維生素C對(duì)人膠質(zhì)瘤SHG44細(xì)胞和裸鼠皮下移植瘤的影響
發(fā)布時(shí)間：2018-07-29 19:30
【摘要】：第一部分：維生素C對(duì)人膠質(zhì)瘤SHG44細(xì)胞的影響作用 目的：通過培養(yǎng)人膠質(zhì)瘤SHG44細(xì)胞,然后采用不同濃度維生素C處理SHG44細(xì)胞,用順鉑處理SHG44細(xì)胞作為陽(yáng)性對(duì)照組,觀察維生素C對(duì)SHG44細(xì)胞增殖、凋亡的影響,以及對(duì)細(xì)胞中HIF-1α和VEGFmRNA表達(dá)水平的影響。探討維生素C對(duì)SHG44細(xì)胞的可能作用機(jī)制。 方法：通過培養(yǎng)人膠質(zhì)瘤SHG44細(xì)胞,將不同濃度維生素C(0、0.5、1.0、2.0、4.0、8.0、10.0mmol/L)作用于處于對(duì)數(shù)生長(zhǎng)期的SHG44細(xì)胞,MTT比色法檢測(cè)細(xì)胞的IC50值；然后將不同濃度維生素C(0.0.5、0.75、1.0、1.25、1.5、1.75、2.0mmol/L)和順鉑(20mg/L)作用于處于對(duì)數(shù)生長(zhǎng)期的SHG44細(xì)胞,采用MTT法檢測(cè)細(xì)胞的生長(zhǎng)抑制率；將維生素C (0、1.5mmol/L)和順鉑(20mg/L)作用于處于對(duì)數(shù)生長(zhǎng)期的SHG44細(xì)胞24小時(shí),采用流式細(xì)胞儀檢測(cè)細(xì)胞周期和凋亡,用RT-PCR法檢測(cè)SHG44細(xì)胞中HIF-1α和、EGFmRNA的相對(duì)表達(dá)。 結(jié)果：維生素C表現(xiàn)為濃度依賴性抑制SHG44細(xì)胞的生長(zhǎng)。在570nm波長(zhǎng),維生素C作用24小時(shí)后,其IC50值為1.695mmol/L;當(dāng)維生素C的濃度大于1.25mmol/L,并作用SHG44細(xì)胞48小時(shí)后細(xì)胞出現(xiàn)明顯的增殖抑制并大量死亡；維生素C和順鉑對(duì)SHG44細(xì)胞的處理都使得細(xì)胞的早期凋亡較空白對(duì)照組顯著增加(P0.05)。維生素C和順鉑的處理都使得S期的SHG44細(xì)胞較空白對(duì)照組減少(P0.05)。與空白對(duì)照組相比,常氧條件下,維生素C和順鉑的處理都能升高SHG44細(xì)胞中HIF-1αmRNA的表達(dá),和降低VEGF mRNA的表達(dá)(P0.05)；維生素C組和順鉑組兩組之間細(xì)胞中VEGFmRNA的表達(dá)其差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論：維生素C能夠抑制人膠質(zhì)瘤SHG44細(xì)胞的增殖,并且促進(jìn)細(xì)胞的凋亡。維生素C的作用機(jī)制可能跟降低細(xì)胞中VEGF mRNA的表達(dá),抑制組織細(xì)胞的血管生成有關(guān)。 第二部分：維生素C對(duì)裸鼠皮下移植瘤的影響作用 目的：通過構(gòu)建人膠質(zhì)瘤SHG44細(xì)胞BALB/c型裸鼠皮下移植瘤模型。采用順鉑作為陽(yáng)性對(duì)照組,觀察不同劑量的維生素C對(duì)裸鼠皮下移植瘤的生長(zhǎng)抑制作用。以及對(duì)移植瘤組織細(xì)胞中HIF-1α和VEGFmRNA表達(dá)的影響。分析維生素C對(duì)膠質(zhì)瘤的潛在作用機(jī)制,為膠質(zhì)瘤的治療提供實(shí)驗(yàn)室依據(jù)。 方法：1、培養(yǎng)人膠質(zhì)瘤SHG44細(xì)胞并進(jìn)行傳代。然后采用處于對(duì)數(shù)生長(zhǎng)期的細(xì)胞,吹打制成密度為2×107個(gè)/ml的單細(xì)胞懸液。抽取0.2m1注射入裸鼠皮下(背部右前肢外側(cè))。在接種后2周檢查,接種部位皮下長(zhǎng)出包塊,測(cè)量移植瘤體積,大約為100mm3,判定膠質(zhì)瘤模型制作成功。2、將4-5周齡的雌性裸鼠28只隨機(jī)分為4組,每組7只�？瞻讓�(duì)照組給予生理鹽水,0.4ml/只,腹腔注射,每?jī)商煲淮?共3周；維生素C低、高劑量組分別給予2g/kg、4g/kg每只,腹腔注射,每?jī)商煲淮?共3周；順鉑組則給予順鉑2g/kg,腹腔注射給藥,每?jī)商煲淮?共3周。在用藥結(jié)束后第二天處死各實(shí)驗(yàn)組小鼠,并計(jì)算腫瘤體積以及抑瘤率。采用RT-PCR法檢測(cè)各實(shí)驗(yàn)組腫瘤組織細(xì)胞中HIF-1α和VEGFmRNA的表達(dá)情況。 結(jié)果：1、成功建立了裸鼠皮下移植瘤模型,腫瘤種植的成功率較高,達(dá)100%。2、維生素C可以抑制裸鼠皮下移植瘤組織的生長(zhǎng),且高劑量組對(duì)腫瘤的抑瘤率其結(jié)果要大于低劑量組。各實(shí)驗(yàn)組的腫瘤體積跟空白對(duì)照組相比具有統(tǒng)計(jì)學(xué)上的差異(P0.05)。3、維生素C低、高劑量組都能夠降低兩種因子的表達(dá),并且高劑量組降低得更明顯,與空白組相比,其差異都具有統(tǒng)計(jì)學(xué)意義(P0.05)。維生素C低、高劑量組和順鉑組比較,其差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論：人膠質(zhì)瘤SHG44細(xì)胞建立裸鼠皮下移植瘤模型確實(shí)可靠,可以用于膠質(zhì)瘤的預(yù)防以及治療的實(shí)驗(yàn)性研究；在一定劑量范圍內(nèi)的大劑量維生素C可以抑制裸鼠皮下移植瘤的生長(zhǎng),表現(xiàn)出一定的抗腫瘤作用。其機(jī)制可能跟維生素C抑制組織細(xì)胞中HIF-1α和VEGFmRNA的表達(dá),降低腫瘤組織的血供相關(guān)。圖14幅,表1個(gè),
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41
本文編號(hào)：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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41
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