本文選題：彌漫性血管內凝血　切入點：脂多糖　出處：《山東大學》2013年博士論文

【摘要】：研究目的：研究骨髓間充質干細胞(BMSCs)預處理對脂多糖(LPS)誘導彌漫性血管內凝血(DIC)模型大鼠的器官保護性作用并探討其機制。 研究方法： 第一部分：取同種異體Wistar大鼠原代骨髓間充質干細胞進行傳代培養(yǎng)。對培養(yǎng)出的骨髓干細胞進行形態(tài)觀察,考察其增殖能力；通過流式細胞儀對其細胞表型進行鑒定；在特定條件下培養(yǎng)BMSCs向內皮細胞,成骨細胞,脂肪細胞的分化,以證實其潛在可塑性,進一步確定其為BMSCS. 第二部分：LPS誘導Wistar大鼠DIC模型的建立。30只成年雄性Wistar大鼠,月齡7周,體重160-170g,分為2組,實驗組和對照組各15只,按劑量30mg/kg腹腔內注射異戊巴比妥鈉麻醉后,實驗組經(jīng)尾靜脈或股靜脈緩慢注射2ml LPS (3mg/kg,1小時),而對照組給予等量生理鹽水。深麻醉下分別在LPS注射前及注射后4小時和8小時經(jīng)下腔靜脈抽取靜脈血,測定以下凝血參數(shù)：血小板(PLT)數(shù)量,纖維蛋白原(Fib),部分凝血活酶時間(APTT),凝血酶原時間(PT),D-2聚體(D-D)。在LPS注射8小時后處死大鼠。取實驗組及對照組大鼠心臟,肝臟,腎臟,肺等主要器官的組織用10%甲醛固定24h,分別行HE染色和Mallory磷鎢酸蘇木精染色(PTAH染色),顯微鏡下觀察各臟器細胞形態(tài),纖維血栓形成情況,并進行對比；同時計數(shù)100個腎小球,觀察其中有纖維蛋白微血栓的腎小球所占比例,以判斷本組實驗LPS能否成功誘導建立DIC模型。 第三部分：慢病毒介導GFP轉染骨髓間充質干細胞及體內檢測。慢病毒載體(PGCL-GFP、 pHelper1.0、 pHelper2.0)包裝后獲取病毒的上清液保存。取第3代生長狀態(tài)良好的大鼠BMSC5×104個,分別與0.25、2.5、5、12.5、25感染復數(shù)(MOI)的病毒液,在Polybrene液中(8g/m1),32℃,1000g離心2小時,然后種于12孔板培養(yǎng),每孔含10%FBS的DMEM1.5ml;12h后換液,此后每3天換液1次并觀察BMSCs熒光表達情況；第5天每個孔取5個視野計算平均轉染率,了解MOI與轉染效率的關系,選擇最佳MOI。取月齡7周,體重160-170g成年雄性Wistar大鼠10只,經(jīng)大鼠尾靜脈或股靜脈注入1m1培養(yǎng)液(含有GFP標記轉染5天后BMSCs數(shù)量約1×106)共3次,每次間隔24h,第3次注射24h后處死大鼠,取大鼠心臟組織,用10%甲醛固定24h,然后行HE及抗GFP過氧化物酶染色。觀察心肌組織內是否有BMSCs存活。 第四部分：上述實驗成功后,取60只成年雄性Wistar大鼠,月齡7周,體重160-170g,按照BMSCs預處理和LPS注射方案不同分為6組,每組各有大鼠10只。處理組1：1ml培養(yǎng)液(不含BMSCs)注射3次,每次間隔24小時,進行預處理。在最后一次預處理結束后24h,注射1ml LPS(劑量,3mg/kg)o處理組2：1ml培養(yǎng)液(含BMSCs1x103)注射3次,每次間隔24小時,進行預處理。在最后一次預處理結束后24h,注射1ml LPS(劑量,3mg/kg).處理組3：1ml培養(yǎng)液(含BMSCs1x104)注射3次每次間隔24小時,進行預處理。在最后一次預處理結束后24h,注射1ml LPS(劑量,3mg/kg).處理組4：1mI培養(yǎng)液(含BMSCs1x105)注射3次每次間隔24小時,進行預處理。在最后一次預處理結束后24h,注射1ml LPS(劑量,3mg/kg).處理組5：1ml培養(yǎng)液(含BMSCs1x106)注射3次,每次間隔24小時,進行預處理。在最后一次預處理結束后24h,注射1ml LPS(劑量,3mg/kg).對照組：1m1培養(yǎng)液,不含BMSCs,注射3次,每次間隔24小時。在最后一次注射后24h,注射生理鹽水1m1(不含LPS)。共6組。分別在LPS(或不含LPS)注入前及注入后4小時和8小時各抽取靜脈血,測量以下參數(shù)：PLT, Fib, D-2聚體(D-D),APTT, PT,腫瘤壞死因子-α(TNF-α),干擾素-γ(IFN-γ),白細胞介素-1β(IL-1β),肌酐(Cr),丙氨酸轉氨酶(ALT),磷酸肌酸激酶同工酶(CK-MB),內皮素(ET)。取心臟,肝臟,腎臟及肺部組織進行HE染色及PTAH染色,檢測各器官組織細胞形態(tài)及微纖維血栓形成情況。為了進一步證明BMSCs預處理對LPS誘導DIC大鼠的保護作用,我們選擇另外60只成年雄性Wistar大鼠,月齡7周,體重160-170g,隨機分為三組,觀察短期內大鼠死亡事件。為更好觀察短期內大鼠死亡事件,我們加大LPS注射劑量至10mg/kg,觀察24小時內BMSCs預處理對LPS誘導DIC的保護作用。第1組：LPS注射,(LPS10mg/kg);第2組：BMSCs預處理,(1×103/次×3次,每次間隔24小時)+LPS注射(10mg/kg,在BMSCs最后一次預處理結束后24h進行)。第3組：BMSCs預處理,(1×105/次×3次,每次間隔24小時)+LPS注射(10mg/kg,在BMSCs最后一次預處理結束后24h進行)。BMSCs經(jīng)股靜脈注入,LPS行腹腔注射。觀察LPS注射后24h內各組大鼠生存情況,統(tǒng)計各組死亡時間,行統(tǒng)計學生存分析,繪制生存曲線。 第五部分：為進一步明確BMSCs預處理的保護機制,我們對不同劑量BMSCs預處理后大鼠外周血淋巴細胞(PBMC)增殖能力的影響進行了測定,并在體外環(huán)境下研究了不同劑量同種異體BMSCs對LPS刺激PBMC增殖的影響,以及對炎性細胞因子產(chǎn)生的影響。 結果：1,第1-5天,細胞快速生長,是增殖最快的時期,約第6天BMSCs增值達到頂峰,此后維持小幅波動,總體顯示BMSCs生長旺盛。經(jīng)分化培養(yǎng),BMSCs細胞可分化成為內皮細胞,成骨細胞,脂肪細胞；經(jīng)流式細胞學檢測,BMSCs CD29, CD44, CD10, CD106表現(xiàn)陽性,而CD34, CD45表現(xiàn)陰性,符合骨髓間充質干細胞分子表型特征；2,LPS注射組和對照組相比,血漿PLT數(shù)量明顯降低(P＜0.01), Fib含量降低(P0.01),D-2聚體量偏高(P0.01),APTT(P0.01)和PT(P0.01)明顯延長,而且腎臟,肺,肝臟,心臟組織切片經(jīng)HE染色和PTAH染色均提示有微血管血栓形成。LPS注射組發(fā)生DIC。3,慢病毒轉染293T細胞48小時后可見大量熒光,轉染效率良好。病毒轉染BMSCs后第5天,可見BMSCs熒光表達,BMSCs生長良好。不同MOI條件下病毒對BMSCs轉染的效率不同,當MOI為12.5時,轉染效率最高。經(jīng)靜脈注射GFP標記BMSCs三次后(每次間隔24h,劑量為1ml培養(yǎng)液含細胞數(shù)1×106),取心肌組織切片,抗GFP過氧化物酶染色呈陽性,提示心肌內有BMSCs細胞存活。4,①和未經(jīng)BMSCs預處理組相比,BMSCs預處理組血漿PLT(P＜0.01)和Fib(P0.05)含量高,而D-2聚體含量偏低(P0.01),而且,APTT (P＜0.05)和PT(P0.01)延長偏短,凝血狀況較好；預處理BMSCs數(shù)量為1×105/次和1×106/次時最好。②與未經(jīng)BMSC預處理組相比,BMSCs預處理組血漿TNF-a (P＜0.01)IFN-γ(P0.01)、 IL-1β(P＜0.05)濃度值均明顯偏低,而且組間比較具有統(tǒng)計學意義；在預處理BMSCs數(shù)量為1×105/次和1×106/次時,TNF-a, IFN-y和IL-1p值最低。③與未經(jīng)BMSCs預處理組相比,BMSCs預處理組血漿ALT, Cr, CK-MB (P0.05)和ET(P0.05)濃度明顯降低；組間比較發(fā)現(xiàn)Cr和ET水平的差異具有統(tǒng)計學意義；預處理BMSCs數(shù)量為1×105/次和1×106/次時Cr和ET水平最低。④各組腎臟,肺,肝臟,心臟組織切片經(jīng)HE染色和PTAH染色均顯示BMSCs預處理組器官纖維微血管血栓形成量較少,器官細胞損傷較小。預處理BMSCs數(shù)量為1×105/次和1×106/次時,纖維微血管血栓形成量最少,器官細胞損傷最輕。⑤BMSCs預處理對LPS誘導DIC大鼠的死亡事件的觀察,結果提示三組大鼠(每組n=20)24小時內總生存率分別為：第一組34.2%,第二組51.3%,第三組64.5%,生存率比較P=0.02,有顯著統(tǒng)計學意義；組間生存率比較,第一組VS第二組P=0.011,第一組VS第三組P=0.001,第二組VS第三組P=0.35。5,①和對照組相比,經(jīng)BMSCs預處理組大鼠PBMC在LPS刺激下的增殖能力明顯減弱。②LPS可明顯刺激PBMC的增殖,但在BMSCs的影響下,這種刺激作用明顯減弱(P0.05)；在Transwell培養(yǎng)下,可達到同樣效果(P0.05)。LPS刺激后,培養(yǎng)液炎性因子的濃度明顯增加,但和BMSCs混合培養(yǎng)后,炎性因子釋放增加的量明顯減少,在Transwell培養(yǎng)中也有同樣的表現(xiàn)。③和BMSCs混合培養(yǎng)時,LPS刺激PBMC的增殖明顯減少,隨著BMSCs數(shù)量越多,LPS刺激PBMC的增殖數(shù)量越來越少。和BMSCs混合培養(yǎng)時,培養(yǎng)液炎性因子的濃度開始減少,隨著BMSCs量的增加,炎性因子的濃度越來越低。當BMSCs增加到一定濃度時,PBMC增殖量不再減少,炎性因子濃度也不在降低。 結論：1,用密度梯度法進行BMSCs分離,貼壁法和機械刮除法進行BMSCs培養(yǎng)和體外擴增,可以獲得形態(tài)較單一的BMSCs樣細胞,細胞生長形態(tài)和狀況良好,經(jīng)流式細胞表型鑒定和多向分化培養(yǎng)鑒定證實為BMSCs.2,用LPS靜脈推注可成功誘導建立Wistar大鼠DIC模型。此法操作簡便,成功率高。LPS靜脈推注可作為建立大鼠DIC模型的可靠方法。3,用慢病毒載體可成功轉染GFP于大鼠骨髓間充質干細胞中,3次靜脈注射(每次間隔24h)本實驗所培養(yǎng)的GFP標記大鼠骨髓間充質量干細胞后,可在體內發(fā)現(xiàn)存活的BMSCS.4, BMSCs預處理可有效減輕LPS誘導大鼠DIC模型的器官損傷,抑制血管內凝血,提高大鼠生存率。這種保護性反應在一定范圍內具有BMSCs注射數(shù)量依賴性,其機制可能與BMSCs對炎性介質分泌的調節(jié)有關。5,在體內和體外實驗中,BMSCs均可抑制淋巴細胞增殖,調節(jié)炎性因子的釋放,這種作用不依賴與細胞與細胞之間的直接接觸,而且在一定BMSCs數(shù)量范圍內,具有數(shù)量依賴性。
[Abstract]:Objective : To study the protective effects of bone marrow mesenchymal stem cells ( MSCs ) on lipopolysaccharide ( LPS ) induced diffuse intravascular coagulation ( DIC ) in rats and to explore its mechanism .

Study method :

In the first part , the bone marrow mesenchymal stem cells of Wistar rats were cultured in vitro . The cultured bone marrow stem cells were observed and their proliferative capacity was investigated .
The cell phenotype was identified by flow cytometry .
The differentiation of bone marrow cells into endothelial cells , osteoblasts and adipocytes was cultured under specific conditions to confirm their potential plasticity and to further determine their potential plasticity .

In the second part : LPS - induced DIC model of Wistar rats was established . 30 adult male Wistar rats were randomly divided into 2 groups : platelet ( PLT ) quantity , fibrinogen ( Fib ) , partial thrombase time ( PT ) , prothrombin time ( PT ) and D - 2 mer ( D - D ) .
At the same time , 100 glomeruli were counted , and the proportion of glomeruli with fibrin microthrombus was observed to determine whether LPS could induce the establishment of DIC model successfully .

The third part : lentivirus - mediated GFP - mediated bone marrow mesenchymal stem cells and in vivo detection . The supernatant of the virus was obtained after packaging with slow virus vector ( PGCL - GFP , pHelper1.0 , pHelper2.0 ) .
On the 5th day , the average transfection rate was calculated from 5 visual field of each well , and the relationship between the infection rate and the transfection efficiency was studied . Ten male Wistar rats weighing 160 - 170g were injected into 1 ml culture medium ( about 1 脳 106 ) at intervals of 24 h and 24 h . After 24 h , the rats were sacrificed . After 24 h , the rats were sacrificed . After 24 h of the third injection , the rats were sacrificed and then HE and anti - GFP peroxidase staining were performed .

In the fourth part , 60 adult male Wistar rats , 7 weeks of age , 160 - 170g , were randomly divided into 6 groups according to the pre - treatment and LPS injection protocol . After the last pretreatment , 1 ml of LPS ( dose , 3 mg / kg ) was injected into 2 : 1 ml culture solution ( containing BMSCs1x103 ) for 3 times . After the last pretreatment , 1 ml of LPS ( dose , 3 mg / kg ) was injected . Treatment group 3 : 1 ml of culture solution ( containing BMSCs1x104 ) was injected three times at each interval for 24 hours , and the pretreatment was performed . After the last pretreatment , 1 ml of LPS ( dose , 3 mg / kg ) was injected . Treatment group 4 : 1 mI culture solution ( including BMSCs1x105 ) was injected three times at intervals of 24 hours for pretreatment . After the last pretreatment , 1 ml of LPS ( dose , 3 mg / kg ) was injected . Treatment group 5 : 1 ml culture solution ( containing BMSCs1x106 ) was injected three times , each time interval was 24 hours , pretreatment was performed . After the last pretreatment , 1 ml of LPS ( dose , 3 mg / kg ) was injected . In order to further demonstrate the protective effect of pre - treatment on LPS - induced DIC rats , the following parameters were measured : PLT , Fib , D - dimer ( D - D ) , interferon - 緯 ( IFN - 緯 ) , interleukin - 1尾 ( IL - 1尾 ) , creatinine ( Cr ) , alanine aminotransferase ( ALT ) , interleukin - 1尾 ( IL - 1尾 ) , creatinine ( Cr ) , alanine aminotransferase ( ALT ) , interleukin - 1尾 ( IL - 1尾 ) , creatinine ( Cr ) , alanine aminotransferase ( ALT ) , creatine kinase isoenzyme ( CK - MB ) and endothelin ( ET ) . Group 3 : pre - treatment , ( 1 脳 105 / time 脳 3 times , 24 hours at each interval ) + LPS injection ( 10 mg / kg , 24 h after the last pre - treatment ) . The survival rate of each group was observed in 24 hours after LPS injection , and the survival curves were drawn by statistical survival analysis .

The fifth part : To further clarify the protective mechanism of pre - treatment , we measured the effects of different doses of bone marrow cells on the proliferation of peripheral blood lymphocytes ( PBMC ) , and studied the effects of different doses of allogeneic bone marrow cells on the proliferation of PBMC stimulated by LPS in vitro and the effects on inflammatory cytokines .

Results : 1 , 1 - 5 days , the rapid growth of cells was the fastest growing period , and the value added at the 6th day reached the peak , after which small amplitude fluctuation was maintained , and the growth of bone marrow cells was increased . After differentiation culture , the cells of bone marrow were differentiated into endothelial cells , osteoblasts and fat cells .
The expression of CD29 , CD44 , CD10 and CD106 in bone marrow cells was detected by flow cytometry .
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