血小板生成素对脓毒症血小板减少小鼠的作用研究
发布时间：2018-08-21 20:05
【摘要】：目的 脓毒症相关性血小板减少在危重病急救医学领域有较高的发病率和死亡率,威胁人类健康,目前,临床治疗上仍未取得突破性进展。血小板生成素(thrombopoietin, TPO)作为巨核细胞和血小板生成的特异性因子,已被成功应用于实体瘤和白血病化疗后所致的血小板减少症以及特发性血小板减少性紫癜等患者的治疗中,虽有报道TPO对脓毒症血小板减少的患者有效,但关于TPO对脓毒症相关性血小板减少的影响有待研究。基于此基础上,本项目以小鼠为研究对象,腹腔注射大肠杆菌脂多糖(lipopolysaccharide, LPS)制备脓毒症相关性血小板减少模型,观察各组小鼠血小板计数、骨髓增殖、炎症免疫及死亡率情况,以及不同剂量TPO对上述相关指标的影响,旨在观察TPO对脓毒症血小板减少小鼠的保护作用,并探讨其可能的机制,为临床提供理论依据。 方法 本研究采用SPF级C57/BL6小鼠100只,均为雄性,体重在18-21g,随机分为5组,分别为：正常对照组(C组)(注射同等剂量生理盐水)、模型组(LPS组)(腹腔注射LPS30mg/kg)、低剂量TPO治疗组(L组)(腹腔注射LPS30mg/kg+每日皮下注射TPO4.5ug/kg)、中剂量TPO治疗组(M组)(腹腔注射LPS30mg/kg+每日皮下注射TPO9ug/kg)、高剂量TPO治疗组(H组)(腹腔注射LPS30mg/kg+每日皮下注射TPO18ug/kg),其中TPO连续注射3天,观察每日小鼠外周血血小板计数的变化,于第4d处死小鼠,采用酶联免疫吸附(ELISA)方法测定血浆TNF-α、IL-6、IL-10等炎性因子水平,无菌条件下取骨髓采用流式细胞术测定巨核细胞CD41/CD61、造血干细胞CD34+表达,取肺组织做石蜡切片HE染色,光学显微镜镜下观察组织病理改变,取胸腺、脾脏等脏器称重并计算脏器指数,观察各组小鼠72h死亡率。 结果 1.LPS组小鼠注射LPS后血小板开始下降,1-4d血小板为C组的15-40%(P0.01)；1-2d时,L组小鼠血小板较M组、H组、LPS组相比较无明显变化(P0.05)；3-4d时,L组小鼠血小板明显高于M组、H组及LPS组(P0.01),M组、H组较LPS组则无升高(P0.05)；L组、M组、H组及LPS组血小板最低值(×109/L)分别为259.4±51.3、169.4+62.4、137.0±39.8、128.2±31.1,L组小鼠血小板最低值高于M组、H组及LPS组(P0.05),M组、H组较LPS组无明显升高(P0.05)。4d时LPS组小鼠骨髓巨核细胞CD41/CD61较正常组高(P0.05),L组小鼠骨髓巨核细胞CD41/CD61明显高于M组、H组、LPS组及C组(P0.01),M组、H组较C组升高(P0.05),较LPS组无升高(P0.05)；4d时LPS组CD34+较C组无变化(P0.05),L组较M组、H组、LPS组及C组增多(P0.05),M组、H组较LPS组及C组均无增多(P0.051。 2.4d时LPS组小鼠4d时TNF-a、IL-6水平较C组无变化(P0.051,L组、M组、H组分别较LPS组及C组均无明显改变(P0.05)；4d时LPS组IL-10水平较C组无变化(P0.05),L组小鼠IL-10水平明显高于M组、H组、LPS组及C组(P0.01),M组、H组较LPS组及C组无明显变化(P0.05)。4d时LPS组胸腺指数、脾指数较C组降低(P0.01),L组较LPS升高(P0.01),并恢复至C组水平(P0.05),M组、H组较C组降低(P0.01),较LPS组无升高(P0.05)。LPS组肺组织严重受损,L组的肺组织病变范围、程度均较M组、H组、LPS组明显减轻；M、H组病变较LPS组无明显改善。 3.LPS组、L组、M组、H组小鼠72h死亡率分别为65%、35%,60%、60%,L组明显低于其它三组(P0.01), M组、H组较LPS组无明显改善(P0.05)。 结论 1.LPS诱导的脓毒症血小板减少小鼠血小板严重下降,低剂量TPO可促进骨髓增殖,升高其血小板水平,提高血小板最低值,中、高剂量TPO则效果不明显； 2.LPS可致小鼠免疫力下降,肺组织严重受损,低剂量TPO可增加脓毒症血小板减少小鼠的免疫,升高抗炎因子水平,并从一定程度上改善肺组织炎症病变,降低72h死亡率,中、高剂量效果不明显； 3.低剂量TPO对LPS诱导的脓毒症血小板小鼠具有保护作用,可能与其升高血小板、提高免疫有关； 4.短期内应用TPO不增加脓毒症血小板减少小鼠的促炎因子水平,但较长时间应用TPO对脓毒症血小板减少小鼠的影响尚需进一步评估。
[Abstract]:objective
Thrombopoietin (TPO), as a specific factor of megakaryocyte and platelet production, has been successfully applied to solid tumors and leukemia. Although TPO has been reported to be effective in the treatment of thrombocytopenia and idiopathic thrombocytopenic purpura after chemotherapy, the effect of TPO on sepsis-related thrombocytopenia remains to be studied. Based on this, this project took mice as the subjects, and injected the large intestine intraperitoneally. Sepsis-related thrombocytopenia model was established by lipopolysaccharide (LPS). The platelet count, bone marrow proliferation, inflammatory immunity and mortality of mice in each group were observed, and the effects of different doses of TPO on the above-mentioned indexes were observed. The aim was to observe the protective effect of TPO on septic thrombocytopenia mice and to explore its possible mechanism. The mechanism provides a theoretical basis for clinical practice.
Method
In this study, 100 SPF C57/BL6 mice weighing 18-21g were randomly divided into five groups: normal control group (group C) (injected with the same dose of normal saline), model group (LPS group) (injected with LPS 30mg/kg intraperitoneally), low-dose TPO treatment group (L group) (injected with LPS 30mg/kg + injected with TPO 4.5ug/kg per day intraperitoneally) and middle-dose TPO treatment group (injected with same dose of normal saline). Group M (intraperitoneal injection of LPS 30mg/kg + daily subcutaneous injection of TPO 9ug/kg), high-dose TPO treatment group (group H) (intraperitoneal injection of LPS 30mg/kg + daily subcutaneous injection of TPO 18ug/kg), in which TPO was continuously injected for 3 days, the changes of peripheral blood platelet counts in mice were observed daily, and the mice were killed on the 4th day. Plasma TNF-a, IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). The levels of IL-10 and other inflammatory factors were measured by flow cytometry. The expression of CD41/CD61 in megakaryocytes and CD34+ in hematopoietic stem cells were detected by flow cytometry. The lung tissues were stained by HE. The histopathological changes were observed by light microscope. The thymus and spleen were weighed and the organ index was calculated. The 72-hour mortality rate of each group was observed.
Result
1. LPS group mice platelet began to decline after injection of LPS, 1-4 days platelet for C group 15-40% (P 0.01); 1-2 days, L group mice platelet compared with M group, H group, LPS group no significant change (P 0.05); 3-4 days, L group mice platelet significantly higher than M group, H group and LPS group (P 0.01), M group, H group than LPS group blood no increase (P 0.05); The platelet lowest value of L group was higher than that of M group. The platelet lowest value of H group and LPS group was higher than that of M group (P 0.05). The CD41/CD61 of bone marrow megakaryocyte of LPS group was higher than that of normal group (P 0.05). The CD41/CD61 of bone marrow megakaryocyte of L group was significantly higher than that of M group (P 0.05). Group H, group H, LPS and group C (P 0.01), group M, group H were higher than group C (P 0.05), group H was not higher than group LPS (P 0.05); group LPS CD34 + was no change than group C (P 0.05), group L was more than group M, group H, group LPS and group C (P 0.05), group M, group H was no more than group LPS and group C (P 0.051.05).
At 2.4 days, the levels of TNF-a and IL-6 in LPS group were not significantly different from those in C group (P 0.051, L group, M group, H group, LPS group and C group, respectively) (P 0.05); at 4 days, the levels of IL-10 in LPS group were not significantly different from those in C group (P 0.05); at 4 days, the levels of IL-10 in L group were significantly higher than those in M group, H group, LPS group and C group (P 0.01), M group, H group, LPS group and C group (P 0.05). Thymus index and spleen index were lower than those of group C (P 0.01), L group was higher than LPS (P 0.01), and recovered to the level of group C (P 0.05), M group and H group were lower than those of group C (P 0.01), but not higher than LPS group (P 0.05).
3. The 72-hour mortality of LPS group, L group, M group and H group were 65%, 35%, 60%, 60% respectively. The mortality of L group was significantly lower than that of the other three groups (P 0.01). There was no significant improvement in M group and H group compared with LPS group (P 0.05).
conclusion
1. LPS-induced thrombocytopenia in septic mice showed severe platelet reduction. Low-dose TPO could promote bone marrow proliferation, elevate platelet level and raise platelet minimum, while medium-dose and high-dose TPO had no significant effect.
2. LPS can decrease the immunity of mice and severely damage the lung tissue. Low-dose TPO can increase the immunity of mice with thrombocytopenia caused by sepsis, increase the level of anti-inflammatory factors, and improve the inflammation of lung tissue to a certain extent, reduce the mortality rate of 72 hours, but the high-dose effect is not obvious.
3. Low dose TPO has protective effect on LPS-induced septic platelet mice, which may be related to the increase of platelet and immunity.
4. Short-term use of TPO does not increase the level of pro-inflammatory factors in septic thrombocytopenia mice, but the effect of long-term use of TPO on septic thrombocytopenia mice needs further evaluation.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R459.7
本文编号：2196213
[Abstract]:objective
Thrombopoietin (TPO), as a specific factor of megakaryocyte and platelet production, has been successfully applied to solid tumors and leukemia. Although TPO has been reported to be effective in the treatment of thrombocytopenia and idiopathic thrombocytopenic purpura after chemotherapy, the effect of TPO on sepsis-related thrombocytopenia remains to be studied. Based on this, this project took mice as the subjects, and injected the large intestine intraperitoneally. Sepsis-related thrombocytopenia model was established by lipopolysaccharide (LPS). The platelet count, bone marrow proliferation, inflammatory immunity and mortality of mice in each group were observed, and the effects of different doses of TPO on the above-mentioned indexes were observed. The aim was to observe the protective effect of TPO on septic thrombocytopenia mice and to explore its possible mechanism. The mechanism provides a theoretical basis for clinical practice.
Method
In this study, 100 SPF C57/BL6 mice weighing 18-21g were randomly divided into five groups: normal control group (group C) (injected with the same dose of normal saline), model group (LPS group) (injected with LPS 30mg/kg intraperitoneally), low-dose TPO treatment group (L group) (injected with LPS 30mg/kg + injected with TPO 4.5ug/kg per day intraperitoneally) and middle-dose TPO treatment group (injected with same dose of normal saline). Group M (intraperitoneal injection of LPS 30mg/kg + daily subcutaneous injection of TPO 9ug/kg), high-dose TPO treatment group (group H) (intraperitoneal injection of LPS 30mg/kg + daily subcutaneous injection of TPO 18ug/kg), in which TPO was continuously injected for 3 days, the changes of peripheral blood platelet counts in mice were observed daily, and the mice were killed on the 4th day. Plasma TNF-a, IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). The levels of IL-10 and other inflammatory factors were measured by flow cytometry. The expression of CD41/CD61 in megakaryocytes and CD34+ in hematopoietic stem cells were detected by flow cytometry. The lung tissues were stained by HE. The histopathological changes were observed by light microscope. The thymus and spleen were weighed and the organ index was calculated. The 72-hour mortality rate of each group was observed.
Result
1. LPS group mice platelet began to decline after injection of LPS, 1-4 days platelet for C group 15-40% (P 0.01); 1-2 days, L group mice platelet compared with M group, H group, LPS group no significant change (P 0.05); 3-4 days, L group mice platelet significantly higher than M group, H group and LPS group (P 0.01), M group, H group than LPS group blood no increase (P 0.05); The platelet lowest value of L group was higher than that of M group. The platelet lowest value of H group and LPS group was higher than that of M group (P 0.05). The CD41/CD61 of bone marrow megakaryocyte of LPS group was higher than that of normal group (P 0.05). The CD41/CD61 of bone marrow megakaryocyte of L group was significantly higher than that of M group (P 0.05). Group H, group H, LPS and group C (P 0.01), group M, group H were higher than group C (P 0.05), group H was not higher than group LPS (P 0.05); group LPS CD34 + was no change than group C (P 0.05), group L was more than group M, group H, group LPS and group C (P 0.05), group M, group H was no more than group LPS and group C (P 0.051.05).
At 2.4 days, the levels of TNF-a and IL-6 in LPS group were not significantly different from those in C group (P 0.051, L group, M group, H group, LPS group and C group, respectively) (P 0.05); at 4 days, the levels of IL-10 in LPS group were not significantly different from those in C group (P 0.05); at 4 days, the levels of IL-10 in L group were significantly higher than those in M group, H group, LPS group and C group (P 0.01), M group, H group, LPS group and C group (P 0.05). Thymus index and spleen index were lower than those of group C (P 0.01), L group was higher than LPS (P 0.01), and recovered to the level of group C (P 0.05), M group and H group were lower than those of group C (P 0.01), but not higher than LPS group (P 0.05).
3. The 72-hour mortality of LPS group, L group, M group and H group were 65%, 35%, 60%, 60% respectively. The mortality of L group was significantly lower than that of the other three groups (P 0.01). There was no significant improvement in M group and H group compared with LPS group (P 0.05).
conclusion
1. LPS-induced thrombocytopenia in septic mice showed severe platelet reduction. Low-dose TPO could promote bone marrow proliferation, elevate platelet level and raise platelet minimum, while medium-dose and high-dose TPO had no significant effect.
2. LPS can decrease the immunity of mice and severely damage the lung tissue. Low-dose TPO can increase the immunity of mice with thrombocytopenia caused by sepsis, increase the level of anti-inflammatory factors, and improve the inflammation of lung tissue to a certain extent, reduce the mortality rate of 72 hours, but the high-dose effect is not obvious.
3. Low dose TPO has protective effect on LPS-induced septic platelet mice, which may be related to the increase of platelet and immunity.
4. Short-term use of TPO does not increase the level of pro-inflammatory factors in septic thrombocytopenia mice, but the effect of long-term use of TPO on septic thrombocytopenia mice needs further evaluation.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R459.7
【引证文献】
相关期刊论文 前1条
1 陈宇辉;;重组人血小板生成素和免疫球蛋白治疗脓毒症相关性血小板减少症的临床疗效观察[J];中国继续医学教育;2015年25期
，本文编号：2196213
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