摘要
目的:采用人宫颈癌细胞模型为研究对象。研究细胞因子诱导的杀伤细胞(cytokine-induced killer cells,CIK细胞)对人宫颈癌细胞及该细胞荷瘤小鼠的体内外抗肿瘤作用。分析将体外培养的CIK细胞回输体内后在不同组织器官的分布特点,为CIK细胞临床应用提供理论依据。
方法:
1 CIK细胞的制备及扩增采集健康志愿者外周血,经Ficoll密度梯度离心法收集外周血单个核细胞(Peripheral blood mononuclear cell, PBMC),按特定顺序加入IFN-γ、IL-2、抗CD3单抗和IL-1α,每3 d更换新鲜无血清培养基并补充IL-2,每7d测定细胞增殖反应,并绘制细胞生长曲线。经14 d培养后收集CIK细胞。
2 CIK细胞表型及产生细胞因子测定用流式细胞技术检测CIK细胞表型分子的表达。用半定量RT-PCR法检测体外培养0、7、14、21、28、35 d时的CIK细胞IFN-γmRNA表达变化。
3 CIK细胞的杀伤活性测定收集PBMC、体外经细胞因子诱导培养14d的CIK细胞及呈对数生长期的Hela-luc细胞,分别将CIK细胞和PBMC作为效应细胞,Hela-luc细胞作为靶细胞,以效靶比为20:1和40:1将两种细胞混合培养48 h,用MTT比色法测定CIK细胞和PBMC对Hela-luc细胞的杀伤作用。
4经瑞氏-姬姆萨染色后,用光学显微镜观察CIK细胞对Hela-luc细胞作用后的细胞形态变化。
5荷瘤小鼠模型的建立收集呈对数生长期的Hela-luc细胞,制成细胞悬液,调整细胞浓度为1×108/ml,于BALB/c裸鼠右侧胸背部皮下注射细胞悬液0.1ml,1×107/只,建立荷瘤小鼠模型。
6 CIK细胞的体内抑瘤作用向裸鼠移植肿瘤细胞7 d后,于荷瘤小鼠的瘤旁分别注射CIK细胞(实验组)或PBS(对照组),每组6只。通过精诺真体内成像系统动态观察肿瘤大小变化及治疗效果。治疗结束后收集小鼠外周血、肿瘤组织、肺、肝、脾组织。
7用ELISA法检测荷瘤小鼠外周血中IFN-γ的水平。
8经HE染色,显微镜下观察肺、肝、脾的组织病理学变化以及有无肿瘤细胞浸润。
9用羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)标记CIK细胞,经激光共聚焦显微镜观察标记情况。
10 CIK细胞在荷瘤小鼠体内的分布检测通过腹腔、瘤旁分别给荷瘤小鼠注射经CFSE标记的CIK细胞,对照组不进行任何处理。分别于注射CIK细胞后3、6、12、24 h留取外周血、肿瘤组织、肺、肝、脾。将上述各待测组织剪碎,制成单细胞悬液,一部分经流式细胞技术检测各组织中CFSE标记的CIK细胞含量;另一部分进行激光共聚焦显微镜观察肿瘤组织中CIK细胞的分布情况。
结果:
1将人外周血单个核细胞经不同细胞因子体外培养后,诱导分化的CIK细胞在培养至第7 d时开始快速增殖,14 d时达高峰,增殖倍数最高达100倍以上。
2在培养过程中,CIK细胞CD3+、CD8+、CD3+CD56+ T细胞的百分率均大幅度增加,14 d时的表达率分别为92%、42.12%、63.34%。CD3+CD56+ T细胞的百分率与最初培养当天相比(1.10%)增加57.5倍。
3体外诱导培养不同时间的CIK细胞IFN-γmRNA表达水平不断增加,在14 d时达到高峰。
4以20:1、40:1的比例将效、靶细胞作用48 h后,CIK细胞对Hela-luc细胞的杀伤率分别为(51.16±2.64)%、(72.14%±4.21)%。PBMC对Hela-luc细胞的杀伤率则分别为(16.33±3.09)%和(21.26±2.71)%。
5经瑞氏-姬姆萨染色显微镜下可见:正常靶细胞呈菱形或多边形生长,形态饱满,细胞核染色均匀;与CIK细胞共培养的靶细胞周围有多个效应细胞围绕,形成花环状,靶细胞膜疏松,胞浆中有空泡。
6注射Hela-luc细胞7 d后,在裸鼠体内全部成瘤(成瘤率达100%),成功建立了荷瘤小鼠模型。
7经CIK细胞治疗4次后,荷瘤小鼠的肿瘤明显小于对照组(p<0.05),经CIK细胞治疗的第5周、第8周,对肿瘤的总抑瘤率分别为47.18%、64.38%。
8 CIK细胞治疗组荷瘤小鼠外周血中IFN-γ水平(61.92±6.49)pg/ml明显高于对照组(34.30±1.78)pg/ml(p﹤0.05)。
9 CIK细胞治疗组和对照组荷瘤小鼠肿瘤组织无明显形态学差异;肝脏均可见完整汇管区,未见癌结节,但有大量炎细胞浸润;脾脏均可见癌结节。CIK细胞治疗组小鼠肺脏未见癌结节,但有炎细胞浸润,对照组小鼠肺脏出现大量癌结节。
10 CFSE标记的CIK细胞经腹腔注射荷瘤小鼠3 h后,可在肺、肝、脾、外周血、肿瘤中观察到绿色荧光,以肺脏的分布浓度最高(32.22%),其次是肝脏,肿瘤组织只有9.44%;注射6 h时,小鼠的肺、肝、脾脏分布浓度逐渐下降,而外周血、肿瘤组织浓度逐渐增加;注射24 h时,肿瘤组织浓度达到最高(20.56%),而肺脏浓度最低(6.73%)。
11 CFSE标记的CIK细胞经瘤旁注射荷瘤小鼠3 h后,同样可在肺、肝、脾、外周血、肿瘤中观察到绿色荧光,肺脏的分布浓度最高(36.83%),其次是肿瘤组织(25.75%);注射6 h时,外周血浓度达到最高(48.02%),其他器官浓度逐渐下降;注射24 h时,外周血浓度为23.41%,肿瘤组织为13.18%,肝、脾、肺中的浓度下降到3%以下。
结论:
1 CIK细胞在体内外对宫颈癌Hela-luc细胞均有较强的杀伤作用,在体内能明显抑制荷瘤小鼠肿瘤的生长,其作用机制可能与促进免疫细胞产生IFN-γ有关。
2将CIK细胞经不同途径注射荷瘤小鼠后,可以广泛分布于全身器官,但在各脏器的分布与输注途径的不同有关。其中腹腔注射可能适用于体腔内肿瘤及恶性积液的治疗,而瘤旁注射可能适用于体表肿瘤的治疗。
Objective: The BALB/c nude mice inoculated with cervical cancer cell line Hela-luc were used as nude mouse xenograft model. To study the anti-tumor activity of cytokine-induced killer cells (CIK cells) against Hela-luc cells in vitro and in vivo. To analyse the characteristics of the distribution of CIK cells in cervical cancer nude mouse xenograft model. This research will provide theoretical for clinical application of CIK cells.
Methods:
1 The preparation and proliferation of CIK cells. The blood samples were obtained from anonymous healthy human volunteers. Peripheral blood mononuclear cells (PBMC) were isolated using standard Ficoll density gradient centrifugation. PBMC were induced to become CIK cells in vitro at the presence of interferon-gamma (IFN-γ), interleukin-2 (IL-2), CD3 monoclonal antibody (CD3McAb) and interleukin- 1α(IL-1α), in a certain sequence. CIK cells were incubated in a culture system that was renewed every 3 days with fresh medium include IL-2. To determinate proliferation response and draw curve of CIK cells growth every 7 days. After 14 days, CIK cells were collected.
2 The phenotype and cytokine production of CIK cells were determined. Flow cytometry analysis was used to analyze the phenotype of CIK cells. The expression level of IFN-γmRNA in CIK cells was detected through semi-quantitative RT-PCR assay in 0, 7, 14, 21, 28, 35 days.
3 Killing activity of CIK cells were determined in vitro. After 14 days, CIK cells by cytokines were collected in vitro. Logarithmic phase of Hela-luc cells were collected. CIK cells and PBMC were used as effector cells, respectively. Hela-luc cells were used as target cells. CIK cells and PBMC were added at specified effector: target (E: T) ratios (20:1, 40:1) and incubated after 48 hours, respectively. Killing activity of CIK cells and PBMC against Hela-luc cells was measured by MTT assay.
4 After Wright-Giemsa’s stainning, morphological images of Hela-luc cells after CIK cells treated were observed by light microscope.
5 The nude mouse was inoculated Hela-luc cells to establish tumor model. Logarithmic phase of Hela-luc cells were collected into single-cell suspension. To adjust cell concentration 1×108/ml. Hela-luc cells were injected in the BALB/c nude mice’s right department of shoulder (0.1ml, 1×107 once) to establish nude mouse xenograft model.
6 Anti-tumor effect of CIK cells in vivo. After nude mice were injected tumor cells 7 days, the CIK cells (experiment group) or PBS (control group) was re-infused through tumor adjacent in tumor barring mice. Every group has 6 mice. Tumor changing and treatment effect were detected using in vivo Xenogen IVIS Imaging System. Peripheral blood of tumor barring mice, tumor tissue, lung, liver and spleen were collected after treatmented in two groups.
7 The level of IFN-γin peripheral blood of tumor barring mice was detected by ELISA assay.
8 After HE staining, the histopathological changed and tumor cells infiltrated of liver, lung and spleen were observed by microscope.
9 CIK cells were labled by 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE), and labled situation observed by laser scanning confocal microscope.
10 Distribution of CIK cells in nude mouse xenograft model. CIK cells labled by CFSE were injected to tumor barring mice via peritoneal cavity or tumor adjacent. Control group didn’t do any treatment. Collected peripheral blood, tumor tissue, lung, liver and spleen in three groups after injected CIK cells 3 hours, 6 hours, 12 hours, 24 hours. Above-mentioned organizations were made of single-cell suspension. A part of single-cell suspension was detected CIK cells concontration by flow cytometry. Another part of single-cell suspension was observed distribution situation of CIK cells in tumor tissue by laser scanning confocal microscope.
Results:
1 The human peripheral blood mononuclear cells were cultured by different cytokines in vitro. Induced differentiation of CIK cells grew rapidly at 7 days and grew up to the peak at 14 days. The multiplication of CIK cells was highest reached 100 times.
2 The CD3+, CD8+, CD3+CD56+ T cells’percentage were largely increased in the cells culturing process. And they expression rates may reach above 92%, 42.12%, 63.34% at 14 days, respectively. CD3+CD56+ T cells’percentage increased 57.5 times than beginning (1.10%).
3 The expression level of IFN-γmRNA in CIK cells was gradually decreased in vitro cultured times. The expression level of IFN-γmRNA in CIK cells up to maximum was 14 days in cultured times. The expression level of IFN-γmRNA in CIK cells was very low in control group.
4 At an effector-target cell ratio of 20: 1, 40: 1 after 48 hours, CIK cells showed higher cytotoxic effect for Hela-luc cells with the results are (51.16±2.64)% and (72.14%±4.21)% as measured by the MTT assay, respectively. The killing cytotoxic were (16.33±3.09)% and (21.26±2.71)% in PBMC, respectively.
5 After Wright-Giemsa stainning, microscope observed: The normal Hela-luc cells were diamond-shaped or polygons, full shape, nuclear stainning were equal. A number of effector cells arounded the target cells to forming flower ring, and the target cell membrane changed loose, and cytoplasm had many vacuole in experiment group.
6 After subcutaneous injected Hela-luc cells 7 days, the nude mice xenograft rate was 100%. Nude mice xenograft model were successful established.
7 The tumor size in experiment group were smaller than that in control group after CIK cells treated (p<0.05). The inhibitory rates of the fifth week, eighth week were 47.18% and64.38%, respectiveily.
8 The level of IFN-γin experiment group (61.92±6.49) pg/ml was higher than that in control group (34.30±1.78) pg/ml (p<0.05).
9 The histopathological changed of tumor had no obviously difference between experiment group and control group. Alveola walls were integrity and cancer nodules didn’t find in liver, but there were many inflammatory cells in two groups. Cancer nodules were found in spleen in two groups. Many cancer nodules didn’t find in lung, but there were many inflammatory cells infiltrated in experiment group. Many tumor nodules were found in control group lung.
10 Lung, liver, spleen, peripheral blood and tumor tissue were observed green fluorescence by laser scanning confocal microscope after injected CIK cells 3 hours via peritoneal cavity. The highest concentration of CIK cells was the lung (32.22%). Second was the liver. The concentration of CIK cells in tumor tissue was only 9.44%. After injected CIK cells 6 hours, the concentrations of CIK cells in lung, liver and spleen were gradually decreased; but the concentrations of CIK cells in peripheral blood and tumor tissue were gradually increased. After injected CIK cells 24 hours, the highest concentration of CIK cells was the tumor tissue (20.56%), and the lowest concentration of CIK cells was the lung (6.73%).
11 Lung, liver, spleen, peripheral blood and tumor tissue were also observed green fluorescence by laser scanning confocal microscope after injected CIK cells 3 hours via tumor adjacent. The highest concentration of CIK cells was the lung (36.83%). Second was the tumor tissue (25.75%). After injected CIK cells 6 hours, the highest concentration of CIK cells was peripheral blood (48.02%), and the concentrations of CIK cells in other organs were gradually decreased. After injected CIK cells 24 hours, the concentration of CIK cells in peripheral blood was 23.41% and the concentration of CIK cells in tumor tissue was 13.18%; the concentration of CIK cells in liver, spleen and lung dropped to below 3%.
Conclusions:
1 CIK cells were highly efficient cytolytic effector cells which have a stronger significant suppression against growth of cervical cancer’s Hela-luc cells in vivo and in vitro. The CIK cells showed potent anti-tumor activity in animal experiment,and its mechanisms was maybe relate with IFN-γ.
2 The CIK cells were extensively distributed to organs after injected via peritoneal cavity or tumor adjacent. There is relationship between distribution situation of CIK cells and infusion ways. Infusion of CIK cells via peritoneal cavity maybe suit for malignant tumor and malignant effusions in body cavity; and application by means of tumor adjacent should suit for body surface tumor.
引文
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