中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2011年
4期
321-326
,共6页
施媛萍%郑璐%罗光华%魏江%张俊%于洋
施媛萍%鄭璐%囉光華%魏江%張俊%于洋
시원평%정로%라광화%위강%장준%우양
原发性肝细胞肝癌%锌指蛋白%逆转录-聚合酶链反应%膜联蛋白V/碘化丙啶染色法%凋亡
原髮性肝細胞肝癌%鋅指蛋白%逆轉錄-聚閤酶鏈反應%膜聯蛋白V/碘化丙啶染色法%凋亡
원발성간세포간암%자지단백%역전록-취합매련반응%막련단백V/전화병정염색법%조망
Hepatocellular carcinoma%Human zinc finger%Reverse transcription-polymerase chain reaction%Annexin V-FITC/PI assay%Apoptosis
目的 分析hZNF23在人HCC组织及HepG2细胞系中的表达情况,以探讨其与HCC临床病理特征和细胞凋亡的关系.方法 采用实时荧光定量RT-PCR法检测37例HCC患者(按Edmondson分为两组,Ⅰ+Ⅱ级17例,Ⅲ+Ⅳ级20例;按临床分为3组,HCC合并肝硬化和HBV感染组31例,HCC合并肝硬化和HCC合并HRV感染组各3例)癌组织和其癌旁肝组织标本中hZNF23及内参GAPDH mRNA的表达水平,并分析其与HCC临床病理特征的关系;体外培养HepG2细胞后,分为4组(对照组、1.25、2.5和5 μg/ml顺铂组)或6组(对照组、1.25、2.5、5、10和20μg/ml顺铂组).采用四唑盐比色法(MTT)和流式细胞仪膜联蛋白V/碘化丙啶染色法检测与观察HepG2细胞的增殖及凋亡情况;实时荧光定量RT-PCR法检测HepG2细胞在不同浓度的顺铂诱导凋亡后,hZNF23及内参GAPDH mRNA的表达水平.结果 37例HCC患者癌组织及其癌旁组织标本中hZNF23相对表达量分别为8.84(3.59～15.05)和22.20(13.85～42.90),差异有统计学意义(U=259.5,P＜0.01).Edmondson Ⅰ+Ⅱ级HCC组织hZNF23 mRNA相对表达量为12.80(4.80～19.50),高于Ⅲ+Ⅳ级的5.01(2.88～11.68),且差异有统计学意义(U=99.00,P＜0.05);合并肝硬化组为9.92(3.80～15.25),未合并肝硬化组为3.21(2.78～3.60)、合并HBV感染组为9.09(3.72～15.25),无HBV感染组为2.48(1.79～12.10),合并肝硬化组与未合并肝硬化组、合并HBV感染组与未合并HBV感染组比较,其hZNF23 mRNA表达量差异均无统计学意义(U值分别为16.00和24.00,P均＞0.05).顺铂作用于HepG2细胞24 h后,用MTT检测,顺铂明显抑制HepG2细胞增殖;膜联蛋白V/碘化丙啶染色法检测细胞凋亡率依次为(0.9±0.2)%、(4.2±0.3)%、(9.8±4.3)%、(23.0±6.0)%,顺铂显著诱导HepG2细胞凋亡,且呈剂量依赖性(F=27.890,P＜0.01).实时荧光定量RT-PCR法检测1.25、2.5、5μg顺铂诱导的HepGR细胞组hZNF23 mRNA相对表达量依次为(10.39±3.08)×10-5、(24.10±2.09)×10-5、(6.90±2.24)×10-4,均显著高于对照组[(9.48±1.80)×10-5,F=6.027,P＜0.01].结论 HCC组织中hZNF23相对表达水平与Edmondson分级密切关联;顺铂对HepG2细胞的凋亡作用可能与hZNF23上调有关.
目的 分析hZNF23在人HCC組織及HepG2細胞繫中的錶達情況,以探討其與HCC臨床病理特徵和細胞凋亡的關繫.方法 採用實時熒光定量RT-PCR法檢測37例HCC患者(按Edmondson分為兩組,Ⅰ+Ⅱ級17例,Ⅲ+Ⅳ級20例;按臨床分為3組,HCC閤併肝硬化和HBV感染組31例,HCC閤併肝硬化和HCC閤併HRV感染組各3例)癌組織和其癌徬肝組織標本中hZNF23及內參GAPDH mRNA的錶達水平,併分析其與HCC臨床病理特徵的關繫;體外培養HepG2細胞後,分為4組(對照組、1.25、2.5和5 μg/ml順鉑組)或6組(對照組、1.25、2.5、5、10和20μg/ml順鉑組).採用四唑鹽比色法(MTT)和流式細胞儀膜聯蛋白V/碘化丙啶染色法檢測與觀察HepG2細胞的增殖及凋亡情況;實時熒光定量RT-PCR法檢測HepG2細胞在不同濃度的順鉑誘導凋亡後,hZNF23及內參GAPDH mRNA的錶達水平.結果 37例HCC患者癌組織及其癌徬組織標本中hZNF23相對錶達量分彆為8.84(3.59～15.05)和22.20(13.85～42.90),差異有統計學意義(U=259.5,P＜0.01).Edmondson Ⅰ+Ⅱ級HCC組織hZNF23 mRNA相對錶達量為12.80(4.80～19.50),高于Ⅲ+Ⅳ級的5.01(2.88～11.68),且差異有統計學意義(U=99.00,P＜0.05);閤併肝硬化組為9.92(3.80～15.25),未閤併肝硬化組為3.21(2.78～3.60)、閤併HBV感染組為9.09(3.72～15.25),無HBV感染組為2.48(1.79～12.10),閤併肝硬化組與未閤併肝硬化組、閤併HBV感染組與未閤併HBV感染組比較,其hZNF23 mRNA錶達量差異均無統計學意義(U值分彆為16.00和24.00,P均＞0.05).順鉑作用于HepG2細胞24 h後,用MTT檢測,順鉑明顯抑製HepG2細胞增殖;膜聯蛋白V/碘化丙啶染色法檢測細胞凋亡率依次為(0.9±0.2)%、(4.2±0.3)%、(9.8±4.3)%、(23.0±6.0)%,順鉑顯著誘導HepG2細胞凋亡,且呈劑量依賴性(F=27.890,P＜0.01).實時熒光定量RT-PCR法檢測1.25、2.5、5μg順鉑誘導的HepGR細胞組hZNF23 mRNA相對錶達量依次為(10.39±3.08)×10-5、(24.10±2.09)×10-5、(6.90±2.24)×10-4,均顯著高于對照組[(9.48±1.80)×10-5,F=6.027,P＜0.01].結論 HCC組織中hZNF23相對錶達水平與Edmondson分級密切關聯;順鉑對HepG2細胞的凋亡作用可能與hZNF23上調有關.
목적 분석hZNF23재인HCC조직급HepG2세포계중적표체정황,이탐토기여HCC림상병리특정화세포조망적관계.방법 채용실시형광정량RT-PCR법검측37례HCC환자(안Edmondson분위량조,Ⅰ+Ⅱ급17례,Ⅲ+Ⅳ급20례;안림상분위3조,HCC합병간경화화HBV감염조31례,HCC합병간경화화HCC합병HRV감염조각3례)암조직화기암방간조직표본중hZNF23급내삼GAPDH mRNA적표체수평,병분석기여HCC림상병리특정적관계;체외배양HepG2세포후,분위4조(대조조、1.25、2.5화5 μg/ml순박조)혹6조(대조조、1.25、2.5、5、10화20μg/ml순박조).채용사서염비색법(MTT)화류식세포의막련단백V/전화병정염색법검측여관찰HepG2세포적증식급조망정황;실시형광정량RT-PCR법검측HepG2세포재불동농도적순박유도조망후,hZNF23급내삼GAPDH mRNA적표체수평.결과 37례HCC환자암조직급기암방조직표본중hZNF23상대표체량분별위8.84(3.59～15.05)화22.20(13.85～42.90),차이유통계학의의(U=259.5,P＜0.01).Edmondson Ⅰ+Ⅱ급HCC조직hZNF23 mRNA상대표체량위12.80(4.80～19.50),고우Ⅲ+Ⅳ급적5.01(2.88～11.68),차차이유통계학의의(U=99.00,P＜0.05);합병간경화조위9.92(3.80～15.25),미합병간경화조위3.21(2.78～3.60)、합병HBV감염조위9.09(3.72～15.25),무HBV감염조위2.48(1.79～12.10),합병간경화조여미합병간경화조、합병HBV감염조여미합병HBV감염조비교,기hZNF23 mRNA표체량차이균무통계학의의(U치분별위16.00화24.00,P균＞0.05).순박작용우HepG2세포24 h후,용MTT검측,순박명현억제HepG2세포증식;막련단백V/전화병정염색법검측세포조망솔의차위(0.9±0.2)%、(4.2±0.3)%、(9.8±4.3)%、(23.0±6.0)%,순박현저유도HepG2세포조망,차정제량의뢰성(F=27.890,P＜0.01).실시형광정량RT-PCR법검측1.25、2.5、5μg순박유도적HepGR세포조hZNF23 mRNA상대표체량의차위(10.39±3.08)×10-5、(24.10±2.09)×10-5、(6.90±2.24)×10-4,균현저고우대조조[(9.48±1.80)×10-5,F=6.027,P＜0.01].결론 HCC조직중hZNF23상대표체수평여Edmondson분급밀절관련;순박대HepG2세포적조망작용가능여hZNF23상조유관.
Objective To detect the expression level of human zinc finger 23 (ZNF23) in hepatocellular carcinoma tissue samples and HepG2 cell lines and investigate the relationship between hZNF23 expression and clinicopathological characteristics of HCC and cell apoptosis. Methods The expression levels of hZNF23 and GAPDH mRNA in 37 cases of HCC were measured by real-time RT-PCR. The association between the expression of hZNF23 and the clinicopathological characteristics of HCC was analyzed. Cultured HepG2 cells were divided into 4 groups ( control group, 1.25 μg/ml , 2.5 μg/ml and 5 μg/ml cisplatin)or 6 groups( control group, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml cisplatin). MTT method was employed to evaluate cell proliferation. Annexin V-FITC assay was used to assess percentage of apoptotic HepG2 cells. The expression levels of hZNF23 and GAPDH mRNA of HepG2 cells after apoptosis induced by cisplatin with a series of concentrations were measured by real-time RT-PCR.Results The median ( quartile1, quartile 3) expression levels of hZNF23 mRNA in 37 HCC tissue samples and adjacent tissue samples were 8.84 (3.59-15.05), 22.20 ( 13.85-42.90 ), respectively. There was significant difference ( U = 259.5, P ＜ 0.01 ). The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in cancer tissue samples with Edmondson stage Ⅰ + Ⅱ [12.80(4.80-19.50)] was much higher than those in stage Ⅲ + Ⅳ [5.01 ( 2.88-11.68 ), U = 99.00, P ＜ 0.05] The median ( quartile1,quartile 3 ) expression levels of hZNF23 mRNA in patients with and without hepatic cirrhosis were 9.92(3.80-15.25) , 3.21 (2.78-3.60), respectively. The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in HBV infection and non-infection patients was 9.09(3.72-15.25 ), 2.48 (1.79-12.10),respectively. There was no significant difference between groups with and without hepatic cirrhosis and between HBV infection and non-infection groups( U = 16. 00 and 24.00, P ＞0.05 ). MTT assay indicated that cisplatin significantly inhibited HepG2 cells proliferation in a dose-dependent manner. Annexin V-FITC/PI assay showed that HepG2 cells apoptosis rates were (0.9 ± 0.2 ) %, ( 4. 2 ± 0.3 ) %, ( 9.8 ± 4. 3 ) %,(23.0 ± 6.0)%, respectively. Cisplatin significantly induced HepG2 cells apoptosis in a dose-dependent manner( F = 27.89, P ＜ 0.01 ). The expression levels of hZNF23 mRNA in cisplatin groups [( 10.39 ±3.08) × 10-5, (24.10 ± 2.09) × 10-5, (6.90 ± 2.24) × 10-4] were significantly lower than that of the controlgroup[(94.80±1.80) ×10-5, F=6.027, P＜0.01]. Conclusions The expression level hZNF23 mRNA is related to Edmondson stage of HCC. The apoptosis effect of cisplatin on HepG2 cells may be associated with the upregulation of hZNF23.
