B7基因转染治疗原发性肝癌的实验研究

Ｂ７基因转染治疗原发性肝癌的实验研究袁爱力张智翔肖丽萍侯淑琴肿瘤细胞缺乏主要组织相容性复合体（ＭＨＣ）－Ⅰ类抗原表达是肿瘤患者免疫监视功能低下的重要原因。实验研究已证实将ＭＨＣ－Ⅰ类基因Ｈ－２Ｋ导入到鼠白血病细胞中后不仅可以表达，而且杀伤活性亦增强［...     

表达大鼠IL-2、B7-1基因CBRH7919细胞的建立

目的:研究携带大鼠IL-2、B7-1目的基因的CBRH7919细胞(即CBRH7919/IL-2/B7-1)体外表达目的基因的能力,方法:RT-PCR扩增Wistar大鼠IL-2、B7-1目的基因,分别克隆至重组逆转录病毒载体pBaBe-puro及pMSCV-neo,构建逆转录病毒载体pBaBe-puro-IL-2及p...     

表达白细胞介素-2质粒DNA抗小鼠肝癌作用

目的探讨瘤内注射表达hIL-2质粒DNA抗小鼠肝癌皮下移植瘤的作用。方法构建真核表达质粒载体pDC511hIL-2;ELISA方法检测质粒载体在真核细胞中的表达;胸腺细胞增殖法(MTT法)检测hIL-2生物学活性。小鼠肝癌H22皮下移植瘤瘤内直接注射质粒DNA后,观察各组小鼠存活时间,肿瘤体积变化;检测小鼠脾脏细胞毒T淋巴细胞(CTL)杀伤活性。在质粒DNA注射后一月进行瘤体组织病理学观察。结果构建的质粒载体能在真核细胞内高效表达hIL-2(3087.18pg/ml.24h.106)。质粒载体表达的hIL-2体外刺激胸腺细胞增殖作用非常明显。hIL-2基因治疗组与空载体组相比,肿瘤生长明显受抑制(F=3.85,P=0.03),小鼠存活期显著延长(x2=4.10,P=0.03),并且小鼠脾细胞CTL杀伤活性增强。pDC511hIL-2DNA瘤内注射一月后,肿瘤病灶炎性细胞浸润明显,病灶内肿瘤细胞广泛坏死。结论瘤内注射hIL-2表达质粒DNA可抑制小鼠肝癌皮下移植瘤的生长,提高荷瘤生存期。     

IL-1与IL-2联合激活的骨髓对白血病细胞的净化作用

目的：体外观察白细胞介素－１（ＩＬ－１）对白细胞介素－２（ＩＬ－２）激活骨髓自然杀伤细胞（ＮＫ）和淋巴因子－激活杀伤细胞（ＬＡＫ）活性的影响及两者联合激活的骨髓对白血病细胞的净化作用。方法：标准４ｈ＾５１Ｃｒ释放实验测定激活骨髓的细胞毒作用;集落培养法观察激活骨髓对ＨＬ－６０和Ｋ５６２细胞的净化作用。结果：１０～２００μｇ／ＬＩＬ－１对正常骨髓的ＮＫ和ＬＡＫ活性无明显影响;ＩＬ－２能够增强骨髓的ＮＫ活性和诱导ＬＡＫ细胞的产生,呈浓度递增趋势;１００μｇ／ＬＩＬ－１能增强１万～１００万Ｕ／ＬＩＬ－２诱导的骨髓ＮＫ及ＬＡＫ活性。在体外净化实验中,１００μｇ／Ｌ ＩＬ－１或１００万Ｕ／Ｌ ＩＬ－２对Ｋ５６２和（或）ＨＬ－６０细胞无直接杀伤,而在掺入１％Ｋ５６２或ＨＬ－６０的正常骨髓中,加入ＩＬ－１和（或）ＩＬ－２,对     

人B7-1转基因肝癌瘤苗的实验研究

用逆转录病毒转移体系建立表达人B7-1(hB7-1)的肝癌瘤苗,瘤苗与外周血淋巴细胞(PBLs)或T细胞共同孵育,观察该瘤苗激活T淋巴细胞的能力.发现所建立的hB7-1转基因肝癌瘤苗可有效刺激PBLs或T细胞增殖并产生特异性免疫反应.认为hB7-1转基因肝癌瘤苗可能是肝癌基因治疗的有效方法之一.     

脂质体-白细胞介素-2基因复合物瘤体内注射治疗肝细胞癌的实验研究

以阳离子脂质体Lipofectin介导含人白细胞介紊-2(IL-2)基因的真核表达质粒pME18S-IL-2转染小鼠肝癌HAC细胞，48小时后在培养上清液中可测得人IL-2活性(30U／m1)，96小时达最高峰(76U／ml)。将Lipofectin-pME18S-IL-2复合物直接瘤体内注射，观察它对荷HAC肝癌实体瘤小鼠的治疗效果，发现治疗后小鼠生存时间明显延长(32．1±9．4无比20．9±7．5天，n=10，P<0．01)。实验组肿瘤体积明显小于对照组(0．65±0．31cm^3比1．076±0．43cm^3，n=12，P<0．01)。取转基因后瘤组织总RNA进行RT—PCR反应，可检出IL-2的mRNA，病理检查见肿瘤中央明显坏死及周围炎性细胞浸润。实验结果表明，脂质体-IL-2基因复合物直接注射入瘤体后可获局部IL-2基因表达，从而诱生机体抗肿瘤效应，这为肝癌基因治疗提供了一种简便实用的模式。     

IL-12与粒细胞-巨噬细胞集落刺激因子基因联合治疗小鼠肝癌的研究

目的 研究L-12与粒细胞-巨噬细胞集落刺激因子(granalocyte-macrophage-colong scimulatingfactor,GM-CSF)联合基因治疗对小鼠皮下肝癌的治疗作用。方法 将皮下接种1×106BNL肝癌细胞的小鼠分四组于肿瘤接种3、6d后分别经尾静脉高压注射总量为25μg的细胞因子编码质粒:(1)pXX-GM-CSF、pXX-IL-12各 12.5μg;以pXX-IL-12 25μg;(3)pXX-GM-CSF 25μg;(4)pXX-Neo 25μg,每组6只小鼠。观察小鼠皮下肿瘤生长情况及所诱导的细胞免疫反应。同时研究了经尾静脉高压注射后小鼠血清IL-12、GM-CSF和IFN-γ浓度变化。结累IL-12与GM-CSF联台基因治疗可以诱导更强的抗肝癌免疫和细胞免疫反应。GM-CSF可以增强IL-12分泌并减少其诱导的IFN-γ分泌。结论IL-12和GM-CSF联台基因治疗可以产生较强的抗瘤作用并能减少IL-12的副作用。     

转4-1BBL基因细胞诱导带瘤宿主抗瘤效应的体内研究

目的 观察协同刺激分子4-1BBLigand(4-1BBL)基因导入小鼠肝癌细胞Hepal-6后在小鼠体内诱导的抗瘤效应。方法 应用逆转录病毒载体，将小鼠4-1BBL导入小鼠肝癌细胞Hepal-6细胞中，Histidinol(HisD)筛选后获高表达4-1BBL分子阳性细胞克隆，经丝裂霉素C（MMC）处理制备成肿瘤细胞瘤苗TCV4-1BBL，观察其对不同动物模型的体内免疫保护作用和免疫治疗作用，结果 （1）能对同系肝癌细胞产生完全的免疫保护作用，并能保持无瘤状态长期存活（100d以上）；（2）对早期（接种7d)形成的肿瘤有强的治疗作用。（3）对晚期（接种14d)肿瘤，有明显的治疗作用。使大部分小鼠的肿瘤消退。结论 4-1BBL修饰的肿瘤细胞疫苗能明显刺激增强带瘤宿主的抗瘤效应。     

IL-1B基因多态性与幽门螺旋杆菌感染后胃癌发病的关系

取35例胃癌(胃癌组)及37例慢性胃炎(对照组)患者全血标本,使用基因芯片检测技术,结合PCR 体外扩增方法,检测人IL1B-31和-511位点基因多态性,并用13C尿素呼气试验、免疫印迹试验检测Hp感染情况.结果胃癌组Hp感染率高于对照组(P＜0.05),胃癌组IL-1B-31位点T携带子的频率高于对照组(P＜0.05).与C/C型比较,T携带子基因型者发生胃癌的风险增加,OR=4.237(95%CI:1.563～10.000);IL-1B-511位点的各基因型频率在两组间差异无统计学意义(P＞0.05).认为IL-1B-31位点基因多态性可能增加Hp感染后中国人发生胃癌的危险性.     

TK基因联合白介素-2/粒、巨噬细胞集落刺激因子基因治疗胃癌的实验研究

目的：用逆转录病毒载体pLxSN转导TK基因,用真核表达载体pIRES转导细胞因子基因,观察TK基因联合白介素－2（mouseII-2)/粒,巨噬细胞集落刺激因子（mouse)GM-CSF)两种细胞因子基因对小鼠胃癌的治疗作用,方法：先用TK基因治疗胃癌细胞株,然后联合mIL-2/mGM-CSF基因治疗小鼠模型胃癌,其“自然基因”与细胞因子基因相互协同,产生显著的抗肿瘤作用,PT－PCR检测基因表达,结果：20％的转基因细胞发了可杀灭70％－80％以上的肿瘤细胞,表现出很强的旁观者效应,实验表明,应用TK／9－丙氧鸟苷（GCV）,即可产生很强的抗肿瘤作用（P＜0．01）,结合mIL-2/mGM-CSF两种细胞因子基因,抗肿瘤作用会得到进一步强化,肿瘤消退速度明显加快,大部分肿瘤完全消退,具有显著抗肿瘤作用,联合IL－2／GM－CSF后,抗肿瘤作用会得进一步强,生明显的抗肿效应。     

Prevention of hepatocellular carcinoma in mice by IL-2 and B7-1 genes co-transfected liver cancer cell vaccines

AIM: To study the immunoprotective effect of liver cancer vaccine with co-transfected IL-2 and B7-1 genes on hepatocarcinogenesis in mice.METHODS: The murine liver cancer cell line Hepal-6 was transfected with IL-2 and/or B7-1 gene via recombinant adenoviral vectors and the liver cancer vaccines were prepared. C57BL/6 mice were immunized with these vaccines and challenged with the parental Hepal-6 cells afterwards.The immunoprotection was investigated and the reactive T cell line was assayed.RESULTS: The immunoprotection of the tumor vaccine was demonstrated. The effect of IL-2 and B7-1 genes cotransfected Hepal-6 liver cancer vaccine (Hep6-IL2/B7vaccine) on the onset of tumor formation was the strongest.When attacked with wild Hepal-6 cells, the median survival period of the mice immunized with Hep6-IL2/B7 vaccine was the longest (68 days, χ2=7.70-11.69, P＜0.05) and the implanted tumor was the smallest (z =3.20-44.10, P＜0.05).The effect of single IL-2 or B7-1 gene-transfected vaccine was next to the IL2/B7 gene co-transfected group, and the mean survival periods were 59 and 54 days, respectively.The mean survival periods of wild or enhanced green fluorescence protein gene modified vaccine immunized group were 51 and 48 days, respectively. The mice in control group all died within 38 days and the implanted tumor was the largest (z=3.20-40.21, P＜0.05). The cellular immunofunction test and cytotoxicity study showed that the natural killer (NK) cell, lymphokine activated killer (LAK) cell and cytotoxic T lymphocyte (CTL) activities were significantly increased in mice immunized with the Hep6-IL2/B7 vaccine, (29.5±2.5%,65.0±2.9%, 83.1±1.5% respectively, compared with other groups, P＜0.05).CONCLUSION: The Hep6-IL2/B7 liver cancer vaccines can induce the mice to produce activated and specific CTL against the parental tumor cells, and demonstrate stronger effect on the hepatocarcinogenesis than single gene modified or the regular tumor vaccine. Therefore, the vaccines may become a novel potential therapy for recurrence and metastasis of HCC.     

Expression of costimulatory molecules, B7-1 and B7-2 on human gastric carcinoma

Costimulation of T cells via B7-1 and B7-2 molecules on a tumor has been shown to be important for eliciting cell-mediated antitumor immunity. We studied the surface expression of B7-1 and B7-2 in 24 cases of gastric carcinoma from the primary locus, 20 cases of metastatic carcinoma from malignant ascites, 20 cases of benign gastric mucosa and 7 gastric carcinoma cell lines by two-color flow cytometry with mAb CD80 and CD86. The B7-1 and B7-2 molecules were expressed by 6 cell lines, and 1 cell line showed the predominant expression of B7-2 but not B7-1. Almost all patients with primary gastric carcinoma and benign gastric mucosa showed high levels of expression of the B7-1 and B7-2, revealing approximately 40%–60% positive cells. However, the percentage of B7-1-positive cells of poorly differentiated primary carcinomas was significantly lower than that of well-differentiated carcinoma and normal mucosa (P<0.01). Furthermore, all of the metastatic carcinoma cells revealed consistently very low or undetectable levels of expression of the B7-1 molecule, only 8% (mean) of cells being positive, despite showing higher levels of B7-2 expression. Thus, it seems likely that decreased or deleted expression of B7-1 correlates with the grade of tumor differentiation, tumor progression and metastasis. These results suggest that the B7-1 molecule on the gastric carcinoma bearing CD80⁺CD86⁺ is abrogated during tumor invasion and/or metastasis, and the tumor finally acquires the CD80⁻CD86⁺ phenotype. Consequently, inadequate B7-1 costimulation may contribute to the escape of tumors from destruction by the host's immune system. Received: 26 December 1997 / Accepted: 2 March 1998     

小鼠免疫共刺激信号B7-1和B7-2联合表达载体的构建及包装

目的构建含小鼠B7-1和B7-2基因的双表达载体pLXSN-mB7-1-IRES-mB7-2,并包装到PA317细胞中。方法1将EcoRI和BamHI酶切下来的pMD18-mB7-2质粒上的mB7-2基因片段克隆到含有IRES片段的MINV质粒上,得到MINV-IRES-mB7-2载体;2MunI酶切载体MINV-IRES-mB7-2,末端钝化,然后BamHI再酶切,得到含钝末端和BamHI粘末端的IRES-mB7-2基因片段;3XhoI酶切质粒pLXSN-mB7-1,末端钝化,然后BamHI再酶切,形成含钝末端和BamHI粘末端的开环pLXSN-mB7-1质粒,将IRES-mB7-2基因片段插入其中,得到pLXSN-mB7-1-IRES-mB7-2双表达载体;并PA317细胞包装,PCR和电镜鉴定。结果经酶切、PCR、测序等鉴定载体构建成功,并包装到PA317细胞内。结论成功得到了双表达载体pLXSN-mB7-1-IRES-mB7-2和包装后的PA317/mB7-1-mB7-2细胞。     

α-干扰素治疗慢性乙型肝炎患者肝组织B7-1表达的变化

目的了解α-干扰素(-αIFN)治疗慢性乙型肝炎(chronic hepatitis B,CHB)患者前后肝组织B7-1的表达特点。方法采用免疫组织化学方法检测18例正规使用-αIFN的CHB患者治疗前、后肝活检组织共刺激分子(B7-1)的表达及其与-αIFN疗效的关系。结果18例CHB患者肝组织B7-1阳性表达颗粒的平均吸光度值治疗后较治疗前显著增加(分别为24.98±4.23和11.77±2.35,t=11.58,P<0.01)。B7-1表达水平与-αIFN近期疗效呈显著正相关(r=0.68,P<0.01)。结论-αIFN可促进肝细胞B7-1表达,B7-1表达水平可能是预测-αIFN近期疗效的重要参数之一。     

慢性乙型肝炎肝组织B7-1表达与α-干扰素近期疗效的关系

目的了解慢性乙型肝炎(CHB)肝组织B7-1表达与α-干扰素(α-IFN)近期疗效的关系。方法对68例使用α-IFN的CHB患者治疗前行肝组织活检,采用免疫组织化学方法观察其B7-1的表达。结果68例CHB中45例(66.2％)B7-1表达阳性,5例正常人肝组织无表达。α-IFN的总应答率,肝组织B7-1表达阳性组为66.7％(30/45),阴性组为39.1％(9/23),二者差异显著(x2=7.20,P<0.01)。随B7-1阳性表达程度的增强,肝组织炎性活动积分(HAI)及血清丙氨酸转氨酶(ALT)异常值明显增加。结论肝组织B7-1表达水平可能是预测α-IFN近期疗效的重要参数之一。     

Anti-gastric cancer active immunity induced by FasL／B7-1 gene-modified tumor cells

AIM:To study the activation of cytotoxic T lymphocytes (CTLs) against gastric cancer cells induced by FasL/B7-1 (FB-11) gene-modified tumor cells,and to explore whether co-expression of FasL and B7-1 in SGC-7901 tumor cells could initiate synergistic antitumor effect. METHODS: FasL and B7-1 genes were transfected into human SGC-7901 gastric cancer cells with adenovirus vectors. The positive clones were selected by G418. FasL and B7-1 genes were detected by flow cytometry and RT-PCR. Abdominal infiltrating lymphocytes and sensitized spleen cells were obtained from mice that were immunized with SGC-7901/FB-11 or wild type SGC-7901 cells intraperitoneally,and cytotoxicity of these CTLs against tumor cells was determined by MTT assay. RESULTS: Flow cytometry and RT-PCR showed that FasL and B7-1 genes were highly expressed. FasL and B7-1 transfected cancer cells had a high apoptosis index. DNA laddering suggested that FasL and B7-1 genes induced gastric cancer cell apoptosis. FasL+/B7-1+SGC-7901 cells (SGC-7901/FB-11) were inoculated subcutaneously in the dorsal skin of C57BL/6 mice and then decreased their tumorigenicity greatly (z=2.15-46.10, P<0.01).SGC-7901/FB-11 cell-sensitized mice obtained protective immune activity against the rechallenge of wild type SGC-7901 cells (z=2.06-44.30, P<0.05). The cytotoxicity of CTLs induced by SGC-7901/FB-11 cells against SGC-7901 was significantly higher than that of CTLs activated by wild-type SGC-7901 cells (84.1±2.4% vs30.5±2.3%, P<0.05). CONCLUSION: FasL and B7-1 genes can effectively promote the activity of CTLs against gastric cancer cells. FasL/B7-1 molecules play an important role in CTL cytotoxicity.     

水流动力学注射介导白细胞介素-12基因治疗小鼠原位移植性肝癌

以细胞因子白细胞介素(IL)-12为基础的抗肿瘤免疫疗法有望成为治疗肝脏肿瘤的有效手段之一.以往的IL-12抗肿瘤基因治疗研究中多采用皮下接种肝癌模型,肿瘤的生长环境与临床实际情况相差甚远.