摘要
一、供体NK细胞对非清髓异基因造血干细胞移植中受体异基因反应的抑制作用及其机制
目的:体外实验系统阐明异基因造血干细胞移植中,供体NK细胞对受体CD8+T细胞的否决作用机制;体内实验系统证明供体NK细胞抑制非清髓性异基因造血干细胞移植中受体的异基因反应从而促进植入。
方法:在体外实验系统中,将C57BL/6(H-2b)小鼠的脾细胞作为反应细胞(模拟受体细胞),BALB/c(H-2d)小鼠的脾细胞经致死剂量照射后作为刺激细胞(模拟供体细胞),建立混合淋巴细胞培养体系(MLR)。3H嵌入法检测反应细胞的增殖情况;细胞毒性试验检测反应细胞对H-2d肿瘤细胞的杀伤作用。在此MLR系统中,加入致死剂量照射的BALB/c(H-2d)小鼠骨髓培养的体外激活的NK细胞(ALAK),模拟供体NK细胞,用同样方法检测C57BL/6(H-2b)反应细胞在H-2d抗原刺激下的繁殖和细胞杀伤功能。在MLR体系中加入TGF-β的阻断抗体,观察其是否有阻断供体NK细胞否决作用的效应;分别加入从Fas突变体小鼠和穿孔素敲基因鼠培养的NK细胞来阐明供体NK细胞否决作用是否通过Fas-FasL和穿孔素介导的细胞毒反应来完成。体内实验系统中,采用BALB/c(H-2d)小鼠做为供体鼠,同时用所获取的造血干细胞体外培养供体NK细胞;采用C57BL/6(H-2b)小鼠做为受体鼠,进行全身性照射的非清髓性移植前预处理。通过尾静脉注射对受体鼠进行异基因造血干细胞移植,共设以下三组:尾静脉注射供体造血干细胞组;尾静脉注射受体NK细胞和供体造血干细胞组;尾静脉注射供体NK细胞和供体造血干细胞组。造血系统再造完成后,取各组受体鼠脾细胞,流式细胞仪检测供体细胞的植入率,检测供体来源的B细胞、NK细胞、巨噬细胞、CD4+T细胞、CD8+T细胞数量;3H嵌入法检测受体脾细胞在H-2d抗原刺激下的增殖情况;细胞毒性试验检测脾细胞对H-2d抗原包被的肿瘤细胞P815的杀伤作用;CTLL-2细胞增殖实验检测脾细胞在H-2d抗原刺激下所表达的IL-2水平。对以上实验结果进行统计学处理分析。
结果:体外实验结果表明,激活的BABL/c小鼠H2d NK细胞可以显著抑制混合反应体系中C57BL/6小鼠来源的H2b反应细胞的增殖效应(p﹤0.01);激活的H2d NK细胞显著抑制H2b反应细胞对H2d抗原包被的肿瘤细胞的杀伤活性(p﹤0.01);相同数目的H2b NK细胞或者H2d脾细胞并未表现出相同的抑制作用;抗TGF-β阻断剂并不能减弱异基因NK细胞的抑制作用;Fas配体突变小鼠来源的NK细胞与野生型小鼠来源的NK细胞抑制作用无显著差别,穿孔素敲基因小鼠来源的NK细胞的抑制作用则有所减弱。体内实验结果显示,尾静脉注射供体NK细胞和供体造血干细胞实验组的供体细胞植入率明显高于其它两组(p﹤0.05);尾静脉注射供体NK细胞和供体造血干细胞实验组小鼠的脾脏细胞中供体来源的B细胞、NK细胞、巨噬细胞、CD4+T细胞、CD8+T细胞数量明显高于其它两组(p﹤0.05);尾静脉注射供体NK细胞和供体造血干细胞实验组的3H嵌入、细胞毒性实验、及IL-2水平的受体异基因反应水平检测结果与另外两实验组相比显著降低(p﹤0.05)。
结论:激活的异基因NK细胞能够特异性抑制异基因反应细胞的增殖效应和杀伤活性;颗粒酶介导的杀伤活性可能是这种抑制效应的机制之一,而Fas-Fas配体通路及TGFβ信号通路并非该抑制效应的作用机制;异基因NK细胞可以通过抑制受体细胞的异基因反应促进非清髓异基因造血干细胞的植入。
二、供体NK细胞及IL-15对非清髓异基因造血干细胞移植的促进作用
目的:探讨供体NK细胞与IL-15在非清髓异基因造血干细胞移植中对植入可能存在的协同促进效应。
方法:采用C57BL/6(H-2b)小鼠做为供体鼠,同时用所获取的造血干细胞体外培养供体NK细胞;采用BABL/c(H-2d)小鼠做为受体鼠,进行全身性照射的非清髓性移植前预处理。通过尾静脉注射对受体鼠进行异基因造血干细胞移植,共设以下四组:尾静脉注射供体造血干细胞组;尾静脉注射供体NK细胞和供体造血干细胞组;尾静脉注射供体造血干细胞并在移植后腹腔注射IL-15组;尾静脉注射供体NK细胞和供体造血干细胞并在移植后腹腔注射IL-15组。构建重组小鼠IL-15基因的慢病毒表达载体,于293T细胞包装并获得病毒,感染供体来源的NK细胞,从而得到自身携带IL-15基因的供体NK细胞。同上建立小鼠非清髓异基因造血干细胞移植模型,实验分组如下:尾静脉注射供体造血干细胞组;尾静脉注射供体NK细胞和供体造血干细胞组;尾静脉注射供体造血干细胞并在移植后腹腔注射IL-15组;尾静脉注射携带IL-15基因的供体NK细胞及供体造血干细胞组。造血系统再造完成后,取以上各组的受体鼠脾细胞,流式细胞仪检测供体细胞的植入率,检测供体来源的B细胞、NK细胞、巨噬细胞、CD4+T细胞、CD8+T细胞数量;3H嵌入法检测脾细胞在H-2b抗原刺激下的增殖情况;细胞毒性试验检测脾细胞对H-2b抗原包被的肿瘤细胞EL4的杀伤作用;CTLL-2细胞增殖实验检测脾细胞在H-2b抗原刺激下所表达的IL-2水平。对以上实验结果进行统计学处理分析。
结果:在采用腹腔注射IL-15的实验体系中,尾静脉注射供体NK细胞和供体造血干细胞并在移植后腹腔注射IL-15组的植入率明显高于其它三组(p﹤0.05);其受体鼠脾细胞中供体来源的B细胞、NK细胞、巨噬细胞、CD4+T细胞、CD8+T细胞数量明显高于其它三组(p﹤0.05);3H嵌入、细胞毒性实验、及IL-2水平的检测结果均与其它三组有显著性差异(p﹤0.05)。在供体NK细胞自身携带IL-15基因的实验体系中,初步实验结果证明:尾静脉注射携带IL-15基因的供体NK细胞及供体造血干细胞组的植入率明显高于其它三组(p﹤0.05);受体鼠脾细胞中供体来源的B细胞、NK细胞、巨噬细胞、CD4+T细胞、CD8+T细胞数量明显高于其它三组(p﹤0.05);3H嵌入、细胞毒性实验、及IL-2水平的检测结果均与其它三组有显著性差异(p﹤0.05)。
结论:在小鼠的非清髓异基因造血干细胞移植模型中,供体NK细胞和IL-15的联合使用能够显著促进植入、增加供者来源的各细胞亚群的百分比,并能够抑制受者的异基因反应,但二者作用是否发生协同尚不明了。携带IL-15基因的供体NK细胞的输注也能同样显著促进植入、移植后免疫重建、抑制受者的异基因反应。
三、小鼠IL-15亚型的发现及其功能的初步研究
目的:探寻在小鼠脾脏细胞免疫激活状态下发现的小鼠IL-15亚型的基因结构、可能的产生机制及功能。
方法:取C57BL/6小鼠脾脏,制备脾细胞悬液及骨髓细胞悬液,通过磁珠分选及CSF刺激原代培养小鼠B细胞及巨噬细胞。收集生长良好的小鼠骨髓瘤细胞SP2/0,小鼠单核巨噬细胞白血病细胞RAW246.7及原代培养的B细胞、巨噬细胞,Trizol-氯仿法提取各种细胞的总RNA,逆转录成cDNA作为模板,利用所设计的特异性引物,进行PCR扩增,得到小鼠IL-15基因的目的片段,2%琼脂糖电泳鉴定扩增产物。在LPS刺激培养12h的小鼠脾脏细胞中加入放线菌酮继续培养12h,并设LPS刺激培养24h的小鼠脾细胞及无任何处理的小鼠脾细胞作为对照,Western Blot检测各种培养条件下小鼠脾细胞中IL-15亚型蛋白的表达。克隆IL-15基因及其亚型,并构建IL-15及其亚型重组的pET43.1原核表达载体,于BL-21表达菌中自发表达,SDS-PAGE电泳鉴定目的蛋白并验证表达蛋白的可溶性,纯化获得有功能活性的IL-15及其亚型蛋白,Western Blot检测所得纯化蛋白。CTLL-2细胞增殖实验体外检测IL-15亚型的功能。
结果:小鼠骨髓瘤细胞SP2/0,小鼠单核巨噬细胞白血病细胞RAW246.7及原代培养的B细胞、巨噬细胞在LPS刺激培养后均可检测到缺失6号外显子的IL-15亚型。放线菌酮的加入可以减少LPS刺激所产生的该种亚型蛋白。重组IL-15及亚型的pET43.1表达载体构建成功,并成功纯化获得有功能的IL-15及IL-15亚型蛋白。该种IL-15亚型可能对正常IL-15蛋白的功能活性起负调控作用。
结论:缺失6号外显子的IL-15亚型蛋白可能由LPS刺激细胞后IL-15mRNA进行选择性剪切而产生,B细胞及巨噬细胞在LPS刺激下均可产生此种亚型。此亚型蛋白可能在免疫系统激活状态下对正常IL-15蛋白的功能活性其负调控作用。
Part ?: Activated allogeneic NK cells as suppressors of alloreactive responses in nonmyeloablative allogeneic hematopoietic stem cell transplantation
Object: The current study attempted to elucidate the mechanism of the veto activity of donor NK cells against host cytotoxic T cell precursors, and evaluate the effect of donor NK cells administration on engraftment and immune reconstitution in a murine nonmyeloablative allogeneic hematopoietic stem cell transplantation (allo-HSCT) model.
Methods: We used H2b splenocytes from C57BL/6 mice as responder cells, and the irradiated H2d splenocytes from BALB/c mice as stimulator cells to set up an in vitro MLR system. Tritiated thymidine was pulsed to detect the proliferation of responder cells, cytotoxicity assay was used to detect the killing capacity of responder cells against the H2d tumor cells. Irradiated H2d NK cells from BALB/c mice were added into the MLR reactions mimicking the donor NK cells. The proliferation and killing capacity of the responder cells were detected the same way as the above. Anti-TGFβblocking antibody was added to the MLR system mentioned above to determine whether the suppressive effects of the donor NK cells were caused by the release of TGFβ. The NK cells from wild type C57BL/6 mice, gld mice (FasL mutant), and pfp mice (perforin KO, both on C57BL/6 background) were used in the MLR assay to further determine the killing mechanisms of the donor NK cells. We further demonstrate that donor NK cells can promote engraftment by suppressing host alloreactive responses in nonmyeloablative allo-HSCT model. Donor NK cells from BALB/c mice were infused into host C57BL/6 mice during nonmyeloablative allo-HSCT. To evaluate the role of donor NK cell administration in transplantation outcomes, donor-recipient pairs were divided into three groups: The recipient C57BL/6 (H-2b) mice conditioned with donor hematopoietic stem cells, the recipient mice ?conditioned with sygeneous hematopoietic stem cells, the recipient mice conditioned with donor hematopoietic stem cells and donor NK cells. After two months, proliferation of the donor hematopoietic stem cells was determined through flow analysis. The counts of donor-derived splenic B, T, NK, and macrophage cells were also measured by flow anlaysis. The proliferation capacity of the splenocytes from host mice was measured with 3H-thymidine assay. The killing capacity of the host splenocytes against the H2d tumor cells was detected by the cytotoxicity assay. The allo-response of the host splenocytes was also measured by the level of IL-2 expression after the three day MLR reaction mentioned above by MTT.
Results: The activated H2d NK cells from BALB/c mice significantly suppressed the proliferation of H2b splenocytes from C57BL/6 mice in mixed lymphocyte responses (MLR) stimulated with irradiated H2d splenocytes from BALB/c mice (p<0.01). The ability for H2b splenocytes to kill H2d tumor targets was also significantly inhibited by activated H2d NK cells (p<0.01). The same number of H2b ALAK cells or H2d splenocytes did not show the same suppressive effect. Anti-TGFβantibody blockade did not diminish this suppressive effect of NK cells. NK cells from gld (FasL mutant) mice suppressed the allo-responses as well as the wild type NK cells. NK cells from pfp (perforin knockout) mice did not completely block the inhibitory effect. The BABL/c mice treated with donor NK cells resulted in higher donor chimerism percentage compared with control groups (p<0.05). The counts of donor derived lymphocyte subsets were increased in the spleen of the recipient mice infused with donor NK cells compared with control groups (p<0.05). The proliferation of the splenocytes from donor NK treated recipient mice was lower than the splenocytes from the control groups in mixed lymphocyte responses (MLR) stimulated with irradiated H2b splenocytes from C57BL/6 (B6) mice (p<0.05). The ability for the mixed splenocyts to kill H2d tumor targets and the level of IL-2 expression during the MLR had also been analyzed, the results of the recipient mice group infused with donor NK cells were lower compared with the control groups (p<0.05).
Conclusion: Our study demonstrates that activated allogeneic NK cells can specifically suppress the alloreactive cells, inhibiting both their proliferation and killing capacities. Granule-mediated killing might be part of the suppressive mechanism, while Fas-FasL pathway and TGFβsignaling are not involved. Donor allogeneic NK cell could promote the engraftment as suppressors of alloreactive responses in nonmyeloablative allogeneic hematopoietic stem cell transplantation.
PartП: Donor NK cells and IL-15 treatment promoted engraftment during nonmyeloablative allo-HSCT
Object: We carried out experiments to determine the effects of combining donor NK cell infusion and IL-15 administration in a murine nonmyeloablative allo-HSCT model.
Methods: Donor NK cells were purified from C57BL/6 mice and expanded in vitro. Donor NK cells were infused into host BALB/c mice during nonmyeloablative allo-HSCT. To evaluate the role of donor NK cell administration in transplantation outcomes, donor-recipient pairs were divided into four groups: The recipient BALB/c (H-2d) mice conditioned with donor hematopoietic stem cells, the recipient mice conditioned with donor hematopoietic stem cells and donor NK cells, the recipient mice conditioned with donor hematopoietic stem cells and IL-15 administration and the recipient mice conditioned with donor hematopoietic stem cells, donor NK cells and IL-15 administration. IL-15 gene was subcloned into the transfer plasmid of the lentivirus system, which was transfected together with the packaging plasmids into 293T cells. Then the recombinant virus was obtained and used to infect donor NK cells. The murine nonmyeloablative allo-HSCT model was established and donor-recipient pairs were also divided into four groups: The recipient BALB/c (H-2d) mice conditioned with donor hematopoietic stem cells, the recipient mice conditioned with donor hematopoietic stem cells and donor NK cells, the recipient mice conditioned with donor hematopoietic stem cells and IL-15 administration and the recipient mice conditioned with donor hematopoietic stem cells and the viral infected donor NK cells. After two months, engraftment of the donor hematopoietic stem cells was determined through flow analysis. The counts of donor-derived splenic B, T, NK, and macrophage cells were also measured by flow anlaysis. The proliferation capacity of the splenocytes from host mice was measured with 3H-thymidine assay. The killing capacity of the host splenocytes against the H2b tumor cells was detected by the cytotoxicity assay. The allo-response of the host splenocytes was also determined by measuring IL-2 secretion after the three day MLR reaction mentioned above by MTT.
Results: The BABL/c mice treated with donor NK cells and hIL-15 resulted in obviously higher donor chimerism percentage compared with control groups (p<0.05). The counts of donor derived lymphocyte subsets were increased in the spleen of the recipient mice infused with donor NK cells and hIL-15 compared with control groups (p<0.05). The proliferation of the splenocytes from donor NK and hIL-15 treated recipient mice was significantly lower than the splenocytes from the control groups in thel MLR stimulated with irradiated H2b splenocytes from C57BL/6 (B6) mice (p<0.05). The ability for the mixed splenocyts to kill H2b tumor targets and the level of IL-2 expression during the MLR had also been analyzed, the results of the recipient mice group infused with donor NK cells and hIL-15 were obviously lower compared with the control groups (p<0.05). In the use of donor NK cells infected by the IL-15 gene recombinant virus experimental system, the primary results show the administration of viral infected donor NK cells could also resulted in higher donor chimerism percentage, higher counts of donor derived lymphocyte subsets and more obviously suppression of the host allo-response.
Conclusion: Donor NK cell and IL-15 treatment could promote the engraftment and the development of donor derived cell subsets and suppress the host allo-response in the nonmyeloablative allo-HSCT murine transplant model.
PartⅢ: The discovery and function studies on IL-15 isoform
Object: To explore the gene structure, origine and function of a newly discovered IL-15 isoform.
Methods: Bone marrow cell and spleen cell suspensions were prepared from C57BL/6 mice. B cells were obtaind from spleen cells by Micro Beads purification, and the macrophages were obtained from the CSF stimulation of the bone marrow cells. Collect the mouse myeloma cells SP2/0, mouse monocyte-macrophage leukemia cells RAW246.7, and the B cells macrophages from primary culture. Total RNA of these cells was extracted and the coding sequence of IL-15 was amplified by RT-PCR. The cycloheximide was added into the splenocytes after stimulated with LPS for 12 hours. Splenocytes were then cultured for another 12 hours. The splenocytes stimulated with LPS for 24 hours and the normal splenocytes were also prepared as control. Western blot was used to detect the expression of IL-15 isoform gene in these splenocytes. The PCR product of IL-15 isoform gene was cloned and sequenced correctly. IL-15 gene was subcloned into the plasmid of pET43.1 vector and expressed in E.coli BL-21(DE3). Detect the solubility of the fusion protein and purify the protein. Western blot was used to detect the puriefied protein and its function was analyzed by the proliferation of CTLL-2 cells.
Results: Our analysis revealed the exon 6 of IL-15 is absent when the mouse myeloma cells SP20, mouse monocyte-macrophage leukemia cells RAW246.7, and the B cells macrophages from primary culture were stimulated with LPS. The infusion of cycloheximide can reduce the expression of this isoform. pET43.1 vector containing IL-15 gene was constructed successfully. The fusion protein was purified successfully. The in vitro data demonstrated this IL-15 isoform may mediate negative control mechanism to regulate the function of normal IL-15.
Conclusion: The IL-15 mRNA isoforms lacking exon 6 may be generated by alternative splicing events of the cells stimulated with LPS. Both of B cells and Macrophages could generate this isoform when stimulated with LPS. In the states of immune activation, this isoform may mediate negative control mechanism to regulate the function of normal IL-15.
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