇的耐藥性相關。抑製HB-EGF蛋白的錶達可有效促進紫杉醇耐藥卵巢癌細胞凋亡,抑製其生長。
목적:검측간소결합표피생장인자(HB-EGF)재자삼순내약란소상피성암(란소암)세포화조직중적표체수평,병탐토HB-EGF여란소암자삼순내약성적관계。방법(1)체외실험:단백인적법검측란소암세포주A2780(자삼순민감)화A2780/Taxol(자삼순내약)세포중HB-EGF단백적표체。체외세포실험분위4조,A2780+CRM197조、A2780/Taxol+CRM197조、A2780조、A2780/Taxol조,채용사갑기우담서람(MTT)비색법검측4조세포적증식능력[이흡광도(A)치표시],류식세포의검측4조세포적세포주기비례,매표의검측4조세포중반광안산천동안산단백매3(caspase-3)단백적활성[이4초기분알(pNa)농도표시]。(2)체내실험:장A2780화A2780/Taxol세포분별접충우라서액하,건립란소암라서이식류모형;면역조화SP법검측라서이식류조직중HB-EGF단백적표체。체내동물실험분위4조,방법동체외세포실험분조,매조5지라서,관찰4조라서이식류적생장정황,병계산억류솔。결과(1)체외실험결과:A2780/Taxol세포중HB-EGF단백적표체수평위2.11±0.41,명현고우A2780세포적0.75±0.20(P<0.01)。A2780+CRM197조、A2780/Taxol+CRM197조세포증식적억제작용수CRM197농도적증가이증강,정명현적농도의뢰성(P<0.01),차당CRM197농도≥1μg/ml시,A2780/Taxol+CRM197조세포증식적억제작용명현고우A2780+CRM197조세포(P<0.05);A2780+CRM197조세포G0/G1기비례[(67±4)%]고우A2780조[(54±6)%],A2780/Taxol+CRM197조세포G0/G1기비례[(72±4)%]고우A2780/Taxol조[(24±8)%],분별비교,차이균유통계학의의(P<0.01);A2780+CRM197조세포중pNa농도[(40±6)μmol/L,즉caspase-3단백활성]고우A2780조[(6±6)μmol/L],A2780/Taxol+CRM197조세포중pNa농도[(66±12)μmol/L]고우A2780/Taxol조[(9±6)μmol/L],분별비교,차이균유통계학의의(P<0.01)。(2)체내실험결과:접충A2780화A2780/Taxol세포적라서이식류조직중HB-EGF단백적표체수평분별위(5.0±2.2)、(10.8±3.3)분,량자비교,차이유통계학의의(P<0.01)。A2780/Taxol+CRM197조종류체적화종류질량분별위(546±85)mm3화(0.56±0.09)g,명현저우A2780/Taxol조적(1840±145)mm3화(1.76±0.23)g,A2780+CRM197조종류체적화종류질량분별위(818±63)mm3화(0.74±0.15)g,명현저우A2780조적(1355±119)mm3화(1.31±0.27)g,분별비교,차이균유통계학의의(P<0.05)。A2780/Taxol+CRM197조적억류솔위68%,고우A2780+CRM197조적43%;A2780/Taxol+CRM197조적억류솔위68%,고우A2780+CRM197조적43%,분별비교,차이균유통계학의의(P<0.01)。결론자삼순내약적란소암세포화조직중HB-EGF단백균고표체,여란소암대자삼순적내약성상관。억제HB-EGF단백적표체가유효촉진자삼순내약란소암세포조망,억제기생장。
Objective To examine the expression of heparin binding-epidermal growth factor-like growth factor (HB-EGF) in paclitaxel-resistant ovarian cancer and elucidate the relationship between HB-EGF and the resistance of ovarian cancer to paclitaxel. Methods The human ovarian carcinoma cell line A2780 and the paclitaxel-resistant human ovarian carcinoma cell line A2780/Taxol were cultured in vitro. Western blot was used to dectect the expression of HB-EGF protein in A2780 and A2780/Taxol groups. The A2780 cells were treated with cross-reacting material 197 (CRM197 and A2780 + CRM197 group) or dimethyl sulphoxide (DMSO;A2780 group), while the A2780/Taxol cells were treated with CRM197 (A2780/Taxol+CRM197 group) or DMSO (A2780/Taxol group). The effects of CRM197 on growth and proliferation was tested by methyl thiazolyl tetrazolium( MTT) and the results were showed as absorbance (A).The effects of CRM197 on cell cycles was tested by flow cytometry, while the effects of CRM197 on apoptosis was examined by caspase-3 activity assay and the results were showed as p-nitroaniline(pNa). In animal experiment, four groups of cells were inoculated to BALB/c nude mouse subcutaneously to observe tumor formation ability following CRM197 treatment. Immunohistochemistry was used to determine the expression of HB-EGF protein in A2780 and A2780/Taxol group. Results The expression level of HB-EGF protein in A2780/Taxol group (2.11±0.41) was significantly higher than that of A2780 group (0.75±0.20;P<0.01). The inhibition effect of CRM197 on the cell growth of A2780+CRM197 and A2780/Taxol+CRM197 group was accompanied by the acceleration of CRM197 concentration(P<0.01). When CRM197≥1 μg/ml, the inhibition effect of CRM197 on the cell growth of A2780/Taxol+CRM197 group was significantly higher than that in A2780/Taxol group(P<0.05). In cell cycle experiment, CRM197 induced the cell-cycle arrest at the G0/G1 phase in A2780+CRM197 cells[(67 ± 4)%] compared with A2780 cells[(54 ± 6)%;P<0.01], while CRM197 significantly induced the cell-cycle arrest at the G0/G1 phase in A2780/Taxol+CRM197 cells [(72± 4)%] compared with A2780/Taxol cells [(24±8)%;P<0.01]. CRM197 treatment in A2780+CRM197 group [(40 ± 6)μmol/L] led to the acceleration of the caspase-3 activity when compared to A2780 group [(6 ± 6)μmol/L;P<0.01], while CRM197 treatment in A2780/Taxol+CRM197 group [(66 ± 12)μmol/L] led to significant acceleration of the caspase-3 activity when compared to A2780 group [(9 ± 6)μmol/L;P<0.01]. In experiments in vivo, the expression scores of HB-EGF protein in A2780/Taxol tumors(10.8 ± 3.3) were higher than that in A2780 tumors (5.0±2.2;P<0.01). The tumor size and tumor weight of the A2780/Taxol+CRM197 group were both higher than those of the A2780+CRM197 group [(546 ± 85) mm3 vs (1 355 ± 119) mm3,(0.56 ± 0.09) g vs (1.31 ± 0.27) g; all P<0.01]. The CRM197 inhibition rate of the A2780+CRM197 and A2780/Taxol + CRM197 group were 43% and 68% respectively, showed that CRM197 significantly suppressed the growth of A2780/Taxol xenografts in vivo(P<0.01). Conclusions HB-EGF is over-expressed in paclitaxel-resistant ovarian cancer and may be contributes to drug resistance. Inhibition of HB-EGF expression potently enhances apoptosis and inhibit the growth of paclitaxel-resistant ovarian cancer, shedding light on the HB-EGF-targeted therapy options for chemoresistant ovarian cancer patients.