【摘要】：目的 觀察體外激素難治性前列腺癌細(xì)胞系冷凍消融后高遷移族蛋白B1 (high mobility group protein B1, HMGB1)表達(dá)的規(guī)律,及其與體外誘導(dǎo)異體外周血來(lái)源的樹(shù)突狀細(xì)胞(dendritic cell, DC)分化成熟的關(guān)系,初步探討HMGB1對(duì)冷凍免疫反應(yīng)的影響。材料和方法 培養(yǎng)激素難治性前列腺癌細(xì)胞系PC-3,經(jīng)冷凍消融(利用Endocare公司氬氦冷凍系統(tǒng)直徑1.7mm冷凍器)裂解細(xì)胞后收集上清液,藉酶聯(lián)免疫吸附試驗(yàn)(ELISA)方法對(duì)冷凍前后上清液中的HMGB1進(jìn)行定量分析。然后應(yīng)用westernblot檢測(cè)不同數(shù)量PC-3細(xì)胞冷凍壞死上清液中HMGB1的釋放情況。采集前列腺癌患者抗凝的新鮮外周血,應(yīng)用淋巴細(xì)胞分離液(Ficoll)用貼壁的方法分離單個(gè)核細(xì)胞(PBMC),經(jīng)重組人粒細(xì)胞-巨噬細(xì)胞集落刺激因子(rhGM-CSF)、重組人白細(xì)胞介素4(rhIL-4),體外誘導(dǎo)培養(yǎng)獲取未成熟DC(imDC)。將PC-3細(xì)胞冷凍壞死上清液直接或加入足量HMHB1(?)(?)和抗體分別與imDC混合,在體外適宜培養(yǎng)條件下進(jìn)行培養(yǎng)。倒置顯微鏡下觀察細(xì)胞形態(tài),進(jìn)一步在共聚焦顯微鏡下觀察DC骨架結(jié)構(gòu)變化。流式細(xì)胞儀測(cè)定DC表而成熟共刺激分了分了CD83、CD86、HLA-DR的表達(dá)。觀察HMGB1刺激與DC表面成熟共刺激分子CD83、CD86和HLA-DR表達(dá)的時(shí)-效關(guān)系及量-效關(guān)系。 結(jié)果 1.流式細(xì)胞術(shù)分析,冷凍消融前PC-3細(xì)胞正常、凋亡、壞死三種狀態(tài)的比例分別為(95.58±1.23)%、(2.00±1.61)%、(0.56±1.09)%,冷凍消融后PC-3正常、凋亡、壞死三種狀態(tài)的比例分別為(12.22±9.53)%、(5.46±1.14)%、(82.70±11.35)%,兩者比較差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 2. ELISA結(jié)果顯示：1×106/ml組、1×107/ml組、1×108/ml組PC-3細(xì)胞上清液中HMGB1濃度分別由冷凍前的(4.59±0.25)ng/ml、(6.04±0.13)ng/ml、(7.39±0.18)ng/ml上升至(14.28±0.84)ng/ml、(71.68±4.62)ng/ml、(329.64±32.89)ng/ml,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。western blot同樣證實(shí)：隨著PC-3細(xì)胞數(shù)量增加,冷凍壞死上清液中的HMGB1濃度也隨之增加。 3.樹(shù)突狀細(xì)胞經(jīng)冷凍壞死上清液刺激后,微絲蛋白有所增加,細(xì)胞體積增大,觸突變長(zhǎng)變粗,應(yīng)力纖維形成、胞漿溶膠增厚,細(xì)胞韌性和強(qiáng)度增加。冷凍壞死上清液中加入HMGB1中和抗體后,可見(jiàn)觸突變短、變細(xì),細(xì)胞膜周?chē)陀|突的熒光強(qiáng)度均減少,應(yīng)力纖維生成減少、胞漿溶膠變薄。 4.冷凍壞死上清液中的HMGB1誘導(dǎo)DC表面成熟共刺激分了高表達(dá)：冷凍刺激組,DC表面成熟共刺激分子CD83、CD86、HLA-DR表達(dá)較對(duì)照組上調(diào)明顯(P0.01)；抗體干預(yù)組,DC表面成熟共刺激分子CD83、CD86、HLA-DR表達(dá)較冷凍刺激組明顯下降(P0.01),但較對(duì)照組仍高(P0.05) 5.冷凍壞死上清液中的HMGB1誘導(dǎo)DC表面成熟共刺激分子表達(dá)的時(shí)-效關(guān)系：用濃度為1×106/ml的PC-3細(xì)胞冷凍壞壞死上清液分別與iDC共培養(yǎng)24h、48h和72h,DC表面成熟共刺激分子CD83、CD86和HLA-DR表達(dá)分別于24～72h明顯上調(diào)(P0.05,P0.01),其中以培養(yǎng)48h組DC表面共刺激分子表達(dá)上調(diào)最為顯著(P0.01)。 6.冷凍壞死上清液中的HMGB1誘導(dǎo)DC表面成熟共刺激分了表達(dá)的量-效關(guān)系：與空白對(duì)照組相比,1×106/ml、1×107/ml組DC表面共刺激分子CD83、CD86、HLA-DR明顯上調(diào)(P0.05,P0.01),1×108/ml組DC表面僅CD83表達(dá)明顯上調(diào)(P0.05),CD86、HLA-DR無(wú)明顯變化(P0.05)結(jié)論 冷凍消融導(dǎo)致腫瘤細(xì)胞壞死,壞死腫瘤細(xì)胞釋放HMGB1作為一種內(nèi)源性危險(xiǎn)信號(hào),可使DC細(xì)胞骨架發(fā)生變化,增強(qiáng)其游走遷移能力,促使免疫突觸形成,方便其進(jìn)入淋巴結(jié),與T細(xì)胞作用引發(fā)免疫反應(yīng),從而啟動(dòng)特異性T細(xì)胞免疫應(yīng)答。同時(shí),HMGB1在冷凍免疫反應(yīng)中具有雙重調(diào)節(jié)作用,低濃度的HMGB1可作為重要的免疫刺激信號(hào),促進(jìn)DC表面成熟共刺激分子高表達(dá),并在一定時(shí)間內(nèi)達(dá)到作用強(qiáng)度峰值；高濃度的HMGB1則使DC表面成熟共刺激分子表達(dá)受到抑制,對(duì)DC免疫功能活化起到一定的抑制作用。
[Abstract]:Purpose To observe the law of high mobility group protein B1 (HMGB1) expression after the cryoablation of the in vitro hormone-resistant prostate cancer cell line and the close-up of the differentiation of the dendritic cells (DC) from the peripheral blood source in vitro A preliminary study on the effect of HMGB1 on the frozen immune response in response to that material and Methods The cell line PC-3 of the hormone-resistant prostate cancer cell line was cultured. After the cells were lysed by cryoablation (with the diameter of 1.7 mm cryostat of the argon-helium cryosystem of Encoare Company), the supernatant was collected and the HMGB1 in the supernatant was determined by enzyme-linked immunosorbent assay (ELISA). Quantitative analysis, and then the release of HMGB1 in the supernatant of different numbers of PC-3 cells was detected by western blot. The patients with prostate cancer were collected with anticoagulated fresh peripheral blood, and the mononuclear cells (PBMC) were isolated by means of adherent method using lymphocyte separation fluid (Ficoll), and the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and the recombinant human interleukin-4 (rhIL) were obtained. -4), in vitro induction culture to obtain immature DC (im DC). The PC-3 cells are frozen and necrotic, and the supernatant is directly or added to a sufficient amount of HMH. B1( ? (?) and the antibody are mixed with the imDC, respectively, and then in the appropriate culture conditions in vitro Line culture. The morphology of the cells was observed under an inverted microscope, and the junction of the DC framework was further observed under a confocal microscope. The expression of CD83, CD86, HLA-DR was determined by flow cytometry. Expression of CD83, CD86 and HLA-DR on the expression of CD83, CD86 and HLA-DR in the mature costimulatory molecules of HMGB1 and DC surface. off-effect Results 1. The proportion of PC-3 cells, apoptosis and necrosis before and after cryoablation was (95.58% 1.23)%, (2.00% 1.61)%, (0.56% 1.09)%, and PC after cryoablation. The three states of normal, apoptotic and necrotic were (12.22, 9.53)%, (5.46, 1.14)%, (82.70, 11.35)%, respectively. (P0.05).2. The results of ELISA showed that the concentration of HMGB1 in the supernatant of PC-3 cells was increased to (14.28 to 0.84) ng/ ml, (7.39-0.18) ng/ ml, (14.28-0.84) ng/ ml, (71.68-4.62) ng/ ml, (329.64-32), respectively, by the pre-freezing (4.59-0.25) ng/ ml, (6.04-0.13) ng/ ml, (7.39-0.18) ng/ ml, respectively. (89) ng/ ml, the difference was of statistical significance (P0.01). western blot also confirmed that as the number of PC-3 cells increased, the HMG in the supernatant of the frozen necrosis 3. After the dendritic cells were stimulated by freezing and necrosis, the microfilament protein increased, the volume of the cells increased, the contact mutation was long, the stress fiber was formed, and the cytoplasmic sol increased. The thickness, the toughness and the strength of the cells were increased. After addition of HMGB1 and the antibody to the supernatant of the frozen necrosis, the visible contact mutation was short, and the fluorescence intensity of the surrounding and the contact surface of the cell membrane was reduced, and the stress fiber 4. The expression of HMGB1 in the supernatant of the frozen necrosis was significantly higher than that of the control group (P0.01). The expression of CD83, CD86 and HLA-DR in the frozen and necrotic supernatant was significantly higher than that in the control group (P0.01). The expression of CD83, CD86 and HLA-DR in the stimulated molecule decreased significantly (P0.01). The time-effect relationship of the expression of the mature costimulatory molecules of the DC surface was induced by the HMGB1 in the supernatant of the frozen necrosis: the PC-3 cells with a concentration of 1-106/ ml were used to freeze the necrosis and necrosis. The supernatant was co-cultured with iDC for 24 h,48 h and 72 h, respectively. The expression of CD83, CD86 and HLA-DR on DC surface was up-regulated at 24-72 h (P0.05, P0.01). The expression of HMGB1 in the supernatant of the frozen necrosis was significantly higher than that of the blank control group (P 0.01). Compared with the blank control group, the DC surface costimulatory molecules CD83, CD86 and HLA-DR in the 1/107/ ml group were significantly up-regulated (P0.05, P0.01), and the DC surface of the 1-(108/ ml) group was only The expression of CD83 was up-regulated (P0.05), CD86 and HL. There was no significant change in A-DR (P0.05). Conclusion The cryoablation leads to the necrosis of the tumor cells and the release of HMGB1 in the necrotic tumor cells as an endogenous risk signal. the force, which causes the formation of the immune synapse, facilitates the entry of the immune synapse into the lymph node and is initiated by the action of the T-cell At the same time, HMGB1 has a double regulation function in the freezing and immune response, and the low concentration of HMGB1 can be used as an important immunostimulating signal to promote the mature costimulatory molecules of the DC surface. High expression and the peak of action intensity within a certain period of time; high concentration of HMGB1 can inhibit the expression of the mature costimulatory molecules of the DC surface
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392;R737.25

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 龔非力;;HMGB1——一種重要的警報(bào)素[J];現(xiàn)代免疫學(xué);2009年03期

，

本文編號(hào)：2502153