【作者】 张沂南；

【导师】 王国民； 陈思锋； 郭剑明；

【作者基本信息】 复旦大学 ， 外科学， 2008， 博士

【摘要】 第一部分非清髓性异基因骨髓移植治疗小鼠前列腺癌的实验研究目的探讨非清髓性异基因骨髓移植对小鼠前列腺癌的治疗效果及移植后移植物抗宿主病（GVHD）情况。方法应用C57小鼠源性前列腺癌株RM-1（2×10⁶/只）制成C57BL小鼠前列腺癌皮下移植瘤模型,分为对照组和异基因骨髓移植组,每组10只。应用FC方案(氟达拉滨30mg/m²,-d^1-5;环磷酰胺300mg/m²,-d^1-3)对异基因骨髓移植组小鼠进行非清髓性预处理。异基因骨髓移植组小鼠经尾静脉注射BALB/c小鼠来源的骨髓细胞5×10⁶个/只,脾细胞1×10⁷个/只。记录两组小鼠肿瘤的体积及生存时间,比较两组肿瘤生长率及生存时间差异,进行冰冻切片HE染色行病理学观察,并记录异基因骨髓移植组GVHD情况。全骨髓培养法培养异基因骨髓移植组濒死小鼠骨髓细胞,观察小鼠前列腺癌骨转移情况。结果小鼠前列腺癌皮下移植瘤模型成瘤率100%,预处理药物对肿瘤生长并无影响（P＞0.05）。异基因骨髓移植显著抑制了小鼠前列腺癌的生长（P=0 028）,并显著延长了受鼠的生存时间（P=0.00001）。异基因骨髓移植组小鼠发生了Ⅲ～Ⅳ级的GVHD。濒死的异基因骨髓移植组小鼠前列腺癌已发生骨转移。结论在目前的非清髓性预处理条件下,荷瘤小鼠可以耐受异基因骨髓细胞及脾细胞移植所诱发的GVHD。异基因骨髓移植明显抑制了小鼠前列腺癌的生长,并延长了受体小鼠的生存时间。异基因骨髓移植延长了受鼠的生存时间,可能是其发生骨转移的主要原因。第二部分非清髓性异基因骨髓间充质干细胞移植治疗小鼠前列腺癌的实验研究目的观察非清髓性异基因骨髓间充质干细胞移植对小鼠前列腺移植瘤的治疗效果及移植后移植物抗宿主病（GVHD）情况。方法应用全骨髓培养法培养BALB/c小鼠骨髓间充质干细胞,通过流式细胞术检测第四代细胞中CD44阳性细胞率,并通过油红染色鉴定由骨髓间充质干细胞分化的脂肪细胞。应用C57小鼠源性前列腺癌株RM-1（2×10⁶/只）制成C57BL小鼠皮下移植瘤模型,分为对照组、异基因骨髓移植组和异基因骨髓间充质干细胞移植组,每组10只。应用FC方案(氟达拉滨30mg/m²,-d^1-5;环磷酰胺300mg/m²,-d^1-3)对非对照组小鼠进行非清髓性预处理。异基因骨髓移植组小鼠经尾静脉注射BALB/c小鼠来源的骨髓细胞5×10⁸个/只,脾细胞1×10⁷个/只。异基因干细胞移植组小鼠经尾静脉注入骨髓间充质干细胞2×10⁶个/只。记录三组小鼠肿瘤的体积及生存时间,比较三组肿瘤生长率及生存时间差异,进行冰冻切片HE染色行病理学观察骨髓间充质干细胞移植组GVHD情况,并与异基因骨髓移植组相比较。结果全骨髓培养法培养的第四代细胞中CD44阳性率为84.29%,油红染色证实由骨髓间充质干细胞自然分化的脂肪细胞存在。与对照组相比,异基因骨髓间充质干细胞移植显著的抑制了小鼠前列腺癌的生长（P=0.047）,并明显延长了异基因骨髓间充质干细胞移植组小鼠的生存时间（P=0.00001）。与异基因骨髓移植组相比,异基因骨髓间充质干细胞移植对小鼠前列腺癌的抑制及对受鼠生存时间的延长效果相似（P值分别为0.46及0.96）。异基因骨髓间充质干细胞移植未能避免GVHD的发生,GVHD程度为,Ⅱ～Ⅲ级,优于异基因骨髓移植后GVHD程度（P=0.0017）。结论建立了较为完善的小鼠骨髓间充质干细胞培养和鉴定的方法。异基因骨髓间充质干细胞移植显著的抑制了小鼠前列腺癌的生长,延长了受体小鼠的生存时间。与异基因骨髓移植相比,异基因骨髓间充质干细胞移植对小鼠前列腺癌生长的抑制及延长受鼠生存时间的作用相似,但诱发的GVHD程度较低。第三部分同种反应性NK细胞联合异基因骨髓移植治疗小鼠前列腺癌的实验研究目的在异基因骨髓移植平台上观察同种反应性NK细胞移植对移植物抗肿瘤效应（GVTR）的增强作用及对移植物抗宿主病（GVHD）的抑制作用。方法应用雄性C57BL小鼠和雌性BALB/c小鼠繁育CB6F1^H-2d/b小鼠（F1代）。免疫磁珠分选法（MACS）分选F1代小鼠NK细胞,并通过流式细胞术鉴定并分选同种反应性NK细胞。将RM-1癌细胞（2×10⁶/只）皮下移植给亲代C57BL父鼠制成小鼠前列腺癌皮下移植瘤模型,分为对照组、异基因骨髓组及同种反应性NK细胞联合异基因骨髓移植组（联合移植组）,每组10只。应用FC方案(氟达拉滨30mg/m²,-d^1-5;环磷酰胺300mg/m²,-d^1-3)对非对照组小鼠进行非清髓性预处理。异基因骨髓移植组经尾静脉注射BALB/c小鼠来源的骨髓细胞5×10⁶个/只,脾细胞1×10⁷个/只;联合移植组首先经尾静脉注射F1代小鼠骨髓细胞5×10⁶个/只,48小时候后再经尾静脉注射同种反应性NK细胞1×10⁶个/只。记录三组小鼠肿瘤的体积及生存时间,比较三组肿瘤生长率及生存时间差异,进行冰冻切片HE染色行病理学观察联合移植组GVHD情况,并与异基因骨髓移植组相比较。全骨髓培养法培养濒死小鼠骨髓细胞,观察并比较异基因骨髓移植组及联合移植组小鼠前列腺癌骨转移情况。结果F1代小鼠NK细胞中Ly49A细胞阳性率为44.98%,即为同种反应性NK细胞。与异基因骨髓移植相比,联合移植更加显著的抑制了小鼠前列腺癌的生长（P＜0.05）,并更加明显延长了联合移植组小鼠的生存时间（P=0.001）。同种反应性NK细胞移植显著的降低了异基因骨髓移植后GVHD程度,联合移植组小鼠GVHD程度为Ⅰ～Ⅲ级,优于单纯异基因骨髓移植（P=0.0003）。濒死小鼠骨髓培养提示,与异基因骨髓移植组比较,联合移植组小鼠骨髓细胞中的肿瘤细胞较少且生长受抑制。结论同种反应性NK细胞联合异基因骨髓移植显著的抑制了小鼠前列腺癌的生长并延长了受体小鼠的生存时间。与异基因骨髓移植相比,联合移植显著的增强了GVTR,因而更加明显的抑制了小鼠前列腺癌的生长;同时降低了GVHD程度,从而延长了受体小鼠的生存时间。同种反应性NK细胞移植对小鼠前列腺骨转移癌可能具有抑制作用。更多还原

【Abstract】 PartⅠNon-myeloablative Allogenetic Bone Marrow Transplantation for the Treatment of Prostate Cancer in Murine ModelObjective The objectives of this part are to explore the graft versus tumor （GVT） effects and the severity of graft versus host disease（GVHD） post allogenetic bone marrow transplantation in murine prostate cancer model.Methods Following nonmyeloablative regimens consisted of fludarabine(30mg/m²,-d^1-5) and cyclophosphamide(300mg/m²,-d^1-3),C57BL mice bearing RM-1（2×10⁶ each） tumors underwent an allogeneic bone marrow cells（5×10⁶ each） and spleen cells（1×10⁷ each） transplantation from BALB/c donors through the tail veins.The tumor volumes and the survival time of the recipients were recorded and compared with the control group and the severity of GVHD was graded by pathology morphological standard.The bone marrow cells from the dying recipients were cultured for the evidence of bone metastasis.Results All C57BL mice were observed formation of transplanted prostate cancer.The nonmyeloablative regimens did not inhibit the growth of the tumors （P＞0.05）.Allogenetic bone marrow transplantation inhibited the growth of the tumors and prolonged the recipients’ survival time significantly（P=0.028 and 0.00001,respectively）.The recipients’ GVHD were gradingⅢ～Ⅳ,and the evidences of bone metastasis were found in the dying recipients.Conclusions Under these given nonmyeloablative regimens,GVHD were well endured by the recipients.Allogenetic bone marrow transplantation inhibited the growth of the tumors and prolonged the recipients’ survival time significantly.The recipients’ significantly prolonged survival time would be the main reason for the prostate cancer bone metastasis.PartⅡNon-myeloablative Allogenetic Bone Marrow Derived Mesenchymal Stem Cell Transplantation for the Treatment of Prostate Cancer in Murine ModelObjective The objectives of this part are to explore the graft versus tumor （GVT）effects and the severity of graft versus host disease（GVHD） post allogenetic bone marrow derived mesenchymal stem cell（MSC） transplantation in murine prostate cancer model.Methods Bone marrow cells were cultured and purified by adherent method to the fourth generation.The MSCs were then identified by flow cytometry for CD44 positive cells and oil red stain for the differentiated fat cells.Following nonmyeloablative regimens consisted of fludarabine(30mg/m²,-d^1-5) and cyclophosphamide(300mg/m²,-d^1-3),C57BL mice bearing RM-1（2×10⁶ each） tumors underwent an allogeneic bone marrow MSCs（2×10⁶ each） transplantation and bone marrow cells（5×10⁶ each） and spleen cells（1×10⁷ each） transplantation from BALB/c donors through the tail veins.The tumor volumes,survival time and the grade of GVHD of the recipients were recorded, and then compared with the allogenetic bone marrow transplantation group.Results The percentage of CD44 positive cells in the fourth generation was 84.29%,and the oil red stain proved the existence of differentiated fat cells. Comparing with the control group,allogenetic bone marrow derived MSC transplantation inhibited the growth of the tumors and prolonged the recipients’ survival time significantly（P=0.047 and 0.00001,respectively）.Comparing with the allogenetic bone marrow transplantation group,allogenetic bone marrow derived MSC transplantation shared the same inhibiting effect of the tumors and prolonging effect of the recipients’ survival time（P=0.46 and 0.96, respectively）.The MSC transplantation did not avoid the occurrence of GVHD, and the recipients’ GVHD were gradingⅡ～Ⅲ,better than these of the allogenetic bone marrow transplantation group’s（P=0.0017）.Conclusions The method of MSC culture and identity was well founded. Allogenetic bone marrow derived MSC transplantation inhibited the growth of the tumors and prolonged the recipients’ survival time significantly.Compared with allogenetic bone marrow transplantation group,the MSC transplantation shared same graft versus tumor effects,but decreased the severity of GVHD.PartⅢAlloreactive NK Cells Transplantation Enhanced GVT Effects and Decreased GVHD in Murine Prostate Cancer Model.Objective The objectives of this part are to explore the enhancement of the graft versus tumor（GVT） effects and reduction of the graft versus host disease （GVHD） post allogenetic bone marrow and alloreactive natural killer（NK） cell transplantation in murine prostate cancer model.Methods The alloreacitve NK cells were isolated from the spleen of CB6F1^H-2d/b（the F1 generation of C57BL male mice and BALB/c female mice） mice,purified by negative magnetic cell sorting（MACS）,and then Ly49A positive cells were identified and collected by flow cytometry.Following nonmyeloablative regimens consisted of fludarabine(30mg/m²,-d^1-5) and cyclophosphamide(300mg/m²,-d^1-3),C57BL mice bearing RM-1（2×10⁶ each） tumors underwent an allogeneic bone marrow cells（5×10⁶ each） and alloreacitve NK cells（1×10⁶ each） transplantation 48 hours later from CB6F1^H-2d/b donors through the tail veins.The tumor volumes and the survival time of the recipients were recorded,the severity of GVHD was graded by pathology morphological standard,and the bone marrow cells from the dying recipients were cultured for the evidence of bone metastasis,and then compared with that of the allogenetic bone marrow transplantation group.Results The percentage of Ly49A positive cells in NK cells isolated from the CB6F1^H-2d/b mice was 44.98%.Compared with allogenetic bone marrow transplantation group,the alloreacitve NK cells transplantation inhibited the growth of the tumors and prolonged the recipients’ survival time more significantly（P＜0.05 and 0.001,respectively）;decreased the severity of the recipients’ GVHD,which gradingⅠ～Ⅲ,better than these of the allogenetic bone marrow transplantation group’s（P=0.0003）;and the bone marrow culture of the dying recipients demonstrated evidences of bone metastasis inhibition.Conclusions Compared with allogenetic bone marrow transplantation, alloreactive NK Cells transplantation inhibited the growth of the tumors, prolonged the recipients’ survival time and decreased the severity of GVHD more significantly.There are evidences that alloreactive NK cells transplantation inhibited the prostate cancer bone metastasis.Therefore,the alloreactive NK cells transplantation enhanced GVT effects and decreased GVHD in murine prostate cancer model.更多还原