siRNA抑制CNN2表達對肝癌細胞生物學行為及裸鼠成瘤的影響
發(fā)布時間：2018-08-02 16:24
【摘要】：目的：建立穩(wěn)定轉染CNN2 siRNA的肝癌細胞株,用以觀察CNN2蛋白在表達沉默時對細胞生物學行為和裸鼠成瘤的影響。方法：(1)將插入有CNN2 siRNA的慢病毒以及空載慢病毒LV3分別轉染SK-hep-1肝癌細胞,通過嘌呤霉素篩選建立穩(wěn)定轉染的“基因表達下調”細胞模型。Quantitative Real Time-PCR (qRT-PCR)和Western-blot法分別觀察分析轉染前后目的基因CNN2在轉錄和翻譯水平的表達變化。(2)通過Transwell實驗檢測細胞的遷移能力和侵襲能力的改變；MTT實驗檢測細胞增殖能力,觀察轉染細胞株與對照組細胞增殖能力的差異；流式細胞儀檢測細胞周期,觀察抑制CNN2表達對細胞周期的影響。(3)構建SK-hep-1-siRNA, SK-hep-1-LV3, SK-hep-1三種細胞株的裸鼠皮下成瘤模型,每組12只裸鼠,雌雄各半。每日觀察裸鼠生活情況,待腫瘤長出后,每周測一次瘤體積,稱裸鼠體重。第4周處死裸鼠,取瘤體測體積,稱重,解剖裸鼠觀察是否有腫瘤轉移。瘤組織石蠟包埋切片,HE染色進行病理觀察,TUNEL法檢測細胞凋亡,免疫組織化學檢測CNN2表達,磷酸化MEK,磷酸化ERK,磷酸化AKT以及MEK, ERK, AKT蛋白表達。結果：(1) SK-hep-1-siRNA細胞株及空載SK-hep-1-LV3細胞株在慢病毒轉染24 h后,帶有綠色熒光,證實轉染成功,用2μg/mL嘌呤霉素篩選72 h后,在熒光顯微鏡下觀察,細胞100%帶有綠色熒光,說明已將未轉染成功的肝癌細胞清除,以及成功構建了穩(wěn)定轉染的SK-hep-1-siRNA和SK-hep-1-LV3細胞株。qRT-PCR、Western-blot顯示SK-hep-1-siRNA細胞株中目的基因CNN2在mRNA和蛋白質水平上表達均較SK-hep-1-LV3細胞株和未轉染細胞株降低(P0.001),轉錄水平CNN2表達抑制率約為68.3%,翻譯水平CNN2表達抑制率約為63.9%。(2)對三種細胞株的觀察表明,CNN2表達沉默能抑制SK-hep-1細胞的增殖,使細胞周期阻滯在S期(P0.01),并使遷移運動(P0.01)和侵襲能力降低(P0.001)。(3)成功構建裸鼠皮下移植瘤模型。相比于SK-hep-1-LV3裸鼠成瘤組和SK-hep-1裸鼠成瘤組,SK-hep-1-siRNA裸鼠成瘤組腫瘤生長速度較慢,瘤體較小,成瘤率降低,4周后剝離瘤組織測得瘤體重量較輕,三組裸鼠瘤體積差異與重量差異均有統(tǒng)計學意義(P0.001)。所有組別的裸鼠均未發(fā)生腫瘤轉移,裸鼠重量三組相當,無顯著性差異。HE染色結果顯示,三組裸鼠瘤組織均呈現(xiàn)典型的腫瘤組織特征,即細胞核大,染色深,細胞排列緊密。TUNEL法測凋亡結果顯示三組瘤組織均未出現(xiàn)細胞凋亡的情況。SK-hep-1-siRNA組瘤組織CNN2、磷酸化MEK、磷酸化ERK表達下調,與陰性對照組和空白對照組相比差異有統(tǒng)計學意義(P0.01,P0.05,P0.01),磷酸化AKT, AKT, MEK, ERK表達在三組之間無顯著性差異。結論：(1)成功構建了SK-hep-1-siRNA細胞株和SK-hep-1-LV3細胞株。(2)CNN2 siRNA慢病毒穩(wěn)定轉染可以抑制肝癌SK-hep-1細胞增殖,遷移,侵襲能力,并使細胞周期阻滯在S期。(3) CNN2 siRNA慢病毒穩(wěn)定轉染可以抑制SK-hep-1細胞成瘤能力。CNN2可能通過提高MAPK通路蛋白MEK, ERK的磷酸化水平而發(fā)揮促進腫瘤發(fā)生發(fā)展的作用。
[Abstract]:Objective: to establish a hepatocellular carcinoma cell line stably transfected with CNN2 siRNA to observe the effect of CNN2 protein on cell biological behavior and tumor formation in nude mice. Methods: (1) transfection of CNN2 siRNA with lentivirus and empty lentivirus LV3 to SK-hep-1 hepatoma cells respectively, and to establish a stable transfected "base" by purinomycin screening. The expression of CNN2 at the transcriptional and translation levels of the target gene CNN2 before and after transfection was observed by.Quantitative Real Time-PCR (qRT-PCR) and Western-blot method. (2) the changes in the cell migration ability and invasion ability were detected by Transwell test. The proliferation ability of the cells was detected by MTT test, and the transfection rate was observed. The cell cycle was detected by flow cytometry and the effect of inhibiting the expression of CNN2 on the cell cycle was observed by flow cytometry. (3) the subcutaneous tumor formation model of SK-hep-1-siRNA, SK-hep-1-LV3, and SK-hep-1 cells in nude mice was constructed, and 12 nude mice in each group were half. The daily life of nude mice was observed, after the tumor grew out. The tumor volume of nude mice was measured once a week, and the nude mice were weighed. Fourth weeks were killed in nude mice. The tumor was measured and weighed and weighed. The tumor metastases were observed in nude mice. The paraffin embedded section of the tumor tissue, HE staining for pathological observation, TUNEL assay of apoptosis, immunohistochemical detection of CNN2 expression, phosphorylated MEK, phosphorylated ERK, phosphorylated AKT, MEK, ERK, Results: (1) (1) the SK-hep-1-siRNA cell line and the unloaded SK-hep-1-LV3 cell line had green fluorescence after 24 h transfection of lentivirus, confirmed the successful transfection. After screening 72 h with 2 mu g/mL puramycin, the cells were observed under the fluorescence microscope, and the cells were 100% with green fluorescence, indicating that the untransfected hepatoma cells were cleared and formed. The stable transfected SK-hep-1-siRNA and SK-hep-1-LV3 cell strain.QRT-PCR were constructed, and Western-blot showed that the expression of target gene CNN2 in SK-hep-1-siRNA cell line was lower than that of SK-hep-1-LV3 cell line and untransfected cell line (P0.001). The inhibition rate of transcription level CNN2 expression was about 68.3%, and the expression level of translation level was inhibited. The observation of the rate of 63.9%. (2) on three cell lines showed that the expression of CNN2 could inhibit the proliferation of SK-hep-1 cells, block the cell cycle at S (P0.01), and reduce the migration (P0.01) and invasion ability (P0.001). (3) the subcutaneous tumor model of nude mice was successfully constructed. Compared to the tumor formation and SK-hep-1 nude mice of SK-hep-1-LV3 nude mice, the tumor cells were successfully constructed. In the group of SK-hep-1-siRNA nude mice, the tumor growth rate was slower, the tumor body was smaller, the tumorigenesis rate decreased. The weight of the tumor body was light in the dissection tissue after 4 weeks. The difference of tumor volume between the three groups of nude mice and the weight difference were statistically significant (P0.001). There was no tumor metastasis in all groups of nude mice, the weight of nude mice was equivalent to three groups, no significant difference.HE The staining results showed that the tumor tissues of the three groups of nude mice showed typical tumor tissue characteristics, that is, the nucleus was large, the staining was deep, and the cells arranged closely.TUNEL method to determine the apoptotic results in the three groups of tumor tissues. The tumor tissue was CNN2, phosphorylated MEK, and phosphorylated ERK expression was down regulated, and the negative control group and the blank were in the blank. There were statistically significant differences (P0.01, P0.05, P0.01), phosphorylated AKT, AKT, MEK, and ERK expression between the three groups. Conclusion: (1) SK-hep-1-siRNA cell line and SK-hep-1-LV3 cell line were successfully constructed. (2) CNN2 siRNA lentivirus stable transfer can inhibit the proliferation, migration, invasion ability of hepatocellular carcinoma SK-hep-1 cells, and make it possible Cell cycle arrest in S phase. (3) CNN2 siRNA lentivirus stable transfection can inhibit the tumorigenesis of SK-hep-1 cells,.CNN2 may play a role in promoting the development of tumor by increasing the phosphorylation level of MAPK pathway protein MEK and ERK.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R735.7
本文編號：2160005
[Abstract]:Objective: to establish a hepatocellular carcinoma cell line stably transfected with CNN2 siRNA to observe the effect of CNN2 protein on cell biological behavior and tumor formation in nude mice. Methods: (1) transfection of CNN2 siRNA with lentivirus and empty lentivirus LV3 to SK-hep-1 hepatoma cells respectively, and to establish a stable transfected "base" by purinomycin screening. The expression of CNN2 at the transcriptional and translation levels of the target gene CNN2 before and after transfection was observed by.Quantitative Real Time-PCR (qRT-PCR) and Western-blot method. (2) the changes in the cell migration ability and invasion ability were detected by Transwell test. The proliferation ability of the cells was detected by MTT test, and the transfection rate was observed. The cell cycle was detected by flow cytometry and the effect of inhibiting the expression of CNN2 on the cell cycle was observed by flow cytometry. (3) the subcutaneous tumor formation model of SK-hep-1-siRNA, SK-hep-1-LV3, and SK-hep-1 cells in nude mice was constructed, and 12 nude mice in each group were half. The daily life of nude mice was observed, after the tumor grew out. The tumor volume of nude mice was measured once a week, and the nude mice were weighed. Fourth weeks were killed in nude mice. The tumor was measured and weighed and weighed. The tumor metastases were observed in nude mice. The paraffin embedded section of the tumor tissue, HE staining for pathological observation, TUNEL assay of apoptosis, immunohistochemical detection of CNN2 expression, phosphorylated MEK, phosphorylated ERK, phosphorylated AKT, MEK, ERK, Results: (1) (1) the SK-hep-1-siRNA cell line and the unloaded SK-hep-1-LV3 cell line had green fluorescence after 24 h transfection of lentivirus, confirmed the successful transfection. After screening 72 h with 2 mu g/mL puramycin, the cells were observed under the fluorescence microscope, and the cells were 100% with green fluorescence, indicating that the untransfected hepatoma cells were cleared and formed. The stable transfected SK-hep-1-siRNA and SK-hep-1-LV3 cell strain.QRT-PCR were constructed, and Western-blot showed that the expression of target gene CNN2 in SK-hep-1-siRNA cell line was lower than that of SK-hep-1-LV3 cell line and untransfected cell line (P0.001). The inhibition rate of transcription level CNN2 expression was about 68.3%, and the expression level of translation level was inhibited. The observation of the rate of 63.9%. (2) on three cell lines showed that the expression of CNN2 could inhibit the proliferation of SK-hep-1 cells, block the cell cycle at S (P0.01), and reduce the migration (P0.01) and invasion ability (P0.001). (3) the subcutaneous tumor model of nude mice was successfully constructed. Compared to the tumor formation and SK-hep-1 nude mice of SK-hep-1-LV3 nude mice, the tumor cells were successfully constructed. In the group of SK-hep-1-siRNA nude mice, the tumor growth rate was slower, the tumor body was smaller, the tumorigenesis rate decreased. The weight of the tumor body was light in the dissection tissue after 4 weeks. The difference of tumor volume between the three groups of nude mice and the weight difference were statistically significant (P0.001). There was no tumor metastasis in all groups of nude mice, the weight of nude mice was equivalent to three groups, no significant difference.HE The staining results showed that the tumor tissues of the three groups of nude mice showed typical tumor tissue characteristics, that is, the nucleus was large, the staining was deep, and the cells arranged closely.TUNEL method to determine the apoptotic results in the three groups of tumor tissues. The tumor tissue was CNN2, phosphorylated MEK, and phosphorylated ERK expression was down regulated, and the negative control group and the blank were in the blank. There were statistically significant differences (P0.01, P0.05, P0.01), phosphorylated AKT, AKT, MEK, and ERK expression between the three groups. Conclusion: (1) SK-hep-1-siRNA cell line and SK-hep-1-LV3 cell line were successfully constructed. (2) CNN2 siRNA lentivirus stable transfer can inhibit the proliferation, migration, invasion ability of hepatocellular carcinoma SK-hep-1 cells, and make it possible Cell cycle arrest in S phase. (3) CNN2 siRNA lentivirus stable transfection can inhibit the tumorigenesis of SK-hep-1 cells,.CNN2 may play a role in promoting the development of tumor by increasing the phosphorylation level of MAPK pathway protein MEK and ERK.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R735.7
【相似文獻】
相關期刊論文 前2條
1 彭志紅,楊建民,唐波,司遂海,房殿春,羅元輝;KAI1基因對MHCC97-H細胞裸鼠成瘤及轉移的影響[J];世界華人消化雜志;2004年04期
2 ;[J];;年期
相關碩士學位論文 前2條
1 康雪晴;siRNA抑制CNN2表達對肝癌細胞生物學行為及裸鼠成瘤的影響[D];廣西醫(yī)科大學;2016年
2 陳吉;激活突變SHP-2酪氨酸磷酸酶促進砷導致的MEFs惡性轉化和裸鼠成瘤風險[D];安徽醫(yī)科大學;2013年
，本文編號：2160005
本文鏈接：http://sikaile.net/yixuelunwen/zlx/2160005.html
最近更新
教材專著
