白细胞介素6基因转染的白血病细胞克隆株的建立及其生物学…

作将人白细胞介素６（ＩＬ－６）基因转汾至ＦＢＬ－３红白血病细胞，建立了高分泌ＩＬ－６的ＦＢＬ－３细胞克隆株，并观察了其生物学特性，采用磷酸钙ＤＮＡ共沉淀法将ＩＬ－６表达载体ＢＣＭＧＮｅｏ－ＩＬ－６转移至ＦＢＬ－３细胞中，通过Ｇ４１８抗性筛选，有限稀释法和上清中ＩＬ－６活性的测定，从多株阳性克隆中筛选到一株高分泌ＩＬ－６的克隆株，体外观察表明，ＩＬ－６基因转染的ＦＢＬ－３细胞体外生长能力，集落形成     

IL—6基因转染的白血病细胞不同条件下小鼠体内外分泌IL—6水…

为了解白细胞介素６（ＩＬ－６）基因转染的高分泌ＩＬ－６的ＦＢＬ－３红白血病细胞（ＦＢＬ－３－ＩＬ－６）株体内外的生物学特性，动态观察在不同剂量＾６０Ｃｏγ射凰其ＩＬ－６的分泌水平，同时动态观察经腹腔、局部皮下注射途径将其移植入Ｃ５７ＢＬ／６小鼠体内后，血清和局部细胞培养上清液中ＩＬ－６含量。结果表明，ＦＢＬ－３－ＩＬ－６细胞以８Ｇｙ照射剂量为制备瘤苗的最佳照射剂量。照射后２周内一直保护较高水平的Ｉ     

IL-6基因转染的白血病细胞不同条件下小鼠体内外分泌IL-6水平的研究

为了解白细胞介素６（ＩＬ－６）基因转染的高分泌ＩＬ－６的ＦＢＬ－３红白血病细胞（ＦＢＬ－３－ＩＬ－６＋）株体内外的生物学特性，动态观察在不同剂量６０Ｃｏγ射线照射后其ＩＬ－６的分泌水平，同时动态观察经腹腔、局部皮下注射途径将其移植入Ｃ５７ＢＬ／６小鼠体内后，血清和局部细胞培养上清液中ＩＬ－６含量。结果表明，ＦＢＬ－３－ＩＬ－６＋细胞以８Ｇｙ照射剂量为制备瘤苗的最佳照射剂量，照射后２周内一直保持较高水平的ＩＬ－６分泌量。体内移植时，经腹腔途径注入后可测得一定浓度的血清ＩＬ－６，皮下注入时注射局部细胞培养上清中亦有一定水平的ＩＬ－６。为将该细胞作为治疗白血病瘤苗及其注射间隔时间提供了实验依据。     

白细胞介素6基因转移的白血病细胞体内诱导小鼠免疫功能的实验研究

作者观察了人白细胞介素６（ＩＬ－６）基因转移的、能高分泌ＩＬ－６的ＦＢＬ－３红白血病细胞对机体抗肿瘤免疫功能的影响。结果发现，高分泌ＩＬ－６的红白血病细胞接种到小鼠体内后，能显著提高小鼠脾脏的细胞毒性Ｔ淋巴细胞、ＮＫ活性及ＩＬ－２诱导的ＬＡＫ活性，小鼠脾细胞诱生的ＩＬ－２、肿瘤坏死因子和粒－巨噬细胞系集落刺激因子水平亦增高，腹腔巨噬细胞杀伤活性明显增强。结果表明，高分泌ＩＬ－６的ＦＢＬ－３红白血病细胞体内接种后能显著地增强机体的抗肿瘤免疫功能。研究结果为白血病的生物治疗以及ＩＬ－６基因转移的新型瘤苗的应用研究提供了实验依据。     

白细胞介素6基因转移的白血病细胞体内诱导小鼠免疫功能的…

作观察了人白细胞介素６（ＩＬ－６）基因转移的、能高分泌ＩＬ－６的ＦＢＬ－３红白血病细胞对机体抗肿瘤免疫功能的影响。结果发现，高分泌ＩＬ－６的红白血病细胞接种到小鼠体内后，能显提高小鼠脾脏的细胞毒性Ｔ淋巴细胞、ＮＫ活性及ＩＬ－２诱导的ＬＡＫ活性，小鼠脾细胞诱生的ＩＬ－２、肿瘤坏死因子和粒－巨噬细胞系集落刺激因子水平亦增高，腹腔巨噬细胞杀伤活性明显增强。结果表明，高分泌ＩＬ－６的ＦＢＬ－３红白血病     

白细胞介素2基因和白细胞介素3基因共转染白血病瘤苗的抗白血病作用的实验研究

目的：研究白细胞介素２（ＩＬ－２）和白细胞介素３（ＩＬ－３）基因共转染的白血病细胞瘤苗对白血病小鼠的治疗效果。方法：用ＩＬ－２重组腺病毒（Ａｄ－ＩＬ－２）和（或）ＩＬ－３重组腺病毒（Ａｄ－ＩＬ－３）转染红白血病细胞株ＦＢＬ－３，６０Ｃｏ照射后制备成瘤苗对实验性白血病小鼠进行治疗，观察肿瘤生长，小鼠生存期及治疗后小鼠腹腔巨噬细胞、ＮＫ细胞、诱导杀伤性Ｔ淋巴细胞（ＣＴＬ）杀伤活性，并与低剂量环磷酰胺（ＣＴＸ）合用，观察其抗肿瘤作用。结果：用ＩＬ－２或ＩＬ－３基因转染瘤苗治疗的白血病小鼠肿瘤生长明显减缓，生存期显著延长（Ｐ＜０．０５），用联合转染ＩＬ－２和ＩＬ－３基因的瘤苗治疗较单独转染ＩＬ－２或ＩＬ－３基因的瘤苗治疗效果更佳，与低剂量ＣＴＸ合用后抗肿瘤效果最佳。治疗后小鼠腹腔巨噬细胞、ＮＫ细胞、ＣＴＬ细胞杀伤活性显著增强。结论：ＩＬ－２、ＩＬ－３基因转染的瘤苗能够有效地通过诱导机体抗肿瘤免疫功能而发挥抗肿瘤作用，与低剂量ＣＴＸ合用后，抗肿瘤效果更佳。     

白细胞介素2基因和白细胞介素3基因共转染白血病瘤苗的抗白…

目的：研究白细胞介素２（ＩＬ－２）和白细胞介素３（ＩＬ－３）基因共转染的白血病细胞瘤苗对白血病的小鼠的治疗效果。方法：用ＩＬ－２重组腺病毒（Ａｄ－ＩＬ－２）和（或）ＩＬ－３重组腺病毒转染红白轿病细胞株ＦＢＬ－３，＾６０Ｃｏ射后制备成瘤苗对实验性白血病小鼠进行治疗，观察肿瘤生长，小鼠生存期及治疗后小鼠腹腔巨噬细胞，ＮＫ细胞，诱导杀伤性Ｔ淋巴细胞（ＣＴＬ）杀伤活性，并与低剂量环磷酰胺合用，观察其抗肿瘤     

人白细胞介素6基因转移的成纤维细胞对自然杀伤活性的增强作用

应用磷酸钙共沉淀法将人白细胞介素６（ＩＬ－６）基因转入成纤维细胞ＨＩＨ３Ｔ３，经Ｇ４１８抗性筛选和有限稀释法，从１２株阳性细胞克隆中选出一株高分泌ＩＬ－６克隆（代号为Ｔ６．６）。Ｔ６．６培养上清能显著增强自然杀伤（ＮＫ）活性，当与ＩＬ－２合用时增强效果更佳，抗ＩＬ－６单抗能阻断这种增强作用。将Ｔ６．６大量扩增后植入小鼠腹腔，４８小时后血清中可检测出较高水平的ＩＬ－６，且脾细胞ＮＫ活性比对照组显著升高。实验结果表明，人ＩＬ－６基因转移的成纤维细胞在体内、外均能显著增强ＮＫ活性，提示成纤维细胞介导的ＩＬ－６基因疗法可应用于抗肿瘤、抗感染和造血功能重建等。     

成纤维细胞介导的α干扰素基因疗法的建立

以成纤维细胞为基因载体细胞，建立。干扰素（ＩＦＮ－α）基因疗法的实验模型，并动态观察实验动物体内ＩＦＮ－α的分泌水平。将６８２ｂｐ的全序列入ＩＦＮ－αｃＤＮＡ插入到携有Ｎｅｏ￣Ｒ基因的表达载体ＢＭＧＮｅｏ的ＸｈｏＩ位点上，对此重组表达载体（ＢＭＧＮｅｏ－ＩＦＮ－α）进行了限制性酶切鉴定。用磷酸钙共沉淀法将ＢＭＧＮｅｏ－ＩＦＮ－α转入ＮＩＨ３Ｔ３成纤维细胞中，通过Ｇ４１８抗性筛选、有限稀释和检测上清中ＩＦＮ－α生物活性，从１４株阳性克隆中筛选到一株高分泌ＩＦＮ－α达１０２４Ｕ／ｍｌ的克隆株（ＮＩＨ３Ｔ３－ＩＦＮ－α￣＋）。将此＊阳性克隆细胞体外大量扩增，包裹入胶原，然后埋植入小鼠腹腔内，于１２小时到１５天可从小鼠血清中检测出ＩＦＮ－α，以７２小时的分泌水平最高，表明成纤维细胞能成功地将人ＩＦＮ－α基因导入体内并稳定表达。     

电转入IL—6受体基因在大鼠急性髓细胞白血病模型中的表达

应用平方波脉冲电转法将编码人ＩＬ－６受体的ｃＤＮＡ转入不表达ＩＬ－６Ｒ的ＢＮＭＬ大鼠急性早幼粒细胞白血病ＩＬ１２细胞中，转染细胞经含６００μｇ／ｍｌ的Ｇ４１８半固体－液体二步法培养，筛出Ｇ４１８抗性细胞；ＦＡＣＳ检测显示该细胞与ＦＩＴＣ标记的抗ＩＬ－６Ｒ单抗呈显的荧光强度，＾１２５Ｉ－ＩＬ－６受体结合实验表明，ｋｄ＝４．７ｎｍ，每个细胞ＩＬ－６Ｒ数为４０００左右，Ｎｏｒｔｈｅｒｎ－Ｂｌｏｔ发现该     

接种IL—6基因转导的白血病细胞后局部病理形态学实验研究

将人白细胞介素6(IL-6)基因转导的高分泌IL-6的FBL-3红白血病细胞克隆株体外扩增后,接种至C57BL/6小鼠皮下,观察肿瘤生长情况,并于不同时间取接种处组织作常规病理、免疫组化及电镜检查。结果表明接受IL-6基因转导的FBL-3细胞接种后的小鼠成瘤晚、生长速度慢,且肿瘤接种局部可见大量粒细胞和部分浆细胞、淋巴细胞的浸润,接种处组织免疫组化显示增殖细胞核抗原阳性率显著降低,提示IL-6基因转导的白血病细胞在体内致瘤性降低与其接种局部某些杀伤细胞浸润而诱导机体抗肿瘤免疫功能增强有关。     

IL—2基因修饰增强白血病抗原冲击的树突状细胞诱导的抗肿瘤？…

目的 研究腺病毒介导的白细胞介素（ＩＬ）２基因修饰能否使抗原冲击的树突状细胞（ＤＣ）在体内诱导出更强的抗肿瘤免疫反应。方法 用小鼠粒－巨噬细胞击落刺激因子（ｍＧＭ－ＣＳＦ）和ｍＩＬ－４扩增上鼠骨髓ＤＣ在ＦＢＬ－３红白血病细胞冻融抗原冲击的同时转染ＩＬ－２腺病毒。观察其体外分泌ＩＬ－２的水平,刺激Ｔ细胞增殖的能力,其体内皮下免疫后小鼠引流淋巴结细胞数和组分的变化以及主艉地产生细胞毒性Ｔ淋巴细胞（ＣＴ     

Therapy of disseminated murine leukemia with cyclophosphamide and immune Lyt-1+,2- T cells. Tumor eradication does not require participation of cytotoxic T cells

The ability of noncytolytic Lyt-1+,2- T cells immune to FBL-3 leukemia to effect eradication of disseminated FBL-3 was studied. Adult thymectomized, irradiated, and T-depleted bone marrow-reconstituted (ATXBM) B6 hosts were cured of disseminated FBL-3 by treatment with 180 mg/kg cyclophosphamide (CY) and adoptively transferred Lyt-1+,2- T cells obtained from congenic B6/Thy-1.1 donors immune to FBL-3. Analysis of the T cell compartment of ATXBM hosts treated and rendered tumor-free by this therapy revealed that the only T cells present in the mice were donor-derived Lyt-1+,2- T cells. In vitro stimulation of these T cells with FBL-3 tumor cells, which express class I but no class II major histocompatibility complex antigens, induced lymphokine secretion, but did not result in the generation of cytotoxic T lymphocytes (CTL). Thus, in a setting in which mice lack Lyt-2+ T cells, and in which no CTL of either host or donor origin could be detected, immune Lyt-1+,2- T cells, in conjunction with CY, mediated eradication of a disseminated leukemia. The results suggest that delayed-type hypersensitivity responses induced by immune T cells represent a potentially useful effector mechanism for in vivo elimination of disseminated tumor cells.     

Augmentation of the anti-tumor therapeutic efficacy of long-term cultured T lymphocytes by in vivo administration of purified interleukin 2

Spleen cells from C57BL/6 mice immunized in vivo with a syngeneic Friend virus-induced leukemia, FBL-3, were specifically activated by culture for 7 d with FBL-3, then nonspecifically induced to proliferate in vitro for 12 d by addition of supernatants from concanavalin A- stimulated lymphocytes containing interleukin 2 (IL-2). Such long-term cultured T lymphocytes have previously been shown to specifically lyse FBL-3 and to mediate specific adoptive therapy of advanced disseminated FBL-3 when used as an adjunct to cyclophosphamide (CY) in adoptive chemoimmunotherapy. Because the cultured cells are dependent upon IL-2 for proliferation and survival in vitro, their efficacy in vivo is potentially limited by the availability of endogenous IL-2. Thus, the aim of the current study was to determine whether exogenously administered purified IL-2 could augment the in vivo efficacy of long- term cultured T lymphocytes. Purified IL-2 alone or as an adjunct to CY as ineffective in tumor therapy. However, IL-2 was extremely effective in augmenting the efficacy of IL-2-dependent long-term cultured T lymphocytes in adoptive chemoimmunotherapy. The mechanism by which IL-2 functions in vivo is presumably by promoting in vivo growth and/or survival of adoptively transferred cells. This assumption was supported by the findings that IL-2 did not enhance the modest therapeutic efficacy of irradiated long-term cultured cells that were incapable of proliferating in the host and was ineffective in augmenting the in vivo efficacy of noncultured immune cells that are not immediately dependent upon exogenous IL-2 for survival.     

转染血管内皮细胞生长因子受体基因的K562细胞在裸鼠体内生长的实验研究

目的 观察人白血病细胞转染可溶性内皮细胞生长因子受体基因（sFlt-1)后,对其在裸鼠体内生长的影响。方法 ①将内皮细胞生长因子受体－1（Flt-1)的配体结合区基因片断与免疫球蛋白重链的恒定区基因片断重组成Flt-Ig融合基因,插入到pcDNA3质粒;②采用电穿方法转染K562细胞,经过筛选、克隆,用RT－PCR检测Flt-1 mRNA的表达;③将表达Flt-Ig基因的细胞和转染对照载体的细胞分别给裸鼠移植,动态观察肿瘤的生长。结果 Flt-Ig融合基因转染K562细胞后,获得5株Flt-Ig mRNA表达阳性的细胞株,取一株扩增后移植给6只裸鼠,移植后第14,21和28天实验组肿瘤的体积约为对照组的1／2,明显小于对照组。结论 转染Flt-Ig融合基因的K562细胞,在裸鼠体内生长受到明显抑制,这可能与转染后的肿瘤细胞表达可溶性Flt-Ig蛋白,中和了肿瘤细胞分泌的内皮细胞生长因子,抑制肿瘤血管新生有关。     

用单倍体相合小鼠FBL-3红白血病细胞株移植建立CB6F1小鼠红白血病模型

本研究探讨建立F1代单倍体相合小鼠FBL-3红白血病模型的可行性,并观察FBL-3细胞在小鼠体内的生物学特性。将FBL-3H2-d小鼠红白血病细胞经尾静脉分别接种给小鼠C57BL/6和CB6F1H-2b/d(C57BL/6×BALB/c),观察两种小鼠的生存时间和染色体变化,并对濒死小鼠的肝、脾、肺和肾进行病理检查,部分进行电子显微镜检查。分析骨髓和脾脏细胞染色体核型及MHC分子表达情况。结果表明:静脉接种103-107个白血病细胞的情况下,C57BL/6小鼠和CB6F1小鼠发病率分别为100%和92.5%;接种的细胞数量与存活时间呈线性关系;接种相同数量FBL-3细胞的CB6F1小鼠平均生存时间较C57BL/6小鼠延长。白血病细胞主要侵及肝、脾、骨髓、肺和肾组织,糖原染色阳性、氯醋酸染色部分阳性,过氧化物酶、碱性磷酸酶、丁酸染色均为阴性。电子显微镜观察到细胞内病毒样颗粒。脾脏和骨髓细胞染色体多数为非二倍体,且H-2b表达率升高,H-2d表达率降低。结论:经尾静脉接种FBL-3细胞可以建立CB6F1小鼠红白血病模型。     

Retroviral immunotoxin gene therapy of leukemia in mice using leukemia-specific T cells transduced with an interleukin-3/Bax fusion protein gene

In past studies, we showed that T cells transduced with retroviral diphtheria immunotoxin (IT) target genes could serve as vehicles for delivering IT to tumors in vivo. We took advantage of the observation that antigen-specific T cells are able to penetrate tumors to design an approach delivering combined cellular and humoral therapy directly to the tumor site. To improve tumor specificity, we selected interleukin (IL)-3 as a ligand because its receptor is selectively overexpressed on myeloid leukemia progenitors. Because Bcl-2 family proteins show structural similarity to diphtheria toxin (DT), we constructed a unique retroviral IT using Bax, a proapoptotic member of the Bcl-2 family, in place of DT. Bax was chosen because several studies showed that its transduction induces lethal apoptosis in different cancers. The retroviral construct for gene therapy included IL-3 positioned downstream of its 80 amino acid leader, and permitted cotranslational protein synthesis of hybrid IL-3/human Bax fusion protein. Other vectors were constructed with IL-3 fused to DT or Pseudomonas exotoxin. Retroviral vectors were used to transiently transduce C8, a CD4(+) T cell clone that specifically recognized FBL-3, a lethal myeloid leukemia. Supernatants collected from transduced cells showed proapoptotic activity and selectively inhibited FBL-3 cells in vitro. Intraperitoneal injection of transduced but not nontransduced C8 into mice with subcutaneous tumors or systemic cancer significantly inhibited tumor growth. These results indicate that retroviral IT made with IL-3 and various toxic proteins may be useful in patients with acute myelogenous leukemia (AML). Furthermore, the Bax construct may be particularly useful as a nonimmunogenic substitute for bacterial toxins in retIT.     

GM—CSF基因修饰,抗原预激的树突状细胞体内诱导白血病细胞分化？…

目的：探讨树突状细胞在肿瘤治疗过程中的作用及机制。方法：以小鼠ＦＢＬ－３红白血病细胞皮下接种Ｃ５７ＢＬ／６小鼠建立荷瘤模型，采用流式细胞仪分析和透射电镜等技术，观察了粒－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）基因修饰的抗原预激的树突状细胞体内治疗作用。结果：采用ＧＭ－ＣＳＦ基因修饰的抗原预激的树突状细胞治疗，可以明显抑制白血病细胞生长；白血病细胞表达ＣＤ３４水平增加，同时ＭＨＣ－Ⅱ，Ｂ７－１，Ｂ７－