mB7-1修饰的B16细胞体外诱导免疫效应

目的探讨mB7-1基因修饰的B16细胞的体外抗瘤效应。方法提取小鼠脾细胞mRNA,PCR法获得mB7-1片段,与pcDNA3连接,并进行酶切鉴定及测序。重组质粒经脂质体介导转染鼠B16细胞,检测基因表达,MTT法检测CTL杀伤作用。结果完成mB7-1-pcDNA3重组载体连接及鉴定,基因测序与GenBank一致,并转染至B16细胞,检测到基因表达。经mB7-1修饰B16细胞可诱导淋巴细胞产生明显抑瘤效应。结论应用本文方法可获得mB7-1修饰B16细胞系,为研究其抗瘤效应奠定了基础。

mB7-1转染大鼠卵巢癌细胞诱导细胞免疫应答及体内致瘤性研究

为探讨转染mB7 1基因大鼠卵巢癌细胞系后体外诱导细胞免疫应答及其致瘤性 ,我们以逆转录病毒为载体将mB7 1基因转染入大鼠低分化卵巢上皮癌细胞株NuTu 19中 ,流式细胞仪检测mB7 1的表达。用同源淋巴细胞肿瘤细胞混合培养实验 (MTLCs)测定淋巴细胞增殖指数 ,MTT法检测细胞毒淋巴细胞的增殖及其杀瘤活性。将NuTu 19/Neu、NuTu 19/mB7 1细胞分别接种于同源大鼠Fischer344皮下 ,观察mB7 1修饰的肿瘤细胞在动物体内的致瘤性。结果显示 ,mB7 1基因成功地转染入大鼠低分化卵巢上皮癌细胞株NuTu 19,流式细胞仪测定mB7 1基因呈阳性表达。NuTu 19/mB7 1体外刺激脾淋巴细胞增殖能力明显高于对照组细胞 (P <0 0 5 )。NuTu 19/mB7 1体外诱导的CTL较对照组对NuTu 19细胞的杀伤率显著增高 (P<0 0 1)。两种细胞体外增殖能力无明显改变 ,NuTu 19/mB7 1组在Fischer344大鼠体内成瘤时间明显晚于对照组 (P <0 0 5 )。由此可见 ,mB7 1基因修饰的鼠卵巢癌细胞可体外诱导细胞免疫应答 ,接种动物后 。

Study of cellular immunity response of mB7-1 gene transfected mouse ovarian cancer cell line and its tumorigenecities in vivo

Objective:To investigate the cellular immunity response in vitro and the tumorigenecities in vivo of mB7 1 gene transfected murine ovarian cancer cell line.Methods:mB7 1 gene was transfected into the NuTu 19 cell line by retrovirus vector, and the expression of mB7 1 gene was confirmed by flow cytometry(FCM).NuTu 19/neo and NuTu 19/mB7 1 cells were injected subcutaneously into syngeneic Fischer 344 rats respectively,and their tumorigenecities were recorded.Proliferation indices of lymphocyte were assayed after syngenieic mixed tumor lymphocyte cultures(MTLCs).The lysis activity of CTL toward tumor cells was determined using methyl thiazolyl tetrazolium(MTT) assay.Results:Successful transfection of mB7 1 gene into NuTu 19 cell line was comfirmed with FCM.In vitro study showed that there was no obvious changes in cell growth of gene transfected cell line,compared with the cell line NuTu 19.NuTu 19/mB7 1 cells could induce more effective proliferation of effector lymphocytes(P<0.05). The lysis activity of CTL activated by NuTu 19/mB7 1 was stronger than that of NuTu 19/neo (P<0.01).Tumor sizes were smaller in the NuTu 19/mB7 1 receptance syngeneic Fischer 344 rats compared with those in the control group.Conclusion:mB7 1 genetically modified ovarian cancer cells could induce the cellular immunity response in vitro and the tumorigenecitiy of NuTu 19 cells was decreased after inoculation with the experimental vaccine.

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。