本文選題：γδT細(xì)胞 + 血液腫瘤��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文
【摘要】：血液腫瘤是血液系統(tǒng)一系列惡性疾病的總稱,包括急性白血病、慢性白血病、多發(fā)性骨髓瘤、骨髓增生異常綜合征、淋巴瘤和惡性組織細(xì)胞病等。在我國(guó),血液腫瘤發(fā)病率較高。雖然聯(lián)合化療方案不斷改進(jìn),支持治療不斷改善,血液腫瘤的初治CR不斷提高,生存率也有明顯提高。即使初始治療達(dá)到完全緩解,但血液腫瘤復(fù)發(fā)率依然很高,尤其急性白血病長(zhǎng)期生存率低。同時(shí)還有部分病例達(dá)不到CR,其長(zhǎng)期生存率更低。復(fù)發(fā)、難治的病例至今仍治療效果有限,缺乏理想的治療手段。傳統(tǒng)的腫瘤治療方法有手術(shù)切除、放療、化療和靶向治療,其中手術(shù)切除、放療、化療均是作用于腫瘤組織,只能局部清除腫瘤細(xì)胞,靶向治療雖然能清除散在的腫瘤細(xì)胞,但存在腫瘤抗原變異和丟失、耐藥等問(wèn)題,均無(wú)法清除全身的腫瘤細(xì)胞,腫瘤的復(fù)發(fā)也就無(wú)法避免。腫瘤的免疫治療作用于人體的免疫系統(tǒng),通過(guò)提高人體免疫系統(tǒng)的清除腫瘤能力來(lái)達(dá)到治療腫瘤的目的。在2013年,《Science》雜志將腫瘤免疫治療評(píng)為年度十大科技突破之首,為血液腫瘤的治療帶來(lái)曙光。過(guò)繼性免疫細(xì)胞治療,是將采自患者的或健康供者的PBMC,體外擴(kuò)增活化后,將獲得的免疫細(xì)胞回輸給患者,利用活化的免疫細(xì)胞抗腫瘤活性來(lái)清除患者體內(nèi)的腫瘤細(xì)胞。目前,過(guò)繼性免疫細(xì)胞治療應(yīng)用的比較多的人體的免疫細(xì)胞有T淋巴細(xì)胞、NK細(xì)胞和DC細(xì)胞。T淋巴細(xì)胞被認(rèn)為是機(jī)體唯一能特異性識(shí)別殺傷腫瘤細(xì)胞的免疫細(xì)胞。根據(jù)表達(dá)不同的TCR,T淋巴細(xì)胞可分為αβT和γδT細(xì)胞。γδT細(xì)胞是表達(dá)TCRγδ的T細(xì)胞亞群,γδT細(xì)胞只占外周T淋巴細(xì)胞1%-5%,但在組織中卻可高達(dá)20-50%。γδT細(xì)胞屬于人體的固有免疫細(xì)胞,在體內(nèi)起著抗腫瘤、抗病毒感染、抗細(xì)菌感染和免疫監(jiān)視等作用,能促進(jìn)未成熟B細(xì)胞分泌Ig A,Ig M,Ig G,調(diào)節(jié)體液免疫,能活化未成熟DC細(xì)胞。γδT細(xì)胞,能以MHC非限制性的方式識(shí)別多種腫瘤相關(guān)抗原,可通過(guò)以下方式殺傷腫瘤細(xì)胞:通過(guò)凋亡誘導(dǎo)蛋白配體途徑Fas-Fas L和TRAILR誘導(dǎo)腫瘤細(xì)胞凋亡;分泌大量的細(xì)胞因子,作用于腫瘤細(xì)胞及其微環(huán)境,直接或協(xié)同其他免疫細(xì)胞殺傷腫瘤細(xì)胞;通過(guò)ADCC和穿孔素-顆粒酶B殺傷腫瘤細(xì)胞;通過(guò)某些膜表面受體如FcγR,經(jīng)ADCC發(fā)揮細(xì)胞毒作用;作為APC,發(fā)揮提呈抗原作用。已有不少臨床試驗(yàn)應(yīng)用γδT細(xì)胞于腫瘤的治療,已在結(jié)直腸癌、腎癌、非小細(xì)胞肺癌、乳腺癌和胰腺癌等腫瘤治療上取得進(jìn)展,取得良好療效和安全性。將γδT細(xì)胞應(yīng)用于血液腫瘤的治療有三個(gè)優(yōu)勢(shì):活化的γδT細(xì)胞對(duì)腫瘤細(xì)胞強(qiáng)大的殺傷活性;γδT細(xì)胞能夠分泌大量細(xì)胞因子和提呈抗原協(xié)同其他免疫細(xì)胞殺傷腫瘤細(xì)胞;血液腫瘤患者常存在免疫低下或缺陷,因此常引發(fā)感染,特別是機(jī)會(huì)感染,γδT細(xì)胞屬于固有免疫細(xì)胞,應(yīng)用于腫瘤的治療時(shí)能在清除腫瘤細(xì)胞的同時(shí),抗病毒和細(xì)菌感染。目的:本研究的目的就是建立γδT細(xì)胞培養(yǎng)體系,探討γδT細(xì)胞對(duì)多種血液腫瘤細(xì)胞的殺傷活性,并初步探討影響γδT細(xì)胞殺傷的一些機(jī)制,為γδT細(xì)胞應(yīng)用于血液腫瘤的治療提供實(shí)驗(yàn)依據(jù)。內(nèi)容:本文的研究?jī)?nèi)容主要分二個(gè)部分。首先是優(yōu)化Zol聯(lián)合IL-2的培養(yǎng)方案,建立γδT細(xì)胞培養(yǎng)體系,比較兩種無(wú)血清T細(xì)胞培養(yǎng)基Op Tmizer和X-VIVO15培養(yǎng)基體外培養(yǎng)γδT細(xì)胞的效果和添加血清對(duì)無(wú)血清Op Tmizer和X-VIVO 15培養(yǎng)基體外培養(yǎng)γδT細(xì)胞的影響;然后是采用流式細(xì)胞術(shù)檢測(cè)γδT細(xì)胞對(duì)Jurkat、THP-1、HL-60、K562、Raji、U-937和RPMI-8226細(xì)胞的殺傷活性,檢測(cè)γδT細(xì)胞分泌IFN-γ和TNF-α的水平,檢測(cè)γδT細(xì)胞表達(dá)CD107a的水平,檢測(cè)延長(zhǎng)與靶細(xì)胞共孵育時(shí)間對(duì)γδT細(xì)胞對(duì)靶細(xì)胞的殺傷活性的影響,檢測(cè)Zol和美伐他汀預(yù)處理對(duì)γδT細(xì)胞殺傷腫瘤細(xì)胞的影響,并比較γδT與NK和CIK細(xì)胞對(duì)K562細(xì)胞的殺傷作用。方法:1、優(yōu)化Zol聯(lián)合IL-2的培養(yǎng)方案,采用RPMI-1640培養(yǎng)基,建立γδT細(xì)胞培養(yǎng)體系,在此基礎(chǔ)上,比較兩種無(wú)血清T細(xì)胞培養(yǎng)基Op Tmizer和X-VIVO15培養(yǎng)基體外培養(yǎng)γδT細(xì)胞的效果,并比較添加血清對(duì)無(wú)血清培養(yǎng)基擴(kuò)增γδT細(xì)胞的影響。2、體外培養(yǎng)Jurkat、THP-1、HL-60、K562、Raji、U-937和RPMI-8226細(xì)胞,倒置顯微鏡下觀察處于對(duì)數(shù)生長(zhǎng)期,分別與γδT細(xì)胞共孵育,效靶比為10:1和20:1,共孵育4 h,用流式抗體標(biāo)記后上流式細(xì)胞儀檢測(cè),比較γδT細(xì)胞對(duì)Jurkat、THP-1、HL-60、K562、Raji、U-937和RPMI-8226細(xì)胞的殺傷活性和不同的效靶比對(duì)γδT細(xì)胞殺傷的影響。將γδT細(xì)胞與K562細(xì)胞按10:1的效靶比共孵育48 h,分別于0、4、16、24、36和48 h用流式細(xì)胞儀檢測(cè)γδT細(xì)胞分泌IFN-γ和TNF-α的水平。將HL-60和K562細(xì)胞分別與γδT細(xì)胞按10:1的效靶比共孵育4 h和20 h后,用流式細(xì)胞儀檢測(cè)延長(zhǎng)共孵育時(shí)間對(duì)γδT細(xì)胞殺傷K562細(xì)胞的影響。用不同濃度的Zol預(yù)處理K562細(xì)胞,分別與γδT細(xì)胞按10:1的效靶比共孵育4 h后,用流式細(xì)胞儀檢測(cè)不同濃度的Zol對(duì)γδT細(xì)胞殺傷K562細(xì)胞的影響。用5μmol/L的美伐他汀預(yù)處理K562細(xì)胞,后與γδT細(xì)胞按10:1的效靶比共孵育4 h后,用流式細(xì)胞儀檢測(cè)美伐他汀對(duì)γδT細(xì)胞殺傷K562細(xì)胞的影響。簽署知情同意書(shū)后,抽取4位淋巴瘤患者的外周血,分離得到PBMC,采用不同的培養(yǎng)體系體外擴(kuò)增γδT與NK和CIK細(xì)胞,后分別與K562細(xì)胞按5:1、10:1和20:1的效靶比共孵育4 h后,用流式細(xì)胞儀檢測(cè),比較γδT與NK和CIK細(xì)胞對(duì)K562細(xì)胞的殺傷作用。結(jié)果:1、采用Zol聯(lián)合IL-2的培養(yǎng)方案可以在體外擴(kuò)增外周血γδT細(xì)胞;Op Tmizer培養(yǎng)基比X-VIVO 15培養(yǎng)基體外培養(yǎng)γδT細(xì)胞的效果好(P0.05),適當(dāng)加入血清能提高擴(kuò)增效率(P0.05)。2、發(fā)現(xiàn)γδT細(xì)胞對(duì)Jurkat、THP-1、HL-60、K562、U-937和RPMI-8226細(xì)胞均有明顯的殺傷活性(P0.05),對(duì)Raji細(xì)胞的殺傷作用較弱;隨著與K562細(xì)胞共孵育時(shí)間的延長(zhǎng),γδT細(xì)胞分泌IFN-γ和TNF-α的水平基本呈現(xiàn)出時(shí)間依賴性增加,TNF-α自8h后逐漸增加;與K562細(xì)胞和HL-60細(xì)胞共孵育后,γδT細(xì)胞CD107a的表達(dá)水平均出現(xiàn)顯著上調(diào)(P0.01)。延長(zhǎng)γδT細(xì)胞與K562和HL-60細(xì)胞共孵育時(shí)間,γδT細(xì)胞對(duì)K562和HL-60細(xì)胞的殺傷活性也明顯增強(qiáng)(P0.01)。Zol能提高γδT細(xì)胞對(duì)K562細(xì)胞的殺傷活性,而美伐他汀減弱了γδT細(xì)胞對(duì)K562細(xì)胞的殺傷活性;4位B細(xì)胞淋巴瘤患者均成功在體外擴(kuò)增出γδT、NK和CIK細(xì)胞。效靶比為5:1時(shí),γδT、NK和CIK細(xì)胞對(duì)K562細(xì)胞的殺傷活性無(wú)統(tǒng)計(jì)學(xué)差異(P0.05),效靶比為10:1和20:1時(shí),γδT細(xì)胞對(duì)K562細(xì)胞的殺傷活性與NK細(xì)胞無(wú)統(tǒng)計(jì)學(xué)差異(P0.05),但明顯強(qiáng)于較CIK細(xì)胞(P0.01)。結(jié)論:Zol聯(lián)合IL-2的培養(yǎng)體系體外成功擴(kuò)增健康人外周血γδT細(xì)胞;Op Tmizer培養(yǎng)基比X-VIVO 15培養(yǎng)基體外培養(yǎng)γδT細(xì)胞的效果好,適當(dāng)加入胎牛血清(FBS)能提高擴(kuò)增效率;γδT細(xì)胞可顯著殺傷多種血液腫瘤細(xì)胞;γδT細(xì)胞對(duì)K562細(xì)胞的殺傷活性與NK細(xì)胞無(wú)統(tǒng)計(jì)學(xué)差異,明顯強(qiáng)于CIK細(xì)胞,;在48 h內(nèi)隨著與k562細(xì)胞共孵育時(shí)間的延長(zhǎng),γδT細(xì)胞分泌IFN-γ的水平呈時(shí)間依賴性增加,而TNF-α自8 h后逐漸增加;與K562和HL-60細(xì)胞共孵育后,γδT細(xì)胞的CD107a分子的表達(dá)有水平明顯上調(diào)增加;延長(zhǎng)γδT細(xì)胞與K562和HL-60細(xì)胞共孵育時(shí)間,γδT細(xì)胞對(duì)K562和HL-60細(xì)胞的殺傷活性也明顯增強(qiáng)(P0.01)。Zol能提高γδT細(xì)胞對(duì)K562細(xì)胞的殺傷活性,而美伐他汀減弱了γδT細(xì)胞對(duì)K562細(xì)胞的殺傷活性�？傊�,Zol聯(lián)合IL-2體外培養(yǎng)的γδT細(xì)胞對(duì)多種血液腫瘤細(xì)胞均具有較高的殺傷活性。與靶細(xì)胞共孵育后,γδT細(xì)胞殺傷靶細(xì)胞的效率提高。提高效靶比和和延長(zhǎng)與靶細(xì)胞共孵育時(shí)間,都能提高γδT細(xì)胞對(duì)靶細(xì)胞的殺傷活性。Zol能提高γδT細(xì)胞對(duì)K562細(xì)胞的殺傷活性,而美伐他汀減弱了γδT細(xì)胞對(duì)K562細(xì)胞的殺傷活性。為血液腫瘤的細(xì)胞免疫治療提供實(shí)驗(yàn)依據(jù)。
[Abstract]:Blood tumor is the general name of a series of malignant diseases of the blood system, including acute leukemia, chronic leukemia, multiple myeloma, myelodysplastic syndrome, lymphoma and malignant histiocytic disease. In China, the incidence of blood tumors is high. Although combined chemotherapy regimens continue to improve, support treatment continues to improve, the initial blood tumor In the treatment of CR, the survival rate also improved. Even if the initial treatment reached complete remission, the recurrence rate of blood tumor was still very high, especially in the acute leukemia, the long-term survival rate was low. At the same time, some cases were less than CR, and the long-term survival rate was lower. The recurrence, refractory cases still had limited treatment effect and lack of ideal treatment. Traditional methods of tumor treatment include surgical resection, radiotherapy, chemotherapy and targeted therapy. Surgical excision, radiotherapy and chemotherapy are all responsible for tumor tissue, only local tumor cells can be removed. Targeting therapy can remove scattered tumor cells, but there are some problems, such as tumor antigen variation and loss, drug resistance, etc. In the 2013,
magazine named the tumor immunotherapy as the first of the ten major scientific and technological breakthroughs of the year and brought the dawn of the treatment of blood tumor. The immune cell therapy is a PBMC that will be taken from a patient or a healthy donor. After in vitro expansion and activation, the acquired immune cells are returned to the patient, and the tumor cells in the patient are removed by the active immune cell antitumor activity. At present, the immune cells of adoptive immunotherapy should use more T lymphocytes in the human body. NK cells and DC cell.T lymphocytes are considered to be the only immune cells that can specifically identify tumor cells. According to the expression of different TCR, T lymphocytes can be divided into alpha beta T and gamma delta T cells. Delta T cells belong to the natural immune cells of the human body. They play the role of anti-tumor, antiviral infection, anti bacterial infection and immune surveillance in the body. It can promote the secretion of Ig A, Ig M, Ig G, modulate the humoral immunity and activate immature DC cells. The gamma delta T cells can identify various tumor related antigens in a MHC and non restrictive way, which can be passed through the MHC and non restrictive way. The following methods kill tumor cells: inducing apoptosis by Fas-Fas L and TRAILR through apoptosis inducing protein ligand pathway; secreting a large number of cytokines, acting on tumor cells and their microenvironment, killing tumor cells directly or in conjunction with other immune cells; killing tumor cells through ADCC and perforin granzyme B; through some membrane tables Surface receptors, such as Fc gamma R, play cytotoxic effects by ADCC; as APC, play an antigenic role. Many clinical trials have applied gamma delta T cells in the treatment of tumors, and have made progress in the treatment of colorectal cancer, kidney cancer, non small cell lung cancer, breast cancer and pancreatic cancer. Good efficacy and safety have been achieved. [delta] T cells are applied to blood. The treatment of tumor has three advantages: activated gamma delta T cells have a strong killing activity against tumor cells; gamma delta T cells can secrete a large number of cytokines and antigen in synergy with other immune cells to kill tumor cells; the patients with blood tumors often have immunodeficiency or defects, and therefore often cause infection, especially opportunistic infection, and gamma delta T cells belong to the tumor cells. The aim of this study is to establish a gamma delta T cell culture system, to explore the killing activity of gamma delta T cells to a variety of blood tumor cells, and to explore some mechanisms for the killing of gamma delta T cells, which should be used for the gamma delta T cells. The experimental basis for the treatment of blood tumor is provided. The content of this study is mainly divided into two parts. First, it is to optimize the culture scheme of Zol combined with IL-2, to establish a T cell culture system, to compare the effect of two kinds of serum free T cell culture medium Op Tmizer and X-VIVO15 culture medium in vitro culture of gamma delta T cells and to add serum to serum-free Op T. The effects of mizer and X-VIVO 15 culture medium on the culture of gamma delta T cells in vitro, and then using flow cytometry to detect the killing activity of gamma delta T cells on Jurkat, THP-1, HL-60, K562, Raji, U-937 and RPMI-8226 cells. The effect of time on the killing activity of gamma delta T cells on target cells was used to detect the effects of Zol and simvastatin on the killing of tumor cells by gamma delta T cells, and to compare the killing effects of gamma delta T and NK and CIK cells on K562 cells. Methods: 1, the culture scheme of Zol combined with IL-2 was optimized, and RPMI-1640 culture medium was used to establish the culture system of delta T cells. On the basis of the comparison of the effects of two serum-free T cell culture medium Op Tmizer and X-VIVO15 medium in vitro culture of gamma delta T cells, the effects of serum on the amplification of.2, Jurkat, THP-1, HL-60, K562, Raji, K562, and cells were observed in the logarithmic growth period under the inverted microscope. The effect target ratio was 10:1 and 20:1, and the target ratio was 4 h. The effect of gamma delta T cells on Jurkat, THP-1, HL-60, K562, Raji, U-937 and RPMI-8226 cells was compared with the effect of different target ratio on the killing of gamma delta cells. The level of IFN- gamma and TNF- alpha secreted by gamma delta T cells was detected by flow cytometry at 48 h, respectively. The effects of HL-60 and K562 cells on 4 h and 20 h were incubated with HL-60 and K562 cells according to the target ratio of 10:1, respectively. The effects of prolonged incubation time on the killing cells of gamma delta cells were detected by flow cytometry. After 4 h cells were incubated with gamma delta T cells according to the target ratio of 10:1 to 4 h, the effect of Zol on the killing of K562 cells by gamma delta T cells was detected by flow cytometry. The K562 cells were pretreated with the statins of 5 mu mol/L, and after 4 h were incubated with the 10:1 target ratio of the gamma delta T cells, the statins were detected by flow cytometer. After the informed consent book was signed, the peripheral blood of 4 patients with lymphoma was extracted and PBMC was isolated. The T and NK and CIK cells were amplified in vitro by different culture systems, and then the K562 cells were incubated with the target ratio of 5:1,10:1 and 20:1 for 4 h, respectively, and were detected by flow cytometry and compared with those of NK and CIK cells. Results: 1, the peripheral blood gamma delta T cells can be amplified in vitro by Zol combined with IL-2, and the effect of Op Tmizer culture based on X-VIVO 15 medium is good (P0.05), and the proper addition of serum can improve the amplification efficiency (P0.05).2. The cells had obvious killing activity (P0.05), and the killing effect on Raji cells was weak. With the prolongation of incubation time with K562 cells, the level of IFN- gamma and TNF- alpha secreted by delta T cells showed a time dependent increase, and TNF- alpha gradually increased since 8h; after incubation with K562 and HL-60 cells, the expression level of CD107a Delta T cell CD107a The incubation time of gamma delta T cells with K562 and HL-60 cells was prolonged, and the killing activity of K562 and HL-60 cells was significantly increased by the gamma delta T cells (P0.01).Zol could increase the killing activity of the delta T cells to K562 cells, while simvastatin weakened the killing activity of gamma delta T cells to the cells; 4 patients with lymphoma cell lymphoma were all Gamma delta T, NK and CIK cells were successfully amplified in vitro. When the target ratio was 5:1, there was no significant difference in the killing activity of T, NK and CIK cells to K562 cells (P0.05). The culture system successfully amplified healthy human peripheral blood gamma delta T cells in vitro; Op Tmizer culture was better than X-VIVO 15 culture medium in vitro culture of gamma delta T cells, and proper addition of fetal bovine serum (FBS) could improve the amplification efficiency; gamma delta T cells could kill a variety of blood tumor cells significantly, and the killing activity of gamma delta T cells to K562 cells was not statistically significant to NK cells. The difference was obviously stronger than that of CIK cells, and the level of IFN- gamma secreted by T cells increased in time with the prolongation of the incubation time with K562 cells in 48 h, while TNF- alpha increased gradually after 8 h, and the expression of CD107a molecules in the gamma delta T cells increased significantly after CO incubation with K562 and HL-60 cells; 60 cell incubation time, the killing activity of gamma delta T cells to K562 and HL-60 cells also increased significantly (P0.01).Zol can increase the killing activity of delta T cells to K562 cells, while simvastatin weakened the killing activity of delta T cells to K562 cells. In a word, the Zol combined with IL-2 in vitro gamma Delta T cells are higher for a variety of blood tumor cells. After incubating with target cells, the efficiency of gamma delta T cells to kill target cells increased. Improving target ratio and prolonging the incubation time with target cells could increase the killing activity of gamma delta T cells against target cells and increase the killing activity of.Zol Delta T cells to K562 cells, while simvastatin weakened the killing of K562 cells by gamma delta T cells. It provides experimental evidence for cellular immunotherapy of hematological malignancies.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733
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本文編號(hào)：1895050
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