Inhibiting colorectal cancer liver metastasis by combined injection of allogeneic bone marrow cells and lymphocytes

doi:10.3969/j.issn.2095-4344.0963

Abstract

Abstract:

BACKGROUND: Clinical and experimental studies on prevention and treatment of hepatic metastasis after colonic cancer treatment by allogeneic hematopoietic stem cell transplantation have yet to be well reported. A mixed chimera is expected to achieve certain effects in patients with low-tumor burden after colorectal cancer resection.
OBJECTIVE: To investigate the inhibitory effect of allogeneic hematopoietic stem cell transplantation on colorectal cancer liver metastasis as well as the effect on chimera level and graft-versus-host disease and to study the anti-tumor mechanism.
METHODS: CB6F1 mice were hybridized by BALB/c×C57BL/6 mice and injected with CT-26 cells to make animal models of colorectal cancer liver metastasis. Then, model mice were randomized into four groups (n=8 per group): control group, cyclophosphamide group, HLA-identical spleen cells plus bone marrow cell transplantation+cyclophosphamide group (HLA-identical transplantation group), and haploidentical spleen cells plus bone marrow cell transplantation+cyclophosphamide group (haploidentical transplantation group). Mixture of spleen cells and bone marrow cells from CB6F1 and C57BL/6 mice were injected via the tail vein followed by a non-myeloablative pretreatment with intraperitoneal injection of cyclophosphamide, and thereafter, lymphocytes were given via infusion. Survival time, tumor liver metastasis and the occurrence of graft-versus-host disease were observed in mice. The chimera was analyzed by flow cytometry, and levels of plasma interleukin-2, interferon-γ, interleukin-4, and transforming growth factor β were determined by ELISA
RESULTS AND CONCLUSION: The survival time and inhibition rate of liver metastasis in the haploidentical transplantation group were significantly prolonged and increased compared with cyclophosphamid and HLA-identical transplantation group, respectively. At 7 days after transplantation, the chimera level in the haploidentical transplantation group was significantly increased up to over 99% at 28 days of transplantation, indicating that donor cells were basically substituted by recipient cells. Significantly enhanced anti-tumor effect was found in the haploidentical transplantation group, while there were no signs of severe graft-versus-host disease. Moreover, the levels of plasma interleukin-2 and interferon-γ in mice were significantly higher than those in the other groups, and the level of transforming growth factor β was significantly reduced. However, there was no significant difference in the interleukin 4 level. To conclude, co-transplantation of cyclophosphamide-pretreated allogeneic bone marrow cells and lymphocytes significantly increases the chimera level, accompanied by the production of obvious anti-tumor effect of the graft that has an inhibitory effect on colorectal cancer liver metastasis. Changes in levels of plasma interleukin-2, interferon-γ, interleukin-4, and transforming growth factor β are associated with tumor growth inhibition.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Colonic Neoplasms, Neoplasm Metastasis, Liver, Bone Marrow Transplantation, Lymphocytes, Chimera, Graft vs Host Disease, Tissue Engineering

CLC Number:

R394.2

Qu Yuan-qian, Zhou Qing, Zhang Jin, Xu San-rong. Inhibiting colorectal cancer liver metastasis by combined injection of allogeneic bone marrow cells and lymphocytes[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 4053-4058.

Figures/Tables 2

2.1 生存时间对照组中位生存期31.5 d，环磷酰胺组中位生存期51.5 d，全相合移植组中位生存期52 d，半相合移植组生存期均> 90 d。半相合移植组生存期较其他3组差异有显著性意义(P < 0.05)。生存时间比较见图1。 2.2 移植物抗宿主病的观察 2.2.1 急性移植物抗宿主病评分移植后第7，14天全相合移植组急性移植物抗宿主病评分明显低于半相合移植组，且差异有显著性意义(P < 0.05)，第28天全相合移植组急性移植物抗宿主病评分与半相合移植组相比差别减小，差异无显著性意义(P > 0.05)，见表2。 2.2.2 肝脏、脾脏、肠道及皮肤的移植物抗宿主病病理表现建模后30 d，病理观察结果显示：半相合移植组小鼠的肝细胞肿胀变性，伴有片状灶性坏死、肝血窦扩张，并伴炎性细胞浸润；脾脏可见淤血性改变；小肠部分腺体及局部黏膜上皮可见坏死；皮肤组织基底细胞层空泡变性，可见散在嗜酸性坏死细胞，周围有淋巴细胞浸润符合移植物抗宿主病病理改变，见图2。 2.3 各组小鼠肝转移抑制率、肝转移灶数目分级、抑瘤率结肠癌肝转移模型建立后第30天时探查各组小鼠肝脏，对照组肝转移率达100.0%，环磷酰胺组、全相合移植组、半相合移植组肝转移率分别为 87.5%，87.5%及0。结果提示：半相合移植组小鼠的肝转移灶数目明显少于其他3组，且差异具有显著性意义(P < 0.05)，肝转移抑制率及抑瘤率明显高于环磷酰胺组、全相合移植组(P < 0.05)。环磷酰胺组和全相合移植组比较差异无显著性意义(P > 0.05)，见表3。 2.4 小鼠外周血嵌合率半相合移植组小鼠在移植后3 d，仍以自体H-2Kd/b双阳性细胞为主，移植后7 d，供鼠H-2Kb单阳性细胞嵌合率增加，达23.6%；移植后14 d，供鼠细胞嵌合率进一步增加，达到82.3%；移植后28 d，供鼠细胞嵌合率达到99.3%，提示基本取代受鼠细胞(H-2Kd/b双阳性细胞)(图3)。 2.5 外周血白细胞介素2、γ-干扰素、白细胞介素4、转化生长因子β水平见图4。 2.5.1 各组血浆白细胞介素2水平对照组为(50.500±10.184) ng/L，环磷酰胺组为(40.625±11.612) ng/L，全相合移植组为(58.750±6.018) ng/L，半相合移植组为(95.750±10.634) ng/L，半相合移植组白细胞介素2的分泌量明显高于其他3组，且差异有显著性意义(P < 0.05)。 2.5.2 各组血浆γ-干扰素水平对照组为(94.125±12.597) ng/L，环磷酰胺组为(88.250±12.725) ng/L，全相合移植组为(103.125±18.419) ng/L，半相合移植组为(185.625±25.612) ng/L，半相合移植组γ-干扰素的分泌量明显高于其他3组，且差异有显著性意义(P < 0.05)。 2.5.3 各组血浆白细胞介素4水平对照组为(9.063±0.275) ng/L，环磷酰胺组为(10.087±0.340) ng/L，全相合移植组为(10.337±0.319) ng/L，半相合移植组为(11.137±0.377) ng/L，各组白细胞介素4的分泌量差异无显著性意义(P > 0.05)。 2.5.4 各组血浆转化生长因子β水平对照组为(10.900±0.373) ng/L，环磷酰胺组为(9.762±0.375) ng/L，全相合移植组为(9.563±0.222) ng/L，半相合移植组为(5.688±0.153) ng/L，半相合移植组转化生长因子β的分泌量明显低于其他3组，且差异有显著性意义(P < 0.05)。"

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