联合重组纤维连接蛋白活化异基因杀伤细胞过继治疗肾癌的实验研究
摘要
目的转移性肾癌的一线治疗包括细胞因子治疗和靶向治疗,过继细胞免疫治疗对一线治疗失败的患者仍有一定疗效,可作为晚期肾癌的挽救性治疗。获得足够的杀伤细胞是过继细胞免疫治疗取得疗效的基本条件,使用重组人纤维连接蛋白(RetroNectin)联合抗人CD3单克隆抗体(CD3mAb)和白细胞介素-2(Interleukin-2,IL-2),在体外培养外周血单个核细胞(Peripheral blood monocytes,PBMC)可获得大量活化的杀伤细胞(RetroNectin activated killer,RAK)。本实验将研究健康人来源的RAK细胞在体外增殖和活化的能力,以及对裸鼠体内人肾肿瘤生长的抑制作用和对人肾癌细胞体外的杀伤作用,为申请开展异基因RAK细胞(Allogeneic-RAK,Allo-RAK)治疗晚期肾癌的临床试验积累资料。
方法①分离健康人PBMC以RetroNectin、CD3mAb和IL-2体外培养产生RAK细胞,以CD3mAb和IL-2培养产生CD3单克隆抗体活化的杀伤细胞(Anti-CD3-activated killer,CD3AK),台盼兰染色计数比较RAK细胞与CD3AK细胞的增殖倍数;观察RAK细胞的活化形态;流式细胞仪测定RAK细胞培养前后细胞表面标志CD3、CD4、CD8、CD25、CD16、CD56等的变化。②同时培养健康人及肾癌患者外周血来源的RAK细胞,均用台盼兰染色计数细胞增殖倍数、流式细胞仪测定细胞表面标志CD3、CD4、CD8、CD25的变化、酶联免疫吸附法(Enzyme-linked Immunosorbent Assay,ELISA)检测培养上清液中干扰素-γ(Interferon gamma,IFN-γ)及肿瘤坏死因子-α(Tumor necrosis factor,TNF-α)的浓度。③用PCR法选择人白细胞抗原A2(Human leucocyte antigens,HLA-A2)阳性的人肾癌细胞系RCC-LSL,将皮下接种RCC-LSL的裸鼠分为4组,分别接受高剂量Allo-RAK(1×10~8个细胞/天)、低剂量Allo-RAK(5×10~7个细胞/天)、IL-2和生理盐水治疗,连续6天,测量各组裸鼠实体瘤体积的变化及荷瘤35天时的瘤重。④用PCR法选择HLA-A2阳性的健康志愿者及肾透明细胞癌患者各2例,将取自2例肾癌患者的肾肿瘤进行肾癌原代细胞培养;采集健康志愿者和肾癌患者外周血分别培养RAK细胞,体外杀伤HLA-A2阳性的肾透明细胞癌细胞系RCC-LSL、RCC-FTL以及2株肾癌原代细胞,乳酸脱氢酶法定量检测RAK细胞体外杀伤肾癌细胞的能力。
结果①RAK培养第14天时细胞呈集落样活化状态,形成以细胞毒性T细胞(CD_3~+CD_8~+)为主(占67%)的细胞群:CD_3~+CD_(25)~+细胞明显增多、CD_(25)~+(IL—2受体)表达上调;RAK细胞的扩增倍数明显高于CD3AK细胞,二者培养第14天时分别扩增423倍及50倍(p＜0.05)。②健康人与肾癌患者来源的RAK细胞培养第14天时,扩增倍数分别为471倍及318倍,CD_3~+CD_8~+T细胞的比例分别为66.68%及55.45%,差异均有统计学意义(p＜0.05);培养上清液中的IFN-γ及TNF-α浓度无显著差异(p分别为0.32及0.40)。③高剂量和低剂量异基因RAK治疗组肿瘤平均体积的增长均低于IL-2对照组和生理盐水对照组;荷瘤35天时高剂量Allo-RAK治疗组裸鼠平均瘤重(0.333±0.263克)明显低于低剂量Allo-RAK治疗组(1.354±0.193克)、IL-2治疗组(1.113±0.254克)及生理盐水治疗组(1.100±0.560克)(p值分别为0.040、0.026及0.048),高剂量Allo-RAK治疗组的抑瘤率为69.7%;低剂量Allo-RAK治疗组瘤重也低于IL-2治疗组及生理盐水治疗组,但差异未达到统计学显著性(p值分别为0.413及0.253);④Allo-RAK对人肾透明细胞癌细胞系RCC-LSL、RCC-FTL及2株肾癌原代细胞具有明显的杀伤作用,效靶比为37.5∶1、75∶1及150∶1时,杀伤肾癌细胞的比率分别为6.80%～71.69%、17.25%～79.98%及27.92%～83.48%。
结论联合重组人纤维连接蛋白体外培养淋巴细胞能显著提高细胞的扩增倍数,获得大量的杀伤细胞;用健康人外周血培养的RAK细胞体外扩增倍数及效应细胞的比例均高于肾癌患者外周血培养的RAK细胞;健康人来源的异基因RAK细胞能在动物体内起到抑制肾肿瘤生长的作用,并在可体外杀伤肾癌细胞。本研究提示异基因RAK治疗方案可能成为治疗转移性肾癌的有效手段。
Obejeetive Cytokines and target therapy are the first line treatments for metastatic renal cell carcinoma(mRCC).Adoptive immunotherapy is effective for mRCC patients with first line treatment failure and could used as salvage regimen.Infusion of a large amount of killer cells is critical factor for achieving curative effect of adoptive immunotherapy.The quantity of killer cells could be significantly increased cultured form peripheral blood monocytes(PBMC) with combining RetroNectin,CD3mAb and Interleukin-2(IL-2)(RetroNectin activated killer,RAK) ex vivo.In this experiment, RAKs cultured form perpheral blood of healthy donors would be investigated for its expansion,activation status,anticancer effect on human renal tumor in nude mice in vivo and cytotoxicity effect on RCC cells in vitro.The data would be accumulated for applying licens of treatment for mRCC with allogenic RAK(Allo-RAK).
Methods①RAK cells and CD3AK(anti-CD3-activated killer) were cultured form perpheral blood monocytes of(PBMC) stimulated by RetroNectin,CD3mAb and IL-2 and by CD3mAb and IL-2 respectively.RAK and CD3AK cell numbers and expansion folds were countered by Typan Blue.Surface markers of RAKs of CD3,CD4, CDS,CD25,CD16 and CD56 before and after culture were examined with fluorescent antibody by flow cytometry.②RAKs were cultured form perpheral blood monocytes of healthy donor and RCC patients stimulated by RetroNectin,CD3mAb and IL-2.The expansion folds was observed and surface markers of RAKs were examined with fluorescent antibody of CD3,CD4,CD8 and CD25 by flow cytometry.Interferon gamma (IFN-γ)and tumor necrosis factor(TNF-α) in RAK culture supernatant were measured by enzyme-linked immunosorbent assay kits.③HLA-A2 positive human renal cell line RCC-LSL was selected by PCR.Nude mice inoculated with RCC-LSL subcutaneously were divided into four groups and treated with high dose AIIo-RAK(1×10~8 cells per day), low dose Allo-RAK(5×10~7 cells per day),IL-2 and saline for six consecutive days.The tumor sizes were measured continuously and tumors were weighted on 35th day after inoculation.④Two healthy donors and two renal cell carcinoma patients of HLA-A2 positive were selected by PCR.Two strains of primary renal cancer cells were cultured form the selected patients.Cytotoxicity effect of RAKs cultured form selected candidates on HLA-A2 positive RCC cell lines RCC-LSL and RCC-FTL and on two strains of primary renal cancer cells was quantitatively analyzed by LDH assay in vitro.
Results①RAK cells formed colonies in which sixty-seven percent were CD_3~+CD_8~+T cells cultured after 14 days.CD_3~+CD_(25)~+ cells and expression of CD_(25)~+ increased significantly in RAKs.The expansion folds of RAKs(423 times) cultured at 14 days was higher than that of CD3AK cells(50 times)(p＜0.05).②The expansion folds were 471 and 318 and the proportion of CD_3~+CD_8~+ were 66.68%and 55.45% respectively of RAKs cultured form healthy donors and RCC patients cultured for 14 days.The difference were statistically significant(p＜0.05).No difference between two groups of IFN-γand TNF-αin culture supematant(p value was 0.32 and 0.40 respectively).③The increase of average sizes of tmors of both high and low dose AIIo-RAK group was lower than that of IL-2 group and saline group.The average tumor weights of high dose Allo-RAK group(0.333±0.263 g) was significantly lower than that of low dose AIIo-RAK group(1.354±0.193 g),IL-2 group(1.113±0.254 g) and saline group(1.100±0.560 g)(p value was 0.040,0.026 and 0.048 respectively).The tumor inhibition rate of high dose Allo-RAK group was 69.7%.The average tumor weight of low dose Allo-RAK group was also lower than that of IL-2 group and saline group,but there was no significant difference(p value was 0.413 and 0.253 respectively).④Allo-RAKs had significant cytotoxicity effects on both RCC cell lines RCC-LSL and RCC-FTL and on two strains of primary renal cancer cells.The lysis rates of RCC cells were 6.80%～71.69%,17.25%～79.98%and 27.92%～83.48%as the effector target ratio were 37.5:1,75:1 and 150:1.
Conclusions The quantity of effector cells could be significantly increased cultured form PBMC with combining RetroNectin with CD3mAb and IL-2 ex vivo.The expansion ability and proportion of effector cells of RAKs form healthy donors were higher than that from RCC patients.The AIIo-RAKs were showed cytotoxicity effect on RCC cells in vitro;Allo-RAK form healthy donors had the function of killing human RCC cells and inhibiting tumor growth in tumor beating nude mice in vivo.This study suggests allo-RAK might be an effective measure for mRCC patients.
方法①分离健康人PBMC以RetroNectin、CD3mAb和IL-2体外培养产生RAK细胞,以CD3mAb和IL-2培养产生CD3单克隆抗体活化的杀伤细胞(Anti-CD3-activated killer,CD3AK),台盼兰染色计数比较RAK细胞与CD3AK细胞的增殖倍数;观察RAK细胞的活化形态;流式细胞仪测定RAK细胞培养前后细胞表面标志CD3、CD4、CD8、CD25、CD16、CD56等的变化。②同时培养健康人及肾癌患者外周血来源的RAK细胞,均用台盼兰染色计数细胞增殖倍数、流式细胞仪测定细胞表面标志CD3、CD4、CD8、CD25的变化、酶联免疫吸附法(Enzyme-linked Immunosorbent Assay,ELISA)检测培养上清液中干扰素-γ(Interferon gamma,IFN-γ)及肿瘤坏死因子-α(Tumor necrosis factor,TNF-α)的浓度。③用PCR法选择人白细胞抗原A2(Human leucocyte antigens,HLA-A2)阳性的人肾癌细胞系RCC-LSL,将皮下接种RCC-LSL的裸鼠分为4组,分别接受高剂量Allo-RAK(1×10~8个细胞/天)、低剂量Allo-RAK(5×10~7个细胞/天)、IL-2和生理盐水治疗,连续6天,测量各组裸鼠实体瘤体积的变化及荷瘤35天时的瘤重。④用PCR法选择HLA-A2阳性的健康志愿者及肾透明细胞癌患者各2例,将取自2例肾癌患者的肾肿瘤进行肾癌原代细胞培养;采集健康志愿者和肾癌患者外周血分别培养RAK细胞,体外杀伤HLA-A2阳性的肾透明细胞癌细胞系RCC-LSL、RCC-FTL以及2株肾癌原代细胞,乳酸脱氢酶法定量检测RAK细胞体外杀伤肾癌细胞的能力。
结果①RAK培养第14天时细胞呈集落样活化状态,形成以细胞毒性T细胞(CD_3~+CD_8~+)为主(占67%)的细胞群:CD_3~+CD_(25)~+细胞明显增多、CD_(25)~+(IL—2受体)表达上调;RAK细胞的扩增倍数明显高于CD3AK细胞,二者培养第14天时分别扩增423倍及50倍(p＜0.05)。②健康人与肾癌患者来源的RAK细胞培养第14天时,扩增倍数分别为471倍及318倍,CD_3~+CD_8~+T细胞的比例分别为66.68%及55.45%,差异均有统计学意义(p＜0.05);培养上清液中的IFN-γ及TNF-α浓度无显著差异(p分别为0.32及0.40)。③高剂量和低剂量异基因RAK治疗组肿瘤平均体积的增长均低于IL-2对照组和生理盐水对照组;荷瘤35天时高剂量Allo-RAK治疗组裸鼠平均瘤重(0.333±0.263克)明显低于低剂量Allo-RAK治疗组(1.354±0.193克)、IL-2治疗组(1.113±0.254克)及生理盐水治疗组(1.100±0.560克)(p值分别为0.040、0.026及0.048),高剂量Allo-RAK治疗组的抑瘤率为69.7%;低剂量Allo-RAK治疗组瘤重也低于IL-2治疗组及生理盐水治疗组,但差异未达到统计学显著性(p值分别为0.413及0.253);④Allo-RAK对人肾透明细胞癌细胞系RCC-LSL、RCC-FTL及2株肾癌原代细胞具有明显的杀伤作用,效靶比为37.5∶1、75∶1及150∶1时,杀伤肾癌细胞的比率分别为6.80%～71.69%、17.25%～79.98%及27.92%～83.48%。
结论联合重组人纤维连接蛋白体外培养淋巴细胞能显著提高细胞的扩增倍数,获得大量的杀伤细胞;用健康人外周血培养的RAK细胞体外扩增倍数及效应细胞的比例均高于肾癌患者外周血培养的RAK细胞;健康人来源的异基因RAK细胞能在动物体内起到抑制肾肿瘤生长的作用,并在可体外杀伤肾癌细胞。本研究提示异基因RAK治疗方案可能成为治疗转移性肾癌的有效手段。
Obejeetive Cytokines and target therapy are the first line treatments for metastatic renal cell carcinoma(mRCC).Adoptive immunotherapy is effective for mRCC patients with first line treatment failure and could used as salvage regimen.Infusion of a large amount of killer cells is critical factor for achieving curative effect of adoptive immunotherapy.The quantity of killer cells could be significantly increased cultured form peripheral blood monocytes(PBMC) with combining RetroNectin,CD3mAb and Interleukin-2(IL-2)(RetroNectin activated killer,RAK) ex vivo.In this experiment, RAKs cultured form perpheral blood of healthy donors would be investigated for its expansion,activation status,anticancer effect on human renal tumor in nude mice in vivo and cytotoxicity effect on RCC cells in vitro.The data would be accumulated for applying licens of treatment for mRCC with allogenic RAK(Allo-RAK).
Methods①RAK cells and CD3AK(anti-CD3-activated killer) were cultured form perpheral blood monocytes of(PBMC) stimulated by RetroNectin,CD3mAb and IL-2 and by CD3mAb and IL-2 respectively.RAK and CD3AK cell numbers and expansion folds were countered by Typan Blue.Surface markers of RAKs of CD3,CD4, CDS,CD25,CD16 and CD56 before and after culture were examined with fluorescent antibody by flow cytometry.②RAKs were cultured form perpheral blood monocytes of healthy donor and RCC patients stimulated by RetroNectin,CD3mAb and IL-2.The expansion folds was observed and surface markers of RAKs were examined with fluorescent antibody of CD3,CD4,CD8 and CD25 by flow cytometry.Interferon gamma (IFN-γ)and tumor necrosis factor(TNF-α) in RAK culture supernatant were measured by enzyme-linked immunosorbent assay kits.③HLA-A2 positive human renal cell line RCC-LSL was selected by PCR.Nude mice inoculated with RCC-LSL subcutaneously were divided into four groups and treated with high dose AIIo-RAK(1×10~8 cells per day), low dose Allo-RAK(5×10~7 cells per day),IL-2 and saline for six consecutive days.The tumor sizes were measured continuously and tumors were weighted on 35th day after inoculation.④Two healthy donors and two renal cell carcinoma patients of HLA-A2 positive were selected by PCR.Two strains of primary renal cancer cells were cultured form the selected patients.Cytotoxicity effect of RAKs cultured form selected candidates on HLA-A2 positive RCC cell lines RCC-LSL and RCC-FTL and on two strains of primary renal cancer cells was quantitatively analyzed by LDH assay in vitro.
Results①RAK cells formed colonies in which sixty-seven percent were CD_3~+CD_8~+T cells cultured after 14 days.CD_3~+CD_(25)~+ cells and expression of CD_(25)~+ increased significantly in RAKs.The expansion folds of RAKs(423 times) cultured at 14 days was higher than that of CD3AK cells(50 times)(p＜0.05).②The expansion folds were 471 and 318 and the proportion of CD_3~+CD_8~+ were 66.68%and 55.45% respectively of RAKs cultured form healthy donors and RCC patients cultured for 14 days.The difference were statistically significant(p＜0.05).No difference between two groups of IFN-γand TNF-αin culture supematant(p value was 0.32 and 0.40 respectively).③The increase of average sizes of tmors of both high and low dose AIIo-RAK group was lower than that of IL-2 group and saline group.The average tumor weights of high dose Allo-RAK group(0.333±0.263 g) was significantly lower than that of low dose AIIo-RAK group(1.354±0.193 g),IL-2 group(1.113±0.254 g) and saline group(1.100±0.560 g)(p value was 0.040,0.026 and 0.048 respectively).The tumor inhibition rate of high dose Allo-RAK group was 69.7%.The average tumor weight of low dose Allo-RAK group was also lower than that of IL-2 group and saline group,but there was no significant difference(p value was 0.413 and 0.253 respectively).④Allo-RAKs had significant cytotoxicity effects on both RCC cell lines RCC-LSL and RCC-FTL and on two strains of primary renal cancer cells.The lysis rates of RCC cells were 6.80%～71.69%,17.25%～79.98%and 27.92%～83.48%as the effector target ratio were 37.5:1,75:1 and 150:1.
Conclusions The quantity of effector cells could be significantly increased cultured form PBMC with combining RetroNectin with CD3mAb and IL-2 ex vivo.The expansion ability and proportion of effector cells of RAKs form healthy donors were higher than that from RCC patients.The AIIo-RAKs were showed cytotoxicity effect on RCC cells in vitro;Allo-RAK form healthy donors had the function of killing human RCC cells and inhibiting tumor growth in tumor beating nude mice in vivo.This study suggests allo-RAK might be an effective measure for mRCC patients.
引文
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