本文選題：結(jié)直腸癌 + NIBP　； 參考：《廣西醫(yī)科大學(xué)》2015年博士論文

【摘要】：背景和目的結(jié)直腸癌是臨床最常見的惡性腫瘤之一,在全世界范圍內(nèi),結(jié)直腸癌的發(fā)病率和死亡率均處于惡性腫瘤的第3位。在我國,隨著生活水平的不斷提高,發(fā)病率呈上升態(tài)勢。結(jié)直腸癌的發(fā)生發(fā)展是遺傳和環(huán)境等多種因素作用導(dǎo)致多基因改變及其相互作用的結(jié)果,其中涉及到多個信號轉(zhuǎn)導(dǎo)通路,比NF-κB信號通路。它們通過NF-kB轉(zhuǎn)錄因子,調(diào)節(jié)炎癥和腫瘤相關(guān)的至關(guān)重要的幾個途徑,影響腫瘤細胞的生存、血管生成、腫瘤細胞的運動性和侵襲力,從而促進腫瘤細胞的增殖,抑制細胞凋亡,并引起腫瘤的侵襲和轉(zhuǎn)移。NIBP是一種支架蛋白,它將NF-κB經(jīng)典途徑中的關(guān)鍵激活子-IKKβ和非經(jīng)典途徑中的關(guān)鍵酶-NIK連接成為三聚體。研究發(fā)現(xiàn),NIBP的表達升高可以促進IKKβ及其下游p65的磷酸化,由此推測,NIBP在NF-κB經(jīng)典途徑中發(fā)揮激活劑的作用。另一方面,抑制NIBP的表達則能減少由NIK和IKKβ分別介導(dǎo)的INF-κB信號通路的活化。此外,NIBP可能提高由TNF-a刺激引起NF-κB信號通路的活化從而促進腫瘤細胞的侵襲能力。最近研究還發(fā)現(xiàn),NIBP在人結(jié)腸癌組織中的表達明顯升高,推測這種升高可能與NF-κB信號通路的活化有關(guān),NIBP可能參與腺瘤向腺癌的演變過程。因此,本研究以NIBP為研究對象,檢測結(jié)直腸癌組織中NIBP、NF-κB活性因子p-p65和c-myc、cyclinD1的表達,了解其表達與結(jié)直腸癌的臨床分期及臨床病理關(guān)系。利用基因工程構(gòu)建NIBP基因沉默載體,在體外試驗中驗證NIBP基因表達水平變化對結(jié)腸癌細胞增殖的影響,探討其在結(jié)直腸癌的發(fā)生、發(fā)展及侵襲轉(zhuǎn)移中的作用和意義。方法1.采用免疫組化SP法分別檢測16例正常結(jié)腸粘膜、21例結(jié)直腸腺瘤以及114例結(jié)直腸癌患者結(jié)直腸組織中NIBP、NF-κB活性因子p-p65、c-myc和cyclinD1的表達,分析NIBP等在結(jié)直腸癌臨床分期和臨床病理的關(guān)系,推測其在結(jié)直腸癌的發(fā)生發(fā)展和侵襲轉(zhuǎn)移中的作用。2.利用基因工程,以人結(jié)腸癌細胞HCT116株為研究對象,設(shè)計并構(gòu)建人NIBP基因的慢病毒表達載體pLenti6.3-EGFP-NIBP-miR-1/2/3;另外,同時還設(shè)計無關(guān)的序列作為陰性對照(negative control, NC);人結(jié)腸癌細胞HCT1 16作為空白對照。用NIBP基因干擾的慢病毒及慢病毒陰性對照病毒液Lenti-EGFP感染靶細胞、空白細胞作對照,抽提細胞的蛋白,WB檢測目的蛋白的表達,篩選出最佳干擾靶點。目的基因干擾靶點慢病毒Lenti-EGFP-NIBP-miR感染HCT 116細胞后加入BSD(Blasticidin, BSD)進行篩選,獲得穩(wěn)定轉(zhuǎn)染NIBP低表達的單克隆HCT116細胞。應(yīng)用QRT-PCR檢測轉(zhuǎn)染效率,選擇NIBP干擾效率高的穩(wěn)轉(zhuǎn)株細胞株用于后續(xù)的細胞功能試驗。3.以空白對照組(blank control,簡稱CON組,即未轉(zhuǎn)染的HCT116細胞株)、陰性對照組(NC組,Lenti-EGFP-miR轉(zhuǎn)染HCT116細胞株)、NIBP低表達組(NIBP組,Lenti-EGFP-NIBP-miR轉(zhuǎn)染HCT 116細胞株)和NIBP低表達組細胞加NF-κB經(jīng)典途徑激活劑TNF-a組(NIBPT組)作為研究對象,采用CCK-8檢測試驗盒檢測各組細胞的增殖能力；流式細胞儀檢測各組細胞的凋亡和細胞周期分布情況；透射電子顯微鏡觀察細胞凋亡情況；QRT-PCR和Western Blot檢測細胞相關(guān)周期蛋白c-myc和cyclinD1的表達情況。結(jié)果1.免疫組化顯示結(jié)直腸癌組織中NIBP、NF-κB活性因子p-p65、c-myc和cyclinD1的陽性表達率均高于結(jié)直腸腺瘤和正常結(jié)腸粘膜組織,P0.05；而且NIBP、NF-κB活性因子p-p65、c-myc和cyclinDl在結(jié)腸癌組織中的表達與腫瘤的分化程度、組織類型、浸潤深度、TNM和Duke's分期、淋巴結(jié)轉(zhuǎn)移及遠處轉(zhuǎn)移相關(guān),P0.05。同時,NIBP、c-myc和cyclinD1與NF-κB活化因子p-p65呈正相關(guān)關(guān)系。2.建立NIBP沉默載體和穩(wěn)定轉(zhuǎn)染HCT116細胞株。我們利用質(zhì)粒載體pLenti6.3-MCS/V5 DEST系列慢病毒表達載體成功構(gòu)建人NIBP基因的慢病毒表達載體Lenti-EGFP-NIBP-miR(NIBP)和Lenti-EGFP-miR(NC);經(jīng)酶切、重組克隆及鑒定等,證實載體構(gòu)建成功。設(shè)計的NIBP基因1#,2#,3#干擾靶點進行慢病毒包裝并感染HCT116細胞后3個靶點的敲減效率均非常明顯,NIBP蛋白均沒有出現(xiàn)明顯的條帶。我們選擇了其中的第3#靶點進行后續(xù)穩(wěn)轉(zhuǎn)株篩選。將這些載體轉(zhuǎn)染HCT116細胞株,放在熒光顯微鏡下觀察,看到綠色熒光；可以篩選出穩(wěn)定轉(zhuǎn)染的單克隆細胞株；通過QRT-PCR和Western Blot試驗證實NIBP干擾效果滿意,HCT116細胞NIBP干擾穩(wěn)轉(zhuǎn)株與陰性對照穩(wěn)轉(zhuǎn)株相比,NIBP干擾效率高。3. NIBP對結(jié)腸癌細胞生長的影響到試驗研究：CCK-8試驗顯示,NIBP轉(zhuǎn)染細胞組和NIBPT組的細胞生長較空白組和陰性對照組減慢,P0.05；細胞凋亡檢測四組細胞的凋亡率差異無統(tǒng)計學(xué)意義,P0.05；細胞周期檢測結(jié)果顯示NIBP和NIBPT組的細胞多處于Go/G1期(分別為73.500%±3.061%和70.133%±5.181%),明顯高于HCT116細胞組和NC細胞組(55.933%±3.963%和52.000%±2.193%),P0.05；NIBP和NIBPT組細胞的S期分別為6.700%±1.249%和9.467%±4.858%,低于HCT116細胞組和NC細胞組(11.300%±3.751%和13.567%±1.563%),差異有統(tǒng)計學(xué)意義,P0.05;NIBP和NIBPT組的細胞處于G2/M期分別為14.967%±1.041%和16.067%±1.380%,明顯低于HCT116細胞組和NC細胞組(29.933%±2.309%和31.200%±2.007%),P0.05；說明NIBP轉(zhuǎn)染后結(jié)腸癌細胞的凋亡無明顯增加,但其增殖能力下降,而且即使使用NF-κB信號通路經(jīng)典途徑激活劑TNF-a也不能使其增殖能力恢復(fù)。透射電鏡觀察發(fā)現(xiàn)NIBP轉(zhuǎn)染細胞的凋亡亦無明顯增加。而QRT-PCR和WB的檢測結(jié)果顯示四組細胞中c-myc和cyclinD1的表達無顯著差異。結(jié)論1、NIBP、p-p65、c-myc和cyclinD1在結(jié)直腸癌組織中高表達。2. NIBP、p-p65、c-myc和cyclinD1的表達與腫瘤的分化程度、浸潤深度、TNM和Duke's分期及有無淋巴結(jié)轉(zhuǎn)移或/和遠處轉(zhuǎn)移有關(guān)。3、NIBP基因促進結(jié)腸癌HCT116細胞的增殖能力,可能是通過激活NF-κB經(jīng)典途徑來實現(xiàn)的。4、NIBP對結(jié)腸癌HCT116細胞株的凋亡無明顯影響。5、NIBP對結(jié)腸癌HCT116細胞株的c-myc和cyclinD1的表達也無明顯影響。
[Abstract]:Background and objective colorectal cancer is one of the most common malignant tumors in clinical. In the world, the incidence and mortality of colorectal cancer are third in the malignant tumor. In China, with the continuous improvement of the living standard, the incidence of colorectal cancer is on the rise. The development of colorectal cancer is caused by many factors such as heredity and environment. The results of multiple gene changes and their interactions involving multiple signal transduction pathways, which are more important than the NF- kappa B signaling pathway. They regulate several important pathways associated with inflammation and cancer through NF-kB transcription factors, affecting the survival of tumor cells, angiogenesis, motility and invasiveness of tumor cells, thus promoting tumor cells. Proliferation, inhibition of apoptosis, and the invasion and metastasis of tumors,.NIBP is a scaffold protein, which connects the key activator -IKK beta in the classical pathway of NF- kappa B and the key enzyme in the non classical pathway, -NIK, to be a trimer. The study shows that the increase of NIBP expression can promote the phosphorylation of IKK beta and its downstream p65, thus speculating that NIBP is in NF- kappa B. On the other hand, inhibition of the expression of NIBP can reduce the activation of the INF- kappa B signaling pathway mediated by NIK and IKK beta, respectively. In addition, NIBP may improve the activation of the NF- kappa B signaling pathway by TNF-a stimulation to promote the invasion of tumor cells. The recent study also found that NIBP is in human colon cancer tissue. The expression in the NF- kappa B signaling pathway may be related to the activation of the signal pathway. NIBP may be involved in the evolution of adenoma to adenocarcinoma. Therefore, this study uses NIBP as a study object to detect the expression of NIBP, NF- kappa B active factor p-p65, c-myc, cyclinD1, and to understand the expression of the colorectal cancer and the clinical stages of colorectal cancer. The effect and significance of the changes of NIBP gene expression level on the proliferation of colon cancer cells and the role and significance of NIBP gene expression level on the occurrence, development and invasion and metastasis of colorectal cancer were examined in vitro by using gene engineering to construct a NIBP gene silencing carrier. Methods 16 cases of normal colon mucosa were detected by the immunization of SP. 21 cases of colorectal adenoma and 114 cases of colorectal cancer, NIBP, NF- kappa B active factor p-p65, c-myc and cyclinD1 expression, analysis of the relationship between NIBP and the clinical staging and clinicopathological features of colorectal cancer, and speculate that its role in the development of colorectal cancer and invasion and metastasis of.2. using genetic engineering, with human colon cancer cell HC The T116 strain was used to design and construct the Lentivirus Expression Vector pLenti6.3-EGFP-NIBP-miR-1/2/3 of human NIBP gene. In addition, unrelated sequences were designed as negative control (negative control, NC); human colon cancer cell HCT1 16 was used as a blank control. Lentivirus and lentivirus negative control virus liquid Lenti-EG with NIBP gene interference were used as control. FP infected target cells, blank cells as control, extract cell protein, WB to detect the expression of target protein, screening out the best interference target. Target gene interfered with lentivirus Lenti-EGFP-NIBP-miR infection HCT 116 cells after BSD (Blasticidin, BSD) screening, obtained stable transfection of low expression of NIBP of the monoclonal HCT116 cells. QRT application of QRT. The transfection efficiency was detected by -PCR, and the stable cell line with high NIBP interference efficiency was selected for subsequent cell function test,.3. was used as a blank control group (blank control, CON group, or untransfected HCT116 cell line), negative control group (NC group, Lenti-EGFP-miR transfected HCT116 cell line), NIBP low expression group (NIBP group, transfected 116) Cell lines and NIBP low expression group cells plus NF- kappa B classical pathway activator TNF-a group (NIBPT group) were used as the research object. CCK-8 test box was used to detect the proliferation ability of each cell. Flow cytometry was used to detect the cell apoptosis and cell cycle distribution; transmission electron microscopy was used to observe the cell apoptosis; QRT-PCR and Weste. RN Blot was used to detect the expression of cell related cyclin c-myc and cyclinD1. Results 1. immunohistochemical staining showed that NIBP, NF- kappa B active factor p-p65, c-myc and cyclinD1 positive rates were higher than those of colorectal adenoma and normal colon mucosa, P0.05. The expression in cancer tissue is related to the degree of differentiation, type of tissue, depth of invasion, TNM and Duke's staging, lymph node metastasis and distant metastasis, P0.05., NIBP, c-myc and cyclinD1 are positively related to NF- kappa B activator p-p65.2.,.2. to establish NIBP silencing carrier and stable transfection HCT116 cell strain. /V5 DEST series lentivirus vector successfully constructed the Lentivirus Expression Vector Lenti-EGFP-NIBP-miR (NIBP) and Lenti-EGFP-miR (NC) of human NIBP gene. Through enzyme digestion, recombinant cloning and identification, it confirmed the success of the vector construction. The designed NIBP gene 1#, 2#, 3# interfered the target of the lentivirus packaging and infected the HCT116 cells after the knockout effect of 3 targets. The NIBP protein had no obvious bands. We selected the 3# target for subsequent stable strain screening. These vectors were transfected into HCT116 cell lines and observed under the fluorescence microscope to see the green fluorescence; the stable transfected monkon cell lines could be screened; the QRT-PCR and Western Blot tests were used. The effect of NIBP interference was satisfactory. Compared with the negative control stable strain of HCT116 cell NIBP interference, the effect of NIBP interference efficiency.3. NIBP on the growth of colon cancer cells was studied. The CCK-8 test showed that the cell growth of NIBP transfected cell group and NIBPT group was slower than that of the blank group and the negative control group, P0.05; the apoptosis detection was four. There was no significant difference in the apoptosis rate of the group cells, P0.05. The cell cycle detection showed that the cells in the NIBP and NIBPT groups were mostly in the Go/G1 phase (73.500% + 3.061% and 70.133% + 5.181% respectively), obviously higher than the HCT116 cell group and the NC cell group (55.933% + 3.963% and 52% + 2.193%), and the S period of the P0.05; NIBP and NIBPT groups was 6.700%, respectively. 1.249% and 9.467% + 4.858%, lower than the HCT116 cell group and the NC cell group (11.300% + 3.751% and 13.567% + 1.563%), the difference was statistically significant. The cells in the group NIBP and NIBPT were 14.967% + 1.041% and 16.067% +, respectively, lower than the HCT116 cell group and NC cell group (29.933% + 2.309% and 31.200% + 3.751%), P0.05. The apoptosis of colon cancer cells was not significantly increased after NIBP transfection, but its proliferation ability decreased, and the proliferation ability could not be restored even with the use of NF- kappa B signaling pathway activator TNF-a. The transmission electron microscopy showed that the apoptosis of NIBP transfected cells was not significantly increased. The results of QRT-PCR and WB detection showed that c-myc in four groups of cells. Conclusion 1, 1, NIBP, p-p65, c-myc and cyclinD1 express the expression of.2. NIBP, p-p65, c-myc and cyclinD1 in colorectal cancer tissues, which are related to the degree of differentiation, depth of invasion, TNM and Duke's stages, and whether there is lymph node metastasis or / and distant transfer. The ability, possibly through the activation of the classical pathway of NF- kappa B, is.4. NIBP has no significant effect on the apoptosis of HCT116 cell line of colon cancer, and NIBP has no significant effect on the expression of c-myc and cyclinD1 in colon cancer HCT116 cell lines.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.34

【相似文獻】

相關(guān)期刊論文 前10條

1 顧晉,彭亦凡,李沛,陸欣欣,李熒,王怡,徐光煒;術(shù)前區(qū)域動脈灌注化療對結(jié)直腸癌組織中血小板衍生的內(nèi)皮細胞生長因子表達的影響[J];實用腫瘤雜志;2001年06期

2 張建兵,何松,繆宏蘭,吳麗華,韓枋,于蘭,楊書云,周建云;耐藥基因蛋白在結(jié)直腸癌組織中的表達及其臨床意義[J];實用癌癥雜志;2004年02期

3 尹慕軍,王杉,葉穎江,姜可偉,崔志榮,梁斌,方偉崗;人結(jié)直腸癌組織中胸腺嘧啶核苷磷酸化酶和二氫嘧啶脫氫酶的活性變化及其意義[J];中華普通外科雜志;2005年07期

4 馮波;鄭民華;馬君俊;蔡劬;張軼;計駿;瞿穎;李健文;陸愛國;王明亮;劉炳亞;朱正綱;;結(jié)直腸癌組織中激肽釋放酶10基因的表達及其與臨床及病理的關(guān)系[J];中華外科雜志;2006年09期

5 張燕;李延青;張尚忠;楊曉云;袁俊華;郭玉婷;盧雪峰;;結(jié)直腸癌組織中葡糖醛酸轉(zhuǎn)移酶2B同工酶和肝細胞核因子的表達研究[J];癌變.畸變.突變;2008年05期

6 章禮久;孟剛;許建明;;結(jié)直腸癌組織淋巴管分布特點與淋巴結(jié)轉(zhuǎn)移及預(yù)后的關(guān)系[J];安徽醫(yī)科大學(xué)學(xué)報;2010年05期

7 馬順茂;賈漪濤;劉紅磊;杜蕓;王永軍;李中信;;結(jié)直腸癌組織中基質(zhì)金屬蛋白酶-10的表達及臨床意義[J];中國全科醫(yī)學(xué);2011年02期

8 趙海霞;牟東;薛世祥;代劍華;袁月;彭貴勇;房殿春;;結(jié)直腸癌組織中核干因子和增殖細胞核抗原的表達與血清葉酸水平的關(guān)系[J];第三軍醫(yī)大學(xué)學(xué)報;2011年10期

9 袁娜娜;盧高峰;唐芙愛;;人結(jié)直腸癌組織中腫瘤相關(guān)巨噬細胞的檢測[J];鄭州大學(xué)學(xué)報(醫(yī)學(xué)版);2013年01期

10 王春雷,張國華,鄧心新,顧倬云,鄭曉非,王彩云,劉海玲;結(jié)直腸癌組織端粒酶活性的表達及臨床價值[J];中華實驗外科雜志;1999年03期

相關(guān)會議論文 前10條

1 傅佶泓;崔龍;陳衛(wèi);;結(jié)直腸癌組織miRNA,mRNA表達譜芯片分析及功能研究[A];中華醫(yī)學(xué)會腫瘤學(xué)分會第七屆全國中青年腫瘤學(xué)術(shù)會議——中華醫(yī)學(xué)會腫瘤學(xué)分會“中華腫瘤 明日之星”大型評選活動暨中青年委員全國遴選論文匯編[C];2011年

2 黃文斌;趙建華;黃悅;李莉;楊小兵;王勁松;張琦;;結(jié)直腸癌組織中腫瘤出芽的臨床病理意義[A];中華醫(yī)學(xué)會病理學(xué)分會2007年學(xué)術(shù)年會暨第九屆全國病理大會論文匯編[C];2007年

3 邢曉明;黃瓊;王英紅;來茂德;;結(jié)直腸癌組織蛋白質(zhì)組學(xué)分析[A];中華醫(yī)學(xué)會病理學(xué)分會2006年學(xué)術(shù)年會論文匯編[C];2006年

4 湯菲;單保恩;;二胺氧化酶抗體的制備及其在人結(jié)直腸癌組織中表達的意義[A];河北省免疫學(xué)會第5次會員代表暨學(xué)術(shù)大會論文摘要集[C];2006年

5 蔡崎;施達仁;陸洪芬;孫孟紅;;CD44v3,v6蛋白在結(jié)直腸癌組織中的表達及其臨床意義[A];2000全國腫瘤學(xué)術(shù)大會論文集[C];2000年

6 張志勇;趙增仁;張麗靜;吳瑜;高艷敏;胡月明;李芳;;結(jié)直腸癌組織中MAC30的表達及其意義[A];中華醫(yī)學(xué)會病理學(xué)分會2009年學(xué)術(shù)年會論文匯編[C];2009年

7 李邦華;鐘瓊;;結(jié)直腸癌組織中ERCC1表達及其臨床意義[A];江西省首屆中西醫(yī)結(jié)合腫瘤學(xué)術(shù)研討會論文集[C];2010年

8 陳積賢;張潔;任振華;薛迪新;吳偉力;張仁虎;余銘;林道浙;林肖;梁美珍;賀先偉;徐平;黃建武;;結(jié)直腸癌組織中p27基因甲基化與其臨床病理的關(guān)系[A];2011年浙江省肛腸外科學(xué)術(shù)大會暨結(jié)直腸肛門疾病診治新進展學(xué)習(xí)班論文匯編[C];2011年

9 方合志;沈麗君;魏佳;丁志囡;白益東;呂建新;;人結(jié)直腸癌組織中線粒體基因D環(huán)區(qū)突變的研究[A];中國遺傳學(xué)會第八次代表大會暨學(xué)術(shù)討論會論文摘要匯編（2004-2008）[C];2008年

10 方合志;沈麗君;魏佳;丁志囡;白益東;呂建新;;人結(jié)直腸癌組織中線粒體基因D環(huán)區(qū)突變的研究[A];第二屆中國醫(yī)學(xué)細胞生物學(xué)學(xué)術(shù)大會暨細胞生物學(xué)教學(xué)改革會議論文集[C];2008年

相關(guān)博士學(xué)位論文 前5條

1 陳德波;結(jié)直腸癌組織差異表達蛋白質(zhì)的鑒定與分析[D];福建醫(yī)科大學(xué);2010年

2 孔斌;水通道蛋白-1（AQP-1）在結(jié)直腸癌組織中的表達及其與腫瘤微血管形成的關(guān)系研究[D];河北醫(yī)科大學(xué);2010年

3 楊光遠;大腸癌上調(diào)表達蛋白NPM1單克隆抗體的制備及初步應(yīng)用[D];吉林大學(xué);2013年

4 李素艷;NIBP在結(jié)直腸癌組織中的表達及其對結(jié)腸癌細胞增殖的影響[D];廣西醫(yī)科大學(xué);2015年

5 李日恒;Livin、Caspase-3在結(jié)直腸癌組織中的表達及意義[D];吉林大學(xué);2007年

相關(guān)碩士學(xué)位論文 前10條

1 宋媛;脂聯(lián)素及其受體在結(jié)直腸癌組織中的表達及相關(guān)研究[D];遼寧醫(yī)學(xué)院;2011年

2 朱萌;內(nèi)皮細胞特異性分子-1在結(jié)直腸癌組織的表達及其與血清癌胚抗原的相關(guān)性[D];重慶醫(yī)科大學(xué);2012年

3 程少會;環(huán)氧化酶-2在結(jié)直腸癌組織中的表達及其臨床意義[D];吉林大學(xué);2005年

4 馮文坡;4.1蛋白在結(jié)直腸癌組織中的表達[D];鄭州大學(xué);2007年

5 王艷富;己糖激酶-Ⅱ在結(jié)直腸癌組織中表達狀況研究[D];復(fù)旦大學(xué);2010年

6 楊樹鋼;DCR3、EGFR和Ki-67在大腸癌中的表達及臨床意義[D];福建醫(yī)科大學(xué);2008年

7 涂金花;結(jié)直腸癌組織HER-2的表達及意義[D];福建醫(yī)科大學(xué);2012年

8 馬小杰;結(jié)直腸癌組織中基因Nob1的表達及臨床意義[D];山西醫(yī)科大學(xué);2014年

9 夏曉天;結(jié)直腸癌組織EphB4受體的表達及臨床意義[D];上海交通大學(xué);2008年

10 吳芳;結(jié)直腸癌組織LV與TAM相關(guān)性分析[D];廣州醫(yī)學(xué)院;2010年

，

本文編號：2115027