藤黄酸对肾癌细胞的抑制作用研究

发布时间：2018-08-03 11:12

【摘要】：第一部分藤黄酸对肾癌细胞RC-2增殖能力及其来源的外核体分泌量及成分的影响目的：探讨藤黄酸对肾癌细胞RC-2增殖能力及其细胞源性外核体（exosomes）的分泌量和成分的影响。方法：MTT法检测藤黄酸作用前后肾癌RC-2细胞株增殖能力变化。选取藤黄酸作用前后肾癌RC-2细胞株上清液，用蔗糖梯度离心法分别提取exosomes，用透射电镜观察其形态差异，观察其分泌量的变化，二喹啉甲酸（BCA）法对比其蛋白浓度变化并计算其总蛋白质含量差异，蛋白免疫印迹(Western Blot)法对比exosomes表面分子及抗原ICAM-1、HSP-70、G250和蛋白Survivin含量差异，采用逆转录-聚合酶链反应(RT-PCR)法对比RC-2细胞藤黄酸处理前后野生型p53基因表达的变化。结果：MTT结果显示藤黄酸能够抑制肾癌细胞株RC-2增殖，而这种抑制呈浓度及时间依赖性。经藤黄酸处理后，透射电镜观察肾癌细胞株RC-2源性exosomes形态未见明显改变，其分泌量和蛋白含量较处理前明显增加（P0.05）,其所含表面分子ICAM-1、HSP-70、G250蛋白质含量较处理前未见明显变化（P0.05），其蛋白Survivin表达明显减少（P0.05），RT-PCR法结果显示藤黄酸作用RC-2细胞后表达野生型p53mRNA较未经藤黄酸作用细胞明显上调（P0.05）。结论：藤黄酸能够以浓度及时间依赖性抑制肾癌细胞株RC-2增殖，其作用肾癌细胞株RC-2后，exosomes所含表面分子及抗原表达无明显变化，但肾癌细胞株分泌exosomes量和蛋白质量明显增加，同时其所含凋亡抑制蛋白Survivin表达明显减少，这些变化可能与藤黄酸引起的肾癌细胞RC-2表达野生型p53mRNA上调有关。第二部分藤黄酸作用后的肾癌细胞RC-2分泌的exosomes对肾癌细胞株RC-2增殖和凋亡的影响目的：观察藤黄酸作用肾癌细胞株RC-2后其分泌的exosomes对肾癌细胞株RC-2（稳定表达hepaCAM基因）增殖和凋亡的影响。方法：用腺病毒感染构建稳定表达hepaCAM基因的肾癌RC-2细胞株，采用RT-PCR法和Western blot检测感染前后hepaCAMmRNA及蛋白表达变化，以藤黄酸处理过的RC-2细胞株分泌的exosomes与RC-2细胞株（稳定表达hepaCAM基因）共同培养组为实验组，未经藤黄酸处理过的RC-2细胞株分泌的等量exosomes与RC-2细胞株（稳定表达hepaCAM基因）共同培养组为对照组，MTT法检测两组细胞株增殖情况，Annexin V-FITC／PI双染色流式细胞术检测细胞凋亡的变化，Western blot检测两组细胞共培养48h后hepaCAM、AKT及p-AKT、p-ERKl/2、ERKl/2蛋白表达变化。以p-AKT抑制剂MK-2206在各时间点加入对照组细胞，观察细胞表达hepaCAM、AKT及p-AKT蛋白的变化。采用RT-PCR法检测58例肾癌及相应癌旁对照组织中hepaCAM、PI3K、AKTmRNA的表达并分析三者的相关性;免疫组织化学法检测hepaCAM、p-AKT蛋白的表达,并分析两者的相关性。结果：腺病毒感染hepaCAM基因后的肾癌RC-2细胞株与非感染组细胞株比较能够稳定表达hepaCAM基因和蛋白（P0.05）,MTT结果显示实验组与对照组exosomes都能呈时间及浓度依耐性促进RC-2细胞株增殖，当exosomes浓度大于100ug/ml,实验组RC-2细胞株增殖力48h、72h与对照组比较明显减弱（P0.05）,凋亡实验显示实验组与对照组exosomes都能呈时间及浓度依耐性抑制RC-2细胞株凋亡，当exosomes浓度大于100ug/ml,实验组RC-2细胞株凋亡抑制率48h、72h与对照组比较明显减弱（P0.05）, Western blot检测两组exosomes作用48h后对照组较实验组细胞株hepaCAM蛋白表达随时间梯度明显减少（P0.05），，p-AKT蛋白表达对照组较实验组表达随时间梯度明显增加(P0.05),而p-ERKl/2、ERKl/2、AKT表达两组变化不具有统计学差异（P0.05）。对照组各时间点加入p-AKT抑制剂MK-2206处理后，exosomes引起的hepaCAM表达抑制较未加入MK-2206处理组明显减弱（P0.05）。RT-PCR法显示肾癌组织中hepaCAMmRNA、PI3KmRNA、AKTmRNA与癌旁对照组织的表达水平相比差异有统计学意义(P0.05),且hepaCAMmRNA与PI3KmRNA、AKTmRNA表达呈负相关(P0.05)。免疫组织化学结果显示p-AKT蛋白在肾癌组织中的表达水平明显高于癌旁对照组织(P0.05),而hepaCAM蛋白在肾癌组织中的表达水平明显低于癌旁对照组织(P0.05)，且hepaCAM、p-AKT蛋白表达水平呈负相关(P0.05)。结论：肾癌细胞RC-2源性exosomes能够促进肾癌细胞株RC-2（稳定表达hepaCAM基因）增殖及抑制其凋亡，经藤黄酸处理过的肾癌细胞RC-2源性exosomes促进肾癌细胞株RC-2（稳定表达hepaCAM基因）增殖能力及抑制凋亡能力明显减弱。肾癌细胞RC-2源性exosomes的促增殖能力很可能以一种p-AKT途径依赖性抑制肾癌细胞株RC-2hepaCAM蛋白表达有关，而经藤黄酸处理过的肾癌细胞RC-2源性exosomes通过p-AKT途径抑制肾癌细胞株RC-2hepaCAM蛋白的表达能力显著降低。HepaCAM表达与PI3K/AKT在肾癌组织的表达具有一定的负相关性。第三部分藤黄酸联合舒尼替尼抑制肾癌细胞786-0的体内外实验研究目的：观察藤黄酸联合舒尼替尼对肾癌细胞增殖及侵袭等生物学行为的影响。方法：单独或联合应用藤黄酸和舒尼替尼分别作用肾癌786-0细胞株，采用MTT法、流式细胞技术分别检测细胞活力和周期变化，采用细胞迁移和侵袭实验检测细胞移动与侵袭能力变化，酶联免疫吸附剂测定(ELISA)细胞分泌VEGF变化，Western blot检测与细胞周期及转移等相关调控蛋白表达变化。用786-0细胞株构建小鼠移植瘤模型，用藤黄酸单独或联合舒尼替尼治疗小鼠移植瘤，观察移植瘤生长情况，免疫组织化学法检测移植瘤生长和血管生长情况。结果：联合应用藤黄酸与舒尼替尼作用786-0细胞后其细胞增殖率为(63.2±5.7)%，比单药组更显著抑制细胞增殖（P0.05），并且更多细胞[(27.43±3.11)%]的细胞周期聚集于sub-G1期(P0.05)，联合用药组与单药组比较能够更显著抑制细胞的迁移和侵袭能力（P0.05），更能显著抑制细胞分泌VEGF[浓度(141.72±3.98pg/mL/105细胞)]（P0.05），Western blot显示其与单药组比较Bcl-2表达显著减少(P0.05)，P21表达显著增加(P0.05)，VEGF显著减少（P0.05）, MMP-2显著减少（P0.05）,而CyclinB1与MMP-9表达未见明显统计学差异（P0.05）。体内实验显示：联合用药组比单药组更能显著抑制小鼠移植瘤的生长及血管的形成（P0.05）。结论：体外实验证实联合应用藤黄酸和舒尼替尼作用肾癌细胞株786-0比单独用药能够更显著抑制细胞生长、侵袭。体内实验证实联合应用藤黄酸和舒尼替尼作用肾癌细胞株786-0小鼠移植瘤比单独用药能够更显著抑制小鼠移植瘤的生长及血管形成。
[Abstract]:Part one
Effects of garcinic acid on proliferation and exosome secretion and components of renal cell carcinoma RC-2 cells
Objective: To investigate the effects of gambogic acid on the proliferation of RC-2 cells and the secretion and composition of exosomes.
Methods: MTT assay was used to detect the proliferation of RC-2 cell lines of renal carcinoma before and after the action of rink acid. The supernatant of RC-2 cell line of renal carcinoma before and after the action of garcinic acid was selected and exosomes was extracted by sucrose gradient centrifugation. The morphological difference was observed by transmission electron microscope, and the changes of its secretion were observed. Two quinoline formic acid (BCA) method was used to compare the change of protein concentration. The difference of total protein content was calculated. The difference of exosomes surface molecules and antigen ICAM-1, HSP-70, G250 and protein Survivin content were compared by Western Blot method. Reverse transcription polymerase chain reaction (RT-PCR) method was used to compare the changes of gene expression of wild type p53 gene before and after the treatment of RC-2 cells.
Results: MTT results showed that RC could inhibit the proliferation of RC-2 cell line in renal cell carcinoma cell line, and this inhibition was dependent on concentration and time. After treatment with RP, the RC-2 derived exosomes morphology of renal cell carcinoma cell line was not obviously changed, and its secretion and protein content increased significantly (P0.05), and its surface molecule ICAM-1, HSP-70, G250 protein content was not significantly changed before treatment (P0.05), and its protein Survivin expression decreased significantly (P0.05). RT-PCR results showed that the expression of wild type p53mRNA was significantly higher than that of untreated cells (P0.05) after the action of garcinic acid in RC-2 cells.
Conclusion: it can inhibit the proliferation of renal cell carcinoma cell line RC-2 in concentration and time dependence, and there is no obvious change in the expression of surface molecules and antigen in exosomes cell line RC-2, but the secretion of exosomes and protein in the renal cell line is obviously increased, and the expression of the apoptosis inhibitor protein Survivin is obviously reduced. These changes may be related to upregulated expression of wild type p53mRNA in RC-2 cells induced by garcinic acid.
The second part
Effects of exosomes produced by RC-2 on renal cell carcinoma RC-2 cell proliferation and apoptosis
AIM: To observe the effect of exosomes secreted by gambogic acid on proliferation and apoptosis of RC-2 cells.
Methods: using adenovirus infection to construct a RC-2 cell line with hepaCAM gene, the expression of hepaCAMmRNA and protein expression before and after infection was detected by RT-PCR and Western blot. The co culture of exosomes and RC-2 cell lines (the stable expression of hepaCAM gene) secreted by the RC-2 cell line of the glandric acid was co cultured as the experimental group. The same amount of exosomes secreted by the acid treated RC-2 cell line and the RC-2 cell line (the stable expression of hepaCAM gene) was the control group. The proliferation of two groups of cell lines was detected by MTT, and the changes of cell apoptosis were detected by Annexin V-FITC / PI double staining flow cytometry. Western blot was used to detect the 48h hepaCAM. The expression of -ERKl/2, ERKl/2 protein was changed. P-AKT inhibitor MK-2206 was added to the control group at all time points to observe the expression of hepaCAM, AKT and p-AKT protein in the cells. The expression of hepaCAM, PI3K, AKTmRNA in 58 cases of renal carcinoma and corresponding paracancerous tissues was detected by RT-PCR method and the correlation of the three were analyzed. Immunohistochemistry was used to detect the hepaCA. The expression of M and p-AKT protein was analyzed and their correlation was analyzed.
Results: the hepaCAM gene and protein (P0.05) could be expressed stably after the adenovirus infected hepaCAM gene RC-2 cell line and the non infected cell line. The MTT results showed that both the experimental group and the control group exosomes could promote the proliferation of RC-2 cell lines in time and concentration, when the concentration of exosomes was greater than 100ug/ml, and the RC-2 cell line of the experimental group was more than 100ug/ml. The proliferation of 48h and 72h decreased significantly with the control group (P0.05). Apoptosis experiments showed that both the experimental group and the control group had time and concentration dependent inhibition of the apoptosis of RC-2 cell lines. When the exosomes concentration was greater than 100ug/ml, the apoptosis inhibition rate of RC-2 cells in the experimental group was 48H, and 72h was significantly weakened (P0.05) with the control group (P0.05), and two groups were detected by Western. Compared with the experimental group, the expression of hepaCAM protein in the control group decreased with the time gradient (P0.05). The expression of p-AKT protein expression in the control group increased significantly with the time gradient (P0.05), while the two groups of p-ERKl/2, ERKl/2, and AKT expression did not have statistical differences (P0.05). The control group was added to the p-AKT inhibitor MK- at each time point in the control group. (P0.05). The expression of p-AKT in the control group was more than the time gradient (P0.05). The control group was added to the p-AKT inhibitor MK- at every time point of the control group. After 2206 treatment, the inhibition of hepaCAM expression induced by exosomes was significantly lower than that in the non MK-2206 treatment group (P0.05).RT-PCR method showed that the expression level of hepaCAMmRNA, PI3KmRNA, AKTmRNA and paracancerous tissue was statistically significant (P0.05), and hepaCAMmRNA and PI3KmRNA, AKTmRNA expression was negatively correlated. The expression of p-AKT protein in renal carcinoma tissue was significantly higher than that of the para cancerous tissue (P0.05), while the expression level of hepaCAM protein in renal carcinoma was significantly lower than that in the paracancerous control tissue (P0.05), and the expression level of p-AKT protein was negatively correlated (P0.05).
Conclusion: RC-2 derived exosomes in renal cell carcinoma cells can promote the proliferation and inhibit the apoptosis of renal cancer cell line RC-2 (hepaCAM gene). The RC-2 derived exosomes of renal cancer cells treated with garcinic acid can promote the proliferation and inhibition of apoptosis of renal cancer cell line RC-2 (the stable expression of hepaCAM gene). The RC-2 derived exo of renal cell carcinoma cells. The proliferation promoting ability of somes is likely to be associated with a p-AKT pathway dependent inhibition of the expression of RC-2hepaCAM protein in renal cell carcinoma cell lines. The expression of RC-2 derived exosomes in renal cancer cells treated by garcinic acid through the p-AKT pathway inhibits the expression of RC-2hepaCAM protein in the renal cell carcinoma cell line and significantly reduces the expression of.HepaCAM and PI3K/AKT in the renal carcinoma tissue. There is a certain negative correlation between DA and da.
The third part
Inhibitory effect of garcinic acid combined with sunitinib on renal cell carcinoma 786-0 in vivo and in vitro
Objective: To observe the effects of garcinic acid combined with sunitinib on the proliferation and invasion of renal cell carcinoma.
Methods: the 786-0 cell lines of renal cancer were treated separately or combined with rnyl, respectively. The cell viability and cycle changes were detected by MTT and flow cytometry. Cell migration and invasion test were used to detect cell migration and invasion ability. The changes of VEGF in ELISA cells were detected by enzyme linked immunosorbent assay (ELISA), and Western blo T was used to detect the changes in the expression of regulatory proteins related to cell cycle and metastasis. The transplanted tumor model of mice was constructed with 786-0 cell lines, and the xenografts were treated with lufhuang acid alone or combined with sulanitinib, and the growth of the transplanted tumor was observed. The growth of the transplanted tumor and the growth of the blood tube were detected by immunohistochemistry.
Results: the cell proliferation rate was (63.2 + 5.7)% (63.2 + 5.7)% after the combined use of the 786-0 cells, which was more significantly inhibited than the single drug group (P0.05), and the cell cycle of more cells [(27.43 + 3.11)%] was clustered in the sub-G1 phase (P0.05). The combination of the combination group and the single drug group could significantly inhibit the migration and invasion of the cells. The concentration of VEGF[(141.72 + 3.98pg/mL/105 cells) was significantly inhibited by P0.05 (P0.05), and Western blot showed a significant decrease in Bcl-2 expression compared with the single drug group (P0.05), the expression of P21 significantly increased (P0.05), VEGF significantly decreased (P0.05), and there was no significant difference in the expression of Bcl-2 (P0.05). 5) in vivo experiments showed that the combined treatment group could significantly inhibit the growth and angiogenesis of transplanted tumor in mice than that in the single drug group (P0.05).
Conclusion: in vitro experiments have proved that the combined use of garcinic acid and suneinib on renal cancer cell line 786-0 can significantly inhibit cell growth and invasion. In vivo experiments have proved that the combined use of garcinic acid and suneinib action of renal cancer cell line 786-0 mice transplanted tumor could significantly inhibit the growth of transplanted tumor in mice. Long and vascular formation.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.11

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