中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (51): 7697-7702.doi: 10.3969/j.issn.2095-4344.2016.51.015

• 组织构建与生物活性因子 tissue construction and bioactive factors • 上一篇    下一篇

过表达人抗菌肽LL37腺病毒构建及转染阴道上皮细胞

申复进,许学先,周利梅   

  1. 武汉大学人民医院妇3科,湖北省武汉市   430060
  • 收稿日期:2016-09-26 出版日期:2016-12-09 发布日期:2016-12-09
  • 作者简介:申复进,男,1978年生,湖北省京山县人,汉族,2008年武汉大学外科学毕业,博士,副主任医师,主要从事女性泌尿生殖系组织工程研究。
  • 基金资助:

    国家自然科学基金青年项目(81100395)

Construction of a recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene and its transfection into vaginal epithelial cells

Shen Fu-jin, Xu Xue-xian, Zhou Li-mei   

  1. First Department of Gynecology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
  • Received:2016-09-26 Online:2016-12-09 Published:2016-12-09
  • About author:Shen Fu-jin, M.D., Associate chief physician, First Department of Gynecology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
  • Supported by:

    the Youth Project of National Natural Science Foundation of China, No. 81100395

摘要:

文章快速阅读:

文题释义:
抗菌肽:原指昆虫体内经诱导而产生的一类具有抗菌活性的碱性多肽物质,相对分子质量在2 000-7 000,由20-60个氨基酸残基组成。这类活性多肽多数具有强碱性、热稳定性以及广谱抗菌等特点。世界上第一个被发现的抗菌肽的是1980年由瑞典科学家G.Boman等人经注射阴沟通杆菌及大肠杆菌诱导惜古比天蚕蛹产生的具有抗菌活性的多肽,定名为Cecropins。
腺病毒:是一种没有包膜的直径为70-90 nm的颗粒,由252个壳粒呈廿面体排列构成。衣壳里是线状双链DNA分子,腺病毒分两个属,共约100余血清型。一般会造成呼吸系统的不适,对啮齿类动物有致癌能力,或能转化体外培养的啮齿类动物细胞,但对人体不出现致癌性。
摘要
背景:
LL-37作为唯一存在人体的抗微生物肽,不仅能促内皮细胞增殖,而且具有较强的促进血管生成能力,在组织修复初期的血管生成中起重要作用。
目的:构建过表达人抗菌肽LL37基因的重组腺病毒,体外转染犬阴道上皮细胞,检测抗菌肽LL37的表达和分泌情况。
方法:PCR法扩增LL37基因片段,构建融合绿色荧光蛋白(EGFP)和LL37的重组腺病毒表达质粒,限制性内切酶酶切和测序法鉴定构建的重组腺病毒表达质粒,采用HEK293包装后反复冻融扩增、纯化过表达LL37的腺病毒,终点稀释法检测腺病毒滴度。体外分离培养犬阴道上皮细胞,转染过表达LL37腺病毒,分别于转染后1,2,3,5和7 d采用荧光显微镜观察细胞转染情况,ELISA法测定细胞培养上清液中LL37的分泌表达情况。
结果与结论:①限制性内切酶酶切鉴定与DNA测序证实成功构建了GV314-LL37腺病毒表达载体,包装纯化后腺病毒滴度达到3×109 pfu/mL;②犬阴道上皮细胞可在体外成功分离培养并稳定传代;③过表达LL37腺病毒可高效转染阴道上皮细胞,转染24 h后荧光显微镜下即可见绿色荧光蛋白表达,随时间延长表达逐渐增强,转染72 h时表达最强,转染效率达89%;④ELISA证实阴道上皮细胞转染过表达LL37腺病毒后表达分泌LL37,转染后细胞培养上清中LL37表达量显著高于空白组,表达呈时间依赖性,转染3 d表达最强,转染后7 d仍持续分泌表达;⑤结果说明,成功构建了过表达人抗菌肽LL37基因的重组腺病毒,转染犬阴道上皮细胞后高效表达分泌LL37,为构建具有抗感染能力和促血管生成的功能性组织工程化阴道奠定了基础。

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松组织工程
ORCID: 0000-0002-5195-8628(申复进)

关键词: 组织构建, 组织工程, 抗菌肽, LL37, 阴道上皮细胞, 腺病毒, 转染, 阴道, 组织工程, 国家自然科学基金

Abstract:

BACKGROUND: LL37, the only antimicrobial peptide of the cathelicidin family identified from the human, not only promotes the proliferation of endothelial cells, but also plays an important role in angiogenesis and re-epithelialization.
OBJECTIVE: To construct a recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene, and to detect the expression and secretion of LL37 after transfected into the canine vaginal epithelial cells.
METHODS: The cDNA encoding LL37 was amplified by PCR. Recombinant adenovirus expression plasmid encoding LL37 and green fluorescent protein (EGFP) was constructed, and identified using restriction endonuclease technology and DNA sequencing method. Adenovirus particles were generated by cotransfecting the 293-packaging cell line. The adenovirus were collected, amplified and concentrated, and viral titers were determined by end-point dilution assay by applying serial dilutions of the purified viruses to 293 cells. Primary cultured canine vaginal epithelial cells were transfected by the recombinant adenovirus. The transfection efficacy was observed by fluorescence microscope, and the cultured supernatant was collected to determine the expression of LL37 by ELISA method at 1, 2, 3, 5 and 7 days after transfection.
RESULTS AND CONCLUSION: The adenovirus vector GV314-LL37 with the titer of 3×109 pfu/mL was successfully constructed and identified by DNA sequencing methods. Canine vaginal epithelial cells were successfully isolated and cultured and grew stably. After transfection, vaginal epithelial cells could express the EGFP and LL37 efficiently in a time-dependent manner detected by fluorescence microscope and ELISA method. The transfection efficacy of EGFP reached to 89% at 72 hours. The level of LL37 in the cell culture supernatant in the transfection group was significantly higher than that in the control group, the highest expression of LL37 was found at 3 days that lasting for 7 days. In conclusion, the recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene is successfully constructed, which can express and secrete LL37 after transfected into canine vaginal epithelial cells, providing a foundation for constructing the tissue-engineered vagina possessing anti-infection and neovascularization.

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松组织工程

Key words: Vagina, Epithelial Cells, Dermcidins, Tissue Engineering

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