维生素C对人胶质瘤SHG44细胞和裸鼠皮下移植瘤的影响
发布时间：2018-07-29 19:30
【摘要】：第一部分：维生素C对人胶质瘤SHG44细胞的影响作用 目的：通过培养人胶质瘤SHG44细胞,然后采用不同浓度维生素C处理SHG44细胞,用顺铂处理SHG44细胞作为阳性对照组,观察维生素C对SHG44细胞增殖、凋亡的影响,以及对细胞中HIF-1α和VEGFmRNA表达水平的影响。探讨维生素C对SHG44细胞的可能作用机制。 方法：通过培养人胶质瘤SHG44细胞,将不同浓度维生素C(0、0.5、1.0、2.0、4.0、8.0、10.0mmol/L)作用于处于对数生长期的SHG44细胞,MTT比色法检测细胞的IC50值；然后将不同浓度维生素C(0.0.5、0.75、1.0、1.25、1.5、1.75、2.0mmol/L)和顺铂(20mg/L)作用于处于对数生长期的SHG44细胞,采用MTT法检测细胞的生长抑制率；将维生素C (0、1.5mmol/L)和顺铂(20mg/L)作用于处于对数生长期的SHG44细胞24小时,采用流式细胞仪检测细胞周期和凋亡,用RT-PCR法检测SHG44细胞中HIF-1α和、EGFmRNA的相对表达。 结果：维生素C表现为浓度依赖性抑制SHG44细胞的生长。在570nm波长,维生素C作用24小时后,其IC50值为1.695mmol/L;当维生素C的浓度大于1.25mmol/L,并作用SHG44细胞48小时后细胞出现明显的增殖抑制并大量死亡；维生素C和顺铂对SHG44细胞的处理都使得细胞的早期凋亡较空白对照组显著增加(P0.05)。维生素C和顺铂的处理都使得S期的SHG44细胞较空白对照组减少(P0.05)。与空白对照组相比,常氧条件下,维生素C和顺铂的处理都能升高SHG44细胞中HIF-1αmRNA的表达,和降低VEGF mRNA的表达(P0.05)；维生素C组和顺铂组两组之间细胞中VEGFmRNA的表达其差异无统计学意义(P0.05)。 结论：维生素C能够抑制人胶质瘤SHG44细胞的增殖,并且促进细胞的凋亡。维生素C的作用机制可能跟降低细胞中VEGF mRNA的表达,抑制组织细胞的血管生成有关。 第二部分：维生素C对裸鼠皮下移植瘤的影响作用 目的：通过构建人胶质瘤SHG44细胞BALB/c型裸鼠皮下移植瘤模型。采用顺铂作为阳性对照组,观察不同剂量的维生素C对裸鼠皮下移植瘤的生长抑制作用。以及对移植瘤组织细胞中HIF-1α和VEGFmRNA表达的影响。分析维生素C对胶质瘤的潜在作用机制,为胶质瘤的治疗提供实验室依据。 方法：1、培养人胶质瘤SHG44细胞并进行传代。然后采用处于对数生长期的细胞,吹打制成密度为2×107个/ml的单细胞悬液。抽取0.2m1注射入裸鼠皮下(背部右前肢外侧)。在接种后2周检查,接种部位皮下长出包块,测量移植瘤体积,大约为100mm3,判定胶质瘤模型制作成功。2、将4-5周龄的雌性裸鼠28只随机分为4组,每组7只。空白对照组给予生理盐水,0.4ml/只,腹腔注射,每两天一次,共3周；维生素C低、高剂量组分别给予2g/kg、4g/kg每只,腹腔注射,每两天一次,共3周；顺铂组则给予顺铂2g/kg,腹腔注射给药,每两天一次,共3周。在用药结束后第二天处死各实验组小鼠,并计算肿瘤体积以及抑瘤率。采用RT-PCR法检测各实验组肿瘤组织细胞中HIF-1α和VEGFmRNA的表达情况。 结果：1、成功建立了裸鼠皮下移植瘤模型,肿瘤种植的成功率较高,达100%。2、维生素C可以抑制裸鼠皮下移植瘤组织的生长,且高剂量组对肿瘤的抑瘤率其结果要大于低剂量组。各实验组的肿瘤体积跟空白对照组相比具有统计学上的差异(P0.05)。3、维生素C低、高剂量组都能够降低两种因子的表达,并且高剂量组降低得更明显,与空白组相比,其差异都具有统计学意义(P0.05)。维生素C低、高剂量组和顺铂组比较,其差异有统计学意义(P0.05)。 结论：人胶质瘤SHG44细胞建立裸鼠皮下移植瘤模型确实可靠,可以用于胶质瘤的预防以及治疗的实验性研究；在一定剂量范围内的大剂量维生素C可以抑制裸鼠皮下移植瘤的生长,表现出一定的抗肿瘤作用。其机制可能跟维生素C抑制组织细胞中HIF-1α和VEGFmRNA的表达,降低肿瘤组织的血供相关。图14幅,表1个,
[Abstract]:Part one: the effect of vitamin C on human glioma SHG44 cells
Objective: to cultivate human glioma SHG44 cells, then treat SHG44 cells with different concentrations of vitamin C, and use cisplatin to treat SHG44 cells as positive control group, observe the effect of vitamin C on SHG44 cell proliferation, apoptosis, and the expression of HIF-1 alpha and VEGFmRNA in cell, and explore the possibility of vitamin C on SHG44 cells. Use the mechanism.
Methods: through the culture of human glioma SHG44 cells, different concentrations of vitamin C (0,0.5,1.0,2.0,4.0,8.0,10.0mmol/L) were acted on the SHG44 cells in the logarithmic growth period, and the IC50 value of the cells was detected by MTT colorimetric method; then the different concentrations of vitamin C (0.0.5,0.75,1.0,1.25,1.5,1.75,2.0mmol/L) and cisplatin (20mg/L) were used in the logarithmic birth. Long term SHG44 cells were used to detect cell growth inhibition rate by MTT; vitamin C (0,1.5mmol/L) and cisplatin (20mg/L) were used as SHG44 cells in the logarithmic growth period for 24 hours. The cell cycle and apoptosis were detected by flow cytometry, and the RT-PCR method was used to detect the relative expression of HIF-1 alpha and EGFmRNA in SHG44 cells.
Results: vitamin C showed a concentration dependent inhibition of the growth of SHG44 cells. At 570nm wavelength, after 24 hours of vitamin C action, the IC50 value was 1.695mmol/L; when the concentration of vitamin C was greater than 1.25mmol/L, and after the action of SHG44 cells for 48 hours, the cells showed significant proliferation inhibition and large amount of death; vitamin C and cisplatin were located in the SHG44 cells. The early apoptosis of the cells increased significantly compared with the blank control group (P0.05). The treatment of vitamin C and cisplatin reduced the SHG44 cells in S phase compared with the blank control group (P0.05). Compared with the blank control group, the treatment of vitamin C and cisplatin increased the expression of HIF-1 a mRNA in SHG44 cells and reduced the mRNA of VEGF. There was no significant difference in the expression of VEGFmRNA between the two groups of vitamin C group and cisplatin group (P0.05). (P0.05).
Conclusion: vitamin C can inhibit the proliferation of human glioma SHG44 cells and promote cell apoptosis. The mechanism of vitamin C may be related to reducing the expression of VEGF mRNA in cells and inhibiting the angiogenesis of tissue cells.
The second part: the effect of vitamin C on subcutaneous transplanted tumor in nude mice.
Objective: to construct a subcutaneous tumor model of human glioma SHG44 cell BALB/c nude mice. Cisplatin was used as a positive control group to observe the inhibitory effect of different doses of vitamin C on the growth of subcutaneous transplanted tumor in nude mice, and the effect on the expression of HIF-1 alpha and VEGFmRNA in the tissue cells of the transplanted tumor. The potential of vitamin C to glioma was analyzed. To provide laboratory evidence for the treatment of glioma.
Methods: 1, the human glioma SHG44 cells were cultured and subcultured. Then the cells in the logarithmic growth period were blown into a single cell suspension with a density of 2 x 107 /ml. The 0.2m1 was injected into the subcutaneous of the nude mice (the lateral right forelimb of the back). After 2 weeks of inoculation, the volume of the tumor was measured under the skin of the inoculated site, and the volume of the transplanted tumor was measured about 100mm3, To determine the successful.2 of glioma model, 28 female nude mice of 4-5 weeks old were randomly divided into 4 groups, with 7 rats in each group. The blank control group was given saline, 0.4ml/ only, intraperitoneal injection, once every two days for 3 weeks. The vitamin C was low, the high dose group was given 2g/kg, 4g/kg each, the abdominal injection, once every two days, for 3 weeks; cisplatin group was given cisplatin 2. G/kg was administered intraperitoneally, once every two days, for a total of 3 weeks. The mice were killed at second days after the end of the drug use, and the tumor volume and the tumor suppressor rate were calculated. The expression of HIF-1 alpha and VEGFmRNA in the tumor tissue cells of the experimental groups was detected by RT-PCR.
Results: 1, the tumor model of nude mice was successfully established. The success rate of tumor planting was high, up to 100%.2. Vitamin C could inhibit the growth of subcutaneous tumor tissue in nude mice, and the tumor inhibition rate in high dose group was greater than that in low dose group. The tumor volume of the experimental group was statistically different from that of the blank control group. (P0.05).3, low vitamin C, high dose group could reduce the expression of two factors, and the high dose group decreased more obviously. Compared with the blank group, the difference was statistically significant (P0.05). The vitamin C was low, and the difference between high dose group and cisplatin group was statistically significant (P0.05).
Conclusion: the model of human glioma SHG44 cells to establish a nude mouse subcutaneous transplanted tumor model is reliable and can be used for the prevention and treatment of glioma. Large dose of vitamin C in a certain dose can inhibit the growth of subcutaneous transplanted tumor in nude mice and show certain anti-tumor effect. The mechanism may be inhibited by vitamin C. The expression of HIF-1 alpha and VEGFmRNA in tissue cells reduced the blood supply of tumor tissues. Fig. 14, table 1.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41
本文编号：2153752
[Abstract]:Part one: the effect of vitamin C on human glioma SHG44 cells
Objective: to cultivate human glioma SHG44 cells, then treat SHG44 cells with different concentrations of vitamin C, and use cisplatin to treat SHG44 cells as positive control group, observe the effect of vitamin C on SHG44 cell proliferation, apoptosis, and the expression of HIF-1 alpha and VEGFmRNA in cell, and explore the possibility of vitamin C on SHG44 cells. Use the mechanism.
Methods: through the culture of human glioma SHG44 cells, different concentrations of vitamin C (0,0.5,1.0,2.0,4.0,8.0,10.0mmol/L) were acted on the SHG44 cells in the logarithmic growth period, and the IC50 value of the cells was detected by MTT colorimetric method; then the different concentrations of vitamin C (0.0.5,0.75,1.0,1.25,1.5,1.75,2.0mmol/L) and cisplatin (20mg/L) were used in the logarithmic birth. Long term SHG44 cells were used to detect cell growth inhibition rate by MTT; vitamin C (0,1.5mmol/L) and cisplatin (20mg/L) were used as SHG44 cells in the logarithmic growth period for 24 hours. The cell cycle and apoptosis were detected by flow cytometry, and the RT-PCR method was used to detect the relative expression of HIF-1 alpha and EGFmRNA in SHG44 cells.
Results: vitamin C showed a concentration dependent inhibition of the growth of SHG44 cells. At 570nm wavelength, after 24 hours of vitamin C action, the IC50 value was 1.695mmol/L; when the concentration of vitamin C was greater than 1.25mmol/L, and after the action of SHG44 cells for 48 hours, the cells showed significant proliferation inhibition and large amount of death; vitamin C and cisplatin were located in the SHG44 cells. The early apoptosis of the cells increased significantly compared with the blank control group (P0.05). The treatment of vitamin C and cisplatin reduced the SHG44 cells in S phase compared with the blank control group (P0.05). Compared with the blank control group, the treatment of vitamin C and cisplatin increased the expression of HIF-1 a mRNA in SHG44 cells and reduced the mRNA of VEGF. There was no significant difference in the expression of VEGFmRNA between the two groups of vitamin C group and cisplatin group (P0.05). (P0.05).
Conclusion: vitamin C can inhibit the proliferation of human glioma SHG44 cells and promote cell apoptosis. The mechanism of vitamin C may be related to reducing the expression of VEGF mRNA in cells and inhibiting the angiogenesis of tissue cells.
The second part: the effect of vitamin C on subcutaneous transplanted tumor in nude mice.
Objective: to construct a subcutaneous tumor model of human glioma SHG44 cell BALB/c nude mice. Cisplatin was used as a positive control group to observe the inhibitory effect of different doses of vitamin C on the growth of subcutaneous transplanted tumor in nude mice, and the effect on the expression of HIF-1 alpha and VEGFmRNA in the tissue cells of the transplanted tumor. The potential of vitamin C to glioma was analyzed. To provide laboratory evidence for the treatment of glioma.
Methods: 1, the human glioma SHG44 cells were cultured and subcultured. Then the cells in the logarithmic growth period were blown into a single cell suspension with a density of 2 x 107 /ml. The 0.2m1 was injected into the subcutaneous of the nude mice (the lateral right forelimb of the back). After 2 weeks of inoculation, the volume of the tumor was measured under the skin of the inoculated site, and the volume of the transplanted tumor was measured about 100mm3, To determine the successful.2 of glioma model, 28 female nude mice of 4-5 weeks old were randomly divided into 4 groups, with 7 rats in each group. The blank control group was given saline, 0.4ml/ only, intraperitoneal injection, once every two days for 3 weeks. The vitamin C was low, the high dose group was given 2g/kg, 4g/kg each, the abdominal injection, once every two days, for 3 weeks; cisplatin group was given cisplatin 2. G/kg was administered intraperitoneally, once every two days, for a total of 3 weeks. The mice were killed at second days after the end of the drug use, and the tumor volume and the tumor suppressor rate were calculated. The expression of HIF-1 alpha and VEGFmRNA in the tumor tissue cells of the experimental groups was detected by RT-PCR.
Results: 1, the tumor model of nude mice was successfully established. The success rate of tumor planting was high, up to 100%.2. Vitamin C could inhibit the growth of subcutaneous tumor tissue in nude mice, and the tumor inhibition rate in high dose group was greater than that in low dose group. The tumor volume of the experimental group was statistically different from that of the blank control group. (P0.05).3, low vitamin C, high dose group could reduce the expression of two factors, and the high dose group decreased more obviously. Compared with the blank group, the difference was statistically significant (P0.05). The vitamin C was low, and the difference between high dose group and cisplatin group was statistically significant (P0.05).
Conclusion: the model of human glioma SHG44 cells to establish a nude mouse subcutaneous transplanted tumor model is reliable and can be used for the prevention and treatment of glioma. Large dose of vitamin C in a certain dose can inhibit the growth of subcutaneous transplanted tumor in nude mice and show certain anti-tumor effect. The mechanism may be inhibited by vitamin C. The expression of HIF-1 alpha and VEGFmRNA in tissue cells reduced the blood supply of tumor tissues. Fig. 14, table 1.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41
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