IL-15联合GM-CSF对急性白血病NK细胞作用的研究

目的选择细胞因子IL-15和GM—CSF作为刺激物，观察其对急性白血病自然杀伤细胞(NK细胞)的激活作用。方法分离健康成人及化疗缓解后的白血病患者外周血单个核细胞(PBMC)，分别加入IL-2、IL-15、GM—CSF培养，MTr法检测PBMC及活化后的PBMC对白血病K562的细胞毒作用。流式细胞仪检测NK细胞并计数NK／PBMC比例。结果正常人及化疗缓解组PBMC经过IL-15、IL-15 GM—CSF分别作用后，细胞均呈现不同程度的增殖。化疗后缓解的增殖程度均小于正常人。正常人及化疗缓解组PB—MC联合应用IL-15和GM—CSF后对k562杀伤活性优于单用IL-15。缓解组对k562的杀伤活性明显低于正常对照组，化疗缓解后病人的NK／PBMC比例低于正常人。经联合应用IL-15、GM—CSF孵育后NK／PB—MC高于正常人。结论正常人及白血病患者PBMC与IL-15和GM—CSF联合孵育，能有效刺激PBMC增殖，提高NK／PBMC的比例。通过正常人及白血病患者的PBMC对K562的杀伤能力，间接反应IL-15和GM—CSF能有效激活外周血NK细胞，增加杀伤活性。     

PATHOLOGICAL STUDIES ON THE ANTI－INVASIVE CHARACTER BY RECOMBINANT HUMAN INTERLEUKIN－6 GENE－TRANSFECTED MOUSE LEUKEMIA CELLS

ＰＡＴＨＯＬＯＧＩＣＡＬＳＴＵＤＩＥＳＯＮＴＨＥＡＮＴＩ－ＩＮＶＡＳＩＶＥＣＨＡＲＡＣＴＥＲＢＹＲＥＣＯＭＢＩＮＡＮＴＨＵＭＡＮＩＮＴＥＲＬＥＵＫＩＮ－６ＧＥＮＥ－ＴＲＡＮＳＦＥＣＴＥＤＭＯＵＳＥＬＥＵＫＥＭＩＡＣＥＬＬＳＧｅＬｉｎｆｕ葛林阜，Ｃａｏ...     

Fibroblast growth factor-2 enhances NK sensitivity of hepatocellular carcinoma cells

The roles of fibroblast growth factor-2 (FGF-2) in the hepatocellular carcinoma (HCC) development are still controversial. In this study, we investigated the expression of FGF-2 in chronic hepatitis (CH) type C patients with or without HCC and the immunoregulation of FGF-2 in NK sensitivity of HCC cells. The FGF-2 expressions were detected in the liver tissues of patients, but not in normal liver. The serum FGF-2 levels of the patients with CH, liver cirrhosis (LC) or HCC were significantly higher than those of healthy volunteers. The serum FGF-2 levels of patients decreased with the progression of chronic liver disease. HCC occurrence of LC patients with high levels of serum FGF-2 was significantly lower than that with low levels of serum FGF-2. Proinflammatory cytokines, such as IL-1β and IL-6, induced FGF-2 expressions in HCC cells and normal hepatocytes. FGF-2 stimulation resulted in increasing the expression of the membrane-bound major histocompatibility complex class I-related chain A (MICA), an NK activating molecule, and decreasing that of human leukocyte antigen (HLA) class I, an NK inhibitory molecule, on HCC cells. This did not occur with normal hepatocytes. Adding anti-FGF receptor-2 neutralizing antibody resulted in inhibiting the change of MICA and HLA class I expressions on FGF-2 stimulated HCC cells. FGF-2 stimulation on HCC cells resulted in increasing NK sensitivity against HCC cells. These findings indicate that FGF-2 produced by HCC cells or normal hepatocytes of chronic liver disease may play critical roles in eliminating HCC cells by innate immunity.     

Neutrophils with protumor potential could efficiently suppress tumor growth after cytokine priming and in presence of normal NK cells

In tumor-bearing state, the function of neutrophils is converted from tumor-suppressing to tumor-promoting. Here we report that priming with IFN-γ and TNF-α could convert the potential of neutrophils from tumor-promoting to tumor-suppressing. The neutrophils with protumor potential have not lost their responsiveness to IFN-γ and TNF-α. After priming with IFN-γ and TNF-α, the potential of the neutrophils to express Bv8 and Mmp9 genes was reduced. Conversely, the tumor-promotional neutrophils recovered the expression of Rab27a and Trail, resumed the activation levels of PI3K and p38 MAPK pathways in response to stimuli, and expressed higher levels of IL-18 and NK-activating ligands such as RAE-1, MULT-1, and H60. Therefore, the anti-tumor function of the neutrophils was augmented, including the cytotoxicity to tumor cells, the capability of degranulation, and the capacity to activate NK cells. Since the function of NK cells is impaired in tumor-bearing state, the administration of normal NK cells could significantly augment the efficiency of tumor therapy based on neutrophil priming. These findings highlight the reversibility of neutrophil function in tumor-bearing state, and suggest that neutrophil priming by IFN-γ/TNF-α might be a potential approach to eliminate residual tumor cells in comprehensive strategy for tumor therapy.     

INDUCTION OF IMMUNE RESPONSE BY IL-6 GENEMODIFIED LEUKEMIA CELLS

ＩＮＤＵＣＴＩＯＮＯＦＩＭＭＵＮＥＲＥＳＰＯＮＳＥＢＹＩＬ６ＧＥＮＥＭＯＤＩＦＩＥＤＬＥＵＫＥＭＩＡＣＥＬＬＳＣａｏＸｕｅｔａｏ曹雪涛ＧｅＬｉｎｇｆｕ葛林阜ＪｕＤｉａｎＷｅｎ鞠佃文ＹｕＹｉｚｈｉ于益芝ＴａｏＱｕｎ陶群ＺｈａｎｇＷｅｉｐｉｎｇ章卫...     

脐血来源的DC增强NK和NKT细胞对白血病细胞的杀伤作用

目的:探讨脐血来源的树突状细胞(DC)增强自然杀伤(NK)细胞和自然杀伤性T(NKT)细胞对白血病细胞杀伤作用的影响机制.方法:提取脐带血单个核细胞(PBMC),体外诱导培养DC、NK和NKT淋巴细胞.以人髓性白血病K-562细胞作为靶细胞,检测NK、NKT、NK+DC和NKT+DC对靶细胞的杀伤效应.检测共培养上清液...     

Transgenic expression of IL-33 activates CD8 T cells and NK cells and inhibits tumor growth and metastasis in mice

IL-33 is a multifunctional cytokine in immune regulation that activates Th1 cells, Th2 cells, CD8⁺ T cells and NK cells. Our study showed that transgenic expression of IL-33 attenuated tumor metastasis in the B16 melanoma and Lewis lung carcinoma (LLC) metastatic models. The percentages and cytotoxicity of CD8⁺ T cells and NK cells and their infiltration into the tumor tissues were significantly increased by the transgenic expression of IL-33 in tumor-bearing mice. Treatment with recombinant IL-33 could also increase the cytotoxicity of CD8⁺ T cells and NK cells in vitro. In addition, depletion of CD8⁺ T cells and NK cells using anti-CD8 or anti-asialo GM1 antibody abolished the pulmonary metastasis inhibition mediated by IL-33. Furthermore, IL-33 stimulated the activation of NF-κB and increased CD69 expression, which is a marker of the activated form of the two cell subsets, in CD8⁺ T cells and NK cells. Our results suggest that IL-33 stimulated NF-κB signaling and promoted the proliferation, activation and infiltration of CD8⁺ T cells and NK cells, which resulted in the inhibition of pulmonary metastasis in B16 melanoma and LLC mice models.     

重组可溶性MICA蛋白负调控NK细胞对白血病细胞的杀伤活性

目的:体外研究重组可溶性MHCⅠ类分子相关A(soluble MHC classⅠchain-related gene A,sMICA)蛋白对自然杀伤细胞(natural killer cell,NK cell)表面活化性受体NKG2D(natural killer group 2 member D)的表达、杀伤白血病细胞的活性和分泌IFN-γ的影响。方法:免疫磁珠分选健康人外周血NK细胞,分为空白对照组、重组sMICA组(200、500、800μg/L三亚组),相互作用24 h后,流式细胞术检测NK细胞NKG2D表达水平,LDH释放法检测NK细胞对白血病细胞K562的杀伤活性,ELISA法检测培养上清中IFN-γ的水平。结果:200、500、800μg/L sMICA作用组与对照组相比,NK细胞表面NKG2D的表达均下调[(68.79±6.87)%,(55.75±9.31)%、(45.14±5.70)%vs(92.75±6.91)%,P<0.05或P<0.01],均抑制NK细胞杀伤白血病细胞K562的活性[(18.67±2.35)%、(15.01±2.25)%、(7.33±2.52)%vs(36.33±2.51)%,P<0.05或P<0.01],均抑制IFN-γ的分泌[(164.48±22.48)、(112.71±10.89)、(70.23±9.64)vs(313.72±16.06)pg/ml,P<0.05,P<0.01]。结论:急性白血病细胞表面脱落的sMICA可下调NK细胞NKG2D的表达和IFN-γ的分泌,抑制了NK细胞对白血病细胞的杀伤活性。     

Combined immunogene therapy of IL-6 and IL-15 enhances anti-tumor activity through augmented NK cytotoxicity

Many tumors evade host immunity by lowering expression of major histocompatibility complex (MHC) molecules. Theoretically, low MHC expression should activate natural killer (NK) cells and in some cases suppress tumor growth; nevertheless, some tumors also produce high concentrations of immunosuppressive cytokines, such as transforming growth factor (TGF)-beta, to inhibit the activity of NK cells. Using a canine transmissible venereal tumor (CTVT) model, we have previously demonstrated that IL-6 is a strong antagonist for TGF-beta. Herein, we found that IL-6 alone was unable to significantly promote TGF-beta-inhibited NK activities. Conversely, IL-15 alone strongly promoted NK activities; however, NK activities were inhibited to baseline levels following the addition of TGF-beta. Therefore, a new strategy using combined immunogene therapy of both IL-6 and IL-15 mediated by electroporation was used in this study. This combined IL-6 and IL-15 treatment effectively relieved the inhibitory effect of TGF-beta and activated NK cell cytotoxicity of lymphokine-activated killer (LAK) cells. Similarly, in isolated DX5+ NK cells, only IL-6 and IL-15 in combination significantly overcame the inhibitory effect of TGF-beta and promoted NK cytotoxicity. The group of BALB/c mice injected with plasmids with IL-6 and IL-15 genes (pIL-6/pIL-15) had the highest percentages of DX5+ NK cells as compared with either the pIL-6 or pIL-15 groups. Further, in SCID mice inoculated with CTVT, electroporation-mediated delivery of pIL-6/pIL-15 was significantly more efficient in suppressing both tumor establishment and tumor growth as compared with pIL-6 or pIL-15 inoculation alone. In addition, the anti-asialo GM-1 antibody abolished NK activities in SCID mice and resulted in outgrowth of the tumors. Together, these results suggest that the TGF-beta-associated inhibition of NK cytotoxicity cannot be adequately restored by simply antagonizing TGF-beta with IL-6: the co-existence of NK activating factors such as IL-15 is also important in restoring TGF-beta-inhibited cytotoxicity. This study highlights the therapeutic potential of the pIL-6/pIL-15 combination by inhibiting TGF-beta activity and enhancing NK cytotoxicity.     

Inhibition of granulocytic differentiation of acute promyelocytic leukemia cells in primary culture by transforming growth factor-β

We investigated the effect of transforming growth factor-β₁ (TGFβ) on the proliferation and differentiation of cultured acute promyelocytic leukemia (APL) cells with the chromosomal t(15;17) translocation obtained from four patients to determine the role of TGFβ on growth and differentiation of APL cells.

DNA synthesis, determined by ³H-thymidine uptake, was inhibited in the presence and absence of granulocyte colony-stimulating factor (G-CSF) in a dose-dependent manner by TGFβ in APL cells obtained from three of the four cases. TGFβ and G-CSF did not significantly affect the differentiation of APL cells, but all-trans retinoic acid (RA) induced morphological and functional differentiation in all APL cells tested. G-CSF markedly enhanced RA-induced granulocytic differentiation in APL cells obtained from all four cases. In cells in which TGFβ inhibited DNA synthesis, it also inhibited RA-induced granulocytic differentiation of APL cells and, to a greater degree, granulocytic differentiation induced by RA plus G-CSF.

These results suggest that TGFβ is a negative regulator of the proliferation and differentiation of APL cells. The significance of TGFβ as an endogenous regulator in differentiation therapy with RA of APL patients is discussed.     

侵袭性NK细胞白血病并发多器官衰竭一例并文献复习

 目的　提高对侵袭性NK细胞白血病（ANKL）及并发多器官功能衰竭（MOF）的认识。方法　报道1例罕见ANKL的诊断、治疗经过并结合文献复习讨论。结果　患者经骨髓、免疫表型等检查确诊ANKL,发病及治疗过程中并发MOF（肝、肾、心、肺等）、代谢性酸中毒、肿瘤溶解综合征、弥漫性血管内凝血（DIC）。予VP方案化疗和相应对症治疗曾一度好转,但很快死于呼吸、循环功能衰竭。结论　ANKL易发生髓外浸润,并发MOF,病情凶险,预后差。     

EFFICIENT ACTIVATION OF ANTITUMOR IMMUNITY BY IL-6 GENE-MODIFIED LEUKEMIA VACCINE IN COMBINATION WITH LOW DOSE CYCLOPHOSPHAMIDE AND LOW DOSEIL-2

ＰｒｏｄｕｃｅｄＩＬ６ｉｓａｃｙｔｏｋｉｎｅｗｉｔｈｍｕｌｔｉｆｕｎｃｔｉｏｎＩｔｉｓｐｏｒｄｕｃｅｄｂｙａｎｕｍｂｅｒｏｆｃｅｌｔｙｐｅｓａｎｄｉｎｖｏｌｖｅｄｉｎｔｈｅｒｅｓｐｏｎｓｅｓｏｆｉｍｍｕｎｅｒｅｓｐｏｎｓｅ，ｈｅｍａｔｏｐｏｉｅｓ...     

In vitro granulocytic stem cell clonogenic capacity of marrow and spleen cells from mice infected with Rauscher leukemia virus.

Bone marrow and spleen cells from early, midstage, and terminal Rauscher leukemia virus (RLV-A)-infected erythroleukemic mice were assessed for granulocyte stem cell (CFU-c) clongenic capacity in the semisolid agar culture assay. It was found that marrow CFU-c concentrations exceeded normal in early stages of this erythroid disease but returned to near normal values during mid- and terminal phases. Splenic CFU-c concentrations, on the other hand, were generally higher than control values for all stages of the disease. These results are discussed with reference to the pathogenesis of RLV-A disease.     

骨髓基质细胞与白血病的关系及其在基因治疗中的研究进展

骨髓基质细胞(bone marrow stromal cells,BMSCs)具有多向分化的干细胞特征,体外易于分离和扩增,回输体内后可归巢于骨髓并通过自我更新而长期存活。骨髓基质细胞可以从多个方面影响白血病细胞的生物学特性,参与白血病的发病过程和病理过程。功能正常的骨髓基质细胞对白血病细胞具有直接的抗瘤效应。骨髓基质细胞具有显著的免疫调节功能。基因修饰的骨髓基质细胞回输体内后可在一定时期表达多种治疗性的外源目的基因蛋白。因此,骨髓基质细胞被认为是一种理想的治疗性细胞载体和白血病基因治疗中的靶细胞。干扰素是能够产生多向性生物学效应的细胞因子家族,在毛细胞白血病、慢性髓细胞性白血病治疗中疗效显著。本文就干扰素基因修饰的骨髓基质细胞对白血病的分子靶向治疗的可能性及前景作一简要综述。     

白血病干细胞与白血病的靶向治疗

  白血病是一类造血系统紊乱性疾病，现在已经认为这种造血紊乱起源于极少量的白血病干细胞。文章对白血病干细胞的认识现状及白血病干细胞学说对开展靶向白血病治疗策略的启示进行了综述。     

肺癌病人外周血T淋巴细胞亚群,NK活性和IL—2水平的研究

目的探讨肺癌不同阶段机体细胞免疫功能状态。方法选择肺癌病人27例（Ⅱ期以下13例，N期以上14例），健康对照14例。取外周静脉血提取单个核细胞（PBMC），单抗致敏红细胞花环法测定T淋巴细胞亚群；以K562细胞为靶细胞，3H－TdR法测定NK活性；用PHA刺激PBMC产生IL－2，IL－2依赖细胞3H－TdR掺入法检测。结果Ⅱ期以下肺癌组CD。百分比（％）、NK活性（u）和IL－2水平（u）分别为：32．92±10．85％；20．56±3．88u；10．62±2．64u。对照组分别为42．70±2．81％；28．50±2．29；19．39±3．13。二组三项指标对比P值均＜0．05．N期以上肺癌组三项指标分别为28．26±8．26％；16．24＋5．66u；9．59±2．70u，与对照组比较P值均＜0．01。结论肺癌病人的细胞免疫功能明显降低，随病情进展，纵膈及远隔器官转移的出现，免疫功能进一步下降。提示肺癌病人采用放疗或化疗的同时，应辅以免疫治疗。     

从白血病干细胞探讨中医中药治疗白血病的思路方法

白血病干细胞是能否治愈白血病的决定因素,白血病细胞可以通过化疗清除,而白血病干细胞却不能。白血病干细胞静止于龛内,不仅耐药,而且逃避免疫杀伤,但它表现出造血干细胞的"干性"、"定向性"和"迁移性"是白血病复发的主要根源。基于这些共识中医中药研究者应重新认识白血病细胞多药耐药和白血病复发,探求有效的白血病中医治疗新方法和新思路。     

从白血病干细胞探讨中医中药治疗白血病的思路方法

白血病干细胞是能否治愈白血病的决定因素,白血病细胞可以通过化疗清除,而白血病干细胞却不能。白血病干细胞静止于龛内,不仅耐药,而且逃避免疫杀伤,但它表现出造血干细胞的＂干性＂、＂定向性＂和＂迁移性＂是白血病复发的主要根源。基于这些共识中医中药研究者应重新认识白血病细胞多药耐药和白血病复发,探求有效的白血病中医治疗新方法和新思路。     

As2O3诱导白血病细胞凋亡过程中Ca2+的变化及其调节

目的研究Ca2 在As2O3诱导白血病细胞系NB4、K562、HL-60凋亡中的作用,及抗氧化剂NAC、NDMS、CAT和Cae 清除剂Quin 2对As2O3引起的Ca2 变化的调控作用.方法细胞内Ca2 用Fluo-3AM标记,并用流式细胞仪检测.结果0.6 μmol/L As2O3作用72 h能诱导NB4发生44.9%的凋亡,提高其浓度至2.7和8.1 μmol/L,也能诱导K562和HL-60分别发生58.3%和62.3%凋亡.As2O3能不同程度降低NB4和HL-60细胞内Ca2 水平,对K562细胞内Ca2 水平影响不大.抗氧化剂NAC、NDMS、CAT和Ca2 清除剂Quin 2对As2O3引起的NB4细胞Ca2 水平下降显示出抑制作用,对HL-60细胞内Ca2 水平下降无明显抑制作用.结论Ca2 水平下降在As2O3诱导的不同白血病细胞凋亡作用过程起着不同的作用.抗氧化剂NAC、NDMS、CAT和Ca2 清除剂Quin 2可不同程度地抑制As2O3诱导的NB4细胞凋亡时细胞内Ca2 水平下降.