中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
Chinese Journal of Experimental Surgery
2015年
11期
2708-2711
,共4页
刘虹%孔刚%龚志军%卿笃桔%李恩就%黎飞
劉虹%孔剛%龔誌軍%卿篤桔%李恩就%黎飛
류홍%공강%공지군%경독길%리은취%려비
结肠癌细胞%KISS-1基因%体外表达
結腸癌細胞%KISS-1基因%體外錶達
결장암세포%KISS-1기인%체외표체
Colon cancercell%KISS-1 gene%In vitro expression
目的 观察KISS-1基因对结肠癌细胞株HT29体外表达的作用和对其生物学功能的影响.方法 通过基因转染法使KISS-1在结肠癌细胞株HT29中稳定表达,应用噻唑蓝(MTT)法和Transwell侵袭实验检测KISS-1表达对结肠癌细胞株HT29的增殖和侵袭能力的作用和影响;通过免疫组织化学法和实时定量聚合酶链反应(Real-time PCR)技术检测KISS-1基因mRNA在体外结肠癌细胞株HT29中的表达水平,并用Real-time PCR测定转染后结肠癌细胞株HT29中c-fos、c-jun mRNA水平表达变化,并通过显微镜、透射电镜等观察凋亡细胞的形态学改变.结果 (1)Real-time PCR和免疫细胞组织化学证实转染后结肠癌细胞中有KISS-1稳定表达.(2)MTt法表明:培养0、24 h时各组HT29细胞数差异无统计学意义(P＞0.05),48 h后,随着培养时间延长,转染组HT29细胞数明显低于空白对照组与阴性对照组(P＜0.05),而空白对照组与阴性对照组HT29细胞数差异无统计学意义(P＞0.05).(3) Transwell侵袭实验结果:转染KISS-1基因的结肠癌细胞HT29穿透Matrigel基底膜的细胞数显著低于空质粒转染组及空白对照组细胞(P＜0.01),侵袭指数显著降低(P＜0.01),侵袭力抑制率达到56.5％,显著低于空质粒转染组及空白对照组细胞(P＜0.01).空质粒转染组及空白对照组细胞比较,侵袭穿膜细胞数差异无统计学意义(P＞0.05).(4)KISS-1作用于结肠癌细胞株HT29 24 h后,形态学观察结肠癌细胞株HT29发生凋亡或坏死.(5)Real-time PCR:转染组c-jun和c-fos吸光度值为0.87 ±0.16和0.44±0.12,阴性对照组吸光度值为1.82±0.39和1.76±0.34,空白对照组吸光度值为1.65±0.36和1.58±0.31.与对照组比较,转染组c-jun和c-fos扩增产物条带吸光度值均显著降低(P＜0.01).结论 KISS-1基因对体外结肠癌细胞株HT29具有抑制增殖和促进凋亡作用,其可能是通过负性调控信号通路降低结肠癌细胞中c-fos、c-jun的表达.
目的 觀察KISS-1基因對結腸癌細胞株HT29體外錶達的作用和對其生物學功能的影響.方法 通過基因轉染法使KISS-1在結腸癌細胞株HT29中穩定錶達,應用噻唑藍(MTT)法和Transwell侵襲實驗檢測KISS-1錶達對結腸癌細胞株HT29的增殖和侵襲能力的作用和影響;通過免疫組織化學法和實時定量聚閤酶鏈反應(Real-time PCR)技術檢測KISS-1基因mRNA在體外結腸癌細胞株HT29中的錶達水平,併用Real-time PCR測定轉染後結腸癌細胞株HT29中c-fos、c-jun mRNA水平錶達變化,併通過顯微鏡、透射電鏡等觀察凋亡細胞的形態學改變.結果 (1)Real-time PCR和免疫細胞組織化學證實轉染後結腸癌細胞中有KISS-1穩定錶達.(2)MTt法錶明:培養0、24 h時各組HT29細胞數差異無統計學意義(P＞0.05),48 h後,隨著培養時間延長,轉染組HT29細胞數明顯低于空白對照組與陰性對照組(P＜0.05),而空白對照組與陰性對照組HT29細胞數差異無統計學意義(P＞0.05).(3) Transwell侵襲實驗結果:轉染KISS-1基因的結腸癌細胞HT29穿透Matrigel基底膜的細胞數顯著低于空質粒轉染組及空白對照組細胞(P＜0.01),侵襲指數顯著降低(P＜0.01),侵襲力抑製率達到56.5％,顯著低于空質粒轉染組及空白對照組細胞(P＜0.01).空質粒轉染組及空白對照組細胞比較,侵襲穿膜細胞數差異無統計學意義(P＞0.05).(4)KISS-1作用于結腸癌細胞株HT29 24 h後,形態學觀察結腸癌細胞株HT29髮生凋亡或壞死.(5)Real-time PCR:轉染組c-jun和c-fos吸光度值為0.87 ±0.16和0.44±0.12,陰性對照組吸光度值為1.82±0.39和1.76±0.34,空白對照組吸光度值為1.65±0.36和1.58±0.31.與對照組比較,轉染組c-jun和c-fos擴增產物條帶吸光度值均顯著降低(P＜0.01).結論 KISS-1基因對體外結腸癌細胞株HT29具有抑製增殖和促進凋亡作用,其可能是通過負性調控信號通路降低結腸癌細胞中c-fos、c-jun的錶達.
목적 관찰KISS-1기인대결장암세포주HT29체외표체적작용화대기생물학공능적영향.방법 통과기인전염법사KISS-1재결장암세포주HT29중은정표체,응용새서람(MTT)법화Transwell침습실험검측KISS-1표체대결장암세포주HT29적증식화침습능력적작용화영향;통과면역조직화학법화실시정량취합매련반응(Real-time PCR)기술검측KISS-1기인mRNA재체외결장암세포주HT29중적표체수평,병용Real-time PCR측정전염후결장암세포주HT29중c-fos、c-jun mRNA수평표체변화,병통과현미경、투사전경등관찰조망세포적형태학개변.결과 (1)Real-time PCR화면역세포조직화학증실전염후결장암세포중유KISS-1은정표체.(2)MTt법표명:배양0、24 h시각조HT29세포수차이무통계학의의(P＞0.05),48 h후,수착배양시간연장,전염조HT29세포수명현저우공백대조조여음성대조조(P＜0.05),이공백대조조여음성대조조HT29세포수차이무통계학의의(P＞0.05).(3) Transwell침습실험결과:전염KISS-1기인적결장암세포HT29천투Matrigel기저막적세포수현저저우공질립전염조급공백대조조세포(P＜0.01),침습지수현저강저(P＜0.01),침습력억제솔체도56.5％,현저저우공질립전염조급공백대조조세포(P＜0.01).공질립전염조급공백대조조세포비교,침습천막세포수차이무통계학의의(P＞0.05).(4)KISS-1작용우결장암세포주HT29 24 h후,형태학관찰결장암세포주HT29발생조망혹배사.(5)Real-time PCR:전염조c-jun화c-fos흡광도치위0.87 ±0.16화0.44±0.12,음성대조조흡광도치위1.82±0.39화1.76±0.34,공백대조조흡광도치위1.65±0.36화1.58±0.31.여대조조비교,전염조c-jun화c-fos확증산물조대흡광도치균현저강저(P＜0.01).결론 KISS-1기인대체외결장암세포주HT29구유억제증식화촉진조망작용,기가능시통과부성조공신호통로강저결장암세포중c-fos、c-jun적표체.
Objective To study the anti-proliferation and inducing apoptosis effects of KISS-1 on HT29 colon cancer cells.Methods Effects of the expression of the gene transfection method for detection of KISS-1 on colon cancer cell line HT29 anti proliferation and biological function;the stable expression in colon cancer cell line KISS-1 by HT29 gene transfection method, by detecting the expression level of KISS-1 gene mRNA immunohistochemical method and real-time quantitative polymerase chain reaction (Real-time PCR) technique in colon cancer cells in vitro in HT29, and the changes of c-fos, c-jun were determined by Real-time PCR mRNA HT29 expression level in colon cancer cell line transfected cells, apoptotic morphological changes under microscope, HE staining, transmission electron microscope.Results Real-time PCR and immune cell histochemistry confirmed after transfection of colon cancer primary KISS-1 stable expression have lesions and metastases in Real-time PCR transfected cells;confirmed colon cancer primary c-fos, c-jun mRNA levels of the lesion and metastatic cells decreased significantly (P ＜ 0.01).There was no significant difference in the number of HT29 cells in culture 0 and 24 h, and the number of HT29 cells in the transfected group was significantly lower than that in control group (P ＜ 0.05), while the number of HT29 cells in control group was significantly lower than that in control group (P ＞ 0.05).The effect of KISS-1 on HT29 24 h colon cancer cell line, morphological observation of colon cancer cell line HT29 apoptosis or necrosis.Real-time PCR confirmed that c-jun and mRNA c-fos levels (0.87 ± 0.16 and 0.44 ± 0.12) were significantly decreased in colon cancer primary lesion and metastasis cells (1.82 ± 0.39 and 1.76 ± 0.34, P ＜ 0.01).Conclusion KISS-1 gene can inhibit proliferation and promote apoptosis effect on colon cancer cells in vitro HT29, which may be decreased expression of colon cancer cells c-fos, c-jun through negative regulation of signal transduction pathway.
