Flt3配体胞外域的原核表达纯化及对脐血CD34~＋细胞的扩增作用

背景：Fms样酪氨酸激酶3配体（Flt3配体）是一种重要的生长因子,通过激活特定的酪氨酸激酶受体,调控造血细胞的生长、生存和/或分化,具有促进造血干细胞体外扩增的应用潜力。目的：构建pET32a（＋）-hFLext原核表达载体,表达、纯化hFLext蛋白,观察其对脐血CD34＋细胞的扩增作用。方法：克隆hFLext,构建pET32a（＋）-hFLext重组表达载体。转化大肠杆菌BL21,IPTG诱导蛋白表达,镍珠亲合层析纯化蛋白。磁珠分选脐血CD34＋细胞,单独加入hFLext或联合干细胞因子、血小板生成素孵育1周,观察体外扩增作用。结果与结论：成功克隆hFLext,并构建了pET32a（＋）-hFLext重组表达载体。在大肠杆菌BL21成功表达Trx-hFLext融合蛋白,经8mol/L尿素变性包涵体蛋白,逐步透析复性,镍珠亲合层析纯化蛋白,成功获得高纯度的Trx-hFLext融合蛋白。Trx-hFLext融合蛋白不仅具有维持及轻度刺激CD34＋细胞体外扩增的作用,并且与干细胞因子及血小板生成素具有协同作用,为造血干/祖细胞体外扩增研究奠定了基础。     

靶基因调控的脐血干/祖细胞体外长期扩增与调控

背景:脐血干细胞是基因治疗最理想的靶细胞之一,但基因转移率低下是目前面临的主要障碍.酪氨酸激酶JAK2在造血干/祖细胞自我更新中扮演着重要的作用,为了克服脐血基因转移率低下的障碍,根据基因调控表达技术原理,是否可开发一个可以靶向扩增JAK2基因修饰的脐血CD34 细胞体系.目的:探讨转基因JAK2介导的脐血干祖细胞长期扩增调控的可行性和安全性. 单位:卫生部北京医院血液科.材料:实验于2003-06/2006-04在卫生部北京医院血液科实验室完成.脐血取自健康、足月、自然分娩后立即断脐的脐血.脐血由北京医院妇产科提供,产妇及家属均知情同意,实验经医学伦理委员会批准.MiniMACS磁性分离仪、免疫磁珠吸附CD34单抗购自德国Miltenyi Biotec公司, 流式细胞仪购自美国FACScalibur,人重组干细胞因子、Flt3配体、人白介素-6、粒细胞-巨噬细胞集落刺激因子、粒细胞集落刺激因子、血小板生成素为PeproTec产品,SPF级裸鼠购自北京医科大学动物中心.方法:构建逆转录病毒载体MGI-F2JAK2,内含有JAK2基因的功能催化区和两个与小分子靶向基因合成药物(AP20187)结合的位点蛋白(2xF36v,F2)组成.AP20187可与F36v特异结合引起JAK2二聚化而激活细胞内信号传导.该载体同时含有绿色荧光蛋白报告基因,用作检测细胞增殖的标记.应用MiniMACS免疫磁珠分选系统纯化分离脐血CD34 细胞,用含JAK2的逆转录病毒上清转染脐血CD34 细胞.转导后的CD34 细胞在集落刺激因子、Flt3配体、血小板生成素、白介素-6细胞因子的联合培养条件下,以不加或加入AP20187分别作为对照组和实验组.主要观察指标:①应用流式细胞仪测定两组CD34 细胞中所含绿色荧光蛋白细胞的百分率,确定基因转移率.②扩增后的脐血祖细胞集落培养结果.③取培养10周的脐血CD34 细胞于裸鼠的胁部皮下注射,30 d后观察成瘤情况.结果:①实验组与对照组均可获得CD34 细胞大量扩增.随着培养时间的延长,实验组扩增的CD34 细胞GFP阳性率由基线水平逐渐上升于第11周时达到95%以上,而对照组绿色荧光蛋白报告基因阳性率逐渐下降到基线水平以下并逐渐消失.②实验组转基因CD34 细胞于12周后仍可产生造血祖细胞集落红系祖细胞、粒单系祖细胞、脐血多向造血祖细胞,所形成的造血祖细胞集落以粒单系祖细胞为主.③裸鼠实验无致瘤特性.结论:转染JAK2 基因的人脐血CD34 细胞协同其他细胞因子可以体外长期扩增脐血干祖细胞,对今后开展干细胞治疗某些遗传性血液病有潜在的应用价值.     

CD26多克隆抗体的制备与鉴定

目的针对CD26酶催化结构域制备多克隆抗体。方法应用RT-PCR技术以人白细胞mRNA为模板,扩增获取编码CD26催化结构域的基因序列,克隆入原核表达载体PET32a后,转化BL21感受态细菌,经IPTG诱导表达得到his-CD26融合蛋白;亲和层析柱纯化并经Western-blot鉴定后,用此重组蛋白免疫2只新西兰纯种大白兔,获取免疫血清共180 ml,经蛋白A柱纯化及抗原抗体亲和纯化后,采用间接ELISA法检测抗体效价,Western-blot及免疫细胞化学染色进行抗体效价及特异性鉴定。结果构建出PET32a/CD26原核表达质粒,并在大肠杆菌BL21中获得高效表达,经his亲合层析纯化后蛋白量达2.3 mg/ml;制备的多抗血清纯化后效价达256 000,经Western-blot证明能特异性的识别CD26重组蛋白,免疫细胞化学染色显示能特异的结合于H9细胞。结论成功制备了能特异性识别CD26的多克隆抗体。     

小鼠Foxp3融合蛋白的原核表达及纯化

目的克隆小鼠Foxp3(MFoxp3)基因,构建含该基因的原核表达载体,并表达、纯化出小鼠的Foxp3融合蛋白。方法RT-PCR方法从小鼠淋巴细胞扩增出小鼠Foxp3基因,连入T Easy Vector进行测序,然后将序列正确的小鼠Foxp3基因用酶切的方法从T Easy vector切下,并将其连接到用相同酶切,含有硫氧还蛋白(含6个组氨酸“标签”)的pET 32a( )原核表达载体中构建pET 32a( )-MFoxp3表达载体。用IPTG诱导转化pET 32a( )-MFoxp3表达载体的大肠杆菌BL21(DE3),并以镍螯合层析法纯化MFoxp3融合蛋白。结果经RT-PCR成功地克隆了1,290 bp的小鼠Foxp3基因,测序正确后转化大肠杆菌原核表达载体pET 32a( ),重组质粒在BL21(DE3)中成功表达出分子质量约为63 kD的融合蛋白,运用Ni-NTA方法纯化出目的蛋白,纯化后的蛋白纯度达80%,并用Western Blotting检测蛋白的灵敏度和特异性。结论构建了小鼠Foxp3的融合蛋白原核表达载体并成功表达与纯化出小鼠Foxp3的融合蛋白。     

重组人体干细胞因子的表达、复性、纯化及其对脐带血干细胞体外扩增作用研究

目的对重组人体干细胞因子（rhSCF）蛋白在脐带血干细胞体外扩增中的作用进行研究。方法通过大肠杆菌表达系统,经IPTG诱导表达获取SCF包涵体,经透析复性、纯化后获得蛋白。从脐带血中提取单核细胞（MNC）,分选其中具有CD34＋抗原表位的细胞,使用一定浓度的血小板生成素（TPO）、FLT-3配体（FL）及rhSCF组合对脐带血干细胞进行7 d体外扩增以研究rhSCF的作用。结果成功使用IPTG在大肠杆菌BL21诱导表达rhSCF,包涵体经复性及CM-Sepharose纯化获得高纯度的rhSCF。干细胞体外扩增实验表明rhSCF、FL及TPO联用,具有协同刺激CD34＋细胞体外扩增的作用。结论成功建立rhSCF原核表达、复性和纯化体系,为造血干细胞体外扩增体系的优化研究奠定了基础,同时为干细胞因子的临床应用提供了有价值的参考。     

原核表达重组人白介素-3及其对脐带血造血干细胞体外增殖的影响

目的研究原核表达重组人白介素-3(rhIL-3)及其对脐带血造血干细胞体外增殖的影响。方法构建pET-43.1-rhIL-3表达载体,克隆至BL21菌株,并进行中试发酵生产,所得包涵体经透析复性后,采用阳离子交换柱纯化,纯化产物送N-端测序。Ficoll密度梯度离心结合免疫磁珠法富集人脐带血中CD34+细胞,以不同浓度rhIL-3及干细胞因子(SCF)、Fms样酪氨酸激酶受体-3配体(FL)和促血小板生成素(TPO)因子组合培养1周,测定细胞总数,计算CD34+细胞增殖倍数。结果在SCF和FL存在下,rhIL-3可明显促进造血干祖细胞增殖,但较无rhIL-3时CD34+细胞占细胞总数的比例有所下降。与SCF、FL共同作用时的最佳浓度在15 ng/mL左右,与SCF、FL、TPO共同作用时的最佳浓度在50 ng/mL左右。结论高效表达及纯化的rhIL-3与SCF、FL和TPO细胞因子组合对脐带血造血干细胞具有良好的增殖作用。     

细胞因子介导的人脐血单个核细胞体外扩增并重建NOD/SCID小鼠造血及免疫功能

背景:细胞因子介导的脐血造血细胞体外扩增有望能解决脐血移植数量不足的问题.目的:实验拟确立在无基质培养条件下体外扩增脐血单个核细胞最合适的细胞因子组合及干细胞因子、FLT3配基协同促聚核细胞生成因子对造血及免疫重建功能的影响.方法:将新鲜脐血标本分离的单个核细胞接种于含有不同细胞因子组合的无血清无基质培养体系中培养7 d,根据不同细胞因子组合分组,将3因子干细胞因子+FLT3配基+促聚核细胞生成因子组合在无血清无基质条件下扩增培养7 d前后的脐血单个核细胞移植给经亚致死量照射的NOD/SCID小鼠.在扩增培养0,7 d检测脐血CD34+细胞数及CD34+CXCR4+,CD34+CD62L+,CD34+CD49d+的细胞数.脐血移植6周后通过流式细胞仪,PCR法检测存活小鼠体内的人源性细胞.结果与结论:移植6周后,存活小鼠体内均可检测到人源性CD45+细胞,扩增脐血移植组的NOD/SCID小鼠存活率和人特异性基因捡出率均高于新鲜脐血移植组和高于生理盐水移植组(P<0.05).扩增脐血组存活NOD/SCID小鼠骨髓中可检测到人髓系细胞(CD33+),T淋巴细胞(CD4+),B淋巴细胞(CD19+)和人造血干细胞成分(CD34+)细胞的表达.结果提示干细胞因子+FLT3配基+促聚核细胞生成因子3因子组合脐血造血细胞体外扩增是最合适的细胞因子组合,其扩增的脐血单个核细胞能够植入并重建NOD/SCID小鼠的造血及免疫功能.     

人BIGH3基因克隆表达对兔角膜基质细胞黏附和迁移的影响

背景:BIGH3蛋白位于角膜上皮和基质层,在基质层高表达,先前研究已经发现BIGH3蛋白能促进角膜上皮创伤愈合,为此进一步探讨其对角膜基质创伤愈合的影响.目的:构建人BIGH3基因的原核表达载体,观察其对角膜细胞与细胞外基质黏附和迁移的作用.方法:PCR扩增BIGH3基因的ORF阅读框,并通过Kpn Ⅰ和Sal Ⅰ插入到原核表达载体pET32a(+)中.经PCR、酶切和序列测定方法鉴定重组质粒.将重组质粒转入BL21(DE3)表达,IPTG诱导表达人BIGH3融合蛋白,并进行SDS-PAGE电泳分析和Western blot检测分析:以Ni-NTA树脂对蛋白纯化与复性,作用于体外培养的兔角膜细胞,用MTT法分析其对细胞与细胞外基质黏附的影响,采用改良的Boyden微孔膜双槽法观察BIGH3蛋白对角膜细胞迁移的影响.结果与结论:重组质粒经PCR、酶切与DNA测序证实插入了pET32a(+)载体中.通过IPTG诱导,成功的表达融合蛋白在包涵体中,经SDS-PAGE电泳分析,出现了一条新生的蛋白条带,Western blot也证实了该蛋白具有与BIGH3抗体特异性的结合能力.MTT法和Boyden微孔膜双槽法分别证明所表达的BIGH3融合蛋白可促兔角膜细胞黏附和迁移.成功构建了人BIGH3重组融合蛋白表达质粒,纯化了该基因的原核表达产物,并通过重组融合蛋白BIGH3能促进兔角膜细胞与细胞外基质黏附及迁移,而验证了重组BIGH3蛋白的活性.     

不同细胞饲养层支持脐血CD34＋细胞体外扩增的效果比较

背景:成纤维细胞饲养层与基质细胞饲养层一样能分泌多种细胞因子,其中包括起正性作用和负性作用的细胞因子,它们对共培养体系内的干/祖细胞起双向调节作用,但何时表现为抑制性或促进性作用目前尚无定论.目的:分析比较骨髓基质细胞及皮肤成纤维细胞饲养层对脐血CD34+细胞体外扩增的影响,以了解共培养体系对脐血CD34+细胞体外扩增的支持作用.设计、时间及地点:细胞学体外对照观察,于2003-08/2004-05在广东医学院附属医院中心实验室完成.材料:原代成纤维细胞、骨髓标本由广东医学院附属医院提供,脐血标本由广东医学院附属医院妇产科及湛江市妇幼保健院妇产科提供.方法:体外分离培养人脐血单个核细胞,应用免疫磁珠(阳性)分选法提取脐血CD34+细胞.设立3组:液体培养对照组为脐血CD34+细胞(3×108L-1)+RPMI 1640培养液(含体积分数为10%牛血清白蛋白、10 μg/L粒-巨噬细胞集落刺激因子、10 μg/L干细胞因子、10 μg/L白细胞介素3),接种于24扎培养板;骨髓基质细胞饲养层组、成纤维细胞饲养层组分别将体外分离培养的第4代人骨髓基质细胞,皮肤成纤维细胞接种于24孔培养板,70%融合时加入等量脐血CD34+细胞及相同成分的RPMI 1640培养液.主要观察指标:脐血CD34+细胞增殖情况与集落形成能力.结果:与骨髓基质细胞饲养层组比较,液体培养对照组、成纤维细胞饲养层组脐血CD34+细胞增殖率及集落形成能力均显著降低(P均＜0.01):后2组间比较差异无显著性意义(P＞0.05).结论:骨髓基质细胞饲养层培养体系能促进脐血CD34+细胞体外扩增,并可以较好地保持脐血CD34+细胞的干细胞特性,效果显著优于成纤维细胞饲养层及液体培养体系.     

抗CD133单链抗体/白喉毒素DAB389融合蛋白的基因构建、表达、纯化及鉴定

目的构建抗CD133单链抗体(scFV)/白喉毒素DAB389融合蛋白,在大肠杆菌里表达,并鉴定及纯化。方法采用PCR的方法,扩增融合基因DAB389-Linker-CD133(scFV),并插入到原核表达载体pET28a中,构建了重组表达载体pET28a-DAB389-CD133,融合蛋白在BL21(DE3)中表达,表达产物经SDS-PAGE鉴定,通过Ni柱纯化目的蛋白,获得了纯度达95%的DAB389-Linker--CD133(scFV)融合蛋白。Western blot鉴定蛋白活性。流式细胞仪检测能否与CD133抗原特异性结合。结果扩增的片段与理论值一致,序列分析正确;纯化得到纯度为95%的重组蛋白。Western blot分析能特异性地与抗白喉抗体结合。流式细胞仪检测能与CD133抗原特异性结合。结论成功构建了抗CD133单链抗体(scFV)/白喉毒素DAB389融合蛋白,具有结合抗原和免疫毒素双重活性,为进一步靶向治疗奠定了基础。     

Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo

Stimulation of Flt3 receptor tyrosine kinase through its cognate ligand expands early hematopoietic progenitor and dendritic cells (DCs) in humans and mice. The exact developmental stages at which hematopoietic progenitors express Flt3, are responsive to its ligand, and subsequently develop to DCs, are not known. Here we show that common lymphoid and common myeloid progenitors, as well as steady state DCs in thymus, spleen, and epidermis, express Flt3. The receptor is down-regulated once definitive B cell, T cell, and megakaryocyte/erythrocyte commitment occurs, and Flt3 is not detectable on other steady state hematopoietic cell populations. Upon in vivo Flt3 ligand (Flt3L) administration, Flt3+ progenitor cells and their progeny DCs are expanded, whereas Flt3- downstream progenitors are not, or are only slightly increased. Transplantation of common lymphoid and common myeloid progenitors and subsequent Flt3L injection increases progeny DCs of both precursor populations. These findings provide a definitive map of Flt3 expression in the hematopoietic hierarchy and directly demonstrate that Flt3L can drive DC development along both the lymphoid and myeloid developmental pathways from Flt3+ progenitors to Flt3+ DCs.     

人胎肝FLT3配基膜外区基因的克隆及其在COS-7细胞中的表达

FLT3是表达于造血干、祖细胞及胎盘、脑和睾丸等组织的一种Ⅲ型酪氨酸激酶受体。FLT3配基(FL)对造血祖细胞有协同刺激作用。本研究用反转录-聚合酶链反应(RT-PCR)方法从胎肝组织克隆了FL膜外片段cDNA,其序列与从T淋巴细胞系及胸腺基质细胞系中获得的FL克隆序列完全相同。用真核细胞表达载体pCMV4在COS-7细胞中表达了FL膜外片段。重组人FL(rhFL)可协同GM-CSF增加骨髓细胞中CFU-GM的产率。     

Bcr/Abl expression stimulates integrin function in hematopoietic cell lines.

Cell adhesion to the extracellular matrix is largely mediated by adhesion molecules of the integrin family and is often diminished upon oncogenic transformation. However, we show here that the chronic myelogenous leukemia oncogene Bcr/Abl has positive effects on VLA-4 and VLA-5 integrin function. The presence of Bcr/Abl in the GM-CSF- or IL-3-dependent hematopoietic cell lines MO7e, 32D, and BaF/3 enhanced cell binding to both soluble and immobilized fibronectin. The effect was due to enhanced function of the VLA-5 integrin fibronectin receptor and not to increased surface expression. In parallel, Bcr/Abl stimulated cell adhesion to the VLA-4 integrin ligand VCAM-1. Stimulation of VLA-5 function directly correlated with induction of Bcr/Abl tyrosine kinase activity in a temperature-sensitive kinase mutant. Thus, Bcr/Abl stimulates integrin-dependent cell adhesion, by a mechanism involving increased ligand binding, with the tyrosine kinase activity of Bcr/Abl likely playing a key role. Consistent with these results, hematopoietic precursor cells from chronic myelogenous leukemia patients also showed increased adhesion to fibronectin.     

粒—巨噬系集落刺激因子和红细胞生成素诱导JAK2酪氨酸磷酸化…

目的：确定与细胞内蛋白质酪氨酸磷酸化相关的激酶。方法：用粒－巨噬系集落刺激因子（ＧＭ－ＣＳＦ）和红细胞生纱（Ｅｐｏ）依赖细胞株ＵＴ－７以及Ｅｐｏ依赖细胞株ＵＴ－７／Ｅｐｏ进行酪氨酸磷酸化的研究。结果：当ＧＭ－ＣＳＦ或Ｅｐｏ刺激其生长依赖细胞时可快速诱导分子量为１４５０００，１３００００，８００００和４００００等多种蛋白质酷氨酸磷酸化。鉴定了分子量为１３００００的酪氨酸磷酸化的蛋白质为ＪＡＫ２，一种     

粒-巨噬系集落刺激因子和红细胞生成素诱导 JAK2 酪氨酸磷酸化并使之激活

目的：确定与细胞内蛋白质酪氨酸磷酸化相关的激酶。方法：用粒－巨噬系集落刺激因子（ＧＭ－ＣＳＦ）和红细胞生成素（Ｅｐｏ）依赖细胞株ＵＴ－７以及Ｅｐｏ依赖细胞株ＵＴ－７／Ｅｐｏ进行酪氨酸磷酸化的研究。结果：当ＧＭ－ＣＳＦ或Ｅｐｏ刺激其生长依赖细胞时可快速诱导分子量为１４５０００，１３００００，８００００和４００００等多种蛋白质酪氨酸磷酸化。鉴定了分子量为１３００００酪氨酸磷酸化的蛋白质为ＪＡＫ２，一种非受体型酪氨酸激酶。同时，ＧＭ－ＣＳＦ或Ｅｐｏ刺激亦可激活ＪＡＫ２激酶活性。酪氨酸磷酸化和激活ＪＡＫ２只发生在ＧＭ－ＣＳＦ或Ｅｐｏ生长依赖细胞。结论：ＪＡＫ２信号传导途径在ＧＭ－ＣＳＦ和Ｅｐｏ诱导的细胞增殖中起重要作用。     

Induction of potent TRAIL-mediated tumoricidal activity by hFLEX/Furin/TRAIL recombinant DNA construct.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to exert selectively cytotoxic activity against many tumor cells but not normal cells. On the other hand, the ligand for the receptor tyrosine kinase Fms-like tyrosine kinase 3 (Flt3L) is a growth factor for hematopoietic progenitors and is a potent stimulating factor for dendritic and NK cells. Previously, we have demonstrated that it is possible to inhibit the outgrowth of primary tumors by the administration of an hFlex (the extracellular domain of the Flt3L) and TRAIL (amino acid residues 95-281) secreted fusion protein. Here, we report that by the insertion of a linker sequence encoding the cleavage site for the Golgi-expressed endoprotease furin between the DNA sequences encoding hFlex and TRAIL, the tumoricidal activity of the cleaved TRAIL protein generated was greatly enhanced in comparison to the hFlex/TRAIL fusion protein. Furthermore, we demonstrate that intratumoral injection of the hFlex/furin/TRAIL DNA, in conjunction with cationic liposomes, significantly suppressed the outgrowth of the human CNE-2 nasopharyngeal tumor xenografts in SCID mice. In situ histological examinations confirmed the expression of TRAIL in the treated tumor nodules and the induction of apoptosis was also evidenced by the presence of numerous pyknotic nuclei.     

Stimulation of Fc gamma RIIIA results in phospholipase C-gamma 1 tyrosine phosphorylation and p56lck activation

Binding of ligand to the alpha subunit of Fc gamma RIIIA(CD16), expressed at the natural killer (NK) cell membrane in association with homo or heterodimers of proteins of the zeta family, results in phosphorylation of several proteins on tyrosine residues. We have analyzed the role of protein tyrosine phosphorylation in the regulation of molecular events induced upon stimulation of Fc gamma RIIIA in NK cells and in T cells expressing the Fc gamma RIII alpha chain in association with endogenous zeta 2 homodimers and devoid of other (CD3, CD2) transducing molecules. Our data indicate that treatment of these cells with protein tyrosine kinase inhibitors prevents not only Fc gamma RIIIA-induced protein tyrosine phosphorylation but also phosphatidylinositol 4,5 diphosphate hydrolysis and increased intracellular Ca2+ concentration, indicating a primary role of tyrosine kinase(s) in the induction of these early activation events. Occupancy of Fc gamma RIIIA by ligand results in phospholipase C (PLC)-gamma 1 tyrosine phosphorylation in NK cells and in Fc gamma RIIIA-transfected CD3-/CD2- T cells, and induces functional activation of p56lck in Fc gamma RIIIA alpha/zeta 2-transfected T cells, suggesting the possibility that the receptor-induced PLC-gamma 1 activation occurs upon phosphorylation of its tyrosine residues mediated by this kinase and is, at least in part, responsible for the signal transduction mediated via CD16 upon ligand binding.     

Phosphatidylinositol-3 kinase activation induced upon Fc gamma RIIIA- ligand interaction

Induced activation of protein tyrosine kinase(s) is a central event in signal transduction mediated via the low affinity receptor for IgG (Fc gamma RIIIA, CD16) in natural killer (NK) cells. Tyrosine phosphorylation may affect the function of several protein directly, or indirectly by inducing their association with other tyrosine phosphorylated proteins. Here, we report that Fc gamma RIII stimulation induces activation of phosphatidylinositol (PI)-3 kinase in NK cells. Phosphotyrosine immunoprecipitates from Fc gamma RIII-stimulated NK cells contain PI-kinase activity and PI-3 kinase can be directly precipitated from them. Conversely, a series of tyrosine-phosphorylated proteins is coprecipitated with PI-3 kinase from the stimulated, but not from control cells. Analogous results obtained using Jurkat T cells expressing transfected Fc gamma RIIIA alpha ligand binding chain in association with gamma 2 or zeta 2 homodimers indicate that both complexes transduce this effect, although the Fc gamma RIIIA-zeta 2 complexes do so with greater efficiency. Accumulation of phosphoinositide D3 phosphorylated products in stimulated cells confirms PI-3 kinase activation, indicating the participation of this enzyme in Fc gamma RIIIA-mediated signal transduction.