IL-27基因转染对小鼠结肠腺癌细胞生物学特性的影响

目的:获得稳定表达小鼠IL-27(mIL-27)基因的小鼠结肠腺癌细胞株(Colon26),研究IL-27基因对小鼠结肠腺癌细胞体内外生物学特性的影响。方法:通过脂质体法将pcDNA3.1( )-His(B)/mIL-27质粒转染结肠腺癌细胞,筛选建立高表达细胞株。采用Western blot、ELISA、RT-PCR法证实IL-27基因成功转染Colon26。用MTT比色法检测IL-27质粒转染后对Colon26细胞生长的影响;流式细胞术检测IL-27质粒转染后对Colon26细胞凋亡的影响;将Colon26-IL-27细胞接种于BALB/c小鼠的右侧背部皮下,观察其致瘤性。结果:Colon26-IL-27细胞可表达IL-27mRNA并分泌IL-27,IL-27的表达在体外对Colon26细胞的增殖、凋亡均无影响,而在体内则具有明显的抗瘤活性。结论:成功制备Colon26-IL-27细胞株,证明IL-27能在体内明显抑制肿瘤的生长,延长荷瘤小鼠的生存期,具有一定的抗肿瘤作用。     

杂色曲霉素对小鼠脾细胞IL-2及IFN-γ分泌和表达的影响

目的：探讨杂色曲霉素(ST)对体外培养的小鼠脾细胞IL-2及IFN-γ mRNA表达及其蛋白分泌的影响。方法：分别采用半定量RT-PCR及ELISA方法，研究5种不同剂量ST(0．125mg／L，0．25mg／L，0．5mg／L，1mg/L，2mg／L)预处理对小鼠脾细胞IL-2及IFN-γ mRNA表达及其蛋白分泌的影响。结果：不同剂量ST预处理2h均可引起小鼠脾细胞IL-2及IFN-γ表达的改变，但不同剂量影响不同，小剂量ST处理组(0．125、0．25mg/L)在ST处理后2h，可诱导脾细胞IL-2及IFN-γmRNA的表达；而大剂量ST处理组(1mg／L、2mg／L)可明显抑制脾细胞IL-2及IFN-γmPuNA表达及其相应蛋白的分泌，尤以ST 1mg/L的抑制作用最明显。结论：ST可影响小鼠脾细胞IL-2及IFN-γ表达和分泌的改变，较小剂量组(0．12．5、0．25mg／L)表现为诱导作用，而较大剂量组(1mg/L、2mg／L)则表现为明显抑制作用。     

稳定转染小鼠IL-17全长基因的结肠癌细胞株的建立及基因转染对C26细胞的影响

目的建立稳定转染小鼠IL-17基因全长的小鼠结肠癌C26细胞株并进行鉴定。方法脂质体法将携带小鼠IL-17基因全长的真核表达载体pc DNA3.1转染小鼠结肠癌细胞C26,经G418筛选出稳定表达IL-17的细胞株,镜下观察细胞形态,RTPCR法、免疫荧光法检测目的基因和蛋白的表达;划痕修复实验检测细胞的迁移能力,MTS法检测细胞体外增殖能力。结果获得1株稳定表达IL-17的C26细胞,C26/IL-17细胞高表达IL-17基因及蛋白,证明该细胞可表达IL-17;IL-17高表达可以增强C26细胞的迁移能力,减弱该细胞的体外增殖能力。结论成功建立了稳定转染IL-17基因的小鼠结肠癌细胞株。     

小鼠白细胞介素17A的原核表达及对RAW264.7细胞分泌炎症因子的影响

目的 在大肠杆菌中高效表达与纯化小鼠白细胞介素17A(mIL-17A),并研究其对巨噬细胞分泌炎症因子的影响.方法 以活化的脾细胞为模板,通过RT-PCR法扩增小鼠IL-17a基因的编码序列,构建重组表达质粒pET28a/mIL-17a,并在大肠杆菌(E.coli)中诱导表达,经亲和层析获得纯化的mIL-17A蛋白.以mIL-17A蛋白刺激体外培养的鼠巨噬细胞RAW264.7,72 h后应用realtime PCR法分析IL-6、巨噬细胞炎症蛋白1(Ccl3)、巨噬细胞炎症蛋白2(Cxcl3)、β防御素2(defensinβ2)的mRNA表达量,并用ELISA法检测培养上清中Ccl3、Cxcl3、IFN-γ、IL-4及IL-6的蛋白质表达水平.结果 在E.coli中成功高效表达了有生物活性的IL-17A蛋白,可促进体外培养的RAW264.7细胞IL-6、Cxcl3、defensin β2 mRNA的表达,并能促进Ccl3、Cxcl3、IFN-γ、IL-4以及IL-6蛋白的表达.结论 成功表达并制备了具备生物学活性的mIL-17A,该蛋白具有刺激巨噬细胞表达趋化因子、防御素及细胞因子的能力.     

小鼠白细胞介素21 cDNA的克隆及真核表达质粒的构建

目的：克隆小鼠自细胞介素2l(IL-21)基因，构建真核表达质粒，用以进行肿瘤的基因治疗。方法：用RT-PCR法。从ConA活化的小鼠T细胞中扩增IL-21 cDNA。克隆人哺乳动物细胞高效表达质粒pcDNA3．1中，构建重组mIL-21真核表达质粒。重组体用载体上的通用引物和PCR下游引物为测序引物，鉴定克隆的正确性。将已鉴定的重组质粒用脂质体法转染Sp2／0细胞，用RT-PCR法鉴定转染细胞中IL-21基因的表达，用MTT比色法检测表达的mIL-21诱导的NK细胞杀伤活性的增强。结果：正确构建了重组真核表达质粒pcDNA3．1／mIL-21，并在转染的细胞中检测出IL-2l的表达，表达的mIL-21可在体外增强NK细胞的杀伤活性：结论：成功地构建了重组真核表达质粒pcDNA3．1／mIL-21，为进一步在肿瘤动物模型中进行IL-21基因治疗及疗效观察奠定了基础.     

PIKA佐剂在体内外诱导小鼠免疫应答的探讨

目的：探讨PIKA佐剂在体内外诱导小鼠的免疫应答。方法：体外将小鼠脾淋巴细胞与不同浓度PIKA佐剂培养后，ELISA检测细胞因子IFN-γ、IL-12p40的产生，FACS检测细胞的增殖及活化。体内将PIKA佐剂经小鼠腹腔注射后，检测不同时间点血清中IFN-γ、IL-12pao、IL-6、TNF-α等细胞因子的产生。结果：PIKA佐剂体外呈剂量依赖性诱导小鼠脾细胞产生IFN-γ和IL-12p40。细胞亚群分析的结果表明，PIKA可显著刺激B细胞和NK细胞活化、增殖。体内注射PIKA佐剂后可诱导细胞因子IFN-γ、IL-12pao、IL-6、TNF-α的产生，但产生的时间点不同。结论：PIKA佐剂体内外直接通过诱导细胞因子的产生，B细胞和NK细胞的活化和增殖，而促进免疫应答反应。     

模拟mIL-18天然生成真核表达体系的构建及生物活性初探

目的：构建模拟小鼠IL-18(mIL-18)天然生成真核表达体系，并探讨其与人IL-2(hIL-2)真核表达载体pVR1012-hIL-2(phIL-2)体内外共转染对细胞免疫功能的影响。方法：分别构建小鼠IL-18前体(mproIL-18)和小鼠IL-1β转换酶(mICE)的真核表达载体pVR1012-mpmIL-18(pmproIL-18)和pEGFP-N1-mICE(pmICE)。两者单独或共转染COS-7细胞，分别在mRNA和蛋白水平检测IL-18的表达；将pmproIL-18、pmICE和phIL-23种真核表达载体分别通过体内、外共转染，初步检测其生物学活性。结果：pmproIL-18及pmICE构建正确，分别转染COS-7细胞后能检测到相应mRNA表达。在pmproIL-18单独或pmproIL-18／pmICE共转染的COS-7细胞中能检测到前体和成熟2种不同形式的mIL-18蛋白表达，并且mIL-18能分泌到上清；所分泌的mIL-18与hIL-2体外联合作用可增强小鼠脾细胞增殖能力。pmproIL-18、pmICE和phIL-23种真核表达载体联合肌肉注射的小鼠脾细胞体外杀伤同基因型肿瘤细胞的能力显著提高。结论：构建的模拟mIL-18天然生成的真核表达体系(pmproIL-18／pmICE)与phiL-2共转染有可能用于肿瘤的基因治疗。     

逆转录病毒介导的小鼠IL-23基因在鼠高转移性乳腺癌细胞系MA-891中的表达

目的：建立表达小鼠IL-23基因的鼠乳腺癌细胞系IL-23／MA-891，探讨外源性IL-23基因对MA-891细胞生长及其生物学性状的影响。方法：将插入IL-23基因的质粒，应用逆转录病毒载体LXSN经感染皿（ecotropic）和PA317（amphotropic）两种包装细胞包装，经G418筛选获得表达IL-23分子的PA317阳性细胞克隆，用IL-23／PA317培养上清转染MA-891细胞，获得表达IL-23的IL-23／MA-891细胞。分别用RT-PCR法、ELISA法和免疫组化染色法分析IL-23／MA-891细胞表达IL-23的mRNA和蛋白的水平，筛选出高表达IL-23的IL-23／MA-891细胞克隆用于下层研究中；用流式细胞仪检测MA-891细胞中MI-ICⅠ、MI-ICⅡ、CD80、CD86以及FAS蛋白的表达、细胞周期变化及细胞凋亡情况；用ELISA法检测IL-23／MA-891细胞培养上清诱导脾细胞分泌IFN-γ的能力，用MTT比色法检测细胞增殖反应。结果：外源性IL-23基因转染的小鼠乳腺癌细胞系IL-23／MA-891，在mRNA水平和蛋白水平均可获得稳定表达；IL-23／MA-891细胞与LXSN／MA-891和MA-891比较，细胞周期及细胞凋亡率无显著性差异（P〉0．05），H-2Kb（MHCⅠ类分子）、Ⅰ—Ab（MHCⅡ／类分子）、CD80、CD86以及FAS蛋白的表达水平无显著性差异（P〉0．05）；IL-23／MA-891细胞的增殖反应与LXSN／MA-891和MA-891细胞相比虽有所降低，但无显著性差异（P〉0．05）。然而，IL-23／MA-891细胞的培养上清可明显促进小鼠脾细胞分泌IFN-γ（P〈0．01）。结论：建立的稳定表达IL-23的IL-23／MA-891细胞，具有较强的免疫学调节活性，其生物学形状与MA-891和LXSN／MA-891细胞相比较没有明显差异，但可进一步用于肿瘤相关免疫基因治疗的研究。     

IL-18基因转染对小鼠卵巢癌OVHM细胞免疫生物学特性的影响

目的 分析转染IL-18基因后,小鼠卵巢癌OVHM细胞的IL-18 mRNA及蛋白表达、培养上清中IL-18含量、IFN-γ诱生能力等免疫生物学特性的变化,初步探讨IL-18基因转染治疗卵巢癌的应用价值.方法 将逆转录病毒携带的小鼠活性IL-18基因转染至OVHM(OVHM/IL-18),以空载体转染的OVHM(OVHM/LXSN)和野生型OVHM作为对照.分别采用RT-PCR及免疫细胞染色法检测3组细胞的IL-18 mRNA及蛋白表达;ELISA方法测定细胞培养上清中IL-18的含量、及其诱生小鼠脾细胞产生IFN-γ的能力.结果 OVHM/IL-18细胞中可检测到IL-18 mRNA及蛋白的表达,而OVHM/LXSN和野生型OVHM细胞中未见表达.OVHM/IL-18细胞可分泌IL-18,于培养24 h时达高峰(138.25 pg/mL±12.36 pg/mL),之后逐渐减弱;OVHM/LXSN和野生型OVHM细胞上清中未检测出IL-18.0VHM/IL-18细胞培养上清诱生IFN-γ的水平明显高于VHM/KXSN和野生型OVHM细胞(分别为33.84 pg/mL±2.36 pg/mL、18.80 pg/mL±1.06 pg/mL、18.43 pg/mL±2.64 pg/mL,P<0.01),VHM/LXSN与野生型OVHM细胞之间未见显著差异(P0.05).结论 IL-18基因转染可有效提高小鼠卵巢癌细胞IL-18mRNA的表达及其蛋白的合成释放、诱导小鼠脾细胞产生IFN-γ.IL-18基因转染可纠正肿瘤细胞的IL-18低表达状态,诱导IL-18的抗瘤效应,可能对抑制肿瘤生长、促进肿瘤消退有一定的应用价值.     

小鼠白细胞介素12基因转染及其在黑色素瘤细胞B16F10中的表达

目的:探索小鼠白细胞介素12(mIL-12)基因在小鼠黑色素瘤B16F10细胞中的表达。方法:应用DNA重组技术将mIL-12基因插入pcDNA3.1真核表达载体中, 通过电穿孔转染B16F10细胞, 筛选出阳性细胞克隆后, 应用PCR、RT-PCR及Westernblot技术检测mIL-12基因在B16F10细胞中的整合及表达。结果:在DNA、mRNA及蛋白质3个水平均证实mIL-12基因已转染到B16F10细胞中并表达。结论:mIL-12基因可成功地转染体外培养的B16F10细胞并表达, 为进一步研究IL-12基因修饰的肿瘤细胞的基因瘤苗奠定了基础。     

Antitumor activity and immune enhancement of murine interleukin-23 expressed in murine colon carcinoma cells

Interleukin （IL）-23, a cytokine composed of p19 and the p40 subunit of IL-12, can enhance the proliferation of memory T cells and production of IFN-γ from activated T cells. It can also induce antitumor effects in murine model. To further evaluate the antitumor activity and immune enhancement of IL-23 in vivo, murine colon carcinoma cells retrovirally transduced with mIL-23 gene were injected subcutaneously （s.c.） into BALB/c mice. Survival time and tumor volume were observed. LDH release assay, [^3H]-TdR incorporation assay and ELISA were used to determine CTL activity, proliferation of splenocytes and level of cytokines, respectively. Number of dendritic cells （DCs） was analyzed by flow cytometry （FCM）. IL-23 secreted by Colon26/IL-23 cells suppressed the growth of tumor and prolonged the survival time of mice, enhanced proliferation of splenocytes, CTL activity, and number of DCs. IL-23 also promoted the production of Thl cytokines such as IFN-γ, IL-12 and TNF-α. However, the level of IL-4 was not enhanced significantly. These data suggested that IL-23 secreted by tumor cells can induce antitumor activitv bv enhancing immune resnonse.     

The secreted form of the p40 subunit of interleukin (IL)-12 inhibits IL-23 functions and abrogates IL-23-mediated antitumour effects

Interleukin (IL)-23 is a heterodimeric cytokine consisting of a novel p19 molecule and the p40 subunit of IL-12. Since secreted p40 can act as an antagonist for IL-12, we investigated whether p40 also inhibited IL-23-mediated immunological functions. p40 did not induce interferon (IFN)-gamma or IL-17 production from splenocytes but impaired IL-23-induced cytokine production by competitive binding to the IL-23 receptors. Furthermore, a mixed population of murine colon carcinoma Colon 26 cells transduced with the p40 gene and those transduced with the IL-23 gene developed tumours in syngenic mice, whereas the IL-23-expressing Colon 26 cells were completely rejected. p40 also suppressed IFN-gamma production of antigen-stimulated splenocytes and IL-23-mediated cytotoxic T-lymphocyte activities in the mice that rejected Colon 26 cells expressing IL-23. p40 can thereby antagonize IL-23 and is a possible therapeutic agent for suppression of IL-23 functions.     

逆转录病毒介导IL-18基因在大鼠胶质瘤细胞C6中的表达

目的 :建立表达IL 18基因的大鼠胶质瘤细胞C6 /IL 18,并探讨外源性IL 18基因对C6细胞生长的影响。方法 :应用逆转录病毒载体 ,将IL 18基因导入C6细胞。经G4 18筛选后 ,获得表达IL 18分子的细胞克隆C6 /IL 18。用RT PCR法检测目的基因mRNA的表达 ;用流式细胞和免疫细胞化学染色法检测目的蛋白的表达。用ELISA法检测C6 /IL 18细胞培养上清诱导脾细胞分泌IFN γ的能力 ,以确定IL 18的生物学活性。用MTT比色法检测细胞体外增殖的状况 ,用流式细胞技术检测细胞的增殖活性 ,并建立大鼠胶质瘤模型 ,观察C6 /IL 18细胞体内致瘤性的改变。结果 :外源IL 18基因于mRNA水平和蛋白水平可获得稳定表达 ,并可诱生大鼠脾细胞分泌IFN γ。同时 ,该细胞系的体外增殖率及体内致瘤性 ,均较亲代C6胶质瘤细胞明显下降。结论 :外源性IL 18基因能部分地抑制C6细胞的体外增殖率和体内致瘤性。建立了可进一步用于相关肿瘤基因免疫和基因治疗研究的大鼠胶质瘤细胞系C6 /IL 18。     

Expression of interleukin-22 in murine carcinoma cells did not influence tumour growth in vivo but did improve survival of the inoculated hosts

Interleukin (IL)-22, a novel cytokine belonging to the IL-10 family, is secreted from activated T and natural killer cells and is possibly involved in inflammatory responses. We examined whether expression of the IL-22 gene in murine colon carcinoma Colon 26 cells (Colon 26/IL-22) could produce any antitumour effects in the inoculated mice. Although growth of Colon 26/IL-22 tumours in syngeneic mice was not different from that of parent tumours, survival of the mice that were subcutaneously or intraperitoneally inoculated with Colon 26/IL-22 tumours was significantly prolonged compared with the mice inoculated with parent tumours. Metastasis was not influenced by IL-22 expressed in tumours. Expression of the IL-22 receptor-specific gene, IL-22R, was not induced in spleen cells stimulated with concanavalin A, anti-CD3 or anti-CD40 antibody, despite constitutive expression of the IL-10R2 gene, which encodes another component of the heterodimeric IL-22 receptor complex. IL-22 thereby does not directly act on immunocompetent cells, and IL-22 expressed in tumours can favour apothanasia of inoculated hosts.     

Preliminary study on mouse interleukin-21 application in tumor gene therapy

lnterleukin-21(IL-21)is a recently characterized T cell-derived cytokine with a significant homology to IL-2,IL-4 and IL-15.To determine whether IL-21 has broad immunoregulatory activity and can stimulate durableantitumour responses,we constructed mouse IL-21(mIL-21) recombinant plasmid and evaluated its antitumorefficacy.Mouse IL-21 cDNA was amplified from Con A-activated mouse T cells by RT-PCR.RecombinantpcDNA3.1/mIL-21 was constructed and transfected into Sp2/0 cells.Mouse IL-21 expression was analyzed byWestern blotting and its activities were detected by ~3H-TdR incorporation and MTT assay.The recombinantpcDNA3.1/mIL-21 was injected s.c.into tumor lump.Tumor size,weight,the activities of CTLs,NK cells andLAK cells and serum IFN-γ,level were measured for evaluating mIL-21 mediated antitumor responses.Theresults indicated that mIL-21 was correctly expressed in Sp2/0 cells and it can improve the proliferation of Tcells and B cells,and enhance NK cytotoxic activity in vitro.The activities of CTL and NK cells,and serumIFN-γlevel were significantly improved,furthermore the tumor growth was obviously suppressed inpcDNA3.1/mIL-21 treated mice.However,the LAK activity did not alter significantly.Taken together,thisstudy suggests that the injection with recombinant plasmid containing mIL-21 is a potential approach fortumor gene therapy.Cellular & Molecular Immunology.2004;1(6):461-466.     

Lactic acid bacterium potently induces the production of interleukin-12 and interferon-gamma by mouse splenocytes

We previously have reported that the lactic acid bacterium, Lactobacillus casei strain Shirota (LcS), shows marked antitumor activities and an ability to modify immune responses. In this study, we examined whether LcS can induce the production of interleukin (IL)-12 and interferon-gamma (IFN-gamma), which are important cytokines for antitumor and antimicrobial immunity, from murine splenocytes in vitro in order to clarify the mechanisms of its immune modification. Stimulation by LcS induced a marked production of IL-12 by X-ray-irradiated splenocytes (X-irr-Spl). The production of IL-12 by X-irr-Spl was independent of the presence of nylon wool column-passed splenocytes (NW-Spl). IFN-gamma was produced by splenocytes by the stimulation with concanavalin A (Con A). LcS showed a synergistic stimulatory effect on the ConA-induced production of IFN-gamma. In addition, X-irr-Spl were required for the IFN-gamma; production by NW-Spl treated with LcS. The IFN-gamma production was reduced by anti-IL-12 antibody treatment. NW-Spl produced IFN-gamma following treatment with recombinant IL-12. Thus, we confirmed that IFN-gamma production by splenocytes was the result of the production of IL-12 from X-irr-Spl stimulated by LcS. Furthermore, in BALB/c mice, the oral administration of viable LcS augmented the production of IFN-gamma but not that of IL-4 or IL-5 by splenocytes. Thus, we suggested that LcS primarily activated X-irr-Spl, probably macrophages, and these cells secreted IL-12. The IL-12 induced by LcS stimulated the production of IFN-gamma.     

Interleukin-18 in combination with IL-2 enhances natural killer cell activity without inducing large amounts of IFN-gamma in vivo.

Interleukin-18 (IL-18) is known to synergistically enhance murine natural killer (NK) cell activity in vitro when combined with either IL-12 or IL-2. However, it has also been demonstrated that simultaneous administration of IL-18 and IL-12 to mice induces extraordinarily large amounts of interferon-gamma (IFN-gamma) in the serum. In this study, we examined the effects of a combination of IL-18 and IL-2 on in vivo NK cell activation in parallel with the induction of toxicity. In contrast to the IL-18 and IL-12 combination, the combined administration of IL-18 and IL-2 to BALB/c mice for 3 days induced neither high levels of IFN-gamma production nor other visible side effects. When compared with treatment with IL-18 or IL-2 alone, the combined treatment resulted in a significant increase in the number of DX-5 (pan-NK cells marker)-positive cells in spleens and a marked enhancement of splenic NK activity, as determined by standard cytotoxicity assays. Enhanced splenic cytotoxicity generated in the mice treated with both IL-18 and IL-2 was also observed against syngeneic Colon 26 adenocarcinoma cells. Consistent with this in vitro observation, combined treatment produced a significantly stronger inhibitory effect on the pulmonary metastases following i.v. injection of Colon 26 tumor cells than treatment with either cytokine alone. These results suggest that IL-18 combined with IL-2 potentiates in vivo NK cell activity and contributes to inhibition of tumor metastasis without inducing significant toxicity.     

抗原特异性Th17细胞的分化与调节

目的: 探讨影响抗原特异性Th17细胞分化和调节的因素.方法: T细胞受体转基因小鼠(DO11.10)CD4 T细胞, 与同背景正常小鼠的脾细胞混合, 经OVA多肽刺激后, 在不同的Th17极化的培养条件下, 观察Th17细胞及细胞因子的产生. 结果: 单纯OVA抗原刺激主要诱导特异性Th1型反应, 在TGF-β和IL-6存在的条件下, 主要诱导Th17反应; 当加入IL-23之后, 促进了Th17细胞的分化.阻断了IFN-γ和IL-4之后, Th17细胞的百分率明显增加.LPS可以促进Th1型细胞因子的产生, 但对抗原特异性Th17细胞的分化没有明显的促进作用.结论: 抗原特异性Th17细胞是与Th1、 Th2相对独立的细胞亚群, TGF-β、 IL-6和 IL-23可诱导或促进Th17的分化, 而Th1和Th2细胞因子抑制其分化.     

Interleukin-12 gene modification exerts anti-tumor effects on murine mammary sarcoma cell line in vivo

The aim of this project was to investigate the anti-tumor effect of an IL-12 gene modified mammary sarcoma murine cell line, EMT6/IL-12, in mouse model. In this study, we transfected the recombinant eukaryotic plasmid encoding IL-12 gene （pcDNA6-p70） into EMT6 and obtained the IL-12 expressing EMT6/IL-12 cell line. Then EMT6/IL-12 cells were s.c. inoculated into mice. The recombinant vector treatment group was set as control. We then evaluated the inhibition of tumor growth and the anti-tumor immunity function in vivo such as cytotoxicity, proliferation of splenocytes and serial IFN-T level. And the percentage of IFN-T producing CD4 or CD8 T cells among splenocytes was also analyzed in tumor bearing mice. Our results showed that the growth of tumors was obviously inhibited in EMT6/IL-12 group. Moreover, the capacities of anti-tumor immunity were all significantly higher in EMT6/IL-12 group compared to the controls. The results of the present investigation support the notion that EMT6/IL-12 could exert gene therapy in tumor model by improving the anti-tumor cellular immunity.     

转IL—2基因的骨髓基质细胞对骨髓移植后免疫功能重建的促进作用

目的探讨了用逆转录病毒载体转入小鼠IL-2基因的基质细胞系QXMSC1对异基因骨髓移植后免疫功能重建的促进作用.方法将小鼠IL-2cDNA片段连接到逆转录病毒载体PLXSN上,构建重组逆转录病毒载体PL2SN(含小鼠IL-2cDNA).用磷酸钙共沉淀法将PL2SN转入单嗜性包装细胞系CRE,获得G418抵抗细胞株后,以上清感染双嗜性包装细胞系CRIP,G418筛选后获得高滴度的包装细胞系CRIPIL-2.感染NIH3T3细胞测定CRIPIL-2培养上清病毒滴度为3.4×105cfu/ml.感染骨髓基质细胞系QXMSC1(H-2d),G418筛选,有限稀释法得10个单克隆细胞株,选择表达IL-2最高的细胞株为实验用细胞QXMSC1IL-2,用于以后的实验.供体小鼠BALB/c(H-2d)骨髓以抗T细胞单抗anti-Thy1.2加补体去除骨髓中T细胞,受体小鼠C57BL/6(H-2b)经γ射线致死剂量照射后,输入供体骨髓1×107/只,同时输入基质细胞QXMSC1IL-25×105/只.在第30天、60天检测BMT小鼠脾细胞对LPS、ConA的反应,脾细胞产生IL-2的能力,BMT小鼠产生抗体生成细胞(PFC)的能力及产生DTH的能力.流式细胞仪检测了QXMSC1IL-2对BMT小鼠T细胞亚群的影响.结果电泳及酶切鉴定证实构建的PL2SN质粒.QXMSC1IL-2细胞系IL-2的分泌量为857U(1×106*24h).异基因骨髓移植,共输入QXMSC1IL-2能明显增加BMT小鼠脾细胞对LPS、ConA的反应.脾细胞产生IL-2的能力增强.PFC数目增加,DTH反应增强.T细胞亚群中CD4+/CD8+的比例有所恢复.结论转入IL-2的骨髓基质细胞系可促进骨髓移植后免疫功能重建.