复方藤梨根制剂调节PG细胞间隙连接通讯功能及Cx43基因表达的研究
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摘要
复方藤梨根制剂调节PG细胞间隙连接通讯功能及C×43基因表达的研究
目的 细胞间隙连接通讯(Gap Junctional Interce-llular Communication，GJIC)是细胞间的一种重要连接方式，在细胞分化、生长控制及维持体内环境平衡等方面起重要作用。近年的研究显示，肿瘤和转化细胞中普遍存在GJIC的缺陷，认为其与肿瘤的发生、发展有着密切的关系。近年来许多学者热衷于研究能上调GJIC的药物，但研究复方中药对GJIC功能影响的有关文献尚未见报道。复方藤梨根制剂(FFTLG)是导师在祖国医学扶正、祛邪两大治疗法则指导下且经长期临床验证的治疗多种肿瘤的效验方。该方抗肿瘤的具体作用机理—在控制肿瘤的生长、抗转移、逆转多药耐药性、对化疗药物的减毒增效作用及广泛的免疫调控作用等方面已经被实验证实。那么，GJIC是否为FFTLG抗肿瘤作用靶点之一是本课题研究的目的。
方法 利用MTT法体外观察不同浓度的FFTLG对PG细胞的杀伤作用；应用激光共聚焦显微镜(LSCM)和流式细胞仪(FCM)观察不同浓度剂量组对PG细胞GJIC功能和C×43表达的影响。
结果 ①FFTLG浓度为10mg／ml即表现出一定的杀伤力，且随药物浓度的增加作用加强；②经不同浓度的FFTLG作用72h后，PG细胞的GJIC功能由对照组的6.47
土4．30逐渐上升到124．51士12．17；CX43表达也随药物
浓度的增加而增加，由对照组的 85．97士5．34 上调为
139．62士4．96。经统计学分析，20＊g／ml、25＊g／＊1、
30ml加 浓度组与空白对照组及 10。g／M组相比均具有
显著性差异（P＜ 0．01）。
结论①一定浓度的FFTLG能抑制PG细胞的生长；
②FFTLG能恢复PG细胞GJIC 功能；③FFTLG有 明显上
调PG细胞CX43的表达。
Objective Gap junction is an important intercellular channel and plays an important role in vital functions, including the regulation of cell growth and cell differentiation, and the maintenance of cell homeostasis. Recent research shows that the Gap Junctional Intercellular Communication (GJIC) is deficient in tumor and conversion cell. So it is closely associated with the occurrence and progression of the tumor. At present, researchers are keen to study the drugs, which can promote GJIC. But research of the traditional Chinese medicine (TCM) mixture effectiveness on GJIC has still not been explored. FuFan TengLiGen(FFTLG) mixture conformed to the treatment principles of TCM, expelling evils and strengthening the body. Clinically, it is a useful formula to treat malignant tumors. Its concrete action mechanisms of inhibiting growth and metastasis of carcinoma; reserving multidrug resistance(MDR); attenuating and increasing of the effects of Chemotherapy and Radiotherapy; and regulating the immunity of the bo
dy. Therefore, it is reasonable to postulate that FFTLG mixture will be effective on regulating Gap Junctional Intercellular Communication.
Methods The anti-proliferation effects of different concentration of FFTLG mixture on PG cell lines were determined with
MTT assay. Laser scanning confocal mircroscopy(LSCM) and flow cytometry(FCM) were used to determine the changes of GJIC and connexin43(Cx43) protein expression after being treated with the different concentration dose of FFTLG mixture.
Results (1)FFTLG mixture began to inhibit the proliferation of PG cell lines when the concentration dose was 10mg/ml. The effect increased following with the concentrations dose. (2) Having been treated with the different concentrations of FFTLG mixture for 72 hours, the GJIC of PG cell line increase from 6.47 4.30 to 124.51 12.17; the Cx43 protein expression also increased from 85.97 5.34 to 139.62 4.96. the above results show that the groups which concentration doses are 20mg/mK 25mg/mK 30ml/ml can promote GJIC and Cx43 protein expression. There is a significant difference in results between the above three groups(20mg/ml, 25mg/ml, 30ml/ml) compared with the lower concentration and the control groups (P<0.01) .
Conclusion (1)FSTLG mixture can inhibit the proliferation of PG cell lines as long as the concentration dose is no less than 10mg/ml;(2)FSTLG mixture can promote GJIC of PG cell lines; (3)FSTLG mixture can obviously up-regulate the Cx43 protein expression of PG cell lines.
目的 细胞间隙连接通讯(Gap Junctional Interce-llular Communication，GJIC)是细胞间的一种重要连接方式，在细胞分化、生长控制及维持体内环境平衡等方面起重要作用。近年的研究显示，肿瘤和转化细胞中普遍存在GJIC的缺陷，认为其与肿瘤的发生、发展有着密切的关系。近年来许多学者热衷于研究能上调GJIC的药物，但研究复方中药对GJIC功能影响的有关文献尚未见报道。复方藤梨根制剂(FFTLG)是导师在祖国医学扶正、祛邪两大治疗法则指导下且经长期临床验证的治疗多种肿瘤的效验方。该方抗肿瘤的具体作用机理—在控制肿瘤的生长、抗转移、逆转多药耐药性、对化疗药物的减毒增效作用及广泛的免疫调控作用等方面已经被实验证实。那么，GJIC是否为FFTLG抗肿瘤作用靶点之一是本课题研究的目的。
方法 利用MTT法体外观察不同浓度的FFTLG对PG细胞的杀伤作用；应用激光共聚焦显微镜(LSCM)和流式细胞仪(FCM)观察不同浓度剂量组对PG细胞GJIC功能和C×43表达的影响。
结果 ①FFTLG浓度为10mg／ml即表现出一定的杀伤力，且随药物浓度的增加作用加强；②经不同浓度的FFTLG作用72h后，PG细胞的GJIC功能由对照组的6.47
土4．30逐渐上升到124．51士12．17；CX43表达也随药物
浓度的增加而增加，由对照组的 85．97士5．34 上调为
139．62士4．96。经统计学分析，20＊g／ml、25＊g／＊1、
30ml加 浓度组与空白对照组及 10。g／M组相比均具有
显著性差异（P＜ 0．01）。
结论①一定浓度的FFTLG能抑制PG细胞的生长；
②FFTLG能恢复PG细胞GJIC 功能；③FFTLG有 明显上
调PG细胞CX43的表达。
Objective Gap junction is an important intercellular channel and plays an important role in vital functions, including the regulation of cell growth and cell differentiation, and the maintenance of cell homeostasis. Recent research shows that the Gap Junctional Intercellular Communication (GJIC) is deficient in tumor and conversion cell. So it is closely associated with the occurrence and progression of the tumor. At present, researchers are keen to study the drugs, which can promote GJIC. But research of the traditional Chinese medicine (TCM) mixture effectiveness on GJIC has still not been explored. FuFan TengLiGen(FFTLG) mixture conformed to the treatment principles of TCM, expelling evils and strengthening the body. Clinically, it is a useful formula to treat malignant tumors. Its concrete action mechanisms of inhibiting growth and metastasis of carcinoma; reserving multidrug resistance(MDR); attenuating and increasing of the effects of Chemotherapy and Radiotherapy; and regulating the immunity of the bo
dy. Therefore, it is reasonable to postulate that FFTLG mixture will be effective on regulating Gap Junctional Intercellular Communication.
Methods The anti-proliferation effects of different concentration of FFTLG mixture on PG cell lines were determined with
MTT assay. Laser scanning confocal mircroscopy(LSCM) and flow cytometry(FCM) were used to determine the changes of GJIC and connexin43(Cx43) protein expression after being treated with the different concentration dose of FFTLG mixture.
Results (1)FFTLG mixture began to inhibit the proliferation of PG cell lines when the concentration dose was 10mg/ml. The effect increased following with the concentrations dose. (2) Having been treated with the different concentrations of FFTLG mixture for 72 hours, the GJIC of PG cell line increase from 6.47 4.30 to 124.51 12.17; the Cx43 protein expression also increased from 85.97 5.34 to 139.62 4.96. the above results show that the groups which concentration doses are 20mg/mK 25mg/mK 30ml/ml can promote GJIC and Cx43 protein expression. There is a significant difference in results between the above three groups(20mg/ml, 25mg/ml, 30ml/ml) compared with the lower concentration and the control groups (P<0.01) .
Conclusion (1)FSTLG mixture can inhibit the proliferation of PG cell lines as long as the concentration dose is no less than 10mg/ml;(2)FSTLG mixture can promote GJIC of PG cell lines; (3)FSTLG mixture can obviously up-regulate the Cx43 protein expression of PG cell lines.
引文
1 林仲翔．间隙连接通讯-生长调控-癌变三者之间的关系．细胞生物学杂志，1998，10：111～115．
2 Chaudhuri R, Sigler K, et al. Gap juncational intercellular communication in mouse lung epithelial call lines:effects of cell transformation and tumor promoters. Cancer Lett, 1993,71(1～3):11.
3 Willecke K, Hennemann H, Dahl E, et al. The diversity of J Cell Biol,1991,56:1～2.
4 陈必良，马向东，王德堂，等．维甲酸对HeLa细胞间隙连接蛋白基因Cx43表达德调节作用．癌症，1999，18(6)：646～648．
5 朱俊东，陈东升，糜漫天，等．β-胡萝卜素对促癌剂抑制细胞间隙连接通讯的影响．第三军医大学学报，1996，18(4)：324～326．
6 陈清勇．粘附分子和细胞间隙连接通讯在肺癌侵袭和转移中的作用及药物的调控研究．浙江大学博士学位论文．2001．
7 林玲，王泽时，俞丽霞．三根去瘤方对Lewis肺癌等的抗肿瘤作用实验研究．浙江中医学院学报 1995，519(1)：37～39．
8 冯正权．复方三根制剂逆转K562／ADR和K562／VCR多药耐药性机制的研究．浙江中医学院硕士研究生学位论文．2000．
9 林玲，王泽时，俞丽霞．三根去瘤方对DNA损伤的保护及对化疗药物减毒增效的研究．浙江中医学院学报 1997，21(5)：30～33．
10 张志谦，林仲翔，吕有勇，等．高转移人肺癌表型去恶化研究．中华肿瘤杂志，1997，19(4)：25．
11 Rush RJ.The role of gap juncational intercellular communication in neoplasis. Ann Clin Lab Sci, 1994, 24(3):216.
12 Willecke K, Hennemann H, Dahl E, et al. The diversity of connexin genes encoding gap juncctional proteins. Eur J Cell Biol, 1991 Oct;56:1～7.
13 Eghbali B, Kessler JA, Reid LM, et al. Involvement of gap junctions in tumorigenesis: transfection of tumor cells with connexin32 cDNA retards growth in vivo. Proc Natl Acad Sci USA, 1991 Dec;88(23):10701～10705.
14 Yamadsaki H, Mesnil M, Omori Y, et al. Intercellular communication and caricinogenesis. Murat Res, 1995,333(1～2):181.
15 Yamadsaki H, Krutovskih V, Mesnil M, et al. Role of connexin(gapjunction) genes in cell growth control and caricinogenesis. C-R-Acad-Sci-Ⅲ, 1999,322(2～3):151.
16 Mckarns SC, Doolittle DJ. limitations of the scarpeloading/dye transfer technique to quantify communication. Cell Biol Toxical, 1992,8(1):89.
17 Carruba G, Webber MM. Bello Deocampo D, et al. Laserscanning analysis of cell communication in cultured human prostate tumor cells. Anal Quant Cytol Histol, 1999, 21(1):54.
18 马向东，王文亮，徐力青，等．肝癌细胞间隙连接蛋白Cx32，Cx43的流式细胞仪分析．细胞分子与免疫学杂志，1999，15，(1)：51．
2 Chaudhuri R, Sigler K, et al. Gap juncational intercellular communication in mouse lung epithelial call lines:effects of cell transformation and tumor promoters. Cancer Lett, 1993,71(1～3):11.
3 Willecke K, Hennemann H, Dahl E, et al. The diversity of J Cell Biol,1991,56:1～2.
4 陈必良，马向东，王德堂，等．维甲酸对HeLa细胞间隙连接蛋白基因Cx43表达德调节作用．癌症，1999，18(6)：646～648．
5 朱俊东，陈东升，糜漫天，等．β-胡萝卜素对促癌剂抑制细胞间隙连接通讯的影响．第三军医大学学报，1996，18(4)：324～326．
6 陈清勇．粘附分子和细胞间隙连接通讯在肺癌侵袭和转移中的作用及药物的调控研究．浙江大学博士学位论文．2001．
7 林玲，王泽时，俞丽霞．三根去瘤方对Lewis肺癌等的抗肿瘤作用实验研究．浙江中医学院学报 1995，519(1)：37～39．
8 冯正权．复方三根制剂逆转K562／ADR和K562／VCR多药耐药性机制的研究．浙江中医学院硕士研究生学位论文．2000．
9 林玲，王泽时，俞丽霞．三根去瘤方对DNA损伤的保护及对化疗药物减毒增效的研究．浙江中医学院学报 1997，21(5)：30～33．
10 张志谦，林仲翔，吕有勇，等．高转移人肺癌表型去恶化研究．中华肿瘤杂志，1997，19(4)：25．
11 Rush RJ.The role of gap juncational intercellular communication in neoplasis. Ann Clin Lab Sci, 1994, 24(3):216.
12 Willecke K, Hennemann H, Dahl E, et al. The diversity of connexin genes encoding gap juncctional proteins. Eur J Cell Biol, 1991 Oct;56:1～7.
13 Eghbali B, Kessler JA, Reid LM, et al. Involvement of gap junctions in tumorigenesis: transfection of tumor cells with connexin32 cDNA retards growth in vivo. Proc Natl Acad Sci USA, 1991 Dec;88(23):10701～10705.
14 Yamadsaki H, Mesnil M, Omori Y, et al. Intercellular communication and caricinogenesis. Murat Res, 1995,333(1～2):181.
15 Yamadsaki H, Krutovskih V, Mesnil M, et al. Role of connexin(gapjunction) genes in cell growth control and caricinogenesis. C-R-Acad-Sci-Ⅲ, 1999,322(2～3):151.
16 Mckarns SC, Doolittle DJ. limitations of the scarpeloading/dye transfer technique to quantify communication. Cell Biol Toxical, 1992,8(1):89.
17 Carruba G, Webber MM. Bello Deocampo D, et al. Laserscanning analysis of cell communication in cultured human prostate tumor cells. Anal Quant Cytol Histol, 1999, 21(1):54.
18 马向东，王文亮，徐力青，等．肝癌细胞间隙连接蛋白Cx32，Cx43的流式细胞仪分析．细胞分子与免疫学杂志，1999，15，(1)：51．