Banin Eyal, Obolensky Alexey, Piontek Elena, Falk Haya, Pikarsky Eli, Pe'er Jacob, Panet Amos, Chowers Itay
Department of Ophthalmology, The Hebrew University-Hadassah Hospital and Medical School, Jerusalem, Israel.
Invest Ophthalmol Vis Sci. 2003 Apr;44(4):1529-33. doi: 10.1167/iovs.02-0529.
To test the feasibility of gene transfer into lacrimal gland tissue in primary culture, using different viral vectors.
Lacrimal glands were dissected from adult Sabra rats and divided by pincers to 0.3-0.4 mm fragments. Tissue was maintained under primary organ culture conditions using the "raft" technique. The ability of three different viral vectors to conduct beta-galactosidase (beta-gal) gene delivery was examined: adenovirus (Ad5CMVLacZ), vaccinia (VSC9), and herpesvirus (tkLTRZ(1)). Tissue fragments were incubated for 60 minutes with one of the viral vectors and transferred to fresh medium. After 3 and 7 days, beta-gal expression was examined by X-gal staining in gross preparations and in histologic sections.
At 3 days, beta-gal expression was observed in 33% of tissue fragments exposed to the vaccinia vector and in 18% and 14% of fragments exposed to the adenoviral and herpes vectors, respectively. After 7 days in culture, successful gene delivery occurred in 77% of vaccinia, 41% of adenovirus, and only 13% of herpesvirus applications. Vector-specific reporter gene expression patterns were observed: With the vaccinia vector, lacrimal duct cells were predominantly stained; in contrast, the adenoviral vector tended to transduce the interacinar areas, with beta-gal expression mainly occurring within the myoepithelial cells.
Vaccinia and adenovirus are efficient vectors for gene transfer into lacrimal gland tissue in primary culture. The specific expression pattern obtained by the vaccinia vector probably reflects its characteristic tissue tropism to lacrimal duct cells. The results presented in this ex vivo system may be a first step toward expressing genes with products that could be continuously delivered to the eye through the tears. Such proteins could include anti-inflammatory, anti-angiogenic, anti-herpetic, anti-bacterial, or anti-glaucomatous agents, among others.
使用不同的病毒载体测试基因转移到原代培养泪腺组织中的可行性。
从成年Sabra大鼠中取出泪腺，用镊子将其分成0.3 - 0.4毫米的碎片。采用“筏”技术在原代器官培养条件下维持组织。检测了三种不同病毒载体进行β-半乳糖苷酶（β-gal）基因传递的能力：腺病毒（Ad5CMVLacZ）、痘苗病毒（VSC9）和疱疹病毒（tkLTRZ(1)）。将组织碎片与其中一种病毒载体孵育60分钟，然后转移到新鲜培养基中。3天和7天后，通过X-gal染色在大体标本和组织切片中检测β-gal表达。
3天时，在暴露于痘苗病毒载体的组织碎片中，33%观察到β-gal表达；暴露于腺病毒和疱疹病毒载体的碎片中，分别有18%和14%观察到β-gal表达。培养7天后，痘苗病毒载体的基因传递成功率为77%，腺病毒为41%，疱疹病毒仅为13%。观察到载体特异性报告基因表达模式：痘苗病毒载体主要使泪管细胞染色；相比之下，腺病毒载体倾向于转导腺泡间区域，β-gal表达主要发生在肌上皮细胞内。
痘苗病毒和腺病毒是原代培养泪腺组织基因转移的有效载体。痘苗病毒载体获得的特异性表达模式可能反映了其对泪管细胞的特征性组织嗜性。本体外系统中的结果可能是朝着表达能通过泪液持续递送至眼部的产物的基因迈出的第一步。这类蛋白质可能包括抗炎、抗血管生成、抗疱疹、抗菌或抗青光眼药物等。
