苦参碱抑制裸鼠皮下移植性胃癌生长及转移机制的研究
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摘要
目的：
研究苦参碱对人胃癌SGC-7901细胞裸鼠皮下移植瘤生长及浸润转移的抑制作用机制,初步探讨其在裸鼠体内抗肿瘤作用的机制,为苦参碱治疗胃癌提供实验理论。
材料与方法：
1.培养人胃癌SGC-7901细胞,调整细胞浓度为1×108／ml,以0.1ml／只注射于每只裸鼠右腋窝皮下,建立人胃癌SGC-7901细胞裸鼠皮下移植瘤模型。
2.选取裸鼠移植瘤体积达到100mm3的裸鼠进行实验,将裸鼠随机分为四组：阴性对照组(NS)、阳性对照组(5-FU)、苦参碱组(Mat)及联合组(5-FU+Mat),每组6只,每日一次按公斤体重给药,分别于腹腔注射等体积的NS、5-FU(50mg／kg)、Mat(75mg／kg)以及5-FU(50mg/kg)+Mat(75mg/kg)。连续给药4周,观察裸鼠移植瘤生长情况。
3.实验结束时,颈椎脱臼处死裸鼠,剥离裸鼠移植瘤,用中性福尔马林固定。病理切片HE染色,光学显微镜下观察裸鼠移植瘤病理形态学的改变。做脱氧核糖核酸末端转移酶介导的端口末端标记法(TDT-mediated dUP nick end labelling, TUNEL)染色来检测裸鼠移植瘤细胞的凋亡,计算凋亡指数(apoptosis index, AI)。用免疫组织化学染色方法(SABC法)检测裸鼠移植瘤细胞间及间质内微血管密度(MVD)以及Bcl-2、Bax、VEGF蛋白的表达情况。
结果：
1.裸鼠皮下移植瘤病理形态学观察结果：光学显微镜下见苦参碱组、5-氟尿嘧啶组以及联合组裸鼠皮下移植瘤细胞均发生不同程度的坏死及凋亡,以联合组最为明显。高倍镜下见坏死为红染的无结构物质,凋亡以膜皱缩形成的凋亡小体为特征。
2.裸鼠皮下移植瘤细胞凋亡的检测结果：病理切片TUNEL染色光学显微镜下见联合组、5-氟尿嘧啶组及苦参碱组均有明显的裸鼠移植瘤细胞凋亡,其AI分别为22.04±1.55、11.06±1.80和10.32±0.98,与阴性对照组比较差异均具有显著性(P<0.01)。同时,联合组与5.氟尿嘧啶组及苦参碱组比较差异具有显著性(P<0.01)。
3.免疫组织化学染色检测结果：苦参碱组、5-氟尿嘧啶组和联合组裸鼠移植瘤细胞MVD、Bcl-2蛋白及VEGF蛋白的表达均较阴性对照组低,而Bax蛋白的表达则升高。经统计学分析,与阴性对照组比较差异均具有显著性(P<0.05)。其中联合组的MVD、Bcl-2蛋白与苦参碱组比较差异具有显著性(P<0.05),但联合组的Bax蛋白、VEGF蛋白与苦参碱组比较差异无显著性。(P>0.05)。
结论：
1.苦参碱能促进人胃癌SGC-7901细胞裸鼠皮下移植瘤细胞的凋亡,抑制其微血管生成。
2.苦参碱促进人胃癌SGC-7901细胞裸鼠皮下移植瘤细胞凋亡可能与下调Bcl-2蛋白及上调Bax蛋白的表达有关。
3.苦参碱抑制人胃癌SGC-7901细胞裸鼠皮下移植瘤细胞微血管生成可能与下调VEGF蛋白的表达有关。
Objective:
This subject mainly researched the inhibitive mechanisms of grwth and metastasis about Matrine on human gastric carcinoma cell line SGC-7901 transplantation tumor in nude mice.The results of the research could provide particular theoretical evidence on the treatment of gastric cancer with Matrine.
Materials and Methods:
1. Adjusted the consistency of human gastric carcinoma cell line SGC-7901 to 1×108/ml, injected 0.1ml in right-oxter in every nude mouse, and then nude mice implanted models of human gastric carcinoma cell line SGC-7901 were established.
2. Selected nude mice whose transplantation tumor cubage reached 100mm3. Those nude mice were divided into four groups at random, which they were NS group、5-Fu group、Matrine group and the combinatian group. Every group were six. NS (isopyknic) or 5-Fu (50mg/kg) or Matrine (75mg/kg) or 5-Fu (50mg/kg)+Matrine (75mg/kg) was injected intraperitoneally respectively per day. Observed the changes that the transplantation tumor were growing. We had observed totally 28 days.
3. Executed nude mice by shedding cervical vertebra when the expriment came to over, took out of transplantation tumors and those tumors were fixed in neutral formalin and stained in the way of HE. Observed tectology changes in human gastric carcinoma cell line SGC-7901 transplantation tumors in nude mice through microscope. Besides, TUNEL was used to detect tumor cell apoptosis through microscope, too. At last we detected the expression level of apoptosis-related proteins Bcl-2 and Bax and microvessel generate-related proteins CD 105 and VEGF in human gastric carcinoma cell line SGC-7901 transplantation tumor in nude mice with IHC.
Results:
1. Results of pathomorphology of transplantation tumor showed that tumor cells in the Matrine group、5-Fu group and combination group had apoptosis which registered as apoptosticbodies and necrosis which registered as red structurelessness matter with different extent in high power lens through microscope, especially in the combination group.
2. The results of apoptosis of transplantation tumor cells:TUNEL coloration showed that the combination group、5-Fu group and Matrine group had characteristics of apoptosis.The A1 was 22.04±1.55 and 11.06±1.80 and 10.32±0.98 separately.Compared with the NS group,the difference was significantly different (p<0.01). Moreeverthe combination group, compared with the Matrine group and 5-Fu group,had apparently statistical significance(P<0.01).
3. Results of Immunohistochemistry (IHC) showed that the expression of MVD、Bcl-2 protein and VEGF protein were down-regulated while Bax protein was up-regulated in transplantation tumor cells in the Matrine group、5-Fu group and combination group. Compared with the NS group, the expression of MVD、Bcl-2 protein、Bax protein andVEGF protein in three groups had statistical significance(p<0.05).The expression of MVD and Bcl-2 protein had statistical significance between Mat group and combination group (p<0.05), while the expression of Bax protein and VEGF protein had no statistical significance between Mat group and combination group (p>0.05).
Conclusion:
1. Matrine could induce the apoptosis and inhibits the microvessel-generated of human gastric carcinoma cell line SGC-7901 transplantation tumor in nude mice.
2. Matrine induces the apoptosis of human gastric carcinoma SGC-7901 cell transplantation tumor maybe through down-regulating the expression of Bcl-2 protein and up-regulating the expression of Bax protein.
3. Matrine inhibites the microvessel-generated of human gastric carcinoma SGC-7901 cell transplantation tumor maybe through down-regulating the expression of VEGF protein.
研究苦参碱对人胃癌SGC-7901细胞裸鼠皮下移植瘤生长及浸润转移的抑制作用机制,初步探讨其在裸鼠体内抗肿瘤作用的机制,为苦参碱治疗胃癌提供实验理论。
材料与方法：
1.培养人胃癌SGC-7901细胞,调整细胞浓度为1×108／ml,以0.1ml／只注射于每只裸鼠右腋窝皮下,建立人胃癌SGC-7901细胞裸鼠皮下移植瘤模型。
2.选取裸鼠移植瘤体积达到100mm3的裸鼠进行实验,将裸鼠随机分为四组：阴性对照组(NS)、阳性对照组(5-FU)、苦参碱组(Mat)及联合组(5-FU+Mat),每组6只,每日一次按公斤体重给药,分别于腹腔注射等体积的NS、5-FU(50mg／kg)、Mat(75mg／kg)以及5-FU(50mg/kg)+Mat(75mg/kg)。连续给药4周,观察裸鼠移植瘤生长情况。
3.实验结束时,颈椎脱臼处死裸鼠,剥离裸鼠移植瘤,用中性福尔马林固定。病理切片HE染色,光学显微镜下观察裸鼠移植瘤病理形态学的改变。做脱氧核糖核酸末端转移酶介导的端口末端标记法(TDT-mediated dUP nick end labelling, TUNEL)染色来检测裸鼠移植瘤细胞的凋亡,计算凋亡指数(apoptosis index, AI)。用免疫组织化学染色方法(SABC法)检测裸鼠移植瘤细胞间及间质内微血管密度(MVD)以及Bcl-2、Bax、VEGF蛋白的表达情况。
结果：
1.裸鼠皮下移植瘤病理形态学观察结果：光学显微镜下见苦参碱组、5-氟尿嘧啶组以及联合组裸鼠皮下移植瘤细胞均发生不同程度的坏死及凋亡,以联合组最为明显。高倍镜下见坏死为红染的无结构物质,凋亡以膜皱缩形成的凋亡小体为特征。
2.裸鼠皮下移植瘤细胞凋亡的检测结果：病理切片TUNEL染色光学显微镜下见联合组、5-氟尿嘧啶组及苦参碱组均有明显的裸鼠移植瘤细胞凋亡,其AI分别为22.04±1.55、11.06±1.80和10.32±0.98,与阴性对照组比较差异均具有显著性(P<0.01)。同时,联合组与5.氟尿嘧啶组及苦参碱组比较差异具有显著性(P<0.01)。
3.免疫组织化学染色检测结果：苦参碱组、5-氟尿嘧啶组和联合组裸鼠移植瘤细胞MVD、Bcl-2蛋白及VEGF蛋白的表达均较阴性对照组低,而Bax蛋白的表达则升高。经统计学分析,与阴性对照组比较差异均具有显著性(P<0.05)。其中联合组的MVD、Bcl-2蛋白与苦参碱组比较差异具有显著性(P<0.05),但联合组的Bax蛋白、VEGF蛋白与苦参碱组比较差异无显著性。(P>0.05)。
结论：
1.苦参碱能促进人胃癌SGC-7901细胞裸鼠皮下移植瘤细胞的凋亡,抑制其微血管生成。
2.苦参碱促进人胃癌SGC-7901细胞裸鼠皮下移植瘤细胞凋亡可能与下调Bcl-2蛋白及上调Bax蛋白的表达有关。
3.苦参碱抑制人胃癌SGC-7901细胞裸鼠皮下移植瘤细胞微血管生成可能与下调VEGF蛋白的表达有关。
Objective:
This subject mainly researched the inhibitive mechanisms of grwth and metastasis about Matrine on human gastric carcinoma cell line SGC-7901 transplantation tumor in nude mice.The results of the research could provide particular theoretical evidence on the treatment of gastric cancer with Matrine.
Materials and Methods:
1. Adjusted the consistency of human gastric carcinoma cell line SGC-7901 to 1×108/ml, injected 0.1ml in right-oxter in every nude mouse, and then nude mice implanted models of human gastric carcinoma cell line SGC-7901 were established.
2. Selected nude mice whose transplantation tumor cubage reached 100mm3. Those nude mice were divided into four groups at random, which they were NS group、5-Fu group、Matrine group and the combinatian group. Every group were six. NS (isopyknic) or 5-Fu (50mg/kg) or Matrine (75mg/kg) or 5-Fu (50mg/kg)+Matrine (75mg/kg) was injected intraperitoneally respectively per day. Observed the changes that the transplantation tumor were growing. We had observed totally 28 days.
3. Executed nude mice by shedding cervical vertebra when the expriment came to over, took out of transplantation tumors and those tumors were fixed in neutral formalin and stained in the way of HE. Observed tectology changes in human gastric carcinoma cell line SGC-7901 transplantation tumors in nude mice through microscope. Besides, TUNEL was used to detect tumor cell apoptosis through microscope, too. At last we detected the expression level of apoptosis-related proteins Bcl-2 and Bax and microvessel generate-related proteins CD 105 and VEGF in human gastric carcinoma cell line SGC-7901 transplantation tumor in nude mice with IHC.
Results:
1. Results of pathomorphology of transplantation tumor showed that tumor cells in the Matrine group、5-Fu group and combination group had apoptosis which registered as apoptosticbodies and necrosis which registered as red structurelessness matter with different extent in high power lens through microscope, especially in the combination group.
2. The results of apoptosis of transplantation tumor cells:TUNEL coloration showed that the combination group、5-Fu group and Matrine group had characteristics of apoptosis.The A1 was 22.04±1.55 and 11.06±1.80 and 10.32±0.98 separately.Compared with the NS group,the difference was significantly different (p<0.01). Moreeverthe combination group, compared with the Matrine group and 5-Fu group,had apparently statistical significance(P<0.01).
3. Results of Immunohistochemistry (IHC) showed that the expression of MVD、Bcl-2 protein and VEGF protein were down-regulated while Bax protein was up-regulated in transplantation tumor cells in the Matrine group、5-Fu group and combination group. Compared with the NS group, the expression of MVD、Bcl-2 protein、Bax protein andVEGF protein in three groups had statistical significance(p<0.05).The expression of MVD and Bcl-2 protein had statistical significance between Mat group and combination group (p<0.05), while the expression of Bax protein and VEGF protein had no statistical significance between Mat group and combination group (p>0.05).
Conclusion:
1. Matrine could induce the apoptosis and inhibits the microvessel-generated of human gastric carcinoma cell line SGC-7901 transplantation tumor in nude mice.
2. Matrine induces the apoptosis of human gastric carcinoma SGC-7901 cell transplantation tumor maybe through down-regulating the expression of Bcl-2 protein and up-regulating the expression of Bax protein.
3. Matrine inhibites the microvessel-generated of human gastric carcinoma SGC-7901 cell transplantation tumor maybe through down-regulating the expression of VEGF protein.
引文
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