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目的:构建携带人SP-B蛋白+1580 SNP不同等位基因的转基因小鼠并进行细菌性肺炎模型的造模。方法:利用受精卵原核注射技术将hSP-B基因整合至小鼠染色体上获得F0代小鼠,将其与mSP-B基因敲除鼠进行交配,逐步去除转基因小鼠体内m SP-B基因。利用PCR技术鉴定小鼠基因型,通过测序确定+1580位点的等位基因。将铜绿假单胞菌经支气管灌注接种至小鼠肺内进行细菌性肺炎造模,对照组注射等量灭菌生理盐水。结果:F2代小鼠只表达人SP-B蛋白而不表达鼠SP-B蛋白,蛋白表达量与人肺内含量相近,即为构建成功的转基因小鼠。3个小鼠家系+1580位点等位基因为T,1个家系为C。细菌接种(1×10~6CFU/mouse)后24小时,小鼠肺泡内炎症渗出明显,大量中性粒细胞浸润,SP-B蛋白含量明显降低,但不同等位基因间在此条件下无明显差异。结果:成功构建只表达人SP-B蛋白的转基因小鼠模型,细菌性肺炎模型造模成功,为今后进一步研究人SP-B蛋白的生理功能及+1580基因多态性与肺疾病的关系提供了有力的工具。
OBJECTIVE: To construct transgenic mice carrying different alleles of human SP-B protein +1580 SNP and to model the bacterial pneumonia model. METHODS: FSP mice were obtained by integrating hSP-B gene into mouse chromosome by pronuclear injection of fertilized egg and mating with mSP-B knockout mice to gradually remove m SP-B gene from transgenic mice. Mouse genotypes were identified by PCR and the alleles at +1580 locus were determined by sequencing. Pseudomonas aeruginosa was inoculated into the lungs of mice via bronchial injection for bacterial pneumonia modeling, and the control group was injected with the same amount of sterile saline. Results: F2 mice only expressed human SP-B protein but not mouse SP-B protein. The expression level of protein was similar to that of human lung, which means the transgenic mice were successfully constructed. Three pedigrees +1580 locus allele T, a family of C. At 24 hours after bacterial inoculation (1 × 10 ~ 6CFU / mouse), the inflammation of alveoli in the mice showed obvious exudation, a large amount of neutrophil infiltration, and the content of SP-B protein decreased obviously. However, among the different alleles, Significant differences. Results: The transgenic mouse model expressing human SP-B protein was constructed successfully and the model of bacterial pneumonia was successfully established. It provides a theoretical basis for further study on the physiological function of human SP-B protein and the relationship between +1580 gene polymorphism and lung diseases A powerful tool.