高迁移率族蛋白B1影响前列腺癌冷冻免疫反应的实验研究
发布时间：2019-06-19 06:02
【摘要】：目的 观察体外激素难治性前列腺癌细胞系冷冻消融后高迁移族蛋白B1 (high mobility group protein B1, HMGB1)表达的规律,及其与体外诱导异体外周血来源的树突状细胞(dendritic cell, DC)分化成熟的关系,初步探讨HMGB1对冷冻免疫反应的影响。材料和方法 培养激素难治性前列腺癌细胞系PC-3,经冷冻消融(利用Endocare公司氩氦冷冻系统直径1.7mm冷冻器)裂解细胞后收集上清液,藉酶联免疫吸附试验(ELISA)方法对冷冻前后上清液中的HMGB1进行定量分析。然后应用westernblot检测不同数量PC-3细胞冷冻坏死上清液中HMGB1的释放情况。采集前列腺癌患者抗凝的新鲜外周血,应用淋巴细胞分离液(Ficoll)用贴壁的方法分离单个核细胞(PBMC),经重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、重组人白细胞介素4(rhIL-4),体外诱导培养获取未成熟DC(imDC)。将PC-3细胞冷冻坏死上清液直接或加入足量HMHB1(?)(?)和抗体分别与imDC混合,在体外适宜培养条件下进行培养。倒置显微镜下观察细胞形态,进一步在共聚焦显微镜下观察DC骨架结构变化。流式细胞仪测定DC表而成熟共刺激分了分了CD83、CD86、HLA-DR的表达。观察HMGB1刺激与DC表面成熟共刺激分子CD83、CD86和HLA-DR表达的时-效关系及量-效关系。 结果 1.流式细胞术分析,冷冻消融前PC-3细胞正常、凋亡、坏死三种状态的比例分别为(95.58±1.23)%、(2.00±1.61)%、(0.56±1.09)%,冷冻消融后PC-3正常、凋亡、坏死三种状态的比例分别为(12.22±9.53)%、(5.46±1.14)%、(82.70±11.35)%,两者比较差异具有统计学意义(P0.05)。 2. ELISA结果显示：1×106/ml组、1×107/ml组、1×108/ml组PC-3细胞上清液中HMGB1浓度分别由冷冻前的(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,差异具有统计学意义(P0.01)。western blot同样证实：随着PC-3细胞数量增加,冷冻坏死上清液中的HMGB1浓度也随之增加。 3.树突状细胞经冷冻坏死上清液刺激后,微丝蛋白有所增加,细胞体积增大,触突变长变粗,应力纤维形成、胞浆溶胶增厚,细胞韧性和强度增加。冷冻坏死上清液中加入HMGB1中和抗体后,可见触突变短、变细,细胞膜周围和触突的荧光强度均减少,应力纤维生成减少、胞浆溶胶变薄。 4.冷冻坏死上清液中的HMGB1诱导DC表面成熟共刺激分了高表达：冷冻刺激组,DC表面成熟共刺激分子CD83、CD86、HLA-DR表达较对照组上调明显(P0.01)；抗体干预组,DC表面成熟共刺激分子CD83、CD86、HLA-DR表达较冷冻刺激组明显下降(P0.01),但较对照组仍高(P0.05) 5.冷冻坏死上清液中的HMGB1诱导DC表面成熟共刺激分子表达的时-效关系：用浓度为1×106/ml的PC-3细胞冷冻坏坏死上清液分别与iDC共培养24h、48h和72h,DC表面成熟共刺激分子CD83、CD86和HLA-DR表达分别于24～72h明显上调(P0.05,P0.01),其中以培养48h组DC表面共刺激分子表达上调最为显著(P0.01)。 6.冷冻坏死上清液中的HMGB1诱导DC表面成熟共刺激分了表达的量-效关系：与空白对照组相比,1×106/ml、1×107/ml组DC表面共刺激分子CD83、CD86、HLA-DR明显上调(P0.05,P0.01),1×108/ml组DC表面仅CD83表达明显上调(P0.05),CD86、HLA-DR无明显变化(P0.05)结论 冷冻消融导致肿瘤细胞坏死,坏死肿瘤细胞释放HMGB1作为一种内源性危险信号,可使DC细胞骨架发生变化,增强其游走迁移能力,促使免疫突触形成,方便其进入淋巴结,与T细胞作用引发免疫反应,从而启动特异性T细胞免疫应答。同时,HMGB1在冷冻免疫反应中具有双重调节作用,低浓度的HMGB1可作为重要的免疫刺激信号,促进DC表面成熟共刺激分子高表达,并在一定时间内达到作用强度峰值；高浓度的HMGB1则使DC表面成熟共刺激分子表达受到抑制,对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
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392;R737.25
本文编号：2502153
[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
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392;R737.25
【参考文献】
相关期刊论文 前1条
1 龚非力;;HMGB1——一种重要的警报素[J];现代免疫学;2009年03期
，本文编号：2502153
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