本文選題：miR-21 + 骨肉瘤��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：研究背景:骨肉瘤是對青、少年危害最大的原發(fā)的惡性骨腫瘤,以往以手術(shù)切除(截肢)治療為主�；颊卟坏惺芤蛑w殘缺造成的肉體和精神的嚴(yán)重創(chuàng)傷,而且其5年生存率不超過20%,對于患者及其家庭來說都是一個(gè)災(zāi)難。即使現(xiàn)在采用各種保肢手術(shù)輔助術(shù)前、術(shù)后的化療,可以大大提高五年生存率,并很大程度的保留患者的肢體功能,從而明顯提高患者的生活質(zhì)量,但仍有近四分之一到三分之一[1](國內(nèi)文獻(xiàn)報(bào)道為40%—50%[2])的患者生存期不超過5年。如何提高化療的有效率,減少骨肉瘤的復(fù)發(fā)和轉(zhuǎn)移,是目前臨床中對骨肉瘤治療的研究重點(diǎn)。近來,很多腫瘤學(xué)的研究把重點(diǎn)放在了腫瘤干細(xì)胞的領(lǐng)域[3]。腫瘤干細(xì)胞具有無限增值,自我更新和多向分化的能力,而且腫瘤干細(xì)胞多處于靜止期,對化療不敏感[4]。經(jīng)化療之后,腫瘤體內(nèi)干細(xì)胞比例明顯提高,是產(chǎn)生化療耐藥性和腫瘤復(fù)發(fā)的主要原因。骨肉瘤保肢手術(shù)聯(lián)合輔助術(shù)前、術(shù)后化療的綜合治療后,骨肉瘤的復(fù)發(fā)與轉(zhuǎn)移是由于骨肉瘤干細(xì)胞的存在的原因。目前,骨肉瘤的診斷和治療發(fā)展處于一個(gè)瓶頸期,如何有效殺傷腫瘤內(nèi)的干細(xì)胞,減少腫瘤體內(nèi)干細(xì)胞的比例,是提高治療效果的有效途徑。CD133是一種跨膜糖蛋白,先后被發(fā)現(xiàn)于造血干細(xì)胞和正常干細(xì)胞[5,6],后來又被發(fā)現(xiàn)于大多數(shù)腫瘤干細(xì)胞膜,現(xiàn)作為廣譜的標(biāo)記物被應(yīng)用于腫瘤干細(xì)胞的分辨與分離。目前,CD133是篩選骨肉瘤中腫瘤干細(xì)胞的常規(guī)公認(rèn)的關(guān)鍵性標(biāo)識[7,8],所以,我們在本實(shí)驗(yàn)中亦把CD133基因的表達(dá)作為腫瘤細(xì)胞呈現(xiàn)干細(xì)胞特性的標(biāo)志。MicroRNA(miRNA)是一族非編碼的小分子RNA(長度平均約為22個(gè)核苷酸),在基因轉(zhuǎn)錄后水平發(fā)揮重要的調(diào)控作用,通常引起翻譯抑制、目標(biāo)退化和基因沉默,對人的生命過程具有重要意義。其中,許多miRNA作為原癌基因或是抑癌基因,在腫瘤的發(fā)生、發(fā)展和轉(zhuǎn)移中扮演重要的角色。miR-21作為致癌的miRNA,在不同的惡性腫瘤的侵襲、血管浸潤和轉(zhuǎn)移中起重要作用[9-11]。有研究證實(shí)miR-21在骨肉瘤細(xì)胞中有高表達(dá),以及miR-21促進(jìn)骨肉瘤的生長和轉(zhuǎn)移。同時(shí),研究證實(shí)miR-21在多種惡性腫瘤的干細(xì)胞中亦有異常表達(dá),并通過蛋白或基因的相互作用來調(diào)節(jié)惡性腫瘤干細(xì)胞的功能[12];另外,有研究證實(shí)miR-21的抑制劑(反義寡核苷酸鏈)可以提高某些腫瘤細(xì)胞對化療藥物的敏感性[13-15]。VEGF是對血管內(nèi)皮細(xì)胞具有特異性的肝素結(jié)合的生長因子,是人體內(nèi)主要的促血管生成因素。在腫瘤生長時(shí),在多種促癌因素作用下腫瘤組織內(nèi)VEGF及其受體的表達(dá)激增,腫瘤組織內(nèi)大量不規(guī)則的新生血管生成,為腫瘤組織生長提供充足養(yǎng)分,同時(shí)為腫瘤的侵襲和轉(zhuǎn)移提供通路,參與到腫瘤的生長、浸潤和轉(zhuǎn)移各個(gè)過程中[16]。研究證實(shí)VEGF在骨肉瘤的發(fā)生發(fā)展和轉(zhuǎn)移中發(fā)揮重要作用;而在其他惡性腫瘤中,miR-21通過增強(qiáng)VEGF表達(dá),從而促進(jìn)腫瘤的生長和轉(zhuǎn)移[17,18]。因此,在我們關(guān)于miR-21在骨肉瘤的發(fā)生、發(fā)展、轉(zhuǎn)移和腫瘤干細(xì)胞的調(diào)控作用的研究中,引入VEGF,一是可以作為對照來分析miR-21的作用,另外可以分析miR-21與VEGF在對骨肉瘤干細(xì)胞的調(diào)控作用的相關(guān)性。目的:本課題主要研究阻斷miR-21和VEGF作用后不同骨肉瘤細(xì)胞VEGF基因和/或CD133基因表達(dá)的變化,以及對骨肉瘤細(xì)胞生長的影響,證實(shí)miR-21對于骨肉瘤及其干細(xì)胞有調(diào)節(jié)作用。并結(jié)合VEGF對骨肉瘤及其干細(xì)胞特性的調(diào)節(jié)作用進(jìn)行對照分析,旨在初步探討miR-21對骨肉瘤及其干細(xì)胞特性的影響及其可能的作用機(jī)制及規(guī)律,以期找出在臨床中能有效抑制骨肉瘤干細(xì)胞的發(fā)生、發(fā)展,減少骨肉瘤腫瘤干細(xì)胞比例的治療措施。方法:1.細(xì)胞及細(xì)胞培養(yǎng):選擇人骨肉瘤CRL-1543 and CRL-1423細(xì)胞系作為實(shí)驗(yàn)細(xì)胞,按照ATCC推薦的細(xì)胞培養(yǎng)液和培養(yǎng)條件及操作步驟進(jìn)行培養(yǎng)。2.實(shí)驗(yàn)一 Realtime-PCR 實(shí)驗(yàn)2.1對兩個(gè)細(xì)胞系的種植培養(yǎng)分3個(gè)實(shí)驗(yàn)組,分別為Oh(初始)對照組,8h阻斷組,24h阻斷組,每組包括2孔1543細(xì)胞和2孔1423細(xì)胞,按照前面所述的細(xì)胞培養(yǎng)方法進(jìn)行培養(yǎng)。2.2對兩個(gè)細(xì)胞系的處理將三組細(xì)胞在C02細(xì)胞培養(yǎng)箱中培養(yǎng)24小時(shí)后,所種植細(xì)胞完全貼壁后收獲初始對照組細(xì)胞。然后,在另兩組細(xì)胞的2孔中分別加入miR-21(has-mir-21-5p)反義寡核苷酸鏈和VEGF受體抑制劑,然后,將兩個(gè)抑制組細(xì)胞的培養(yǎng)板放回培養(yǎng)箱繼續(xù)培養(yǎng),分別于miR-21 inhibitor轉(zhuǎn)染和VEGF受體抑制劑處理后8小時(shí)和24小時(shí)收集細(xì)胞,作為8小時(shí)抑制組和24小時(shí)抑制組。對各實(shí)驗(yàn)組的各個(gè)細(xì)胞樣品分別提取RNA,然后反轉(zhuǎn)錄制備cDNA,進(jìn)行real time-PCR實(shí)驗(yàn),檢測各樣本的VEGF和CD133基因的表達(dá)。3.實(shí)驗(yàn)二細(xì)胞免疫組化實(shí)驗(yàn)3.1對兩個(gè)細(xì)胞系的種植培養(yǎng)取兩個(gè)8孔chamber slider(腔室載玻片),分別標(biāo)記為CD133和VEGF。每個(gè)chamber slider均培養(yǎng)兩排細(xì)胞,上面一排植入1543細(xì)胞,下面一排植入1423細(xì)胞,每個(gè)培養(yǎng)孔內(nèi)植入10000個(gè)細(xì)胞,并加入相應(yīng)的培養(yǎng)液0.5ml,放在孵化器中培養(yǎng)24小時(shí),待細(xì)胞完全貼壁。3.2對兩個(gè)細(xì)胞系的處理然后將每排四個(gè)培養(yǎng)孔的細(xì)胞中第一個(gè)孔作為空白對照,第二個(gè)孔作為陰性對照,第二個(gè)孔的作為VEGF(-)加入VEGF受體抑制劑,第四個(gè)孔作為miR-21(-)轉(zhuǎn)染miR-21(has-mir-21-5p)阻斷劑。將處理過的細(xì)胞再放入培養(yǎng)箱中培養(yǎng)24h,然后進(jìn)行免疫組化染色實(shí)驗(yàn),檢測各細(xì)胞樣品中VEGF基因和CD133基因的表達(dá)。4.實(shí)驗(yàn)三細(xì)胞劃痕實(shí)驗(yàn)4.1對兩個(gè)細(xì)胞系的種植培養(yǎng)分三個(gè)實(shí)驗(yàn)組,分別為空白對照組,miR-21阻斷組,miR-21阻斷+VEGF抑制組。每組包括2孔1543細(xì)胞和2孔1423細(xì)胞,按照前面所述的細(xì)胞培養(yǎng)方法進(jìn)行培養(yǎng)。4.2對兩個(gè)細(xì)胞系的處理所有細(xì)胞在培養(yǎng)箱中培養(yǎng)24小時(shí),待細(xì)胞完全貼壁。然后,在第一組四個(gè)培養(yǎng)孔內(nèi)不加任何阻斷劑,第二組四個(gè)培養(yǎng)孔內(nèi)加入miR-21的阻斷劑,第三組四個(gè)培養(yǎng)孔內(nèi)加入miR-21阻斷劑和VEGF受體的抑制劑。然后,將十二孔細(xì)胞培養(yǎng)板放回培養(yǎng)箱中培養(yǎng)細(xì)胞,待細(xì)胞基本布滿培養(yǎng)孔底部,在每個(gè)培養(yǎng)孔底部用p200 Pipet tip,經(jīng)過圓心垂直底面標(biāo)記線劃一道線,用倒置顯微鏡觀察并在劃痕上與六條標(biāo)記線的交點(diǎn)處留取照片;然后繼續(xù)在培養(yǎng)箱中培養(yǎng),于4h,8h,24h,48h分別留取照片,測量這6個(gè)點(diǎn)位置劃痕兩側(cè)邊緣間的距離,取平均值作為這個(gè)培養(yǎng)孔內(nèi)細(xì)胞在這個(gè)時(shí)間點(diǎn)的劃痕兩側(cè)邊緣間距離,每個(gè)細(xì)胞系的每組有兩個(gè)重復(fù)試驗(yàn)樣品,所以再取平均值即為該細(xì)胞系該組細(xì)胞該時(shí)間點(diǎn)劃痕兩邊緣間的距離,分別計(jì)算各個(gè)時(shí)間點(diǎn)兩邊緣間距離縮小的比例,分析各個(gè)細(xì)胞增生和長入的能力的差別。結(jié)果1.real-time PCR 實(shí)驗(yàn)結(jié)果:1.1各細(xì)胞系VEGF基因和CD133基因表達(dá)的總體變化:抑制VEGF后,1543細(xì)胞和1423細(xì)胞的VEGF的表達(dá)有所減低(p0.05);而CD133的表達(dá)均有明顯增高(p0.05)。阻斷mmiR-21后,1543細(xì)胞的VEGF基因和CD133基因的表達(dá)均有所下降(p0.05);而1423細(xì)胞的VEGF基因和CD133基因的表達(dá)均有顯著提高(p0.05)。1.2各細(xì)胞系VEGF基因和CD133基因表達(dá)隨時(shí)間變化的規(guī)律:(1)抑制VEGF后,1543細(xì)胞對VEGF和CD133基因的表達(dá)均是先有所升高,隨著時(shí)間的進(jìn)展表達(dá)逐漸降低,VEGF基因的表達(dá)到24h時(shí)已經(jīng)低于初始對照組;在阻斷miR-21后,1543細(xì)胞對VEGF和CD133基因的表達(dá)均有所下降,而且隨著時(shí)間的進(jìn)展表達(dá)無明顯變化。(2)1423細(xì)胞在被抑制VEGF后CD133基因的表達(dá)和阻斷miRNA-21后VEGF和CD133基因的表達(dá),在兩個(gè)時(shí)間點(diǎn)的變化差別均比較顯著。其中,抑制VEGF后CD133基因的表達(dá)在8小時(shí)達(dá)到初始對照組的4.13倍,而在24小時(shí)則急劇降低到初始對照組的0.79。2.細(xì)胞免疫組化實(shí)驗(yàn)結(jié)果:2.1 1543 細(xì)胞為 VEGFlow/CD133high細(xì)胞,而 1423 細(xì)胞為 VEGFhi8h/CD133low細(xì)胞。2.2 1543細(xì)胞在抑制VEGF后表現(xiàn)為VEGF基因表達(dá)有所下降,而CD133基因表達(dá)有所上升;在阻斷miR-21后表現(xiàn)為VEGF和CD133基因表達(dá)均下調(diào)。2.3 1423細(xì)胞在抑制VEGF后也表現(xiàn)為VEGF基因表達(dá)有所下降,而CD133基因表達(dá)有所上升;但在阻斷miR-21后表現(xiàn)為VEGF和CD133基因表達(dá)均明顯上升。3.細(xì)胞劃痕實(shí)驗(yàn)結(jié)果:3.1 1543細(xì)胞增殖速度明顯快于1423細(xì)胞。3.2 1543細(xì)胞兩個(gè)實(shí)驗(yàn)組(抑制miR-21組和同時(shí)抑制miR-21和VEGF組)細(xì)胞均較對照組生長速度減慢(p0.05),兩個(gè)實(shí)驗(yàn)組細(xì)胞的生長速度之間無明顯差別。3.3 1423細(xì)胞經(jīng)miR-21抑制劑處理后較對照組細(xì)胞生長速度減慢(p0.05),而添加兩種抑制劑(miR-21和VEGF受體)的細(xì)胞生長速度最慢,差別具有統(tǒng)計(jì)學(xué)意義(p0.05)。結(jié)論1.阻斷miR-21對于VEGF基因低表達(dá)的骨肉瘤細(xì)胞有較明顯的調(diào)控作用,而抑制VEGF對于VEGF基因高表達(dá)的骨肉瘤細(xì)胞有較明顯的調(diào)控作用。2.抑制VEGF后兩種骨肉瘤細(xì)胞株CD133基因表達(dá)均上調(diào),VEGF可能在骨肉瘤細(xì)胞中抑制CD133基因的表達(dá)。3.miR-21在VEGF低表達(dá)的骨肉瘤中有促進(jìn)骨肉瘤干細(xì)胞的發(fā)生和增殖,從而增加腫瘤體內(nèi)腫瘤干細(xì)胞比例的作用。在這一部分細(xì)胞中阻斷miR-21能更顯著的降低骨肉瘤細(xì)胞干細(xì)胞標(biāo)志物CD133的表達(dá),可能具有減少腫瘤干細(xì)胞的作用。4.對于VEGF高表達(dá)的1423細(xì)胞,抑制VEGF可以雙向調(diào)控CD133基因的表達(dá),表現(xiàn)為短時(shí)間的促進(jìn)后長時(shí)間的抑制。意義:我們的研究發(fā)現(xiàn)在低表達(dá)VEGF的骨肉瘤細(xì)胞中轉(zhuǎn)染miR-21的反義寡核苷酸鏈,可以顯著下調(diào)骨肉瘤細(xì)胞VEGF和CD133的表達(dá)。VEGF是促進(jìn)血管新生的關(guān)鍵生長因子,在腫瘤轉(zhuǎn)移和血管新生中發(fā)揮關(guān)鍵作用;而CD133是重要的干細(xì)胞標(biāo)志物,反映了腫瘤細(xì)胞的干細(xì)胞特性。這兩個(gè)基因表達(dá)下降,提示腫瘤細(xì)胞促進(jìn)血管新生的能力和干細(xì)胞的比例下降。這提示抑制miR-21的表達(dá)可能發(fā)揮抑制腫瘤的生長和血管新生以及轉(zhuǎn)移,同時(shí)減少腫瘤干細(xì)胞比例的作用,可以有效提高化療效果。而VEGF受體抑制劑能有效的抑制高表達(dá)VEGF的骨肉瘤細(xì)胞的生長、侵襲和轉(zhuǎn)移,減少瘤體內(nèi)腫瘤干細(xì)胞比例,從而提高腫瘤對化療的敏感性。綜上所述,我們的研究為不同分子特征的骨肉瘤的治療提供了有價(jià)值的基礎(chǔ)研究信息,為指導(dǎo)臨床用藥和治療提供了幫助�；谖覀兊难芯拷Y(jié)果,可以對不同分子特征(VEGF高表達(dá)或VEGF低表達(dá))的骨肉瘤使用相應(yīng)的治療藥物,有助于實(shí)現(xiàn)精準(zhǔn)用藥和精準(zhǔn)治療。
[Abstract]:Background: osteosarcoma is a primary malignant bone tumor that is most harmful to young people. Surgical excision (amputation) is the main treatment in the past. Patients not only have to suffer severe physical and mental trauma caused by limb disability, but their 5 year survival rate is not more than 20%. It is a disaster for the patients and their families. Even now, it is a disaster. Preoperative chemotherapy can greatly improve the five year survival rate and greatly retain the patient's limb function, thus significantly improving the patient's quality of life, but there are still nearly 1/4 to 1/3 [1] (the domestic literature is reported to 40% - 50%[2]) for less than 5 years of survival. How to improve the chemotherapy Efficiency, reducing the recurrence and metastasis of osteosarcoma is the focus of the current clinical research on osteosarcoma. Recently, many oncology studies have focused on the infinite value added, self renewal and multidirectional differentiation of [3]. tumor stem cells in the field of cancer stem cells, and the tumor stem cells are mostly at stationary phase and are not sensitive to chemotherapy. After chemotherapy, the proportion of stem cells in the tumor is significantly increased, which is the main cause of chemotherapeutic resistance and tumor recurrence. The recurrence and metastasis of osteosarcoma is due to the existence of osteosarcoma stem cells after the combined surgery of osteosarcoma and postoperative chemotherapy combined with adjuvant chemotherapy, and the diagnosis and treatment of osteosarcoma at present. The exhibition is in a bottleneck period. How to kill the stem cells in the tumor effectively and reduce the proportion of the stem cells in the tumor is an effective way to improve the effect of the treatment..CD133 is a kind of transmembrane glycoprotein, which has been found in hematopoietic stem cells and normal stem cells, [5,6], and later found in most of the tumor stem cell membranes, which are now widely used as a broad spectrum marker. It is applied to the resolution and separation of cancer stem cells. Currently, CD133 is a commonly recognized key marker [7,8] for screening tumor stem cells in osteosarcoma. Therefore, in this experiment, the expression of CD133 gene expression as a marker of stem cell characteristics of tumor cells (miRNA) is a group of non coded small molecules RNA (miRNA). About 22 nucleotides, which play an important regulatory role at the post transcriptional level, usually cause translation inhibition, target degradation and gene silencing, which are important for human life process. Many of them play an important role in the development and metastasis of cancer as the proto oncogene or tumor suppressor gene,.MiR-21 as an important role in the development and metastasis of cancer. The miRNA of cancer plays an important role in the invasion, invasion and metastasis of different malignant tumors. [9-11]. studies have confirmed that miR-21 has high expression in osteosarcoma cells, and miR-21 promotes the growth and metastasis of osteosarcoma. Meanwhile, research has confirmed that miR-21 has abnormal expression in the stem cells of a variety of malignant tumors and through protein or gene. Interaction to regulate the functional [12] of malignant tumor stem cells; in addition, studies have shown that the inhibitor of miR-21 (antisense oligonucleotide chain) can improve the sensitivity of some tumor cells to chemotherapeutic drugs [13-15].VEGF is a specific heparin binding growth factor for vascular endothelial cells, and is the major vascular generation in the human body. Factors. In the growth of tumor, the expression of VEGF and its receptor in tumor tissue is increasing under the action of a variety of cancer promoting factors. There are a lot of irregular neovascularization in the tumor tissue, providing sufficient nutrients for tumor tissue growth, and providing access to tumor invasion and metastasis, and [16 in the process of tumor growth, infiltration and metastasis. Studies have confirmed that VEGF plays an important role in the development and metastasis of osteosarcoma, and in other malignant tumors, miR-21 promotes the growth and transfer of [17,18]. by enhancing VEGF expression, thus introducing VEGF in our study of the regulatory role of miR-21 in the occurrence, development, metastasis and tumor stem cells of osteosarcoma. One can be used as a contrast to analyze the role of miR-21 and to analyze the correlation between miR-21 and VEGF in the regulation of osteosarcoma stem cells. Objective: this topic mainly studies the alteration of the VEGF gene and / or CD133 gene expression in different osteosarcoma cells after blocking the action of miR-21 and VEGF, and the effect on the growth of osteosarcoma cells. MiR-21 has a regulatory effect on osteosarcoma and its stem cells. Combined with the regulation of VEGF on osteosarcoma and its stem cell characteristics, the effect of miR-21 on the characteristics of osteosarcoma and its stem cells and its possible mechanism and rules are discussed in order to find out the effective inhibition of osteosarcoma stem cells in the clinic. Methods: 1. cells and cell culture: the selection of human osteosarcoma CRL-1543 and CRL-1423 cell line as the experimental cell, the cell culture solution recommended by the ATCC, the culture conditions and the operation steps for the cultivation of.2. to test the planting of a Realtime-PCR experiment and the cultivation of two cell lines. The culture was divided into 3 experimental groups, which were Oh (initial) control group, 8h block group and 24h blockage group. Each group included 2 holes 1543 cells and 2 hole 1423 cells. In accordance with the cell culture method mentioned above, the two cell lines were cultured and three cells were cultured for 24 hours in the C02 cell culture box, and the cells were harvested and harvested at the beginning of the harvest. Then, miR-21 (has-mir-21-5p) antisense oligonucleotide chain and VEGF receptor inhibitor were added to the 2 cells of the other two groups. Then, the culture plates of two inhibitory groups were returned to culture box to continue to be cultured, and cells were collected for 8 hours and 24 hours after miR-21 inhibitor transfection and VEGF receptor inhibitor treatment, respectively. For 8 hour inhibition group and 24 hour inhibition group, RNA was extracted from each cell sample of each experiment group, then cDNA was reversed, real time-PCR experiment was carried out, and VEGF and CD133 genes were detected in each sample,.3. experiment two cell immuno histochemistry experiment 3.1 and two 8 hole chamber slider (chamber loading) were taken for two cell lines. Two rows of cells were cultured for each chamber slider, which were labeled as CD133 and VEGF. respectively. The top row was implanted with 1543 cells, the lower one was implanted 1423 cells, 10000 cells were implanted in each culture hole, and the corresponding culture medium 0.5ml was added to the incubator for 24 hours, and the cells completely adhered to the wall.3.2 to two cell lines. The first hole in each row of four cells was taken as a blank control, second holes were used as negative control, second holes were used as VEGF (-) to add VEGF receptor inhibitor and fourth holes as miR-21 (-) transfected miR-21 (has-mir-21-5p) blocker. The treated cells were then placed in the incubator and cultured for 24h, and then immunohistochemical staining was performed. The experiment was to detect the expression of VEGF gene and CD133 gene in each cell sample.4. experiment three cell scratch test 4.1 to two cell lines in three experimental groups, which were blank control group, miR-21 blockage group and miR-21 blocking +VEGF inhibition group. Each group consisted of 2 holes 1543 cells and 2 holes 1423 cells, according to the cell culture mentioned above. All the two cell lines treated by.4.2 were cultured for 24 hours in the culture box, and the cells were completely adhered to the wall. Then, no blockers were added in the first four culture holes, the second groups and four culture holes were added to the blocking agent of miR-21, and the third groups were added to the inhibitor and the inhibitor of the miR-21 blocker and the VEGF receptor in the four culture holes. After that, the twelve cell culture plate was put back in the culture box, and the cells were basically covered with the bottom of the culture hole. P200 Pipet tip was used at the bottom of each culture hole. The line was marked through the vertical bottom of the center. The photo was observed with the inverted microscope and the intersection of the six marking lines on the scratch, and then continued to be cultured in the incubator. 4h, 8h, 24h, and 48h were taken respectively to measure the distance between the two sides of the scratches at the 6 points, and the average value was taken as the distance between the two sides of the scratch in this time point of the cell. Each cell line had two repeated test samples, and the refetching average was the time point of the cell line. The distance between the two sides of the scratch was calculated and the distance between the two edges of each time point was reduced, and the differences in the ability to proliferate and grow in each cell were analyzed. Results 1.real-time PCR experimental results: 1.1 the overall changes in the expression of VEGF and CD133 genes in each cell line: after the inhibition of VEGF, the expression of VEGF in 1543 cells and 1423 cells was reduced. Low (P0.05), and the expression of CD133 increased significantly (P0.05). After blocking mmiR-21, the expression of VEGF and CD133 genes in 1543 cells decreased (P0.05), and the expression of VEGF and CD133 genes in 1423 cells increased significantly (P0.05).1.2 cell lines and the regularity of the expression of the gene expression with time: (1) The expression of VEGF and CD133 genes increased first, and the expression of VEGF and CD133 decreased with time. The expression of VEGF gene was lower than that of the initial control group. After blocking miR-21, the expression of VEGF and CD133 genes in the 1543 cells decreased, and there was no obvious change with the development of time. (2) 1423 cells. The expression of CD133 gene after inhibition of VEGF and the expression of VEGF and CD133 gene after blocking miRNA-21 were significantly different at two time points. Among them, the expression of CD133 gene was 4.13 times as high as that of the initial control group at 8 hours after the inhibition of VEGF, and at 24 hours it decreased sharply to the 0.79.2. cell immunohistochemistry experiment of the initial control group. Results: 2.11543 cells were VEGFlow/CD133high cells, while 1423 cells were VEGFhi8h/CD133low cells.2.2 1543 cells showed a decrease in the expression of VEGF gene after inhibition of VEGF, and the expression of CD133 gene increased. After blocking miR-21, the expression of VEGF and CD133 gene expressed in.2.3 1423 cells were also shown as VE after VEGF. The expression of GF gene decreased, but the expression of CD133 gene increased, but the expression of VEGF and CD133 gene expression increased obviously after blocking miR-21. The proliferation rate of 3.11543 cells was significantly faster than that of the two experimental groups of the 1423 cell.3.2 1543 cells (inhibiting the miR-21 group and simultaneously inhibiting the miR-21 and VEGF groups). The growth speed of the group was slow (P0.05). There was no significant difference between the growth speed of the cells in the two experimental groups. The growth speed of.3.3 1423 cells was slower than that of the control group (P0.05), while the growth speed of the cells added with two inhibitors (miR-21 and VEGF receptor) was the slowest, and the difference was statistically significant (P0.05). Conclusion 1. blocked MI. R-21 has obvious regulation effect on osteosarcoma cells with low expression of VEGF gene, while inhibition of VEGF has a significant regulatory effect on osteosarcoma cells with high expression of VEGF gene, CD133 gene expression of two osteosarcoma cell lines is up regulation after.2. inhibition VEGF. VEGF may inhibit the expression of CD133 gene in osteosarcoma cells,.3.miR-21 in VEGF The low expression of osteosarcoma promotes the occurrence and proliferation of osteosarcoma stem cells and increases the proportion of tumor stem cells in the tumor. In this part of the cells, blocking miR-21 can significantly reduce the expression of CD133, a stem cell marker of osteosarcoma cells, which may have the effect of reducing the effect of.4. on the high expression of VEGF by 14 23 cells, inhibition of VEGF can regulate the expression of CD133 gene in two direction, showing a short period of inhibition. Significance: our study found that the transfection of miR-21 antisense oligonucleotide chain in the osteosarcoma cells with low expression of VEGF can significantly reduce the expression of VEGF and CD133 in osteosarcoma cells, which is the key to promoting angiogenesis. Growth factors play a key role in tumor metastasis and angiogenesis, and CD133 is an important marker of stem cells, reflecting the stem cell characteristics of tumor cells. These two genes are reduced, suggesting that the tumor cell's ability to promote angiogenesis and the proportion of stem cells decrease. This suggests that inhibition of the expression of miR-21 may play a role in inhibiting tumor. The growth and angiogenesis and metastasis, as well as reducing the proportion of tumor stem cells, can effectively improve the effect of chemotherapy. VEGF
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R738.1

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