本文選題：4-1BBL + 雙向信號(hào)　； 參考：《蘇州大學(xué)》2010年碩士論文

【摘要】： 4-1BBL(CD137L)是Ⅱ型跨膜糖蛋白分子,與其受體4-1BB同為共刺激分子TNF/TNFR超家族中的重要成員。4-1BBL分子表達(dá)在各種活化的免疫細(xì)胞表面,如:B細(xì)胞、巨噬細(xì)胞、樹突狀細(xì)胞、活化的T細(xì)胞等免疫細(xì)胞,但在未活化的T細(xì)胞上不表達(dá)。另有研究顯示,在某些腫瘤細(xì)胞表面也有4-1BBL分子的表達(dá)。4-1BBL的表達(dá)與惡性血液病有關(guān):如惡性血液病患者的血清中可溶性4-1BBL(s4-1BBL)含量異常升高;另外,在某些T和B腫瘤細(xì)胞系,雖然4-1BBL/4-1BB呈較低的構(gòu)成性共表達(dá),但4-1BBL/4-1BB信號(hào)均可促進(jìn)白血病細(xì)胞增殖并參與白血病細(xì)胞耐受抗腫瘤藥物的機(jī)制。4-1BBL分子與其受體4-1BB結(jié)合后可介導(dǎo)雙向信號(hào)。研究表明,兩者相互作用產(chǎn)生的正向信號(hào)可促進(jìn)活化的T細(xì)胞增殖,這一過程并不依賴CD28信號(hào);而通過膜4-1BBL傳遞的逆向信號(hào)可以抑制活化T細(xì)胞增殖并誘導(dǎo)其凋亡;4-1BBL逆向信號(hào)還能誘導(dǎo)單核細(xì)胞活化,促進(jìn)其IL-6、IL-8和TNF-α的分泌以及M-CSF的產(chǎn)生,并延長(zhǎng)其生存;逆向信號(hào)還能刺激CD34+造血干細(xì)胞來源的DC成熟等,如4-1BBL逆向信號(hào)可以上調(diào)DC細(xì)胞上CD86、CD83的表達(dá)以及IL-12的分泌。因此,4-1BBL/4-1BB介導(dǎo)雙向信號(hào)在T淋巴細(xì)胞、單核細(xì)胞及樹突狀細(xì)胞(DC)介導(dǎo)的免疫調(diào)節(jié)中發(fā)揮重要的作用。但這種4-1BB/4-1BBL雙向信號(hào)在免疫應(yīng)答和免疫調(diào)節(jié)中的作用和確切機(jī)理尚待深入研究和全面理解。為此,制備特異性抗人4-1BBL分子單克隆抗體,具備重要的研發(fā)價(jià)值。本實(shí)驗(yàn)制備了一株鼠抗人4-1BBL單克隆抗體39A8,能夠識(shí)別多種腫瘤細(xì)胞株,同時(shí)以表達(dá)4-1BBL分子的人單核白血病細(xì)胞株U937、THP-1及膠質(zhì)瘤細(xì)胞株675321為研究對(duì)象,初步研究了單克隆抗體39A8對(duì)腫瘤細(xì)胞體外生長(zhǎng)的影響。本論文共分為兩個(gè)部分。 1.鼠抗人4-1BBL單抗的雜交瘤細(xì)胞株的制備 以基因轉(zhuǎn)染細(xì)胞L929/4-1BBL作為免疫原,免疫BALB/c小鼠,采用B淋巴細(xì)胞雜交瘤技術(shù),將免疫小鼠的脾臟和小鼠骨髓瘤細(xì)胞SP2/0進(jìn)行細(xì)胞融合,用HAT選擇培養(yǎng)基培養(yǎng)。以L929/4-1BBL為陽性篩選細(xì)胞株,以L929細(xì)胞為陰性對(duì)照,通過間接免疫熒光標(biāo)記法對(duì)雜交瘤細(xì)胞株進(jìn)行篩選。用有限稀釋法進(jìn)行陽性雜交瘤的亞克隆化培養(yǎng)。經(jīng)過多次亞克隆及反復(fù)篩選,最終得到一株持續(xù)分泌抗人4-1BBL單克隆抗體的雜交瘤細(xì)胞株(39A8),經(jīng)快速定性試紙法鑒定,39A8的重鏈IgG2a,輕鏈為κ鏈。雜交瘤細(xì)胞株經(jīng)體外連續(xù)傳代培養(yǎng),液氮凍存半年后復(fù)蘇,仍生長(zhǎng)良好,穩(wěn)定分泌抗體。 2.鼠抗人4-1BBL單克隆抗體的體外生物學(xué)特性研究 間接免疫熒光法標(biāo)記U937、THP-1、Hela、PC-3、Daudi、M231、SHG44等細(xì)胞,用流式細(xì)胞術(shù)分析細(xì)胞膜熒光標(biāo)記百分率和熒光強(qiáng)度。結(jié)果顯示,4-1BBL能不同程度地在某些腫瘤細(xì)胞株上表達(dá)。將不同濃度的4-1BBL單抗分別加入到天然表達(dá)4-1BBL分子的U937、THP-1、675321細(xì)胞的培養(yǎng)體系中,分別在培養(yǎng)24h、48h、72h和96h,觀察細(xì)胞的生長(zhǎng)狀態(tài)、計(jì)數(shù)細(xì)胞數(shù)量并繪制生長(zhǎng)曲線。研究結(jié)果顯示:4-1BBL單抗39A8可促進(jìn)上述三種細(xì)胞的生長(zhǎng)與增殖。 綜上所述,本研究成功制備了一株特異性的鼠抗人4-1BBL單克隆抗體,進(jìn)而鑒定了其生物學(xué)特性;利用抗人4-1BBL單抗初步探討了4-1BBL逆向信號(hào)對(duì)腫瘤細(xì)胞的生物學(xué)效應(yīng)。因此,這株功能性鼠抗人4-1BBL單克隆抗體的成功制備為其以后的生物學(xué)功能和信號(hào)途徑的研究以及臨床診斷試劑盒的研制提供了有效的物質(zhì)基礎(chǔ)。
[Abstract]:4-1BBL (CD137L) is a type II transmembrane glycoprotein, and its receptor 4-1BB is an important member of the.4-1BBL molecular TNF/TNFR superfamily expression on the surface of various activated immune cells such as B cells, macrophages, dendritic cells, activated T cells and other immune cells, but not expressed in non activated T cells. Other research shows that in some tumor cells also have the expression of.4-1BBL 4-1BBL molecules associated with malignant hematological diseases: such as serum of patients with malignant hematological diseases in 4-1BBL (s4-1BBL) levels were higher; in addition, T and B in some tumor cell lines, while 4-1BBL/4-1BB was a low CO expression, but 4-1BBL/4-1BB signal can promote two-way signal mediated molecular mechanism of.4-1BBL proliferation of leukemia cells and leukemia cells in tolerance of antitumor drugs after binding to its receptor 4-1BB. The results show that two The positive signals generated by the interaction can promote the proliferation of activated T cells, this process is not dependent on the CD28 signal; and reverse signal transfer through the membrane of 4-1BBL can inhibit the activation of proliferation and induce apoptosis of T cells; 4-1BBL reverse signaling can induce monocyte activation and promote its IL-6, IL-8 and TNF- secretion and M-CSF the produce, and prolong its survival; reverse signal can stimulate CD34+ hematopoietic stem cell derived DC mature, such as 4-1BBL reverse signal can up regulate DC cell CD86, CD83 expression and secretion of IL-12. Therefore, 4-1BBL/4-1BB mediated bidirectional signaling in T lymphocytes, monocytes and dendritic cells (DC) mediated immunity play an important role in the regulation of 4-1BB/4-1BBL. But the effect of this two-way signal in immune response and immune regulation and the exact mechanism still needs further research and comprehensive understanding. Therefore, the preparation of specific anti Human 4-1BBL monoclonal antibody, has important research value. A mouse anti human 4-1BBL monoclonal antibody 39A8 was prepared in this experiment, it can identify a variety of tumor cell lines, and 4-1BBL expression in human monocytic leukemia cell lines U937, THP-1 and glioma cell line 675321 as the research object, we studied the influence of monoclonal 39A8 antibody on the growth of tumor cells in vitro. This paper is divided into two parts.
Preparation of hybridoma cell line of 1. mouse anti human 4-1BBL monoclonal antibody
In transfected cells L929/4-1BBL as immunogen, BALB/c mice were immunized with B lymphocyte hybridoma technique, the immunized mouse spleen and mouse myeloma cell SP2/0 cell fusion by HAT selective medium. L929/4-1BBL was used as positive screening cell line, L929 cells as negative control. The hybridoma cell lines were screened by indirect immunofluorescence positive hybridoma by limited dilution of a Longhua culture. After many times of subcloning and repeated screening, finally get a continuous secretion of anti human 4-1BBL monoclonal antibody hybridoma cell line (39A8), the rapid qualitative identification method, the heavy chain IgG2a 39A8, light chain kappa chain. Hybridoma cell lines in vitro cultured and cryopreserved in liquid nitrogen after half a year, still grew well and stably secreting antibodies.
In vitro biological characteristics of 2. mouse anti human 4-1BBL monoclonal antibodies
Indirect immunofluorescence labeling of U937, THP-1, Hela, PC-3, Daudi, M231, SHG44 cell, analysis of cell membrane fluorescence labeling percentage and fluorescence intensity by flow cytometry. The results showed that 4-1BBL at different level of expression in some tumor cells. Different concentrations of 4-1BBL were added to the natural expression of 4-1BBL monoclonal antibody molecular U937 culture system of THP-1675321 cells, respectively in cultured 24h, 48h, 72h and 96h, observe cell growth state and the number of cells and the growth curve. The results showed that 4-1BBL mAb 39A8 could promote the growth and proliferation of the three cell types.
In summary, a strain specific mouse anti human 4-1BBL monoclonal antibody was prepared successfully in this study, and then identified its biological characteristics; using anti human 4-1BBL monoclonal antibody to investigate the biological effects of 4-1BBL on tumor cell reverse signal. Therefore, this strain was successfully functional mouse anti human 4-1BBL monoclonal antibody provides a material basis the prepared for the research the biological function and signaling pathways in the future and development of clinical diagnostic kit.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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本文編號(hào)：1738757