Barka T, Gresik E W, van Der Noen H
Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, New York 10029, USA.
J Histochem Cytochem. 2000 Nov;48(11):1453-60. doi: 10.1177/002215540004801102.
We have studied the transduction of TAT-HA-beta-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6-21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate beta-galactosidase activity. Transduction of the fusion protein into A5 and C6-21 cells was concentration- and time-dependent. Therefore, the intensity of the beta-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-beta-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for beta-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.
我们利用组织化学方法来显示β-半乳糖苷酶活性，研究了TAT-HA-β-半乳糖苷酶融合蛋白转导进入两种大鼠唾液腺来源的细胞系A5和C6-21、器官培养的胎鼠下颌下腺细胞以及逆行导管注射后的大鼠下颌下腺。融合蛋白转导进入A5和C6-21细胞是浓度和时间依赖性的。因此，与长达24小时的暴露相比，暴露1小时后细胞质中β-半乳糖苷酶染色的强度较低。然而，融合蛋白转导进入了两种类型培养细胞的100%。当妊娠13天的小鼠胎儿外植体暴露于融合蛋白时，上皮细胞和间充质细胞均被该酶染色，反应产物在核周细胞质区域明显积聚。与上皮细胞相比，间充质细胞的组织化学染色更强。TAT-HA-β-半乳糖苷酶融合蛋白也通过逆行导管注射递送至大鼠下颌下腺。对注射腺体制备的冰冻切片进行β-半乳糖苷酶活性的组织化学染色显示，融合蛋白的转导也是时间和剂量依赖性的。在逆行注射后10分钟至1小时处死的大鼠腺体中，基本上所有腺泡细胞和导管细胞均显示细胞质染色。然后染色强度下降，在注射融合蛋白24小时后处死的大鼠腺体中未见到染色。这些结果表明，全长活性TAT融合蛋白在体外和体内均可靶向唾液腺细胞，以分析生理、发育和病理生理过程。
