中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
Chinese Journal of Hepatology
2015年
11期
837-843
,共7页
癌,肝细胞%细胞凋亡%慢病毒%自杀基因%肿瘤抑素
癌,肝細胞%細胞凋亡%慢病毒%自殺基因%腫瘤抑素
암,간세포%세포조망%만병독%자살기인%종류억소
Carcinoma,hepatocellular%Apoptosis%Lentivirus%Suicide gene%Tumstatin
目的 观察重组慢病毒Lv-hTERTp-TK与Lv-hTERTp-tumstatin在肝癌HepG2细胞中的靶向表达,探讨二者联合在体内外对HepG2细胞的抑制作用. 方法 用不同MOI重组慢病毒Lv-hTERTp-TK、 Lv-hTERTp-tumstatin转染肝癌细胞HepG2、正常肝细胞L02,72 h后荧光显微镜观察转染情况;逆转录PCR检测TK、 tumstatin mRNA在HepG2、 L02细胞中的表达;四甲基偶氮唑蓝法观察两种细胞增殖情况;流式细胞术检测两种细胞凋亡情况;RT-PCR、 Western blot检测HepG2细胞bcl-2、血管内皮生长因子(VEGF) mRNA和蛋白的表达;将HepG2细胞接种于30只BALB/c裸鼠皮下,构建动物移植瘤模型,随机分为5组,空白对照组、空载体组、tumstatm组、TK组及联合组,每组6只,瘤内分别注射磷酸盐缓冲溶液、空载体慢病毒Lv-hTERTp-eGFP、重组慢病毒Lv-hTERTp-tumstatin、重组慢病毒Lv-hTERTp-TK和重组慢病毒Lv-hTERTp-tumstatin+Lv-hTERTp-TK;4周后处死裸鼠,观察各组移植瘤体积及质量的变化;行HE染色进行各组肿瘤组织及主要脏器的形态学观察;免疫组织化学法检测肿瘤内微血管密度;TUNEL法检测肿瘤细胞凋亡率.数据采用方差分析. 结果 转染后TK、 tumstatin基因在HepG2细胞中特异表达,在L02细胞中无表达.TK组、tumstatin组及联合组均对HepG2细胞生长有抑制作用,联合组抑制率明显高于单基因作用组(F=731.679,P＜0.05),各组L02细胞抑制率差异无统计学意义(F=0.774,P＞0.05);联合组HepG2细胞总凋亡率达72.61％±3.29％,明显高于TK组(53.63％±3.06％)、tumstatin组(45.13％±2.15％)、空白对照组(13.29％±1.15％)(F=365.022,P＜0.05),各组L02细胞凋亡率差异无统计学意义(F=0.391,P＞0.05);与对照组相比,实验组HepG2细胞bcl-2及VEGF表达水平均下降(bcl-2 mRNA:F=322.780,P＜0.05;VEGF mRNA:F=561.136,P＜0.05;bcl-2蛋白:F=34.203,P＜0.05;VEGF蛋白:F=32.988,P＜0.05),其中联合组bcl-2及VEGF表达水平均低于单基因组(P值均＜0.05).tumstatin组、TK组和联合组的抑瘤率分别为40.68％、 44.96％和72.09％,联合组抑瘤效果最显著(P＜0.05);HE染色显示联合组肿瘤细胞凋亡最明显.与空白对照组相比,其他各组裸鼠体内肝、脾和肾等主要脏器组织形态学均无显著变化;空白对照组、空载体组、TK组、tumstatin组和联合组的微血管密度分别为28.4±4.28、29.2±2.59、20.8±3.35、 14.2±4.87、5.8±2.77,tumstatin组抗肿瘤血管生成作用强于TK组(P＜0.05),且联合组作用最明显(P＜0.05).联合组肿瘤细胞凋亡率为70.11％±5.67％,明显高于tumstatin组(23.75％±5.21％)、TK组(48.30％±4.99％)、空白对照组(3.89％±1.13％)(P＜0.05),其中TK组的促凋亡作用较tumstatin组显著(P＜0.05). 结论 hTERT启动子驱动TK、 tumstatin在HepG2细胞中靶向表达,两种基因联合在体内外显著抑制HepG2细胞增殖和促进HepG2细胞凋亡,作用明显强于单基因,其机制可能与bcl-2及VEGF表达下调有关.
目的 觀察重組慢病毒Lv-hTERTp-TK與Lv-hTERTp-tumstatin在肝癌HepG2細胞中的靶嚮錶達,探討二者聯閤在體內外對HepG2細胞的抑製作用. 方法 用不同MOI重組慢病毒Lv-hTERTp-TK、 Lv-hTERTp-tumstatin轉染肝癌細胞HepG2、正常肝細胞L02,72 h後熒光顯微鏡觀察轉染情況;逆轉錄PCR檢測TK、 tumstatin mRNA在HepG2、 L02細胞中的錶達;四甲基偶氮唑藍法觀察兩種細胞增殖情況;流式細胞術檢測兩種細胞凋亡情況;RT-PCR、 Western blot檢測HepG2細胞bcl-2、血管內皮生長因子(VEGF) mRNA和蛋白的錶達;將HepG2細胞接種于30隻BALB/c裸鼠皮下,構建動物移植瘤模型,隨機分為5組,空白對照組、空載體組、tumstatm組、TK組及聯閤組,每組6隻,瘤內分彆註射燐痠鹽緩遲溶液、空載體慢病毒Lv-hTERTp-eGFP、重組慢病毒Lv-hTERTp-tumstatin、重組慢病毒Lv-hTERTp-TK和重組慢病毒Lv-hTERTp-tumstatin+Lv-hTERTp-TK;4週後處死裸鼠,觀察各組移植瘤體積及質量的變化;行HE染色進行各組腫瘤組織及主要髒器的形態學觀察;免疫組織化學法檢測腫瘤內微血管密度;TUNEL法檢測腫瘤細胞凋亡率.數據採用方差分析. 結果 轉染後TK、 tumstatin基因在HepG2細胞中特異錶達,在L02細胞中無錶達.TK組、tumstatin組及聯閤組均對HepG2細胞生長有抑製作用,聯閤組抑製率明顯高于單基因作用組(F=731.679,P＜0.05),各組L02細胞抑製率差異無統計學意義(F=0.774,P＞0.05);聯閤組HepG2細胞總凋亡率達72.61％±3.29％,明顯高于TK組(53.63％±3.06％)、tumstatin組(45.13％±2.15％)、空白對照組(13.29％±1.15％)(F=365.022,P＜0.05),各組L02細胞凋亡率差異無統計學意義(F=0.391,P＞0.05);與對照組相比,實驗組HepG2細胞bcl-2及VEGF錶達水平均下降(bcl-2 mRNA:F=322.780,P＜0.05;VEGF mRNA:F=561.136,P＜0.05;bcl-2蛋白:F=34.203,P＜0.05;VEGF蛋白:F=32.988,P＜0.05),其中聯閤組bcl-2及VEGF錶達水平均低于單基因組(P值均＜0.05).tumstatin組、TK組和聯閤組的抑瘤率分彆為40.68％、 44.96％和72.09％,聯閤組抑瘤效果最顯著(P＜0.05);HE染色顯示聯閤組腫瘤細胞凋亡最明顯.與空白對照組相比,其他各組裸鼠體內肝、脾和腎等主要髒器組織形態學均無顯著變化;空白對照組、空載體組、TK組、tumstatin組和聯閤組的微血管密度分彆為28.4±4.28、29.2±2.59、20.8±3.35、 14.2±4.87、5.8±2.77,tumstatin組抗腫瘤血管生成作用彊于TK組(P＜0.05),且聯閤組作用最明顯(P＜0.05).聯閤組腫瘤細胞凋亡率為70.11％±5.67％,明顯高于tumstatin組(23.75％±5.21％)、TK組(48.30％±4.99％)、空白對照組(3.89％±1.13％)(P＜0.05),其中TK組的促凋亡作用較tumstatin組顯著(P＜0.05). 結論 hTERT啟動子驅動TK、 tumstatin在HepG2細胞中靶嚮錶達,兩種基因聯閤在體內外顯著抑製HepG2細胞增殖和促進HepG2細胞凋亡,作用明顯彊于單基因,其機製可能與bcl-2及VEGF錶達下調有關.
목적 관찰중조만병독Lv-hTERTp-TK여Lv-hTERTp-tumstatin재간암HepG2세포중적파향표체,탐토이자연합재체내외대HepG2세포적억제작용. 방법 용불동MOI중조만병독Lv-hTERTp-TK、 Lv-hTERTp-tumstatin전염간암세포HepG2、정상간세포L02,72 h후형광현미경관찰전염정황;역전록PCR검측TK、 tumstatin mRNA재HepG2、 L02세포중적표체;사갑기우담서람법관찰량충세포증식정황;류식세포술검측량충세포조망정황;RT-PCR、 Western blot검측HepG2세포bcl-2、혈관내피생장인자(VEGF) mRNA화단백적표체;장HepG2세포접충우30지BALB/c라서피하,구건동물이식류모형,수궤분위5조,공백대조조、공재체조、tumstatm조、TK조급연합조,매조6지,류내분별주사린산염완충용액、공재체만병독Lv-hTERTp-eGFP、중조만병독Lv-hTERTp-tumstatin、중조만병독Lv-hTERTp-TK화중조만병독Lv-hTERTp-tumstatin+Lv-hTERTp-TK;4주후처사라서,관찰각조이식류체적급질량적변화;행HE염색진행각조종류조직급주요장기적형태학관찰;면역조직화학법검측종류내미혈관밀도;TUNEL법검측종류세포조망솔.수거채용방차분석. 결과 전염후TK、 tumstatin기인재HepG2세포중특이표체,재L02세포중무표체.TK조、tumstatin조급연합조균대HepG2세포생장유억제작용,연합조억제솔명현고우단기인작용조(F=731.679,P＜0.05),각조L02세포억제솔차이무통계학의의(F=0.774,P＞0.05);연합조HepG2세포총조망솔체72.61％±3.29％,명현고우TK조(53.63％±3.06％)、tumstatin조(45.13％±2.15％)、공백대조조(13.29％±1.15％)(F=365.022,P＜0.05),각조L02세포조망솔차이무통계학의의(F=0.391,P＞0.05);여대조조상비,실험조HepG2세포bcl-2급VEGF표체수평균하강(bcl-2 mRNA:F=322.780,P＜0.05;VEGF mRNA:F=561.136,P＜0.05;bcl-2단백:F=34.203,P＜0.05;VEGF단백:F=32.988,P＜0.05),기중연합조bcl-2급VEGF표체수평균저우단기인조(P치균＜0.05).tumstatin조、TK조화연합조적억류솔분별위40.68％、 44.96％화72.09％,연합조억류효과최현저(P＜0.05);HE염색현시연합조종류세포조망최명현.여공백대조조상비,기타각조라서체내간、비화신등주요장기조직형태학균무현저변화;공백대조조、공재체조、TK조、tumstatin조화연합조적미혈관밀도분별위28.4±4.28、29.2±2.59、20.8±3.35、 14.2±4.87、5.8±2.77,tumstatin조항종류혈관생성작용강우TK조(P＜0.05),차연합조작용최명현(P＜0.05).연합조종류세포조망솔위70.11％±5.67％,명현고우tumstatin조(23.75％±5.21％)、TK조(48.30％±4.99％)、공백대조조(3.89％±1.13％)(P＜0.05),기중TK조적촉조망작용교tumstatin조현저(P＜0.05). 결론 hTERT계동자구동TK、 tumstatin재HepG2세포중파향표체,량충기인연합재체내외현저억제HepG2세포증식화촉진HepG2세포조망,작용명현강우단기인,기궤제가능여bcl-2급VEGF표체하조유관.
Objective To observe targeted expression of recombinant lentivirus-mediated (Lv)-hTERTp-TK and Lv-hTERTp-tumstatin in HepG2 cells, and explore the inhibitory effect of their combination on HepG2 cells both in vitro and in vivo.Methods Lv-hTERTp-TK and Lv-hTERTptumstatin were used to infect HepG2 and L02 cells at different MOIs.Transfection efficiency was observed by fluorescence microscopy.Expression of TK and tumstatin mRNA was detected by reverse-transcriptase PCR.Proliferation and apoptosis were detected by MTT and flow cytometry, respectively.The HepG2 cells were examined by real time-PCR and western blotting to determine expression level of bcl-2 and VEGF mRNA and protein.A murine hepatocellular carcinoma model was established by injecting 1 × 107 HepG2 cells into 30 BALB/c nude mice.The modeled mice were randomly divided into a control group, mock group, Lv-hTERTp-tumstatin group, Lv-hTERTp-TK group, and combination group for four weeks of injections at regular intervals of PBS, Lv-hTERTp-null, Lv-hTERTp-tumstatin, Lv-hTERTp-TK, and Lv-hTERTp-tumstatin plus Lv-hTERTp-TK, respectively.Changes in tumor volume and weight, and cell morphology of tumor and major organs, were assessed by hematoxylin-eosin staining.Microvascular density of tumor tissue and cell apoptosis were assessed by immunohistochemical and TUNEL staining, respectively.Results The Lv-infected HepG2 cells, and not the Lv-infected L02 cells, expressed TK and tumstatin.Lv-hTERTp-TK and Lv-hTERTp-tumstatin, alone or in combination, inhibited proliferation and increased apoptosis of the HepG2 cells, but the combination was more effective than either alone (P ＜ 0.05).None of the treatments affected proliferation or apoptosis of the L02 cells (P ＞ 0.05).The combination also led to a greater reduction of bcl-2 and VEGF than either alone (all, P ＜ 0.05).Tumor growth was significantly inhibited by the combination (P ＜ 0.05).In vivo, the combination treatment induced the greatest amount of apoptosis of the HepG2 cells.Cell morphology of major organs such as liver, spleen and kidney were similar to the control group.The combination also produced the most significant effect on tumor microvascular density (P ＜ 0.05) and the highest apoptosis index (P ＜ 0.05).Conclusion The HTERT promoter can induce targeted expression of TK and tumstatin in HepG2 cells.Lv-hTERTp-TK combined with Lv-hTERTp-tumstatin can significantly inhibit proliferation and induce apoptosis of human HepG2 cells in vitro and in vivo, which may be related to down-regulation ofbcl-2 and VEGF.
