血小板生成素對膿毒癥血小板減少小鼠的作用研究
發(fā)布時間：2018-08-21 20:05
【摘要】：目的 膿毒癥相關(guān)性血小板減少在危重病急救醫(yī)學(xué)領(lǐng)域有較高的發(fā)病率和死亡率,威脅人類健康,目前,臨床治療上仍未取得突破性進(jìn)展。血小板生成素(thrombopoietin, TPO)作為巨核細(xì)胞和血小板生成的特異性因子,已被成功應(yīng)用于實(shí)體瘤和白血病化療后所致的血小板減少癥以及特發(fā)性血小板減少性紫癜等患者的治療中,雖有報(bào)道TPO對膿毒癥血小板減少的患者有效,但關(guān)于TPO對膿毒癥相關(guān)性血小板減少的影響有待研究。基于此基礎(chǔ)上,本項(xiàng)目以小鼠為研究對象,腹腔注射大腸桿菌脂多糖(lipopolysaccharide, LPS)制備膿毒癥相關(guān)性血小板減少模型,觀察各組小鼠血小板計(jì)數(shù)、骨髓增殖、炎癥免疫及死亡率情況,以及不同劑量TPO對上述相關(guān)指標(biāo)的影響,旨在觀察TPO對膿毒癥血小板減少小鼠的保護(hù)作用,并探討其可能的機(jī)制,為臨床提供理論依據(jù)。 方法 本研究采用SPF級C57/BL6小鼠100只,均為雄性,體重在18-21g,隨機(jī)分為5組,分別為：正常對照組(C組)(注射同等劑量生理鹽水)、模型組(LPS組)(腹腔注射LPS30mg/kg)、低劑量TPO治療組(L組)(腹腔注射LPS30mg/kg+每日皮下注射TPO4.5ug/kg)、中劑量TPO治療組(M組)(腹腔注射LPS30mg/kg+每日皮下注射TPO9ug/kg)、高劑量TPO治療組(H組)(腹腔注射LPS30mg/kg+每日皮下注射TPO18ug/kg),其中TPO連續(xù)注射3天,觀察每日小鼠外周血血小板計(jì)數(shù)的變化,于第4d處死小鼠,采用酶聯(lián)免疫吸附(ELISA)方法測定血漿TNF-α、IL-6、IL-10等炎性因子水平,無菌條件下取骨髓采用流式細(xì)胞術(shù)測定巨核細(xì)胞CD41/CD61、造血干細(xì)胞CD34+表達(dá),取肺組織做石蠟切片HE染色,光學(xué)顯微鏡鏡下觀察組織病理改變,取胸腺、脾臟等臟器稱重并計(jì)算臟器指數(shù),觀察各組小鼠72h死亡率。 結(jié)果 1.LPS組小鼠注射LPS后血小板開始下降,1-4d血小板為C組的15-40%(P0.01)；1-2d時,L組小鼠血小板較M組、H組、LPS組相比較無明顯變化(P0.05)；3-4d時,L組小鼠血小板明顯高于M組、H組及LPS組(P0.01),M組、H組較LPS組則無升高(P0.05)；L組、M組、H組及LPS組血小板最低值(×109/L)分別為259.4±51.3、169.4+62.4、137.0±39.8、128.2±31.1,L組小鼠血小板最低值高于M組、H組及LPS組(P0.05),M組、H組較LPS組無明顯升高(P0.05)。4d時LPS組小鼠骨髓巨核細(xì)胞CD41/CD61較正常組高(P0.05),L組小鼠骨髓巨核細(xì)胞CD41/CD61明顯高于M組、H組、LPS組及C組(P0.01),M組、H組較C組升高(P0.05),較LPS組無升高(P0.05)；4d時LPS組CD34+較C組無變化(P0.05),L組較M組、H組、LPS組及C組增多(P0.05),M組、H組較LPS組及C組均無增多(P0.051。 2.4d時LPS組小鼠4d時TNF-a、IL-6水平較C組無變化(P0.051,L組、M組、H組分別較LPS組及C組均無明顯改變(P0.05)；4d時LPS組IL-10水平較C組無變化(P0.05),L組小鼠IL-10水平明顯高于M組、H組、LPS組及C組(P0.01),M組、H組較LPS組及C組無明顯變化(P0.05)。4d時LPS組胸腺指數(shù)、脾指數(shù)較C組降低(P0.01),L組較LPS升高(P0.01),并恢復(fù)至C組水平(P0.05),M組、H組較C組降低(P0.01),較LPS組無升高(P0.05)。LPS組肺組織嚴(yán)重受損,L組的肺組織病變范圍、程度均較M組、H組、LPS組明顯減輕；M、H組病變較LPS組無明顯改善。 3.LPS組、L組、M組、H組小鼠72h死亡率分別為65%、35%,60%、60%,L組明顯低于其它三組(P0.01), M組、H組較LPS組無明顯改善(P0.05)。 結(jié)論 1.LPS誘導(dǎo)的膿毒癥血小板減少小鼠血小板嚴(yán)重下降,低劑量TPO可促進(jìn)骨髓增殖,升高其血小板水平,提高血小板最低值,中、高劑量TPO則效果不明顯； 2.LPS可致小鼠免疫力下降,肺組織嚴(yán)重受損,低劑量TPO可增加膿毒癥血小板減少小鼠的免疫,升高抗炎因子水平,并從一定程度上改善肺組織炎癥病變,降低72h死亡率,中、高劑量效果不明顯； 3.低劑量TPO對LPS誘導(dǎo)的膿毒癥血小板小鼠具有保護(hù)作用,可能與其升高血小板、提高免疫有關(guān)； 4.短期內(nèi)應(yīng)用TPO不增加膿毒癥血小板減少小鼠的促炎因子水平,但較長時間應(yīng)用TPO對膿毒癥血小板減少小鼠的影響尚需進(jìn)一步評估。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R459.7
本文編號：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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R459.7
【引證文獻(xiàn)】
相關(guān)期刊論文 前1條
1 陳宇輝;;重組人血小板生成素和免疫球蛋白治療膿毒癥相關(guān)性血小板減少癥的臨床療效觀察[J];中國繼續(xù)醫(yī)學(xué)教育;2015年25期
，本文編號：2196213
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