本文關(guān)鍵詞： 重組人p53腺病毒(rAd-p53) 樹突狀細(xì)胞 彌漫性大B細(xì)胞淋巴瘤　出處：《蘭州大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:樹突狀細(xì)胞(dendritic cell,DC)是目前已知唯一能激活初始T細(xì)胞(naive Tcells)功能最強(qiáng)的抗原遞呈細(xì)胞(antigen presenting cells,APC),因其強(qiáng)大的抗原遞呈功能和激活抗原后特異性的細(xì)胞毒性T淋巴細(xì)胞(cytotoxic T lymphocytes CTL)反應(yīng),成為腫瘤生物免疫治療的重要載體,而淋巴瘤源性DC(L-DC)既帶有淋巴瘤瘤細(xì)胞抗原,同時(shí)又是抗原提呈細(xì)胞,來源于患者本人,不存在MHC限制性,能有效地激活特異的抗腫瘤免疫,在淋巴瘤的生物免疫治療中發(fā)揮重要作用。但L-DC與正常人CD14~+或CD34~+前體細(xì)胞來源的CD83~+的DC相比,其遷移、吞噬作用及抗原加工遞呈功能減弱,使抗腫瘤免疫應(yīng)答能力下降,同時(shí)由于腫瘤細(xì)胞釋放細(xì)胞免疫抑制因子,引起DC的免疫耐受,這樣就限制了DC在淋巴瘤免疫治療中的作用,如何獲得更具抗原遞呈功能的DC,打破免疫耐受,成為目前研究的熱點(diǎn)。有研究表明,p53基因突變和過度表達(dá)與NHL的發(fā)生發(fā)展有關(guān)[1],在彌漫性大B細(xì)胞淋巴瘤中,其異常表達(dá)率可高達(dá)50%[2]。目前,體內(nèi)和體外試驗(yàn),應(yīng)用重組人p53腺病毒(rAd-p53),把wt-p53基因?qū)雽?shí)體瘤中或與實(shí)體瘤細(xì)胞株共培養(yǎng),可產(chǎn)生瘤細(xì)胞的生長抑制和誘導(dǎo)凋亡,同時(shí)增強(qiáng)了放療的敏感性,從而使rAd-p53成為治療實(shí)體瘤另外一種途徑,為腫瘤的治療提供了一種新的“武器”[3-10]。并且發(fā)現(xiàn)腺病毒p53修飾DC后可使其表達(dá)內(nèi)源性抗原,上調(diào)DC共刺激分子表達(dá),產(chǎn)生特異性CTL效應(yīng)。因此,如何獲得臨床上需要的有功能的L-DC及糾正瘤細(xì)胞內(nèi)的突變的p53基因的功能,是急需解決的問題。目前,雖然分子靶向治療藥物的問世,如CD20單抗(美羅華)等,使得DLBCL的治愈率提高了很多,但在淋巴瘤的治療中如何解決微小殘留病灶(MRD)、DC免疫耐受等問題,仍然是目前臨床治療淋巴瘤遇到的難題,因此,我們利用rAd-p53能否誘導(dǎo)淋巴瘤瘤細(xì)胞為成熟DC,作為腫瘤疫苗治療淋巴瘤及解決MRD、DC免疫耐受問題是本實(shí)驗(yàn)?zāi)康乃凇?方法:采集經(jīng)病理確診的彌漫性大B細(xì)胞淋巴瘤的淋巴結(jié)和外周血的單個核細(xì)胞(MNC)進(jìn)行培養(yǎng)。分為3組:實(shí)驗(yàn)組A(淋巴結(jié)源性DC轉(zhuǎn)染rAd-p53,L-rAd-p53-DC),實(shí)驗(yàn)組B(外周血源性DC轉(zhuǎn)染rAd-p53,P-rAd-p53-DC);對照組A(淋巴結(jié)源性DC轉(zhuǎn)染rAd,L-rAd-DC),對照組B(外周血源性DC轉(zhuǎn)染rAd,P-rAd-DC);空白對照組A(淋巴結(jié)源性DC未轉(zhuǎn)染,L-N-DC),空白對照組B(外周血源性DC未轉(zhuǎn)染,P-N-DC),以上各組均用GM-CSF+IL-4培養(yǎng),至第7天,實(shí)驗(yàn)組加rAd-p53;對照組加rAd;空白對照組未加任何物質(zhì);各組于培養(yǎng)第8天加入TNF-a+CD40mAb繼續(xù)培養(yǎng)48小時(shí)后,流式細(xì)胞儀檢測DC免疫表型、酶聯(lián)免疫吸附法(ELISA)進(jìn)行上清夜IL-12水平測定,四甲基偶氮唑鹽比色(MTT)法觀察各組刺激T淋巴細(xì)胞增殖能力(MLR)及乳酸脫氫酶(LDH)法針對淋巴瘤瘤細(xì)胞的細(xì)胞毒作用(CTL效應(yīng))。在分離外周血過程中,貼壁的進(jìn)行DC培養(yǎng),非貼壁的培養(yǎng)成效應(yīng)細(xì)胞。 結(jié)果:淋巴結(jié)源性和外周血源性的單個核細(xì)胞,經(jīng)培養(yǎng)后均成功獲得具有樹突狀突起的細(xì)胞,但以實(shí)驗(yàn)組樹突狀突起明顯。結(jié)果顯示:DC的表型(CD_(1a)除外)CD83、CD80、CD86和HLA-DR實(shí)驗(yàn)組均較對照組及空白對照組明顯增高(p＜0.05)。上清液中IL-12分泌水平實(shí)驗(yàn)組均較對照組及空白對照組明顯增高(p＜0.05)。實(shí)驗(yàn)組具有明顯的刺激自體淋巴細(xì)胞增殖的能力,且刺激能力隨rAd-p53-DC與淋巴細(xì)胞比例的增加而升高。對照組也能刺激同種自體淋巴細(xì)胞增殖的能力,但較實(shí)驗(yàn)組組差(p＜0.05)。對靶細(xì)胞的細(xì)胞毒作用(CTL效應(yīng))結(jié)果顯示,實(shí)驗(yàn)組介導(dǎo)的細(xì)胞殺傷率顯著高于對照組及空白對照組(P＜0.05)。且實(shí)驗(yàn)組A的CTL效應(yīng)明顯高于實(shí)驗(yàn)組B,兩者之間有顯著性差異(P＜0.05)。 結(jié)論:利用rAd-p53可以成功地誘導(dǎo)彌漫性大B細(xì)胞淋巴瘤瘤細(xì)胞和外周血來源單個核細(xì)胞為樹突狀細(xì)胞,形態(tài)學(xué)觀察及細(xì)胞表型檢測證實(shí)均為成熟DC,其IL-12分泌水平及介導(dǎo)的MLR和CTL效應(yīng)明顯強(qiáng)于非rAd-p53組所誘導(dǎo)的DC組。提示以rAd-p53-DC為基礎(chǔ)的腫瘤疫苗有可能在解決淋巴瘤的MRD、DC免疫耐受等問題上發(fā)揮強(qiáng)大的治療作用,為淋巴瘤的治療研究提供了一個新的思路和策略。
[Abstract]:Objective: dendritic cells (dendritic, cell, DC) is currently the only known to activate naive T cells (naive Tcells) the most potent antigen-presenting cells (antigen presenting cells, APC), because of its powerful antigen-presenting function and activation of antigen specific cytotoxic T lymphocytes (cytotoxic T lymphocytes CTL) the reaction, become an important carrier for tumor immunotherapy, and lymphoma derived DC (L-DC) is a tumor cell antigen, and antigen presenting cells from the patients themselves, without limitation of MHC, can effectively activate specific antitumor immunity and play an important role in the biological treatment of lymphoma. But compared with normal CD14~+, L-DC or CD34~+ precursor cells derived CD83~+ DC migration, phagocytosis and antigen processing and presentation function, make the antitumor immune response ability, at the same time due to the tumor Cellular immune cells release inhibiting factor, DC induced immune tolerance, thus limiting the role of DC in lymphoma treatment, how to get more antigen-presenting function of DC, to break the immune tolerance and become the hotspot of research. Studies have shown that p53 gene mutation and overexpression is related to carcinogenesis and development of [1] NHL and in diffuse large B cell lymphoma, the abnormal expression rate can be as high as 50%[2]. at present, in vivo and in vitro experiments using recombinant human p53 adenovirus (rAd-p53), wt-p53 gene was transfected into solid tumors or tumor cell lines were cultured, can produce tumor cell growth inhibition and apoptosis induction, at the same time to enhance the sensitivity of radiotherapy, so rAd-p53 has become the treatment of solid tumors another way for tumor therapy provides a new "weapon" [3-10]. and adenovirus p53 DC modified by the expression of endogenous antigen, DC up-regulated the expression of costimulatory molecules to produce specific CTL effect. Therefore, how to obtain the p53 gene mutations in tumor cell L-DC and correct the clinical need of function within the function, is an urgent problem. At present, although the molecular targeted therapy drugs, such as CD20 (Mei Luohua), the monoclonal antibody the cure rate of DLBCL has improved a lot, but in the treatment of lymphoma in how to solve the minimal residual disease (MRD), DC immune tolerance and other issues, is still the current clinical treatment of lymphoma problem, therefore, we use rAd-p53 can induce lymphoma tumor cells into mature DC, as a tumor vaccine for the treatment of lymphoma and MRD. The immune tolerance of DC problem is the purpose of this study lies.
鏂規(guī)硶:閲囬泦緇忕梾鐞嗙‘璇婄殑寮ユ極鎬уぇB緇嗚優(yōu)娣嬪反鐦ょ殑娣嬪反緇撳拰澶栧懆琛，

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