【摘要】：第一部分 藤黃酸對腎癌細(xì)胞RC-2增殖能力及其來源的外核體分泌量及成分的影響 目的：探討藤黃酸對腎癌細(xì)胞RC-2增殖能力及其細(xì)胞源性外核體（exosomes）的分泌量和成分的影響。 方法：MTT法檢測藤黃酸作用前后腎癌RC-2細(xì)胞株增殖能力變化。選取藤黃酸作用前后腎癌RC-2細(xì)胞株上清液，用蔗糖梯度離心法分別提取exosomes，用透射電鏡觀察其形態(tài)差異，觀察其分泌量的變化，二喹啉甲酸（BCA）法對比其蛋白濃度變化并計算其總蛋白質(zhì)含量差異，蛋白免疫印跡(Western Blot)法對比exosomes表面分子及抗原ICAM-1、HSP-70、G250和蛋白Survivin含量差異，采用逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)法對比RC-2細(xì)胞藤黃酸處理前后野生型p53基因表達的變化。 結(jié)果：MTT結(jié)果顯示藤黃酸能夠抑制腎癌細(xì)胞株RC-2增殖，而這種抑制呈濃度及時間依賴性。經(jīng)藤黃酸處理后，透射電鏡觀察腎癌細(xì)胞株RC-2源性exosomes形態(tài)未見明顯改變，其分泌量和蛋白含量較處理前明顯增加（P0.05）,其所含表面分子ICAM-1、HSP-70、G250蛋白質(zhì)含量較處理前未見明顯變化（P0.05），其蛋白Survivin表達明顯減少（P0.05），RT-PCR法結(jié)果顯示藤黃酸作用RC-2細(xì)胞后表達野生型p53mRNA較未經(jīng)藤黃酸作用細(xì)胞明顯上調(diào)（P0.05）。 結(jié)論：藤黃酸能夠以濃度及時間依賴性抑制腎癌細(xì)胞株RC-2增殖，其作用腎癌細(xì)胞株RC-2后，exosomes所含表面分子及抗原表達無明顯變化，但腎癌細(xì)胞株分泌exosomes量和蛋白質(zhì)量明顯增加，同時其所含凋亡抑制蛋白Survivin表達明顯減少，這些變化可能與藤黃酸引起的腎癌細(xì)胞RC-2表達野生型p53mRNA上調(diào)有關(guān)。 第二部分 藤黃酸作用后的腎癌細(xì)胞RC-2分泌的exosomes對腎癌細(xì)胞株RC-2增殖和凋亡的影響 目的：觀察藤黃酸作用腎癌細(xì)胞株RC-2后其分泌的exosomes對腎癌細(xì)胞株RC-2（穩(wěn)定表達hepaCAM基因）增殖和凋亡的影響。 方法：用腺病毒感染構(gòu)建穩(wěn)定表達hepaCAM基因的腎癌RC-2細(xì)胞株，采用RT-PCR法和Western blot檢測感染前后hepaCAMmRNA及蛋白表達變化，以藤黃酸處理過的RC-2細(xì)胞株分泌的exosomes與RC-2細(xì)胞株（穩(wěn)定表達hepaCAM基因）共同培養(yǎng)組為實驗組，未經(jīng)藤黃酸處理過的RC-2細(xì)胞株分泌的等量exosomes與RC-2細(xì)胞株（穩(wěn)定表達hepaCAM基因）共同培養(yǎng)組為對照組，MTT法檢測兩組細(xì)胞株增殖情況，Annexin V-FITC／PI雙染色流式細(xì)胞術(shù)檢測細(xì)胞凋亡的變化，Western blot檢測兩組細(xì)胞共培養(yǎng)48h后hepaCAM、AKT及p-AKT、p-ERKl/2、ERKl/2蛋白表達變化。以p-AKT抑制劑MK-2206在各時間點加入對照組細(xì)胞，觀察細(xì)胞表達hepaCAM、AKT及p-AKT蛋白的變化。采用RT-PCR法檢測58例腎癌及相應(yīng)癌旁對照組織中hepaCAM、PI3K、AKTmRNA的表達并分析三者的相關(guān)性;免疫組織化學(xué)法檢測hepaCAM、p-AKT蛋白的表達,并分析兩者的相關(guān)性。 結(jié)果：腺病毒感染hepaCAM基因后的腎癌RC-2細(xì)胞株與非感染組細(xì)胞株比較能夠穩(wěn)定表達hepaCAM基因和蛋白（P0.05）,MTT結(jié)果顯示實驗組與對照組exosomes都能呈時間及濃度依耐性促進RC-2細(xì)胞株增殖，當(dāng)exosomes濃度大于100ug/ml,實驗組RC-2細(xì)胞株增殖力48h、72h與對照組比較明顯減弱（P0.05）,凋亡實驗顯示實驗組與對照組exosomes都能呈時間及濃度依耐性抑制RC-2細(xì)胞株凋亡，當(dāng)exosomes濃度大于100ug/ml,實驗組RC-2細(xì)胞株凋亡抑制率48h、72h與對照組比較明顯減弱（P0.05）, Western blot檢測兩組exosomes作用48h后對照組較實驗組細(xì)胞株hepaCAM蛋白表達隨時間梯度明顯減少（P0.05），，p-AKT蛋白表達對照組較實驗組表達隨時間梯度明顯增加(P0.05),而p-ERKl/2、ERKl/2、AKT表達兩組變化不具有統(tǒng)計學(xué)差異（P0.05）。對照組各時間點加入p-AKT抑制劑MK-2206處理后，exosomes引起的hepaCAM表達抑制較未加入MK-2206處理組明顯減弱（P0.05）。RT-PCR法顯示腎癌組織中hepaCAMmRNA、PI3KmRNA、AKTmRNA與癌旁對照組織的表達水平相比差異有統(tǒng)計學(xué)意義(P0.05),且hepaCAMmRNA與PI3KmRNA、AKTmRNA表達呈負(fù)相關(guān)(P0.05)。免疫組織化學(xué)結(jié)果顯示p-AKT蛋白在腎癌組織中的表達水平明顯高于癌旁對照組織(P0.05),而hepaCAM蛋白在腎癌組織中的表達水平明顯低于癌旁對照組織(P0.05)，且hepaCAM、p-AKT蛋白表達水平呈負(fù)相關(guān)(P0.05)。 結(jié)論：腎癌細(xì)胞RC-2源性exosomes能夠促進腎癌細(xì)胞株RC-2（穩(wěn)定表達hepaCAM基因）增殖及抑制其凋亡，經(jīng)藤黃酸處理過的腎癌細(xì)胞RC-2源性exosomes促進腎癌細(xì)胞株RC-2（穩(wěn)定表達hepaCAM基因）增殖能力及抑制凋亡能力明顯減弱。腎癌細(xì)胞RC-2源性exosomes的促增殖能力很可能以一種p-AKT途徑依賴性抑制腎癌細(xì)胞株RC-2hepaCAM蛋白表達有關(guān)，而經(jīng)藤黃酸處理過的腎癌細(xì)胞RC-2源性exosomes通過p-AKT途徑抑制腎癌細(xì)胞株RC-2hepaCAM蛋白的表達能力顯著降低。HepaCAM表達與PI3K/AKT在腎癌組織的表達具有一定的負(fù)相關(guān)性。 第三部分 藤黃酸聯(lián)合舒尼替尼抑制腎癌細(xì)胞786-0的體內(nèi)外實驗研究 目的：觀察藤黃酸聯(lián)合舒尼替尼對腎癌細(xì)胞增殖及侵襲等生物學(xué)行為的影響。 方法：單獨或聯(lián)合應(yīng)用藤黃酸和舒尼替尼分別作用腎癌786-0細(xì)胞株，采用MTT法、流式細(xì)胞技術(shù)分別檢測細(xì)胞活力和周期變化，采用細(xì)胞遷移和侵襲實驗檢測細(xì)胞移動與侵襲能力變化，酶聯(lián)免疫吸附劑測定(ELISA)細(xì)胞分泌VEGF變化，Western blot檢測與細(xì)胞周期及轉(zhuǎn)移等相關(guān)調(diào)控蛋白表達變化。用786-0細(xì)胞株構(gòu)建小鼠移植瘤模型，用藤黃酸單獨或聯(lián)合舒尼替尼治療小鼠移植瘤，觀察移植瘤生長情況，免疫組織化學(xué)法檢測移植瘤生長和血管生長情況。 結(jié)果：聯(lián)合應(yīng)用藤黃酸與舒尼替尼作用786-0細(xì)胞后其細(xì)胞增殖率為(63.2±5.7)%，比單藥組更顯著抑制細(xì)胞增殖（P0.05），并且更多細(xì)胞[(27.43±3.11)%]的細(xì)胞周期聚集于sub-G1期(P0.05)，聯(lián)合用藥組與單藥組比較能夠更顯著抑制細(xì)胞的遷移和侵襲能力（P0.05），更能顯著抑制細(xì)胞分泌VEGF[濃度(141.72±3.98pg/mL/105細(xì)胞)]（P0.05），Western blot顯示其與單藥組比較Bcl-2表達顯著減少(P0.05)，P21表達顯著增加(P0.05)，VEGF顯著減少（P0.05）, MMP-2顯著減少（P0.05）,而CyclinB1與MMP-9表達未見明顯統(tǒng)計學(xué)差異（P0.05）。體內(nèi)實驗顯示：聯(lián)合用藥組比單藥組更能顯著抑制小鼠移植瘤的生長及血管的形成（P0.05）。 結(jié)論：體外實驗證實聯(lián)合應(yīng)用藤黃酸和舒尼替尼作用腎癌細(xì)胞株786-0比單獨用藥能夠更顯著抑制細(xì)胞生長、侵襲。體內(nèi)實驗證實聯(lián)合應(yīng)用藤黃酸和舒尼替尼作用腎癌細(xì)胞株786-0小鼠移植瘤比單獨用藥能夠更顯著抑制小鼠移植瘤的生長及血管形成。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.11

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