本文選題：防御素 + 黑色素瘤��； 參考：《南方醫(yī)科大學(xué)》2010年博士論文

【摘要】：前言: 天然免疫曾經(jīng)被認(rèn)為是機體免疫系統(tǒng)應(yīng)答外界刺激的低級形式,但目前研究表明天然免疫反應(yīng)不僅能夠直接而快速對抗外界刺激,而且還對機體的獲得性免疫反應(yīng)起直接的激活和導(dǎo)向作用�？咕氖巧矬w天然免疫反應(yīng)的重要因子,它不僅能夠直接殺傷入侵的病原微生物,中和有害的微生物產(chǎn)物,抑制病原微生物入侵靶細(xì)胞,而且,還可通過作用于多種白細(xì)胞亞群,加強抗原特異性T淋巴細(xì)胞及B淋巴細(xì)胞的活化等免疫調(diào)節(jié)功能而增強機體的應(yīng)激能力,從而間接發(fā)揮保持機體內(nèi)環(huán)境穩(wěn)態(tài)的作用。目前,已有若干研究表明,抗菌肽的抗菌功能對生理條件下的鹽離子濃度高度敏感,而且還可受到血清成分的直接抑制作用；而其免疫調(diào)節(jié)功能卻不受生理條件下,生理濃度的鹽離子及血清成分等因素的影響,因此,有人主張極有可能免疫調(diào)節(jié)功能才是抗菌肽的主要功能。 防御素是抗菌肽中重要的一類,它普遍存在于高等生物,不僅對病原微生物具有廣譜的毒殺效應(yīng),而且還具有重要的免疫調(diào)節(jié)活性。鼠源性β-防御素2(mBD2)是防御素中具有代表性的一種,研究表明,它可通過趨化因子受體CCR6趨化未成熟樹突狀細(xì)胞,并作為TLR4的內(nèi)源性配體促進樹突狀細(xì)胞的成熟,產(chǎn)生一系列促炎癥因子、趨化因子,上調(diào)共刺激分子表達(dá)。用編碼非免疫原性淋巴瘤抗原與mBD2融合蛋白的DNA質(zhì)粒免疫小鼠,可在小鼠體內(nèi)誘發(fā)出明顯的抗腫瘤活性�？梢�,mBD2作為天然免疫反應(yīng)的組成部分,還可協(xié)助非免疫原性抗原在小鼠體內(nèi)誘導(dǎo)出有效的特異性獲得性免疫反應(yīng),是一種天然的免疫佐劑。mBD2是連接天然免疫反應(yīng)和獲得性免疫反應(yīng)的橋梁,提示了一種新的腫瘤免疫治療策略。利用mBD2能夠增強抗原特異性免疫反應(yīng)的特性,可研究開發(fā)以mBD2為基礎(chǔ)的腫瘤疫苗。目前,盡管人類已經(jīng)研究了多種免疫佐劑,但是除了鋁制劑以外,大多數(shù)免疫佐劑都由于具有嚴(yán)重的毒副作用而不能夠直接應(yīng)用于人體；而鋁制劑雖然可用,但是它的免疫增強活性有限,且傾向于引發(fā)TK2型免疫反應(yīng),因此具有很大的局限性。mBD2作為一種內(nèi)源性天然活性物質(zhì),具有上述強烈的免疫調(diào)節(jié)活性,我們將mBD2應(yīng)用于惡性黑色素瘤的免疫治療,期望它能夠克服目前鋁制劑存在的局限性。 惡性黑色素瘤簡稱“惡黑瘤”,是一種來源于黑色素細(xì)胞的惡性腫瘤,其惡性程度高、起病隱匿、誤診率高、預(yù)后很差,是全世界范圍內(nèi)發(fā)病率增長最快的惡性腫瘤之一。惡性黑色素瘤對一般的綜合性治療措施：如放療、化療都表現(xiàn)為耐受,早期惡黑瘤的治療主要以手術(shù)為主,晚期惡黑瘤預(yù)后很差,尚無有效的治療手段,主要以個體化的綜合治療為原則。由于惡性黑色素瘤是一種免疫原性很強的腫瘤,因此特異性免疫治療一直以來都是惡性黑色素瘤治療的重要手段之一。盡管免疫治療在許多惡性黑色素瘤小鼠模型中都能夠誘導(dǎo)出明顯的抗腫瘤免疫保護作用,但臨床實驗的結(jié)果表明,人體對惡性黑色素瘤免疫治療的反應(yīng)率遠(yuǎn)遠(yuǎn)不如實驗動物有效,僅10～30%。目前,還不清楚人體對惡性黑色素瘤免疫治療產(chǎn)生個體差異的確切原因,但是,普遍認(rèn)為疫苗的臨床有效性不僅決定于其種類和制備方法,還取決于用于增強疫苗的有效性而選擇的免疫佐劑。 本文將防御素mBD2的免疫調(diào)節(jié)活性應(yīng)用于惡性黑色素瘤的免疫治療,制備mBD2基因修飾的惡性黑色素瘤細(xì)胞疫苗、觀察其體內(nèi)抗腫瘤免疫效果,并探討其抗腫瘤免疫保護作用的機制。 實驗方法： 1.采用RT-PCR技術(shù)從C57BL/6小鼠腎臟組織中擴增出小鼠β-防御素2(mBD2)成熟肽基因片段,采用overlap PCR方法將小鼠IgK信號肽序列加在mBD2成熟肽基因序列的5’端,T-A克隆后,構(gòu)建含有IgK信號肽的mBD2分泌性真核表達(dá)載體pcDNA3.1(+)-IgK-mBD2,并通過PCR、酶切、測序等方法鑒定其正確性。 2.采用脂質(zhì)體轉(zhuǎn)染法分別將空載體pcDNA3.1(+)及含有目的基因的真核表達(dá)載體pcDNA3.1(+)-IgK-mBD2轉(zhuǎn)染惡性黑色素瘤B16細(xì)胞,G418篩選后,獲得穩(wěn)定表達(dá)細(xì)胞株,分別命名為B16,p和B16-mBD2。RT-PCR從mRNA水平鑒定G418抗性基因neo基因的表達(dá),從而證明pcDNA3.1(+)-IgK-mBD2和pcDNA3.1(+)等質(zhì)粒的成功轉(zhuǎn)染；RT-PCR從mRNA水平鑒定mBD2基因水平的表達(dá)；Western-Blot從蛋白水平鑒定mBD2分子的表達(dá)。 3.采用MTT法測定三株細(xì)胞：B16、B16-p、B16-mBD2在120小時內(nèi)的生長曲線；采用PI染色流式細(xì)胞術(shù)檢測對數(shù)生長期三株細(xì)胞的細(xì)胞周期分布及凋亡情況；采用FITC標(biāo)記的抗小鼠CD80、CD86、MHCⅠ、MHCⅡ等抗體染色,流式細(xì)胞術(shù)檢測三株細(xì)胞上述CD80、CD86、MHCⅠ、MHCⅡ等相關(guān)分子的表達(dá)變化。 4.采用輻射方法將三株細(xì)胞B16、B16-p、B16-mBD2制備成細(xì)胞疫苗,分別在C57BL/6小鼠體內(nèi)進行免疫預(yù)防和免疫治療動物實驗。設(shè)立生理鹽水對照組、B16對照組、B16-p對照組及B16-mBD2實驗組。 5.免疫預(yù)防實驗：6～8周齡雌性C57BL/6小鼠,隨機分成4組,每組10～12只,分別為生理鹽水對照組、B16對照組、B16-p對照組和B16-mBD2實驗組。各組所有動物,按照各自的分組,分別于左腋下皮下注射給予0.1mL生理鹽水及106個輻射的B16、B16-p和B16-mBD2等細(xì)胞疫苗。各組動物免疫七天后,各組所有小鼠均給予5×104個對數(shù)生長期的野生型B16細(xì)胞致瘤,逐日觀察各組小鼠生存狀態(tài)、制作腫瘤體內(nèi)生長曲線、進行生存期分析及HE染色觀察各免疫預(yù)防組腫瘤組織及重要臟器,如：肝臟、腎臟、脾臟、肺臟等的組織學(xué)變化。 6.免疫治療實驗：6～8周齡雌性C57BL/6小鼠,隨機分成4組,每組10～12只,分別為生理鹽水對照組、B16對照組、B16-p對照組和B16-mBD2實驗組.,各組所有動物分別于左腋下皮下注射給予105個對數(shù)生長期野生型B16細(xì)胞致瘤,同時各組所有小鼠按照各自的分組,于同一天開始,左腋下皮下注射分別給予O.1mL生理鹽水及106個輻射的B16、B16-p和B16-mBD2等細(xì)胞疫苗進行治療,每周兩次,連續(xù)兩周。逐日觀察各組小鼠生存狀態(tài)、制作腫瘤體內(nèi)生長曲線、進行生存期分析及HE染色觀察各免疫治療組腫瘤組織及重要臟器,如：肝臟、腎臟、脾臟、肺臟等的組織學(xué)變化。 7.采用ELISA方法測定B16、B16-p和B16-mBD2等細(xì)胞疫苗免疫小鼠后,各組小鼠脾臟淋巴細(xì)胞培養(yǎng)上清中IFN-γ,、IL-12、IL-4等含量的變化；采用非放射性細(xì)胞毒性分析方法測定B16、B16-p和B16-mBD2等細(xì)胞疫苗免疫小鼠后,各組小鼠NK細(xì)胞殺傷活性及CTL細(xì)胞殺傷活性。 8.采用SPSS 13.0進行統(tǒng)計學(xué)分析。細(xì)胞生長曲線、動物實驗中的腫瘤體內(nèi)生長曲線等采用重復(fù)測量因素的方差分析；細(xì)胞周期分布、凋亡率、細(xì)胞膜表面免疫相關(guān)分子的表達(dá)變化等采用單向方差分析；方差齊性時,多重比較采用LSD法,方差不齊時,多重比較采用Dunnett T3法。CTL細(xì)胞殺傷活性、NK細(xì)胞殺傷活性等采用析因設(shè)計,運用單因素方差檢驗(one-way ANOVA)LSD法進行數(shù)據(jù)分析。P0.05表示有統(tǒng)計學(xué)意義。 實驗結(jié)果： 1.成功構(gòu)建含有鼠源性IgK信號肽序列的mBD2真核分泌性表達(dá)載體pcDNA3.1(+)-IgK-mBD2,并鑒定正確。 2.pcDNA3.1(+)及pcDNA3.1(+)-IgK-mBD2等載體轉(zhuǎn)染B16細(xì)胞后,G418篩選得到穩(wěn)定表達(dá)細(xì)胞株,分別命名為B16-p和B16-mBD2。 3.細(xì)胞生長曲線實驗證實,與野生型B16細(xì)胞及B16-p相比較,B16-mBD2細(xì)胞的增殖速度顯著減慢(F=144.256,P0.05)；與野生型B16細(xì)胞相比較,B16-p的增殖速度沒有顯著變化。 4.流式細(xì)胞儀檢測結(jié)果顯示,接種24小時后,與對數(shù)生長期的野生型B16細(xì)胞及B16-p相比較,對數(shù)生長期的B16-mBD2細(xì)胞的細(xì)胞周期發(fā)生S期輕度阻滯(F=8.952,P0.05)；三株細(xì)胞B16、B16-p和B16-mBD2的細(xì)胞膜表面分子CD80、CD86、MHCⅠ、MHCⅡ等表達(dá)沒有顯著性差異。 5.在免疫預(yù)防實驗中,生理鹽水對照組、B16對照組、B16-p對照組所有小鼠在49天內(nèi)全部死亡,組間沒有顯著性差異,中位生存期分別為35天、37天、33天；而B16-mBD2免疫預(yù)防組小鼠腫瘤體內(nèi)生長速度顯著緩慢(F=118.387,P0.05),小鼠生存期顯著延長(X2=18.857,P0.05),中位生存期為55天,同期生存狀態(tài)比其他各個對照組明顯良好。至實驗結(jié)束,即致瘤后第150天為止,仍有37.5%的小鼠實現(xiàn)無瘤生存。 6.在免疫治療實驗中,生理鹽水對照組、B16對照組及B16-p對照組所有小鼠均在44天內(nèi)全部死亡,組間沒有顯著性差異,中位生存期分別為32天、34天、31天；B16-mBD2免疫治療組小鼠腫瘤體內(nèi)生長速度顯著緩慢(F=289.615,P0.05),小鼠生存期顯著延長(X2=22.006,P0.05),中位生存期為59天,同期生存狀態(tài)比其他各個對照組明顯良好。至實驗結(jié)束,即致瘤后第150天為止,仍有25%的小鼠實現(xiàn)無瘤生存。 7.在免疫預(yù)防和免疫治療實驗中,B16-mBD2疫苗免疫組均誘導(dǎo)淋巴細(xì)胞大量浸潤到腫瘤組織,脾臟淋巴小結(jié)增多,生發(fā)中心反應(yīng)性增生、增大。生理鹽水對照組、B16對照組、B16-p對照組均不能誘導(dǎo)這種主動性抗腫瘤免疫反應(yīng)。 8.B16-mBD2細(xì)胞疫苗免疫后,可促進IFN-γ,產(chǎn)生顯著增多(F=506.814,P0.05),促進IL-12產(chǎn)生顯著增多(F=83.637,P0.05),與B16對照組、B16-p對照組比較差異有統(tǒng)計學(xué)意義,但對IL-4的含量沒有影響。B16-mBD2細(xì)胞疫苗可誘導(dǎo)小鼠針對B16細(xì)胞的特異性CTL殺傷活性顯著增強(F=44.376, P0.05)、NK細(xì)胞殺傷活性顯著增強(F=119.750,P0.05),與B16對照組、B16-p對照組比較差異有統(tǒng)計學(xué)意義。 9.B16-mBD2細(xì)胞疫苗免疫小鼠后,對小鼠的主要臟器如：肝臟、腎臟、脾臟、肺臟等的HE染色形態(tài)學(xué)觀察顯示,各個主要臟器未見明顯的病理改變,提示B16-mBD2疫苗相對安全。 結(jié)論： mBD2基因轉(zhuǎn)染惡性黑色素瘤B16細(xì)胞后,制備的轉(zhuǎn)基因細(xì)胞疫苗具有明顯的抗小鼠惡性黑色素瘤B16效應(yīng),該疫苗免疫后,通過誘導(dǎo)小鼠NK細(xì)胞殺傷活性增強、CTL殺傷活性增強、促進IFN-γ、IL-12等Thl型細(xì)胞因子產(chǎn)生增加、誘導(dǎo)大量淋巴細(xì)胞浸潤到腫瘤組織內(nèi)部以及誘發(fā)小鼠脾臟淋巴小結(jié)增生、增大等多種機制發(fā)揮抗惡性黑色素瘤免疫保護作用,且安全無毒副作用。mBD2基因修飾的B16細(xì)胞疫苗免疫后對Th2型細(xì)胞因子IL-4的含量沒有影響,提示該疫苗抗腫瘤免疫保護作用可能與Th2型免疫反應(yīng)無關(guān)�？傊�,mBD2基因修飾的B16細(xì)胞疫苗能夠同時激活天然免疫(NK細(xì)胞活性)和獲得性免疫(CTL細(xì)胞殺傷活性)、腫瘤局部免疫(腫瘤組織內(nèi)淋巴細(xì)胞浸潤)和系統(tǒng)免疫(誘導(dǎo)IFN-γ、IL-12等細(xì)胞因子含量增高)來發(fā)揮抗惡性黑色素瘤作用,為惡性黑色素瘤的免疫治療開拓了新的思路。
[Abstract]:Preface:
Natural immunity has been considered as a low-grade form of immune response to external stimuli. However, the present study shows that the natural immune response can not only directly and rapidly antagonize external stimuli, but also directly activate and guide the immune response of the body. Antimicrobial peptides are an important factor in the natural immune response of organisms. It can not only kill the pathogenic microorganism of the invasion directly, neutralize the harmful microbial products, inhibit the pathogenic microorganism to invade the target cells, but also can enhance the stress of the body by strengthening the immune regulation function such as the activation of the antigen specific T lymphocyte and the B lymphocyte. At present, some studies have shown that the antibacterial function of antimicrobial peptides is highly sensitive to the concentration of salt ions under physiological conditions, and can also be directly inhibited by the serum components, but the immune regulation function is not affected by physiological conditions, the physiological concentration of salt ions and serum components. Therefore, it has been suggested that immunomodulation function is the main function of antimicrobial peptides.
Defensin is an important class of antimicrobial peptides. It exists widely in higher organisms. It not only has broad-spectrum toxicity to pathogenic microorganisms, but also has important immunoregulatory activity. The mouse derived beta defensin 2 (mBD2) is a representative of the defensin. It is shown that it can be used to chemotaxis the immature tree through the chemokine receptor CCR6. Protrusion cells, which act as endogenous ligands for TLR4, promote the maturation of dendritic cells and produce a series of pro-inflammatory factors, chemokines and up regulation of the expression of CO stimulator molecules. Immunization of mice with DNA plasmid encoding non immunogenic lymphoma antigen and mBD2 fusion protein can induce obvious antitumor activity in mice. Visible, mBD2 The component of natural immune response can also assist non immunogenic antigen in inducing effective specific acquired immune response in mice. A natural immune adjuvant,.MBD2, is a bridge linking natural immune response and acquired immune response, suggesting a new strategy for tumor immunotherapy. The use of mBD2 can enhance resistance. The characteristics of the primary specific immune response can be used to develop a mBD2 based tumor vaccine. At present, although a variety of immune adjuvant has been studied, most of the immune adjuvant, in addition to aluminum, can not be directly applied to the human body because of its serious toxic and side effects; although aluminum is available, it is immune to it. The enhanced activity is limited and tends to trigger the TK2 type immune response. Therefore, it has a great limitation of.MBD2 as an endogenous natural active substance, which has the above strong immunomodulatory activity. We apply mBD2 to the immunotherapy of malignant melanoma, and expect it to overcome the limitations of the present aluminum preparation.
Malignant melanoma, called "evil black tumor", is a malignant tumor derived from melanocytes. Its malignant degree is high, the disease is concealed, the misdiagnosis rate is high, and the prognosis is poor. It is one of the fastest growing malignant tumors in the world. The malignant melanoma is resistant to general comprehensive treatment, such as radiotherapy and chemotherapy. The treatment of early malignant melanoma is mainly based on operation, and the prognosis of late malignant melanoma is very poor. There is no effective treatment. It is mainly based on the principle of individualized comprehensive treatment. Because malignant melanoma is a kind of tumor with strong immunogenicity, specific immunotherapy has always been one of the important means for the treatment of malignant melanoma. Although immunotherapy can induce obvious antitumor immune protection in many malignant melanoma mice models, the results of clinical trials show that the response rate of the human body to malignant melanoma is far less effective than that of experimental animals. At present, it is not clear that the human immune treatment for malignant melanoma is not clear at the present time of 10 to 30%.. The exact cause of individual differences is produced, but it is generally believed that the clinical effectiveness of the vaccine is not only determined by its species and preparation methods, but also depends on the immune adjuvant used to enhance the effectiveness of the vaccine.
In this paper, the immunomodulatory activity of defensin mBD2 was applied to the immunotherapy of malignant melanoma, to prepare the mBD2 gene modified melanoma cell vaccine, to observe the anti-tumor immune effect in the body and to explore the mechanism of its anti-tumor immunity protection.
Experimental methods:
1. RT-PCR technique was used to amplify the mouse beta defensin 2 (mBD2) mature peptide gene fragment from the C57BL/6 mouse kidney tissue, and the IgK signal peptide sequence of mice was added to the 5 'end of the mBD2 mature peptide gene sequence by overlap PCR method. After T-A was cloned, the mBD2 secreting eukaryotic expression vector containing IgK signal peptide was constructed, and pcDNA3.1 (+) -IgK-mBD2 was constructed. PCR, enzyme digestion, sequencing and other methods were used to identify their correctness.
2. liposome transfection was used to transfect the empty carrier pcDNA3.1 (+) and the eukaryotic expression vector containing the target gene pcDNA3.1 (+) -IgK-mBD2 into the malignant melanoma B16 cells. After G418 screening, the stable expression cell lines were obtained, which were named B16, P and B16-mBD2.RT-PCR, respectively, to identify the neo gene of the G418 resistance gene from mRNA level, thus proving PC. DNA3.1 (+) -IgK-mBD2 and pcDNA3.1 (+) and other plasmids were transfected successfully; RT-PCR was identified from mRNA level and the expression of mBD2 molecules was identified from the protein level by Western-Blot.
3. MTT method was used to determine the growth curve of three cells: B16, B16-p, B16-mBD2 in 120 hours. The cell cycle distribution and apoptosis of three cells in the logarithmic growth period were detected by PI staining flow cytometry; the anti body staining of CD80, CD86, MHC I and MHC II was used as a FITC marker, and the CD80 of three cells was detected by flow cytometry. C cells were tested for CD80, C Expression changes of D86, MHC I, MHC II and other related molecules.
4. the three cells B16, B16-p, and B16-mBD2 were prepared by radiation method, and the immunotherapy and immunotherapy in C57BL/6 mice were carried out. The control group of physiological saline, the B16 control group, the B16-p control group and the B16-mBD2 experimental group were set up.
5. immunoprophylaxis experiment: 6~8 weeks old female C57BL/6 mice were randomly divided into 4 groups, 10~12 in each group, respectively, the saline control group, the B16 control group, the B16-p control group and the B16-mBD2 experimental group. All the animals were subsubcutaneously injected into the left axillary saline and 106 radiation B16, B16-p and B16-mBD2 under the left axillary subaxillary injection. Seven days after seven days of immunization, all mice in each group were given 5 * 104 logarithmic long-term wild type of wild type of tumor. The survival state of each group was observed day by day, the growth curve of the tumor in vivo was made, the survival time analysis and HE staining were used to observe the tumor tissues and important organs of the immunization prevention groups, such as liver, kidney and spleen. Histological changes of the dirty, lung and so on.
6. immunotherapy experiment: 6~8 weeks old female C57BL/6 mice were randomly divided into 4 groups, with 10~12 rats in each group, respectively, the saline control group, the B16 control group, the B16-p control group and the B16-mBD2 experimental group. All the animals in each group were subcutaneously injected under the left axillary subcutaneous injection to induce the tumor of the wild type B16 cells of the 105 logarithmic growth period, and all the mice in each group were in accordance with each group. Each group, on the same day, was injected under the left subaxillary subaxillary injection to give O.1mL saline and 106 radiation B16, B16-p and B16-mBD2 cell vaccines for two times a week for two weeks. The survival state of the mice in each group was observed day by day, the growth curve of the tumor in vivo was made, the survival time analysis and HE staining were used to observe the immunotherapy. Histological changes of tumor tissues and important organs, such as liver, kidney, spleen and lungs.
7. ELISA method was used to determine the changes of IFN- gamma, IL-12, IL-4 in the spleen lymphocyte culture supernatant of mice after immunization with B16, B16-p and B16-mBD2 cells. The cytotoxicity of B16, B16-p and B16-mBD2 vaccines in mice were determined by non radioactive cytotoxicity analysis method. Cell killing activity.
8. the SPSS 13 was used for statistical analysis. The cell growth curve, the growth curve of the tumor in the animal experiment, the variance analysis of the repeated measurement factors, the cell cycle distribution, the apoptosis rate, the change of the expression of the immune related molecules on the surface of the cell membrane were analyzed by one-way ANOVA; when the variance was homogeneous, the multiple comparison was compared with the LSD method and variance. In the absence of homogeneity, multiple comparison used Dunnett T3.CTL cell killing activity, NK cell killing activity by factorial design and statistical significance of data analysis using single factor variance test (one-way ANOVA) LSD method for data analysis.P0.05.
Experimental results:
1. mBD2 eukaryotic secretory expression vector pcDNA3.1 (+) -IgK-mBD2 containing the IgK signal peptide sequence of rat origin was successfully constructed and identified correctly.
After transfection of B16 cells with 2.pcDNA3.1 (+) and pcDNA3.1 (+) -IgK-mBD2, G418 was screened for stable expression of cell lines, named B16-p and B16-mBD2. respectively.
The 3. cell growth curve experiment showed that the proliferation rate of B16-mBD2 cells was significantly slower than that of wild type B16 cells and B16-p (F=144.256, P0.05), and the proliferation rate of B16-p was not significantly changed compared with that of wild type B16 cells.
4. flow cytometry showed that, after 24 hours of inoculation, compared with the wild type B16 cells and B16-p of the logarithmic growth period, the cell cycle of B16-mBD2 cells in logarithmic growth period had a mild block of S phase (F=8.952, P0.05), and the expression of the membrane surface molecules CD80, CD86, MHC I, etc. of the three cells B16, B16-p and B16-mBD2 were not significant. Sexual differences.
5. in the immunoprophylaxis experiment, all the mice in the saline control group, the B16 control group and the B16-p control group were all dead within 49 days. There was no significant difference between the groups and the median survival period was 35 days, 37 days and 33 days respectively. The growth rate of the mice in the B16-mBD2 immunization group was significantly slow (F=118.387, P0.05), and the survival period of the mice was significantly prolonged (X 2=18.857, P0.05), the median survival period was 55 days, and the survival state of the same period was better than that of the other control groups. Until the end of the experiment, 37.5% of the mice were still alive without tumor until 150th days after the tumor.
6. in the immunotherapy experiment, all the mice in the saline control group, the B16 control group and the B16-p control group all died within 44 days, and there was no significant difference between the groups. The median survival period was 32 days, 34 days and 31 days respectively. The growth rate of the mice in the B16-mBD2 immunotherapy group was significantly slow (F=289.615, P0.05), and the survival period of the mice was significantly prolonged. (X2=22.006, P0.05), the median survival period was 59 days, and the survival state of the same period was better than that of the other control groups. Until the end of the experiment, 25% of the mice were still alive without tumor at 150th days after the tumor.
7. in the immunoprophylaxis and immunotherapy experiments, the B16-mBD2 immunization group induced a large number of lymphocytes infiltrating into the tumor tissue, the increase of the lymph nodes in the spleen, the reactive hyperplasia of the germinal center, and the increase. The control group of the physiological saline, the B16 control group and the B16-p control group all did not induce this active anti-tumor immune response.
After immunization of 8.B16-mBD2 cell vaccine, IFN- gamma could be promoted and increased significantly (F=506.814, P0.05), and IL-12 production was significantly increased (F=83.637, P0.05). Compared with B16 control group, the B16-p control group had a significant difference, but the content of IL-4 did not affect the specific activity of.B16-mBD2 cell vaccine to induce specific killing activity against B16 cells. Sex increased significantly (F=44.376, P0.05), and NK cell killing activity increased significantly (F=119.750, P0.05), compared with B16 control group and B16-p control group, the difference was statistically significant.
After the 9.B16-mBD2 cell vaccine was immunized with mice, the HE staining of the main organs of the mice, such as liver, kidney, spleen and lung, showed that there was no obvious pathological changes in the main organs, suggesting that the B16-mBD2 vaccine was relatively safe.
Conclusion:
After transfection of mBD2 gene to malignant melanoma B16 cells, the transgenic cell vaccine prepared by the vaccine has obvious anti murine melanoma B16 effect. After immunization, the mouse NK cells are induced by the vaccine.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R739.5

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