抑癌基因TSLC1对人前列腺癌T_3B细胞增殖、侵袭和转移能力影响的实验研究
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摘要
目的：(1)建立稳定表达肺癌抑癌基因—1(TSLC1)的人前列腺癌T_3B细胞系并对其进行鉴定；(2)探讨TSLC1基因对人前列腺癌T_3B细胞增殖和侵袭的生物学特性影响；(3)研究TSLC1对人前列腺癌T_3B细胞成瘤和转移能力的影响。
方法：(1)脂质体转染的方法将pCI-TSLC1质粒稳定转染至T_3B细胞系，经克隆化培养以及G418筛选，建立了稳定表达TSLC1蛋白的细胞系。分别从核酸和蛋白水平对转染进行鉴定，并对其细胞纯度、遗传稳定性等生物学特性进行鉴定。(2)以稳定表达外源基因TSLC1的人前列腺癌T_3B细胞为实验组，以转染空质粒pCI-neo的T_3B细胞为对照组，野生型T_3B细胞为空白组。MTT法检测细胞增殖，FACSort流式细胞仪检测细胞周期，AnnexinV／PI双染法检测细胞凋亡情况，Tanswell法检测细胞体外侵袭能力，细胞划痕实验检测细胞迁移能力。(3)将培养的实验组、对照组、空白组三组肿瘤细胞分别以4.0×10~6／200μL浓度注入裸鼠皮下，观察各组成瘤情况。按上述分组以4.0×10~6／200μL浓度进行尾静脉注射观察建立转移模型。将三组细胞分别以2.0×10~6／10μL进行骨原位注射建立骨转移瘤模型，观察各组骨转移率。
结果：(1)成功获得高表达TSLC1的稳定细胞系，核酸和蛋白水平鉴定都说明TSLC1基因成功整合到细胞基因组中。生物学性状研究结果表明该转基因细胞系纯度好，遗传性状稳定。(2)与对照组和空白组相比，实验组细胞株细胞生长速度减慢，增殖受到明显抑制；实验组细胞周期发生了明显的G_0／G_1期阻滞；实验组细胞早期凋亡率、晚期凋亡率和总凋亡率均明显升高(P＜0.01)；体外侵袭和迁移能力受到较大抑制(P＜0.05)。(3)实验组皮下瘤出现明显晚于对照组和空白组，而且瘤体也明显小于对照组和空白组(P＜0.01)；尾静脉注射注射后实验组体内肺转移形成时间晚，转移灶数目少；实验组的骨转移率为20％，明显低于对照组(100％)和空白组(100％)，P＜0.05。
结论：(1)成功建立了稳定表达TSLC1的T_3B细胞系，该细胞系遗传性质稳定，为进一步研究TSLC1在前列腺癌中发挥的作用奠定了基础。(2) TSLC1基因能明显抑制T_3B细胞的增殖和侵袭，并诱导细胞发生凋亡。(3) TSLC1基因明显抑制T_3B细胞的成瘤和转移能力。
Objective 1. To establish the prostatic carcinoma cell subline which expressingTSLC1 protein and to identify its biological characteristics. 2. To elucidate the effect ofTSLC1 on proliferation and invasion of human prostatic carcinoma cell line T_3B. 3. Tostudy the effect of TSLC1 on tumorigenicity and metastasis of human prostatic carcinomacell line T_3B.
Methods 1. The recombinant plasmid pCI-TSLC1 was stably transfected into T_3Bcells with Lipofectamine 2000. The positive clones were developed by selection by G418.Biological characteristics of T_3B cells which were stably transfected with pCI-TSLC1 werestudied. 2. The T_3B cells stably transfected with exogenous gene TSLC1 (experimentalgroup) and those treated with pCI-neo vector (control group) and without any treatment(blank group) were compared. Cell proliferation was analyzed with MTT assay. FACSortflow cytometry analysis was performed to assess the cell cycle distribution and apoptosis.Transwell was performed to assess the ability of invasion. The speed of cell migration wasdetected by cell scratch method. 3. The experimental group, control group and blank groupcells were injected respectively into nude mices subcutaneously with cell concentration of4.0×10~6/200μL. The growth of tumor was examined in three groups. The three groups cellconcentration of 4.0×10~6/200μL was injected into nude mice through vena caudalis toobserve metastasis. The three groups cell concentration of 2.0×10~6/10μL were injectedin-situ to establish osseous metastasis model. We examined the rate of osseous metastasis inthree groups.
Results 1. The stable cell subline highly expressing TSLC1 protein was obtained. The ectogenous gene TSLC1 has integrated into the genomes of T_3B cells which wereidentified from both nucleic acid and protein levels. The genetic stability and purity of thecell population were confirmed. 2. The growth of TSLC1-transfected cells was significantlysuppressed in vitro compared with those of the control and blank groups, displaying that theamount of G_0/G_1 cells increased and the amount of S phase cells decreased significantly,which suggest a G_0/G_1 cell cycle arresting. The number of cells in early, late and total phaseapoptosis were significantly higher than those of the control and blank groups (P＜0.01).The ability of invasion and migration were significantly suppressed compared with those ofthe control and blank groups (P＜0.05). 3. The subcutaneous tumor of the experimentalgroup occurred later and smaller than the control and blank groups(P＜0.01). Lungmetastases of nude mice occurred later and less than the control and blank groups. The rateof osseous metastasis was 20% in experimental group, which decreased significantly(P＜0.05) compared with the control group (100%) and the blank group (100%).
Conclusions 1. The stable prostatic carcinoma cell subline highly expressingTSLC1 has successfully established, which provided a basis for further exploring the rolesof TSLC1 in prostatic carcinoma. 2. TSLC1 strongly inhibits the proliferation and invasionof T_3B cells in vitro and induces apoptosis of cells. 3. TSLC1 strongly inhibits thetumorigenicity and metastasis of T_3B cells
方法：(1)脂质体转染的方法将pCI-TSLC1质粒稳定转染至T_3B细胞系，经克隆化培养以及G418筛选，建立了稳定表达TSLC1蛋白的细胞系。分别从核酸和蛋白水平对转染进行鉴定，并对其细胞纯度、遗传稳定性等生物学特性进行鉴定。(2)以稳定表达外源基因TSLC1的人前列腺癌T_3B细胞为实验组，以转染空质粒pCI-neo的T_3B细胞为对照组，野生型T_3B细胞为空白组。MTT法检测细胞增殖，FACSort流式细胞仪检测细胞周期，AnnexinV／PI双染法检测细胞凋亡情况，Tanswell法检测细胞体外侵袭能力，细胞划痕实验检测细胞迁移能力。(3)将培养的实验组、对照组、空白组三组肿瘤细胞分别以4.0×10~6／200μL浓度注入裸鼠皮下，观察各组成瘤情况。按上述分组以4.0×10~6／200μL浓度进行尾静脉注射观察建立转移模型。将三组细胞分别以2.0×10~6／10μL进行骨原位注射建立骨转移瘤模型，观察各组骨转移率。
结果：(1)成功获得高表达TSLC1的稳定细胞系，核酸和蛋白水平鉴定都说明TSLC1基因成功整合到细胞基因组中。生物学性状研究结果表明该转基因细胞系纯度好，遗传性状稳定。(2)与对照组和空白组相比，实验组细胞株细胞生长速度减慢，增殖受到明显抑制；实验组细胞周期发生了明显的G_0／G_1期阻滞；实验组细胞早期凋亡率、晚期凋亡率和总凋亡率均明显升高(P＜0.01)；体外侵袭和迁移能力受到较大抑制(P＜0.05)。(3)实验组皮下瘤出现明显晚于对照组和空白组，而且瘤体也明显小于对照组和空白组(P＜0.01)；尾静脉注射注射后实验组体内肺转移形成时间晚，转移灶数目少；实验组的骨转移率为20％，明显低于对照组(100％)和空白组(100％)，P＜0.05。
结论：(1)成功建立了稳定表达TSLC1的T_3B细胞系，该细胞系遗传性质稳定，为进一步研究TSLC1在前列腺癌中发挥的作用奠定了基础。(2) TSLC1基因能明显抑制T_3B细胞的增殖和侵袭，并诱导细胞发生凋亡。(3) TSLC1基因明显抑制T_3B细胞的成瘤和转移能力。
Objective 1. To establish the prostatic carcinoma cell subline which expressingTSLC1 protein and to identify its biological characteristics. 2. To elucidate the effect ofTSLC1 on proliferation and invasion of human prostatic carcinoma cell line T_3B. 3. Tostudy the effect of TSLC1 on tumorigenicity and metastasis of human prostatic carcinomacell line T_3B.
Methods 1. The recombinant plasmid pCI-TSLC1 was stably transfected into T_3Bcells with Lipofectamine 2000. The positive clones were developed by selection by G418.Biological characteristics of T_3B cells which were stably transfected with pCI-TSLC1 werestudied. 2. The T_3B cells stably transfected with exogenous gene TSLC1 (experimentalgroup) and those treated with pCI-neo vector (control group) and without any treatment(blank group) were compared. Cell proliferation was analyzed with MTT assay. FACSortflow cytometry analysis was performed to assess the cell cycle distribution and apoptosis.Transwell was performed to assess the ability of invasion. The speed of cell migration wasdetected by cell scratch method. 3. The experimental group, control group and blank groupcells were injected respectively into nude mices subcutaneously with cell concentration of4.0×10~6/200μL. The growth of tumor was examined in three groups. The three groups cellconcentration of 4.0×10~6/200μL was injected into nude mice through vena caudalis toobserve metastasis. The three groups cell concentration of 2.0×10~6/10μL were injectedin-situ to establish osseous metastasis model. We examined the rate of osseous metastasis inthree groups.
Results 1. The stable cell subline highly expressing TSLC1 protein was obtained. The ectogenous gene TSLC1 has integrated into the genomes of T_3B cells which wereidentified from both nucleic acid and protein levels. The genetic stability and purity of thecell population were confirmed. 2. The growth of TSLC1-transfected cells was significantlysuppressed in vitro compared with those of the control and blank groups, displaying that theamount of G_0/G_1 cells increased and the amount of S phase cells decreased significantly,which suggest a G_0/G_1 cell cycle arresting. The number of cells in early, late and total phaseapoptosis were significantly higher than those of the control and blank groups (P＜0.01).The ability of invasion and migration were significantly suppressed compared with those ofthe control and blank groups (P＜0.05). 3. The subcutaneous tumor of the experimentalgroup occurred later and smaller than the control and blank groups(P＜0.01). Lungmetastases of nude mice occurred later and less than the control and blank groups. The rateof osseous metastasis was 20% in experimental group, which decreased significantly(P＜0.05) compared with the control group (100%) and the blank group (100%).
Conclusions 1. The stable prostatic carcinoma cell subline highly expressingTSLC1 has successfully established, which provided a basis for further exploring the rolesof TSLC1 in prostatic carcinoma. 2. TSLC1 strongly inhibits the proliferation and invasionof T_3B cells in vitro and induces apoptosis of cells. 3. TSLC1 strongly inhibits thetumorigenicity and metastasis of T_3B cells
引文
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