本文選題：血管生成素 + 膀胱癌細胞　； 參考：《重慶醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的構(gòu)建血管生成素（ANG）基因siRNA干擾質(zhì)粒，并檢測其對人膀胱癌T24細胞增殖、凋亡的影響以及初步探討其分子機制。 方法 1.設(shè)計并構(gòu)建針對人ANG基因mRNA的2個特異性siRNA表達載體和1個無同源性的陰性對照載體，通過SalI酶切和DNA測序鑒定載體后，再轉(zhuǎn)染膀胱癌T24細胞，G418篩選穩(wěn)定表達的細胞株，分別命名為T24-siANG1、T24-siANG2、T24-NC，經(jīng)Q-RCR，Western Blot檢測干擾的有效性。 2.HE染色觀察細胞形態(tài)；MTT檢測細胞增殖能力；流式細胞術(shù)檢測細胞周期；激光共聚焦檢測ANG與RI相互關(guān)系；細胞免疫熒光檢測蛋白ANG、RI、p-AKT、p-GSK3β、p-mTOR表達水平。 3.流式細胞術(shù)檢測細胞凋亡率；Tunel和Hochest33342凋亡試劑盒檢測細胞凋亡；Western blot檢測蛋白ANG、RI，細胞凋亡相關(guān)蛋白Bax、Caspase-3、Bcl-2，信號通路蛋白AKT、GSK3β、mTOR、p-AKT、p-GSK3β、p-mTOR表達水平。 4.構(gòu)建裸鼠移植瘤模型，觀察移植瘤生長并計算抑制率；CD31抗體，HE染色觀察腫瘤微血管生長以及肺組織自發(fā)轉(zhuǎn)移情況；組織免疫熒光檢測蛋白RI、ANG、p-AKT、p-GSK3β、p-mTOR表達水平；免疫組織化學(xué)檢測瘤組織中蛋白ANG、RI、Bax、Caspase-3、Bcl-2、p-AKT、p-GSK3β、p-mTOR、AKT、GSK3β、mTOR表達水平。 結(jié)果 1.測序結(jié)果顯示：重組質(zhì)粒構(gòu)建成功；Q-PCR，Western Blot分析T24-siANG1組ANG的mRNA，蛋白表達顯著抑制，與T24-NC組相比（p0.05）。 2.HE染色結(jié)果顯示：與對照組相比，實驗組T24-siANG1組的細胞惡性程度降低，表現(xiàn)為細胞不重疊生長，細胞核變小，核質(zhì)比減少，呈弱嗜堿性細胞質(zhì)；MTT結(jié)果顯示：T24-siANG1組較T24-NC及T24組細胞增殖能力明顯下降；流式細胞分析結(jié)果表明：T24-siANG1組較兩對照組細胞生長阻滯于G1期，S期和G2期降低；激光共聚焦檢測ANG與RI蛋白具有共定位關(guān)系；細胞及組織免疫熒光檢測結(jié)果顯示：ANG，信號通路關(guān)鍵蛋白p-AKT，p-GSK3β，p-mTOR表達降低，而RI的表達增高。 3.流式細胞分析結(jié)果表明：流式細胞儀檢測結(jié)果顯示：T24-siANG1組細胞有（30.23±15.81）％的凋亡細胞，而T24-NC組和T24細胞組僅有(5.44±3.09)％和(3.96±1.58)％的凋亡細胞，T24-siANG1組較T24-NC組細胞凋亡率明顯增高（p0.01）；TUNEL檢測結(jié)果顯示：T24-siANG1組出現(xiàn)大量凋亡細胞；Hochest檢測結(jié)果顯示：T24-siANG1組出現(xiàn)典型凋亡形態(tài)特征：染色質(zhì)凝集，核碎裂和明亮的藍色熒光等；Western blot結(jié)果顯示：與T24-NC組相比，T24-siANG1組Bcl-2的表達明顯降低(p＜0.05),而Bax和激活的Caspase3的表達明顯升高(p＜0.05)；T24-siANG1組較T24-NC組的信號通路蛋白p-AKT，p-GSK3β，p-mTOR的表達顯著降低(p＜0.01)，而RI蛋白表達水平明顯升高(p＜0.05)。 4.裸鼠在皮下注射各組細胞后，與T24-NC組相比，T24-siANG1組的移植瘤重明顯降低；組織免疫熒光CD31及HE染色實驗結(jié)果均表明：與兩對照組相比，T24-siANG1組瘤組織中微血管明顯減少。肺組織HE染色結(jié)果顯示：T24-siANG1組肺組織未見明顯轉(zhuǎn)移，而兩對照組在大血管附近可見細胞核增大且染色較深的細胞，，表明已發(fā)生遠處轉(zhuǎn)移。免疫組織化學(xué)檢測結(jié)果顯示：T24-siANG1組瘤組織中蛋白RI， Bax，Caspase-3表達明顯升高，而Bcl-2，ANG，p-AKT，p-GSK3β，p-mTOR表達明顯降低， AKT，GSK3β，mTOR的表達無變化，與Western blot結(jié)果一致。 結(jié)論體內(nèi)外實驗均證實：成功構(gòu)建的ANG siRNA干擾質(zhì)粒通過調(diào)控ANG信號通路及凋亡蛋白抑制膀胱癌T24細胞的增殖并促進其凋亡。
[Abstract]:Objective To construct siRNA interfering plasmid of angiopoietin ( ANG ) gene , and to examine its effect on proliferation and apoptosis of human bladder cancer cell line 24 , and to explore its molecular mechanism .

method

1 . Two specific siRNA expression vectors and one non - homologous negative control vector were designed and constructed for human ANG gene mRNA .

2 . HE staining was used to observe the morphology of cells .
MTT assay was used to detect cell proliferation .
Flow cytometry was used to detect cell cycle .
laser confocal detection ANG is related to RI ;
The levels of ANG , RI , p - 1 , p - GSK3 & beta ; , p - mtor expression were detected by immunofluorescence assay .

3 . Flow cytometry was used to detect apoptosis rate .
Apoptosis was detected by Tunel and Hooks 33342 apoptosis kit .
Western blot was used to detect the expression level of Bax , Caspase - 3 , Bcl - 2 , signal pathway proteins , GSK3 尾 , mtor , p - 1 , p - GSK3 尾 , p - mtor expression in protein ANG , RI , cell apoptosis related protein Bax , Caspase - 3 , Bcl - 2 , signal pathway protein 3 .

4 . The nude mice transplanted tumor model was constructed , and the growth of transplanted tumor was observed and the inhibition rate was calculated .
CD31 antibody and HE staining were used to observe the growth of tumor microvessels and spontaneous metastasis of lung tissues .
Tissue immunofluorescence assay protein RI , ANG , p - 1 , p - GSK3beta , p - mtor expression level ;
The levels of protein ANG , RI , Bax , Caspase - 3 , Bcl - 2 , p - 1 , p - GSK3 尾 , p - mtor , T , GSK3 尾 and mtor were detected by immunohistochemistry .

Results

1 . Sequencing results showed that the recombinant plasmid was constructed successfully .
The mRNA and protein expressions of ANG mRNA and protein were significantly inhibited by Q - PCR and Western Blot .

2 . The results of HE staining showed that the malignant degree of cells in the experimental group 24 - siANG1 was decreased compared with the control group , which showed that the cells did not overlap and grow , the nucleus became smaller , the nuclear mass ratio decreased , and showed weak basophilic cytoplasm ;
The results showed that the cell proliferation ability was significantly decreased in the 24 - siANG1 group than that in the 24 - NC group .
The results of flow cytometry showed that the growth arrest of the cells was decreased in G1 phase , S phase and G2 phase compared with the control group .
Laser cofocus detection ANG has a co - located relationship with RI protein .
The results of immunofluorescence assay showed that the expression of the key protein of ANG , signaling pathway was decreased , and the expression of p - GSK3 尾 , p - mtor decreased , while RI increased .

3 . The results of flow cytometry showed that there were ( 30.23 鹵 15.81 ) % apoptotic cells in 24 - siANG1 group , but only ( 5.44 鹵 3.09 ) % and ( 3.96 鹵 1 . 58 ) % of apoptotic cells were found in 24 - NC group and 24 - cell group .
TUNEL assay showed that there appeared a significant number of apoptotic cells in 24 - siANG1 group .
The results showed that the typical apoptotic morphological features : chromatin condensation , nuclear fragmentation and bright blue fluorescence appeared in the 24 - siANG1 group .
Western blot showed that the expression of Bcl - 2 was significantly decreased ( p < 0 . 05 ) , but Bax and the activated caspase3 increased significantly ( p < 0 . 05 ) .
The expression of p - gp , p - GSK3 尾 , p - mtor decreased significantly ( p < 0 . 01 ) , while RI protein expression increased significantly ( p < 0 . 05 ) .

4 . After subcutaneous injection of each group of cells in nude mice , the transplanted tumor weight was significantly decreased in the 24 - siANG1 group compared with the 24 - NC group .
The results of HE staining showed that the expression of protein RI , Bax , Caspase - 3 in 24 - siANG1 group was significantly higher than that in the control group , but the expression of Bcl - 2 , ANG , p - 1 , p - GSK3 尾 , p - mtor was significantly decreased .

Conclusion Both in vivo and in vivo experiments confirm that the ANG siRNA interfering plasmid constructed successfully inhibits the proliferation of bladder cancer cell line 24 and promotes its apoptosis by regulating ANG signal pathway and apoptosis protein .

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.14

【參考文獻】

相關(guān)期刊論文 前1條

1 高娟;康煒;陳俊霞;朱軍;潘湘陽;;siRNA沉默整合素連接激酶對人膀胱癌細胞凋亡的影響[J];第三軍醫(yī)大學(xué)學(xué)報;2012年21期

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