重组人γ-干扰素在耐多药结核病小鼠治疗作用中的研究

Study on the therapeutic effect with recombinant human IFN-？ on multidrug-resistant Mycobacterium tuberculosis in- fected mice

目的探讨重组人γ干扰素（rh-IFN-γ）在MDRTB小鼠中的治疗作用及其免疫学机制。方法72只成年雄性Balb／c小鼠，用含耐多药Mtb的气溶胶感染，感染后第2天处死4只以确定小鼠肺脏植入的菌量，感染后21d（设定治疗0周）脱颈处死4只小鼠作空白对照；余下64只小鼠采用随机数字表法分为对照组、rh-IFN-γ组、莫西沙星组和联合治疗组（莫西沙星＋rh-IFN-γ每组16只，于治疗0周开始给药。治疗4、8、16、20周各组分别脱颈处死4只，以测定小鼠的肺、脾质量指数和肺、脾菌落形成单位（CFU）、8周血清细胞因子IFN-γ和白细胞介素（IL）-10的水平。两组间比较采用t检验，多组间比较采用单因素方差分析，组内两两比较采用SNK法和Games-Howell法。结果肺脾质量指数：莫西沙星组8周肺指数为“5．99±0．72”、低于对照组的“8．01±0．91”（F=6．28，P〈0．01）；莫西沙星组8周脾指数为“2．87±0．15”，低于对照组的“3．87±0．41”（F=8．37，P〈0．01）。肺、脾CFU：莫西沙星组、联合治疗组4周肺组织CFU分别为（4．37±0．20）lgcFu／ml和（4．35±0．18）lgCFU／ml，均低于对照组[（5．30±0．21）lgCFU／ml]和rh-IFN-7组[（5．29±0．13）lgCFU／m1]（F=35．55，P〈0．01），莫西沙星组、联合治疗组8周肺组织CFU分别为（2．86±0．29）lgCFU／ml和（2．63±0．08）lgCFu／ml，均低于对照组[（5．00±0．23）lgCFU／m1]和rh_IFN-γ组[（4．82±0．55）lgCFU／ml]（F=56．83，P〈0．01），且均于16周始无菌化（即无Mtb生长）；rh-IFN-γ组20周脾组织CFU为（3．21±0．40）lgCFU，低于对照组的（4．31±0．06）lgCFU／ml（t=5．45，P〈0．01）；莫西沙星组和联合治疗组的脾组织从4周始无菌化。治疗8周小鼠血清r干扰素和IL-10的水平：rh-IFN-γ组、莫西沙星组和联合治疗组血清IFN-γ水平分别为（3．40±0．64）ng／L、（1．32±0．53）ng／L和（0．47±0．44）ng／L，低于对照组的（10．34±2．09）ng／L（F=55．973，P〈0．01），莫西沙星组和联合治疗组血清IFN_7水平分别为（1．32±0．53）ng／L和（0．47±0．44）ng／L，低于rh-IFN-γ组的（3．40±0．64）ng／L（F=55．973，P〈0．01）。对照组、rh-IFN-γ组、莫西沙星组和联合治疗组血清IL-10水平分别为（6．68±1．30）ng／L、（9．76±3．97）ng／L、（8．74±4．48）ng／L和（21．34±17．58）ng／L，差异无统计学意义（F=2．013，P〉0．05）。结论rh-IFN-γ可降低MDR-TB小鼠脾组织的菌量负荷，下调小鼠血清γ-干扰素水平，但对降低肺组织的菌量负荷和减轻肺脾组织炎症上无明显作用。rh-IFN-7辅助莫西沙星治疗耐多药结核病小鼠，在降低病变器官的菌量负荷和减轻炎症方面无辅助作用。

Objective To study the therapeutic effect and immunological mechanism of recombinant human interferon 7 （rh-IFN-7） in treating the mice with multidrug resistant tuberculosis （MDR TB）. Methods Seventytwo adult male Balb/c mice were infected with multidrug-resistant Mycobacterium tuberculosis via aerosol. Four mice were sacrificed at second day after infection to determine the colony forming units （CFUs） implanted in the lungs. Four mice were sacrificed at 21- day post infection （0 week of treatment）. The remaining 64 mice were randomly divided into control group, rh IFN-7 group, moxifloxacin group and combination therapy group （moxifloxacinq- rh-IFN-γ）, 16 mice in each group, and started to give medicine treatment at 0 week of treatment. Four mice per group were sacrificed at 4-, 8-, 16- and 20 weeks of treatment, to observe the mass index and live bacteria counting （CFUs） of lung and spleen at each time-point,and to detect the levels of IFN γ and IL-10 in sera at 8 weeks of treat ment. Comparison between the two groups used t test. Comparison among groups were tested by One-Way analysis of variance （ANOVA）,comparison in pairs within group used SNK method and Games Howell method. Results Lung and spleen mass index： the lung mass index of moxifloxacin group （5.99±0.72）at 8 weeks were significantly lower than that of control group（8.01±0.91）（F=6.28,P〈0.01）. The spleen mass index of moxifloxacin group （2.87＋0.15） at 8 weeks were significantly lower than that of control group （3.87--0.41）（F=8.37,P〈0.01）. Lung and spleen CFUs： the lung CFUs of moxifloxacin group [（4.37±0.20）lg/ml] and combination therapy group [ （ 4.35 ± 0. 18） lg/ml] at 4 weeks respectively were significantly lower than that of control group [ （5.30±0.21） lg/ml2 and rh-IFN-7 group [（5.29±0.13）lg/ml] （F--35.55,P〈0.01）, the lung CFUs of moxifloxacin group [（2.86± 0.29） lg/ml2 and combination therapy group E （2.63 ±0. 08） lg/ml] at 8 weeks respectively were significantly lower than that of control group [（5.00---4-_0.23）lg/ml~ and rh-IFN-7 group [-（4.82±0.55）lg/ml]（F=56.83,P〈0.01）, and were sterile from 16 weeks （without the growth of Mycobacterium tuberulosis）. Spleen CFUs of rh-IFN-7 group E（3.21±0.40）lg/ml] at 20 weeks were significantly lower than that of control group [（4.31 ± 0.06）lg/ml] （t= 5.45, P〈0.01）. Spleen tissue of moxifloxacin group and combination therapy group were sterile from 4 weeks. IFN- 3＇ and IL-10 levels in the sera at 8 weeks., the serum IFN-7 levels of rh-IFN-7 group E（3. 40±0.64）ng/L], moxi foxacin group [（1. 32±0. 53） ng/L] and combination therapy [（0.47 ± 0.44） ng/L] were significantly lower than that of control group [-（10.34±2.09）ng/L]（F=55. 973,P%0.01）, the serum IFN 3＇ levels of moxifloxacin group and combination therapy group respectively were obviously lower than that of rh-IFN γ group （F = 55. 973, P〈 0.01）; the serum ILl0 levels had no significant difference among control group [（6.68--4-1.30）ng/L], rh-IFN-＇/ group [（9.76±3.97）ng/L] ,moxifloxacin group [-（8.74±4.48）ng/L] and combination therapy group [（21.34± 17.58） ng/L] （F= 2.013, P〉0.05）. Conclusion Rh-IFN-γ can reduce the number of Mycobacterium tuberculosis in splenic tissue and lowered serum IFN-）＇ levels of MDRTB mice, but had no significant effect on reducing the number of Mycobacterium tuberculosis and the inflammation in lung tissue of MDR-TB mice. Rh-IFN-γ in combination with moxifloxacin had also no auxiliary role on the treatment of MDR-TB, and on reducing bacteria loads and inflammation of organs in mice.