Jiao S, Williams P, Safda N, Schultz E, Wolff J A
Department of Pediatrics, University of Wisconsin, Madison 53705, USA.
Cell Transplant. 1993;2(3):185-92. doi: 10.1177/096368979300200302.
We have previously proposed the use of primary muscle cells as a "platform," or "vehicle" for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat brains for at least 6 mo without any decrease in graft size, or tumor formation. Stable, but moderate levels of intracerebral transgene expression were obtained by transplanting plasmid-transfected myotubes in culture. In the present study, high and stable levels of intracerebral transgene expression were achieved by the co-transplantation of plasmid-transfected myoblasts and myotubes in culture. Approximately 5 X 10(5) myoblasts and myotubes were transfected with 10 micrograms pRSVL plasmid DNA, and 30 micrograms Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, and much higher than the levels in myotube grafts, or co-grafts of myoblasts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains transplanted with co-graft of myoblasts, and myotubes transfected with pRSVLac-Z were stained immunofluorescently for beta-galactosidase activity. The muscle grafts contained beta-galactosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expression after postnatal gene transfer have rarely been achieved. Primary muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.
我们之前曾提出将原代肌细胞用作脑内转基因表达的“平台”或“载体”。切碎的肌肉或培养的肌细胞脑移植在大鼠脑中持续至少6个月，移植体大小没有任何减小，也没有形成肿瘤。通过在培养物中移植质粒转染的肌管，可获得稳定但适度的脑内转基因表达水平。在本研究中，通过在培养物中共移植质粒转染的成肌细胞和肌管，实现了脑内转基因的高稳定表达水平。分别用10微克pRSVL质粒DNA和30微克脂质体（BRL）转染约5×10⁵个成肌细胞和肌管。将它们混合在一起（细胞总数为100万），并立体定向注射到成年大鼠脑的尾状核中。转基因表达产物荧光素酶的活性至少4个月保持稳定，且远高于肌管移植或成肌细胞与切碎肌肉共移植中的水平。据推测，肌管充当了成肌细胞可在其上形成肌管的框架。用转染了pRSVLac-Z的成肌细胞和肌管共移植的脑切片进行免疫荧光染色以检测β-半乳糖苷酶活性。移植后4个月，肌肉移植体中含有β-半乳糖苷酶阳性肌纤维。出生后基因转移后如此高且稳定的体内表达水平很少能实现。原代肌细胞是脑内转基因表达的有用载体，对退行性神经疾病的基因治疗可能具有重要价值。
