无绿藻感染小鼠皮肤模型的构建

Establishment of a mouse model of cutaneous protothecosis

目的构建无绿藻感染小鼠皮肤模型。方法BALB/c小鼠分为4组，高浓度接种免疫抑制组为免疫抑制小鼠，腹部皮下接种1×10，孢子／ml中型无绿藻波多黎各变种悬液组；低浓度接种免疫抑制组为免疫抑制小鼠，腹部皮下接种1×10e孢子／ml悬液组；高浓度接种健康组为健康小鼠，腹部皮下接种1×10^9孢子／ml悬液组；对照组为健康小鼠腹部皮下接种生理盐水溶液，每个部位接种量均为200μl。接种后第7、14、28天时观察各组小鼠腹部皮损变化，并进行腹部皮肤病理及真菌检查。结果实验组小鼠接种处均出现丘疹、脓肿，部分出现溃疡、结痂，感染率均为100％。3组小鼠的腹部皮损直径，组内各时间点比较差异均有统计学意义（均P〈0．05），均在第7天时最大，分别为（6．75±1．09）mm、（5．88±1．17）mm和（5．96±0．99）mm；组间相比，在接种后第7、14天时差异无统计学意义（P〉0．05），在第28天时差异有统计学意义（F=8．91，P〈0．05），高浓度接种免疫抑制组最大为（4．38±0．86）mm。3个实验组基本病理改变为坏死、脓肿、肉芽肿形成，HE染色和过碘酸雪夫（PAS）染色均可见孢子，皮损直接镜检可见孢子，培养为无绿藻生长。对照组未感染无绿藻。结论腹部皮下接种免疫抑制和健康小鼠均可构建无绿藻病皮肤感染模型。

Objective To develop a mouse model of cutaneous protothecosis. Methods Totally, 48 BALB/c mice were randomly and equally divided into four groups： high- and low-concentration immunosuppressive groups-immunosuppressed mice inoculated with P. zopfii vat. portorieensis suspension of 1 ×10^9 and 1 × 10^6 colony forming units （CFU） conidia/ml respectively, high-concentration healthy group-heahhy mice inoculated with P. zopfii var. portoricensis suspension of 1×10^9 CFU conidia/ml, and control group-healthy mice inoculated with sodium chloride physiological solution. The P. zopfii suspension or sodium chloride physiological solution was subcutaneously inoculated to the abdominal skin of mice, with the inoculation volume being 200 μl. Skin appearance at the inoculation site was observed, and four mice were sacrificed in each group on day 7, 14 and 28 after the inoculation. Skin specimens were resected from the inoculation sites of mice and subjected to pathological and mycological examinations. Results Mice in the three experiment groups inoculated with the P.zopfii suspension were all infected, with the appearance of papules and abscess at the inoculation sites of all mice as well as ulcer and crusts in some mice. Meanwhile, no mice were infected in the control group. Significant differences were noted in the diameter of skin lesions between the three time points in these experiment groups （all P 〈 0.05）, and the largest diameter of lesions was observed on day 7, which was （6.75 ＋ 1.09） mm in the high-concentration immunosuppressive group, （5.88 ＋ 1.17） mm in the low-concentration immunosuppressive group, and （5.96 -＋ 0.99） mm in the high-concentration healthy group. Comparisons of lesion diameter between the three experiment groups revealed a statistical difference on day 28 （F = 8.91, P 〈 0.05）, with the largest lesion diameter observed in the high-concentration immunosuppressive group （4.38 ＋ 0.86 mm）, but no statistical difference was found on day 7 or 14 （both P 〉 0.05）. Pathology of skin specimens consistently revealed necrosis, abscess and granuloma formation in the three experiment groups. Spores were found in the lesions of infected mice by haematoxylin-eosin staining, periodic acid-Schiff staining, and direct microscopy. Culture of tissue samples from the experiment groups grew Prototheca. Conclusion The mouse model of protothecosis can be established by subcutaneous inoculation of Prototheea suspension in the abdominal skin of healthy or immunosuppressed mice.