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目的:开展重组腺病毒-胸苷激酶基因制剂(adenovirus-mediated delivery of herpes simplex virus thymidine kinase gene,ADV-tk)局部或静脉用药后于动物体内的分布行为的研究。方法:分别采用绿色荧光蛋白(green fluorescent protein,GFP)和原位杂交法检测分析重组腺病毒及胸苷激酶于肝脏、脾脏、淋巴结、肠系膜、肾上腺、胸腺、肾脏、甲状腺、前列腺、卵巢等组织分布的情况。结果判断采用免疫组织化学评分法(immuno-histochemical scores,IHS),并结合阳性细胞百分比及阳性细胞染色强弱2个方面进行评价。结果:(1)3种不同给药途径的组织分布特征具有相似性,ADV或tk阳性表达的组织脏器从强到弱分布依次为:肝脏、脾脏、淋巴结、肠系膜、肾上腺、胸腺、肾脏、甲状腺、前列腺、卵巢。ADV或tk mRNA的丰度具有一致性。但不同给药途径在组织分布丰度上有明显区别,由强至弱分别为:局部注射>腹腔注射>静脉注射,在组织分布特征上具有一致性。(2)小鼠腹腔注射后,ADV及tk的各组织分布动态变化上具有一致性,腹腔注射后1 d ADV及tk即具有高表达,7 d表达最强,14 d下降60%左右,21 d残留20%左右,32 d基本消失。(3)随着ADV-GFP给药剂量的增强,ADV的组织分布表现为剂量依赖性逐步增强。剂量为病毒颗粒数每1 kg为6.7×108时,ADV主要分布于肝脏、脾脏、淋巴结和肠系膜,分布较低。当剂量达到病毒颗粒数每1 kg为6×109时,ADV在10个器官的分布以肝脏、脾脏、淋巴结、肠系膜和肾上腺分布增加最为明显。当剂量增加到病毒颗粒数每1 kg为1×1012时,ADV在10个器官的分布继续增加;在病毒颗粒数每1 kg为1×1013的大剂量病毒剂量情况下,肝脏的转染效率增加不太显著,但在其他阳性器官的分布增加。结论:本实验提示,采用每周用药1次,病毒颗粒数每1 kg为1×1012,局部或局部血管用药的给药方案可以得到肝脏有效转染强度,有望为今后指导制定临床的最佳给药方案提供借鉴与参考。
OBJECTIVE: To study the distribution behavior of adenovirus-mediated delivery of herpes simplex virus thymidine kinase gene (ADV-tk) in animals after local or intravenous administration. Methods: The recombinant adenovirus and thymidine kinase were detected by green fluorescent protein (GFP) and in situ hybridization in liver, spleen, lymph node, mesentery, adrenal, thymus, kidney, thyroid, prostate and ovary Distribution of the situation. Results The results were judged by immunohistochemical score (IHS), combined with the percentage of positive cells and staining intensity of positive cells. Results: (1) The tissue distribution characteristics of three different routes of administration were similar. The distributions of ADV and tk positive tissue from strong to weak were as follows: liver, spleen, lymph node, mesentery, adrenal gland, thymus, kidney, Thyroid, prostate, ovary. The abundance of ADV or tk mRNA is consistent. However, different routes of administration have obvious differences in the distribution of tissue distribution, from strong to weak, respectively: local injection> intraperitoneal injection> intravenous injection, and have the same characteristics in the distribution of tissues. (2) After intraperitoneal injection, the dynamic distribution of ADV and tk in each tissue was consistent. At 1 d after intraperitoneal injection, ADV and tk were highly expressed, the expression was highest on the 7th day and decreased about 60% on the 14th day d left about 20%, 32 d disappeared. (3) With the dose of ADV-GFP increased, the tissue distribution of ADV showed a dose-dependent increase. At a dose of 6.7 × 108 per 1 kg of virus particles, ADV was mainly distributed in the liver, spleen, lymph nodes and mesentery with a low distribution. The distribution of ADV in 10 organs increased most obviously in the liver, spleen, lymph nodes, mesentery and adrenal gland when the dose reached 6 × 109 per 1 kg of virus particles. The distribution of ADV in 10 organs continued to increase when the dose was increased to 1 × 1012 per 1 kg of virus particles. In the case of large dose of virus dose of 1 × 1013 per 1 kg of virus particles, the transfection efficiency of liver Increase is not significant, but distribution increases in other positive organs. Conclusion: This experiment suggests that once weekly administration, the number of particles per 1 kg of virus particles 1 × 1012, local or regional blood drug administration program can be obtained in the liver effective transfection intensity, is expected to guide the development of clinical best Dosing regimen to provide reference and reference.