發(fā)布時(shí)間：2018-04-19 15:16

  本文選題：樹突狀細(xì)胞 + 乳腺癌�。� 參考：《泰山醫(yī)學(xué)院》2010年碩士論文

【摘要】：背景 乳腺癌是女性最常見的惡性腫瘤,且發(fā)病率呈逐年上升趨勢,當(dāng)前,乳腺癌的治療主要治療手段包括手術(shù)治療、化療、放療及內(nèi)分泌治療,即便如此,術(shù)后仍有很高的復(fù)發(fā)率,預(yù)后仍不理想。近年來,生物治療逐漸受到人們重視,人們開始利用制備腫瘤疫苗的方法治療惡性腫瘤。腫瘤疫苗因其以較低的抗原刺激就能激發(fā)強(qiáng)烈的免疫反應(yīng)、腫瘤特異性、自體耐受和安全性以及長時(shí)間的免疫記憶能避免腫瘤復(fù)發(fā)等優(yōu)點(diǎn)被越來越多的用來治療各種惡性腫瘤,并且取得了良好的效果。同樣,以樹突狀細(xì)胞(dendritic cells,DC)為基礎(chǔ)構(gòu)建的乳腺癌治療疫苗也成為乳腺癌生物治療的研究熱點(diǎn)；其中,直接利用裂解的腫瘤細(xì)胞抗原負(fù)載樹突狀細(xì)胞制備的腫瘤疫苗因腫瘤抗原易于制備和療效確切,被廣泛應(yīng)用；人們利用裂解腫瘤細(xì)胞抗原負(fù)載樹突狀細(xì)胞制備的腫瘤疫苗治療非霍奇金淋巴瘤、骨髓瘤、惡性黑色素瘤、非小細(xì)胞肺癌、前列腺癌、結(jié)腸直腸癌等,在臨床試驗(yàn)中均取得了一定效果,但是,因?yàn)槎鄶?shù)腫瘤的免疫原性很弱,使得樹突狀細(xì)胞不能正常呈遞抗原以有效激活特異性細(xì)胞毒T淋巴細(xì)胞(cytotoxic T cell s,CTL),從而限制了樹突狀細(xì)胞腫瘤疫苗的療效不能進(jìn)一步提高,為了提高腫瘤的免疫原性,提高樹突狀細(xì)胞腫瘤疫苗的效價(jià),人們想到了在抗原中加入佐劑(如熱休克蛋白、綠膿桿菌菌毛等)的方法,并且已經(jīng)在體外實(shí)驗(yàn)中取得了可喜的成果。熱休克蛋白(HSPs)又稱分子伴侶,是機(jī)體細(xì)胞受到各種應(yīng)急刺激時(shí)產(chǎn)生的一類保護(hù)性蛋白,參與有關(guān)蛋白的折疊、裝配、細(xì)胞內(nèi)運(yùn)輸及蛋白質(zhì)降解等過程,調(diào)節(jié)這些蛋白的活性和功能,具有免疫佐劑和激活DC的功能。本研究我們分別采用熱休克法和凍融法制備小鼠乳腺癌樹突狀細(xì)胞腫瘤疫苗,并對采用不同方法制備的腫瘤疫苗治療小鼠乳腺癌進(jìn)行療效對比。目的 1.研究利用粒-巨噬細(xì)胞集落刺激因子(Granulocyte-Macrophage Clone stimulating factor, GM-CSF)、白細(xì)胞介素-4(Interleukin-4,IL-4)和腫瘤壞死因子-a(Tumor necrosis factor-a, TNF-a)聯(lián)合培養(yǎng)小鼠骨髓來源的成熟樹突狀細(xì)胞。 2.分別利用熱休克和凍融法處理小鼠EMT6乳腺癌細(xì)胞,研究所獲得的乳腺癌細(xì)胞裂解物當(dāng)中熱休克蛋白-70(Hot Shock Protein-70,HSP70)的表達(dá)情況。 3.將獲得全細(xì)胞抗原與培養(yǎng)的樹突狀細(xì)胞混合培養(yǎng),制備小鼠樹突狀細(xì)胞乳腺癌腫瘤疫苗。分別利用以上制備的不同的小鼠乳腺癌樹突狀細(xì)胞腫瘤疫苗治療小鼠移植瘤,對比熱休克法和凍融法制備的小鼠乳腺癌樹突狀細(xì)胞腫瘤疫苗對荷瘤小鼠的療效。 4.21天時(shí)處死小鼠,稱量小鼠腫瘤體積及質(zhì)量,并取血比較不同治療組小鼠血清中細(xì)胞因子(cytokine)干擾素-γ(Interferon-γ,INF-γ)、白細(xì)胞介素-2(Interleukin-2,IL-2)和白細(xì)胞介素-12(P70)(Interleukin-12,IL-12(P70))的水平。 方法 1.定期通過光學(xué)顯微鏡觀察培養(yǎng)的樹突狀細(xì)胞的形態(tài),利用流式細(xì)胞儀對樹突狀細(xì)胞的表型進(jìn)行分析。 2.用免疫組織化學(xué)法(immunohistochemical method)檢測不同方法制備的小鼠乳腺癌腫瘤細(xì)胞中HSP70的表達(dá)量的不同。 3.建立小鼠乳腺癌動物模型并分組,應(yīng)用不同的樹突狀細(xì)胞腫瘤疫苗進(jìn)行治療。 4.至21天,,處死小鼠,稱量小鼠腫瘤體積和質(zhì)量,并取血利用酶聯(lián)免疫吸附測定(enzyme linked immunosorbent assay,ELISA)法測定各組小鼠血清中細(xì)胞因子INF-γ、IL-2、IL-12(P70)的水平。 結(jié)果 1.通過對骨髓源性樹突狀細(xì)胞形態(tài)的觀察及流式細(xì)胞儀(flow cytometry)對細(xì)胞表面表型分析,利用GM-CSF、IL-4和TNF-a聯(lián)合培養(yǎng)的小鼠骨髓來源的樹突狀細(xì)胞為典型的成熟樹突狀細(xì)胞。 2.經(jīng)免疫組織化學(xué)法檢測,熱休克法制備的小鼠EMT6乳腺癌細(xì)胞裂解物中HSP70的表達(dá)量遠(yuǎn)遠(yuǎn)大于凍融法制備的小鼠EMT6乳腺癌細(xì)胞裂解物中HSP70的表達(dá)量。 3.與凍融法疫苗治療組小鼠腫瘤相比較,熱休克法疫苗治療組小鼠的腫瘤質(zhì)量及體積均受到明顯抑制(p0.05)。 4.用ELISA法測得的熱休克法疫苗治療組小鼠體內(nèi)INF-γ、IL-2和IL-12(P70)的水平均顯著高于凍融法疫苗治療組(p0.05)和空白對照組(p0.05)。 結(jié)論 由于熱休克蛋白的免疫佐劑作用,熱休克法制備的DC腫瘤疫苗較凍融法制備的DC腫瘤疫苗能在小鼠體內(nèi)激發(fā)更強(qiáng)的免疫反應(yīng),對小鼠乳腺癌有更好的治療效果。3
[Abstract]:background
Breast cancer is the most common malignant tumor in women, and the incidence of the disease is increasing year by year. At present, the main treatment methods of breast cancer are surgical treatment, chemotherapy, radiotherapy and endocrine therapy. Even so, there are still high recurrence rates and poor prognosis after operation. In recent years, biological therapy has gradually been paid attention to, and people have begun to use it. The tumor vaccine is used to treat malignant tumor. The tumor vaccine can stimulate strong immune response with lower antigen stimulation, tumor specificity, self tolerance and safety, and long time immune memory can avoid tumor recurrence. It has been used more and more to treat various malignant tumors and has achieved good effect. Also, the treatment vaccine of breast cancer, based on dendritic cells (DC), has also become a research hotspot in the biological treatment of breast cancer. The tumor cell antigen loaded dendritic cells are used to treat non Hodgkin's lymphoma, myeloma, malignant melanoma, non small cell lung cancer, prostate cancer, colorectal cancer and other clinical trials. However, because the immunogenicity of most of the tumor is weak, the dendritic cells can not be normal. In order to effectively activate the specific cytotoxic T lymphocyte (cytotoxic T cell s, CTL), the efficacy of the dendritic cell tumor vaccine can not be further improved. In order to improve the immunogenicity of the tumor and increase the titer of the dendritic cell tumor vaccine, an adjuvant (such as heat shock protein, green pus) is thought to be added to the antigen. HSPs, also known as molecular chaperone, is a kind of protective protein produced by various emergency stimuli, and participates in the process of protein folding, assembly, intracellular transport and protein degradation, and regulates the activity of these proteins. In this study, we used heat shock and freeze-thaw method to prepare mouse breast cancer dendritic cell tumor vaccine, and to compare the effect of tumor vaccine prepared by different methods in the treatment of breast cancer in mice.
1. the study used Granulocyte-Macrophage Clone stimulating factor (GM-CSF), interleukin -4 (Interleukin-4, IL-4) and tumor necrosis factor -a (Tumor necrosis factor-A) to cultivate the mature dendritic cells from the bone marrow of mice.
2. the mice EMT6 breast cancer cells were treated with heat shock and freeze-thaw respectively. The expression of heat shock protein -70 (Hot Shock Protein-70, HSP70) in the lysates of breast cancer cells was studied.
3. the murine dendritic cell breast cancer vaccine was prepared by mixed culture of total cell antigen and cultured dendritic cells. The mouse breast cancer dendritic cell tumor vaccine was used to treat mice transplanted tumor, and the mouse breast cancer dendritic cell tumor vaccine was compared with the heat shock method and the freeze thawing method. The effect of tumor bearing mice.
Mice were killed at 4.21 days, and the tumor volume and quality of mice were weighed and the serum levels of cytokines (cytokine) interferon gamma (Interferon- gamma, INF- gamma), interleukin -2 (Interleukin-2, IL-2) and interleukin -12 (P70) (Interleukin-12, IL-12 (P70)) were compared in the serum of different treatment groups.
Method
1. the morphology of cultured dendritic cells was observed by optical microscope regularly, and the phenotype of dendritic cells was analyzed by flow cytometry.
2. immunohistochemical method (immunohistochemical method) was used to detect the expression of HSP70 in mouse breast cancer cells prepared by different methods.
3. establish mouse mammary cancer animal models and divide them into different dendritic cell tumor vaccines.
4. to 21 days, mice were killed, the volume and quality of the mice were weighed, and the serum levels of cytokine INF- gamma, IL-2, IL-12 (P70) were measured by enzyme linked immunosorbent assay (ELISA).
Result
1. through the observation of the morphology of bone marrow derived dendritic cells and the analysis of cell surface phenotypes by flow cytometry (flow cytometry), the dendritic cells from bone marrow derived from GM-CSF, IL-4 and TNF-a were typical mature dendritic cells.
2. the expression of HSP70 in EMT6 breast cancer cell lysates prepared by heat shock method was far greater than the expression of HSP70 in EMT6 breast cancer cell lysates prepared by freezing and thawing method.
3. compared with the frozen thawed vaccine treatment group, the tumor mass and volume of the mice in the heat shock vaccine treatment group were significantly inhibited (P0.05).
4. the levels of INF- gamma, IL-2 and IL-12 (P70) in the mice treated with ELISA method were significantly higher than those of the freeze-thaw vaccine treatment group (P0.05) and the blank control group (P0.05).
conclusion
Due to the immune adjuvant of heat shock protein, DC tumor vaccine prepared by heat shock method can stimulate more immune response in mice than DC tumor vaccine prepared by freeze-thaw method, and has a better therapeutic effect on mouse breast cancer,.3

【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R737.9

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