本文選題：血管生成素 + 膀胱癌細(xì)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(xì)胞增殖、凋亡的影響以及初步探討其分子機(jī)制。 方法 1.設(shè)計(jì)并構(gòu)建針對人ANG基因mRNA的2個(gè)特異性siRNA表達(dá)載體和1個(gè)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(xì)胞，G418篩選穩(wěn)定表達(dá)的細(xì)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(xì)胞形態(tài)；MTT檢測細(xì)胞增殖能力；流式細(xì)胞術(shù)檢測細(xì)胞周期；激光共聚焦檢測ANG與RI相互關(guān)系；細(xì)胞免疫熒光檢測蛋白ANG、RI、p-AKT、p-GSK3β、p-mTOR表達(dá)水平。 3.流式細(xì)胞術(shù)檢測細(xì)胞凋亡率；Tunel和Hochest33342凋亡試劑盒檢測細(xì)胞凋亡；Western blot檢測蛋白ANG、RI，細(xì)胞凋亡相關(guān)蛋白Bax、Caspase-3、Bcl-2，信號(hào)通路蛋白AKT、GSK3β、mTOR、p-AKT、p-GSK3β、p-mTOR表達(dá)水平。 4.構(gòu)建裸鼠移植瘤模型，觀察移植瘤生長并計(jì)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R(shí)I、ANG、p-AKT、p-GSK3β、p-mTOR表達(dá)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(dá)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(dá)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(shí)驗(yàn)組T24-siANG1組的細(xì)胞惡性程度降低，表現(xiàn)為細(xì)胞不重疊生長，細(xì)胞核變小，核質(zhì)比減少，呈弱嗜堿性細(xì)胞質(zhì)；MTT結(jié)果顯示：T24-siANG1組較T24-NC及T24組細(xì)胞增殖能力明顯下降；流式細(xì)胞分析結(jié)果表明：T24-siANG1組較兩對照組細(xì)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(xì)胞及組織免疫熒光檢測結(jié)果顯示：ANG，信號(hào)通路關(guān)鍵蛋白p-AKT，p-GSK3β，p-mTOR表達(dá)降低，而RI的表達(dá)增高。 3.流式細(xì)胞分析結(jié)果表明：流式細(xì)胞儀檢測結(jié)果顯示：T24-siANG1組細(xì)胞有（30.23±15.81）％的凋亡細(xì)胞，而T24-NC組和T24細(xì)胞組僅有(5.44±3.09)％和(3.96±1.58)％的凋亡細(xì)胞，T24-siANG1組較T24-NC組細(xì)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(xì)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(lán)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(dá)明顯降低(p＜0.05),而Bax和激活的Caspase3的表達(dá)明顯升高(p＜0.05)；T24-siANG1組較T24-NC組的信號(hào)通路蛋白p-AKT，p-GSK3β，p-mTOR的表達(dá)顯著降低(p＜0.01)，而RI蛋白表達(dá)水平明顯升高(p＜0.05)。 4.裸鼠在皮下注射各組細(xì)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(shí)驗(yàn)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(xì)胞核增大且染色較深的細(xì)胞，，表明已發(fā)生遠(yuǎn)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R(shí)I， Bax，Caspase-3表達(dá)明顯升高，而Bcl-2，ANG，p-AKT，p-GSK3β，p-mTOR表達(dá)明顯降低， AKT，GSK3β，mTOR的表達(dá)無變化，與Western blot結(jié)果一致。 結(jié)論體內(nèi)外實(shí)驗(yàn)均證實(shí)：成功構(gòu)建的ANG siRNA干擾質(zhì)粒通過調(diào)控ANG信號(hào)通路及凋亡蛋白抑制膀胱癌T24細(xì)胞的增殖并促進(jìn)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(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.14

【參考文獻(xiàn)】

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(xì)胞凋亡的影響[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2012年21期

本文編號(hào)：1789315