Research on a highly-efficient and tumor-specific gene therapy using autologous NK/NKT cells as gene carriers

我们检查了实验原发性肿瘤模型中的肿瘤浸润细胞群，其中将3LL或B16 F10细胞注射到C57 BL 6小鼠的侧腹。3LL肿瘤周围可见大量浸润细胞。这些细胞中的大多数是CD 3 +/NK1.1- Trills和中性粒细胞，其余包括NK细胞和MHC II类+细胞（巨噬细胞和树突状细胞）。培养后NK 1.1 + CD 3-细胞的比例从几个%上升到10-15%。将这些培养的脾细胞注射到3LL或B16肿瘤周围（直径约10 mm），并对肿瘤浸润细胞进行化学染色观察。在脾细胞感染后48小时内，在瘤周组织中发现明显的NK 1.1 + CD 3-细胞浸润。用RTPCR和Western免疫印迹法检测肿瘤细胞系中p16 INK 4的表达。p16 INK 4在两种细胞中均有mRNA表达，但蛋白表达均低于可检测水平。通过cDNA克隆获得p16 INK 4的全长cDNA，并将其插入质粒表达载体。在3LL和B16 F10的p16 INK 4转染子中观察到可检测的表达。p16 INK 4过表达导致这些细胞系的显著生长抑制。目前，我们正在构建p16 INK 4腺病毒载体，并利用另一种腺病毒载体对p16 INK 4腺病毒载体在小鼠NK细胞中的转运进行初步实验。

We examined the population of tumor infiltrating cells in the experimental primary tumor model where 3LL or B16F10 cells were injected to the lateral flank of C57BL6 mice. A number of infiltrating cells were found around the 3LL tumor. The majority of these cells were CD3+/NK1.1- Trills and neutrophils, and the remaining includes NK cells and MHC class II+ cells (macrophages and dendritic cells).Murine splenocytes were obtained and cultured for seven days in the presence ofIL-2/1, -12. The proportion of NK1.1+CD3- cells was elevated from a few % to 10-15% after the culture. These cultured splenocytes were injected around the 3LL or B16 tumors (approximately 10 mm in diameter) and the tumor infiltrating cells were immunohistochemically observed. Marked infiltration of NK1.1+CD3- cells was found in the peritumoral tissue within 48 hours after the splenocyte infection. Similar results were obtained from cultured bone marrow cells of Thalidomide-treated mice.Then the expression of p16INK4 in the tumor cell lines was examined by RTPCR and Western immunoblotting. mRNA expression of p16INK4 was observed in the both cells, but its protein expression was almost under the detectable level. Full length cDNA of p16INK4 was obtained by cDNA cloning, and inserted to a plasmid expression vector. A detectable expression was observed in the p16INK4-transfectant of 3LL and B16F10. p16INK4 overexpression resulted in a marked growth suppression of these cell lines. Now we are working for the construction of p16INK4 adenovirous vector and performing preliminary experiments using another adenovirus vector on the transport of the vectors by murine NK cells in vitro.